United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/9-78-037
November 1978
Research and Development
x>EPA
Short-Term Tests for
Health and Ecological
Effects
Part 1: Program Overview
Part 2: Directory of Tests
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/9-78-037
November 1978
SHORT-TERM TESTS
FOR HEALTH AND ECOLOGICAL EFFECTS
PART'I: PROGRAM OVERVIEW
Sponsored by the
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington, District of Columbia 21040
Hosted by the
"Genetic Toxicology Program
Biochemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use. Men-
tion of specific contractors does not necessarily indicate either exclusive
engagement or recommendation.
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinoge' esis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory participates in the development and
revision of air quality criteria documents on pollutants for which national
ambient air quality standards exist or are proposed, provides the data
for registration of new pesticides or proposed suspension of those already
in use, conducts research on hazardous and toxic materials, and is primarily
responsible for providing the health basis for non-ionizing radiation
standards. Direct support to the regulatory function of the Agency is
provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of
health care and surveillance of persons having suffered imminent and
substantial endangerment of their health.
Historically, the Health Effects Research Laboratory has made a strong
commitment to the development and implementation of short-term tests for
potential health effects of environmental agents.
This commitment is evidenced by the formation within this laboratory of
.two new programs; the Genetic Toxicology Program and the Neurotoxicology
Program. These programs will require redirection and commitment of new
resources to develop techniques that rapidly evaluate pure chemicals and
complex environmental samples for possible genotoxic and neurotic hazard.
Research on short-term tests for health and ecological effects is ad-
vancing rapidly throughout the Office of Health and Ecological Effects. This
document should prove of significant value in maintaining coordination of the
research program during this growth period.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
I I !
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PREFACE
At the request of Dr. Delbert Barth, Deputy Assistant Administrator for
the Office of Health and Ecological Effects (OHEE), the first OHEE Workshop on
Short Term Tests for Health and Ecological Effects was held January 18-20, 1978,
at the U.S. Environmental Protection Agency (EPA), Health Effects Research
Laboratory, Research Triangle Park (HERL-RTP), North Carolina.
The need for such a meeting, especially in the area of genetic toxicology,
was suggested by Dr. Alexander Malcolm of the U.S. Environmental Protection
Agency at Narrangansett, Rhode Island. The Workshop was planned and coordinated
by Dr. Frode Ulvedal and Dr. George Armstrong of OHEE with the Assistance of
Dr. Malcolm and the staff of the Biochemistry Branch, HERL-RTP.
The following objectives of the Workshop were established and transmitted
with Dr. Barth's letter of invitation to the seven OHEE laboratories and to the
National Center for Toxicological Research:
• Produce a directory listing OHEE's screening system efforts with the
address and telephone numbers of the key people associated with these
efforts;
• Initiate a continuing dialogue among the various investigators as well
as visitations among sister laboratories;
• Formulate an agreement for the coordination of the Agency's efforts in
health and ecological bioassays; and
• Identify OHEE's needs for future research in this area.
The following Proceedings of the Workshop reflect the presentations given
by the representatives of the OHEE laboratories and the National Center for
ToxicologicaI Research. These statements describe the research interests and
capabilities of the participating laboratories to perform short-term bioassays
i v
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PREFACE (continued)
for potential health and ecological effects of environmental agents. It is
hoped that additional workshops will continue to improve coordination among
the EPA laboratories involved in short-term tests for health and ecological
effects.
A major initiative of the Workshop was the preparation of a directory of
the short-term tests currently being performed throughout OHEE. This document,
the Directory of Short-Term Tests for Health and Ecological Effects, provides
information on the tests themselves, the laboratories where they are being per-
formed, and the key individuals involved. It should prove to be an important
Agency and interagency reference to this rapidly growing and challenging field
of scientific investigation. The Directory also seeks to further the intent
of the Workshop, namely to enhance communication, collaboration, understanding,
and appreciation of a major component of the U.S. Environmental Protection
Agency's research program.
Michael D. Waters, Ph.D.
Coordinator, Genetic Toxicology Program
Chief, Biochemistry Branch
Health Effects Research Laboratory
Research Triangle Park, North Carolina
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CONTENTS
Foreword i i i
Preface iv
Acknowledgment VIM
List of Attendees ix
Introductory opeech, by Delbert Earth, Ph.D.
Overview of Research on Short-Term Predictive Methods at the
Environmental Research Laboratory-DuIuth,
by James M. MoKim, Ph.D 5
Overview of Short-Term Testing at the Environmental Research
Laboratory-Corva I I is, by William Miller II
Overview of Short-Term Testing at the Environmental Research
Laboratory-Narragansett, by Alexander R. Maloolm, Ph.D 19
Overview of Short-Term Testing at the Environmental Research
Laboratory-Gulf Breeze, by Steven C. Sohimmel 27
Overview of Short-Term Testing at the Health Effects Research
Laboratory-Research Triangle Park, by Stephen Nesnow, Ph.D 31
Screening Activities at the Health Effects Research Laboratory-
Cincinnati, by Richard J. Bull, Ph.D 43
Overview of Short-Term Bioassays Performed at the National
Center for Toxicologica1 Research, by Daniel A. Casciano, Ph.D.. . 49
Directory of Short-Term Tests for Health and Ecological Effects 53
vii
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ACKNOWLEDGMENT
The oral presentations and written overview papers of each speaker, the
participation of each attendee, and the editing and final typing by Northrop
Services, Inc., are gratefully acknowledged. Dr. Stephen Nesnow was respon-
sible for the technical editing of the Program Overview.
vi i i
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LIST OF ATTENDEES
NAME
Delbert Barth
Frode Ulvedal
George Armstrong
John Knelson
WiI I jam Durham
James McKim
Robert Drummond
Richard Anderson
Steven Hedtke
WiI I Jam Mi
Jay Gi le
ler
Alexander Malcolm
Norman Richards
Steven Schimmel
Michael Waters
Joel I en Hu i si ngh
Stephen Nesnow
Shahbeg Sandhu
Lawrence Reiter
Diane Courtney
Jeffery Charles
Nei I Chernoff
Robert Chadwick
Richard Bui I
Bernard Daniel
Dan Casciano
AFFILIATION
U.S. EPA/ORD/OHEE
U.S. EPA/ORD/OHEE
U.S. EPA/ORD/OHEE
U.S. EPA/ORD/OHEE
U.S. EPA/HERL-RTP
U.S. EPA/ERL-DUL
U.S. EPA/ERL-DUL
U.S. EPA/ERL-DUL
U.S. EPA/ERL-DUL
U.S. EPA/ERL-COR
U.S. EPA/ERL-COR
U.S. EPA/ERL-NAR
U.S. EPA/ERL-GB
U.S. EPA/ERL-GB
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-RTP
U.S. EPA/HERL-CIN
U.S. EPA/HERL-CIN
NCTR
IX
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Introductory Speech
by
Uelbert Barth, Deputy Assistant Administrator
Office of Health and Ecological Effects
U.S. Environmental Protection Agency
Washington, District of Columbia
This morning, I hope to be able to set the stage for what you will do over
the course of the next day or two. I wiI I give you some of my impressions as
to what the most important things are that should transpire. In short, what
we have to do is to unify the Office of Health and Ecological Effects (OHEE);
once that is accomplished, we will have to unify the Office of Research and
Development (ORD), and then the Environmental Protection Agency (EPA). Many
different people within EPA are carrying out many different activities that go
in many different directions. These activities are not being adequately co-
ordinated. I feel that even in our own office, the Office of Health and Ecolog-
ical Effects, we are not fully coordinated. That is the principal purpose for
this first meeting. Once we are organized, we will be prepared to sponsor
meetings of this sort with a much broader attendance; people from other offices
will attend, particularly from such programs as the Office of Toxic Substances
(OTS) which is obviously interested in the entire subject.
The importance of biological testing within OHEE is twofold. First, these
tests may be used as screening devices to set priorities for additional and
more indepth studies. Certainly, we are concerned with short-term acute effects,
The effects that we are most concerned about, however, are the chronic ones.
Ultimately, very expensive and time-consuming tests are necessary to arrive at
the chronic effects from long-term exposure to various kinds of environmental
pollutants. If we can devise short-term tests capable of predicting where to
look first for chronic effects, our program for chronic effects will be greatly
helped.
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Secondly, these types of tests may be applied to the entire area of risk
assessment. When I say "risk assessment", I am talking in the broadest possible
terms. We must define what the data needs are and how to interpret the data in
order to conduct a meaningful risk assessment. In dealing with batteries of
tests, we are on quite infirm ground if we arrive at the risk associated with
various environmental pollutants solely on the basis of a single test or even
on the basis of a few tests. Perhaps someday we will be able to do so. At pre-
sent we must define the capabilities and limitations of the various tests, also
the quality assurance procedures that will have to be related to these tests.
Ultimately, these tests will have to be written up in "cook-book" fashion in
the Federal Register; they must be capable of being conducted by industrial
groups on the basis of the descriptions found in the Federal Register.
The objectives of this particular meeting are fourfold. First and foremost
we should have an information exchange. We should bring to each other's atten-
tion all of the work that is going on in our respective laboratories in this
area. After finding out what these efforts are, we should develop mechanisms
to coordinate the ongoing work in all of the various laboratories. Then we
should develop the research needs that we perceive in this area. And finally,
we should develop a joint planning mechanism to accomplish those research needs
in the future.
The very foundation for cooperation has to be laid in the planning stage.
This cooperation will be very difficult if we each go and plan independently,
start a project moving in a certain direction, and then try to make mid-course
corrections in order to avoid overlaps with other programs. It would be much
better to avoid the necessity for such corrections before commencing any of the
studies.
I will outline some topics that you might consider for special emphasis.
Some of these I have already alluded to. Certainly, the capabilities and limi-
tations of all of the tests that we are presently working with will have to be
established. When I say "capabilities and limitations," 1 mean in the broad
sense. In short, what we need are error terms. How many times are we going to
get false positive results with these tests, or how many times are we going to
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get false negative results? We need a Research and Development (R and D) program
to investigate these questions for the various tests. Within ORD and EPA we
also need a mechanism for carrying out routine testing. Once we have established
the procedures, I personally do not want to see our research laboratories en-
cumbered with large numbers of routine tests. It is entirely possible that this
could be done by a contractor; a captive contract laboratory with a project
officer on the R and D side would perhaps suffice. In addition, I foresee the
need for a rapid turnaround on aquiring results with respect to some pollutant-
of-the-day or pollutant-of-the-month that has been identified for concern.
Immediately, everybody wants to know what the effects of this pollutant are and
quite often we have virtually no data. Obviously, we need some kind of a
procedure to obtain results quickly from at least some of the shorter tests and
then to indicate whether further tests are needed. Then, someone must decide
whether or not to go ahead with further tests, particularly if they become
extremely expensive.
In this whole area of testing, a clear statement assigning responsibilities
has not been made by the agency as a whole, i.e., by EPA. There are obvious
requirements for such a statement. The Office of Pesticide Programs is very
much concerned with this matter as is the Office of Air Programs for the Fuel
and Fuel Additive Program. And, of course, OTS is involved. ORD has not
clearly specified who does what to whom, when and why in this area. And we
haven't done it in OHEE. Hopefully, some draft statements will result from
this meeting, designating where these responsibilities should lie. We need
some kind of a draft statement to at least say that here is what we think OTS
should be doing. Here is what we think ORD should be doing. Here's what OHEE
should be doing. Here's what Air, Land, and Water Use should be doing. Here
is what the Office of Energy, Minerals, and Industry (OEMI) should be doing,
etc. You will not be able to reach any final decision on this as people out-
side of OHEE are involved. But somewhere, someone has to develop a "straw man"
that suggests how these responsibilities should be assigned. As it is now,
everybody is going off in his own direction; consequently, these efforts lack
coordination.
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Finally, we will need a five-year R and D plan for this entire problem
area of coordination. I don't expect you to do it at this particular meet ing,
but perhaps you may be able to outline some of the pieces. This plan shquld .„:*.
set forth the kind of objectives that should be met, the kind of approaches* ......
that should be taken to meet these objectives, how time lines and various
milestones should t?e cjeveloped over the course of the five years, and how the,
resources needed to carry out this R and D plan should be assigned.
That is the end product. However, as I said earlier, I do not expect a
polished plan to. come out of this; you can't do everything in a single meeting.
Whatever you can do in the nature of at least outlining the plan would be ex-
tremely helpful in order to build future meetings of this sort with a 'broader
attendance. Once we unify OHEE, then as I indicated, we will be able to bring
other people in.
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OVERVIEW OF RESEARCH ON SHORT-TERM PREDICTIVE METHODS
AT THE ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
by
James M. McKim, Ph.D., Chief
Physiological Effects of Pollutants Section
Research Branch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Duluth, Minnesota
The Environmental Research Laboratory-Duluth (ERL-DUL) has devoted a
considerable amount of research effort toward the development of tests involv-
ing aquatic animals. These tests seek to determine the subtle toxic effects
caused by low levels of environmental contaminants.
The initial efforts involved the development and laboratory use of chronic
toxicity tests, exposing a particular species throughout an entire life-cycle
and through all stages of development. Initially only fish were utilized, but
more recently several invertebrate species were included. The biological end-
points observed and measured during these chronic tests were survival, growth,
deformities, reproduction, and residue buildup. These tests provided the
best predictive information available on the concentration of a waterborne
pollutant necessary to cause detrimental effects to specific animal popula-
tions; these tests are considered at this time, the ultimate laboratory test
for predicting "no effect" environmental contaminant concentrations for aquatic
animals. Further, a five-year field validation study by this laboratory revealed
the chronic toxicity test to be a valid useful tool in establishing environmental
concentrations of selected chemicals.
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However, these life-cycle tests present several problems:
• Time —each test requires I to 2 years to complete,
• Cost - $35v009.00 to $70,000.00 -per test, •
• Few Species —only four to five freshwater species can be
utilized successfully in a life-cycle test, and
• High Risk — it becomes diffieulj; tqNcoftti,n^an exposure for a year
or more without dfsease or mechanical problems interrupting the test.
Therefore, to eliminate these p,ro,bl^m,| a,n.4 ''t
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The remainder of this overview wiI I contain a series of short summaries
on each of the six predictive methods outlined in Figure I. For more detailed
information, please contact the ERL-DUL researcher associated with the sum-
mary write-up.
FRESHWATER LABORATORY ECOSYSTEMS FOR TOXICOLOGICAL TESTING (Steven Hedtke)
Traditional aquatic toxicology determines the impact of chemicals on
isolated single species. However, in order to evaluate the effects due to
the influence of other species, multi-species systems and laboratory ecosystems
can be useful. Information on biomagnification, changes in competition and
predator-prey interactions, modification of a toxicant's chemical form due to
the activity of other species, and changes in ecosystem processes can be ob-
tained. In addition, simultaneously testing several species can be more cost-
effective in obtaining fate and effects data.
Specific tests have been utilized or are being developed to monitor
changes in periphyton community structure, the uptake of compounds through
food/water, and the effect of toxicants on the carbon budget of laboratory
ecosystems.
INVERTEBRATE SHORT-TERM TESTS (Richard Anderson, Ph.D.)
Toxicological test systems using invertebrates are as diverse as the
animal's habitats. Invertebrate research at ERL-DUL has been directed toward
understanding the life cycle of and developing test procedures for cladocerans,
amphipods, and aquatic insects. Recent research has centered on the insects.
To develop toxicological tests, an understanding of the biology of the test
species is required. Two chironomids are now being reared in the laboratory
and egg-to-egg test systems are being developed. Tests that expose the animal
during specific developmental events are being developed. These tests may
decrease the exposure time without decreasing the biological implications of
the data. The first series of tests are examining the hatching process. Test
systems for mayflies, caddisflies, and stoneflies are also being developed.
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TESTS WITH EARLY LIFE STAGES OF FISH FOR PREDICTING LONG-TERM TOXICITY
(James McKIm, Ph.D.)
Partial and complete life-cycle toxicity tests with fish, involving all
developmental stages, have been used extensively to establish water-quality
criteria for aquatic life. During extended chronic exposures of fish to
selected toxicants, certain developmental stages have frequently shown a
greater sensitivity than others. In 56 life-cycle toxicity tests completed
during the last decade with 34 organic and inorganic chemicals and four species
of fish, the embryo-larval and early juvenile life stages were the most, or
among the most, sensitive. Tests with these stages can be used to estimate
the maximum acceptable toxicant concentration (MATC) within a factor or two
in most cases. Therefore, toxicity tests with these early life stages of
fish should be useful in establishing water-quality criteria and in screening
large numbers of chemicals. Work is now under way to further develop both
laboratory and field methods for conducting these tests, and to develop new
biological endpoints permitting shorter exposure times.
RESPIRATORY IMPAIRMENT IN FISH AS A SHORT-TERM INDICATOR OF TOXICANT STRESS
(Robert Drummond)
Various behavioral and physiological aberrations of fish have been used
to indicate stress due to the presence of toxic material. Changes in cough
(gill purge) frequency, opercular rate, oxygen consumption, feeding patterns,
locomotor activity, fin movements, eye movements, reproductive displays,
swimming ability, avoidance, and the ability to respond to a stimulus have
all been used as a measure of such stress. Of these, we believe that change
in cough frequency has wide potential application as a bioassay tool for the
following reasons:
• the response occurs in a variety of fish,
• many chemical substances are known to elicit the response,
• the endpoint is rapid and sensitive,
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• the degree of response usually occurs in direct proportion to the
toxicant concentration, and
• it is predictive of long-term adverse effects at levels near the
MATC values for most of the single toxicants investi'gated to date.
QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS (Gilman Veith, Ph.D.)
The application of structure-activity correlations to estimate the
toxic effects of organic chemicals to aquatic organisms is being evaluated
using a variety of biological test endpoints. A series of phenols are being
tested for lethality using 96- and 192-hour acute tests with fathead minnows,
30-day embryo-larvel tests, and cough response tests. The effects from the
respective tests are correlated with such structural parameters of the
chemicals as the n-octano!/water partition coefficient and the Hammet con-
stants. Present research is seeking to determine the precision of toxicity
predictions as well as the range of chemicals which can be included in a
single correlation.
Additional studies have shown that the chemical residues in fish exposed
to a given concentration in water can be estimated using a proportionality
factor called the bioconcentration factor. This work has shown a strong
correlation between the bioconcentration factor and the n-octanoI/water parti-
tion coefficient. Current efforts are attempting to develop an instrumental
screening technique through this structure — bioconcentration correlation —
to minimize the need for costly bioconcentration testing.
Simultaneously, a structure-activity data base is being compiled and
appropriate computer software developed to make the data base available on
the National Institutes of Health (NIH) and EPA computer systems.
STANDARD METHODS DEVELOPMENT (Charles Stephan)
About 1970 many aquatic toxicoIogists realized that a need existed both
for more cooperation and exhange of information between interested persons
as well as for the standardization of terminology and procedures. Today,
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through the American Society for Testing and Materials (ASTM), interested
persons from governmental, industrial, contract, state, and academic organi-
zations are working together on an equal basis to bring about such standardi-
zation in aquatic toxicology. The principle concerns at present are procedures
for conducting tests, terminology, testing schemes for using various tests,
and the culture of test organisms. The results to date show that standardiza-
tion by consensus of all interested persons does take time and effort, but is
probably the most efficient process for producing a product that has wide
acceptance.
10
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OVERVIEW OF SHORT-TERM TESTING
AT THE ENVIRONMENTAL RESEARCH LABORATORY-CORVALLIS
by
William Miller, Research Environmentalist
Special Studies Branch
Assessment Criteria Development Division
and
Jay Gile, Research Biologist
Terrestrial Ecology Branch
Ecological Effects Research Division
Environmental Research Laboratory
U.S. Environmental Protection Agency
Corvallis, Oregon
As a result of the U.S. Environmental Protection Agency's need to
establish test procedures and regulatory guidelines for specific inorganic,
organic, and complex waste pollutants, the Environmental Research Laboratory-
Corvallis (ERL-COR) has established a strong bioassay (screening) program.
This research involves both aquatic and terrestrial environments. The screen-
ing for ecological effects can and should occur at both the species and
community level. Not only should the effects on specific components of an
ecosystem be evaluated, but effects on community processes such as nutrient
cycling and respiration should also be examined.
AQUATIC RESEARCH
The Aquatic Research Program is classified into freshwater and estuarine-
marine categories. The freshwater research centers on the application of the
Algal Assay:Bottle Test (AA:BT), the estuarine-marine research on the estuarine-
marine version of the AA:BT, the Marine Algal Assay Procedure (MAAP). Both
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AA:BT and MAAP define the impact of point and non-point pollutants upon a-
quatic productivity by measuring algal growth. Estuarine-marine research
also includes the Marine Benthos Bioassay.
The AA:BT developed by ERL-COR has been used to evaluate the following
three areas:
• nutrient limitation,
• heavy metal toxicity, and
• the inhibitory/stimulatory effects of complex industrial wastes.
Nutrient limitation, defined in natural waters by the AA:BT, may be due
to nitrogen, phosphorus, or trace element deficiency. The growth response of
Selenastrum capricornutujn to single and combined additions of the above-men-
tioned pollutants is measured by incorporating an internal check and balance
system. The responses are evaluated to ascertain the trophic state of the
natural water and the limiting nutrients. The AA:BT has proven quite reliable
in predicting the algal growth potential of natural water.
The determination of heavy metal toxicity arose out of the evaluation
of the nutrient limitation. When a test water, based upon its chemical analysis
fails to attain the predicted yield or nutrient limitation status, the
presence of toxicants is usually indicated. The standard addition of 1.0 mg
of Na2EDTA l"1 to these test waters prior to the assay, is used to define the
extent of heavy metal toxicity.
Research conducted at ERL-COR indicated that the inhibition of specific
heavy metals upon the growth of Sejenastrum capricornuturn may be linear
(0 to 100$) with increased zinc content in the test waters, but nonlinear for
increased copper and cadmium content beyond 20 and 40/6, respectively. The
growth response of Selenastrum capricornutum to conditions of heavy metal
stress in natural waters is in essence a "biological response model" of complex
physical and chemical Interactions.
12
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Current research activity objectives are to determine if the growth re-
sponse of the test alga under standard laboratory conditions reflects the
response of sensitive indigenous species to recent discharges of heavy metals.
These responses may be used to establish realistic heavy metal water quality
criteria.
The AA:BT can also be used to define the potential stimulatory/inhibitory
effects of complex wastes on algal growth. In one experiment, seven techniques
evaluated twenty-three textile waste samples (representing eight manufactur-
ing processes) in order to define their toxic properties. The bioassessment
organisms included freshwater and marine algae, crustaceae, fish, and mammals.
A comparison of the sensitivity of these bioassays (see Table I) showed that
the AA:BT, using Selenastrum capricornuturn, was one of the most sensitive tests
used in the textile waste survey. This test not only identified toxic wastes;
it also identified those that were stimulatory.
The AA:BT protocol has been included in the |4th edition of Standard
Methods for the Examination of Water and Wastewater as well as in the EPA
Biological Methods Manual. This method is also under review by the American
Society of Testing and Materials (ASTM).
When the Toxic Substances Control Act (TSCA) outlined in Public Law
94-4(59 was established in October of 1976, test procedures and effluent guide-
lines for pollutants had to be established. A flurry of bioassessment activity
was stimulated, as only the bioactive components of the pollutants are re-
sponsible for the regulation of biological productivity in natural waters. At
the same time, the chemical analysis of specific constituents within the
complex wastes (i.e., zinc, copper, cadmium, phenol, PCB, aniline) continued to
be accepted as the primary reference standard for the legislation of ecological
response criteria; this procedure proved to be both unwise and misleading.
Only concurrent evaluation of both chemical analysis and bioassay results can
provide the scientific base necessary to establish realistic water quality
c r i te r i a.
13
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TABLE 1. COMPARATIVE BIOTEST RESPONSES FOR TEXTILE EFFLUENTS*t
Freshwater ecology
Textile
Plant
A
B
c
D
E
F
G
H
J
K
L
M
N
P8
R
S
T
U
V
w
X
Y
z
Fathead
Minnow
(96-hr LC50).
X secondary
effluent
19.0
NATb
46.5
NAT
NAT
NAT
64.7
c
NAT
NAT
23.5
NAT
48.8
NAT
16.5
NAT
46.5
NAT
36.0
55.2
NAT
NAT
NAT
Daphnia
(48-hr EC50)
X secondary
effluent
9.0
NAT
41.0
NAT
7.8
81.7
62.4
40$ dead at 100)
concentration
NAT
NAT
28. 0
60.0
100$ dead at all
dl lutlons
NAT
8.0
NSAd
NAT
12.1
9.4
6.3
NAT
NAT
42.6
series
Selenaatrwn
(14-day EC 50),
% secondary
effluent
11.3
-
-
-
< 2.0
-
-
7.8
-
-
12.0
-
< 2.0
.
8.8
-
-
-
-
1.0
-
-
15.5
Recommended
• Interpretation •
Selenoetnm
20% secondary
effluent
% Im | * Su
53
83
- 187
- 100
95°
598
- 390
92
76
57
61
149
95e
38
95
382
1911
377
232
95
163
261
84
Marine ecology series
Sheepshead
Minnow
(96-hr LC50),
X secondary
effluent
62.0
NAT
69.5
f
NAT
NAT
NAT
f
f
NAT
NAT
f
47.5
f
f
NAT
68.0
NAT
f
37.5
NAT
f
f
Grass
Shrimp
(96-hr LC50),
X secondary
effluent
21.2
NAT
12.8
f
NAT
NAT
NAT
1
f
NAT
NAT
f
26.3
f
f
NAT
34.5
NAT
f
I9.6
NAT
f
f
Algae
(96-hr EC50),
X secondary
effluent
f
g
90.0
f
10 to 50
85.0
59.0
f
•f
77.0
1. 7
f
2.3
9.0
t
g
70.0
9
94.0
50.0
g
f
f
•--•
"Sample Inadvertently collected prior to settling pond. No acute toxlclty. cDlseased batch of fish nullified this
analysis. No statistical analysis because heavy solids concentration obscured the analysis; the sample does not
appear to be acutely toxic. e95$ growth Inhibition In 2t solution of secondary effluent. Analysis not performed
on this sample. 96rowth Inhibition < 50)1 In 100$ solution of secondary effluent. *No chemical mutagen was detected
by the 10 nlcroblal strains. No rat mortality after 14 days due to maximum dosage of I0~ tn'/kg body weight (LD50).
However, six samples (B, C, F, L, N, and S) showed potential body weight effects, and sample R resulted In eye Irri-
tation.
14
-------
The Marine Algal Assay Procedure (MAAP) defines in estuarine-marine
waters the aquatic primary productivity of algal growth under the influence
of such major nutrients as nitrogen, phosphorus, and silica. This test,
using the green flagellate Dunaliejla tertiolecta and the green alga SeIe-
nastrum capricornutum, can also indicate the nutrient limitation and the po-
tential primary productivity from 0 to ~55% salinity under a wide range of
nutrient conditions. The MAAP has been included in the 14th edition of Stan-
dard Methods for the Examination of Water and Wastewater.
The MAAP has been used to delineate the change from phosphorus-1imi ted
freshwater to nitrogen-)imited marine water in estuaries, as well as to
measure the effect of dredge spoil using the soluble fraction of the Standard
Elutriate Test, the toxicity and biostimulation of detergent formulations,
and the organochlorine residual in secondary effluents. The test has also
monitored the nutrient/physical relationships in Oregon estuaries. Currently,
the MAAP is being adapted for use with suspect toxic compounds under the TSCA
program.
The Acute Benthos Toxicity Bioassay developed at ERL-COR's Newport Field
Station roughly simulates the dumping of dredged materials into the sea.
Since the benthos is affected by the settleable fraction of dredged sediments,
five macrobenthic invertebrates were selected as the test species. Mean sur-
vival compared to the control after 10 days of exposure, is the primary
response criterion. Bioassays have been conducted for sediments from the
Atlantic, Gulf, and Pacific coasts of the United States. The procedure has
been published by the U.S. Corps of Engineers in its Implementation Manual for
Ocean Dumping Regulations.
The bioassay also has potential in determining the toxici.ty of sediment
at sewage and dredge material dump sites. An experiment is planned to com-
pare the benthic impact of sediments from the New York Bight sludge dump site
and relatively clean areas of the Bight. The results will be correlated with
the benthic community structure survey.
15
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TERRESTRIAL RESEARCH
The Terrestrial Research Program at ERL-COR focuses on the development and
refinement of short-term tests on single plant species, portions of ecosys-
tems (e.g., soil and litter), and whole simulated ecosystems.
ERL-COR is currently working on two single plant bioassays. One is the
Stress Ethylene Bioassay and the other the Acetylene Reduction Test.
The first plant bioassay is based on a plant's normally low production
level of hormonal ethylene. However, when plants are subjected to environ-
mental stresses, plant ethylene production increases. During the test, the
plants are maintained in a greenhouse in sealed exposure chambers and the
pollutant is metered into the chamber.
In a study of ozone stress, for example, the increase in the amount of
ethylene produced was influenced by both the amount of ozone applied and the
plant species. The stress ethylene test not only appeared to be subject to
less variability than visual assessment, but it also seemed more sensitive as
evidenced by the lack of visible foliar injury associated with increased
ethylene production on pine and potato. The induction of stress ethylene by
ozone was a short-lived phenomenon with a duration of up to 48 hours following
exposure.
The second plant bioassay relies on the ability of a nodulated plant to
reduce acetylene to ethylene. The process is monitored to indicate the acti-
vity of the enzyme nitrogenase.
Three types of soil microcosms are in various stages of development at
ERL-COR. Research Is also being conducted on a whole simulated ecosystem
called the Terrestrial Microcosm Chamber.
The first two microcosm assays emphasize the decomposition processess
occurring in a soil/litter ecosystem. These systems allow the investigators
to examine changes In respiration over time as a result of:
-------
• the variation between "identically" prepared sets of microcosms,
• the soil/litter moisture effects on microcosm respiration,
• the salt quality/quantity effects on microcosm respiration and
certain other pollutant effects such as:
— the effects of microcosm age and time of pollutant addition
on microcosm respiration,
— the effects of high concentrations of various metals and
nonmetaIs on microcosm respiration,
— the effects on respiration of combinations of two toxic
substances, and
— the effects on respiration of toxic substances on organic
m?vter amended soil/litter microcosms.
The third soil system under development at ERL-COR is an adaption of
earlier systems designed at Oak Ridge Laboratory. The underlying rational
for this system is that intact excised soil cores most reliably represent the
natural system since they retain the soil structure and closely simulate the
abiotic and biotic complexity of the ecosystem.
The fourth system used at ERL-COR is the Terrestrial Microcosm Chamber.
Within a glass box I x 3/4 x 2/3 m high with a plexiglass lid, it has been
attempted to simulate a whole ecosystem including sun cycle, temperature
ranges, a "rain", "spring", and "groundwater" system, membrane and charcoal-
filtered air, a local top soil, biotic components comprising primarily alfalfa
and perennial ryegrass, and fauna. The fauna include representatives from
soil invertebrates, scavengers, pests, and the gray-tailed vole from the
mamma Is.
The Terrestrial Microcosm Chamber provides information on disposition and
metabolism within the system. The following effects can be examined:
• chronic and acute phytotoxicity,
• acute and subacute insecticidal action,
• bioaccumulation,
17
-------
• fetal transfer of residues,
• altered soil respiration,
• interference with nutrient retention,
• mammlfan toxicity, and
• behavioral effects.
Future plans for microcosm research include comparison of the various
soil systems with soil processes in the larger microcosms and the incorpora-
tion of a soil core system within a larger microcosm for testing such toxic
i I
substances as CU , CH3HgCI, 2, 4-D, and DDT.
18
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OVERVIEW OF SHORT-TERM TESTING
AT THE ENVIRONMENTAL RESEARCH LABORATORY-NARRAGANSETT
by
Alexander R. Malcolm, Ph.D., Team Leader
Genetic Toxicology Team
Toxicology Branch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Narragansett, Rhode Island
Within the Research Division, primary responsibility for the development
and application of screening and other short-term tests lies with the Toxi-
cology Branch. Of the four teams comprising this branch, Marine Toxicology,
Response Parameters, Genetic Toxicology, and BioaccumuI at ion/Kinetics, the
first three are directly involved with short-term endpoints. Several other
areas (Mussel Watch, Histopathology, Coastal Environmental Assessment Station
[CEAS]), benthic aspects of the response parameters program, and selected
assays for bi©accumulation designed primarily for long-term assessments,
possess the capacity to provide certain types of short-term, toxicological
data. These may be appropriately introduced at a future time. For the purposes
of this initial meeting, the overview shall be limited to those systems specifi-
cally designed for short-term evaluations.
MARINE TOXICOLOGY TEAM
The Marine Toxicology Team has responsibility for the development of the
bioassay techniques that permit the interaction of test organism and pollutant
in a manner closely approximating their natural encounter. The program has and
shall continue to focus on the development of methodology for a wide variety
of estuarine species of both ecological and commercial importance. Species
representing different communities, trophic levels, and feeding types are
19
-------
selected so as to permit the evaluation of impact on a wide scale. Although
emphasis has been on short-term techniques, efforts have been expanded to in-
clude the whole life cycle, utilizing sensitive, early life histories. This
permits evaluation and comparison of the short-term and reproductive effects
necessary for projections of population viability.
The program is also strongly directed toward problems of bioaccumulation
and the significance of body burden to both public health standards and to
effects on reproduction and population integrity. Bioassay designs should
reflect more closely actual exposure profiles, thus permitting more realistic
estimates of impact.
Current research efforts involve the development of whole life-cycle
bioassays on molluscs, polychaete annelids, and oppossum shrimp. The life-cycle
stages presently being utilized are fertilization, developing embryo, larval,
post-larval metamorphosis, juvenile, and adult. Pollutant stress indicators
include survival, growth-molting, fecundity, hatchabiMty/embryo viability,
feeding rate, respiration, motile behavior, histopathology, cytogenetics,
skeletal abnormalities, and biochemical composition. The major assays are
summarized in Table I.
Other areas of interest include the design of an exposure system allowing
modulation and the simulation of environmental variables such as salinity and
temperature. Methodologies developed by the Marine Toxicology Team and other
teams of the Toxicology Branch have been incorporated into the Region I I Ocean
Disposal Permit Testing Program, the EPA Ocean Disposal Manual, and Standard
Methods.
RESPONSE PARAMETERS TEAM
A major objective of the Response Parameters Team is to elucidate a series
of sublethal response parameters for motile invertebrate larvae and zooplankton.
Parameters being studied include patterns and rates of swimming, motile responses
to specific stimuli (light, gravity, food, other chemical cue), feeding and ener-
gy budget, and histology. To date, indices of larval viability and physiological
20
-------
TABLE 1. BIOASSESSMENT METHODS FOR HAZARD EVALUATION IN THE MARINE ENVIRONMENT
Organism
Phytoplankton
Mac roa 1 gal
Zooplankton
Crustacean
Larvae
Grass Shrimp
My s Ids
Sheepshead
Minnow
Embryo/Fry
Mol luscan
Larvae and
Adu 1 ts
Acute
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Chronic
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Life Cycle
Yes
No
Possible
Yes
Yes
Yes
Yes
No
Yes
Bloaccum
Poss I b 1 e
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Contract
Yes
No
Yes
Possible
Yes
Yes
Yes
Yes
Yes
Lab
Yes .
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Field
Yes
Possible
No
No
No
No
No
No
No
Comments
Excellent short-term
screen; readily available.
New method; promising but
not yet ava liable.
Sensitive but difficult
method; needs work in
1 Ife-cycle area.
Potentially useful screen;
not routinely used.
New method; limited in-
formation.
Excellent new technique;
moderately sensitive
and readily available.
Excellent for chronic
studies; moderate sensi-
tivity.
Excellent measure of
sens 1 1 i ve 1 i f e stage
effects.
Excel lent assays for
chronic and bl ©accumulation;
not often contracted; larval
stages quite sensitive.
Polychaetes
Yes
Yes
Possible
Yes
Yes
Yes Possible
New assays not yet widely
applied; offer potential
for sediment bound toxicants
and food chain studies.
-------
condition have been limited primarily to survival, development to post-larval
stage, rate of development, stage size, and morphological normality. Preliminary/
experiments suggest that the selected sublethal parameters proposed above may
be sufficiently sensitive to permit the short-term detection of adverse toxicant
effects in invertebrate larvae at, or significantly below, levels indicated by
general developmental parameters alone.
Present efforts are devoted to exploratory studies to select the principle
study species and specific behaviors. The behaviors of choice will be those
clearly typical of the field situation and capable of being related to population
success. Behavior which can be tested simply is pursued first. Selection of
the principal study species for subsequent, indepth investigations is based on:
• duration and season of larvae or zooplankton availability,
• possession of swimming patterns that can be analyzed by the Bug-
watcher System (see Figure I), and
• high sensitivity of organisms to various toxicants (metals, oils,
etc.).
Table 2 lists some potential study species and their season of availability.
WINTER
DISPLAY
SCOPE
COMPUTER
D*u G*ntr*l Echpw S/200
KEYBOARDS
TV
CAMERA
BUGWATCHER VIDEO TAPE RECORDER
BUGS
Figure I. Diagram of the Bugwatcher System.
22
-------
TABLE 2. POTENTIAL INVERTEBRATE LARVAL AND ZOOPLANKTON STUDY SPECIES
Organism
Season of availability
Crustacean larvae
Balanus balanoides
Chthamalus
B. improvisus
B. amphitrite
Libina
Cancer
Crangon
Pa Iaemonetes
March-Apri I
May-August
summer — year round
summer — year round
June-September
January-August
May-October
June-September
in laboratories
in laboratories
Zooplankton
Eurytemora affinus
Mysidopsis bahia
M. bigelowei
year round cultures
year round cultures
year round cultures
Mollusc Larvae
Crepidula
Nassarium
Mu Ii nea
Crassostrea
MytiI us
Rotifera
Brachionus plicatilis
June-September/October — year round in
laboratories
June-August — January-August in labora-
tories
June-August — January-August in labora-
tories
June-August — January-August in labora-
tor i es
June-August — January-August in labora-
tories
year round (low-salinity species not
of common occurrence in marine systems)
23
-------
The specific behaviors that are being explored include typical swimming
behavior (locomotory rate and patterns) and shifts in locomotory responses to
specific environmental stimuli (e.g., light, gravity, temperature, salinity,
chemicals-foods, sex pheromone, and alarm substance). All motile behavior
studies utilize the computerized behavioral analysis system (Bugwatcher).
This system is used to quantify the movement and behavior of small, experimental
organisms. With this system, alterations of locomotory or other behavioral
patterns of organisms induced by sub lethal concentrations of the toxicant can
be detected and described. This methodology could represent an early warning
system for stress prior to irreversible damage to or death of the animals.
The system may also permit rapid screening of suspected toxicants.
GENETIC TOXICOLOGY TEAM
The research conducted by the Genetic Toxicology Team encompasses both
mammalian cell and marine assays. At the present time considerable emphasis Is
being given to the development and application of in vitro mammalian cell
assays as indicator systems to identify and assess the DNA-damaging agents in
laboratory studies. This program, initially confined to a single assay for
gene mutation using nutritional markers, has recently been expanded to include
assays for DMA repair and cytogenetic effects. Plans have been formulated to
add a transformation test in the near future. In combination, these short-term
endpoints span the major types of effects induced by genetic toxicants and
represent tests certain to play an increasing role In the regulatory process.
Immediate goals Include the standardization of each assay with known compounds
and the addition of in vitro metabolizing capabilities. The various systems
will then be applied to:
• validation,
• improvement of methods for application,
• problem identification, and
• specific research projects to be determined in consultation with
other OHEE laboratories.
24
-------
The mammalian cell assays may also be utilized in conjunction with various
monitoring programs such as CEAS and Mussel Watch.
The Gene Mutation Research Program is under the direction of A.R. Malcolm
and K.O. Cooper. Current emphasis is on the establishment of an HGPRT locus
assay in Chinese Hamster Ovary (CHO) cells and on improvement of the assay
employing nutritional markers (CHO eel I with 5-bromodeoxyuridine-visible
light selection). In the case of the CHO Cell/BrdU-VL system, the major
problem is the loss of mutants due to the effects of starvation during selec-
tion. Because there are so few markers for measuring mutation in mammalian
cells, further development of this system would be justified.
Research on Cytogenetic Effects is being carried out by G.G. Pesch and
M.M. Barry. An assay for Sister Chromatid Exchange (SCE) is presently being
established with the CHO cell. This cell appears to be we 11-suited for SCE
analysis because of a relatively small chromosome number — 20 — as well as ease
of propagation. It is anticipated that this portion of the program will be
extended to include some of the more traditional types of chromosomal effects.
The assay for SCE has been developed to the point where dose response has been
demonstrated with alkylating agents such as ethyl methane sulfonate. Standard-
ization is continuing with mytomycin C and other compounds.
DMA Repair Research is being conducted by E.H. Jackim and W. Lindblad.
A test for DNA repair, as measured by unscheduled DNA synthesis, is being im-
plemented with the CHO cell. Although the CHO cell may not be the ultimate
cell of choice for the repair test, it has been possible to demonstrate a
significant repair response in populations exposed to ultra-violet radiation
and certain carcinogenic chemicals. A measurable but less than significant
response has been observed with ethyl methane sulfonate. Other standard com-
pounds are being evaluated and the assay is still being refined and modified.
A.R. Malcolm and K.O. Copper will also be responsible for Cell Transfor-
mation Research. The transformation test has not yet been initiated, but work
with the BALB/c3T3 system is scheduled to begin in June of this year.
25
-------
The research in Marine Assays is the responsibility of G.G. Pesch and
staff. It is well known that coastal environments are often impacted with
industrial and domestic discharges containing substantial quantities of
metals, organochlorines, oils, etc., many of which may be genetic toxicants.
Such toxicants not only accumulate in edible marine organisms where they be-
come a direct threat to man, but may also have a detrimental impact on the
genetic structure of natural populations. Initial objectives for the marine
portion of the program include:
*
• the development of short-term assays that can be coupled with long-
term, life-cycle assays, and
• the development of methods for in situ monitoring.
The latter should prove particularly valuable for the identification and
management of point source problems.
Present research efforts are directed toward the development of metho-
dology for the detection of spontaneous and induced genetic damage in the eggs
and larvae of selected marine species under both laboratory and field conditions
Cytogenetic parameters are emphasized. Eggs and larvae comprise the material
of choice for technical reasons. These early, rapidly dividing growth stages
are particularly sensitive to the action of mutagenic agents and are well-suited
to analysis with cytogenetic techniques. An organism of particular monitoring
interest is the marine annelid Neanthes. This animal is receiving considerable
attention as a bioassay tool in several laboratories- and has been found to
possess a small number — 16 to 18 —of large, metacentric chromosomes.
26
-------
OVERVIEW OF SHORT-TERM TESTING
AT THE ENVIRONMENTAL RESEARCH LABORATORY-GULF BREEZE
by
Steven C. Schimmel, Research Aquatic Biologist
Environmental Experiments Branch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Gulf Breeze, Florida
The major mission of the Environmental Research Laboratory-Gulf Breeze
(ERL-GB) is to determine the effects of toxic organic chemicals on estuarine
biota. The major groups of toxic organics being tested include pesticides, in-
dustrial effluents, and carcinogens. ERL-GB is leasing an offshore laboratory
12 miles from land in 110 feet of water. The offshore laboratory provides a
capability for toxicity testing in a simulated open ocean environment. Results
of studies on these chemicals are published in technical journals, the EPA
Research Series, and in the form of written reports to U.S. Environmental
Protection Agency Headquarters, Region and State Agencies.
Research at ERL-GB is defined within the framework of two branches: the
Experimental Environments Branch, and the Processes and Effects Branch. In
addition, a research team has been formed to investigate the impact of carcino-
genic materials on estuarine organisms. The Experimental Environments Branch
considers the uptake and direct toxic effects of organic chemicals on es-
tuarine plants and animals. The Processes and Effects Branch studies the de-
gradation and fate of toxic organics and the effects of these chemicals on
laboratory estuarine ecosystems.
27
-------
EXPERIMENTAL ENVIRONMENTS BRANCH
Within the Experimental Environments Branch, the complexity of testing
ranges from the static, single species bioassay to the multi-species effort
of the larval recruli^rtf.^eKffite'itx^urtWy-^tid'fe's. The static 96-hour
tests prov'hdfiHah leMJJ-ril^te '. t
overall evaluation of shifts in community structure can therefore be determined
28
-------
PROCESSES AND EFFECTS BRANCH
The emphasis of the Processes and Effects Branch is on the development of
methods to evaluate changes in ecosystem compartments exposed to hazardous
inorganic pollutants. The tests under development range from systems that
investigate the fate of pollutants in an estuarine environment to systems that
focus on how pollutants effect specific behavioral responses. The following
systems are being developed:
• Environmental Fate Screening System. The effects of a pollutant are
closely related to its availability and chemical form. This system
allows investigators to determine the environmental processes and
compartments most influential to the movement and transformation of
polIutants.
• Eco-Core System. This technique is used to isolate indigenous micro-
organisms' from intact environmental sediment-water cores and to eval-
uate their degradative potential. The effects of pollutants on mi-
crobial ecology are also studied with this system.
• Continuous Flow Systems. These large- and small-scale systems incor-
porate flowing water and allow the investigation of dynamic environ-
mental processes.
• Aquatic Gradient Avoidance Response System. This system, using
behavior as an indicator of chemical exposure, allows quantification
of the avoidance responses of estuarine organisms to different pollut-
ant concentration gradients.
• Benthic Bioassay System. Changes in sediment surface features pro-
duced by lugworms (Arenicola cristata) and exposed to toxicants are
used in this system to monitor the effects of pollutants on a benthic
infaunal organism.
CARCINOGENESIS RESEARCH TEAM
Energy-related research under the direction of N. Richards, is also being
conducted at ERL-GB. Short-term In vivo and |JT_ vitro tests are being developed
and validated to detect compounds with mutagenic, teratogenic, and carcinogenic
properties from complex environmental mixtures such as shale oil, drilling and
packer fluids and cuttings, and biocides. The program consists of the develop-
ment of methods for the concentration, separation, activation, and detection of
29
-------
A - i' <;I.^?'i3i>oq
toxicants. Concentration and separation techniques are being developed to
increase the detection limits of toxicity tests and to separate the interferina
,.-,.._.: -, , -. - - . - - ': N > - - •',' - • 4- -^ 1- T \ ' '. ,-] ,.- -- -' 1 --, -S V - - "" , -« ' J- * . ' -• - '.-..- - '• ^
substances that are common in tissue residues and other complex environmental
mixtures.
The' mefabo!'ic acViv'ation sVstems^ be i ng 'deve I oped include mi crosomaT"
preparations from a Variety of micro-organisms, fish, algae, and crustaceans-
these are coupled to the SaJjnoneJ I a typh i mu r i urn '( Ames )" M i crdsbrne fe'sf. At'Yhe
enzyme level, tests are being developed for biphenyl hydroxylase and benzo(a)-
pyrene' jrippbxyge.nase, Tpdgct.ion^ '-feraf^ritiz t^s^/ [Tictu;de; Hrnfr; regeneration i n
ar>throp©d-'s, and t-erato'genlc' screen- tests' •vrtth1 isogehic h^r:maphfodytic fish.
Numerous colonies of" isogenic inbred fish a"re being screened in prder to
develop reference fish for in vivo carcinogen bioassays. Other approaches
" "" "' " '' -•
• the genetic manipulation of fish to' produce hybrids with increased
. . .carcinogen; suscep.ti bj I ity, ,. ^ , .,.-,.'- ; ^
• the" use of mutant fish selected for carcjhos|^Vsvsceptib.i I ity, and
• an .evaluation of,.the, use of,Jflb^ed..homQzygQ:us.?!. .wild type, het^ro-
. Zygous, and un|§exag| r^ptoWs^liat.'are^^ and
,- trnplo.id unlsexuals,, ,.'--. »- ~. ...... . .t:-
30
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OVERVIEW OF SHORT-TERM TESTING
AT THE
HEALTH EFFECTS RESEARCH LABORATORY-RESEARCH TRIANGLE PARK
Edited by
Stephen Nesnow, Ph.D., Chief
Metabolic Effects Section
Biochemistry Branch
Environmental Toxicological Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
The short-term bioassays performed at the Health Effects Research Labora-
tory-Research Triangle Park (HERL-RTP) constitute an integrated in vitro and
in vivo approach to identifying and assessing hazardous toxicants and geno-
toxicants present in the environment. Three of the four divisions within the
laboratory are Involved In in this research: Environmental Toxicology, Experi-
mental Biology, and Clinical Studies Divisions. Their fields of inquiry range
from cytotoxicity to carcinogenesis/mutagenesis/teratogenesis to neurobiology
and behavior, immunology and host defense. In the ensuing text, the activities
conducted by each division are highlighted and summarized according to branch;
the names of the investigators who contributed to each summary are Iisted
after the branch title.
ENVIRONMENTAL TOXICOLOGY DIVISION
Biochemistry Branch (Michael Waters, Ph.D., Chief, Biochemistry Branch;
Stephen Nesnow, Ph.D., Chief, Metabolic Effects Section;
Joellen Huisingh, Ph.D., Chief, Cellular Biology Section)
The short-term bioassay program of the Biochemistry Branch, Environmental
Toxicology Division, encompasses three general areas of investigation:
31
-------
• mutagenesis,
• cellular oncogenic transformation (careinogenesis), and
• eel Iular toxicity.
Each area is addressed in an integrated intramural-extramuraI program that in-
cludes basic research, systems development and validation, and systems applica-
tion components. This program is based on the growing concern for detection
of environmental genotoxic agents that may result in an increased incidence of
genetic disease and cancer.
The test systems being developed and implemented under the Biochemistry
Branch program constitute an integrated battery of methods applicable in
screening and mechanistic studies of pure chemicals as well as complex environ-
mental effluents. These bioassay systems are of value both in detecting
potentially hazardous materials and in establishing priorities for the testing
of suspect substances by conventional (whole animal) toxicological methods.
The application of short-term bioassay systems under the program follows
a multi-level approach. The emphasis in Level I testing for mutagens and
potential carcinogens and other toxicants is on detection. These systems
include gene mutation bioassays in bacteria (e.g., Salmonella typhimurium,
Escherichia coli, Saccharomyces cerevisciae) or Neurospora. Bioassays for
primary DNA damage are included in Level I testing (e.g., Escherichia coli
pol A , Bacillus subtil is rec"). These simple qualitative and quantitative
J_n_ vj_trp_ assays for mutagenesis and potential care inogenes is employ a battery
of microbial indicator strains, with an without mammalian microsomal activa-
tion systems. The Salmonella typhimurium (Ames) Microsome Test, for example,
is both an important pre-screening bioassay and an "indicator system", which
can be applied to the examination of body fluids and tissues of exposed animals
for the presence of mutagens.
An important preliminary component of any battery of short-term tests
designed to identify hazardous agents is a search of the literature pertaining
to prior testing of chemically related agents.. Certain test systems are re-
32
-------
fractory towards specific chemicals or classes of chemicals due to the nature
of the organism or the kind of damage being evaluated. Prior knowledge of the
weaknesses of specific bioassays should be incorporated into the decision-
making processes for selection of test systems or conditions. It is well
known, for example, that chlorinated aliphatic and some aromatic agents are
refractory in the Salmonella typhimurium (Ames) Microsome Test. In this case,
other short-term bioassays would be selected or conventional long-term tests
initiated directly.
Level 2 testing emphasizes confirmation of Level I results in appropriate
test cells or organisms. The endpoints of mutagenesis and potential carcino-
genesis are nov separated for greater endpoint definition. Mutagenesis tests
in Level 2 involve, for instance, the use of mammalian cells in culture (e.g.,
L5I78Y, V79, CHO cells). Carcinogenesis testing in vitro involves morphologic,
cellular neoplastic transformation studies (e.g., Syrian hamster embryo cells,
C3HIOTI/2 or Balb/c3T3 mouse embryo fibroblasts). Level 2 also includes bio-
assays for chromosomal alterations (iji vjtro and in vivo cytoqenetics and
sister chromatid exchange in mammalian cells, and gene mutation in Drosophila,
as well as unscheduled DNA synthesis using human cells in culture).
Syrian hamster embryo cells have been used as target cells to assess the
carcinogenic!ty of environmental agents. This neoplastic transformation system
has been validated with over 29 known non-carcinogens and 69 known carcinogens.
These carcinogens were composed of direct alkylating agents, polycyclic aromatic
hydrocarbon, nitrosamines and amides, aromatic amines, azo dyes, and metal
salts. There is a 92% concurrence between the \n vitro results and the in_
vivo data. Of the eight carcinogens which represented the false negatives,
seven were subsequently identified as positive when an exogenous source of
metabolic activation was added to the bioassay. This gave an overall false
negative rate of \%, a false positive rate of Q%, and an overall concurrence
of 99$ for the Syrian Hamster Neoplastic Transformation Bioassay.
Level 3 testing, which concludes the toxicological evaluation process,
involves the use of conventional whole animal methods. Emphasis is placed on
quantitative risk assessment. The in vitro methods are valuable in guiding the
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application of conventional \_r\_ vivo methodology. In turn, the in vivo procedure^
provide the required validation of m vivo methods.
All of these bioassay systems are available through a combined intramural-
extramural program.
The short-term bioassay program in mutagenesis includes:
• basic mechanistic studies using L5I78Y mouse lymphoma cells,
• the development of new assay techniques using Salmonella typhimurium.
• the evaluation of plant mutagenesis assays,
• the evaluation of primary rat hepatocyte DMA repair assays,
• the evaluation of the mutagenicity of diesel exhaust and ambient air
samples, and
• the verification, validation, and application of short-term bioassays
in batteries of test systems.
In addition, the urine of pesticide treated animals is being examined to
evaluate whether or not whole animal metabolism produces mutagenic metabolites.
The development of special techniques for use in the bacterial tester
strains for mutagenesis has provided two new environmental monitoring methods:
• the "well test" analyzes extremely small samples with and without
mammalian activation systems on a single petri plate;
• the new gas trapping technique using bacteriological methods
shows promise for the testing of small ambient air gas samples.
The short-term bioassay program in carcinogenesis centers around the
development and use of oncogenic transformation systems (C3HIOTI/2CL8 and
Balb/c3T3 cells to identify carcinogens, cocarcinogens, tumor initiators, and
tumor promoters. The coupling of metabolic activation systems to these systems
is currently underway.
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The carcinogenesis program has addressed the question as to whether ex-
posure to pesticides increases the carcinogenic or mutagenic potential of
environmental genotoxicants. A combination of J_n_ vj_vo_ and short-term bioassay
test systems indicate that certain pesticides do increase the mutagenic poten-
tial of benzola]pyrene, a ubiquitous environmental carcinogen and mutagen.
Further studies are in progress to evaluate the effects of pesticides on the
carcinogenic potential of benzo[a]pyrene.
A new bioassay using mammalian cells in culture, the Balb/c3T3 Mutagenesis
and Oncogenic Transformation Bioassay, has been developed as part of an overall
program to develop a battery of short-term test systems that would identify
hazardous environmental agents; both the mutagenic and carcinogenic potential
of environmental agents are measured. To validate this new system, a number of
known carcinogens and mutagens, each representive of a different chemical
class, have been used.
The isolated lung perfusion model is assessing the effects of environ-
mental contaminants on the pulmonary metabolism and on the distribution of the
environmental carcinogen benzo[a]pyrene. This research is attempting to clarify
whether a change in metabolic rate, metabolic pathway, or distribution in the
tissues could account for differences in the carcinogenic response.
The short-term bioassay program in toxicity is based on the use of primary
cell strains and multiple biochemical endpoints for identifying and categoriz-
ing the potency of particulates, environmental effluents, and pure agents.
Pevelopmental research has included:
• the evaluation of primary liver cells as carcinogen/mutagen
metabolizer cells to be used with existing short-term bioassays;
• the comparison of the carcinogen metabolizing and promutagen activating
capabilities of primary liver and lung (alveolar macrophage) cells;
and
• the evaluation of the toxicity of a series of organic and inorganic
toxic substances using enzymatic and cytological parameters.
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In order to effectively integrate the available test systems within the
Biochemistry Branch, in vitro and in vivo metaboli sm must be biochemically
well defined. The ability of detection systems to distinguish positives and
negatives in chemical screening depends largely upon the extent to which the
in vitro metabolism of compounds accurately reflects the in vivo conversions.
Knowledge of the ability of cell systems to respond to environmental chemicals
by inducing enzymes that activate and detoxify mutagens and carcinogens, is
essential in evaluating potential genotoxic effects. For this reason, bio-
chemical indicators that provide information critical to the evaluation of
toxicity as well as potential carcinogenicity and mutagenesis, are being
developed in conjunction with the in vitro systems described above; they are
also being compared to similar indicators in the intact animal.
Toxic Effects Branch (Diane Courtney, Ph.D.)
The Teratology Research Program resides in both the Toxic Effects Branch
Environmental Toxicology Division, and the Developmental Biology Branch,
Experimental Biology Division.
Teratology is a beginning research field that needs further exploration.
Its short-term testing program is not yet as well developed as in other areas
of toxicology. Embryonic and fetal development are much more complex than a
single tissue growing in culture. Consequently, the achievement of three-
dimensional growth will present some technical problems. The process of solv-
ing some of these problems will, however, lead to a broader understanding of
embryonic development and ma I development.
One approach to short-term teratology testing is a combined ir^ vivo and
in vitro procedure in which the pregnant rat is treated from day 6 through 8 of
gestation; the embryos are then removed and cultured in vitro for 48 hours.
In this approach the embryo is exposed in_ utero to the toxic agent and any
metabolites that might be produced. Advantages of this technique are:
36
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• each embryo serves as its control for development;
• growth can be determined by measuring DNA, protein, and other
natural constituents;
• three-dimensional development of the cranial flexures can be
observed;
• organ development can be observed by somite counts; and
• observations can be made over time, so that initial lags with
subsequent growth can be detected.
This test correlates very well with in vivo testing of teratogenic agents and
could become a very valuable testing procedure in teratology.
EXPERIMENTAL BIOLOGY DIVISION
Developmental Biology Branch (Neil Chernoff, Ph.D.)
As mentioned above, the Teratology Research Program is conducted jointly
by the Developmental Biology Branch of the Experimental Biology Division and
the Toxic Effects Branch of the Experimental Toxicology Division.
Progress in teratogenicity short-term testing has been hampered by a number
of factors central to this branch of science. The little that is known about
basic mechanisms in teratology has indicated that there is no single trigger
for the production of birth defects. One cannot point to a small number of
specific preliminary events as predictive of birth defect formation. One
problem which short-term teratology testing encounters is the enormous difficulty
in duplicating the maternal-fetal relationship between the metabolism of the
mother and the placenta I interface. In vitro or submammalian systems do not
allow for these factors and may therefore be impossible to extrapolate.
Another reason for the lack of development of short-term testing is that
standard teratology testing is considerably shorter than a standard two-year
carcinogenicity study, generally taking 3 to 4 months to complete.
One short-term teratogenesis assay that this branch is developing, exposes
mouse eggs in vitro to various agents. The four compounds 5-fluorouraciI,
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cadmium, cacodylic acid, and sodium chloride have been examined. Their effect
on the following processes was compared:
• rate of cleavage,
• rate of development,
• survival after re-implantation into the dams, and
• uptake of the amino acid leucine.
These tests were not very predictive of tests done in vivo.
NeurobioTogy Branch (Lawrence Reiter, Ph.D.)
Short-term testing in neurobiology and behavior is aimed at evaluating the
effects of environmental pollutants on the functional integrity of the nervous
system. In order to thoroughly examine toxicant-induced changes in the central
nervous system (CNS) function, scientific investigation must proceed at various
levels of neural organization:
• behavioral,
• neurochemical, and
• neurophysiological.
Behavioral analysis provides information on the consequences of toxicant
exposure upon the functional output of the nervous system; neurochemical and
neurophysiological analysis directly measure nervous system activity.
Within this framework, the Neurotoxicology Program has provided two re-
search functions:
• toxicity testing, and
• methods development and validation.
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For toxfcity testing, currently existing methods have been utilized to systemat-
ically collect information on various neurotoxicants. Methods development and
validation, which has proceeded in unison with toxicity testing, focuses on the
development of sensitive indices for neurotoxicity that are also simple, rapid,
and cost-effective. In the area of behavioral testing, methods range from
basic reflexes to naturalistic behaviors to complicated controlled schedules of
reinforcement. The short-term behavioral testing procedures evaluate locomotor
function, CNS excitability, learning and performance, and social behavior.
Neurochemtcal analyses are primarily concerned with b ioenergetics and neurotrans-
mitter function. Finally, the neurophysiologicaI evaluation of CNS function
utilizes both electroencephalogram and evoked potential measurements.
CLINICAL STUDIES DIVISION
Biomedical Research Branch (Donald Gardner, Ph.D., Chief)
Short-term tests performed in the Biomedical Research Branch, Clinical
Studies Division, use model systems to measure subtle alterations in host de-
fense against airborne toxic substances. Complex cooperative interactions
comprising both mechanical and biological components maintain the sterility of
the lower airways. Test results indicate that the inhalation of pollutants
capable of compromising the functional integrity of any of the individual
elements of the pulmonary defense system increase susceptibility to respiratory
infection. This increase may be effected by the activation of latent infec-
tions once surveillance and defense mechanisms are compromised, or by the
failure of host defense systems to respond adequately to subsequent bacterial
invasion. Certain populations such as the elderly would be threatened as a
result of the decrease in resistance to respiratory infection.
Owing to the problems inherent to the study of the effects of pollutants
on the susceptibility of the human respiratory system to microbial infection,
animal model systems have been developed to investigate various hypotheses that
can later, through appropriate epidemiological surveys, be tested on man. The
animal model should reflect a summation of the varied responses of the respira-
tory tract, including:
39
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• edema,
• eellular disruption,
• reduced macrophage function,
• inflammation, and
• immuno-suppression.
The influence of atmospheric pollutants on the microbe-host interaction is
difficult to predict as the test substance may increase, decrease, or have no
effect on the susceptibility of the host to infection. The host response is
also dependent on the concentration of the pollutant, the physiological state
of the host and of the microbial development on and in the host, and the .
length of time between pollutant exposure and microbial exposure.
A number of different experimental approaches have been used to demonstrate
the potential of chemical agents for altering host susceptibility to respira-
tory infectious agents. One of the most sensitive methods used by a number of
laboratories is called the Infectivity Model. Briefly, animals are randomly
selected for exposure either to filtered room air or to the test substance.
After the cessation of this exposure, the animals from both chambers are com-
bined into a third chamber where they are exposed for approximately 15 min to
an aerosol of viable micro-organisms. Micro-organisms that have been employed
include: Streptococcus pyogenes Group C, Diplococcus pneumoniae. Klebsiella
pneumoniae, Escherichia cojj, Salmonella typhimurium. and influenza virus. At
the termination of this exposure some animals from each group are sacrificed
and examined, using standard microbiological techniques, to determine the
number of inhaled micro-organisms. The remainder of the animals are returned
to clean, filtered room air, and the rate of mortality in the two groups is
determined during a 15-day holding period. This system can also be expanded to
permit observation of altered pathogenic modes, relative mean survival time
and cytological indices of isolated lung cells. Separable elements of the res-
piratory defense system which can be studied include:
40
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• a Iveolar macrophage function,
• mucociliary function,
• ciliary beating, and
• mucous physical properties.
Baterial inactivation ar)d killing by alveolar macrophages and rates of
clearance can be ascertained by using radiolabeled bacteria. Alveolar macro-
phages function as primary effectors of host defense against inhaled particulate
matter such as bacteria, viruses, and fungi, which are ingested, killed, and
degraded by the macrophages. Nonbiological particles such as dusts, soot,
etc., are also ingested and degraded to varying extents, unless their chemical
composition makes them cytotoxic to macrophages, in which case the particles
are released from dead cells to be taken up by other macrophages; the cycle is
then repeated. The capacity of a specific pollutant to affect lung macrophage
function can also be screened by exposing isolated macrophages in vitro.
Specific parameters measured in the macrophage test system include: phagocy-
tosis, viability, cell number, ATP concentration, depression of respiration,
and lysosomal enzyme activities.
One of the primary defense systems of the respiratory tract involves
mucociliary function. The mucociliary escalator is responsible for the removal
of inhaled bacteria and particles deposited in the conducting airways in the
respiratory surfaces. The separate mechanisms involved in mucociliary func-
tions are all extremely vulnerable to inhaled pollutants. Ciliary beating has
been shown to be inactivated by cigarette smoke, while mucociliary transport is
inhibited by a number of substances such as acrolein, formaldehyde, ozone,
sulfuric acid, nickel, acetone, cadmium, sulfur dioxide, nitrogen dioxide,
ammonia, and cigarette smoke. Assays of ciliastatic activity of various agents
are performed j_n_ v 11rp_ fol lowing in vitro or _MT_ vivo exposure of the trachea.
In summary, the short-term bioassays performed at the Health Effects
Research Laboratory-Research Triangle Park respond to the diverse needs of the
U.S. Environmental Protection Agency in the program areas of Pesticides,
Toxic Substances, Carcinogenesis, Transportation, Criteria, Non-Criteria,
41
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Energy, Environmental Assessment, and Multi-Route. This overall laboratory
research effort examines the effects of environmental agents on a multitude of
biological species at various levels of biological organization using many
biological points. This program focuses on short-term bioassays both as re-
search tools to provide a greater insight into basic mechanisms of toxicity and
genotoxicity and as screening tools to aid in the identification of potentially
hazardous agents present in our environment.
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SCREENING ACTIVITIES AT THE
HEALTH EFFECTS RESEARCH LABORATORY-CINCINNATI
by
Richard J. Bull, Ph.D., Chief
Toxicological Assessment Branch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio
At the Health Effects Research Laboratory in Cincinnati, Ohio (HERL-CIN),
short-term screening activities related to air, water, energy, wastewater reuse,
and sludge disposal problems are in progress. The bulk of the effort, however,
addresses problems in the water programs.
Screening activities in the water program are required due to:
• a need to acquire epidemiological data indicating relationships
between chemical water quality and chronic disease;
• the necessity to identify a wide variety of chemical compounds
in finished tap waters:
— more than 600 organic compounds comprise only 5 to \5% of the
total organic carbon;
— inorganic constituents, including toxic trace metals and
asbestos, vary considerably in differing water supplies;
and
• the inadequacy of analytical chemistry methods to deal with the major
portion of organic material in drinking water, suggesting a need for
developing a comprehensive battery of bioassay techniques.
Among those compounds that have been identified are a number of known
carcinogens, cardiovascular, hepatic, renal, and hematopoietic toxins. A very
large number of the compounds are associated with central nervous system
43
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activity; in a few cases the effects have been shown to be irreversible at
high doses. Consequently, there is a need for a comprehensive screening
program geared to a wide variety of toxicologicaI endpoints.
A fairly comprehensive screening program for damage to deoxyribonculeic
acid (DNA) has been in progress for a number of years. The Salmonella
typhlmuri urn (Ames) Microsome Test is being employed in field studies for
correlation with epidemiologicaI data, in the testing of organic concentrates
and other environmental samples, as well as individual compounds. This effort,
in itself, cannot yield the kind of information required for regulatory deci-
sions. Cell transformation assays are also being explored; however, there
is some question as to whether they will add significantly to the solution
because of the difficulties involved in their application and the fact that
they have many of the same limitations as bacterial systems.
The critical question to be answered in this area is the extent to which
the results of any of these tests relate to human disease. The conflict be-
tween negative screening test findings with chloroform versus the positive J_n_
vivo bioassay results from the National Cancer institute (NCI), also with
chloroform, illustrate that these methods cannot be wholly depended upon in
circumstances demanding regulatory decisions. For this reason, work that
promises to considerably shorten the in vivo assessment of damage to genetic
material has been emphasized this past year. This seems to be the only reason-
able way to take into account the impact of pharmacokinetic, metabolic, and
repair factors in order to estimate the degree of risk and modeling dose-
response. Information at this level is essential to define the reliability
and limitations of simpler test systems. This definition is in turn required
for a screening program that can deal reasonably with the problems at hand.
The work also incorporates the idea that the methods developed should be appli-
cable to the human for use In prospective epidemiological studies. This final
set of information is essential to the validation of simpler test systems. In
addition to these efforts, work is being concluded that is specifically de-
signed to determine both the influence of secondary factors such as the immune
response in modifying the course of tumorigenesis in experimental animals, and
the comparative metabolism of compounds by varying species, including man.
44
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Several epidemic logical studies are underway to see if cancer rates may
be associated with specific water supplies. Besides both organic and inorganic
chemical analyses, the finished drinking waters are being subjected to the
Ames test for mutagenesis. On some projects other screening tests for muta-
genesis/carcinogenesis are being used. If relationships with cancer rates
are found, the screening test may provide an empirical test for the potability
of water.
Within the relatively diverse mutagenesis screening programs the need for
quality control is obvious. Although the NCI has begun a large quality
control program, it appears primarily aimed at standardizing the Ames test.
Furthermore, the data from this work will not be available for I 1/2 to 2
years. There appears to be some advantage in evaluating the diverse systems
available to us now, not so much to decide which is the better system, but to
get an objective feel for the range of activities that may be detected and the
sensitivities of simple bacterial and cell transformation assays. Cor\se-
quently, HERL-CIN has designed a quality control program based upon putative
mechanisms of mutagenic action. It wiI I be offered as an annual service to
anyone interested in participating.
Despite this emphasis on mutagenic effects, these are not the effects for
which the best epidemiological data exist. The relationship of cardiovascular
disease to chemical water quality has been with us since the 1950's. It has s
been repeated in a number of studies around the world. Estimates are that up
to \5% reduction in mortality could result from the identification and elimi-
nation of the so-called "water factor." Since the best correlation between
water hardness and cardiovascular disease is a negative one, two studies have
been set up in whole animals to assess the effects of the major inorganic con-
stituents that change with water hardness. However, if these studies fail to
account for the relationship, a severe need for rapid screening techniques
will become apparent to deal with the permutations related to the differences
in the organic constituencies of hard and soft waters. Although a number of
systems may be applicable to this problem, they have not yet been exploited.
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A second area that has received little attention beyond gross terato-
logies I screening are the effects of chemicals on development and aging. This
is an acute problem in the nervous system, since its development in the human
is substantially postnatal and by definition falls outside the realm of terato-
genic effects. Although some view the area of teratogenesis, development, and
aging as being related to mutational events, the bulk of the evidence would
suggest that mutational events do not constitute the major mechanism involved.
HERL-CIN has developed spectral methods that measure the biochemical develop-
ment of the brain directly in the tissues. We have found that brain develop-
ment is significantly and substantially delayed in neonatal rats with peak
blood lead (Pb) concentrations of 36 mg Pb/dl, both from a biochemical and
morphological point of view. These findings corroborate earlier findings of
delayed neurophysiological development of the brain in animals treated with
Pb, paralleled by delays in behavioral development. Blood Pb concentrations
of this order are certainly within the range encountered in human young in
this country. If these data apply to man, as some epidemic logical data sug-
gest they do, environmental Pb is having a much greater impact than previously
appreciated. Such studies are not long-term, at least in terms of biochemical
measurements, involving 3 weeks of lactation. They do demand some devotion of
manpower that can, however, be decreased if smaller species with shorter
lifespans are used. This could also allow for a more immediate assessment of
the long-term functional deficits resulting from such delays in development.
It is imperative that such systems be developed in a comparative manner be-
cause of possible differences in the internal factors involved in regulating
tissue differentiation. However, as these factors are much better understood
in mechanistic terms in plants and lower animals than in mammals and humans, a
more general approach to chemical Iy-induced developmental problems including
teratogenesis may result.
A consideration of utmost importance is the development of biological
methods that can be applied to unconcentrated drinking water and source water
samples. This sensitivity is required because all current concentration
methods delete significant fractions of the material present and all others in
the process. As .mentioned above, the utilization of the Ames test under such
circumstances is already being explored. HERL-CIN is currently investigating
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those aquatic species that have been used to define ecological hazards associat-
ed with chemicals in order to broaden the scope of testing that may be applied.
Such systems would be used in a monitoring mode to determine water treatment
efficiency, safety of shellfish and crops grown with wastewater and sludge
application, as well as to develop criteria for the regulation of individual
compounds.
The air program at HERL-CIN has screening requirements that to a large
extent are similar to those in the water program. However, specialized needs
must be recognized that involve the portal of pollutant entry. Consequently,
there is more emphasis on screening for effects directly on the lung and on
exploring the pharmocokinettcs of the inhalation route. The principle short-
term, specialized methods being used include a test assessing the suscepti-
bility of respiratory infections to microbial pathogens and _in_ vivo induction
of lung tumors on strain A mice. Shorter term systems indicated in the water
program have also been used in inhalation exposures; others will be employed
as they become vaIi dated.
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OVERVIEW OF SHORT-TERM BIOASSAYS PERFORMED AT THE
NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH
by
Daniel A. Casciano, Ph.D., Research Biologist
Department of Health, Education, and Welfare
Food and Drug Administration
National Center for Toxicological Research
Jefferson, Arkansas
The primary mission of the National Center for Toxicological Research
(NCTR) is to conduct research programs designed to increase the understanding
of the biological mechanisms of potentially toxic chemical substances found in
man's environment. The research programs are directed toward:
• the development of methodologies and test protocols that evaluate the
safety of chemical toxicants;
• the development of data that will facilitate the extrapolation of
toxicological data from laboratory animals to man;
• the determination of the basic biological processes for chemical
toxicants in animal organisms; and
• the study of the biological effects of potentially toxic chemical
substances found in man's environment, with emphasis on the deter-
mination of adverse health effects resulting from long-term, low-level
exposure to chemical toxicants.
In the past 5 years, the majority of the research effort at NCTR has been
oriented toward utilizing in vivo mammalian models, primarily the mouse, to
help understand the dose-response relationship, especially at very low doses.
In addition, in-house research include studies of the metabolism of toxic
compounds in cell-free systems and in in^ vivo systems. Studies have also been
initiated to develop/modify short-term bioassay systems that would complement
the in vivo and cell-free studies. The short-term bioassays presently employed
at NCTR include:
49
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• the Salmonella typhimurium (Ames) Microsome Test;
• the Chinese Hamster Ovary (CHO/HGPRT) Mutagenesis Test; and
• the DNA Repair Activity Test (Unscheduled DNA Synthesis).
The Salmonella typhimurium (Ames) Microsome Test is basically that de-
scribed by Ames except for, in some cases, the source of the metabolic activat-
ing enzymes, S9 — prepared from uninduced mouse liver. Since the majority of
the in vivo experimentation at NCTR uses the mouse as a model, S9 serves as
the metabolic activating system. This assay system has been used to study the
mutagenic activity of a variety of chemical classes Including aromatic amines
polycyclic aromatic hydrocarbons, nitrosamines, direct alkylating agents, and
others.
The Chinese Hamster Ovary Mutagenesis Test is basically that described by
Hsie, et al. The CHO cells are ideally suited for mutagenesis studies because
they are genetically well-characterized, exhibit high cloning efficiency, have
a stable karyotype, and are capable of growing in a relatively well-defined
medium on a glass or plastic substratum or in suspension with a short population*
doubling time of 12 to 13 hours. The induced mutants are selected by virture of
resistance to the purine analogue 6-thioguanine. This resistance is due to an
alteration at the structural gene which confers hypoxanthine-guanine phosphoribosvl
transferase activity. This system has been used, coupled with an exogenous
metabolic activating system, to detect promutagens/procarcinogens as well as
direct acting mutagens/carcinogens. This assay system has been used (mainly by
Dr. A, Hsie through an interagency Agreement [IAG]) to study the mutagenic
activity of metals, nitrosamines, aromatic amines, polycyclic aromatic hydro-
carbons, alkylating agents, and others.
The DNA Repair Activity Test performed at NCTR measures the induced
unscheduled DNA synthesis in rodent hepatocytes. Viable hepatocytes are
isolated from the livers of rat or mouse by in situ collagenase perfusion and
placed in primary culture, exposed to a physical or chemical mutagen/carcinogen-
DNA repair activity is then measured as induced unscheduled DNA synthesis. |n_
duced DNA repair activity is assessed by measuring the incorporation of
^H-thymidine using direct scintillation counting of acid precipitable material
50
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cesium chloride gradient analysis, and quantitative autoradiography. The
hepatocyte culture system is very useful because the cells retain many of the
liver's enzymatic capabilities, thereby acting as an endogeneous source of
metabolic activating enzymes and as the target cell. The system has been
used to study the DMA repair inducing capabilities of several chemicals from
a variety of chemical classes including aromatic amines, polycyclic aromatic
hydrocarbons, mycotoxins, alkylating agents, and others.
Other mutagenesis activities at NCTR are in vivo, long-term bioassays
that include the heritable translocation assay and the assay of gene products
(enzymes) found in the somatic tissue of the offspring of the treated population,
detecting induced mutations in male mouse germ cells.
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SHORT-TERM TESTS
FOR HEALTH AND ECOLOGICAL EFFECTS
PART II: DIRECTORY OF TESTS
Prepared for the
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington, District of Columbia 21040
By the
Genetic Toxicology Program
Biochemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
53
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CONTENTS
Abbreviations v
Acknowledgment vi
Note Regarding the Test Systems vi i
1000 Short-Term Tests for Health Effects I
1100 General Toxicity I
I 110 In-Vitro Systems/Acute I
1120 In-Vivo Systems/Acute 21
1130 In-Vivo Systems/Subacute 25
1140 In-Vivo Systems/Inhalation Toxicology 35
1150 In-Vivo Systems/Neurobehavioral 47
1200 Genotoxiclty 57
1210 Mutagenesis 57
1220 DNA Damage 73
1230 Carcinogenesis 83
1240 Marine Applications 91
1300 Perinatal Toxicology 99
2000 Short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
2200 Freshwater Animals and Plants Ill
2210 Fish Toxicity Ill
2220 Invertebrate Toxicity 117
2230 Plant Toxicfty/Residue 123
2240 Ecosystem 125
(continued)
I i i
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CONTENTS (continued)
2300 Marine Animals . . . .
2310 Fish Toxicity. .
2320 Invertebrate Toxicity.
2330 Ecosystem
127
127
tG i
l.n dices :,
.N J
.IV
V
XI
XI I
XI I I
XIV
XV:
2400. Terrestrial Animals and Plants.
. 241.0. Plants.. ,. ..
2420 Ecosystem
2500'
. , . ;-jfi) '-AX ' •'mi '•?.•••':, C"'^:V-;'i 0 | ' •
Test Sys.te/ns, i.n .Numerical -Ord^n v-'.''1i.tT.v;. .'.v >'--.-. p",.i •.
Test S.ys.tejns. i,n Alphab,et-tcal::0rxf&r«' \i ^. :. .'i.''-." . . '..
Experime.nl^k ^uib^eiot'iLiiS^lTig!^!.'^^,.:^! ..v-.-^'.-•;'s. :-v!.
Contact Pe.rson/ie.1 ihfmA'Kph&beticaf Orrcfen . ci\- i.V-,-':. .- ^.M.
U.S. EPA Laboratories in Alphabetical Order,.;., ..4. fc-, ..-,,--.,.
U.S. EPA Departmental Listing in AlphaJpe^rcaJ^flrider-,;.. ;.
Grant/Contract Laboratories in Alphabetj^j^-Order .-,-»-,.
Status of Development of the Test Syp^eirisr,.,n ^ ,f -t .,,,,..,
Endpoints of the General and PerjpataJ. Tpxi.cjty ,•
' ' ' ' ' ' "' "'' -'' "
Test Systems
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Iv
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ABBREVIATIONS
ERL-COR Environmental Research Laboratory-CorvaI I is, Oregon
ERL-DUL Environmental Research Laboratory-Du1uth, Minnesota
ERL-GB Environmental Research Laboratory-Gulf Breeze, Florida
ERl-NAR Environmental Research Laboratory-Narrangansett, Rhode Island
HERL-CIN Health Effects Research Laboratory-Cincinnati, Ohio
HERL-RTP Health Effects Research Laboratory-Research Triangle Park, North
Carolina
NCTR National Center for Toxicological Research, Jefferson, Arkansas
OAWM Office of Air and Waste Management
OEMI Office of Energy, Minerals, and Industry
OHEE Office of Health and Ecological Effects
OPP Office of Pesticide Programs
ORD Office of Research and Development
QJS Office of Toxic Substances
OWHM Office of Water and Hazardous Materials
U.S. EPA United States Environmental Protection Agency
-------
The coopeWlbri -dM y^l%r^tf<^'c|tf each Directory contrfbutor
a re g ra te f u I I y- j
Special thanks are extended to the three science editors, Dr. Shahbeg
SandhU of HERt-^TP/ W^^e^^V'CKaWe^^ 'HEkt-'RfP/ "arid ^''Dr.' James Mckim" of
ERL-DUL, for thjs^rf e^t^q-s;! v-e; ^foft^icinvSQr.eero'.ng! antt'Cdbi-dlTiating the confHi-
butions to the subject areas of Health Effects-Genetic Tpxipalqgy, Health
Ef fects-General and Perinatal Toxicology, and Eco I ogi carl.-1 Effects, respectively
Our sincere appreciation is also extended to Northrop Services, Inc.,
for indexing, editing, and typi hg "fhe^Cn' reStory/ and in particular to Olga
Wierbicki, who coordinated-,the^ /sf fe/rt. t
v-f
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NOTE REGARDING THE TEST SYSTEMS
The test system Information contained in this document was provided by
the workshop participants and their colleagues. The science editors. Dr.
Shahbeg Sandhu, Dr. Jeffrey Charles, and Dr. James Mckim, combined data where
possible, questioned obvious errors and missing information, and insured
uniformity to the extent possible in data sheets and indices. No effort was
made to restrict inclusion of any test system or related information. Selec-
tion and interpretation of terms related to status of development, applica-
tions, complexity (simplest to most complex on a scale of I to 4), program
office support, etc., were the responsibility of the submitters of the in-
formation.
vi i
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CONTENTS: TEST SYSTEMS
1000 Short-Term Tests for Health Effects I
1100 General Toxicity I
I I 10 In-Vitro Systems/Acute I
MM Integrated Systems: Lung Organ Culture System,
Tissue Homogenates, Purified Enzyme Systems . . 2
1112 Integrated System: Tissue Homogenates, Purified
Enzyme Systems 3
1113 Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues 4
1114 Tracheal Mucociliary Transport 6
1115 Platelet Secretion Measured by ATP Release. ... 8
1116 Mammalian Platelet and Fibroblast Impairment. . . 9
III? Lymphocyte Cytotoxicity 10
1118 Impairment of Neutrophil Phagocytosis II
1119 Human Lung Fibroblasts (WI38) 12
IIIIO Chinese Hamster Ovary (CHO) Clonal Toxicity ... 14
HIM Rabbit Alveolar Macrophage (RAM) 16
II112 Rat Hepatocyte (Liver Cell) 18
II113 Chinese Hamster Ovary (CHO) Cytoxicity and
Mutagenicity 19
1120 In-Vlvo Systems/Acute 21
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1111 INTEGRATED SYSTEM: LUNG ORGAN CULTURE SYSTEM, TISSUE HOMOGENATES
PURIFIED ENZYME SYSTEMS
Biological Activity Detected: Toxicity.
Principle: Enzyme inhibition and/or induction.
Endpoints: Qua Iitative : Alteration in enzyme activities and/or
concentration of metabolites. Quantitative: Degree of alteration
in enzyme activities and/or concentration of metabolites.
Strengths: Excellent indicator for pulmonary fibrosis; Very sensitive
early indicators.
Weaknesses: Lacks in specificity in some cases; Terminal; Difficult
to extrapolate to human situation.
Status of Development: Being implemented.
Describe: Test systems have been fully developed. Data are being
collected.
Applications: Multimedia.
Samples: Pure Chemicals: N02, SOg, Hg, Cd, Mn, Zn, Cu. CompI ex
Mixtures: Transportation Related - diesel.
Duration: 5 years ending in 1979.
Cost/sample or chemical: $85.
Interpretation: This system is a very sensitive measure for the
degree of alteration relative to pulmonary fibrosis.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch, Biochemistry Section.
Persons to Contact: S.D. Lee, U.S. EPA, HERL-CIN, 26 W. St. Clair St
Cincinnati, OH 45268, (FTS 684-7442). *'
Grant/Contract Laboratory Involved and Principal Investigators:
U. of California Medical Center, San Francisco, CA 94132,
R.S. Bhatnagar.
Program Office Support: OPP; OEMI.
References: I) Bhatnagar, R.S. The Role of Superoxide in Oxldant-
Induced Pulmonary Fibrosis. In: Biochemical Effects of Environ-
mental Pollutants. S.D. Lee, ed., Ann Arbor Science Publishers,
Ann Arbor, Ml, 1977. 2) Hussain, M.Z., R.S. Bhatnagar, and S.D!
Lee. Biochemical Mechanisms of Interaction of Environmental
Metal Contaminants with Lung Connective Tissue. In: Biochemical
Effects of Environmental Pollutants. Ann Arbor Science Publishers
Ann Arbor, Ml, 1977.
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1112 INTEGRATED SYSTEM: TISSUE HOMOGENATES, PURIFIED ENZYME SYSTEMS
Biological Activity Detected: Toxicity.
Principal: Enzyme inhibition and/or induction.
Endpoints: Qua! itative: Alteration in enzyme activity and/or con-
centration of metabolites. Quantitative: Degree of alteration
in enzyme activity and/or concentration of metabolites.
Strengths: Early indicators; Probably more sensitive than any other
method.
Weaknesses: Lacks in specificity in many cases; Terminal; Difficult
to extrapolate to human situation.
Status of Development: Validated.
Describe: Data are being accumulated to validate toxic effects
of specific pollutants.
Applications: Multimedia.
Samples: Pure Chemicals: 03, N02, Hg, Cd, S02. Complex Mixtures:
Ambient - 03 + S02.
Duration: 3 years ending June 1978.
Cost/sample or chemical: $75.
Interpretation: This system provides sensitive early indicators for
metabolic/cellular injury and recovery.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Biochemistry Section.
Persons to Contact: S.D. Lee, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7442).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of California School of Medicine, Los Angeles, CA 90032,
M.G. Mustafa.
Program Office Support: OHEE; OPP.
References: I) Mustafa, M.G., and S.D. Lee. Pulmonary Biochemical
Alterations Resulting from Ozone Exposure. Ann. Occup. Hyg.,
19:17-26, 1976. 2) Mustafa, M.G., A.D. Hacker, J.J. Ospital,
N. Elsayed, and S.D. Lee. Prophylactic Effect of Dietary Vitamin
E on the Metabolic Response of Lung Tissue to Low-Level Ozone
Exposure. Amer. Rev. Resp. Dis., 113:98, 1976. 3) Hacker, A.D.,
N. Elsayed, M.G. Mustafa, J.J. Ospital, and S.D. Lee. Effects
of Short-Term Nitrogen Dioxide Exposure on Lung Collagen Syn-
thesis. Amer. Rev. Resp. Dis., 113:107, 1976. 4) Ospital, J.J.,
N. Elsayed, A.D. Hacker, M.G. Mustafa, and D.F. Tierney. Altered
Glucose Metabolism in Lungs of Rats Exposed to Nitrogen Dioxide.
Amer. Rev. Resp. Dis., 113:108, 1976. 5) Lee, S.D., and M.G.
Mustafa. Influence of Dietary Antioxidants in Low Level Oxidant
Exposure. Presented at 4th International Clean Air Congress,
Tokyo, Japan, May, 1977. 6) Mustafa, M.G., A.D. Hacker, J.J.
Ospital, M.Z. Hussain, and S.D. Lee. Biochemical Effects of
Environmental Oxidant Pollutants in Animal Lungs in Biochemical
Effects of Environmental Pollutants. S.D. Lee, ed., Ann Arbor
Science Publishers, Ann Arbor, Ml, 1977. 7) Mustafa, M.G., and
S.D. Lee. Biological Effects of Environmental Pollutants:
Methods for Assessing Biochemical Changes. In preparation.
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1113 INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND METABOLIC DAMAGE TO
TARGET TISSUES
Biological Activity Detected: Toxicity.
Principle: Increased functional activity of a tissue requires energy.
Consequently, if a tissue's functional activity is stimulated,
ATP is hydrolyzed to ADP and P. which in turn stimulates oxida-
tion of substrate and resynthesis of ATP. These metabolic changes
may be observed as increases in oxygen consumption, substrate
utilization or as metabolic transients induced in the electron
carriers directly in tissues, in-vitro. The kinetics of these
metabolic responses to stimulation are sensitive to a wide variety
of chemical agents with varying mechanisms of action with both
in-vitro and in-vivo treatments.
Endpoints: To this point in time the test has only been developed
for brain tissue. Responses are measured in response to elec-
trical pulses or elevation in K concentrations. Qua Iitative;
Quantitative: Transient redox changes in NAD(P)H, fp, cyt a, b
c; Substrate utilization; Oxygen consumption; Lactic acid output-
Neuretransmitter release; Amino acid metabolism; Electrical
threshold; Frequency response.
Strengths: In-vitro results may be directly confirmed in-vivo with
same parameters; Applicable to a wide variety of mechanisms;
Applicable to all aerobic tissues; Involves measurement of the
kinetics of going from a resting to an excited state rather than
the steady state, thereby greatly increasing sensitivity; Ap-
plicable to very small tissue samples (2 to 3 mg).
Weaknesses: Does not lend itself to immediate identification of
mechanisms unless there is a direct effect on energy metabolism
proper.
Status of Development: Validated.
Describe: The test has been validated with a wide variety of
inhibitors of energy and membrane active compounds such as ouabain
and saxitoxin, In-vitro and in-vivo treatments with lead, methyl
mercury, and a Iky It in compounds indicate equivalent or more
sensitive measures of effect than other parameters which have
been applied to these problems.
Applications: Multimedia.
Samples: Pure Chemicals; All classes. CompI ex M i xtu res: Indus-
trial ; Energy Related; Transportation Related.
Duration: 2 weeks/compound in-vitro.
Cost: Not precisely established, estimate $1,000 to $2,000/compound
Interpretation: The test basically assesses potential for neuro-
toxicity. It indicates the effect of a chemical somewhere be-
tween functional activity and the metabolism induced by functional
activity. Further studies are required to determine if the effect
is on function or metabolism.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
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1113 INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND METABOLIC DAMAGE TO
TARGET TISSUES (continued)
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Bull, R.J., and A.J. Trevor. J. Neurochem., 10:999-
1009, 1972. 2) Bull, R.J., and A.J. Trevor. J. Neurochem.,
19:1011-1022, 1972. 3) Cummins, J.T., and R.J. Bull. Biochem.
Biophys. Acta, 253:29-38, 1971. 4) Bull, R.J., and J.T. Cummins.
J. Neurochem., 21:923-937, 1973. 5) Bull, R.J., and S.D. Lutkenhoff.
Neuropharmacol., 14:351-359, 1975. 6) Bull, R.J. J. Neurochem.,
26:149-156, 1976. 7) Bull, R.J., P.M. Stanaszek, J.J. O'Neill,
and S.D. Lutkenhoff. Envir. Hlth. Perspect., 12:89-95, 1975.
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1114 TRACHEAL MUCOCILIARY TRANSPORT
Biological Activity Detected: Toxicity.
Principle: Particulates or gases which are deposited on the ciliated
epithelium of conducting airways could adversely affect the major
functions of this tissue —that of clearance of particulates and
effete cells from the lung.
Endpoints: Qua Iitative; Cytological and histological examination
of trachea exposed to air pollutants in-vivo or in-vitro.
Quantitative: Measurement of ciliary beating frequency after
in-vitro or in-vivo exposure.
Strengths: The in-vitro exposure model permits rapid dose-response
studies for ranking of toxicity which can be followed with rapid
dose-response studies after in-vivo exposure; The test is quite
sensitive; The in-vivo exposure testing dose-response effects
permits results to be used for standard setting and regulatory
purposes; The in-vitro exposure model requires small amounts of
polIutant sample.
Weaknesses: For the inhalation exposure, relatively large amounts
of pollutant sample are required.
Status of Development: Validated.
Describe: Both the in-vitro and in-vivo exposure model have been
successfully used for Ni, Cd, H2SOi+, and carbon, and pollutant
mixtures. The model has not been used for screening purposes.
AppIications: Ai r.
Samples: Pure Chemicals: Any chemical likely to be deposited
on conducting airways. Complex Mixtures: Ambient; Industrial;
Energy Related; Transportation Related; Other - any gas, partic-
ulate or combination thereof.
Duration: In-vitro exposure: 2 weeks/dose-response of I chemical;
In-vivo exposure: 4 weeks/dose-response of I chemical.
Cost: Approx. $4,000/dose-response of I chemical in-vivo; $2,000/
dose-response of I chemical in-vitro.
Interpretation: Positive result predictive of damage to clearance
mechanisms of lung.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: J.A. Graham, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
NT Research Institute, 10 West 35th Street, Chicago, IL 60616,
L. Schiff; Northrop Services, Inc., P. 0. Box 12313, Research
Triangle Park, NC 27709, B. Adkins; Ball State University,
Muncie, IN 47306, D. Adalis; U. of North Carolina, Chapel Hf[|
NC 27514, A. Col Iier.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: DAdalis, D., D.E. Gardner, F.J. Miller, and D.L. Coffin.
Toxic Effects of Cadmium on Ciliary Activity Using a Trachea!
Ring Model System. Envir. Res., 13:111-120, 1977. 2) Collier,
A.M., and J.B. Baseman. Organ Culture Techniques with Mycoplasma
Ann. N.Y. Acad. Sci., 225:277-289, 1973. 3) Donnelly, G.M., H.F."
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1114 TRACHEAL MUCOCILIARY TRANSPORT (continued)
McKean, C.S. Heird, and J. Green. Ciliostasis as a Bioassay,
Arch. Envir. Hlth., 28:350-355, 1974.
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1115 PLATELET SECRETION MEASURED BY ATP RELEASE
Biological Activity Detected: Toxicity; Pharmacologic modulation.
Principle: Platelet function is important in thrombosis, shock, and
most inflammatory reactions. Platelet secretion accompanies the
more commonly measured aggregation response, and secretion is
more easily measured than aggregation.
End points: Qua!itative: N/A. Quantitative; ATP release
from platelet suspensions is measured by Lucifirin-luciferase
assay.
Strengths: Fast; Reproducible; Does not require expensive equipment-
Applicable to studies of human blood; Can employ both in-vivo and
in-vitro exposures.
Weaknesses: Have not yet been determined.
Status of Development: Developmental.
Describe: Procedures have not been fully developed.
Applications: Air; Water; Food.
Samples: Pure Chemicals; Most classes. Complex Mixtures;
Ambient; Industrial; Energy Related; Transportation Related.
Duration: I month to develop In-vitro technique; 3 months to
develop in-vivo technique; approx. I week to implement.
Cost: For development $15,000; to test chemical less than $5,000/
chemical.
Interpretation: A positive result is predictive of possible platelet
dysfunction.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: G. Hatch, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Charo, O.F., R.D. Feinman, and T.C. Detwiler. J.
din. Invest., 60:866-873, 1977.
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1116 MAMMALIAN PLATELET AND FIBROBLAST IMPAIRMENT
Biological Activity Detected: Toxicity.
Principle: Contaminant is added to platelets in-vitro. The relative
amounts of energy metabolism intermediates are measured. C14
adenine is used as a precursor.
Endpoints: Qua Iitattve; Ratios of ATP, ADP, and AMP. Quantitative:
N/A.
Strengths: Quantitative; Rapid; Capable of direct interpretation.
Weaknesses: Sensitivity.
Status of Development: Developmental.
Describe: Testing with dilutions of pure compounds and extracts
of wastewater.
Applications: Water.
Samples: Pure Chemicals: Hydrocarbons. Complex Mixtures; Am-
bient - rivers; Other - wastewaters.
Duration: 3 years.
Cost/sample or chemical: $125.
Interpretation: Positive result suggests a possible interference
of platelet function in-vivo.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch.
Persons to Contact: H. Pahren. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Colorado Medical Center, 4200 E. 9th Ave., Denver, CO
80262, C.C. Solomons.
Program Office Support: OHEE.
References: Not yet available.
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1117 LYMPHOCYTE CYTOTOXICITY
Biological Activity Detected: Toxicity.
Principle: Lymphocytes (T cells) have been shown to have cytotoxic
effector actions against neoplastic or other cells. The potential
exists that pollutants could adversely affect this function,
thereby increasing the risk of the host to the development of
neoplastic disease.
Endpoints: Qua Iitative: N/A. Quantitative; Measurements .
of lymphocyte cytotoxic activity and lectin induced transforma-
tion will be made following in-vitro pollutant exposure.
Strengths: The in-vitro model would permit rapid screening for a
significant health parameter; Dose-response studies would permit
ranking of pollutant effects; Relatively small quantities of
pollutant would be required.
Weaknesses: The in-vitro model is not yet validated. Even after
validation, results would have to be confirmed with in-vivo
exposure studies, possibly of a chronic nature, before the data
could be useful for regulation.
Status of Development: Developmental.
Describe: Highly developmental. No pollutants have yet been
tested. Model to be completed March, 1979.
I AppIications: Multimedia.
Samples: Pure Chemicals: Potentially, any chemical. CompI ex
Mixtures; Ambient; Industrial; Energy Related; Transportation
Related; Other - any mixture that does not include gas.
Duration: Not yet determined.
Cost: $l75,000/model development.
Interpretation: In-vitro model not validated at present.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: J.A. Graham, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
Rockefeller U., 1230 York Ave., New York, NY 10021, M. Bowers.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Kirchner, H., and R.M. Blaese. In Lymphocyte Recogni-
tion and .Effector Mechanisms. Acad. Press, N.Y., 1974. pp.
357-361. 2) Light-body, J., and J.C. Rosenberg. In Lymphocyte
Recognition and Effector Mechanisms. Acad. Press, N.Y., 1974.
pp. 363-367. 3) Perlman, P., and G. Holm. Adv. Immunol., ||'
I 17, 1969.
10
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1118 IMPAIRMENT OF NEUTROPHIL PHAGOCYTOSIS
Biological Activity Detected: Toxicity.
Principle: Functional and metabolic disturbance of neutrophils
are measured after exposure to contaminant.
Endpoints: Qua Iitajiive: Percent phagocytosis and percent killing
power are measured. Quantitative_: N/A.
Strengths: Rapid; Dose-response of procedure has been shown.
Weaknesses: Sensitivity may be a possible problem if low PPB
exposure is necessary.
Status of Development: Developmental.
Describe: Testing with dilutions of pure compounds and extracts
of wastewater.
Applications: Water.
Samples: Pure Chemicals: Hydrocarbons. Complex Mixtures: Am-
bient - rivers; Other - wastewaters.
Duration: 3 years.
Cost/sample or chemical: Not yet determined.
Interpretation: Positive result is predictive of possible damage
to neutrophils in-vivo.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
log'cal Assessment Branch.
Persons to Contact: H. Pahren, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Colorado Medical Center, 4200 E. 9th Ave., Denver, CO
80262, W.L. Weston.
Program Office Support: OHEE.
References: I) Tan, J.S., et al. A Modified Assay of NeutrophtI
Function: Use of Lysostrophin to Differentiate Defective
Phagocytosis from Impaired Intrace Ilular KM I ing. J. Lab. Clin.
Med., 78:316, 1971.
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1119 HUMAN LUNG FIBROBLASTS (WI38)
Biological Activity Detected: Toxicity.
Principle: Toxicants alter biosynthetic processes leading to a
reduction in cell growth and division.
Endpoints: Qua Iitative: Morphology. Quantitative: Cell number
and viability; Total cell protein and DNA; Cell adenosine
triphosphate; Incorporation of radio-label led thymidine,
uridine, and leucine.
Strengths: Relatively inexpensive; Rapid; Fewer samples required
than for conventional whole animal bioassays; One of the best
characterized diploid human cells available for toxicity bioassays
Weaknesses: Not representative of intact animals, providing only
preliminary information about the potential health hazards of the
test chemicals; May be replaced by other cell strains as supp|jes
dwindle; The system currently can not be coupled with mutagenecitv
testing unlike other mammalian cell systems.
Status of Development: Being implemented.
Describe: A number of pure compounds have been evaluated.
Applications: Air; Water.
Samples: Pure ChemicaIs; Inorganics, Organics, Heavy Metals.
Complex Mixtures: Industrial; Energy Related - fly ash;
Other - AWT effluent, metal-coated fly ash.
Duration: 20 hr.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Alterations in the basic metabolic processes
and cellular structure indicate the potential toxicity of
the agent.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch; HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch.
Persons to Contact: N.E. Kowal. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7477); M.D. Waters. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
J.L. Huisfngh, U.S. EPA, HERL-RTP, Research Triangle Park, NC 277|i
(FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
Gulf South Research Institute, P. 0. Box 26518, New Orleans, LA
70186, N. Gruener; Northrop Services, Inc. P.O. Box 12313,
Research Triangle Park, NC 27709, N.E. Garrett.
Program Office Support: OHEE; OEM I.
References: I) Campbell, J.A., H.F. Stack, M.R. Williams, D. Tillery
N. Custer, B.F. Russell, S.W. King, E.B. Siegel, and N.E. Garrett*
Cellular Toxicity of Four Liquid Effluent Samples from Textile
Mills: Studies on the Rabbit Alveolar Macrophage, WI38 Human
Fibroblast and Chinese Hamster Ovary Cell In-Vitro. Contract
Report ESG-TR-78-04 to the U.S. Environmental Protection Agency
Northrop Services, Inc., Research Triangle Park, NC. February '
1978. 2) Garrett, N.E., J.A. Campbell, J.L, Huisingh, and M.D.
Waters. The Use of Short-Term Bioassay Systems in the Evaluation
of Environmental Particulates. In: Proceedings of the Symposium
12
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1119 HUMAN LUNG FIBROBLASTS (W138) (continued)
on the Transfer and Utilization of Particulate Control Technology.
Denver, CO, July 24, 1978. In press. 3) Waters, M.D., T.O.
Vaughan, D.J. Abernathy, H.R. Garland, C.C. Cox, and'D.L. Coffin.
Toxicity of Platinum (IV) Salts on Cells of Pulmonary Origin.
Envir. Hlth. Perspect., 12:45-56, 1975. 4) Waters, M.D., D.R.
Abernathy, H.R. Garland, and D.L. Coffin. Toxic Effects of Selected
Metallic Salts on Strain WI38 Human Lung Fibroblasts. In-Vitro,
10:342, 1974. 5) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The
Cellular Toxicity of Complex Environmental Mixtures. In: Proceedings
of the Symposium on the Application of Short-Term Bioassays in the
Fractlonation and Analysis of Complex Environmental Mixtures.
WiIliamsburg, VA, 1978.
13
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11110 CHINESE HAMSTER OVARY (CHO) CLONAL' TOXlCltY
Bi 61 pg i ca I, Act i:vJ ty Detected: •.-., To>d c,} ty,;;;
Pr.icic:ip;j,
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11110 CHINESE HAMSTER OVARY (CHO) CLONAL TOXICITY (continued)
4) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The Cellular
Toxicity of Complex Environmental Mixtures. In: Proceedings of
the Symposium on the Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures.
WiI Iiamsburg, VA, 1978.
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11111 RABBIT ALVEOLAR MACROPHAGE (RAM)
Biological Activity Detected: Toxicity.
Principle: Toxic agents alter basic metabolic processes and cellular
structure of the macrophage.
Endpoints: Qua Iitative; Morphology. Quantitative: Cell number and
viability; Cell adenosine triphosphate; Phagocytic activity Tot I
cell protein; Hydrolytic enzyme specific activities.
Strengths: The alveolar macrophage plays an important role in the
defense of the lung against inhaled particulate materials- Thl
cell type receives direct exposure to environmental toxicants
Weaknesses: This in-vitro cell system approximates the response whl K
might be observed in the intact animal. c"
Status of Development: Being implemented.
Describe: The response of the RAM system to a variety of indus-
trial and energy-related particulates has been studied. ""
Applications: Air; Water.
Samples: Pure Chemicals; Metal chlorides and suI fates, Metallic
oxides. Complex Mixtures: Industrial - textile effluents
aluminum refinery, copper smelter; Energy Related - coal gaslfl
tion and fluidized bed combustion; Other - metal-coated fly ash "~
Duration: 20 hr.
Cost/sample or chemical: $500 to $900.
Interpretation: Changes in cellular adenosine triphosphate and
viability by dye exclusion indicate potential toxicity of tested
substance.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch.
Persons to Contact: M.D. Waters. HERL-RTP, Research Triangle Park
NC 27711, (FTS 629-2693); J.L. Huisingh. HERL-RTP, Research '
Triangle Park, NC 27711, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
NT Research Institute, 10 West 35th Street, Chicago IL
60616, C. Aranyi: Northrop Services, Inc., P.O. Box 12313
Research Triangle Park, NC 27709, N.E. Garrett. '
Program Office Support: OHEE; OEM); OAWM.
References: I) Huisingh, J.L., J.A. Campbell, and M.D. Waters.
Evaluation of Trace Element Interactions Using Cultured Alveola
Macrophages. In: Pulmonary Macrophage and Epithelial Cells
Conf-760972, Sanders, C.L., R.P. Schneider, G.E. Dag|e< and H A
Hagan, eds. ERDA Symposium Series 43, Technical Information CeiU-x
Energy Research and Development Administration, 1977. pp 34* •*l5r
2) Waters, M.D., J.L. Huisingh, and N.E. Garrett. The Cellular
Toxicity of Complex Environmental Mixtures. In: Proceedings of
the Symposium on the Application of Short-Term Bioassays in the
Fractionation and Analysis of Complex Environmental Mixtures
WiIliamsburg, VA, 1978. 3) Waters, M.D., D.E. Gardner, and D I
Coffin. Cytotoxic Effects of Vanadium on Rabbit Alveolar Macr
phage In-Vitro. Toxicol. Appl. Pharmacol., 28:253-263, (974 °~
4) Waters, M.D., T.O. Vaughan, J.A. Campbell, F.J. Miller, and •
D.L. Coffin. Screening Studies on Metallic Salts Using the Rabbr
16
-------
11111 RABBIT ALVEOLAR MACROPHAGE (RAM) (continued)
phage In-Vitro. Toxicol. Appl. Pharmacol., 28:253-263, 1974.
4) Waters, M.D., T.O. Vaughan, J.A. Campbell, F.J. Miller, and
D.L. Coffin. Screening Studies on Metallic Salts Us-ing the Rabbit
Alveolar Macrophage. In-Vitro, 10:342-343, 1974. 5) Waters, M.D.,
D.E. Gardner, C. Arnyi, and D.L. Coffin. Metal Toxicity for
Rabbit Alveolar Macrophages In-Vitro. Envir. Res., 9:32-47, 1975.
6) Waters, M.D., T.O. Vaughan, D.J. Abernathy, H.R. Garland, C.C. Cox,
and D.L. Coffin. Toxicity of Platinum (IV) Salts for Cells of Pul-
monary Origin. Envir. Hlth. Perspect., 12:45-56, 1975.
-------
11112 RAT HEPATOCYTE (LIVER CELL)
Biological Activity Detected: Toxicity.
Principle: Toxic agents alter basic metabolic processes and cellular
structure and function of the hepatocyte.
Endpoints: Qua Iitative; Morphology. Quantitative: Cellular via-
bility; Adenosine triphosphate content; Tyrosine aminotransferase
activity; Total cell protein.
Strengths: These primary liver parenchyonal cells resemble the adult
liver cell in-vivo morphologically and in many of the biochemical
parameters evaluated.
Weaknesses: These cells do not divide and must be isolated from a
rat prior to each assay. Since there is rat-to-rat variation
cells from several rats should be used to evaluate each chemical
Status of Development: Developmental; Being implemented.
Describe: New endpoints are being developed; however, the ussay
is now being implemented with both inorganic and organic chemicals
Applications: Multimedia.
Samples: Pure Chemicals; Inorganic salts, Organic solvents, Organic
sol ids. Complex Mixtures; N/A.
Duration: 20 hr.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Alterations in the basic metabolic processes and
cellular structure and function of the liver cells determine the
potential toxicity of the agent.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division
Biochemistry Branch, Cellular Biology Section. '
Persons to Contact: J.L. Huisingh, HERL-RTP, Research Trianqle Park
NC 27711, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE; OTS. "
References: I) Huisingh, J.L., J.P. Inmon, L.C. King, K. Williams
and M.D. Waters. The Use of Rat Liver Parenchyma! Cells in *
Evaluating Cellular Response to Toxic Metals and Carcinogenic
PolycycIic Aromatic Hydrocarbons. In-Vitro, 13:182, 1977.
2) Waters, M.D., and J.L. Huisingh. In-Vitro Testing for Chemi-
cal Toxicity: Mammalian Target Cells. In-Vitro, 13:192, 1977
18
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11113 CHINESE HAMSTER OVARY (CHO) CYTOTOXICITY AND MUTAGENICITY
Biological Activity Detected: Toxicity; Mutagenicity.
Principle: Toxicity is evaluated from effects on clonal growth.
Mutation at the hypoxanthine-guanine phosphor!bosyI transferase
(HGPRT) locus is determined from mutants isolated in 6-thioguanine
containing media.
Endpoints: Qua Iitative: N/A. Quantitative: Colony formation
(cytotoxicity); Mutation frequency.
Strengths: Cytotoxicity and mutagenicity may be studied simultaneously.
Weaknesses: The cell type may not be representative of metabolically
active cells which receive exposure to environmental toxicants.
Status of Development: Developmental.
Describe: This assay has been shown to be useful in studies of
70 individual environmental agents related to energy technologies
and 3 subtractions of a crude synthetic oil.
Applications: Air; Water.
Samples: Pure Chemicals: Polycyclic hydrocarbons, Metallic compounds,
Nitrosamines, Quincline compounds, Physical agents, A Iky I at ing
agents, Nitrogen mustards, and Aromatic amines. Complex Mixtures:
Energy Related - synthetic fuel.
Duration: 18 days.
Cost/sample or chemical: $500 to $1,000.
Interpretation: Decreasing clonal growth after exposure of CHO cells
indicates potential toxicity of test substance. Increasing numbers
of mutants with increasing concentration of the test substance in-
dicate the substance is a potential mutagen.
LeveI of CompI ex i ty: 2
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section.
Persons to Contact: J.L. Huisingh, HERL-RTP, Research Triangle Park,
NC 2771 I, (FTS 629-2537).
Grant/Contract Laboratory Involved and Principal Investigators:
Northrop Services, Inc., P.O. Box 12313, Research Triangle Park,
NC 27709, N.E. Garrett.
Program Office Support: OHEE; OEM I.
References: I) Hsie, A.W., et al. Quantitative Mammalian Cell Genetic
Toxicology: Study of the Cytotoxicity and Mutagenicity of Seventy
Individual Environmental Agents Related to Energy Technologies
and Three Subfractions of a Crude Synthetic Oil in the CHO/HGPRT
System. In: Proceedings of the Symposium on Short-Term Bioassays
in the Fractionation and Analysis of Complex Environmental Mix-
tures. Wi I Iiamsburg, VA, 1978. 2) O'Neill, J.P., P.A. Brimer,
R. Machanoff, G.P. Hirsch, and A.W. Hsie. A Quantitative Assay of
Mutation Induction at the Hypoxanthine-Quanine PhosphoribosyI
Transferase Locus in Chinese Hamster Ovary Cells: Development
and Definition of the System. Mutat. Res., 45:91-101, 1977.
3) O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian,
P.A. Brimer, and A.W. Hsie. A Quantitative Assay of Mutation Induc
tion at the Hypoxanthine-Guanine PhosphoribosyI Transferase Locus
in Chinese Hamster Ovary Cells (CHO/HGPRT system): Utilization
with a Variety of Mutagenic Agents. Mutat. Res., 45:103-109, 1977.
19
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CONTENTS: TEST SYSTEMS
20 In-Vivo Systems/Acute 21
I 121 LD50 22
1122 Whole Animal LD50-Oral, Dermal 23
130 In-Vivo Systems/Subacute 25
21
-------
1121 LD50
Biological Activity Detected: Toxicity.
Principle: Based on preliminary range finding tests, 10 dose-levels
of the test compound are selected in the range from Q% to \QQ%
mortality. Each group would consist of 7 animals. The animals
receive one dose and then are observed for a period of 14 days
After the 14-day period, a dose-response (cumulative mortality)
curve is plotted and the dose level producing 50% mortality is
interpolated.
Endpolnts: Qua IItative; Clinical signs of toxicity. Quantitative-
Cumulative mortality. ~'
Strengths: Can determine a dose-response curve for nearly all test
compounds and from that establish a maximum tolerated dose level
to use in a multiple dose, longer-ranged study.
Weaknesses: Toxfcity of vehicle; Solubility; Vehicle-compound
synergism and antagonism.
Status of Development: Validated.
Describe: The chemical is administered by route of interest
condition of animals standardized, and observation period '
specified. Decisions, however, must be made concerning the
type of solvent or vehicle that is most appropriate.
Applications: Multimedia.
Samples: Pure Chemicals: Most classes. Complex Mixtures: Drink-
ing water concentrates.
Duration: Variable.
Cost/sample or chemical: $500.
Interpretation: The establishment of a maximum tolerated dose.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Toxicological Assessment Branch.
Persons to Contact: R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St C|a?r
St., Cincinnati, OH 45268, CFTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE. ' *'
References: I) Loomis, T.A. Essentials of Toxicology, 2nd Ed lea
and Feblger, Philadelphia, PA, 1974. pp. 17-25. "
22
-------
1122 WHOLE ANIMAL LD50 - ORAL, DERMAL
Biological Activity Detected: Toxicity.
Principle: Administration of test substance to a sufficient number
of rats, over a dosage range resulting in 0% to \00% effects.
Endpoints: Qua Ii tat i ve: Clinical effects. Quantitative; Effective
dosage to produce effect, LD50 value.
Strengths: Estimates relative toxicity; Economical; Simple to
conduct.
Weaknesses: May not apply to all species; May not correspond with
data from other laboratories.
Status of Development: Being implemented.
Describe: Tests currently being conducted on limited basis on
relevant selected compounds.
Applications: Multimedia.
Samples: Pure Chemicals: AM classes. Complex Mixtures: Other -
technical grade materials, formulated products.
Duration: 3 months.
Cost: $5,000/chemical for full battery of tests.
Interpretation: A rating of the toxicity of the various chemicals
is obtained.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology
Division.
Persons to Contact: R. Linder. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2701).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP.
References: I) Gainer, T.B. Acute Toxicity of Pesticides. Toxicol.
Appl. Pharmacol., I960.
23
-------
CONTENTS: TEST SYSTEMS
1130 In-Vfvo Systems/Subacute 25
113! Integrated System: General Clinical
Pathology 26
1132 Sleep-Time Study 27
1133 Metabolism of Chlorinated Hydrocarbons in
Subhuman Primates 28
1134 Metabolic Profiles 29
1135 Model Substrate MetaboI ism 30
1136 Xenobiotic Mechanisms ..... 32
I 137 Oxidant Production by Leukocytes and Alveolar
Macrophages Measured by Chemi Iuminescence ... 33
1138 Cyclic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages 34
1140 In-Vivo Systems/Inha I at ion Toxicology 35
25
-------
1131 INTEGRATED SYSTEM: GENERAL CLINICAL PATHOLOGY
Biological Activity Detected: Systemic effects.
Principle: Measurement of serum constituents, proteins, enzyme
activities, hematological parameters such as cell counts cell
morphology in peripheral blood and in bone marrow, measurement
of special endocrino logic parameters such as thyroid and adrenal
functions, pituitary hormones, etc. Diagnosis of malignancies
by measurement of tumor marker proteins, serum isoenzyme patterns
etc. Measurement of urinary constituents. '
Endpoints: Qua IItative; Technique dependent. Quantitative: Tech-
nique dependent.
Strengths: High degree of quality control; Great amount of informa-
tion is available as to the diagnostic implications of abnormal
f indings.
Weaknesses: Often not capable of signaling asymptomatic preclinical
toxic effects; Selection of tests must be done with care and
proper planning to ensure maximal effectivity.
Status of Development: Validated.
Describe: This entry encompasses approximately 800 different
varieties of tests that may be performed on animals in-vivo or
on biological specimens in-vitro. Usually, a battery of tests
will be performed examining various organ functions.
AppIi cat i ons: Mu11 i med i a.
Samples: Pure Chemicals; N/A. Complex Mixtures:
Other - biological specimens: blood, urine, bone marrow.
Duration: Continuous.
Cost/sample or chemical: Test dependent.
Interpretation: The totality of a battery is aimed at detecting
organ-specific toxic effects.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Toxicologlcal Assessment Branch, Systemic and Genetic Effects
Group; HERL-CIN, Field Studies Division, Toxicological Assessment
Branch.
Persons to Contact: R.J. Bull. U.S. EPA, HERL-CIN, 26 W. St Clair
St., Cincinnati, OH 45268, (FTS 684-7213); J.P. Bercz U S EPA
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684
7480).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE.
References: Theories and practical techniques as compiled in clinic
pathology textbooks: I) Henry and Davidson. Clinical Diagnosis8
by Laboratory Methods. 2) Tietz. Clinical Chemistry. 3) Win-
trobe. Clinical Hematology.
26
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1132 SLEEP-TIME STUDY
Biological Activity Detected: Identifies the biological act-
ivity of a compound indicating a potential for interaction with
other compounds.
Principle: Chemicals that induce or inhibit MFO will alter the
pharmacological effects of drugs metabolized by MFO. Hexabarbital
and zoxogoI amin are depressant drugs whose properties are well
known in this respect. Animals are given a single dose or multiple
doses of the test compound at a tolerated but effective level. Two
hours after the final dose they are challenged with an anesthetic
dose of hexobarbitaI. A control group receiving no test compound
also receives the hexobarbital. The time is measured from the
instant the animals lose their "righting reflex" (ability to right
themselves when laid flat on their back) to the time they regain
it.
Endpoints: Qua Iitative; Induction or inhibition of liver enzyme
activity. 9uan'^'^a'*''ve; Measured sleep-time.
Strengths: Fast and presumptive assay to determine whether a compound
will induce or inhibit Iiver enzymes; Useful in planning more
extensive metabolism studies.
Weaknesses: Changes in rates of metabolism must be documented to
conclude the effect mediated via MFO.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures;
Industrial; Energy Related.
Duration: Variable depending on projected properties of test compounds.
Cost: Approx. $200/compound.
Interpretation: The results determine the ability of a compound to induce
or inhibit enzymatic systems.
LeveI of CompI ex i ty: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicologies! Assessment Branch; HERL-RTP, Clinical Studies
Division, Biomedica! Research Branch.
Persons to Contact: R.J. Bui I. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213); D.E. Gardner. U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Conney, A.H., et al. Adaptive Increases in Drug
Metabolizing Enzymes Induced by Phenobarbital and Other Drugs.
J. Pharmacol. Exp. Ther., 130:1-8, I960. 2) LaDu, B.N., H.G.
Mandel, and E.L. Way. Fundamentals of Drug Metabolism and Drug
Disposition. The Williams and Wilklns Co., Baltimore, MD, 1971.
27
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1133 METABOLISM OF CHLORINATED HYDROCARBONS IN SUBHUMAN PRIMATES
Biological Activity Detected: Comparative metabo I ism.
Principle: In drug metabolism studies, the primate as a model more
often represents humans than any other animal model. it is
likely that this is the case for environmental contaminants
also. !> '
Endpoints: Qua Iitative: Comparison of metaboIites from various
animal species. (Quantitative; Quantitative analysis of meta-
bolites from various animal species.
Strengths: The prftnate Is most likely to be representative of man
In Its metabolic activity toward environmental contaminants.
Weaknesses: Expense and difficulty of working with monkeys.
Status of Development: Validated.
Describe: After dosage, specific enzymes are tested for activity.
Excreta samples are chemically analyzed for metabolites.
Applications: Multimedia.
Samples: Pure Chemicals; Chlorinated aliphatic hydrocarbons,
Chlorinated aromatic hydrocarbons. Complex mixtures; N/A.
Duration: 3 months.
Cost: $15,000 to $18,000.
Interpretation: Comparison of metabolism from various animal systems.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Exposure Evaluation Branch,
Organlcs Metabolism Section.
Persons to Contact: P.P. Lingg. U.S. EPA, HERL-CIN, 26 W. St Clalr
St., Cincinnati, OH 45268, (FTS 684-7463).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Cincinnati, Cincinnati, OH 45221, C. Smith, (Commercial
513 872-5700).
Program Office Support: OHEE.
References: I) Smith, C., R.D. Lingg, and R.G. Tardiff. Comparative
Metabolism of Haloethers. Ann. N.Y. Aca. Sci., 298:111, 1977.
28
-------
1134 METABOLIC PROFILES
Biological Activity Detected: Toxicity; Biotransformation; Compara-
tive metabolism.
Principle: Identification and quantitative analysis of major meta-
bolites are obtained through the use of modern analytical tech-
niques. From these results, a metabolic pathway for a selected
compound can be proposed. The potential of a compound for inter-
action with macromolecules is better understood.
Endpoints: Qua Iitative: Identification of major metabolites.
Quantitative; Quantitative analysis of major metabolites.
Strengths: Leads to an understanding of the structural differences
in compounds which affect their metabolic disposition and
tox i c i ty.
Weaknesses: Difficult to extrapolate to the human condition.
Status of Development: Being implemented.
Describe: Most separation and derivatization techniques have
been validated using 3-Chloroethers and Trichlorobenzenes as
model compounds. Additional work needs to be done on processing
mass spectral data.
Applications: Multimedia.
Samples: Pure Chemicals; Halogenated aliphatic hydrocarbons,
Halogenated aromatic hydrocarbons. CompI ex M i xtu res; N/A
Duration: 3 months.
Cost/sample or chemical: $10.,000 to $15,000.
Interpretation: The determination of metabolic pathways leads to
an understanding of the differences in the toxication of a
selected chemical in various animal species.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Exposure Evaluation Branch,
Oraanics Metabolism Section; HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch, Metabolic Effects Section,
Persons to Contact: R.D. Lingg, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7463); S. Nesnow. U.S. EPA,
HTRL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) R.D. Lingg, et al. Fate of Bis (2-ChtoroethyI) Ether
in Rats after Acute Oral Administration. To be presented at the
Seventeenth Annual Meeting of the Society of Toxicology, Mar.
12-16, 1978, San Francisco, CA. 2) R.D. Lingg, W. Kaylor,
S.M. Pyle, and R.G. Tardlff. Thiodiglycolic Acid: A Major
Metabolite of Bis (2-ChloroethyI) Ether. Submitted to Toxicol.
and Appl. Pharmacol. Dec., 1977.
29
-------
1135 MODEL SUBSTRATE METABOLISM ' f-s
Biological Activity Detected: Toxicity. ....
Principle: The model substrate assay attempts to determine the effect
of environmental chemicals on metabolic pathways in the living
animal, This test involves repeated exposure of the experimental
animals (6/treatment group) to the toxicants under investigation
After this pretreafment period the animals receive a single oral*
dose of the model substrate, 11+C-I indane. A useful variation in
this assay involves,..the simultaneous administration of lindane
with .the toxicants being studied.
Endpoints: Qua I-1 tat lye; Altered roetabolite profi|es serve as a
"fingerprint of toxicant exposure" since xenobiotics have charac-
teristic, effects, on.-the metabol ic pathways of the model substrate
Quantitative: Induced or inhibited metabolism are measured by
GLC analysis and liquid scintillation counting of excreted pro-
ducts.
Strengths: Analysis of excreted metabolites provide direct measure
of overall metabolism in Iiving animal; Changes in enzyme activity
due to disruption of phospholipid membranes, loss of permeability
. barriers, and loss of local charge effect are avoided; Anomalous
enzyme alterations caused by accumulation of substrate and/or
metabolites are avoided since normal clearance mechanisms are
functional; Economical and practical since comparative activity
of multiple pathways are determined on individual animals; Dallv
changes In the metabolic pathways of animals repeatedly exposed
to toxicants can be determined.
Weaknesses: Requires GLC standards of the model substrate metabolit
being determined. es
Status of Development: Being implemented.
Describe: The modeJ substrate assay has been successfully
employed.to study interactions between a variety of environmental
chemicals, nutritional stress, and the enzyme systems that detoxlf
or enhance the toxlcjty of xenoblptics. The correlation between ^
exposure to xenobiotics^ whose metabolites produce the same
lesion, and their Induction of a model substrate metabolite
profile with a common.eIement is being investigated.
Applications: Multimedia. - •
Samples: Pure Chemicals; Ca, Organochlorine pesticides, Herbicides
Insecticides, Fungosides, Drugs, Toxic.substances. '
Complex Mixtures; Not yet tested.
Duration: Test: 2-week exposure; Analysis: 3 weeks.
Cost: $2,700. . .
Interpretation: It is thought that the generation of unusual altera-
tions in the relative activity of various metabolic pathways of
a model substrate can signal toxic interactions.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology
Division, Biochemistry Branch.
30
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1135 MODEL SUBSTRATE METABOLISM (continued)
Persons to Contact: R.W. Chadwick, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2750); M.F. Copeland.
U.S. EPA, HERL-RTP, Research Triangle Park, NC 27711,
(FTS 629-2678).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OTS.
References: I) Chadwick, R.W., C.J. Chadwick, J.J. Freal, and C.C.
Bryden. Comparative Enzyme Induction and Llndane Metabolism in
Rats Pre-treated with Various Organochlorlne Pesticides. Xeno-
biotlca, 7:235-246, 1977. 2) Chadwick, R.W., W.S. Simmons, C.C.
Bryden, L.T. Chuang, L.M. Key, and C.J. Chadwick. Effect of
Dietary Liquid and Dimethyl Sulfoxlde on Llndane Metabolism.
Toxicol. and Appl. Pharmacol., 59:391-410, 1977. 3) Chadwick,
R.W., M.F. Copeland, and C.J. Chadwick. Enhanced Pesticide
Metabolism, a Previously Unreported Effect of Dietary Fiber in
Mammals. Food and Cosmetics Toxicol., 1978. In press.
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1136 XENOBIOTIC MECHANISMS
iow I ng tr>ev aose reg imen rr\& an iman s-^rer sac
, $m^f£W+il0n' f^^he^y^eP^I%^f ^
Cnnjs: yyarn rariVH;j;. redcap arijai cyjr^po: revei-s^'eytPc reductase
Q-tieffiethyl aW,, aji^'bjfner- mi cro'sbrnsfl' fen'^yrne ac¥l v'HVes. Quantitative
tWafc'ar'lT£' I' cytP-450;' Iev&) s',' cytFQf'iHe'dtictas-6,• 'Cf^d'e'rii^thy I ase, and
6t'he'r:!in1l;cr6s'bjtia 1° bh±yme' ab'tl v:i'tTesJ.rifr> °'u^' -: '.'"'^^ • '-
I iver
l-ang^nUrtiWi^'of ahfrta^i "
Stat-'tis of' Devel'opmenf:' Va I idafe'd:'
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: Xenobiotics. Complex Mixtures: N/A
Duration: Variable, I week to chronic, depending on whether testinq
is acute or chronic.
Cost/sample or chemical: Variable, depending on test. I week at
$35,000/manyear equals approximately $700.
Interpretation: This test determines the potential for synergisms
and/or antagonisms mediated through altered xenobiotic metabolUmc
Level of Complexity: 2. s*
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Toxicological Assessment Branch; HERL-RTP, Environmental Toxi-
cology Division, Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: R.J. Bui I. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213); S. Nesnow. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693). *
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE.
References: I) Hayakwa, T. A Simple Radioisotope Assay for Micro-
somal Aryl Hydroxylase. Anal. Biochem., 51:501-509, 1973. 2)
Dallner, G. Studies on the Structural and Enzymatic Organization
of Liver Microsomes. Acta Path. Scand., 166:7-41, 1963. 3) Neal
R.A. Studies on the Mechanisms of Detoxification of Cholinerqic '
Phosphorothioates. J. Pharmacol. Exp. Therap., 148:185-192, (955
4) Lucier, G.W. Microsomal Rat Liver UDP Glucuronyl Transferase-*
Effects of Piperonyl Butoxide. Arch. Biochem. and Biophys., 145!
520-530, 1971. 5) Omura, T. The Carbon Monoxide Binding Pigment
of Liver MIcrosomes: I. Evidence for its Hemoprotein Nature
J. Biol. Chem., 239;2370-2378, 1964.
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1137 OXIDANT PRODUCTION BY LEUKOCYTES AND ALVEOLAR MACROPHAGES MEASURED
BY CHEMILUMINESCENCE
Biological Activity Detected: Toxicity.
Principle: Oxidant production in alveolar macrophage microbicidal
activity is due to alveolar macrophage metabolic activation. Oxi-
dants are measured by measuring light produced by oxidation re-
actions.
Endpoints: Qua Iitative; N/A. Quantitative; Production
of light as measured in an ATP photometer. The amount of light
can be measured in response to purified macrophage stimulates
and particles.
Strengths: Very reproducible; Fast; Simple; Does not kill cells;
Requires very few cells; Applicable to human blood cells; Several
types of oxidant can be measured; In-vivo or in-vitro dose-response
tests can be made; In-vivo tests are suitable for standard-setting
anr" regulatory purposes.
Weaknesses: Chemiluminescent reactions are susceptible to many
quenching effects and competing reactions; Adequate controls are
necessary; For in-vivo exposures, relatively large amounts of
pollutant sample are required.
Status of Development: Being impIemented.
Describe: Effects of 03, NC>2, and various particles are being
determined.
Applications: Multimedia.
Samples: Pure Chemicals; All transparent chemicals which are
not readily oxidized. Complex Mixtures: All materials except
those which are very opaque.
Duration: In-vitro exposure: 2 weeks/dose-response of I chemical; in-
vivo exposure: 4 weeks/dose-response of I chemical.
Cost: In-vItro exposure: $5,000/chemical; In-vivo exposure: $10,0007
chemical.
Interpretation: Defect in oxidant production predicts possible micro-
bicidal defect in the macrophages.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: G. Hatch. U.S. EPA, HERL-RTP, Biomedical
Research Branch, Research Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Hatch, G.E., D.E. Gardner, and D.B. Menzel. J. Exp.
Med., 147;183-195, 1978. 2) Rosen, H.f and S.J. Klebanoff. J.
Clin. Invest., 58:50, 1976.
33
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1138 CYCLIC NUCLEOTIDE CONCENTRATIONS' IN LEUKOCYTES AND ALVEpLAR1
Biological Activity Detected: Toxicity; Presumptive, oncogenicity•
Pharmacologic modulation of promotion.
Principle: Cyclic AMP and Cyclic GMP are. j'ntracel I ular hormones
which .modulate cellular functions, Including cell proliferation
secretion, and movement. The hormones are measured by radio- '
immunpassay. . .
Endpo.ihts: Qua!ttative: N/A. Quantitative: Concentration
.of cyclic nucleotide per eel I.correlate with cellular activity.
Strengths: Cyclic nucleotide concentrations are easi ly altered by*
subtle means;.Their concentrations are of central importance to
eellutar act}vity; Applicable to human blood cells; In-vivo or
in-vitro dose-response, studies can be done; In-vivo tests would.
be suitable for standard setting and regulatory purposes.
Weaknesses: Tfme consuming; Tedious; Requires a lot of cells; Dif-
ficult to establish steady basal or control values; In-vivo
exposures wiI 1 require relatively I arcje amounts of pollutant
samples.
Status of Development: "Being Implemented.
Describe: Effects of N02, 03, and NO are being tested for effectc
on Cyclic AMP and'Cyclic GMP.
Applications: Air; Water; Food; Multimedia.
Samples: Pure Chemicals; Most chemicals. Complex Mixtures:
Ambient; Industrial; Energy Related; Transportation Related.
Duration: In-vitro exposure: 2 weeks/chemical for dose-response-
In-vivo exposure: 4 weeks/chemical for dose-response. '
Cost: In-vitro exposure: $5,000/chemicaI; In-vivo exposure: $IO,000/
chemical.
Interpretation: Detection of alteration in cellular hormone metabo 11
which fs important to homeostasis.
Level of Complexity: 2. . . ;
OHEE Laboratory Involved: . HERL-RJP, Cl infcal Studies Division,
Blomedical Research Branch.
Persons to Contact: G, Hatch. U.S. EPA, HERL-RTP, Biomedlcal
Research Branch, Research Tr,iangle Park, NC; 2771 I, (FTS 629-2531)
Grant/Cpntract Laboratory Involved and Principal Investigators* N/A
Program Off Ice Suppprt; OHEE; OPP; OEMI; OtS,
References: I) Hatch, .G.E., ^.K. Nlchols/'and H.R. Hi 11. j lmmunr>i
I I9:450-456, 1977,: :; : ,.. '' / ;'.. v * uno'-,
34
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CONTENTS: TEST SYSTEMS
1140 In-Vivo Systems/InhaI at ion Toxicology 35
1141 Toxicity of Aerosolized Pollutants/Acute and
Subacute 36
I 142 Toxicity of Gaseous and Vapor Phase/Acute and
Subacute 37
1143 Inhalation LC50 Tests 38
I 144 Deposition and Clearance of Radioactive
Materials Following Inhalation Exposure .... 39
1145 Integrated System: Pulmonary Function and
Pulmonary Metabolism 40
1146 Pulmonary Function in Rats 41
1147 Pulmonary Function of Unanesthetized
Guinea Pigs 42
1148 Arterial Blood Gas Measurement in
Conscious Rats 43
1149 Infectivity Model 44
I 1410 In-Vivo Alveolar Macrophage Cytotoxicity 45
1150 In-Vivo Systems/Neurobehavioral 47
35
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1141 TOXICITY OF AEROSOLIZED POLLUTANTS/ACUTE AND SUBACUTE
Biological Activity Detected: Toxicity.
Principle: Exposure begins with a relatively high concentration
which results in 100$ mortality in I to 4 hours. The concentratio
is decreased by factors of 10 until no deaths occur. Performed
in rats.
Endpoints: Qua Iitative: N/A. Quantitative: Deaths of
animals being exposed.
Strengths: Non-ambiguous positive or negative results.
Weaknesses: For moderately or low toxic substances the determinat?
of a LC50 may be impractical.
Status of Development: Being implemented.
Describe: The equipment required for aerosol generation in the
respirable range is being evaluated.
Applicat ions: Air.
Samples: Pure Chemicals: Pesticides. CompI ex Mi xtu res: N/A
Duration: Variable, I week to 2 months.
Cost: $10,000 to $20,000/analysis.
Interpretation: A rating of the relative toxicity in mammalian
is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology
Toxic Effects Branch, Inhalation Toxicology Group.
Persons to Contact: J. Charles, U.S. EPA, HERL-RTP, Research Tn- ,
Park, NC 27711, (FTS 629-2696). ir'ang|e
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OPP; OTS. "/A-
References: I) Hinners, R.G., J.K. Burkhart, and C.L Punte Ar
Envir. Hlth., 16:194-206, 1968. ' ' cn>
36
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1142 TOXICITY OF GASEOUS AND VAPOR PHASE/ACUTE AND SUBACUTE
Biological Activity Detected: Toxicity.
Principle: Exposure begins with a relatively high concentration
which results in 100/5 mortality in I to 4 hours." The concentrations
are decreased by factors of 10 untiI no deaths occur. Performed
primarily in rats. Physiologic and biochemical parameters are
measured.
Endpoints: Qua Iitative; N/A. Quantitati ve; Death of
animals being exposed. Biochemical parameters, enzymes, and
substrate levels are assayed for in surviving animals.
Strengths: Non-ambiguous positive or negative results.
Weaknesses: For moderately or low toxic substances the determination
of an LC50 may be impractical.
Status of Development: Being implemented.
Describe: 5 chambers, 20 animals/chambers are in "operation.
Applications: Air.
Samples: Pure Chemicals; Gases, Vaporizabte liquids, Pesticides,
Toxic substances in general. Complex Mixtures: N/A,
Duration: Variable, I week to 2 months.
Cost: $10,000 to $20,000/analysis.
Interpretation: A rating of the relative toxicity in mammalian
species is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Toxic Effects Branch, Inhalation Toxicology Group.
Persons to Contact: J. Charles. U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, CFTS 629-2696).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP; OTS.
References: I) Drew, R.T., and S. Laskin. Methods of Animal Experi-
mentation, Vol. 4. N.Y. Academic Press, pp. 1-41. 2) Hinners,
R.G., J.K. Burkhart, and C.L. Punte. Arch. Envir. Hlth., 16:194-
206, 1968.
37
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1143 INHALATION LC50 TESTS
Biological Activity Detected: Toxicity.
Principle: Animals are exposed to different concentrations of the
agent, and lethality is determined over a preset period of time.
Endpoints: Qua I itative: N/A. Quantitative: Concentration required
to cause death in 50% of the exposed animals.
Strengths: Provides initial data on toxicity.
Weaknesses: Cannot be used to determine subtle changes associated
with low level exposure.
Status of Development: Validated.
Describe: Most chemicals and complex mixtures can be examined-
extremely toxic or carcinogenic samples cannot be examined as
HERL-CIN does not have the necessary facilities.
Applications: Multimedia.
Samples: Pure Chemicals: Most chemicals. Complex Mixtures: Ambient
Industrial; Energy Related; Transportation Related; Other. *
Duration: 14 days/study.
Cost: $4,000 to $10,000, test dependent.
Interpretation: A rating of the relative toxicity in mammalian
species is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Functional Pathology Branch; HERL-RTP, Clinical Studies Division
Biomedical Research Branch. *
Persons to Contact: W. Moore, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7431); D.E. Gardner u S
EPA, HERL-RTP, Research Triangle Park, NC 27711, (FTS 1329-2531)
Grant/Contract Laboratory Involved and Principal Investigators: N/A*
Program Office Support: ORD.
References: I) Moore, W., M. Malanchuk, W. Crocker, D. Hysell A
Cohen, and J.S. Stara, Whole Body Retention in Rats of Different
191Pt Compounds Following Inhalation Exposures. Envir. Hlth
Perspec., 12:35, 1973. 2) Moore, W., J.S. Stara, W. Crocker*
M. Malanchuk, and R. IItis. Comparison of Cadmium Retention*
in Rats Following Different Routes of Administration. Envir
Res., 6:473, 1973.
38
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1144 DEPOSITION AND CLEARANCE OF RADIOACTIVE MATERIALS FOLLOWING INHALATION
EXPOSURE
Biological Activity Detected: Deposition; Trans location clearance.
Principle: Animals are exposed to radioactive aerosol for 15 to 30
minutes; then they are counted at various intervals of time.
Tissues are also taken for analysis.
Endpoints: Qua Iitative: N/A. Quantitative; Data on clearance,
trans location, distribution, and excretion as a factor of time.
Strengths: Provides metabolic parameters on the agent under study.
Weaknesses: Agents used are usually radioactive.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: Radioactive chemicals. Complex Mixtures:
N/A.
Duration: Variable, depending on the goals of the study.
Cost/sample: $3,000 to $5,000, depending on cost of compound.
Interpretation: A measure of the residence tissue in the lung and
body tissues for the compound is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch; HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: W. Moore, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7431); D.E. Gardner. U.S.
EPA, HERL-RTP, Research Triangle Park, NC 27711 (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: I) Moore, W., D. Hysell, W. Crocker, and J.S. Stara.
Biological Fate of 10^Pd in Rats Following Different Routes of
Exposure. Envir. Res., 8:234, 1974.
39
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1145 INTEGRATED SYSTEM: PULMONARY FUNCTION AND PULMONARY METABOLISM
Biological Activity Detected: Toxicity.
Principle: Structural changes in lung airways and parenchyma can
be evaluated in-vivo by use of appropriate tests of pulmonary
function. Concommitant metabolic changes can be estimated in-
vitro.
Endpoints: Changes can be measured in intact animals. Qua Ii tat i ve
a_nd_ Quantitative: Changes in pulmonary mechanics, spirometry and
diffusion can be evaluated. Metabolic changes of lung tissue*
can be estimated in-vitro to establish dose-response relationships
Strengths: Measurements of pulmonary function accomplished by non-
invasive methods; In-vivo dose-response effects more applicable
for standard-setting and regulatory purposes.
Weaknesses: Expensive equipment; Time consuming; Requires several
pairs of hands; Some tests are still being validated; Parenchyma I
changes (i.e., development of pulmonary fibrosis, pulmonary
emphysema or chronic bronchitis) are of great interest in terms
of health effects, but they are usually chronic diseases.
Status of Development: Developmental; Being implemented; Validated
Describe: Different tests are in different stages of development
We are presently measuring lung volumes and capacities (ILL VC
1C, FRC, RV, V-j.), breathing frequency, minute ventilation, dif-'
fusing capacity for carbon monoxide, nitrogen washout, and quasi -
static pressure volume relationships of the lung and chest wall
We are developing methods to measure dynamic compliance and re-"
si stance, maximum flow volume relationships, compliance charac-
teristics of excised lungs, and the single breath oxygen test.
Applications: Air.
Samples: Pure Chemicals: Oxides of N and S, Pulmonary toxicants
(e.g. Paraquat), Pesticides. Complex Mixtures; Ambient - NO?
S02, 03; Energy Related - particulates and N0x, SO , 03, organfcs
others; Transportation Related - diesel; Other - Toxic substances'
Any compound which changes pulmonary physiology. *
Duration: 3 month/dose-response of I chemical.
Cost: $30,000/dose-response of I chemical.
Interpretation: These tests are sensitive and can detect small
changes in lung physiology.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division
Biomedical Research Branch.
Persons to Contact: J.J. O'Ne?I, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2711/2531).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Koo, K.W., et al. Respiratory Mechanics in Normal
Hamsters. J. Appl. Physio I., 40:936-942, 1976. 2) Diamond, |_
and M. O'Donnell. Pulmonary Mechanics in Normal Rats. J ADD!*
Physlol., 43:942-948, 1977. 3) Takezawa, J., F. Miller, and '
J.J. O'Neil. Single Breath Diffusing Capacity and Lung'v0|umes
in Small Laboratory Animals. J. Appl. Physiol., 1979. |n
preparation.
40
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H46 PULMONARY FUNCTION IN RATS
Biological Activity Detected: Toxicity.
Principle: Residual volume of the lung increases with many types of
obstructive and destructive lung damage. The slope of the static
compliance of the pressure-volume curve of the lungs increases
with fibres is and decreases with diseases such as emphysema
that destroy alveolar tissue.
Endpoints: gualitative: N/A. Quantitative; Residual
volume (cc) and the slope of the static compliance curves.
Strengths: The measurements are sensitive indicators of lung volume;
They are relatively easy to perform.
Weaknesses: The measurements are conducted with rats; Pulmonary
anatomy and susceptibility of these animals may differ somewhat
from man.
Status of Development: Validated.
Describe: Rats have shown large changes in residual volume and
in the slope of the static compliance curve, after I to 14 days
of exposure to either 0.75 or 1.0 ppm ozone, A manuscript de-
scribing these results is being prepared.
Applications: Air; Water; Food.
Samples: Pure Chemicals: SuI fates, Oxtdants, Heavy metals.
Complex Mixtures: Ambient - air; Industrial - coal dust; Energy
Related - stack gases; Transportation Related - diesel and gasoline
exhaust; Other - asbestos.
Duration: I day to 30 days.
Cost/sample or chemical: $1,000/animal.
Interpretation: An increase in the slope of the static compliance
curve and/or an increase in residual volume is indicative of
either pulmonary edema or emphysema,
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch.
Persons to Contact: W.E. Pepelko, U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7437).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) J. Appl. Physio I., 26:738-744, 1966. 2) Chest, 475-
481, 1967.
41
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1147 PULMONARY FUNCTION OF UNANESTHETIZED GUINEA PIGS
Biological Activity Detected: Toxicity.
Principle: The method allows for measurement of respiratory and
cardiovascular response of an unanesthetized guinea pig to a
control led test atmosphere.
Endpoints: Qua Iitatfve: ECG. Quantitative: Airway resistance
lung compliance, tidal volume, minute volume, breathing rate*
heart rate.
Strengths: This system is most suitable for I to 3 hour exposures
to gases, aerosols, drug response, complex pollutants, etc.-
Each animal serves as its own control during testing.
Weaknesses: The system can be used for measuring response to a long
term exposure where chronic breathing damage is expected. How-
ever, there is wide variation between animals. So for reliable
results either a dramatic response is needed or a very large
number of animals.
Status of Development: Being implemented.
Describe: System has been used for catalyticaIly altered auto
exhaust study, sulfur dioxide exposure study, and diesel engine
auto exhaust study. Results are presently being evaluated.
Applications: Air; Multimedia.
Samples: Pure Chemicals: Sulfur dioxide. Complex Mixtures;
Transportation Related - catalytically altered car exhaust
diesel auto emissions.
Duration: 5 hours/animal.
Cost/sample or chemical: $500/sample. This refers only to the test
animals needed.
Interpretation: Reliable results require dramatic response difference
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN.
Persons to Contact: M.J. Wiester, U.S. EPA, HERL-CIN, 26 W. St
Clair St., Cincinnati, OH 45268, (FTS 684-7424).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Amdur, M.O., and J. Mead. Mechanics of Respiration
In Unanesthetized Guinea Pigs. Am. J. Physiol., 192:364, (953
42
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1148 ARTERIAL BLOOD GAS MEASUREMENT IN CONSCIOUS RATS
Biological Activity Detected: Toxicity.
Principle: Pollutant inhalation resulting in lung damage can
decrease the ability of the animal to oxygenate the blood and
remove C02.
Endpoints: Qua I itati_y_e; N/A. Quantitative: Arterial blood Pn , Prn
pH, bicarbonate. U2 UU
Strengths: A sensitive indicator of lung damage; An important
parameter as adequate PQ levels are necessary to support life;
A sample can be collected quickly and fairly easily.
, Weaknesses: Rats are the only small laboratory animal to which this
> • method can be adapted; Extrapolation of results from rats to
• • humans may be subject to criticism; Each animal can be used
only once.
Status of Development: Validated.
. . Describe: Arterial blood gas measurements were carried out after
I, 3, 7, and 14 days exposure to either 0.75 or 1.0 ppm ozone.
Arterial blood PQ declined in proportion to lung damage. A
manuscript is in preparation.
Applications: Air; Water; Food.
•; ^- '• Samples: Pure Chemicajs: Su I fates, Oxidants, Trace metals.
Complex Mixtures; Ambient - air; Industrial - coal and rock dust;
Energy Related - stack gas emission; Transportation Related -
J auto and diesel emission; Other - food and water born pollutants
v; such as Paraquat.
Duration: 24 hours to 30 days.
Cost/sample or chemical: $200/animal.
Interpretation: A decrease in arterial PQ or an increase in PCQ
Indicates lung damage. The type of damage is not specified
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Functional Pathology Branch.
Persons to Contact: W.E. Pepelko, U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7437).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: D J. Appl. PhysioI., 38:581-587, 1975. 2) Ann. Rev.
Pharmacoi. andToxicol., 16:465-486, 1976. 3) Am. Rev. Resp.
Dis., 113:531-559, 1976.
43
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1149 INFECTIVITY MODEL
Biological Activity Detected: Toxicity.
Principle: Inhalation of a variety of gases and particulates has
been shown to increase susceptibility to infectious pulmonary
disease. Evaluation of the infectivity model used for these
tests indicates that the model reflects the effects of a pol-
lutant on a number of host defense systems, thereby increasing
its sensitivity for detecting effects.
Endpoints: Qua IItative; N/A. Quantitative; Mortality
from laboratory-induced bacterial pneumonia is measured.
Strengths: The model is established for inhalation toxicology; prjo
work has shown its exquisite sensitivity; The test is rapid- A
battery of related follow-up tests are available; Whole animal
inhalation exposures and dose-response studies are more direct I
applicable to standard setting and regulatory action. ^
Weaknesses: Relatively large amounts of pollutant sample are re-
quired for inhalation studies.
Status of Development: Validated.
Describe: The model has been successfully used for Oa, NO
Cd, Ni, suI fates and pollutant combinations. Even though a*
great potential exists it has not been used for screenfna nn.-r*
Applications: Air. 9 Reposes.
Samples: Pure Chemicals; Any chemical likely to reach gaseous
exchange areas of the lung. Complex Mixtures: Ambient; Indus-
trial; Energy Related; Transportation Related; Other - any othT
particulate, gas or combination of same. er
Duration: 8 weeks/dose-response of I chemical.
Cost: $30,000 to $35,000/dose-response of I chemical.
Interpretation: Estimation of enhanced susceptibility to infectio
diseases due to pollutant. Us
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division
Biomedical Research Branch. '
Persons to Contact: D.E. Gardner, U.S. EPA, HERL-RTP Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators-
NT Research Institute, 10 West 35th St., Chicago, IL 60616
R. Ehrlich; Northrop Services, Inc., P.O. Box 12313 Research
Triangle Park, NC 27709, B. Adkins. n
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I) Coffin, D.L., and D.E. Gardner. Ann. Occup H\/n
15:219-234, 1972. 2) Coffin, D.L., et al. Envir. Hlth P!;:*
13:11-15, 1976. 3) Ehrlich, R. Bacteriol. Rev., 30:604-614 7o •
4) Ehrlich, R., et al. Internet. Conf. Photo. Oxid Pollut ' *
Its Control, Proc. Vol I, EPA-600/3-77-OOIa, 1977. *pp 565
44
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11410 IN-VIVO ALVEOLAR MACROPHAGE CYTOTOXICITY
Biological Activity Detected: Toxicity.
Principle: Cytotoxic effects of inhalation of environmental chemicals
will be measured using isolated alveolar macrophages. Any altera-
tion in these cells could increase the potential risk of respi-
ratory infections.
Endpoints: Qua 11 tatjyej N/A. Quantitative; The fol lowing
measurements can be made: viability, phagocytic functioning,
bacteriocidal activity, enzymatic profile, morphology and other
biochemical parameters.
Strengths: A sensitive indicator of cytotoxicity using an in-vivo
model system; Data generated quickly, which can be used to vali-
date in-vitro cytotoxicity testing; Can serve as criteria for
standard setting and regulatory purposes.
Weaknesses: Requires a substantial quantity of the test chemicals
for generation of'aerosols.
Status of Development: Validated.
Describe: .The model has been successfully used for ©3, NC>2, Cd,
Ni, Mn, cigarette smoke, and other metals.
Applications: Air.
Samples: Pure Chemicals: Particulates. Gases, Mists, Any chemical
likely to reach gaseous exchange areas of the lung. CompI ex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Related.
Duration: 8 weeks/dose-response of I chemical.
Cost: $30,000/dose-response of I chemical.
Interpretation: Depending upon the particular endpoint, alteration
would indicate enhanced susceptibility to infectious disease
or potential for alteration of lung tissue.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Clinical Studies Division,
Biomedical Research Branch.
Persons to Contact: D.E. Gardner, U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2531).
Grant/Contract Laboratory Involved and Principal Investigators:
NT Research Institute, 10 West 35th St., Chicago, IL 60616,
C. Aranyi; U. of California, Davis, CA 95616, E. Goldstein;
Southwest Research Institute, San Antonio, Texas 78284, E.
Gause.
Program Office Support: OHEE; OPP; OEM I; OTS.
References: I)- Gardner, D.E., et al. J. Bacteriol., 98:1041-1043,
1969. 2) Hurst, D.J., et al. J. Reticuloendothel. Soc., 8:288-
300, 1970. 3) Bingham, E. Arch. Envir. Hlth., 25:406-414, 1972.
4) Gardner, D.E. Thesis, U. of Cincinnati, 1971. 5) Warshauer,
D., et al. J. Lab. Clin. Med., 83:228-240, 1974. 6) Kass, E.H.,
et al. Bacteriol. Rev., 30:488-497, 1966. 7) Kim, M., et al.
J. Infect. Dis., 133:310-320, 1976.
45
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CONTENTS: TEST SYSTEMS
1150 In-Vivo Systems/Neurobehavioral 47
1151 Integrated System: Neurobehavioral
Toxicological Assessment 48
I 152 Integrated System: The Effects of Selected
Organic Contaminants in Drinking Water
on the Functions of the Reproductive,
Nervous, and Immune Systems 49
1153 Computer Automated Analysis of Patterned
Behavior in the Primate 51
1154 Computer Automated Analysis of Learning and
Memory in the Primate 52
1155 Integrated System: Behavioral Analysis of
Rats— Developmental, Locomotor, Explor-
atory, and Learned Behavior 53
1156 Integrated System: Instrumental Methods of
Detecting Functional and Metabolic Damage
to the Nervous System 54
1200 Genotoxicity 57
1210 Mutagenesis 57
47
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1151 INTEGRATED SYSTEM: NEUROBEHAVIORAL TOXICOLOGICAL ASSESSMENT
Biological Activity Detected: Toxicity.
Principle: A battery of tests are being implemented which evaluate
various aspects of CNS functions.
Endpoints: Qua I itative: Evaluate CNS Function. Qua Iitative-
These include: locomotor activity, neuromotor function CNS
excitability, learning and memory.
Strengths: Provides rapid and sensitive broad spectrum evaluation
of CNS function.
Weaknesses: Requires skilled personnel; Real possibility of false
negatives.
Status of Development: Validated.
Describe: This battery has been implemented and is current Iv
being validated using known neurotoxins.
AppIi cat i ons: Mu11 i med i a.
Samples: Pure Chemicals: Heavy metal, Pesticides, Nonionizing
radifilm. Complex Mixtures; Nl/A.
Duration: Test: 2 weeks, assuming acute exposure; Analysis: 3 week
Cost: $2,000/sample, assuming acute exposure.
Interpretation: The profile of change in the various functional
tests provide information on neurotoxicity of test compounds.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Divi-
sion, Neurobiology Branch.
Persons to Contact: L. Reiter. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2671).
Grant/Contract Laboratory Involved and Principal Investigators-
Program Office Support: OHEE. *
References: I) Reiter, L., et al. Residential Maze. Envir Hlth
Perspect., 12:119-123, 1975. 2) Archer, J. Anim. Behav* 2l-?n«?
235, 1973, 3) Dunham, N.W., and T.S. Miya. Roto Rod. j* of
Amer. Pharmaceu. Asso., 46(3):208-209, 1957. 4) Gait, S.' R
Rushton, and H. StelIberg. Anim. Behav. and Drug Action"*207-
223, 1964. 5) Tremors, R.R., G.K. Chalmers, and W. Yim ' Proc
Soc. Exp. Bfol. Med., 109:202-205, 1962. 6) Hornston, M Star-H
Reflex. Physio, and Rev., 3:839-844, 1968. 7) Reiter, L., et |
Passive Avoidance Test. Toxicol. and Appl. Pharm., 25:582-588
1973. 8) Miczek, K., and H. Barr, II. Social Behavior Behav
Pharmacol., 176-257, 1976. ' *
48
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1152 INTEGRATED SYSTEM: THE EFFECTS OF SELECTED ORGANIC CONTAMINANTS IN
DRINKING WATER ON THE FUNCTIONS OF THE REPRODUCTIVE, NERVOUS, AND IMMUNE
SYSTEMS
Biological Activity Detected: Reproductive, immune-, neuro-, and
behavioral toxicity; Mutagenicity.
Principle: Determine effects of experimental exposure to certain
selected organic contaminants on: I) Immune system and host re-
sistance capabilities; 2) NeurochemicaI processes and dynamics
in the brain; 3) Behavior; 4) Male reproductive function (includ-
ing dominant lethal mutagenicity assay); 5) General toxicity end-
points (lethality, body-organ weights, hematology, etc.).
Endpoints: Qua!itative; N/A. Quantitative; I) Immune response and
host resistance: Humoral - serum antibody production to S. aureus;
Cell mediated - response to C. parvum; RES activity - global pha-
gocyt. index (vase, clearance) and tissue distribution of *kC S.
aureus; Susceptibility to pathogens (bact., virus, fungus) and
transplant tumor; 2) Brain neurochemistry and dynamics: In-vtvo
and in-vitro (synaptosome) systems; Endogenous levels, uptake, re-
lease, metabolism...in Norepinephrine, dopamine, serotonin and
AcCh systems; 3) Behavioral toxicology: Operant behavior - sched-
uled and learned performance; Learning ability; Behavior develop-
ment; Other - el em. screen, spont. mot. activ., visual, swim,
maze, etc; 4) Reproductive performance and dom. lethal mutagen.
asso.: AntifertiIity, reversibility, mutagenic potential, mode
of action, penetration of BT barrier (in-vivo); Penetration mech-
anism, spermatic nucleo/protein synth. spermatic uptake (in-vitro);
5) Preliminary and range finding shorter term toxicology: Leth-
ality, body/organ weights, hematology, etc.
Strengths: Provides much information on several toxicologic aspect areas in
coordinated manner; Readiness reduces time turnaround.
Weaknesses: Expensive.
Status of Development: Being Implemented.
Describe: The tests are being implemented. Some are still
developmental, but most are validated. Current work applicable
to and aimed at drinking water contaminants, but techniques and
inferences applicable to other media.
Applications: Water.
Samples: Pure Chemicals; Trihalomethanes; Benzenes; Ethers;
Phenyls; Pesticides; PCS; Dioxin; 2, 4-DNT; (Many chemicals, but
not all are under test by all the test systems under present
project). Complex Mixtures: Not now under test but could be
implemented.
Duration: Existing grants, about 2 years, covering multiple chemical;
Individual tests involve short (I to several days) to long-term
(3 months) exposures.
Cost/sample or chemical: Approx. $120,000 for the entire integrated
multidisciplinary workup involving multiple dose levels and ex-
posure periods lasting up to a year for completion of all phases.
However, application of selected portions, reduced exposures or
fewer dose levels, etc., would be less expensive.
49
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1152 INTEGRATED SYSTEM: THE EFFECTS OF SELECTED ORGANIC CONTAMINANTS IN
DRINKING WATER ON THE FUNCTIONS OF THE REPRODUCTIVE, NERVOUS AND
SYSTEMS (continued)
Interpretation: Toxicity interpreted in terms of potential human
health hazards.
Level of Complexity: Complexity levels range from 3 to 5.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Toxicologic Assessment Branch. '
Persons to Contact: K. I. CampbelI, U.S. EPA, HERL-CIN 26 W St PI
St., Cincinnati, OH 45268, (FTS 684-7481). ' ' la"
Grant/Contract Laboratory Involved and Principal Investigators-
Medical College of Virginia, Richmond, VA 23298: J.F. Borzel le
Project Manager (Coordinator) and Reproduction Studies- A E ^'
Munson, Immune Systems, etc.; R.L. Balster, Behavioral'Toxico-
logy; W.L. Dewey, Neurochemistry.
Program Office Support: ORD.
References: I) Grant document and report: R80470I. 2) Grant doc
ment and report: R804290. 3) Deichmann, W., et a I ToxFcol
Appl. Pharmacol., 5:201, 1963. 4) Ball, H. J. Nat. Can Inst
44:1070, 1966. 5) Szakol, L., and H. Hanna. Nat. Can. Inst
Monographic., 35:173, 1972 6) For additional related references
contact investigator. »
50
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1153 COMPUTER AUTOMATED ANALYSIS OF PATTERNED BEHAVIOR IN THE PRIMATE
Biological Activity Detected: Toxicity.
Principle: Animal behavior is structured (patterned) and chemicals
which affect CNS function will disrupt this patterned behavior.
Endpoints: Using closed-circuit TV and on-line computing, various
components of primate behavior are defined and measured for
frequency, duration, and sequences. Qua Iitative: N/A.
Quantitative: Frequency of various motor items in experimental
period; Duration of various motor items per occurrence; Sequencing
of behavior.
Strengths: Should provide sensitive index of neurotoxicity which is
fully automated and applicable to a wide range of pollutant test-
ing; Also no pre-training is required.
Weaknesses: Expensive; Specialized equipment is required.
Status of Development: Developmental.
Describe: System has been developed and is being tested with
known psychoactive drugs.
Applications: Multimedia.
Samples: Pure Chemicals: Heavy metals. Complex Mixtures: N/A.
Duration: Test: I week, assuming acute exposure; Analysis: 2 weeks.
Cost: $200/subject.
Interpretation: Changes in frequency, duration, or patterning of
behavior are indicative of neurotoxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology
Division, Neurobiology Branch.
Persons to Contact: L. Reiter. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2671).
Grant/Contract Laboratory Involved and Principal Investigators:
Iowa State U., Ames, IA 50010, W.E. Lloyde.
Program Office Support: OEMI.
References: I) Norton, S. Physio I. Behav., 11:181-186, 1973. 2)
Norton, S. J, Theoret. Blot., 21:229-243, 1962.
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1154 COMPUTER AUTOMATED ANALYSIS OF LEARNING AND MEMORY IN THE PRIMATE
Biological Activity Detected: Toxicity.
Principle: Chemicals affecting CNS function may interfere with the
learning process. The fact that primates rely heavily on the
visual sensory modality is utilized to determine pollutant
effects on visual discrimination learning.
Endpoints: Qua Iitative; N/A. Quantitative: Two choice non-spatial
visual discrimination tasks examine animals' ability to distin-
guish between different visual patterns; Delayed response task-
evaluates memory coordination utilizing visual stimuli.
Strengths: Provides system index of chemical effects on learninq
and memory; Provides information on behavioral effects in primat
Weaknesses: Expensive; Requires skilled personnel to perform test- 8S
Used only for toxicity testing of pure compounds when specific'
information on primates is required.
Status of Development: Developmental.
Describe: System has been developed and is being tested with
psycho-active drugs.
Applications: Multimedia.
Samples: Pure Chemicals: Heavy metals. Complex Mixtures: N/A
Duration: Test: 6 weeks, assuming acute exposure; Analysis- 2 week
Cost: $200/subject. ' Ks*
Interpretation: Alterations in performace are indicative of neuro-
tox i c i ty.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division
Neurobiology Branch. '
Persons to Contact: L. Reiter, U.S. EPA, HERL-RTP, Research Trian«i
Park, NC 27711, (FTS 629-2671). 'angle
Grant/Contract Laboratory Involved and Principal Investigators-
Iowa State U., Ames, IA 50010, W.E. Lloyde.
Program Office Support: OEM I.
References: I) Fletcher, H.J. The Delayed-Response Problem. |n-
Behavior of Nonhuman Primates, A.M. Schrier, H.F. Harlowj and'
F. Stollnitz, eds. Academic Press, New York, 1965. pp.'l29-
165. 2) Miles, R.C. Discrimination-Learning Sets. In: Be-
havior of Nonhuman Primates, A.M. Schrier, H.F. Harlow,'and
F. Stollnitz, eds. Academic Press, New York, 1965. pp. 53-54
52
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1155 INTEGRATED SYSTEM: BEHAVIORAL ANALYSIS OF RATS - DEVELOPMENTAL,
LOCOMOTOR, EXPLORATORY, AND LEARNING BEHAVIOR
Biological Activity Detected: Toxicity; Behavioral.
Principle: Various levels of lead are known to affect the maturation
of energy metabolism in the cerebral cortex of rats. Studies
have shown that delays in the development of the nervous system
may affect behavioral responses observable in both the young and
adult animal. Specific affects need to be tested on the above
Iisted indices.
Endpoints: Qua Ii tati ve: Expression of learned behavior through use
of a water T-Maze - percentage of correct trials and amount of
time to reach criteria; Expression of locomotor activity and
exploratory behavior through the use of a Berylene Box - primarily
a measurement of frequency and duration spent on each parameter;
Expression of developmental behavior through locomotor activity
and use of ultrasonics - the measurement of general activity pat-
terns and monitoring of communication abilities dependent upon
development. Quantitative: Maze activity; Benylene box; General-
ized and specific locomotor activity; Frequency and duration of
ultrasonic vocalizations.
Strengths: Should provide sensitive behavioral tests which can then
be correlated with physiological data collected in previous
studies.
Weaknesses: Requires specialized instrumentation for testing; Time-
consurning to run the battery of tests for each designated dosage
I eveI.
Status of Development: Being implemented.
Describe: Most of the instrumentation has been constructed and
subjects are being treated with designated lead doses.
Applications: Water.
Samples: Pure Chemicals: Trace metals, Organic chemicals.
" Complex Mixtures; Industrial; Energy Related.
Duration: I to 3 months.
Cost: Not yet established.
Interpretation: Altered physiological or neurological function as a
result of treatment chemical would indicate toxicity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxtcological Assessment Branch.
Persons to Contact: R.J. Bull. U.S. EPA, HERL-CIIM, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory involved and Principal Investigators: N/A.
program Office Support: OHEE; OWHM.
References: 1) Reiter, L.W., et al. Envir. Hlth. Perspect., 12:119-
123, 1975. 2) Sobotka, T.J., etal. Toxicol., 5:175-191, 1975.
3) Brown, D.R. Toxicol. Appl. Pharmacol., 32:628-637, 1975. 4)
Padich, R., et al. Pharmacol. Biochem. Behav., 6:371-375, 1977.
5) Fox, D.A., et al. Toxicol. Appl. Pharmacol., 40:449-461, 1977.
6) Robbins, T., et al. Psychopharmacologia, 28:155-164, 1972.
7) Amsel, A., et al. Science, 197:786-788, 1977.
53
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1156 INTEGRATED SYSTEM: INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND
METABOLIC DAMAGE TO THE NERVOUS SYSTEM
Biological Activity Detected: Toxicity.
Principle: Increased functional activity of a tissue requires energy
Consequently, if a tissue's functional activity is stimulated
ATP is hydro Iyzed to ADP and P., which in turn stimulates oxida-
tion of substrate and resynthesis of ATP. These metabolic chanqes
may be observed as increases in oxygen consumption, substrate
utilization, or as metabolic transients induced in the electron
carriers directly in tissues, in-vitro. The kinetics of these
metabolic responses to stimulation have been shown sensitive to
a wide variety of chemical agents with varying mechanisms of
action with both in-vitro and in-vivo treatments.
Endpoints: To this point in time, an endpoint has only been develoo
for brain tissue. Responses are measured in response to electrl I
pulses (10 s) or elevation in K concentrations (3 to 30 mM).
Qua Ii tat lye: N/A. Quantitative: Transient redox changes in
NAD(P)H, fp, cyt a, b, c.; Substrate utilization; Oxygen con-
sumption; Lactic acid output; Neurotransmitter release; Amino
acid metabolism.
Strengths: In-vitro results may be directly confirmed in-vivo with
the same parameters; Applicable to a wide variety of mechanisms-
Applicable to all aerobic tissues; Involves measurement of the '
kinetics of going from a resting to an excited state rather than
the steady state greatly increasing sensitivity; Applicable to
very small tissue samplers (2 to 3 mg).
Weaknesses: Does not lend itself to immediate identification of
mechanisms unless there is a direct effect on energy metabolism
proper.
Status of Development: Validated.
Describe: The test system has been validated with a wide variet
of inhibitors of energy metabolism and membrane active compound ^
In-vitro and in-vivo treatments with lead, methyl mercury, and S*
alkylation compounds indicate equivalent or more sensitive measu
of effect than other parameters which have been applied to these ^
p rob I ems.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures; Indus-
trial; Energy Related; Transportation Related.
Duration: 2 weeks to 3 months.
Cost: Not yet established.
Interpretation: This is a general system for determining neurotoxicl-K
It is capable of detecting non-specific damage to a variety of "^*
systems (e.g., decreased membrane excitability, altered respons
to neurotransmitters, direct effects on energy metabolism). |t°S
has also been used to detect delays in brain development, which
were subsequently confirmed by morphologic and behavioral method
Level of Complexity: 3. °
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1156 INTEGRATED SYSTEM: INSTRUMENTAL METHODS OF DETECTING FUNCTIONAL AND
METABOLIC DAMAGE TO THE NERVOUS SYSTEM (continued)
Persons to Contact: R.J. Bui I, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Bull, R.J., and A.J. Trevor. J. Neurochem., 19:999-
1009, 1972. 2) Bull, R.J., and A.J. Trevor. J. Neurochem., 19:
1011-1022, 1972. 3) Cummins, J.T., and R. Bull. Biochem. Bio-
phys. Acta, 253:29-38, 1971. 4) Bull, R.J., and J.T. Cummins.
J. Neurochem., 21:923-937, 1973. 5) Bull, R.J., and S.D. Lutken-
hoff. Neuropharmacol., 14:351-359, 1975. 6) Bull, R.J. J.
Neurochem., 26:149-156, 1976. 7) Bull, R.J., P.M. Stanaszek,
J.J. O'Neill, and S.D. Lutkenhoff. Envir. Hlth. Perspect., 12:
89-95, 1975.
55
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CONTENTS: TEST SYSTEMS
1200 Genotoxicity 57
1210 Mutagenesis 57
1211 Salmonella typhimurium (Ames) .... 58
1212 Escherichia col i (WP2) 60
1213 Saccharomyces cerevislae, Schi zosaccharomyces
pombe, Forward and Reverse Mutation 6!
1214 Body Fluid Analysis 62
1215 Bacterial Plasmids 63
1216 Mouse Lymphoma (L5I78Y) 64
1217 Chinese Hamster Ovary Cells (CHO) Drug
Resistance. 65
1218 Chinese Hamster Cells (CHO) Nutritional
Competency. 66
1219 Chinese Hamster Lung Cells (V79) 67
I2MO Drosophi la melanoqaster, Sex Linked
Recess 1 ve Letha I 68
1211 I Tradescantia Stamen Hair 69
12112 Maize Waxy Locus Assay 70
12113 In-Vivo Cytogenetics in Mice 71
1220 DMA Damage 73
57
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1211 SALMONELLA TYPHIMURIUM (AMES)
Biological Activity Detected: Mutagenicity; Presumptive oncogenIcitv
Principle: Histidine dependent strains of Sal none I la genet i<-ai |v
engineered to increase their sensitivity and specificity are
exposed to a test substance in the presence of mammalian'meta-
bolic activating enzymes. The formation of bacterial colonies
in a histidine-free medium after treatment is considered a
criteria of the effectiveness of a test substance as a mutagen
Endpoints: Qua Ii tat i ve: Spot test and plate incorporation test
are considered to be qualitative in nature. In spot tests no
attempt is made to count the number of colonies per plate. In
plate incorporation the number of colonies are counted but not
expressed as a fraction of survival. Quantitative: The induced
mutational frequencies may be expressed on the basis of units
of test material and survival in suspension.
Strengths: Genetically we I I-character!zed system; Rapid; Inexpensive-
Well validated as a test for gene mutation; Works well with In- *
vitro metabolizing microsome fractions; Can be used as indicator
organism in host-mediated assays.
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance; Prokarvotl
organization of genetic material. c
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: All major classes of chemicals except
metals and hormones. Complex Mixtures; Ambient - air particulat
drinking water, and water concentrates; Industrial - effluents- 6S*
Energy Related - alternate effluents, shale; Transportation Re-
lated - auto/truck fuels; Other - human body fluids, extracts
from crops treated with sludge.
Duration: 3 weeks/study.
Cost: $300 to $650/compound for plate test; $1,000 to $1,200/compound
for suspension test.
Interpretation: The growth of colonies in a histidine-deficient
medium Indicates genetic alteration.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Dlvisi
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field °n*
Studies Division, Toxicological Assessment Branch; ERL-GB- Natt
Center for Toxicological Research, Division of Mutagenesis
Somatic Cell Section.
Persons to Contact: J.L. Huisinqh. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2948); L. Claxton. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2942)- '
M.D. Waters. U.S. EPA, HERL-RTP, Research Triangle Park NC' 2771
(FTS 629-2693); J.P. Bercz. U.S. EPA, HERL-CIN, 26 W. St Clair ii'
Cincinnati, OH 45268, (FTS 684-7432); H.R. Pahren. U.S/EPA
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684
7217); L.J. McCabe. U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
58
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1211 SALMONELLA TYPHIMURIUM (AMES) (continued)
Persons to Contact (continued): Cincinnati, OH 45268, (FTS 684-7211)
J.F. Stara, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-7407); N. Richards, U.S. EPA, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011); E. Lazear, NCTR,
Jefferson, AR 72079, (FTS 740-4573); D.A. Casciano, NCTR,
Jefferson, AR 72079, (FTS 740-4495).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA, V.F. Simmons; Litton
Biometics, Inc., Nicholson Lane, Kensington, MD, D.T. Brusick;
U. of Cincinnati Medical Center, J. Loper; Louisiana State U.
Medical School, W. Pelon; U. of Missouri, Columbia, MO, C.
Marianseld; U. of West Florida, J. Bazlis; U. of Texas, Medical
Branch, Galveston, TX 77550, M. Legator.
Program Off ice Support: OHEE; OPP; OEM I; OTS.
References: I) Ames, B.N., et al. Mutation Res., 31:347-364, 1975.
2) McCann, et al. Proc. Natl. Acad. Sci., 70:782-786, 1975. 3)
McCann, and B.N. Ames. Proc. Nati. Acad. Sci., 73:950-954, 1976.
59
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1212 ESCHERICHIA COLI (WP2)
Biological Activity Detected: Mutagenicity.
Principle: Tryptophan dependent strains of Escherichia cojj_ geneti-
cally engineered to increase their sensitivity and specificIty
are exposed to a test substance in the presence of mammalian *
metabolic activating enzymes. The formation of bacterial colon!
in a tryptophan-free medium after treatment indicates the effec-
tiveness of a test substance as a mutagen.
Endpoints: Qua Iitative: Growth in a tryptophan-free medium.
Quantitative: N/A.
Strengths: Rapid; Inexpensive; Well validated test for gene mutatfo
Works well with in-vitro metabolizing microsomal enzymes; Can b
used as indicator organism in host-mediated assays. '
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance; Prokarv +t
organization of genetic material; Not as well characterized nor
as sensitive as Salmonella/microsome assay (1211). |f detects
only base pair substitutions.
Status of Development: Validated,
Describe: N/A.
AppIi cat i ons: Mu11 i med i a.
Samples: Pure Chemicals; All major classes of chemicals except
metals and hormones. Complex Mixtures: Ambient - air p
drinking water, and water concentrates; Industrial - eff
Energy Related - alternate effluents, shale; Transportation
lated - auto/truck fuels; Other - human body fluids, extracts
from crops treated with sludge.
Duration: 3 weeks.
Cost: $450/chemical.
Interpretation: The growth of tester strains in a tryptophan free
medium after treatment with a test substance indicates muta+i
Level of Complexity: I. 'anon.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Tri=
Park, NC 27711, (FTS 629-2693); L. Claxton. U.S. EPA, HERL-RTP
Research Triangle Park, NC 27711, (FTS 629-2942); S.S Sandh,
U.S. EPA, HERL-RTP, Research Triangle Park, NC 277T1 (FTS -
629-2693); J.L. Huisingh, U.S. EPA, HERL-RTP, Research Trianoi
Park, NC 27711, (FTS 629-2948). gie
Grant/Contract Laboratory Involved and Principal Investigators-
Stanford Research Institute, Menlo Park, CA 94205, V F slmmr.
Program Off ice Support: OHEE; OPP. ' ' ""tons.
References: I) Bridges, B.A. Lab Practice, 21:413-416, 1972
2) Bridges, B.A., et al. Mutation Res., 19:295-303^ 1973*
3) Bridges, B.A., etal. Chem/Blol. Interactions, 5:77-84, (972
60
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1213 SACCHAROMYCES CEREVISIAE, SCHIZOSACCHAROMYCES POMBE, FORWARD AND REVERSE
MUTATION
Biological Activity Detected: Mutagenicity.
Principle: Gene mutation is detected by loss of function resulting
in nutritional requirements or resistance to toxic chemicals and
shift in color of cell colonies.
Endpoints: Qua Ii tative: Growth in a selective medium; Change in
colony pigmentation. Quantitative: Mutation frequencies may be
adjusted for cytotoxic effects.
Strengths: Both forward and reverse mutation can be studied; Eukar-
yotic organization of genetic material; Fast; Relatively inexpen-
sive; Cells can be cultured as haploids; Fairly wide spectrum of
genetic events can be scored; Can be used as indicator organism
in host-mediated assays.
Weaknesses: Requires exogenous metabolic activation which has not
worked well with yeast systems; Lacks pharmacological relevance;
Chromosomes are too small to permit direct cytological observa-
tion; Relatively insensitive to some chemicals.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
- Samples: Pure Chemicals: Alkylating agents, Halogenated hydro-
carbons, Polycyclics, Carbamates. Complex Mixtures; Ambient -
water concentrated; Other - extracts from crops treated with
sludge, human body fluids.
Duration: 3 weeks.
Cost: $400 to $700/compound, depending on the test.
Interpretation: The appearance of plgmented colonies and growth In
selective mutation after treatment with test compound Indicates
mutation.
LeveI of CompI exIty: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, HERL-CIN, Field Studies Division,
Tox!colog lea I Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
J.P. Bercz, U.S. EPA, HERL-CIN, 26 W. St. Clalr St., Cincinnati,
OH 45268, (FTS 684-7432); H.R. Pahren, U.S. EPA, HERL-CIN, 26 W.
St. Clalr St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F, Simmons.
Program Off ice Support: OHEE; OPP; OEMI.
References: I) ZImmermann, F.K. In: Chemical Mutagens: Principles
and Methods for Their Detection. Vol. 3. A. Hoilaender, ed.
Plenum Press., NY, 1973. pp. 209-239. 2) Parry, J.M. Muta-
tion Res., 46(3):165-176, 1977. 3) Bruslck, D.J., and V.W.
Mayer. Envir. Hlth. Perspect., 6:83-96.
61
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1214 BODY FLUID ANALYSIS
Biological Activity Detected: Mutagenicity.
Principle: Promutagens which need mammalian metabolic activation
are biotransformed in the intact animal and are tested for muta-
genic activity in Salmons!la, yeast, and Chinese hamster ovarv
eel I test systems. y
Endpoints: Qua Iitative: Appearance of revertant colonies in a
selective medium. Quantitative: The number of prototrophic
colonies in a histidine deficient medium, adjusted for cytotoxfc
effects of the test chemical.
Strengths: Combines in-vivo metabolic activation with the in-vitr
microbial test system.
Weaknesses: Limited number of bacteria exposed; Recovery of bacterl
is problematic; Exposure time has not been standardized; DiffiCu?+
to quantitate the response. '
Status of Development: Being implemented.
Describe: The basic experimental procedure for pure chemicals
has been established. But protocol for testing for mixtures has
not been developed. Furthermore, the test system needs valida-
tion by testing a wide variety of chemicals.
Applications: Multimedia.
Samples: Pure Chemicals; Mycotoxins, Nitrosamines, Aromatic
amines, Aromatic hydrocarbons. Complex Mixtures: Not yet test d
Duration: 4 weeks.
Cost: $1,000 to $l,200/chemical.
Interpretation: The positive response indicates the ability of the
test chemical to be transformed into reactive electrophi le by th
intact mammalian metabolic activation system. 6
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divisi
Biochemistry Branch; HERL-CIN, Field Studies Division, Toxicolo-
gical Assessment Branch.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Tri
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693)-'
L. Claxton. U.S. EPA, HERL-RTP, Research Triangle Park NC '?~J1\\
(FTS 629-2942); J.F. Stara; U.S. EPA, HERL-CIN, 26 W St Glair
St., Cincinnati, OH 45268, (FTS 684-7407).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Texas, Medical Branch, Gaiveston, TX 77550, M. Legator
Stanford Research Institute, Menlo Park, CA 94205 V F Simmon
Program Office Support: OHEE; OPP; OEMI. ' ' S'
References: I) Legator, M., et al. Mutation Res, 26:456, 1974
2) Legator, M., et al. In: Chemical Mutagens: Principles'and
Methods for Their Detection. Vol. 4. A. Hollander, ed
Press, NY, 1976. pp. 171-190. '
62
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1215 BACTERIAL PLASMIDS
Biological Activity Detected: Mutagenicity.
Principle: Purified plasmid DNA is exposed to potential mutagen in
a cell-free system and is then analyzed for ability to infect a
host bacterium.
Endpoints: Qua Iitative: Ability of treated plasmid DNA to infect
bacterial cells. Quantitative: The number of colonies produced
per unit of test materials.
Strengths: May avoid problems of extreme cytotoxicity of many
chemicals since the exposure is in a cell-free system; Rapid;
EconomicaI.
Weaknesses: Problems with exposing purified DNA; Still early in
developmental stage; Requires exogenous metabolic activation
systems.
Status of Development: Developmental.
Describe: Initial pilot work with MNNG is encouraging.
Applications: Multimedia.
Samples: Pure Chemicals: Organics. CompI ex M i xtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: I to 2 days.
Cost: Undetermined.
Interpretation: Loss of infectivity suggests damage to DNA.
Level o' Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: N. Clarke, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7411); B. Daniel. U.S.
EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS
684-7482).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: Not yet available.
63
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1216 MOUSE LYMPHOMA (L5178Y)
Biological Activity Detected: Mutagenicity.
Principle: Forward mutation from thymidjne kinase competency
to thymidine kinase incompetency (TK ), resulting in resistance
to the base analogs BUdR or TFT.
Endpoints: Gene mutation. Qua Iitative: Formation of cell colonies
in a selective medium. Quantitative: Induced mutation frequencie
are based on cell survival and cloning efficiency. S
Strengths: Both forward and reverse mutation can be measured; Cell
will grow in suspension culture; Cells have short generation time-
Cells have stable, near-diploid chromosome number; High plating '
efficiency; High recovery of mutant cells.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Requires additional validation; Lacks pharmacological
relevance; PPLO contamination is a serious problem and the cells '
must be continuously monitored to ensure that they are PPLO-free
Status of Development: Being implemented.
Describe: Problems of expression time and metabolic activation
have yet to be resolved. This bioassay has been contracted out
for validation by the National Cancer Institute.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Halogenated hydro-
carbons, Inorganic derivatives, N-Nltroso compounds, Metals
Mycotoxins. Complex Mixtures: Ambient; Industrial.
Duration: 3 weeks.
Cost: $3,000/compound.
Interpretation: Growth of heterozygous thymidine competent cells In
a medium containing TFT or BUdR suggests mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch, Cellular Biology Section.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S.
EPA, HERL-RTP, Research Triangle Park, NC ^771 I, (FTS 629-
2693); M.M. Brown. U.S. EPA, HERL-RTP, Research Triangle Park
NC 27711, (FTS 629-2693). '
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A Mitchell
Program Office Support: OHEE; OPP; OTS.
References: I) Clive, D., and J.F.S. Spector. Laboratory Procedure
for Assessing Specific Locus Mutations at the TK Locus in Cul-
tured L5I78Y Mouse Lymphoma Cells. Mutation Res 31 • \~j 29
1975.
64
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-J217 CHINESE HAMSTER OVARY CELLS (CHO) DRUG RESISTANCE
Biological Activity Detected: Mutagenicity; Presumptive oncogenicity.
Principle: Forward mutation assay measuring drug resistance at hypo-
xanthine-guanine-phosphoribolyItransferase (HGPRT) locus. The
HGPRT competent cells in presence of mammalian metabolic activa-
tion enzymes are exposed to a test substance. Induced frequency
of HGPRT deficiency is determined using a selective medium
containing base analogue 8-azaguanine (8 AZ) or 6-thioguanine
(6 TH).
Endpoints: Gene mutation. Qua Iitative; Formation of cell colonies
in a selective medium. Quantitative: Induced mutation fre-
quencies are based on cell survival and cloning efficiency.
Strengths: Mammalian organization of genetic material; Forward muta-
tion assay; Fast generation time; Stable karyotype; Easy to cul-
ture; May be particularly well-suited to quantitation as HGPRT
locus mutants may not show replicative advantages/disadvantages
over wild-type cells under nonselective conditions.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Needs additional validation; Lacks pharmacological
relevance; Fairly high spontaneous mutation rate; Long optimal
expression period (7 days).
Status of Development: Being implemented.
Describe: Basic system with metabolic activation has been de-
scribed. Several compounds representing diverse classes of
chemicals have been tested. However, additional chemicals by at
least two laboratories have to be tested before It could be
considered as validated.
Applications: Multimedia.
Samples: Pure Chemicals: Alkylating agents, Nitrosamines, Organics,
PNA's, Metallic compounds. Complex Mixtures; Not yet determined.
Duration: 3 weeks/compound.
Cost: $2,000 to $3,000/compound, Including dose-response.
Interpretation: Growth of HGPRT competent cells In a medium contain-
ing 8 AZ suggests mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch; National Center for Toxicological Research,
Division of Mutagenesls, Somatic Cell Section.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); S.5. Sandhu. U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2693); D.A. Casciano.
NCTR, Jefferson, AR 27079, (FTS 740-4495). ~~
Grant/Contract Laboratory Involved and Principal Investigators:
Oak Ridge National Laboratory, Biology Division, P.O. Box Y,
Oak Ridge, TN 37830, A. Hsie.
Program Office Support: OHEE.
References: I) O'Neill, P.J., et al. Mutation Res., 45:91-101, 1977.
2) O'Neill, P.J., et al. Mutation Res., 45:103-109, 1977.
65
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1218 CHINESE HAMSTER CELLS (CHO) NUTRITIONAL COMPETENCY
Biological Activity Detected: Mutagenicity.
Principle: Isolation of auxotrophic mutants using 5-bromodeoxyuridIne
(BUdR) and visible light as selective agents.
Endpoints: Gene and chromosomal mutation. Qua I itativej Formation
of auxotrophic eel I colonies. Quantitative: Can be used to
determine induced mutation frequencies based on cell survival
and cloning efficiency.
Strengths: System is clean (survivors are either auxotrophs or thev
are not); Several loci are available for detection of genetic
changes; Forward mutation assay with very low spontaneous muta-
tion frequencies at available loci; Test populations can be
easily freed of spontaneous auxotrophs by growing cells three
days in minimal medium; Mammalian system in terms of organizatlo
of genetic materials.
Weaknesses: Assay very insensitive (95% of auxotrophs lost during
selection to effects of starvation); Mutant identification is
tedious and not amenable to screening; Experiments require five
weeks to complete; Requires metabolic activation; Requires valid
tion; Lacks pharmacological relevance; Nutritional mutants appear"
to be at a replicative disadvantage in mixed populations under
nonselective conditions.
Status of Development: Developmental.
Describe: Improvement of technique is necessary for increased
sensitivity. Also necessary is the addition of an in-vitro meta-
bolic activation.
Applications: Multimedia.
Samples: Pure Chemicals; Alkylating esters, Base analogs, Nltro-
samines. Acridine, Mustards, Heavy metals, Hydroxyfamine.
Complex Mixtures: Energy Related - petroleum oil extracts-
Transportation Related - jet fuel extracts; Other - ultraviolet
radiation, x-rays.
Duration: 5 weeks.
Cost: $2,000/assay.
Interpretation: Test agents inducing significant numbers of auxotrooh
may be regarded as potential mutagens/carcinogens for animals and
man.
Level of Complexity: 4 to 5.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Genetic Toxi-
cology Team.
Persons to Contact: A.R. Malcolm. U.S. EPA, ERL-NAR, South Ferrv Rd
Narrangansett, Rl 02882, (FTS 838-4843 X247 or X238). '
Grant/Contract Laboratory Involved and Principal Investigators' IM/A
Program Office Support: OHEE. ' /A'
References: I) Kao, F.T., and T.T. Puck. Induction and Isolation
of Auxotrophic Mutants in Mammalian Cells. In: Methods in Cell
Biology. Vol. 3. D. Prescott, ed. Academic Press, NY 1974
pp. 23-39.
66
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T219 CHINESE HAMSTER LUNG CELLS (V79)
Biological Activity Detected: Mutagenicity.
Principle: Forward mutation assay measuring drug resistance at hypo-
xanthine-guanine-phosphoribolyItransferase (HGPRT) locus. The
HGPRT competent cells in presence of mammalian metabolic activa-
tion enzymes are exposed to a test substance. Induced frequency
of HGPRT deficiency is determined by using a selective medium
containing base analogues 8-azaguanine (8 AZ).
Endpoints: Gene mutation. Qua Iitative: Formation of cell colonies
in a selective medium. Quantitative: Induced mutation fre-
quencies are based on cell survival and cloning efficiency.
Strengths: Mammalian organization of genetic material; Forward muta-
tion assay; Fast generation time; Stable karyotype; Easy to
culture; May be particularly well-suited to quantitatlon as
HGPRT locus mutants may not show replicative advantages/disad-
vantages over wild-type cells under nonselective conditions.
Weaknesses: Mutation measured at a single locus; Requires metabolic
activation; Requires additional validation; Lacks pharmacological
relevance; Fairly high spontaneous mutation rate (l~5 x I0~6);
Long optimal expression period (7 days).
'Status of Development: Being implemented.
Describe: Needs additional validation.
Applications: Multimedia.
Samples: Pure Chemicals: N-Nitroso compounds, A Iky I atIng agents,
Primari ly organics mixtures. Complex Mixtures: Ambient; Indus-
trial; Energy Related.
Duration: 4 weeks.
Cost: $3,000.
Interpretation: Growth of the treated HGPRT competent cells in a
selective medium suggests genetic change.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemical Branch; HERL-CIN, Field Studies Division, Toxicologi-
cal Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
N.E. Kowal, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati,
OH 45268, (FTS 684-7477).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A. Mitchell;
Gulf South Research Institute, P.O. Box 26518, New Orleans, LA
70186, N. Gruener.
Program Office Support: OHEE.
References: I) Krahn, D.F., and C. Heldelburger. Proc. Nat I, Acad.
Sci., 73:188-192, 1977. 2) Artlett, et al. Mutation Res,, 33:
261-278, 1975.
67
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12110 DRQSOPHILA MELANOGASTER, SEX LINKED RECESSIVE LETHAL
Biological Activity Detected: Mutagenicity.
Principle: Wild type males are treated with the test chemical and
mated with untreated females with a marked chromosome. Fi females
are sib-mated and the progeny are scored for the presence of
x-I inked recessive lethals.
Endpoints: Qua Iitative; Change in sex ratios in F2 generation.
Quantitative: Point mutations and small delections may be scored
in germ eel Is.
Strengths: Higher organism, genetically well characterized; Multiple
loci available for detection of genetic alterations; Some metabolic
processes similar to that of mammals; Small number of large chromo
somes; Broad spectrum of genetic events can be detected and
scored; Wealth of mutant strains makes possible detailed analysts
of induced genetic changes.
Weaknesses: Short life span makes organism unsuitable for chronic
exposure studies; Limited use for testing pesticides due to ex-
treme toxicity.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Nitrosamines,
Halogenated ethers. Complex Mixtures: Protocols not yet devel-
oped.
Duration: 4 to 6 weeks.
Cost: $4,000 to $6,500.
Interpretation: Change in the sex ratio in the F2 progeny of the
test population indicates mutation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divis?
Biochemistry Branch, Cellular Biology Section. Sl°n,
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Trian t
Park, NC 27711, (FTS 629-2693); S.S. Sandhu: U.S. EPA HERL RTP
Research Triangle Park, NC 27711, (FTS 629-2693). ' '
Grant/Contract Laboratory Involved and Principal Investigators-
Stanford Research Institute, Menlo Park, CA 94205 A Mitchell
Program Off ice Support: OHEE. * '*
References: I) Vogel, E., and F.H. Sobels. The Function of
in Genetic Toxicology Testing. In: Chemical Mutagens-
and Methods for Their Detection. Vol.4. A. Hollaender ed
Plenum Press, NY, 1976. pp. 93-132. 2) Sobels, F.H., and E'
Vogel. The Capacity of Drosophila for Detecting Relevant Gene-H
Damage. Mutation Res., 41:95-106, 1976. 3) Legator, M.S., and
S. Zimmering. Gen. Toxicol. Ann. Rev. Pharmacol., 387-$OQ| (975
68
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12111 TRADESCANTIA STAMEN HAIR
Biological Activity Detected: Mutagenicity.
Principle: Mutation in petals and stamen hair in clones heterozygous
for flower color is detected as a change in pigmentation.
Endpoints: Qua Ii tat!ve: Change in stamen hair cells' color from
blue to pink. Quanti tative: Mutational events per stamen hair
and dose-response relationship can be established.
Strengths: Can detect broad spectrum of genetic events; Can be used
to monitor in situ environment; Can detect mutagens in the gaseous
phase; Eukaryotic organization of genetic material; Many muta-
licnal events can be observed directly; System appears highly
sensitive to physical and chemical mutagens.
Weaknesses: Lacks pharmacological relevance; May not be suitable
for evaluating many compounds requiring mammalian metabolic
activation.
Status of Development: Developmental.
Describe: The Tradescantia system was initially developed (and
is well-suited) for study of radiation effects. The system is
applicable to at least some chemical mutagens and is currently
under development for that purpose.
Applications: Air; Water.
Samples: Pure Chemicals: Organics, Nitroso derivatives, Poly-
el ichydrocarbons, Nucleic acid bases, and analogs, Hydroxyl-
amines, Hydrasine. Complex Mixtures: Ambient - drinking water,
air; Industrial - soiI.
Duration: 2 to 5 weeks.
Cost: $500 to $700, depending upon the test protocol.
Interpretation: The change in stamen hair cells' color from blue to
pink suggests mutagenicity.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field
Studies Division, ToxicologicaI Assessment Branch.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
L.J. McCabe, U.S. EPA, HERL-CIN, 26 W. St. Clair St., Cincinnati
OH 45268, (FTS 684-721 I).
Grant/Contract Laboratory Involved and Principal Investigators:
Brookhaven National Laboratories, Long Island, New York, L. Shirer;
U. of Missouri, Columbia, MO 65201, J.T. O'Connor; Stanford
Research Institute, Menlo Park, CA 94205, G. Newell.
Program Office Support: OHEE; OPP.
References: I) Sparrow, A.M., et al. Mutation Res., 26:265-276,
1974. 2) Underbrink, A.G., et al. In: Chemical Mutagens:
Principles and Methods for Their Detection. Vol. 3. A. Hollaender,
ed. Plenum Press, NY, 1973. pp. 171-207. 3) McNulty, P.J.,
et al. Mutation Res., 44:235-246, 1977.
69
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12112 MAIZE WAXY LOCUS ASSAY
Biological Activity Detected: Mutagenicity.
Principle: The assay is based on the change from the ability of the
plants to synthesize amylose to the inability to synthesize this
compound. Pollen from the treated plants is stained with iodine
Mutated pollen grains are stained purple.
Endpoints: Qua Iitative: Change in pollen grain color from yellow
to purple. Quantitative: Induced mutation frequency is expressed
Strengths: Test is performed in-vivo representing relevant conditions*
of exposure; Damage to germ cells is measured.
Weaknesses: Time consuming.
Status of Development: Developmental.
Describe: Few pure compounds or mixtures have been tested in
this system. This test system appears promising but needs
vaIi dation.
Applications: Air; Soil; Multimedia.
Samples: Pure Chemicals: Pesticides. Complex Mixtures: Industrial
Duration: 8 weeks.
Cost: Unknown.
Interpretation: Change in pollen grain color after staining suggests
mutation.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division
Biochemistry Branch, Cellular Biology Section. '
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693).'
Grant/Contract Laboratory Involved and Principal Investigators-'
U. of Illinois, Urbana, IL 61801, M. Plewa.
Program Office Support: OPP.
References: I) Plewa, M., and J. Gentile. Maize Cooper. Newsletter
50:44, 1976. 2) Plewa, M., and J. Gentile. Mutation Res
38:287-292, 1976.
70
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12113 IN-VIVO CYTOGENETICS IN MICE
Biological Activity Detected: Mutagenicity.
Principle: Chemicals are administered into mice through various
routes. After a specific period, treated animals are sacrificed.
Bone marrow and spermatogonial cells are analyzed for chromosomal
aberrations.
Endpoints: Qua Iitative: Observation of chromosomal and chromatid
breaks. Quantitative: Number of breaks/eel I as compared to the
control, provides a quantitative assessment of treatment response.
Strengths: In-vivo bioassay provides the benefit of intact pharmo-
kinetics.
Weaknesses: Expensive; Requires a we I I trained personnel to perform
the test and interpret the data.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I ating agents. Complex Mixtures:
Protocol not yet developed.
Duration: 3 weeks.
Cost: $3,000 to $6,500/chemical.
Interpretation: A significant increase in chromosomal aberrations
over the control suggests mutation.
Level of Complexity: 5.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Cellular Biology Section; HERL-CIN, Field
Studies Division, Toxicological Assessment Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693);
L. Claxton, U.S. EPA, HERL-RTP, Research Triangle Park, NC
27711, (FTS 629-2942); J.F. Stara. U.S. EPA, HERL-CIN, 26 W. St.
Clair St., Cincinnati, OH 45268, (FTS 684-7407).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Texas, Medical Branch, Galveston, TX 77550, M. Legator;
Stanford Research Institute, Menlo Park, CA 94205, G. Newell.
Program Office Support: OHEE, OPP.
References: I) Evans, H.J. In: Chemical Mutagens: Principles and
Methods for Their Detection. Vol. 4. A. Hollander, ed. Plenum
Press, NY, 1977. 2) Schmid, W. In: Chemical Mutagens:
Principles and Methods for Their Detection. Vol. 4. A. Hollander,
ed. Plenum Press, NY, 1977. pp. 31-52.
71
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CONTENTS: TEST SYSTEMS
1220 DNA Damage 73
1221 Baci I I us subti 1 is rec" 74
1221 Escherichi_a coli, Pol A" 75
1223 Mitotic Recombination and Gene Conversion
in Saccaromyces cerevisiae. 75
1224 Unscheduled DNA Synthesis (UDS) 77
1225 Sister-Chromatid Exchange Formation (SCE) .... 78
1226 In-Vivo Assessment of DNA Damage 79
1227 Intact Rodent Hepatocytes in Primary
Culture 80
1228 In-Vivo DNA Binding 81
1229 Chinese Hamster Cells (CHO) Unscheduled
DNA Synthesis (UDS) 82
1230 Carcinogenesis 83
73
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1221 BACILLUS SUBTILIS REC"
Biological Activity Detected: Toxicity; Primary DMA damage.
Principle: The DNA recombinational repair deficient and proficient
strains are streaked out along the intersecting lines. The test
chemical is spotted at the intersection. The differential kill!
between the repair deficient and proficient strains is used as °^
criteria of DNA damage. a
Endpolnts: Qua Iitative: A comparison is made between the zone of
growth inhibition for the repair deficient and proficient strata
Quantitative: N/A. ""rains.
Strengths: Very rapid and versatile bioassay; Inexpensive.
Weaknesses: Requires fairly large amount of test substances for
testing; Not suitable for substances which do not diffuse read?I
In agar. ^
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I ating agents, NItroso compounds
Polynuclear aromatics, Nitroso derivatives; Pesticides.
Mixtures: Ambient; Industrial; Energy Related;
Related; Other.
Duration: 2 to 3 weeks.
Cost: $200.
Interpretation: Relative sizes of killing zones in repair-proficle
and repair-deficient stains indicate primary damage to DNA
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicoloqv D!\/r i
Biochemistry Branch. ay 'vision,
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Trt
angle Park, NC 27711, (FTS 629-2693); L. Claxton. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2942)-
S.S. Sandhu. U.S. EPA, HERL-RTP, Research Triangle Park NC'
27711, (FTS 629-2693); J.L. Huisinqh. U.S. EPA, HERL-RTP Resftar. u
Triangle Park, NC 27711, CFTS 629-2948). ' Kesearch
Grant/Contract Laboratory Involved and Principal Investigators*
Stanford Research Institute, Menlo Park, CA 94205 V F Slmm^
Program Office Support: OHEE; OPP. ' ' ' oimmor>s.
References: I) Kada, T., K. Tutikawa, and Y. Sadaie. Mutation
16:165-174, 1972.
74
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1222 ESCHERICHIA COLj, Pol A".
Biological Activity Detected: Toxicity; Primary DNA damage.
Principle: The assay measures the differential killing between DNA
repair proficient Escherichia coli strain (W3I10, Pol A ) and
DNA repair deficient strain (P3478, Pol A ) as affected by environ-
mental toxicants.
Endpoints: Qua Iitative: Differential killing between DNA repair
proficient and deficient strain after treatment with a test-
substance. Quantitative: Liquid suspension test provides a
quantitative measure of primary DNA damage.
Strengths: The assay is well suited for detecting chemicals causing
frame shift mutations; Adequate data base is present on this
bioassay; Genetically wel(-characterized system; Rapid; Inexpensive;
Well validated as a test for gene mutation; Works well with in-
vitro metabolizing microsome fractions; Can be used as indicator
organism in host-mediated assays.
Weaknesses: Reverse mutation assay requiring several strains to
permit detection of a broad spectrum of compounds; Requires
metabolic activation; Lacks pharmacological relevance, Prokaryotic
organization of genetic material.
Status of Development: Validated.
Describe: N/A.
Applications: Multimedia.
Samples: Pure Chemicals: A Iky I at ing agents, Mitroso compounds,
Polynuclear aromatics, Nitroso derivatives, pesticides. Complex
Mixtures: Ambient; Industrial; Energy Related; Transportation
Related; Other.
Duration: 2 to 4 weeks.
Cost: $500.
Interpretation: Relative sizes of killing zones In repair-proficient
and repair-deficient stains indicate primary damage to DNA.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch.
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, CFTS 629-2693); L. Claxton. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2942).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, V.F. Simmons.
Program Office Support: OHEE; OPP.
References: I) Rosenbranz, H.S., et al. Mutation Res., 41:61-70,
1976. 2) Rosenbranz, H.S. Ann. Res. Mlcrobiol., 27:383-401,
1973. 3) Rosenbranz, H.S. Cancer Res., 33:458-459, 1973.
75
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1223 MITOTIC RECOMBINATION AND GENE CONVERSION IN SACCHAROHYCES CEREVISIAF
Biological Activity Detected: Primary DNA damage.
Principle: Recombination of reciprocal type, mitotic recombination
and the non-reciprocal type mitotic gene conversion are used '
for assessing the DNA damaging potential of environmental chem-
icals. ~"
Endpoints: Qua)itative: Appearance of twin spots and growth in
selective medium. Quantitative: The degree of mitotic cross In
over is evaluated by the frequency of twin spot sectors and tha?~
of mitotic gene conversion by the differential growth in a sele
tlve medium. c"
Strengths: The diploid cells with eukaryotic chromosomal oraan?»»+i
Rapid; Inexpensive. ya"izaTion;
Weaknesses: Less versatile due to problems associated with cell
wall permeability and with coupling of metabolic activation-
Inadequate data base showing the reliability of this assay svs+
Status of Development: Being implemented. ein*
Describe: This test system needs to be validated with a varletv
of classes of compounds. Further work is needed in understand In
the mechanism and significance of mitotic recombination and
crossing-over.
Applications: Multimedia.
Samples: Pure Chemicals: A IkyI ating agents, Nftrosamfnes, Carbarn
Nucleic acid bases and analogs, Aromatic
Protocol not yet developed.
Duration: 2 to 3 weeks.
Cost: $200 to $500.
Interpretation: The appearance of twin spots and growth in a se
medium suggests mutation.
Level of Complexity: I.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicoloav Div/r
Biochemistry Branch. sy UIV|sion»
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research T i
angle Park, NC 27711, (FTS 629-2693); S.S. Sandhu. u.S EPA
HERL-RTP, Research Triangle Park, NC 27711, CFTS 629-2693) '
Grant/Contract Laboratory Involved and Principal Investigators-'
Stanford Research Institute, Menlo Park, CA 94205 V F ^S
Program Office Support: OHEE; OPP. ' ' ' 3imfnons.
References: I) ZImmermann, F.K. Mutation Res., 31:71-86 1975
2) Brusfck, D.J., and-V.W. Mayer. Envir. Hlth. Prosoect fi o*
1973. H ' °'83.
76
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1224 UNSCHEDULED DNA SYNTHESIS (UDS)
Biological Activity Detected: Primary DNA.
Principle: This assay evaluates the test compounds for their ability
to induce unscheduled DNA synthesis (UDS) in human diploid WI38
fibroblasts blocked in the Gj phase.
Endpoints: Qua Iitative: Incorporation of 3H thymidine. Quantitative:
dpm 3H thymidine per yg or umole of DNA.
Strengths: DNA repair can be measured in human cells in culture;
Similar studies can be performed in animals using peripheral
leucocytes; The latter permit comparison between in-vitro and in-vivo
exposures to carcinogens or mutagens.
Weaknesses: The precise type of molecular binding between carcinogens
and DNA which triggers excision repair is unknown; DNA repair
synthesis does not measure residual damage to DNA.
Status of Development: Validated.
Describe: The mechanism of UDS is still not completely understood.
Applications: Multimedia.
Samples: Pure Chemicals: Alkylalois; AIkyI at ing agents, Nitroso
compounds, Polynuclear aromatics. Complex Mixtures: Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: 4 to 6 weeks.
Cost: $350 to $2,000.
Interpretation: The incorporation of labeled nucleotide precursors
into the cells arrested in the <5l phase after treatment is used
as a criteria of the ability of the test material to cause pri-
mary DNA damage.
Level of Complexity: 2,
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Biochemistry Branch.
Persons to Contact: M.D. Waters. U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Stanford Research Institute, Menlo Park, CA 94205, A, Mitchell.
Program Office Support: OHEE; OPP.
References: I) Stich, H., and S. Laighes. DNA Repair and Chemical
Carcinogenesis. Pathobiol. Ann., 3:342-376, 1973. 2> San, R.H.,
and H.F. Stich. DNA Repair Synthesis of Cultured Human Ceils as
a Rapid Bioassay for Chemical Carcinogens. Int. J. Cancer, 16:
284-291, 1975. 3) Williams, G.M. Detection of Chemical Carcino-
gens by Unscheduled DNA Synthesis in Rat Liver Primary Ceil
Cultures. Cancer Res., 37:1845-1851, 1977. 4) Simmon, V.F.,
A.D. Mitchell, and T.A. Jorgenson. Evaluation of Selected
Pesticides as Chemical Mutagens: In-vitro ancf In-vfvo Studies.
Ann. Rep,, Envir. Hlth. Effects Series, EPA-600/I-77-028, 1977.
77
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1225 SISTER-CHROMATID EXCHANGE FORMATION (SCE)
Biological Activity Detected: Primary DMA damage.
Principle: SCE involves a reciprocal exchange between sister-chro-
matids which does not result in a change in the overall chromo-
sonal morphology. SCE may be observed as darkly staining and
lightly staining chromatids after growth in BUdR for two succes-
sive cell generations and subsequent-staining with fIuorochrome
dyes.
Endpoints: Qua Iitative: Observation of sister-chromatid exchanges
of metaphase. Quantitative: SCE/eel I are expressed.
Strengths: Rapid; Relatively economical; Very sensitive; Can be
tested in-vivo or in-vitro.
Weaknesses: Mechanism and significance of SCE is not understood-
No clear relationship between SCE and chromosomal breaks has*
been established.
Status of Development: Being implemented.
Describe: The validation of this system is near completion In
several laboratories.
Applications: Multimedia.
Samples: Pure Chemicals: Alkylating agents, Mycotoxins, Halo-
genated hydrocarbons, Ureas and Thioureas, Nitro derivatives
Complex Mixtures: Protocol not yet developed.
Duration: 2 to 4 weeks.
Cost: $1,000 to $1,200.
Interpretation: Test agents inducing signigicant numbers of sister-
chromatid exchanges may be regarded as potential DNA-damagJnq
agents for animals and man.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divis?
Biochemistry Branch; ERL-NAR, Toxicology Branch, Genetic Toxicol°n*
Team. °9V
Persons to Contact: M.D. Waters, U.S. EPA, HERL-RTP, Research Trl
angle Park, NC 27711, (FTS 629-2693); S.5. Sandhu. U.S. EPA
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2693)-'
A.R. Malcolm. U.S. EPA, ERL-NAR, South Ferry Rd., Narraqanse++ t>,
02882, CFTS 838-4843); G.G. Pesch. U.S. EPA, ERL-NAR, South
Ferry Rd., Narragansett, Rl 02882, (FTS 838-4843).
Grant/Contract Laboratory Involved and Principal Investigators-
Stanford Research Institute, Menlo Park, CA 94205 V F Sfmmr>
Program Off ice Support: OHEE; OPP. ' ' ' immons-
References: I) Perry, P., and H.J. Evans. Nature, 258;121-125
1975. 2) Latt, S.A. Proc. Natl. Acad. Sci., 70:3395-3399 'i
3) Popescur, N.C., et al. F. Natl. Cancer Inst., 59:289-293
i y / / •
78
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1226 IN-VIVO ASSESSMENT OF DNA DAMAGE
Biological Activity Detected: Primary DNA damage.
Principle: Detection of in-vivo DNA repair activity that is stimulate
by chemical carcinogens.
Endpoints: Qua Iitative: Initial endpoint-measurement of DNA molecular
weight change. Quantitative: Molecular weight distribution of
cleaved DNA strands.
Strengths: Detects DNA damage in-vivo; Assay is done on biopsy
material; Non-destructive, animal sampled can later be scored
for tumors to validate assay.
Weaknesses: Not yet apparent; Mainly technical.
Status of Development: Developmental.
Describe: Still in early stages of development.
Applications: Multimedia.
Samples: Pure Chemicals: Organic compounds, Inorganics, Heavy
metals. Complex Mixtures: Ambient - water; Industrial; Energy
Related; Transportation Related; Other.
Duration: Variable, but not to exceed 3 months once validated.
Cost: Not yet determined.
Interpretation: Mutagenic and carcinogenic agents act through damage
of DNA. This method will quantitate DNA damage.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: R.J. Bui I. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
Ohio State University, Chemical Biomedical Environmental Research
Group, Columbus, OH 43210, R.W. Hart, (FTS 940-9375).
Program Office Support: OHEE; OWHM.
References: I) Brash, et al. N.Y. Acad. J.C., 1977. 2) Brash and
Hart, R.W. Envir. Hlth. Perspect., 1978. In press.
79
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1227 INTACT RODENT HEPATOCYTES IN PRIMARY CULTURE
Biological Activity Detected: Toxicity; Presumptive mutagenicity
Oncogen!city; DNA damage.
Principle: Detects interaction of chemical agents which result in
DNA damage. This interaction is detected as unscheduled DNA
synthesis using radiotracer, centrifugal and autoradiographic
techniques.
Endpoints: Qua Iitative: A positive response suggests potential to
act as a mutagen or carcinogen. Quantitative; Can quantify the
number of grains/nucleus which increases with increased dose
Strengths: Rapfd; Economical; The entire genome is the target*
Maintains several functions of tissue of origin, thereby activat-
ing many different chemicals; Not limited to direct acting com-
pounds.
Weaknesses: Represents only a single target organ; System lacks
validation; May not detect promoters or co-carcinogens.
Status of Development: Developmental.
Describe: This system is now in the process of validation In
several laboratories. It has not yet been applied to unknown
compounds.
Applications: MuI timed fa.
Samples: Pure Chemicals: Aromatic amines. Polyeye lies, Alky-
lators, Hormones. Complex Mixtures: Not yet determined.
Duration: 2 days to 2 weeks, depending on technique.
Cost: $500 to $2,000.
Interpretation: This test is considered a reliable screen for
prforltization of chemical testing In more complex in-vlvo
systems. It suggests that the chemical reaches the genome and
requires further analysis.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: National Center for Toxicological Rese
Division of Mutagenesis Research, Somatic Cell Section; HERL-RTP
Environmental Toxicology Division, Biochemistry Branch' CeliTi
Biology Section. ' «uuiar
Persons to Contact: D.A. Cascia.no, National Center for Toxicolo t
Research, Jefferson, AR 72079, (FTS 740-4573); J.L. Hulslnnh
U.S. EPA, HERL-RTP, Research Triangle Park, NC 277M—TFfqfeo
2948). ' bZ9~
Grant/Contract Laboratory Involved and Principal Investigators-
American Health Foundation, Naylor Dana Institute for Disease
Prevention, Hammond House Road, Valhalla, NY 10595 G M wt i i
Program Off ice Support: OHEE. ' ' ' "«i»S,
References: I) Kltagawa, T., et al. Cancer Res., 35:3682-3692
1975. 2) Mlchalopoulos, G., etal. Life Sciences, 18-|139*i
1976. 3) Williams, G.M. Cancer Letters, 1:231-236, 1976. 4
San, R.H.C., and H.F. Stich. Intl. J. Cancer, 7:65-74 |Q7i
80
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1228 1N-VIVO DNA BINDING
Biological Activity Detected: Binding of chemical to DNA.
Principle: This test system attempts to correlate binding with DNA
repair and tumorigenicity. Various rodent stains will be treated
with the chemical carcinogen DNA from presumptive target tissue
analyzed for DNA-carcinogen adducts by radiometric and/or fIuoro-
metric techniques, measuring the rate of excision of the bound
chemicaI.
Endpoints: Qua Iitative; Adducts will be determined by chromatographic
procedures. Quantitative: Can quantify amount of carcinogen
bound/unit DNA (u mole carcinogen/mole DNA-phosphate).
Strengths: A direct measurement of the extent of carcinogen inter-
action with DNA; Allows measurement of total DNA damage.
Weaknesses: A considerable portion of the DNA-carcinogen adducts may
have little or no biological relevance.
Status of Development: Developmental.
Describe: Initial experiments are being carried out to provide
background for development of in-vitro DNA repair assay.
Applications: Multimedia.
Samples: Pure Chemicals: Organic-metallic compounds, Organics.
Complex Mixtures: N/A.
Duration: 2 weeks.
Cost: Not yet determined.
Interpretation: This test measures the direct interaction of the
test compound with genetic material as an indication of carcino-
genic and mutagenic activity.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxIcological Assessment Branch.
Persons to Contact: B. Daniel. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7482).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OWHM.
References: Mot yet available.
-------
1229 CHINESE HAMSTER CELLS (CHO) UNSCHEDULED DNA SYNTHESIS (UDS)
Biological Activity Detected: DNA Repair.
Principle: Repair of induced damage to DNA is detected as unscheduled
DNA synthesis via incorporation of 3|H thymidine.
Endpoints: Qua Iitative: Unscheduled DNA synthesis is measured and
compared to controls. Quantitative: Amount of unscheduled DNA
synthesis per cell may be determined.
Strengths: Rapid; Relatively low cost; DNA repair is probably a
more sensitive detector of DNA damage than are chromosomal
aberrations.
Weaknesses: The in-vitro cell assay lacks pharmacological relevance-
Requires metabolic activation. "
Status of Development: Being implemented.
Describe: The test is presently being applied to standard com-
pounds and some selected unknowns. It is still being refined
and modified.
Applications: Water; Multimedia.
Samples: Pure Chemicals: Organics (EMS), Heavy metals, UV radla
tion. Complex Mixtures; Energy Related - JP-5 jet fuel,
Duration: 2 to 3 weeks.
Cost: $1,000 to $1,200/compound.
Interpretation: Agents inducing significant unscheduled DNA synthesi
represent potential carcinogens/mutagens for animals and man. S
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Genetic
Tox i coIogy Team.
Persons to Contact: E. Jackim, U.S. EPA, ERL-NAR, South Ferry Rd
Narragansett, Rl 02882, (FTS 838-4843 X229, X3IO); A,R. Malcolm
U.S. EPA, ERL-NAR, South Ferry Rd., Narragansett, Rl02882—*
(FTS 838-4843 X238, X247).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE. ' '
References: San, R.H.C., and H.F. Stitch. Int. J. Cancer, 16-284-
291, 1975. 2) Martin, C.N., et al. Cancer Letters, 2:355-L360~
1977. 3) Trosko, J.E., and J.D. Yager. Exp. Cell Res 88*47*
55, 1974. " '~
82
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CONTENTS: TEST SYSTEMS
1230 Carcinogenesis 83
1231 C3HIOTI/2CL8 Mouse Embryo Fibroblast
Oncogenic Transformation with Exogenous
Metabolic Activation 84
1232 C3HIOTI/2CL8 Mouse Embryo Fibroblast
Oncogen ic Transformation 85
1233 BHK-21 Mammalian Cell Oncogenic
Transformation 86
1234 Syrian Hamster Embryo Oncogenic
Transformation (Focus Assay) 87
1235 BALB 3T3 Oncogenic Transformation and
Mutagenesis with Exogenous Metabolic
Activation 88
1236 BALB 3T3 Oncogen ic Transformation 89
1237 Pulmonary Adenoma Bioassay in Mice 90
1240 Marine Applications 91
83
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1231 C3H10T1/2CL8 MOUSE EMBRYO FIBROBLAST ONCOGENIC TRANSFORMATION WITH
EXOGENOUS METABOLIC ACTIVATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Normal cells in log phase are treated with the test
agent. Four weeks after the cells have attained confluence they
are scored for morphologically transformed foci (clones of cells)
These transformed cells will give rise to tumors when injected
into immunosupressed syngeneic animals.
Endpoints: Qua Iitative: Appearance of morphologically transformed
foci. Quantitative: Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and trans-
formation is then adjusted for that toxicity.
Strengths: This system is easy to score; Has an extremely low back-
ground of spontaneous transformation; Is particularly sensitive to
PAH and their derivatives; Has metabolic activation capability-
Can be used to detect tumor promoters and initiators.
Weaknesses: These mouse embryo cells are aneuploid; Requires 6 weeks
to complete the experiment; Seems to be somewhat refractory to
the carcinogenic effects of a Iky I ating agents, aflatoxin Bj and
some aromatic amines.
Status of Development: Developmental.
Describe: The C3HIOTI/2 system is being modified by the addition
of exogenous metabolic activation capability, giving, therefore
increased sensitivity towards a broad range of chemical carcinoaA
Applications: Air. 9 ns<
Samples: Pure Chemicals: Polycyclic aromatic hydrocarbons.
Complex Mixtures: N/A.
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000.
Interpretation: The appearance of morphologically altered clones
of cells indicates oncogenic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Dlvlslo
Biochemistry Branch, Metabolic Effects Section. *
Persons to Contact: S. Nesnow. U.S. EPA, HERL-RTP, Research
Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
Microbiological Associates, Bethesda, MD 20014, R. Kouri,'
L. Schectman.
Program Office Support: OEM I.
References: I) Reznikoff, et al. Cancer Res., 33:3231-3249, 1973
2) Nesnow, et al. Cancer Res., 36:1801-1808, 1976. 3) Mondal"
et al. Cancer Res., 36:2254-2260, 1976. 4) Bertram. Cancer
37:514-523, 1977. 5) Benedict, et al. Cancer Res., 37-2202-
2208, 1977.
84
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1232 C3H10T1/2CL8 MOUSE EMBRYO FIBROBLAST ONCOGENIC TRANSFORMATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Normal cells in log phase are treated with the test agent.
Four weeks after the cells have attained confluence they are
scored for morphologically transformed foci (clones of cells).
These transformed cells will give rise to tumors when injected
into immunosupressed syngeneic animals.
Endpoints: Qua Iitative: Appearance of morphologically transformed
foci. Quantitative: Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and the
transformation is then adjusted for that toxicity.
Strengths: This system is easy to score; Has an extremely low back-
ground of spontaneous transformation; Is particularly sensitive
to PAH and their derivatives; Has metabolic activation capability;
Can be used to detect tumor promotors and initiators.
Weaknesses: These mouse embryo cells are aneuploid; Requires 6 weeks
tc complete the experiment; Seems to be somewhat refractory to the
carcinogenic effects of alkylating agents, aflatoxin Blt and some
aromatic amines.
Status of Development: Being implemented.
Describe: The C3HIOTI/2 system Is being modified so that its
metabolic activation capability is increased, giving, therefore,
increased sensitivity towards a broad range of chemical car-
cinogens.
Applications: Multimedia.
Samples: Pure Chemicals: Polyeye lie aromatic hydrocarbons,
Aromatic azo dyes, aromatic amines, pesticides. Complex Mixtures;
Model particulates.
Duration: 5 to 6 weeks.
Cost/sample: $5,700 to $7,000.
Interpretation: The appearance of morphologically altered clones
indicates oncogenic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: S. Nesnow. U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE; OPP.
References: I) Reznikoff, et al. Cancer Res., 33:3231-3249, 1973.
2) Nesnow, et a I. Cancer Res., 36:1801-1808, 1976. 3) Mondal,
et al. Cancer Res., 36:2254-2260, 1976. 4) Bertram. Cancer Res..
37:514-523, 1977. 5) Benedict, et al. Cancer Res., 37-2202-
2208, 1977.
85
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1233 BHK-21 MAMMALIAN CELL ONCOGENIC TRANSFORMATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Normal baby hamster kidney fibroblasts do not grow in
soft agar. After treatment with carcinogens these cells do grow
in soft agar and will give tumors when injected into syngeneic
animals.
Endpoints: Qua Iitative: Cells which grow in soft agar are considered
transformed. Quantitative; Colony formation is scored.
Strengths: Not yet known.
Weaknesses: Not yet known.
Status of Development: Developmental.
Describe: Testing of unconcentrated and concentrated wastewater
samples.
Applications: Water.
Samples: Pure Chemicals: PAH. CompI ex M i xtu res_: Wastewaters.
Duration: 3 weeks.
Cost/sample or chemical: Not yet known.
Interpretation: A positive result suggests a possible carcinogenic
materia I.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
logical Assessment Branch.
Persons to Contact: H. Pahren, U.S. EPA, HERL-CIN, 26 W. St Clair
St., Cincinnati, OH 45268, (FTS 684-7217).
Grant/Contract Laboratory Involved and Principal Investigators:
Syracuse Research Corporation, Merrill Lane, Syracuse, NY
13210, J. Saxena.
Program Office Support: OHEE.
References: I) Bouck, N., and G, diMayorca. Nature, 264:722-727
1976.
86
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1234 SYRIAN HAMSTER EMBRYO ONCOGENIC TRANSFORMATION (FOCUS ASSAY)
Biological Activity Detected: Presumptive oncogenicity.
Principle: Freshly isolated cells from hamster fetuses are seeded
into dishes, passaged twice, and then treated for two consecutive
three drug treatments with the test agents. Toxicity is scored
I to 2 days after the experiment is begun and the transformation
(appearance of morphologically transformed foci) is scored 10 days
after toxicity.
Endpoints: Qua Iitati ve: Appearance of morphologically transformed
foci. Quantitative: Simultaneous cytotoxicity experiments are
performed to obtain the lethal toxicity of the agent and the trans-
formation is then adjusted for that toxicity.
Strengths: These cells have high metabolic activation capability;
Are diploid; Respond to a wide variety of different chemical
agents; Few false positives are known.
Weaknesses: Variability within the assay due to variations in
obtaining and preparing viable primary cell cultures; Observable
spontaneous transformation background; Difficulty in scoring.
Status of Development: Being Implemented.
Describe: The Syrian hamster embryo biassay is being evaluated
for use in the evaluation of particulate samples.
Applications: Air.
Samples: Pure Chemicals: Polycyclic aromatic hydrocarbons.
Complex Mixtures; Model particulates.
Duration: I month.
Cost/assay: $4,000 to $5,000.
Interpretation: The appearance of morphologically altered clones
of cells indicates oncogenic transformation.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division,
Biochemistry Branch, Metabolic Effects Section.
Persons to Contact: S. Nesnow. U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2693); M.D. Waters. U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, CFTS 629-2693).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Casto, B.C., N. Janosko, and J.A. DIPaolo. Cancer Res.,
37:3508-3515, 1977.
87
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1235 BALB 3T3 ONCOGENIC TRANSFORMATION AND MUTAGENESIS WITH EXOGENOUS
METABOLIC ACTIVATION
Biological Activity Detected: Mutagenicity; Presumptive oncogenicity
Principle: Mammalian cell clones of BALB 3T3 clone AI3 undergo
malignant transformation upon treatment with known carcinogens.
Extent of transformation is expressed in focus formation and al-
tered morphology, increased saturation density and enhanced
plating efficiency in soft agar. Simultaneously these cells also
undergo a permanent genetic change at the ouabain locus. Tumor
induction is observed in recipient animals by reinjecting trans-
formed eel Is.
Endpoints: Qua Iitative: Appearance of morphological transformed
foci for transformation and clones of cells which grow in ouabain
supplanted media. Quantitative: Number of observed foci adjust
for the cytotoxic effect of the agent.
Strengths: 8ALB 3T3 cells are mouse embryo fibroblasts which have
the capability to activate 3-methyIcholanthrene to metabolites
which transform these cells; Direct acting alkylating agents such
as MNNG are also effective transforming agents.
Weaknesses: These cells are not transformed by the carcinogens
benzo[a]pyrene or 6-aminochrysene and possibly other potent car-
cinogens unless exogeneous metabolic activation is provided
Status of Development: Developmental.
Describe: The addition of rat liver preparations to activate
carcinogens and mutagens to make this assay system more sensi-
tive is currently underway.
AppIications: Alr.
Samples: Pure Chemicals: PAH, Aromatic amines. Complex Mixtnroc;.
Transportation Related. "~ " *
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000
Interpretation: Morphologically altered clones of cells indicate
oncogenlc transformation. Cells growing in the presence of
ouabain are indicative of a mutagenic change.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology DIvisI
Biochemistry Branch, Metabolic Effects Section. *
Persons to Contact: S. Nesnow, U.S. EPA, HERL-RTP, Research Triannl
Park, NC 27711, (FTS 629-2693). 9'6
Grant/Contract Laboratory Involved and Principal Investigators:
Microbiological Associates, Bethesda, MD 20014, L. Schectman
R. Kourl. '
Program Office Support: OEMI.
References: I) Kakunaga, T. A Quantitative Assay for Malignant
Transformation by Chemical Carcinogens Using Clone from BALB
3T3. Int. J. Cancer, 12:463, 1973.
88
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1236 BALB 3T3 ONCOGENIC TRANSFORMATION
Biological Activity Detected: Presumptive oncogenicity.
Principle: Mammalian cell clones of BALB 3T3 clone AI3 undergo
malignant transformation with known carcinogens.. Extent of trans-
formation is expressed in focus formation and altered morphology,
increased saturation density, and enhanced plating efficiency in
soft agar. Tumor induction is observed in recipient animals by
reinjectir.g transformed eel Is.
Endpoints: Qua!itative: Appearance of morphological transformed foci.
Quantitative: Number of observed foci adjusted for the cytotoxic
effect of the agent.
Strengths: BALB 3T3 cells are mouse embryo fibroblasts which have
the capability to activate 3-methyIcholanthrene to metabolites
which transform these cells; Direct acting alkylating agents such
as MNNG are also effective transforming agents.
Weaknesses: These eel Is are not transformed by the carcinogens benzo-
(s)pyrene or 6-aminochrysene and possibly other potent carcinogens,
Status of Development: Being implemented.
Describe: Currently BALB/T3 cell culture is used for routine
testing, but other cell lines (e.g. epithelial) are being in-
vestigated in an effort to increase sensitivity.
Applications: Water.
Samples: Pure Chemicals: Organics. Complex Mixtures: Ambient -
drinking water; Other - advanced waste treatment, concentrate
effluent.
Duration: 5 to 6 weeks.
Cost/sample or chemical: $5,000 to $7,000.
Interpretation: A positive result indicates possible carcinogenesis.
Level of Complexity: 2.
OHEE Laboratory Involved: HERL-CIN, Field Studies Division, Toxico-
loglcal Assessment Branch; HERL-CIN, Laboratory Studies Division,
Toxicological Assessment Branch.
Persons to Contact: J.P. Bercz, U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7432); N.E. Kowal. U.S. EPA,
HERL-CIN, 26 W. St. Clair St., Cincinnati, OH 45268, (FTS 684-
7477); R.J. Bui I. U.S. EPA, HERL-CIN, 26 W. St. Clair St.,
Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
U, of Cincinnati, Medical Center, Cincinnati, OH 45221,
J.C. Loper and D. Lang; Gulf South Research Institute, P.O. Box
26518, New Orleans, LA 70186, N. Gruener.
Program Office Support: OHEE.
References: I) Kakunaga, T. A Quantitative Assay for Malignant
Transformation by Chemical Carcinogens Using Clone From BALB
3T3. Int. J. Cancer, 12:463, 1973.
89
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1237 PULMONARY ADENOMA BIOASSAY IN MICE
Biological Activity Detected: Presumptive oncogenicity.
Principle: After 13 weeks no untreated strain A mice develop lung
tumors; however, after I year 100$ develop lung tumors. When
treated with a carcinogen, lung tumors start developing within
13 weeks.
Endpoints: Qua Iitative: Tumor formation. Quantitative: Average
number of tumors/mouse lung in the treated animals as compared
to the positive controls (urethane) and untreated controls.
Strengths: Relatively rapid in-vivo carcinogenesis bioassay.
Weaknesses: The adenoma (pulmonary) has no counterpart in human
neoplastic pathology.
Status of Development: Being implemented.
Describe: The system has been developed and used in other
laboratories, see references.
Applications: Multimedia.
Samples: Pure Chemicals; Metalic compounds, Food additives,
chemotherapeutic agents. Complex Mixtures: Industrial; Trans-
portation Related - diesel participate.
Duration: 13 to 30 weeks.
Cost/sample or chemical: Not yet known.
Interpretation: The formation of visually observable lung nodules
Indicates oncogenicity of the test substance.
Level of Complexity: 4.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division
Functional Pathology Branch.
Persons to Contact: J. Orthoefer. U.S. EPA, HERL-CIN, 26 W St Clai
St., Cincinnati, OH 45268, (FTS 684-7434). ' r
Grant/Contract Laboratory Involved and Principal Investigators: N/A
Program Office Support: OHEE.
References: I) Shimkin and Stoner. Lung Tumors in Mice: Application
to Carcinogenesis Bioassay. Adv. in Cancer Res., 21:1-58, 1975
2) Stoner, et al. Test for Carcinogenicity of Metallic Compounds
by the Pulmonary Tumor Response in Strain A Mice. Cancer Res
36:1744-1747, May 1976.
90
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CONTENTS: TEST SYSTEMS
1240 Marine Applications 91
1241 Tumor Induction in Massive Crustaceans,
Molluscs, and Teliost Fish 92
1242 MFO Induction as an Indicator of Toxicity
Exposure 93
1243 Limb Regeneration System 94
1244 Isogenfc Fish 95
1245 Integrated System: Development of Mutagen/
Carcinogen Activation, Concentration,
Separation, and Weathering Systems 95
1246 Biphenyl Hydroxylase 97
1300 Perinatal Toxicology 99
-------
1241 TUMOR INDUCTION IN MASSIVE CRUSTACEANS, MOLLUSCS, AND TELIOST FISH
Biological Activity Detected: Presumptive oncogenicity.
Principle: Animals are exposed to known carcinogens under laboratory
conditions and histopathology is performed. Also, feral animals
are surveyed for histopathological abnormalities and correlations
are established with tissue residues and water concentrations
Endpoints: Qua Iitative: Not supplied. Quantitative: Correlation
of exposure/response.
Strengths: May have value as sentinel system for water quality and
as a model system.
Weaknesses: Field correlation requires laboratory validation.
Status of Development: Being implemented.
Describe: Not supplied.
Applications: Water.
Samples: Pure Chemicals: PNA's. Complex Mixtures: Ambient
water; Industrial - water.
Duration: Not supplied.
Cost: Not supplfed.
Interpretation: Not supplied.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: J. Couch. U.S. EPA, ERL-GB, Sabine Island Gulf
Breeze, FL 32561, (FTS 686-9011). '
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Oregon, Eugene, OR 97403, M. Mix; U. of Southern MlssU«Urv i
Hattiesburg, Ml 39401, B.J. Martin. aii>5'pp!,
Program Office Support: OEM I; OHEE.
References: Not supplied.
92
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1242 MFO INDUCTION AS AN INDICATOR OF TOXICITY EXPOSURE
Biological Activity Detected: Toxicity; Mutagenicity; Presumptive
oncogenicity.
Principle: Fetal and laboratory reared animals are exposed to ambient
environmental conditions as sentinal organisms.'
Endpoints: Qua Iitative: Not supplied. Quantitative: Degree of
induction of MFO.
Strengths: Pre-pre screen method for water quality; Provides guidance
for chemical analysis programs.
Weaknesses: Nonspecific.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water.
Samples: Pure Chemicals: PNA's; Complex Mixtures; Ambient -
estuar i ne/mar i ne.
Duration: Not supplied.
Cost: Not supplied.
Interpretation: Positive test indicates that the test animal has
recently been exposed to inducer(s) of MFO systems.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011); P. Schoor. U.S. EPA, ERL-
GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of West Florida, Pensacola, FL 32504, R. Rao.
Program Office Support: OHEE.
References: Not supplied.
93
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1243 LIMB REGENERATION SYSTEM
Biological Activity Detected: Differentiation/teratology.
Principle: Crustacean limbs are removed at predetermined breakpoints
and regenerated by a precise sequence of biochemical events
Endpoints: Qua Iitative; Interference with limb regeneration -"
determined by gross morphology and histopathology. Quantitative-
Not supplied. -—'
Strengths: Appropriate for marine samples.
Weaknesses: Difficult to extrapolate to humans.
Status of Development: Being implemented.
Describe: Not supplied.
Applications: Water.
Samples: Pure Chemicals: PCP, Colchicine, PNA's. CpmpI ex
Mixtures: Ambient - estuarine/marine; Energy Related - drill In
fluids. 9
Duration: Not supplied.
Cost: Not suppIied.
Interpretation: Pre-screen for teratogens.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards. U.S. EPA, ERL-GB, Sabine Island
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators-
U. of West Florida, Pensacola, FL 32504, R. Rao.
Program Office Support: OEMI.
References: Not supplied.
94
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1244 ISOGENIC FISH
Biological Activity Detected: Presumptive oncogenicity; Teratology.
Principle: Isogenic fish provide uniform progeny with predictable
life stages, and an opportunity to genetically engineer genotypes
for susceptabiIity to carcinogens.
Endpoints: Qua Iitative: Not supplied. Quantitative: Interference
with development; Melanoma/melanin proliferation.
Strengths: In-vivo; Rapid.
Weaknesses: Toxic substances may kill test animal before oncogenic/
carcinogenic response is elicited; Substances may not be permeable
to eggs.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water.
Samples: Pure Chemicals: PNA's, Nitrosamines, Aflotoxin, etc.
Complex Mixtures: Not supplied.
Duration: I month.
Cost: Not yet determined.
Interpretation: Test results indicate presumptive teratogen, presump-
tive carcinogen, or toxic substance.
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of North Carolina, Chapel Hill, NC 27514, D. Humm.
Program Office Support: OEMI.
References: Not supplied.
95
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1245 INTEGRATED SYSTEM: DEVELOPMENT OF MUTAGEN/CARCINOGEN ACTIVATION
CONCENTRATION, SEPARATION, AND WEATHERING SYSTEMS
Biological Activity Detected: Toxicity; Mutagenicity.
Prfn/cple: The above procedures are being developed for use with
quick screen tests. Biological, physical, and chemical methods
are being used to concentrate, separate, and activate compounds
which interfere with testing.
Endpoints: Qua Iftative; Sample concentrated, freed of interferin
substances, activated, prepared for testing. Quantftativa- M?4-
supplied. '
Strengths: Separation of toxic components from complex mixtures
will allow detection; Concentration of dilute genotoxics will
allow their detection; Weathering may assist in prediction of th
environmental fate; Marine activation systems aid in predfctin
biological fate and food web relationships. ^
Weaknesses: Extensive exploratory research is required to validat
the methods.
Status of Development: Developmental.
Describe: Not supplied.
Applications: Water; Food.
Samples: Pure Chemicals; PNA's; Complex Mixtures: Ambient -
estuarine/marine H20; Energy Related - shale; Other - tissue
residues.
Duration: Not supplied.
Cost: Not supplfed.
Interpretation: These methods may be useful for all in-vitro and
in-vivo methods to expand their application to complex samples
Level of Complexity: Not supplied.
OHEE Laboratory Involved: ERL-GB.
Persons to Contact: N. Richards. U.S. EPA, ERL-GB, Sabine l<$i» A
Gulf Breeze, FL 32561, (FTS 686-901M. 'si and,
Grant/Contract Laboratory Involved and Principal Investigators-
Gulf South Research Institute, E. Kline; U. of West Florida
Pensacola, FL 32504, R. Rao. '
Program Office Support: OEM I; OHEE.
References: Not supplied.
96
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-|246 BIPHENYL HYDROXYLASE
Biological Activity Detected: Presumptive oncogenicity.
Principle: Pre-pre screen for carcogenicity depends on correlation
of interference with hydroxylation.
Endpoints: Qua!itative: Not supplied. Quantitative: Interference
with enzyme reaction.
Strengths: Extremely rapid; Inexpensive.
Weaknesses: Requires extensive development, modification and valida-
tion; Current data based on correlation only; Mechanism unknown.
Status of Development: Developmental.
Describe: Requires extensive development, modification, and
validation.
Applications: Water; Multimedia.
Samples: Pure Chemicals: Multiple classes being screened.
Complex Mixtures: Not supplied.
Duration: Not supplied.
Cost: Not supplied.
Interpretation: After validation and development, the test may be
useful as a rapid, inexpensive pre-pre screen.
Level of Complexity: 0.
OHEE Laboratory Involved: ERL-6B.
Persons to Contact: N. Richards. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-901 I).
Grant/Contract Laboratory Involved and Principal Investigators:
Denver Research Institute, Denver, CO 80210, J. Schmidt-CoderIs.
Program Office Support: OEM I.
References: Not supplied.
97
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CONTENTS: TEST SYSTEMS
1300 Perinatal Toxicology 99
1301 Mammalian Teratology 100
1302 Perinatal Toxicology 101
1303 Fetal Toxicity in Rats, Mice, Guinea Pigs/
Hamsters 102
1304 Developmental Toxicity in Neonatal Rats 103
1305 Teratology In-Vivo/ln-Vitro 104
1306 Direct Spectral Measurement of the
Biochemical Development of the
Nervous System 105
Q0 short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
99
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1301 MAMMALIAN TERATOLOGY
Biological Activity Detected: Teratology.
Principle: Determine the potential of the compound to produce
anatomical malformations.
Endpoints: Qua Iitative: N/A. Quantitative: Number and type of
ma I formations.
Strengths: Biologically active compounds are readily detected-
Same techniques used for most species.
Weaknesses: Only gross anatomical malformations are noted
Status of Development: Validated.
Describe: Protocols and methods we I I estabIished. Compounds
are being tested on a routine basis.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures: Ambient-
Industrial, Energy Related; Transportation Related; Other. '
Duration: 6 weeks for small rodents, start to report. Additional
gestational time needed for larger animals.
Cost: $6,000/specie/compound.
Interpretation: The teratogenic potential of compounds can be
estabIished.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Toxic Effects Branch, Perinatal Toxicology Section
Persons to Contact: K.D. Courtney. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Invest Iqators-
Program Office Support: OPP. '
References: I) OPP Guidelines for Teratology.
100
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•I302 PERINATAL TOXICOLOGY
Biological Activity Detected: Postnatal development.
Principle: Establish biochemical markers to determine normalacy of
postnatal development. Isozyme profiles of lactic dehydrogenase
(LDH) and creative phosphokinase (CPK) show definite develop-
mental patterns from day 7 to 14-in the postnatal mouse.
Endpoints: Qua Iitative; N/A. Quantitative: LDH and CPK total
activities and isozyme profiles are determined in postnatal mice
after prenatal and/or actational exposure. Results are entered
into computer by key punch cards and analyzed by established pro-
gram.
Strengths: Establish a measurable parameter of postnatal development
that can be quantified; Determining the cardiac isozymes permits
correlation with clinical human data; Comparative species studies
use same techniques and interpretations.
Weaknesses: Must wait for animals to be born.
Status of Development: Validated.
Describe: The postnatal isozyme profiles of LDH and CPK have
been established and treated animals are being evaluated.
Applications: Multimedia.
Samples; Pure Chemicals: All classes. Complex Mixtures; Ambient;
Industrial; Energy Related; Transportation Related; Other.
Duration: Gestation plus 4 weeks, start to report.
Cost: $3,OOO/compound.
Interpretation: Postnatal development is evaluated with the same
techniques that are used in human medicine so that results are
directly comparable.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Divi-
sion, Toxic Effects Branch, Perinatal Toxicology Section.
Persons to Contact: K.D. Courtney. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP.
References: Not yet aval I able.
101
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1303 FETAL TOXICITY IN RATS, MICE, GUINEA PIGS/HAMSTERS
Biological Activity Detected: Fetal toxicity including teratogenicity.
Principle: Pregnant females are treated with chemical (or other
agent) during the period of major organogenesis of their litters.
The animals are sacrificed before term and the fetuses subsequently
examined for signs of toxicity including visual and skeletal de-
fects.
Endpoints: Qua Iitative: Descriptions of defects and anomalies
encountered. Quantitative: Degree of fetal toxicity, and
incidence of defects encountered.
Strengths: A fairly rapid test, 30 to 45 days to final report; Much
historical background.
Weaknesses: Difficulty in assessing form and degree of fetal toxi-
city; Difficulty in extrapolation of data to human species.
Status of Development: Validated.
Describe: Standardized protocols have been available for over
10 years.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures: Drinking
water contaminants.
Duration: 45 days from initiation of treatment to completion of
ana lysis.
Cost: $IO,000/species.
Interpretation: A positive response in a species that has some
metabolic similarities to man would suggest that the compound
or mixture in question has the potential to induce abnormal
development in humans.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division
Neurobiology Branch.
Persons to Contact: N. Chernoff. U.S. EPA, HERL-RTP, Research Tri-
angle Park, NC 27711, (FTS 629-2326); R. Kavlock. U.S. EPA,
HERL-RTP, Research Triangle Park, NC 27711, (FTS 629-2326)!
Grant/Contract Laboratory Involved and Principal Investigators:
Contract currently under negotiation for the testing of 5
pesticides/year by established protocols.
Program Office Support: OPP; ORD.
References: I) Pesticide Registration Guidelines. In preparation.
2) Testing of Chemicals for Carcinogenic!ty, Mutagenicity, and'
Teratogenicity. Published by Minister of Health and Welfare
Canada, 1973.
102
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1304 DEVELOPMENTAL TOXICITY IN NEONATAL RATS
Biological Activity Detected: Developmental toxicity.
Principle: Pregnant rats are treated with compounds from the time
of implantation continuing through lactation. The neonatal rats
are then examined for a number of developmental milestones.
This design allows for the continuous exposure of a mammalian
organism through its most sensitive periods of development.
Endpoints: Qua Iitative; Growth and viability of neonatal rats.
Quantitatlve: Measurement of developmental milestones in early
postnatal life including reflex and morphological development;
Also measurement of open field behavior in young adults.
Strengths: Exposure to organisms during the perinatal period
maximizes the possibility of producing alterations in the
morphological and behavioral aspects of the exposed animal.
Weaknesses: Testing is a labor-intensive operation; Lack of
standardized procedures by various investigations; Difficult to
extrapolate to humans.
Status of Development: Developmental.
Describe: Investigators in this field are evaluating the
reliability and sensitivity of various test procedures.
Applications: Air; Water; Food.
Samples: Pure ChemicaIs: All classes. Complex Mixtures: N/A.
Duration: 3 to 4 months, start to report.
Cost: $IO,000/species.
Interpretation: The production of growth disturbances/behavioral
anomalies in the young postnatal animal is one of the more
sensitive indicators of developmental toxicity. The implication
of this test is that positive results may point to the induction
of behavioral disturbances in humans subsequent to perinatal
exposure. This link-up to human effects, however, has yet to
be empirically demonstrated.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Biology Division,
Neurobiology Branch.
Persons to Contact: R. Kavlock, U.S. EPA, HERL-RTP, Research Triangle
Park, NC 27711, (FTS 629-2326); N. Chernoff. U.S. EPA, HERL-RTP,
Research Triangle Park, NC 27711, (FTS 629-2326).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OPP; ORD.
References: I) Wiess, B., and J. Spyker. Behavioral Implications
of Prenatal and Early Postnatal Exposure to Chemical Pollutants.
Pediatrics, 53(5):Part III, May 1974. 2) Final Report of the
Committee on Postnatal Evaluation of Animals Subjected to Insult
During Development. C. Kimmel, Chairperson. Funded by the
Nat. Inst. of Envir. Hlth. Sci., Mar. 1977.
103
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1305 TERATOLOGY IN-VIVO/IN-VITRO
Biological Activity Detected: Teratology; Toxicology.
Principle: Treat pregnant rats from day 6 to 9 of gestation, then
remove embryos and grow them in tissue culture for 24 to 48 hrs.
During the culture interval, determinations of anatomical and
biochemical growth are made.
Endpoints: Qua Ii tati ve: Determine degree and normalacy of neurala-
tion and somite development; Quantitative: Measure growth in-
dicators, DMA, and protein.
Strengths: The maternal animal is treated; The embryo is accessible
for a long enough period to make some measurements; Zero time
measurements can use embryos from the same litter; Delays in
growth during this gestational stage could result in malformations
or abnormal development such as extra ribs; Embryos from the
same litter are available for residue determination.
Weaknesses: The culture of the embryos is limited to 48 hours.
Status of Development: Being implemented.
Describe: Embryos from treated animals are being grown in culture
Methods for growing control embryos have been established.
Applications: Multimedia.
Samples: Pure Chemicals: All classes. Complex Mixtures: Ambient-
Industrial; Energy Related; Transportation Related; Other.
Duration: 4 weeks, start to report.
Cost: $2,000/compound.
Interpretation: Toxic as well as teratogenic evaluators are deter-
mined for the embryo.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-RTP, Environmental Toxicology Division
Toxic Effects Branch, Perinatal Toxicology Section. *
Persons to Contact: K.D. Courtney. U.S. EPA, HERL-RTP, Research
Triangle Park, NC 27711, (FTS 629-2370).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Michigan, School of Medicine, Department of Anatomy,
Ann Arbor, Ml 48104, A. Beaudoin.
Program Office Support: OTS.
References: Not yet available.
104
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1306 DIRECT SPECTRAL MEASUREMENT OF THE BIOCHEMICAL DEVELOPMENT OF THE
NERVOUS SYSTEM
Biological Activity Detected: Delayed and arrested development.
Principle: A large change in the capacity of the nervous system for
oxidative metabolism occurs with development. This increase may
be followed directly in slices of tissue by taking advantage of
the known spectral properties of the cytochromes using dual-wave-
length spectroscopy. During brain development there are quantita-
tive and qualitative differences in the way the tissue will re-
spond to stimulation (e.g., electrical, elevated K ) metabolicaIly
that may be observed using polarographic, spectral, and enzymatic
analyses.
Endpoints: Qua Iitative; Developmental changes in: Cytochrome levels;
Oxygen uptake in response to stimulation; Lactic acid output;
Uptake and release of neurotransmitters; Amino acid concentrations;
Redox changes produced in tissue pyridine nucleotides, flavopro-
te'ns, and cytochromes in response to standard stimulants.
Quantitative: Measurements of: Cytochrome levels; Oxygen uptake
in response to stimulation; Lactic acid output; Uptake and re-
lease of neurotransmitters; Amino acid concentrations; Redox
changes produced in tissue pyridine nucleotides, flavoproteins,
and cytochromes in response to standard stimulants.
Strengths: Spectral measurements can be applied to as little as 3 mg
of tissue; Responses of tissues dependent upon the integrity of
a wide variety f systems within the tissue (e.g., cell excit-
ability, Na , K -ATPase, neurotransmittance, uptake, release and
intrinsic activity, glycolytic and TCA cycle enzymes, etc,). The
responses to K are multiphasic, one phase probably being appli-
cable to neuronal responses, the other to glial responses. Con-
sequently, the system is unique in that it will detect a wide
variety of types of damage.
Weaknesses: Principle weakness is that it does not lend itself to
immediate identification of mechanism unless there is a direct
effect on energy metabolism proper so that crossovers can be
identified. However, this disadvantage is overcome by the fact
that delays in development resulting from early exposure to Pb
can be almost perfectly correlated with delays in morphological
development (e.g., synaptogenesis).
Status of Development: Being implemented.
Describe: The basic developmental patterns for the cerebral
cortex have been established for the spectral measurements of
cytochrome concentrations and form of the metabolic responses.
Spectral changes induced by K have been correlated with respira-
tory changes, lactic acid output, and changes in tissue adenine
nucleontides. Delays in development have been demonstrated in
these parameters with Pb and correlated with delayed synaptogenesis
i n the rat.
Applications: Multimedia.
Samples: Pure Chemicals; Organics; Inorganics. Complex Mixtures:
Industrial; Energy Related; Transportation Related; Other.
105
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1306 DIRECT SPECTRAL MEASUREMENT OF THE BIOCHEMICAL DEVELOPMENT OF THE
NERVOUS SYSTEM (continued)
Duration: As short as 3 weeks to as long as 3 months if animals are
to be taken to sexual maturity.
Cost: $2,500 to $25,000, depending upon duration and number of
rep Iications.
Interpretation: Delays in biochemical development of the brain have
been well correlated with delays in morphological development
(synaptogenesis) and behavioral development (activity away from
mother during lactation) in lead-treated animals. With these data
as support, delayed biochemical development may be interpreted as
presumptive evidence of retarded mental development.
Level of Complexity: 3.
OHEE Laboratory Involved: HERL-CIN, Laboratory Studies Division,
ToxicologicaI Assessment Branch.
Persons to Contact: R.J. BuI I. U.S. EPA, HERL-CIN, 26 W. St. Clair
St., Cincinnati, OH 45268, (FTS 684-7213).
Grant/Contract Laboratory Involved and Principal Investigators:
In-house.
Program Office Support: OHEE.
References: I) Himwich, W.A. Developmental Neurobiology. C.C.
Thomas, Springfield, IL, 1970. pp. 22-46, 311-330, 331-369,'
370-392. 2} Bull, R.J., and S.D. Lutkenhoff. J. Neurochem!,
21:913-922, 1973. 3) Bull, R.J., and J.T. Cummins. J. Neuro-
chem., 21:923-937, 1973. 4) Bull, R.J., and J.J. O'Neill.
Psychopharmacol. Commun., 1:109-115, 1975. 5) Bull, R.J., P.M.
Stanaszek, and S.D. Lutkenhoff. Envir. Hlth. Perspect., 12:
89-95, 1975. 6) Bull, R.J. J. Neurochem., 26:149-156, 1976.
106
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CONTENTS: TEST SYSTEMS
2000 Short-Term Tests for Ecological Effects 107
2100 Freshwater and Marine Algae 107
2101 Freshwater Algal Assay Bottle Test 108
2102 Marine Algal Assay Bottle Test 109
2200 Freshwater Animals and Plants Ill
2210 Fish Toxicity , Ml
107
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2101 FRESHWATER ALGAL ASSAY BOTTLE TEST
Biological Activity Detected: Toxicity; Stimulation.
Principle: Standard test alga (Selenastrum caprfcornutum) is added to
a test water, cultured under standard light, temperature, and gas
exchange conditions, and evaluated for inhibitory (toxic) or
stimulatory response to pollutant stress.
Endpoints: Qua Iitative: Can be used to screen stimulatory or
inhibitory properties of point and non-point source pollutants.
Quantitative: Response in mg dry wt |~* of test alga either
stimulatory (% S^) or inhibited (% I14) at day 14. Results are
expressed as % stimulation or % inhibition as compared to control
vs waste concentration.
Strengths: Detection of bioreactive components in a test water
or of waste discharge; Identification of toxic or stimulatory
components.
Weaknesses: Insufficient application of test to relate to potential
health effects of a pollutant.
Status of Development: Validated.
Describe: N/A.
Applications: Water.
Samples: Pure Chemicals: Nutrients, NC>3, N02, NHs, Ortho-P,
Tot-P, Heavy metals, Pesticides, Herbicides, Insecticides.
Complex Mixtures: Ambient - receiving water; Industrial - waste
discharges; Energy Related - coal storage, leachates; Other - new
product formulations, i.e., detergents.
Duration: Test: 14 days; Analysis: 5 days; Total: 21 days maximum
in most cases.
Cost: $400 to evaluate a compound or complex waste.
Interpretation: Test data can be used to define nutrient limitation
heavy metal toxicity, and inhibitory or stimulatory properties of
complex wastes, as they effect ecology of aquatic systems.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Assessment Criteria Development
Division, Special Studies Branch; ERL-DUL, Newtown Fish Toxi-
cology Station.
Persons to Contact: W.E. Mi Iler. U.S. EPA, ERL-COR, 200 SW 35th St
Corvallis, OR 97330, (FTS 420-4775, Commercial 503 757-4775). "'
T. Shirovama. U.S. EPA, ERL-COR, 200 SW 35th St., Corvallis OR
97330, (FTS 420-4776, Commercial 503 757-4776); J.C. Greene! U S
EPA, ERL-COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4764
Commercial 503 757-4764); E. Robinson. U.S. EPA, ERL-DUL, New- '
town Fish Toxicology Station, 3411 Church St., Cincinnati OH
45244, (FTS 684-8601).
Grant/Contract Laboratory Involved and Principal Investigators- N/A
Program Office Support: OHEE; OTS.
References: I) Algal Assay Procedure Bottle Test. U.S. EPA, Aug
1971. 2) Standard Methods for the Examination of Water and
Wastewater. 14th edition, 1975. To be published by ASTM.
108
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2102 MARINE ALGAL ASSAY BOTTLE TEST
Biological Activity Detected: Toxicity; Stimulation.
Principle: Standard test algae are added to a test water, cultured
under standard light, temperature, and gas exchange conditions
and evaluated for inhibitory (toxic) or stimulatory response to
polIutant stress.
Endpoints: Qua Iitative: Can be used to screen potential stimulatory
or toxic_properties of pollutants. Quantitative: Response in mg
dry wt I * of the stimulatory (% S^) or inhibitory (% lltt)
response as compared to control vs control to which a waste
concentration has been added.
Strengths: Detection of stimulatory and/or inhibitory bioreactive
components in a test water, and of waste discharge.
Weaknesses: Insufficient application of test (round Robin); Inade-
quate to predict possible health effects of.specific pollutants.
Status of Development: Validated.
Describe: Inter laboratory calibration is necessary, as are broad
application studies, i.e., complex wastes, organic compounds,
etc.
Applications: Water.
Samples: Pure Chemicals: Nutrients, i.e., Nitrogen and Phosporous,
Heavy metals, RGB's, Chloramines, Free chlorine. Complex Mixtures:
Ambient - receiving waters; Industrial - waste discharges, dredge
spoil, monochlorinated organics detergent builders.
Duration: Test: 14 days; Analysis: 5 days; Total: 21 days.
Cost: $400 to evaluate a compound of complex waste.
Interpretation: Test data can be used to define nutrient limitation,
heavy metal toxicity, and inhibitory or stimulatory properties of
complex wastes, as they effect ecology of marine ecosystems.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-NAR, Toxicology Branch, Marine Toxi-
cology Team; ERL-COR, Marine and Freshwater Branch; ERL-GB,
Experimental Environments Branch.
Persons to Contact: J. Gentile. U.S. EPA, ERL-NAR, South Ferry Rd.,
Narragansett, Rl 02882, (FTS 838-4843 X244); D. Specht. ERL-
COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4675);
G. Walsh, U.S. EPA, ERL-GB, Sabine Island, Gulf Breeze, FL 32561
(FTS 686-901 I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) Marine Algal Assay Procedure Bottle Test. U.S. EPA,
Dec. 1974. Bloassay Procedures for the Ocean Disposal Permit
Program. EPA-600/9-78-010, U.S. EPA, 1978.
109
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CONTENTS: TEST SYSTEMS
2200 Freshwater Animals and Plants
2210 Fish Toxicity,
2211 Acute Fish Toxicity Test, Static and
Flow-Through, All Freshwater Species 112
2212 Subchronic Embryo-Larval, Early Juvenile
Fish Toxicity Test 113
22!3 Chronic Fish Toxicity Test, American
Flagfish (Jordanella floridae) 114
2214 Fish Respiratory Activity Toxicity Test,
Electrode Chamber Method 115
2215 Fish Avoidance Test, Gradient Tanks 116
2220 Invertebrate Toxicity 117
I I
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2211 ACUTE FISH TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL FRESHWATER SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 96-hr LC50 of various pure compounds and
complex mixtures on freshwater organisms.
Endpoints: Qua I itative: Behavior. Quanti tati ve; Mortality (LC50).
Strengths: A "hard" number on lethality.
Weaknesses: The LC50 is not protective of aquatic species. There-
fore, one must estimate or test for lower, no-effect concentra-
tions.
Status of Development: Validated.
Describe: The 96-hr flow-through and static aquatic toxicity
tests have been conducted for many years with some cross valida-
tion between laboratories; Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals
PCB's. Complex Mixtures: Industrial - effluents; Energy Related -
driI I ing muds, oils.
Duration: Test: 96 hours; Analysis: 96 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determined.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan. U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975
*This test Is also applied to marine fish. See 2311
12
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2212 SUBCHRONIC EMBRYO-LARVAL, EARLY JUVENILE FISH TOXICITY TEST
Biological Activity Detected: Toxicity.
Principle: This test involves a 30-day exposure of the embryo-larval,
early juvenile stages of development.
Endpoints: Qua Iitative: Behavior. Quantitative: Survival; Growth;
Deformities; Determination of an estimated maximum acceptable
toxicant concentration (MATC).
Strengths: Direct measure of an MATC; Gives an excellent estimate
of chronic toxicity in I month. Chronic tests themselves would
require 12 months to complete.
Weaknesses: Requires 30 days to complete test; Requires a supply of
fish embryos.
Status of Development: Validated.
Describe: These tests have been run successfully by several
contract laboratories as well as routinely by several EPA
laboratories.
Applications: Water.
Samples: Pure Chemicals: Inorganics, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: 30 days; Analysis: Included in the 30 days.
Cost: $6,000/test.
Interpretation: This test gives an excellent estimate of the chronic
(life-cycle) toxicity of individual toxicants or complex mixtures
to fish.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Toxicants Section.
Persons to Contact: J.M. McKlm, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9567, Commercial 218 727-6692 X567);
W.A. Brunqs. U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth, MN
55804, (FTS 783-9546, Commercial 218 727-6692 X546).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) McKlm, J.M. Evaluation of Tests with the Early Life-
Stage of Fish for Predicting Long-Term Toxicity. J. Fish Res.
Bd. Can., 34(8):I 148-I 154, 1977. 2) U.S. EPA. Proposed Recom-
mended Bioassay Procedure for Egg and Fry Stages of Freshwater
Fish (manuscript). U.S. EPA, Duluth, MN. 3) Sauter, S., K.S.
Buxton, K.J. Macek, and S.F. Petrocelli. Effects of Continuous
Exposure to Lead, Chromium, Copper, and Cadmium on Eggs and Fry
of Selected Freshwater Fish. Ecol. Res. Series, U.S. EPA, Duluth,
MN, 1976.
-------
2213 CHRONIC FISH TOXICITY TEST, AMERICAN FLAGFISH (JORDANELLA FLORIDAE)
Biological Activity Detected: Toxicity.
Principle: Determine the impact of toxicants on survival, growth,
and reproduction of a freshwater fish with a rapid life cycle.
Endpoints: Qua Iitative: Behavior. Quantitative: Growth; Survival-
Reproduction success (fecundity, hatchabi Iity); Determination of
a maximum acceptable toxicant concentration (MATC).
Strengths: Similar in sensitivity to commonly tested freshwater fish
i.e., brook trout and fathead minnow; Short life cycle compared
to most fish; Data generated on all stages of life cycle including
those stages considered to be most sensitive.
Weaknesses: Fish is semitropical and may not have direct application
to most U.S. waters.
Status of Development: Validated.
Describe: Many people at ERL-DUL have run this test successfully
Applications: Water.
Samples: Pure Chemicals: Pesticides, Metals. Complex Mixtures:
Waste oil.
Duration: Test: 6 months; Analysis: Included in 6 months.
Cost: $16,000.
Interpretation: This test can be used as an indicator of potential
chronic fish effects.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Toxicants Section.
Persons to Contact: W.A. Brungs, U.S. EPA, ERL-DUL, 6201 Congdon Blvd
Duluth, MN 55804, (FTS 783-9546, Commercial 218 727-6692 X546)- "'
R. Spehar. U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth, MN 55804
(FTS 783-9521, Commercial 218 727-6692 X52I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A
Program Office Support: OHEE; ORD,
References: I) Smith, W.E. A Cyprinodont Fish, Jordanella florldae
as a Reference Animal for Rapid Chronic Bioassays. J. FisKT'*
Res. Bd. Can., 39:329-330, 1973. 2) Spehar, R.L. Cadmium and
Zinc Toxicity to Jordanella floridae. J. Fish. Res. Bd. Can.
33:1939-1945, 1976. 3) U.S. EPA Committee on Aquatic Bioassays.
Recommended Bioassay Procedures for Brook Trout, Blueglll, Fat-'
head Minnow, and Flagfish Chronic Tests. U.S. EPA, Duluth,
MN, 1972.
I 14
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2214 FISH RESPIRATORY ACTIVITY TOXICITY TEST, ELECTRODE CHAMBER METHOD
Biological Activity Detected: Toxicity.
Principle: Bioelectric signals generated by respiratory activities
are electronically amplified and recorded on stripchart records.
Endpoints: Gill purge (cough) and ventilation rates. Qua Iitative:
Behavior. Quantitative: Rate of increase/unit of time.
Strengths: Rapid; Predictive.
Weaknesses: Stripchart records must be interpreted.
Status of Development: Being implemented.
Describe: The test results have been published (see references)
and other researchers are starting to use this method.
Applications: Water.
Samples: Pure Chemicals: Multi. Complex Mixtures: Industrial.
Duration: Test: 4 days; Analysis: I day.
Cost: $750/chemical.
Interpretation: Concentrations which do not cause a statistically
significant increase in gill purge rates are not likely to cause
long-term adverse effects.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Pollutants Section.
Persons to Contact: R. Drummond. U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9511, Commercial 218 727-6692
X5II; R. Carlson, U.S. EPA, ERL-DUL, 6201 Congdon Blvd., Duluth,
MN 55804, (FTS 783-9591, Commercial 218 727-6692 X59I).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Spoor, W.A., et al. Trans. Am. Fish. Soc., 1971. 2)
Drummond, R. , et al. J. Fish. Res. Bd. Can., 1973. 3) Drummond,
R., et al. Trans. Am. Fish. Soc., 1974. 4) Carlson, R., and
R. Drummond. Water Res., 1978. 5) Drummond, R., and R. Carlson.
Ecol. Rep. Series, U.S. EPA, 1978.
15
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2215 FISH AVOIDANCE TEST, GRADIENT TANKS
Biological Activity Detected: Toxicity; Physical environment.
Principle: Levels of environmental variables and toxicants avoided
by fish are determined.
Endpoints: gual itative: Avoidance behavior. Quantitative: Loco-
motor activity/unit of time.
Strengths: Short-term tests; Endpoints easy to determine; Collection
of data can be automated.
Weaknesses: Needs validation.
Status of Development: Developmental,
Describe: Laboratory investigation underway.
Applications: Water.
Samples: Pure Chemicals: Dissolved gases, Oxygen, Metals, Organics.
Complex Mixtures: Industrial; Energy Related.
Duration: Test: 4 hours; Analysis: I hour.
Cost: $150.
Interpretation: Avoidance has ecological significance.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Office of the Director.
Persons to Contact: W.A. Spoor, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9506, Commercial 218 727-6692 X506).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: I) Spoor, W.A., and R. Drummond. Trans. Am. Fish. Soc.,
101:714-715, 1972.
116
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CONTENTS: TEST SYSTEMS
2220 Invertebrate Toxicity 117
2221 Acute Invertebrate Toxicity Test, Static
and Flow-Through, All Freshwater Species ... Ii8
2222 Subchronic Invertebrate Toxicity Test,
Stream Insects 119
2223 Subchronic Invertebrate Toxicity Tests,
Chironomid (Tanytarsus dissimilis) 120
2224 Chronic Invertebrate Toxicity Test,
Water Flea (Daphnia maqna) '21
2230 Plant Toxicity/Residue 123
17
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2221 ACUTE INVERTEBRATE TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL
FRESHWATER SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 48-hr LC50 or EC50 of various pure com-
pounds and complex mixtures on freshwater organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50)
or Immobilization EC50.
Strengths: A "hard" number on lethality, or measurable effect is
determi ned.
Weaknesses: The LC50 or EC50 is not protective of aquatic species.
Therefore, one must estimate or test for lower, no-effect con-
centrations.
Status of Development: Validated.
Describe: The 48-hr and 96-hr flow-through and static aquatic
toxicity tests have been conducted for many years. Some methods
were written by Standard Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals,
PCB's. Complex Mixtures; Industrial - effluents; Energy Related
driI I ing muds, oi Is.
Duration: Test: 48 or 96 hours; Analysis: 48 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests, the lethal effects of a toxicant
along with a statistically valid 95% confidence Interval can be
determi ned.
LeveI of CompI ex i ty: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch;
ERL-DUL, Extramural Program Branch.
Persons to Contact: C. Stephan. U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570); K.E. Biesinger, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9524, Commercial 218 727-6692 X524).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Biesinger, K.E., and G, Christensen. Effects of
Various Metals on Survival, Growth, Reproduction, and Metabolism
of Daphnia magna. J. Fish Res. Bd. Can., 29(2):1691-1700, 1972.
2) Methods for Acute Toxicity Tests with Fish, Macro invertebrates
and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test is also applied to marine invertebrates. See 2321
18
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2222 SUBCHRONIC INVERTEBRATE TOXICITY TEST, STREAM INSECTS
Biological Activity Detected: Toxicity.
Principle: The insects are exposed for 4 weeks. The time length
allows for a good estimate of toxicity.
Endpoints: Qua Iitative: Behavior. Quantitative: Survival LC50;
Bioaccumulation.
Strengths: Allows testing of stream invertebrates.
Weaknesses: Requires a "clean" collecting site near test facility;
Requires good water source since it is a flow-through system.
Status of Development: Developmental.
Describe: System is developed and can be used if the need was
established (i.e., could be implemented but isn't).
Applications: Water.
Samples: Pure Chemicals: Heavy metals, Pesticides. CompI ex
Mixtures: Not tested.
Duration: Test: 4 week exposures; Analysis: Could be included in
test time.
Cost: $2,800 at 4 weeks at $35,000/manyear.
Interpretation: It is a procedure which allows exposure for a time
longer than acute (4-day) tests. This allows a better prediction
of toxic effects.
Level of Complexity: I to 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physical Pol-
lutant Section.
Persons to Contact: R.L. Anderson, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9565, Commercial 218 727-6692
X565).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Spehar, R.L., R.L. Anderson, and J.T. Fiandt. Toxi-
city and Bl©accumulation of Cadmium and Lead in Aquatic Inverte-
brates. Envir. Pollut., 15:195, 1978. 2) Anderson, R.L., and
D. DeFoe. Toxicity and BioaccumuI at ion of Endrin and Methoxychlor
by Aquatic Invertebrates. 1979. In press.
I 19
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2223 SUBCHRONIC INVERTEBRATE TOXICITY TEST, CHIRONOMID (TANYTARSUS DISSIMILIS)
Biological Activity Detected: Toxicity.
Principle: The animals are exposed from egg to 2nd or 3rd instar
for 10 to 12 days of total exposure.
Endpoints: Qua Iitative: N/A. Quantitative: Survival; Growth;
LC50 and EC values.
Strengths: Exposure through molting; Can
Weaknesses: Static, has only been tested
Status of Development: Developmental.
Describe: A report is being prepared regarding exposure to cadmium,
lead, copper, and zinc.
Applications: Water.
Samples: Pure Chemicals: Heavy metals.
Duration:
also measure growth effects.
wfth metals.
Complex Mixtures:
Ana lysis: Up to 2
N/A.
weeks
Test: 10 to 12 days exposure;
including preparation and clean-up.
Cost: 2 weeks at $35,000/manyear equals approximately $1,400; Chemical
analysis might be additional if complex compounds were used.
Interpretation: The system exposes an insect during embryogenesis,
hatching, growth, and molting. These 4 events are critical to
the survival of the animal.
Level of Complexity: 2 to 3
OHEE Laboratory Involved: ERL-DUL,
lutant Section.
Persons to Contact: R.L. Anderson.
Blvd., Duluth, MN 55804, (FTS
X565).
Grant/Contract Laboratory Involved and Principal Investigators:
Program Office Support: OHEE; ORD.
References: None, in-house development.
Research Branch, Physical Pol-
U.S. EPA, ERL-DUL, 6201 Congdon
783-9565, Commercial 218 727-6692
N/A.
120
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2224 CHRONIC INVERTEBRATE TOXICITY TEST, WATER FLEA (DAPHNIA HAGNA)
Biological Activity Detected: Toxicity; Reproduction.
Principle: Animals are exposed for 3 to 4 weeks. The exposure period
includes molting and reproduction.
Endpoints: Qua Iitative: N/A. Quantitative: Survival (LC50);
Reproduction.
Strengths: Low equipment cost; Manpower requirements.
Weaknesses: Renewal system.
Status of Development: Being implemented.
Describe: Procedure has been published and is now being used
with variation in many places.
Applications: Water.
Samples: Pure ChemicaIs: Metals, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: 3 to 4 weeks may be a good estimate. This would
include data analysis and perhaps chemical analysis if single
o: low number mixtures are used.
Cost: 3 weeks at $35,000/manyear equals approximately $2,100.
Interpretation: Test allows fairly rapid screening of pollutants
with a zooplankton representative.
Level of•Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan, U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9510, Commercial 218 727-6692 X5IO).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Biesinger, K., and G. Christensen. Effects of Various
Metals on Survival, Growth, Reproduction and Metabolism of Daphnia
magna. J. Fish. Res. Bd. Can., 29(12):1691-1700, 1972. 2) ASTM
DRAFT. Proposed Standard Practice for Conducting Life-Cycle
Toxicity Tests with the Daphnid, Daphnia magna.
121
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CONTENTS: TEST SYSTEMS
2230 Plant Toxicity/Residue 123
2231 Acute Plant Toxicity Test,
Duckweed (Lemma mi nor) 124
2240 Ecosystem 125
123
-------
2231 ACUTE PLANT TOXICITY TEST, DUCKWEED (LEMMA MINOR)
Biological Activity Detected: Toxicity; Residue.
Principle: Exposure of growing plants for I week.
Endpoints: Qua IItative: N/A. Quantitative: Growth as reflected in
frond count; EC values.
Strengths: Only flow-through system available for aquatic plants;
Fast.
Weaknesses: Not developed to a point where weakness can be adequately
descri bed.
Status of Development: Developmental.
Describe: Exposure to copper has been completed. Exposure with
other compounds is projected. Completion of procedure is pro-
jected for 1979.
Applications: Water.
Samples: Pure Chemicals: Metals, Organics. Complex Mixtures:
Industrial; Energy Related.
Duration: Test: I week exposure, perhaps I to 2 weeks for prepara-
tion and clean-up.
Cost: 2 weeks at $35,000/manyear equals approximately $1,400; Analysis
cost may be included if simple compounds are used.
Interpretation: This is the only system which allows exposure of an
aquatic plant. Toxicity and bioaccumulation data should be
obtainable from the procedure.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physical Pol-
lutant Section.
Persons to Contact: R.L. Anderson. U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804, (FTS 783-9565, Commercial 218 727-6692
X565).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Wai bridge, C.T. A Flow-Through Testing Procedure with
Duckweed (Lemna minor). Ecol, Res. Series, EPA 600/3-77-108,
U.S. EPA, 1977.
124
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CONTENTS: TEST SYSTEMS
2240 Ecosystem 125
2241 Laboratory Ecosystem 126
2300 Marine Animals 127
2310 Fish Toxicity 127
125
-------
2241 LABORATORY ECOSYSTEM
Biological Activity Detected: Toxicity.
Principle: To determine the effects of toxicants on ecosystem
processes.
Endpoints: Qua Iitative: N/A. Quantitative: Modifications in rates
and components of organic carbon budget includes water inflow
and outflow; System photosynthesis and respiration; Periphyton
growth; Benthic macro!nvertebrates; Sediment accumulation and
degradation; Macrophyte decomposition; MATC, based on change In
the ecosystem processes.
Strengths: More realistic than single species testing; Includes
species and environment interaction; Includes impact of environ-
ment on toxicant; Can follow accumulation of toxicants in food
web; Includes some organisms not traditionally included in fresh-
water toxicology.
Weaknesses: Labor intensive; Difficult to obtain degree of replica-
tion necessary for statistical treatment of data; Complex inter-
actions make data evaluation and interpretation difficult; Must
extrapolate to natural situations.
Status of Development: Developmental.
Describe: Development of preliminary non-toxicant methods has
just been initiated.
Applications: Water.
Samples: Pure Chemicals: Inorganics, Organics. Complex Mixtures:
N/A.
Duration: Test: 3 to 5 months; Analysis: An additional 2 to 3 months.
Cost: $35,000.
Interpretation: Data can be used to identify sensitive ecosystem
processes as well as to determine potential assimilation capacity.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-DUL, Newtown Fish Toxicology Station.
Persons to Contact: S.F. Hedtke. U.S. EPA, ERL-DUL, Newtown Fish
Toxicology Station, 3411 Church Street, Cincinnati, OH 45244,
(FTS 684-8601).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: ORD.
References: Work is presently in developmental stages. Information
on test system has not been published. See Persons to Contact.
126
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CONTENTS: TEST SYSTEMS
2300 Marine Animals 127
2310 Fish Toxicity 127
2311 Acute Fish Toxicity Test, Static and
Flow-Through, All Marine Species 128
2312 Subchronic Embryo-Larval Fish Toxicity Test,
Sheepshead Minnow (Cyprinodon variegatus). . . . 129
2313 Chronic Fish Toxicity Test, Sheepshead
minnow (Cyprinodon variegatus) 130
2320 Invertebrate Toxicity 131
127
-------
2311 ACUTE FISH TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL MARINE SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 96-hr LC50 of various pure compounds and
complex mixtures on marine organisms.
Endpoints: Qua Ii tative: Behavior. Quantitative^ Mortality (LC50).
Strengths: A "hard" number on lethality.
Weaknesses: The LC50 is not protective of aquatic species. There-
fore, one must estimate or test for lower, no-effect concentra-
tions.
Status of Development: Validated.
Describe: The 96-hr flow-through and static aquatic toxicity
tests have been conducted for many years with cross validation
by many laboratories; Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, Heavy Metals,
PCB's. Complex Mixtures: Industrial - effluents; Energy Related
driI I ing muds, oiIs.
Duration: Test: 96 hours; Analysis: 96 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence Interval can be
determined.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephen, U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X570).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test is also applied to freshwater fish. See 2211
128
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2312 SUBCHRONIC EMBRYO-LARVAL FISH TOXICITY TEST, SHEEPSHEAD MINNOW
(CYPRINODON VARIEGATUS)
Biological Activity Detected: Toxicity; Growth; Pathologic effects.
Principle: To determine the effects of a toxicant on the early life
stages of the sheepshead minnow.
Endpoints: Determine concentrations of a toxicant which affect
survival, growth, behavior, and pathologic effects. Qua Iitative:
Behavior. Quantitative: LC50 values; Significant differences can
be established from experimental and control survival, and growth;
Determination of an estimated maximum acceptable toxicant con-
centration (MATC).
Strengths: A good estimate of toxicity, particularly chronic toxicity,
can be made in many instances.
Weaknesses: Duration is generally 28 days, or more.
Status of Development: Being implemented.
Describe: Embryo/fry studies have been conducted by ERL-GB and
a private laboratory.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides, PCB's,
Pentachlorophenol. Complex Mixtures: N/A.
Duration: Test: 28 days; Analysis: 28 to 40 days if a chemical analysis
is required for bioconcentration.
Cost: $6,000 to $7,000, depending upon whether or not chemical analyses
a re req u i red.
Interpretation: From these tests the concentrations of a pollutant
that affects survival, growth, etc. of a sensitive life stage of
an estuarine fish can be determined.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.J. Hansen. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507,
P.R. Parrish.
Program Office Support: OHEE; OPP; OWHM.
References: I) Schlmmel, S.C., P.R. Parrtsh, D,J. Hansen, J.M.
Patrick, Jr., and J. Forester. Endrin: Effects on Several
Estuarine Organisms. Proc. 28th Annu. Conf. Southeast. Asso.
Game Fish Comm., 1974.
129
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2313 CHRONIC FISH TOXICITY TEST, SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
Biological Activity Detected: Toxicity; Growth; Pathologic effects;
Fecundity.
Principle: To determine the chronic effects of a pollutant on an
estuarine fish.
Endpoints: Qua I?tative: Behavior. Quantitative: Growth; Survival;
Fecundity; Pathologic effects.
Strengths: A good estimate of toxic affects.
Weaknesses: Cost; Time.
Status of Development: Being implemented.
Describe: Both ERL-GB and Bionomics, Pensacola, a private labora-
tory, have conducted the tests, although both laboratories have
not completed full chronic tests on the same chemical for com-
parison.
Applications: Water.
Samples: Pure Chemicals: Insecticides, Herbicides. CompI ex
Mixtures: N/A.
Duration: Test: 4 to 5 months; Analysis: 6 to 7 months.
Cost: $35,000 with analytical back-up.
Interpretation: One of the best available estimates of the effects
of a pollutant on an estuarine fish is obtained.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.J. Hansen. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507.
P.R. Parrish.
Program Off ice Support: OHEE; OPP; OWHM.
References: I) Hansen, D.J., S.C. Schimmel, and J. Forester. Endrin:
Effects on the Entire Life Cycle of a Salt Water Fish. J.
Toxicol. Envir. Hlth., 3:721-733, 1977.
130
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CONTENTS: TEST SYSTEMS
2320 Invertebrate Toxicity 131
2321 Acute Invertebrate Toxicity Test, Static
and Flow-Through, All Marine Species 132
2322 Acute Toxicity Test, Benthic Assemblages 133
2323 Chronic Invertebrate Toxicity Test,
Estuarine Shrimp (Pa laemonetes puqio) 134
2324 Chronic Invertebrate Toxicity Test,
Estuarine mysid (Mysidopsis bahia) 135
2330 Ecosystem 137
131
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2321 ACUTE INVERTEBRATE TOXICITY TEST, STATIC AND FLOW-THROUGH, ALL
MARINE SPECIES*
Biological Activity Detected: Toxicity.
Principle: Determine the 48-hr LC50 or E050 of various pure com-
pounds and complex mixtures on marine organisms.
Endpoints: Qua Iitative: Behavior. Quantitative: Mortality (LC50)
or EC50.
Strengths: A "hard" number on lethality, or measurable effect, is
determined.
Weaknesses: The LC50 or EC50 is not protective of aquatic species.
Therefore, one must estimate or test for lower, no-effect con-
centrations.
Status of Development: Validated.
Describe: The 48-hr flow-through and static aquatic toxicity
tests have been conducted for many years with cross validation
by many laboratories. Some methods were written by Standard
Methods and ASTM.
Applications: Water.
Samples: Pure Chemicals; Insecticides, Herbicides, Heavy Metals,
RGB's. Complex Mixtures: Industrial - effluents; Energy Related
dri11 ing muds, oiIs.
Duration: Test: 48 hours; Analysis: 48 hours.
Cost: Static test: $300; Flow-through test: $650.
Interpretation: From the tests the lethal effects of a toxicant
along with a statistically valid 95% confidence interval can be
determined.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-DUL, Technical Assistance Branch.
Persons to Contact: C. Stephan. U.S. EPA, ERL-DUL, 6201 Congdon
Blvd., Duluth, MN 55804 (FTS 783-9510, Commercial 218 727-6692
X5IO).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, Inc., Wareham, MA, S. Sauter and K.J. Macek.
Program Office Support: OHEE; ORD.
References: I) Methods for Acute Toxicity Tests with Fish, Macro-
Invertebrates and Amphibians. EPA-600/3-75-009, U.S. EPA, 1975.
*This test Is also applied to freshwater invertebrates. See 2221
132
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2322 ACUTE TOXICITY TEST, BENTHIC ASSEMBLAGES
Biological Activity Detected: Toxicity.
Principle: The acute effects of toxic substances on a macrofunal
genthic microcosm representative of natural benthic assemblages
in the Pacific Northwest will be examined.
Endpoints: Qua Iitative: Behavior, observe animals able to bury in
dredge sediment. Quantitative: Count of survivors and count
of animals able to bury in substrate in relation to water con-
centration provides EC values.
Strengths: Test organism is a sensitive amphipod species, Paraphoxus
epistomus.
Weaknesses: New species not frequently used in aquatic testing.
Status of Development: Developmental.
Describe: This test procedure has only been conducted at the
Newport, Oregon, faci Iity.
AppM ,ations: Water; Soil.
Samples: Pure Chemicals; Heavy metals, i.e., Ca, Zn, Cr.
Complex Mixtures: Dredge sediment; Complex wastes.
Duration: Test: 96 hours.
Cost: Not yet determined.
Interpretaton: From this test one can determine if a dredge soi I
is acutely toxic to estuarine benthic animals.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Marine and Freshwater Branch, Newport Field Station.
Persons to Contact: R. Swartz, U.S. EPA, ERL-COR, Newport Field
Station, Marine Science Center, Newport, OR 97365, (FTS 423-4111,
Commercial 503867-4041).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OTS.
References: I) U.S. EPA, Corps of Engineers. Appendix F, Guidance for
Performing Solid State BFoassays. In: Report on Ecological
Evaluation of Proposed Discharge of Dredged Material on Ocean
Waters. Tech. Committees on Criteria for Dredge and Fill
Material. U.S. Army Waterways Station, Vtcksburg, MS, July 1977.
133
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2323 CHRONIC INVERTEBRATE TOXICITY TEST, ESTUARINE SHRIMP (PALAEMONETES PUGIO)
Biological Activity Detected: Toxicity; Growth; Reproduction.
Principle: Determine chronic effects of a pollutant on the entire
life cycle of Grass Shrimp, Pa Iaemonetes pugio.
Endpoints: Qua Iitative: N/A. Quantitative: LC50 values;
Significant differences in survival, growth, and fecundity in
experimental animals compared to controls.
Strengths: LC50 values; Significant differences in survival; Deter-
mination of growth and reproduction. Data give good indication
of the effects of a toxicant over the animal's entire life cycle.
Weaknesses: Duration is long, about 5 months for chronic test; 2 to
3 months for partial-chronic.
Status of Development: Developmental.
Describe: Chronic tests have only been conducted at ERL-GB.
Applications: Water.
Samples: Pure Chemicals: Pesticides. Complex Mixtures: N/A.
Duration: Test: Approximately 5 months.
Cost: $25,000, including analytical back-up.
Interpretation: A maximum acceptable toxicant concentration can be
established for this estuarine invertebrate.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.B. Tyler-Schroeder, U.S. EPA, ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Tyler-Schroeder, D.B. Use of the Grass Shrimp,
Pa Iaemonetes gjjqio. In a Life Cycle Toxicity Test. Symposium
on Aquatic Toxicology and Hazard Evaluation. ASTM, 1978. In
press. 2) Bioassay Procedures for the Ocean Disposal Permit
Program. EPA-600/9-78-010, U.S. EPA, 1978.
134
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2324 CHRONIC INVERTEBRATE TOXICITY TEST, ESTUARINE MYSID (MYSIDOPSIS BAHIA)
Biological Activity Detected: Toxicity; Fecundity; Growth.
Principal: Determine the chronic effects of toxicants on the entire
life cycle of the crustacean species.
Endpoints: Qua Iitative: N/A. Quantitative: LC50 values;
Significant differences in growth, reproduction, and survival.
Strengths: An estimate of chronic toxicity can be determined.
Weaknesses: Relatively high cost and duration of test; Animals are
not always available throughout the year.
Status of Development: Being implemented.
Describe: ERL-GB and Bionomics EG & G, a private laboratory,
have conducted these tests.
Applications: Water.
Samples: Pure Chemicals: Pesticides; Metals. Complex Mixtures:
N/A.
Duration: Test: Approximately 28 days.
Cost: $7,000, including analytical back-up on pure chemicals.
Interpretation: A maximum acceptable toxicant concentration (MATC)
can be established for a marine/estuarine invertebrate in this
test.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: D.W. Nimmo. U.S. EPA, ERL-GB, Sabine Island,
Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics EG 4 G, Rt. 6, Box 1002, Pensacola, FL 32507, P.R.
Parrish.
Program Office Support: OHEE; OWP.
References: I) Nlmmo, D.W., L.H. Bhaner, R.A. Rigby, J.M. Sheppard,
and A.J. Wilson. Mysidopsis bahj_a; An Estuarine Species Suitable
for Life-Cycle Toxicity Tests to Determine the Effects of a
Pollutant. In: Aquatic Toxicology and Hazard Evaluation,
Mayer, Hamelink, eds. ASTM, STP 634:109-1 16, 1977.
135
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CONTENTS: TEST SYSTEMS
2330 Ecosystem 137
2331 Estuarine Microcosym I 138
2332 Estuarine Micrccosmy II 139
2333 Estuarine Communities 140
2400 Terrestrial Animals and Plants 141
2410 Plants 141
137
-------
2331 ESTUARINE MICROCOSMY I
Biological Activity Detected: Fate.
Principle: This test assesses transport and degradation potential
of aquatic pollutants.
Endpoints: Qua Iitative: Types of transport; Localization; Mechanisms
of degradation and character of degradation products. Quantitative:
Rates of above processes; Effect of environmental parameters
on rates; EC values.
Strengths: Use of environmental substrates; Mass balance analysis
with radiolabeled pollutants; Versatility; Short turn-around
time.
Weaknesses: Sealing factors from laboratory systems to the environ-
ment.
Status of Development: Developmental.
Describe: Systems have been designed and are in operation. Fate
of pesticides has been tested. Optimization of systems are now in
progress. Field validation is being initiated.
Applications: Water.
Samples: Pure Chemicals: Pesticides, Toxic Organics. CompI ex
Mixtures: Industrial - effluents; Energy Related - oil.
Duration: Test: 4 to 8 weeks/chemical pollutant.
Cost: $2,000/month, not including senior investigator time.
Interpretation: This test supplies data on transport and degrada-
tion in natural system.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Processes and Effect Branch.
Persons to Contact: A.W. Bourquin, ERL-GB, Sabine Island, Gulf
Breeze, FL 32561, (FTS 686-9011); R.L. Garnas. ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011); P.M. Pritchard.
ERL-GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; OPP; OTS; OEM I.
References: I) Bourquin, A.W., R.L. Garnas, P.H. Pritchard, F.G.
Wilkes, C.R. Cripe, and N.I. Rubinstein. Interdependent
Microcosms for the Assessment of Pollutants in the Marine
Environment. Internet. J, of Envir. Studies., 1978. In press.
138
-------
2332 ESTUARINE MICROCOSMY II
Biological Activity Detected: Toxicity.
Principle: This test assesses the toxicity of pollutants to microbial
growth and microbial degradation processes.
Endpoints: Qua Iitative: Types of microorganisms affected; Total
biomass reduction; Selection of species; Physiological indies
affected; Mechanisms of toxicity. Quantitative: EC values.
Strengths: Quick screen which uses natural assemblages of micro-
organ i sms.
Weaknesses: Extrapolation from laboratory systems to the environ-
ment; Requires analytical supports.
Status of Development: Being implemented.
Describe: Pesticides and toxic organics have been tested.
Applications: Water.
Samples: Pure ChemicaIs; Pesticides, Toxic organics, Heavy
metals. Complex Mixtures: Industrial - effluents; Energy Re-
lated -oil.
Duration: Test: 4 to 8 weeks/chemical pollutant.
Cost: $1,500/month, not including senior investigator time.
Interpretation: This test determines toxicant effects on microbial
assemblages.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-GB, Processes and Effect Branch.
Persons to Contact: A.W. Bourquin, ERL-GB, Sabine Island, Gulf
Breeze, FL 32561, (FTS 686-9011); R.L. Garnas. ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011); P.M. Pritchard.
ERL-GB, Sabine Island, Gulf Breeze, FL 32561, (FTS 686-9011).
Grant/Contract Laboratory Involved and Principal Investigators:
Georgia State U., Atlanta, GA 30303, D.G. Ahearn; Gulf Coast
Research Laboratory, P.O. Box 26518, New Orleans, LA, W. W. Walker.
Program Office Support: OEM I.
References: I) Bourquin, A.W., P.M. Pritchard, and W.R. Mahaffey.
Effects of Kepone on Estuarine Microorganisms. Developments in
Industrial Microbiology. 1978. In press. 2) Bourquin, A.W.
Effects of Ma lath ion on Microorganisms of an Artificial SaIt-Marsh
Environment. J. Envir. Quality, 6:383-378, 1977.
139
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2333 ESTUARINE COMMUNITIES
Biological Activity Detected: Toxicity.
Principle: This test determines concentrations of a toxicant which
offset the settling and development of benthic estuarine com-
munities. These communities develop in sand substrate from
larvae in unfiltered seawater.
Endpoints: Qua Iitative: N/A. Quantitative: Measure of species
diversity; Number of sensitive phyla, biomass, growth, and total
number of species; EC values.
Strengths: From this test we can see how a toxicant changes the
community makeup by limiting sensitive groups and promoting
growth of others.
Weaknesses: Duration is long (2 to 4 months); The investigator
must have a good taxonomic background.
Status of Development: Developmental.
Describe: Only ERL-GB and one contractor have completed this
type of community study.
Applications: Water.
Samples: Pure Chemicals; Pesticides. Complex Mixtures: Energy
Related - drilling muds.
Duration: Test: 2 to 4 months to conduct test; Analysis: another
2 to 4 months to identify the animals.
Cost: $6,000.
Interpretation: From these studies we are able to predict the con-
centration of a toxicant that will adversely affect the recruit-
ment and development of estuarine benthic communities.
Level of Complexity: 3 to 4.
OHEE Laboratory Involved: ERL-GB, Experimental Environments Branch.
Persons to Contact: M. Taqatz, ERL-GB, Sabine Island, Gulf Breeze,
FL 32561, (FTS 686-901 I).
Grant/Contract Laboratory Involved and Principal Investigators:
Florida State U., Tallahassee, FL 32306, B. Glasson.
Program Office Support: OHEE; ORD.
References: I) Hansen, D.J. Aroclor 1254: Effect on Composition
of Developing Estuarine Animal Communities in the Laboratory.
Marine Sci., 18:19-33, 1974.
140
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CONTENTS: TEST SYSTEMS
2400 Terrestrial Animals and Plants 141
2410 Plants 141
2411 Stress Ethylene Bioassay in Plants 142
2412 Measurement of NItrogenase Activity by
Acetylene Reduction in Nodulated Plants .... 143
242 J Ecosystem 145
141
-------
2411 STRESS ETHYLENE BIOASSAY IN PLANTS
Biological Activity Detected: Alteration of physiological process.
Principle: Environmental stresses cause plants to produce large
amounts of ethylene. The elevated ethylene occurs before and
often in the absence of visual injury.
Endpoints: Qua Iftative: Observation of increase of ethylene produc-
tion; In some cases modification of plant growth form.
Quantitative: Extent of increase for comparison with reference
plants and chemicals.
Strengths: More sensitive than visual assessment; Less subject
to variabiIity.
Weaknesses: Short duration of the phenomena (approx. 48 hours);
The test is designed as a flow-through test and not as a static '
system.
Status of Development: Being implemented.
Describe: The test has been used to examine effects of ozone and
chlorine on a variety of plants ranging from pine trees to
potatoes.
AppIications: Ai r.
Samples: Pure Chemicals: Gaseous pollutants: Ozone, S02, NO ,
CO. Complex Mixtures: Ambient - air; Industrial - air poflutants;
Energy Related - air pollutants; Transportation Related - air
pollutants; Other - acid rain.
Duration: Test: 3 to 5 days exclusive of plant rearing; Analysis:
Simultaneous with test.
Cost: $2,500.
Interpretation: This test provides evidence of tissue injury. Excess
ethylene production may result in defoliation.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: D.T. TInqey. ERL-COR, 200 SW 35th St., Corvallis,
OR 97330, (FTS 420-4621).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE,
References: I) Tingey, D.T., C. Standley, and R.W. Field. Stress
Ethylene Evolution, a Measure of Ozone Effects on Plants.
Atmos. Envir., 10:969-974, 1976.
142
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2412 MEASUREMENT OF NITROGENASE ACTIVITY BY ACETYLENE REDUCTION IN
NODULATED PLANTS
Biological Activity Detected: Alteration of physiological process.
Principle: Measure reduction of acetylene to ethylene.
Endpoints: Qua Iitative: N/A. Quantitative: Amount of ethylene
produced in relation to pollutant concentration.
Strengths: Requires minimal facilities or equipment; Rapid analysis.
Weaknesses: Needs further evaluation.
Status of Development: Being implemented.
Describe: This test is used to assess the impact of heavy metals
(cadmium) on soybean, alder, and alfalfa nitrogen fixation systems,
AppI[cations: SoiI.
Samples: Pure Chemicals: Heavy metals. Complex Mixtures:
Industrial - sludge.
Duration: Test: 10 to 15 days exclusive of time to grow plants;
Analysis; Immediately following test.
Cost: $5,000/chemical.
Interpretation: This test provides information on the ability of
nodulated plants to fix nitrogen in the presence of stress.
Level of Complexity: I.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: C. WIckliff, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4622).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) Wfckliff, C. PhD Thesis, Oregon State U. 1977.
2) Fishbeck, K., H.J. Evans, and L.L. Boersma. Agronomy J.,
65:429-433, 1973. 3) Huang, Chi-Ying, F.A. Bazzaz, and L.N.
Vanderhoff. Plant Physiology, 54:122-124, 1974.
143
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CONTENTS: TEST SYSTEMS
2420 Ecosystem 145
2421 Terrestrial Microcosm Chamber 146
2422 Soil Core Microcosm 147
2423 Soil/Litter Microcosm 148
2500 Physical-Chemical/Bio-accumulation 149
145
-------
2421 TERRESTRIAL MICROCOSM CHAMBER
Biological Activity Detected: Toxicity; Bioaccumulation; Biomagnifi-
cation; Community processes.
Principle: Radio labeled pesticides at or below accepted field ap-
plication rates are applied to assess fate, species and population
effects.
Endpoints: Qua Iitatfve; Microcosms should be viewed as a tool
which at present provide only trends in fate or effects.
Quantitative; N/A.
Strengths: Higher link between bench and field; Lower cost than
field studies; Provides indices of distribution.
Weaknesses: Cost/unit; Ambiguity of results; Not validated.
Status of Development: Developmental.
Describe: At present ERL-COR is developing a testing protocol
consisting of not one system, but a methodology that utilizes
"benchmark" data as well as various microcosms depending on the
information required.
Applications: Air; Water; Soil.
Samples: Pure Chemicals; Heavy metals, Pesticides, Gaseous
pollutants. Complex Mixtures: Industrial - effluents; Energy
Related - air pollutants; Transportation Related - air pollutants.
Duration: Test: 2 months; Analysis: 3 months.
Cost: $50 to $100,000, depending on the compound.
Interpretation: Fate results utilized ecological mag: bioaccumulation,
biodegradabiIity and degradation. Effects' results are still open
to discussion other than acute toxfcity.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: J.D. Glle, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4649); J.W. GiIlett. U.S. EPA,
ERL-COR, 200 SW 35th St., Corvallis, OR 97330, (FTS 420-4622).
Grant/Contract Laboratory Involved and Principal Investigators:
U. of Michigan, Ann Arbor, Ml 48104, E. Goodman; U. of Wisconsin,
Madison, Wisconsin 53706, P. Lichtenstein.
Program Office Support: OHEE; OPP.
References: I) Gillett, J.W., and J.D. Glle. Pesticide Fate in
Terrestrial Laboratory Ecosystems. Intern. J. Envir. Studies,
10:15-22, 1976.
146
-------
2422 SOIL CORE MICROCOSM
Biological Activity Detected: Monitor community processes.
Principle: The use of an intact system provides a more realistic
representation of a natural system. The test relies on the
production of C02 and nutrient loss as indicators of community
f itness.
Endpoints: Qual i tative: N/A. Quantitative: Rate of C02 production;
Loss of nutrients through leachate.
Strengths: Small size; Low cost/unit; Simple design.
Weaknesses: High degree of variability between units, due to natural
variabi I i ty.
Status of Development: Developmental.
Describe: To date, only inorganics have been examined in the
soil core. ERL-COR is currently evaluating the system for use
with a broad spectrum of toxics.
Appl ications: So i I .
Samples: Pure Chemicals: Heavy metals, Pesticides. Comp I ex
Industrial - waste effluents.
__
Duration: Test: 3 months; Analysis: I to 2 months.
Cost: $2,000 to $3,000.
Interpretation: This test reflects the ability of a community to
decompose material. It also reflects the impact of a chemical
on nutrient cycling.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: J. Gl le, U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4649).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OTS.
References: I) Draggon, S. Soil Core Microcosm. Proceedings of
EPA Workshop on Terrestrial Microcosms. J.W. G! I lett, ed.
Corvallis, OR, 1977. In press.
147
-------
2423 SOIL/LITTER MICROCOSM
Biological Activity Detected: Monitor decomposition process.
Principle: Carbon dioxide production and 02 consumption are monitored as
indicators of microbial respiration.
Endpoints: Qua Iitative: 02; CC^; Microbiota; MIcroarthropods; Nema-
todes. Quantitative: Change in respiratory rate with changing
chemical concentration.
Strengths: Low cost/unit; Simple analytical technique and equipment;
Overall simplicity of system.
Weaknesses: Limited type of data generated.
Status of Development: Developmental.
Describe: Soil is tested in I qt. mason jars with respiration
C02 and 02 measured by simple titrametric or gasometric techniques.
AppIications: Sol I.
Samples: Pure Chemicals: Heavy metals, Pesticides. CompI ex
Mixtures: Ambient; Industrial; Energy Related.
Duration: Test: I to 4 weeks; Analysis: Simultaneous with test.
Cost: $500/chemical.
Interpretation: Changes in respiration may reflect the ability of a
community to decompose organic matter.
Level of Complexity: 3.
OHEE Laboratory Involved: ERL-COR, Ecological Effects Research
Division, Terrestrial Ecology Branch.
Persons to Contact: B. Lighthart. U.S. EPA, ERL-COR, 200 SW 35th St.,
Corvallis, OR 97330, (FTS 420-4832).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE.
References: I) SoiI/Litter Microcosm. In: Proceedings of EPA
Workshop on Terrestrial Microcosms. J.W. Glllett, ed. U.S. EPA,
Corvallis, OR, 1977. In press.
148
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CONTENTS: TEST SYSTEMS
2500 Physica l-Chemica I/Bio-accumul ation 149
2501 Bioconcentration Studies, Marine and
Freshwater Animals 150
2502 Reverse-Phase High Pressure Liquid
Chromatography (HPLC) 151
Indices 153
149
-------
2501 BIOCONCENTRATION STUDIES, MARINE AND FRESHWATER ANIMALS
Biological Activity Detected: Bioconcentration.
Principle: To determine the uptake and depuration rates of pure
chemicals in tissues.
Endpoints: Qua Iitative: Flesh tainting. Quantitative: Uptake
rates; Depuration rates; Bioconcentration factors (uptake of
a pure chemical in tissues divided by the concentration of the
chemical in the exposure water).
Strengths: A good estimate of the uptake, depuration rates of a
pure chemical, plateau level, and bioconcentration factors.
Weaknesses: Duration at least I month; Requires complicated chemical
analyses; Does not always reflect a true picture of bioconcentration
in the field because of uptake from other sources (of food and
sed iment).
Status of Development: Being implemented.
Describe: Various laboratories within EPA and private industry
are now using this method. The procedure has not yet been
accepted by ASTM as it is still being revised.
Applications: Water.
Samples: Pure Chemicals; Insecticides, Herbicides, PCB's,
Chlorinated hydrocarbons, Radiolabeled material. Complex Mixtures:
N/A.
Duration: Test: 30 to 60 days; Analysis: 30 to 60 days.
Cost: $4,000.
Interpretation: From the above tests the potential of contaminants
reaching man's seafoods can be determined.
Level of Complexity: 2.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Chemical Pollutant
Section; ERL-GB, Experimental Environments Branch.
Persons to Contact: G.D. Veith. ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9534); S.C. Schimmel. ERL-GB, Sabine
Island, Gulf Breeze, FL 32561, (FTS 686-9011), .
Grant/Contract Laboratory Involved and Principal Investigators:
Bionomics, EG & G, Rt. 6, Box 1002, Pensacola, FL 32507,
P.R. Parrish.
Program Office Support: OHEE; OPP.
References: I) ASTM Committee on Bioconcentration of Test Materials
in Fishes and Oysters. (E-35 Committee on Pesticides). In prepara-
tion.
50
-------
2502 REVERSE-PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
Biological Activity Detected: Bioconcentration.
Principle: HPLC retention time correlates with the logarithm of the
partition coefficient which correlates with the bioconcentration
factor of organic chemicals in fish tissue.
Endpoints: Qua Iitative: N/A. Quantitative: Provides bioconcentra-
tion potential of organic chemicals in animal tissue.
Strengths: Rapid; Inexpensive.
Weaknesses: Requires a HPLC.
Status of Development: Being implemented.
Describe: Chemical analysis is utilizing HPLC to obtain Log P
values, which can be correlated with water solubility and bio-
concentration factors for organic chemicals.
Applicat ions: Water.
Samples: Pure Chemicals; Organic; Inorganic. Complex Mixtures;
IndustriaI.
Duration: Test: 10 to 20 min; Analysis: 10 to 20 min.
Cost: $100.
Interpretation: This work sets forth a rapid, inexpensive method for
screening chemicals for their bioconcentration potential in the
envi ronment.
Level of Complexity: 0.
OHEE Laboratory Involved: ERL-DUL, Research Branch, Physiological
Effects of Pollutants Section.
Persons to Contact: G.D. Veith. U.S. EPA, ERL-DUL, 6201 Congdon Blvd.,
Duluth, MN 55804, (FTS 783-9534, Commercial 218 727-6692 X534).
Grant/Contract Laboratory Involved and Principal Investigators: N/A.
Program Office Support: OHEE; ORD.
References: I) Veith, G.D., and Morris. Ecol. Rept. Series, U.S. EPA,
1978. In press. 2) Lee, Huges, and G.D. Veith. Water, Air, and
Soil Pollution, 8:749-484, 1977. 3) Veith, G.D., and N.M.
Austin. Detection and Isolation of Bioaccumulable Chemicals
in Complex Effluents. In: Identification and Analysis of
Organic Pollutants in Water. L.A. Keith, ed. Ann Arbor
Science Publishers, Ann Arbor, Ml, 1976. pp. 297-304.
-------
INDICES
153
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER
NUMBER^ NAME OF TEST SYSTEM PAGE
Mil Integrated System: Lung Organ Culture System,
Tissue Homogenates, Purified Enzyme Systems. .'.... 2
1112 Integrated System: Tissue Homogenates, Purified
Enzyme Systems 3
1113 Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues 4
1114 Trachea I Mucociliary Transport 6
1115 Platelet Secretion Measured by ATP Release 8
1116 Mammalian Platelet and Fibroblast Impairment 9
1117 Lymphocyte Cytotoxiclty 10
IMS Impairment of Neutrophil Phagocytosis II
1119 Human Lung Fibroblasts (WI38) 12
I I 110 Chinese Hamster Ovary (CHO) Clonal Toxicity 14
Mill Rabbit Alveolar Macrophage (RAM) 16
I 1112 Rat Hepatocyte (Liver Cell) 18
I II13 Chinese Hamster Ovary (CHO) Cytotoxicity and
Mutagenicity 19
V 1121 LD50 22
1122 Whole Animal LD50 - Ora I , Dermal 23
V
J 1131 Integrated System: General Clinical Pathology 26
I 132 Sleep-Time Study 27
||33 Metabolism of Chlorinated Hydrocarbons in
•Subhuman Primates 28
(continued)
154
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM PAGE
1134 Metabolic Profiles 29
1135 Model Substrate Metabolism 30
) 136 ' Xenobiotic Mechanisms 32
1137 Oxidant Production by Leukocytes and Alveolar
Macrophages Measured by Chemiiuminescence 33
1138 Cyclic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages 34
1141 Toxicity ot Aerosolized Pollutants/Acute and
Subacute 36
"*^| M2 Toxicity of Gaseous and Vapor Phase/Acute and Subacute. . 37
^"1143 Inhalation LC50 Tests 38
^-\\AA Deposition and Clearance of Radioactive Materials
Fo I lowing Inhalation Exposure 39
1145 Integrated System: Pulmonary Function and Pulmonary
Metabolism 40
I 146 Pulmonary Function in Rats 41
1147 Pulmonary Function of Unanesthetized Guinea Pigs 42
1148 Arterial Blood Gas Measurement in Conscious Rats 43
1149 Infectivity Model 44
^ 11410 In-Vivo Alveolar Macrophage Cytotoxicity 45
1151 Integrated System: NeurobehavioraI Toxicological
Assessment 48
1152 Integrated System: The Effects of Selected Organic
Contaminants in Drinking Water on the Functions
of the Reproductive, Nervous, and Immune Systems. ... 49
1153 Computer Automated Analysis of Patterned Behavior
in the Primate 51
(continued)
155
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM PAGE
_^______
1154 Computer Automated Analysis of Learning and Memory
in the Primate 52
1155 Integrated System: Behavioral Analysis of Rats —
Developmental, Locomotor, Exploratory, and Learned
Behavior 53
I 156 Integrated System: Instrumental Methods of Detecting
Functional and Metabolic Damage to the Nervous
System 54
1211 Salmonella typhimurium (Ames) 58
1212 Escherichia col i (WP2) 60
1213 Saccharomyces cerevisiae, Schfzosaccharomyces pombe,
Forward and Reverse Mutation 61
1214 Body Fluid Analysis 62
1215 Bacterial PI asm ids 63
1216 Mouse Lymphoma (L5I78Y) 64
1217 Chinese Hamster Ovary Cells (CHO)
Drug Resistance 65
1218 Chinese Hamster Cells (CHO)
Nutriti.onal Competency 66
1219 Chinese Hamster Lung Cells (V79) 67
12110 Drosophila melanogaster, Sex Linked Recessive Lethal ... 68
121 I I Tradescantia Stamen Ha i r 69
121 12 Maize Waxy Locus Assay 70
121 13 In-Vivo Cytogenetics in Mice 71
1221 Baci I I us subti I is rec~ 74
1222 Escherichia coli, Pol A~ 75
1223 Mitotic Recombination and Gene Conversion in
Saccaromyces cerevisiae. 76
(continued)
156
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM PAGE
1224 Unscheduled DMA Synthesis (UDS) ' 77
1225 Sister-Chromatid Exchange Formation (SCE) 78
£-•"1226 In-Vivo Assessment of DNA Damage 79
1227 Intact Rodent Hepatocytes in Primary Culture 80
1228 In-Vivo UNA Binding 81
1229 Chinese Hamster Cells (CHO)
Unscheduled DNA Synthesis (UDS) ... 82
1231 C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic Trans-
formation with Exogenous Metabolic Activation 84
1232 C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic Trans-
formation 85
1233 BHK-21 Mammalian Cell Oncogenic Transformation 86
1234 Syrian Hamster Embryo Oncogenic Transformation
(Focus Assay) 87
1235 BALB 3T3 Oncogenic Transformation and Mutagenesis
with Exogenous Metabolic Activation 88
1236 BALB 3T3 Oncogenic Transformation 89
"' 1237 Pulmonary Adenoma Bioassay in Mice 90
1241 Tumor Induction in Massive Crustaceans, Molluscs,
and Tel iost Fish 92
1242 MFO Induction as an Indicator of Toxicity Exposure. ... 93
1243 Limb Regeneration System 94
1244 Isogenic Fish 95
1245 Integrated System: Development of Mutagen/Carcinogen
Activation, Concentration, Separation, and
Weathering Systems 96
1246 Biphenyl Hydroxylase 97
(continued)
157
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
NAME OF TEST SYSTEM
1301 Mammalian Teratology ................... 100
1302 Perinatal Toxicology ................... 101
1303 Fetal Toxic ity in Rats, Mice, Guinea Pigs/
Hamsters ........................ 102
1304 Developmental Toxicity in Neonatal Rats ......... 103
1305 Teratology In-Vi vo/ln-Vitro ............... 104
1306 Direct Spectral Measurement of the Biochemical Develop-
ment of the Nervous System ............... 105
2101 Freshwater Algal Assay Bottle Test ............ 108
2102 Marine Algal Assay Bottle Test .............. 109
2211 Acute Fish Toxicity Test, Static and Flow-Through,
All Freshwater Species ................. 112
2212 Subchronic Embryo-Larval, Early Juvenile Fish Toxicity
Test .......................... 113
2213 Chronic Fish Toxicity Test, American Flagfish
(Jordanella floridae) ................. 114
2214 Fish Respiratory Activity Toxicity Test, Electrode
Chamber Method ..................... 115
2215 Fish Avoidance Test, Gradient Tanks ........... 116
222| Acute Invertebrate Toxicity Test, Static and Flow-
Through, A I I Freshwater Species ............ I 18
2222 Subchronic Invertebrate Toxicity Test, Stream Insects . . I 19
2223 Subchronic Invertebrate Toxicity Test, Chironomid
(Tanytarsus d i ss i m i I [s ) ................ 120
2224 Chronic Invertebrate Toxicity Test, Water Flea
(Daphnia^ maqna) .................... 121
2231 Acute Plant Toxicity Test, Duckweed ( Lemma_ mi nor) .... 124
2241 Laboratory Ecosystem ................... 126
(cont i nued)
158
-------
INDEX I: TEST SYSTEMS IN NUMERICAL ORDER (continued)
,MDrEM NAME OF TEST SYSTEM PAGE
NUMBER ^
2311 Acute Fish Toxicity Test, Static and Flow-Through,
Al I Marine Species 128
2312 Subchronic Embryo-Larval Fish Toxicity Test, Sheepshead
Minnow (Cyprinodon variegatus) 129
2313 Chronic Fish Toxicity Test, Sheepshead Minnow
(Cyprinodon varieqatus) 130
2321 Acute Invertebrate Toxicity Test, Static and Flow-
Through, All Marine Species 132
2322 Acute Toxicity Test, Benthic Assemblages 133
2323 Chronic Invertebrate Toxicity Test, Estuarine Shrimp
(Pa laemonetes pugio) 134
2324 Chronic Invertebrate Toxicity Test, Estuarine Mysid
(Mysidopsis bahia) 135
2331 Estuarine Microcosmy I 138
2332 Estuarine Microcosmy II 139
2333 Estuarine Communities 140
2411 Stress Ethylene Bioassay in Plants 142
2412 Measurement of Nitrogenase Activity by Acetylene
Reduction in Nodulated Plants 143
2421 Terrestrial Microcosm Chamber 146
2422 Soil Core Microcosm 147
2423 Soil/Litter Microcosm 148
2501 Bioconcentration Studies, Marine and Freshwater Animals . 150
2502 Reverse-Phase High Pressure Liquid Chromatography
(HPLC) 151
159
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER*
NAME OF TEST SYSTEM NUMBER™ PAGE
Acute Fish Toxicity Test, Static and Flow-Through,
Al I Freshwater Species 22 I I .... 112
Acute Fish Toxicity Test, Static and Flow-Through,
All Marine Species 2311 .... 128
Acute Invertebrate Toxicity Test, Static and Flow-
Through, All Freshwater Species 2221 .... 118
Acute Invertebrate Toxicity Test, Static and Flow-
Through, Al I Marine Species 2321 .... 132
Acute Plant Toxicity Test, Duckweed (Lemma minor) . . . 2231 .... 124
Acute Toxicity Test, Benthic Assemblages 2322 .... 133
Algal Assay Bottle Test, Freshwatert 2101 .... 108
Algal Assay Bottle Test, Marinet 2102 .... 109
Alveolar Macrophage Cytotoxfcity, In-Vivot 11410 .... 45
(Ames) or Salmonella typhimuriumt 1211 .... 58
Arterial Blood Gas Measurement in Conscious Rats. ... 1148 .... 43
Baci I I us subti I is rec" 1221 .... 74
Bacterial Plasmids 1215 .... 63
BALB 3T3 Oncogen ic Transformation 1236 .... 89
BALB 3T3 Oncogenic Transformation and Mutagenesis
with Exogenous Metabolic Activation 1235 .... 88
(continued)
^Integrated Systems are listed twice: once under "Integrated Systems" and once
under the title proper.
fThis test is listed under two titles.
160
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Behavioral Analysis of Rats —Developmental, Loco-
motor, Exploratory, and Learned Behavior:
Integrated System I 155 .... 53
BHK-21 Mammalian Gel! Oncogen ic Transformation 1233 .... 86
Bioconcentra'. < >r\ Studies, Marine and Freshwater
Animals 2501 .... 150
Biphenyl Hydroxylase 1246 .... 97
Body Fluid Analysts 1214 .... 62
C3HIOTI/2CL8 louse Embryo Fibroblast Oncogenic
Transforms r ion 1232 .... 85
C3HIOTI/2CL8 Mouse Embryo Fibroblast Oncogenic
Transformation with Exogenous Metabolic
Activation 1231 . .j . . 84
Chinese Hamster Cells (CHO)
Nutritional Competency 1218 . .1 . . 66
\
Chinese Hamster Cells (CHO)
Unscheduled DNA Synthesis CUDS) 1229 .... 82
Chinese Hamster Lung Cells (V79) 1219 .... 67
Chinese Hamster Ovary Cells (CHO)
Clonal Toxicity Assay MHO .... 14
Chinese Hamster Ovary Cells (CHO)
Cytotoxicity and Mutagenicity Assay IIM3 .... 19
Chinese Hamster Ovary Cells (CHO)
Drug Resistance 1217 .... 65
Chronic Fish Toxicity Test, American Flagfish
(Jordanel la floridae) 2213 .... ||4
Chronic Fish Toxicity Test, Sheepshead Minnow
(Cyprinodon variegatus) 2313 .... 130
Chronic Invertebrate Toxicity Test, Estuarine Mysid
(Mysidopsis bahia) 2324 .... 135
(continued)
161
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Chronic Invertebrate Toxicity Test, Estuarine Shrimp
(Pa laemonetes pugio) 2323 .... 134
Chronic Invertebrate Toxicity Test, Water Flea
(Daphnia magna) 2224 .... 121
Computer Automated Analysis of Learning and Memory
in the Primate I I 54 .... 52
Computer Automated Analysis of Patterned Behavior
in the Primate I 153 .... 5)
Cylic Nucleotide Concentrations in Leukocytes
and Alveolar Macrophages 1138 .... 34
Cytogenetics in Mice, In-Vivot 121 13 .... 7|
Deposition and Clearance of Radioactive Materials
Following Inhalation Exposure 1144 .... 39
Development of Mutagen/Carcinogen Activation,
Concentration, Separation, and Weathering
Systems: Integrated System 1245 .... 95
Developmental Toxicity in Neonatal Rats 1304 .... 103
Direct Spectral Measurement of the Biochemical
Development of the Nervous System 1306 .... 105
DNA Binding, In-Vivot 1228 .... 81
DMA Damage, In-Vivo Assessmentt 1226 .... 79
Drosophila melanogaster. Sex Linked Recessive Lethal. . 12110 .... 68
Effects of Selected Organic Contaminants in Drinking
Water on the Functions of the Reproductive, Ner-
vous, and Immune Systems: Integrated System 1152 .... 49
Escherfchia col i (WP2) 1212 .... 60
Escher ich ia col f, Pol A 1222 .... 75
Estuarine Communities 2333 .... 140
Estuarine Microcosmy 1 2331 .... 133
(continued)
162
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™PAGE
Estuarine Microcosmy II 2332 .... 139
Fetal Toxicity in Rats, Mice, Guinea Pigs/Hamsters. . . 1303 .... 102
Fish Avoidance Test, Gradient Tanks 2215 .... |16
Fish Respiratory Activity Toxicity Test, Electrode
Chamber Method 2214 .... 115
Freshwater Algal Assay Bottle Testt 2101 .... 108
General Clinical Pathology: Integrated System 1131 .... 26
Human Lung Fibrob lasts (WI38) I 119 .... |2
Impairment of NeutrophiI Phagocytosis . 1118 .... n
Infectivity Model 1149 .... 44
Inhalation LC50 Tests 1143 .... 38
Instrumental Methods of Detecting Functional and
Metabolic Damage to the Nervous System:
Integrated System 1156 .... 54
Instrumental Methods of Detecting Functional and
Metabolic Damage to Target Tissues. . 1113 .... 4
Intact Rodent Hepatocytes on Primary Culture 1227 .... 80
Integrated System: Behavioral Analysis of Rats —
Developmental, Locomotor, Exploratory, and
Learned Behavior 1155 .... 53
Integrated System: Development of Mutagen/Carcinogen
Activation, Concentration, Separation and Weathering
Systems 1245 .... 96
Integrated System: The Effects of Selected Organic
Contaminants in Drinking Water on the Functions of the
Reproductive, Nervous, and Immune Systems 1152 .... 49
Integrated System: General Clinical Pathology .... 1131 .... 26
(cont i nued)
163
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM
FEST SYSTEM
NUMBER
PAGE
Integrated System: Instrumental Methods of Detecting
Functional and Metabolic Damage to the Nervous
System I 156 .... 54
Integrated System: Lung Organ Culture System, Tissue
Homogenates, Purified Enzyme Systems MM .... 2
Integrated System: NeurobehavioraI Toxicological
Assessment 1151 .... 48
Integrated System: Pulmonary Function and Pulmonary
Metabolism 1145 .... 40
Integrated System: Tissue Homogenates, Purified
Enzyme Systems 1112 .... 3
Isogenic Fish 1244 .... 95
In-Vivo Alveolar Macrophage Cytotoxicityt I 1410 .... 45
In-Vivo Assessment of DNA Damaget 1226 .... 79
In-Vivo Cytogenetics in Micet . ..... 12113 .... j\
In-Vivo DNA Bindingt 1228 .... si
Laboratory Ecosystem 2241 .... 125
LD50 1121 .... 22
Limb Regeneration System 1243 .... 94
Lung Organ Culture System, Tissue Homogenates,
Purified Enzyme Systems: Integrated System MM .... 2
Lymphocyte Cytotoxicity III? .... |0
Maize Waxy Locus Assay 121 12 .... 70
Mammalian Platelet and Fibroblast Impairment 1116 .... 9
Mammalian Teratology 1301 .... JQQ
Marine Algal Assay Bottle Testt 2102 .... 109
(conti nued)
164
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™ PAGE
Measurement of Nitrogenase Activity by Acetylene
Reduction in Nodulated Plants 2412 .... 143
Metabolic Profiles 1134 .... 29
Metabolism of Chlorinated Hydrocarbons in Subhuman
Primates 1133 .... 28
MFC Induction as an Indicator of Toxicity Exposure. . . 1242 .... 93
Mitotic Recombination and Gene Conversion in
Saccaromyces cerevisiae 1223 .... 76
Model Substrata Metabolism 1135 .... 30
Mouse Lymphoma (L5I78Y) 1216 .... 64
Neurobehavioral ToxicologicaI Assessment:
Integrated System 1151 .... 48
Oxidant Production by Leukocytes and Alveolar Macro-
phages Measured by Chemi luminescence 1137 .... 33
Perinatal Toxicology 1302 .... 101
Platelet Secretion Measured by ATP Release 1115 .... 8
Pulmonary Adenoma Bioassay in Mice 1237 .... 90
Pulmonary Function and Pulmonary Metabolism:
Integrated System 1145 .... 40
Pulmonary Function in Rats 1146 .... 41
Rabbit Alveolar Macrophage (RAM) INN .... |6
Rat Hepatocyte (Liver eel I) II I 12 .... \Q
Pulmonary Function of Unanesthetized Guinea Pigs. . . . 1147 .... 42
Reverse-Phase High Pressure Liquid Chromatography
(HPLC) 2502 .... |5I
Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Forward and Reverse Mutation 1213 .... 61
(continued)
165
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
NAME OF TEST SYSTEM ^NUMBER™
Salmonella typhimurium (Ames)t 1211 .... 53
Sister-Chromatid Exchange Formation (SCE) 1225 .... 78
Sleep-Time Study I 132 .... 27
Soi I Core Microcosm 2422 .... 147
Soil/Litter Microcosm 2423 .... 143
Stress Ethylene Bioassay in Plants 2411 .... 142
Subchronic Embryo-Larval, Early Juvenile Fish
Toxicity Test 2212 .... 113
Subchronic Embryo-Larval Fish Toxicity Test,
Sheepshead Minnow (Cyprinodon variegatus) 2312 .... 129
Subchronic Invertebrate Toxicity Test, Chironomid
(Tanytarsus d issimiI I is) 2223 .... 120
Subchronic Invertebrate Toxicity Test, Stream Insects . 2222 .... 119
Syrian Hamster Embryo Oncogenic Transformation
(Focus Assay) 1234 .... 87
Teratology In-Vivo/ In -Vitro 1305 .... |Q4
Terrestrial Microcosm Chamber 2421 .... 145
Tissue Homogenates, Purified Enzyme Systems:
Integrated System . 1112 .... 3
Toxicity of Aerosolized Pollutants/Acute and Subacute . 1141 .... 35
Toxicity of Gaseous and Vapor Phase/Acute and Subacute. 1142 .... 37
Tracheal Mucociliary Transport 1114 .... 5
Tradescantia Stamen Hair 121 I I .... 59
Tumor Induction in Massive Crustaceans, Molluscs, and
Tel lost Fish 1241 .... 92
Unscheduled DNA Synthesis (DOS) 1224 .... 77
(continued)
166
-------
INDEX II: TEST SYSTEMS IN ALPHABETICAL ORDER (continued)
~ NAME OF TEST SYSTEM ^NUMBER™ PAGE
Whole Animal LD50 -Oral and Dermal 1122 .... 23
Xenobiotic Mechanisms 1136 .... 32
167
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING
ORGANISM - SPECIES
IN-VITRO
TEST TYPE
IN-VfVO
"OTHER"
BACTERIA
BaciI I us subtiI is
Escherichia col i
SaImonelI a typhimurium
PLANTS
AI gae
Duckweed
Ma ize
Plants - genera
Tradescantia
Yeast
INSECTS
Fruit Fl ies
Stream Insects
FISH
American Flagfish
Freshwater - general
1221
1212
1222
1211
1214
1215
2101
2102
1215
1214
1223
2502
(cont i nued)
TEST SYSTEM NUMBER*
2231
121 12
241 I
2412
1213
1214
1223
12110
2222
2213
2211
2212
2214
2215
2501
*An underlined number indicates that the test system is being applied to more
than one organism/species/test type.
168
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
ORGANISM - SPECIES
TEST TYPE
IN-VITRO
IN-VIVO
OTHER
FISH (continued)
Marine - general
Sheepshead Minnow
Teliost
INVERTEBRATES
Chi ronomi d
Crustaceans
Estuarine Mysid
Estuarine Shrimp.
Freshwater - general
Marine - general
Mo I I use
Water Flea
MAMMALS
Chinese Hamster
Guinea Pig
Hamster
Man
Mouse
2502
TEST SYSTEM NUMBER
1242
2311
2501
UNO
11113
1217
1218
1219
1225
1229
1233
I I 19
1224
1216
1224
1225
1227
(continued)
169
2312
2313
1241
2223
1241
2324
2323
2221
2501
2321
2501
1241
2224
I 145
1147
1303
1303
121113
1226
1237
1301
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
nrcpANT^M CPEPTFC ""^ TYPE
UKbANibM uitLit., IN-VITRO IN-VIVO OTHER
TEST SYSTEM NUMBER
MAMMALS (continued)
Mouse 1228 1302
123 1303
1232 1305
1235
1236
Rabbit
Rat 1
12
13
14
15
16
17
18
112
Subhuman Primates
II 1 1
137
138
1410
121
122
131
132
134
135
136
141
142
143
144
145
146
148
149
151
152
153
155
156
303
304
306
133
153
154
Syrian Hamster
OTHER
Benthic Assemblage
Estuarine Community
(cont i nued'.
170
2322
2333
-------
INDEX III: EXPERIMENTAL SUBJECT LISTING (continued)
"ORGANISM-SPECIES " IN.VITRQ —^TYPE
TEST SYSTEM NUMBER
OTHER (continued)
Estuarine Microcosmy 2331
2333
Laboratory Ecosystem 2241
Soil Core Microcosm 2422
SoiI/Litter Microcosm 2423
Terrestrial Microcosm Chamber 2421
171
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER
CONTACT PERSONNEL U,
Anderson, R. L.
Bercz, J.P.
Biesinger, K.E.
Bourquin, A.W.
Brown, M.M.
Brungs, W.A.
Bui 1, R.J.
Campbel 1 , K. 1 .
Carlson, R.
Casciano, 0.
Chadwick, R.W.
Charles, J.
Chernoff, N.
Clarke, N.
Claxton, L.
Copeland, M.F.
Couch, J.
Courtney, K.D.
Daniel , B.
Drummond, R.
Gardner, D.E.
.S. EPA LABORATORY
ERL-DUL
HERL-CIN
ERL-DUL
ERL-GB
HERL-RTP
ERL-DUL
HERL-CIN
HERL-CIN
ERL-DUL
NCTR
HERL-RTP
HERL-RTP
HERL-RTP
HERL-CIN
HERL-RTP
HERL-RTP
ERL-GB
HERL-RTP
HERL-CIN
ERL-DUL
HERL-RTP
(continued)
TEST SYSTEM NUMBER*
2222,
1 131,
1236.
2221.
2331,
1216.
2212,
1 1 13,
1 132,
1156,
1306.
1152.
2214.
121 1,
1135.
1141,
1303,
1215.
1211,
12113,
1 135.
1241.
1301,
1215,
2214.
1 132,
1 144,
2223.
1211, 1213,
2332.
2213.
1121, 1131,
1136, 1155.
1226, 1236,
1217, 1227.
1142.
1304.
1212, 1214,
1221, 1222.
1302, 1305.
1228.
1136, 1143,
1149, M4IO.
*An underlined number indicates that the test system is used by more than one
of the above indicators.
172
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER (continued)
CONTACT PERSONNEL
Garnas, R.L.
Genti le, J .
Gi le, J.D.
Gi 1 left, J.W.
Graham, J .A.
Green, J .C.
Hansen, D.J .
Hatch, G.
Hedtke, S.F.
Huisingh, J.L.
Jackim, E.
Kavlock, R.
Kowal , N.E.
Lazear, E.
Lee, S.D.
Lighthart, B.
Linder, R.
Lingg, R.D.
Malcolm, A.R.
McCabe, L.J.
McKim, J.M.
Mi 1 ler, W.E.
Moore, W.
Nesnow, S.
N i mmo , D . W .
O'Nei 1, J.J.
Orthoefer, J.
Pahren, H.
U.S. EPA LABORATORY
ERL-GB
ERL-NAR
ERL-COR
ERL-COR
HERL-RTP
ERL-COR
ERL-GB
HERL-RTP
ERL-DUL, NFTS
HERL-RTP
ERL-NAR
HERL-RTP
HERL-CIN
NCTR
HERL-CIN
ERL-COR
HERL-RTP
HERL-CIN
ERL-NAR
HERL-CIN
ERL-DUL
ERL-COR
HERL-CIN
HERL-RTP
ERL-GB
HERL-RTP
HERL-CIN
HERL-CIN
TEST SYSTEM NUMBER*
2331,
2102.
2421,
2421.
II 14,
2101.
2312,
1 1 15,
2241.
1 1 19,
11112,
1212,
1229.
1303,
1119,
1211.
Ill),
2423.
1122.
1133,
1218,
121 1,
2212.
2101.
1143,
1134,
1234,
2324.
1145.
1237.
1116,
1213,
2332.
2422.
1 1 17.
2313.
1137, 1138.
II 1 10, Mill,
11113, 1211,
1221.
1304.
1219, 1236.
1112.
1 1 34.
1225, 1229.
I2III.
1 144.
1231, 1232,
1235.
1118, 1211,
1233.
(continued)
173
-------
INDEX IV: CONTACT PERSONNEL IN ALPHABETICAL ORDER (continued)
CONTACT PERSONNEL U.S. EPA LABORATORY
Pepelko, W.E.
Pesch, G.G.
Pritchard, P.M.
Reiter, L.
Richards, N.
Robinson, E.
Sandhu, S.S.
Schimmel, S.C.
Schoor , P.
Shiroyama, T.
Specht, D.
Spehar, R.
Spoor, W.A.
Stara, J.F.
Stephan, C.
Swartz, R.
Tagatz, M.
Tingey, D.T.
Tyler-Schroeder, D.B.
Veith, G.D.
Walsch, G.
Waters, M.D.
Wickl iff, C.
Wiester, M.J.
HERL-CIN
ERL-NAR
ERL-GB
HERL-RTP
ERL-GB
ERL-DUL, NFTS
HERL-RTP
ERL-GB
ERL-GB
ERL-COR
ERL-COR
ERL-DUL
ERL-GB
HERL-CIN
ERL-DUL
ERL-COR
ERL-GB
ERL-COR
ERL-GB
ERL-DUL
ERL-GB
HERL-RTP
ERL-COR
HERL-CIN
TEST SYSTEM NUMBER*
1 146,
1225.
2331,
1151,
121 1 ,
1244,
2101 .
1212,
1216,
121 10,
12113,
1225.
2501.
1242.
2101.
2102.
2213.
2215.
\2\±,
2211,
2311,
2322..
2333.
2411.
2323.
250j_,
2102.
II 19,
1212,
1216,
121 10,
121 13,
1223,
_I234.
2412.
1 147.
1 148.
2332.
1153, 1154.
1242, 1243,
1245, 1246.
1213. 1214,
1217, 1219,
I2III, 121 12,
1221, 1223.
1214. I2M3.
2221, 2224,
2321.
2502.
Mill . 1211,
1213, T21T,
1217, 1219,
I2III, 12112,
1221, T2227
1224, T225",
174
-------
INDEX V: U.S. EPA LABORATORIES IN ALPHABETICAL ORDER
U.S. EPA LABORATORY TEST SYSTEM NUMBER*
Env
ironmental Research Laboratory- 2101, 2
102,
Corvallis, Oregon 2421, 2422,
Env
ironmental Research Laboratory- 2101, 2211,
Duluth, Minnesota 2215, 2221,
2231, 2241,
Env
2502.
ironmental Research Laboratory- 1211, 1
Gulf Breeze, Florida 1245, 1
241,
246,
2323, 2324,
Env
Hea
Hea
2501.
ironmental Research Laboratory- 1218, 1
Narrangansett, Rhode Island
Ith Effects Research Laboratory-
Cincinnati , Ohio
_
1th Effects Research Laboratory-
Research Triangle Park,
North Carol ina
HI,
119,
134,
147,
211,
2111,
236,
!I4,
till,
134,
141,
149,
211,
217,
2113,
1225
1235,
1304.
National Center for Toxicotogica 1 1211,
Research-Jefferson, Arkansas
225,
M2,
121,
136,
148,
213,
21 13,
238,
H5,
1112,
135,
142,
1410,
212,
1219,
1221,
1227,
1301,
1217,
2322,
2423.
2212,
2222,
2311,
1242,
2102,
2331,
1229,
II 13,
1131,
1143,
1152,
1214,
1226,
1306.
117,
1113,
136
143,
151,
1213,
12110,
1222,
1231,
1302,
227.
241 1,
2213,
2223,
2321,
1243,
2312,
2332,
2102.
1 16,
132,
144,
155,
1215,
1228,
119,
122,
137,
144,
153,
1214,
I2III,
1223,
1232,
303,
2412,
2214,
2224,
2501,
1244,
2313,
2333,
1 1 18,
1 133,
1146,
1156,
1219,
1233,
1 1 1 10,
1 132,
1 138,
1 145,
1 154,
1216,
12112,
1224,
1234,
1304,
*An underlined number indicates that the test system is used by more than one
laboratory.
175
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
ENVIRONMENTAL RESEARCH LABORATORY-CORVALLIS
200 SW 35th Street
Corval I is, Oregon 97330
Assessment Criteria Division
Special Studies Branch J.C. Green
W.E. Mi Iler
T. Shi royama
Ecological Effects Research Division
Marine and Freshwater Branch D. Specht
Newport Field Station R. Swartz
Terrestrial Ecology Branch S. Draggon
J.D. Gile
J.W. GiIlett
B. Lighthart
D.T. Tingey
C. Wickliff
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
6201 Congdon Boulevard
Duluth, Minnesota 55803
Extramural Program Branch K.E. Biesinger
Newtown Fish Toxicology Station S.F. Hedtke
E. Robinson
Research Branch
Chemical Pollutant Section G.D. Veith
Physical Pollutant Section R. L. Anderson
Physical Effects of Toxicants Section W.A. Brungs
R. Carlson
R. Drummond
J.M. McKim
R. Spehar
(continued)
176
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH '
(continued)
Technical Assistance Branch C. Stephan
ENVIRONMENTAL RESEARCH LABORATORY-GULF BREEZE
Sabine Island
Gulf Breeze, Florida 32561
Experimental Environments Branch D.J. Hansen
D.W. Nimmo
S.C. Schimmel
M. Tagatz
D.B. Tyler-Schroeder
C. Walsch
Office of the Director J. Couch
(Carcinogenic Research Team) N. Richards
VI.A. Spoor
Processes and Effects Branch A.W. Bourquin
R.L. Garnas
P.M. Pritchard
ENVIRONMENTAL RESEARCH LABORATORY-NARRANGANSETT
South Ferry Road
Narrangansett, Rhode Island 02882
Toxicology Branch
Genetic Toxicology Team E. Jackim
A. R. Ma I co I m
G.G. Pesch
Marine Toxicology Team J. Gentile
HEALTH EFFECTS RESEARCH LABORATORY-CINCINNATI
26 West St. Clair Street
Cincinnati, Ohio 45268
Field Studies Division
Toxicological Assessment Branch J.P. Bercz
N.E. Kowal
L.J. McCabe
H. Pahren
J.F. Stara
(continued)
177
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
HEALTH EFFECTS RESEARCH LABORATORY-CINCINNATI
(continued)
Laboratory Studies Division
Exposure Evaluation Branch R.D. Lingg
Functional Pathology Branch S.D. Lee
J. Orthoefer
W. Moo re
W.E. Pepelko
M.J. Wiester
Toxicological Assessment Branch R.J. Bull
K.I. CampbelI
N. Clarke
B. Daniel
HEALTH EFFECTS RESEARCH LABORATORY-RESEARCH TRIANGLE PARK
Research Triangle Park
North Carolina 2771 I
Clinical Studies Division
Biomedical Research Branch D.E. Gardner
J.A. Graham
G. Hatch
J.J. O'NeiI
Experimental Biology Division
Developmental Biology Branch N. Chernoff
R. Kavlock
Neurobiology Branch L. Reiter
Environmental Toxicology Division
Biochemistry Branch M.M. Brown
R.W. Chadwick
L. Claxton
M.F. Cope land
J.L. Huisingh
S. Nesnow
S.S. Sandhu
M.D. Waters
(continued)
178
-------
INDEX VI: U.S. EPA DEPARTMENTAL LISTING IN ALPHABETICAL ORDER (continued)
DEPARTMENT U.S. EPA LABORATORY CONTACT PERSONNEL
HEALTH EFFECTS RESEARCH LABORATORY-RESEARCH TRIANGLE PARK
(continued)
Toxic Effects Branch J. Charles
K.D. Courtney
R. Linden
NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH
Jefferson, Arkansas 72079
Division of Mutagenesis Research
Somatic Cell Section D.A. Casciano
E. Lazear
179
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
American Health Foundation
Nay lor Dana Institute for
Disease Prevention
Hammond House Road
Valhalla, New York 10595
Ball State University
Muncie, Indiana 47306
Bionomics EG & G
Route 6, Box 1002
Pensacola, Florida 32507
Bionomics, Inc.
Wareham, Massachusetts
Brookhaven National Labo-
ratories
Long Island, New York
California, University of
Davis, California 95616
California, University of
School of Med icine
Los Angeles, California 90032
California, University of
Medical Center
San Francisco, California 94132
Cincinnati, University of
Cincinnati, Ohio 45221
Cincinnati University of
Medical Center
Cincinnati, Ohio 43221
G.M. W!I Iiams
1227,
D. Adalis
P.R. Parrish
K. S. Macek
S. Sauter
L. Shirer
E. Goldstein
M.G. Mustafa
R.S. Bhatnagar
C. Smith
J.C. Loper
D. Lang
(conti nued)
I I 14.
2501, 2312, 2313,
2324.
2211, 2212, 2221,
2311, 2321.
2211, 2212, 2221
2311, 2321.
121 I I.
1410.
I I 12.
I 133.
1211, 1236.
1236.
180
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Colorado, University of
Medical Center
4200 East 9th Avenue,
Denver Colorado 80262
Denver Research Institute
Denver, Colorado 80210
Florida State University
Tallahassee, Florida 32306
Georgia State University
Atlanta, Georgia 30303
Gulf Coast Research Laboratory
P.O. Box Drawer AG
Ocean Springs, Mississippi 39564
Gulf South Research Institute
P.O. Box 26518
New Orleans, Louisiana
I IT Research Institute
10 West 35th Street
Chicago, Illinois 60616
11 Iinois, University of
Urbana, 11linois 61801
Iowa State University
Ames, Iowa 50010
Litton Biometics, Inc.
Nicholson Lane
Kensington, Maryland
Louisiana State University
Medical School
New Orleans, Louisiana 70112
Medical College of Virginia
Richmond, Virginia 23298
C.C. Solomons
W.L. Weston
J. Schmidt-Coder is
B. Glasson
D.G. Ahearn
W.W. Walker
N. Gruener
E. Kline
C. Aranyi
R. Ehrlich
L. Schiff
M. Plewa
W.E. Lloyde
D.T. Brusick
W. Pel on
R.L. Balster
J.F. Borzelleca
W.L. Dewey
A.E. Munson
(continued)
I I 16.
I I 18.
1246.
2333.
2332.
2332.
1119, 1219, 1236.
1245.
HIM, 11410.
I 149.
! I 14.
12112.
1153, 1154.
1211.
121 I.
1152.
1152.
I 152.
1 152.
181
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Michigan, University of
Ann Anbor, Michigan 48104
Microbiological Associates
Bethesda, Maryland 20014
Missouri, University of
Columbia, Missouri 65201
North Carolina, University of
Chapel Hill, North Carolina 27514 D. Humm
A. Beaudoin
E. Goodman
R. Kouri
L. Schectman
C. Marianseld
J.T. O'Conner
A. Col Iier
Northrop Services, Inc.
P.O. Box 12313
Research Triangle Park,
North Carolina 27709
Oak Ridge National Laboratory
P.O. Box Y
Oak Ridge, Tennessee 37830
Ohio State University
Columbus, Ohio 43210
Oregon, University of
Eugene, Oregon 97403
Rockefeller University
1230 York Avenue
New York, New York 10021
Southern Mississippi,
University of
Hattiesburg, Mississippi 39401
Southwest Research Institute
San Antonio, Texas 78284
Stanford Research Institute
Menlo Park, California 94025
B. Adkins
N.E. Garrett
A. Hsie
R.W. Hart
M. Mix
M. Bowers
B.J. Martin
E. Cause
A. MitchelI
G. Newel I
V.F. Simmons
(continued)
1305.
2421.
1231, 1235, 1237.
1231, 1235, 1237.
121 I.
121 II.
I I 14.
1244.
1114, ||49.
1119, IIIIO, Mill,
III 13.
1217.
1226.
1241.
1117.
1241.
11410.
1216, 1219, 12110,
1224.
I2IM, 12113.
1211, 1212, 1213,
1214, 1221, 1222,
1223, 1225.
182
-------
INDEX VII: GRANT/CONTRACT LABORATORIES IN ALPHABETICAL ORDER (continued)
GRANT/CONTRACT LABORATORY
PRINCIPAL INVESTIGATOR
TEST SYSTEM NUMBER
Syracuse Research Corporation
Merri I I Lane
Syracuse, New York 13210
University of Texas
Medical Branch
Galveston, Texas 77550
University of West Florida
Pensacola, Florida 32504
Wisconsin, University of
Madison, Wisconsin 53706
J. Saxena
M. Legator
J. Bazl is
R. Rao
P. Lichtenstein
1233.
1211, 1214, 12113,
121 I.
1242, 1243, 1245.
2421.
183
-------
INDEX VIII: STATUS OF DEVELOPMENT DISTRIBUTION OF THE TEST SYSTEMS
STATUS OF DEVELOPMENT
Deve lopmenta 1 :
Being implemented:
Val i dated:
1 t 15,
1215,
1233,
2222,
2421,
1 1 II,
1 137,
1152,
1229,
1307,
2412,
1 1 12,
1143,
1212,
1302,
2311,
1 1 16,
1218,
1234,
2223,
2322,
II 19,
I 138,
1155,
1232,
2214,
2501 ,
1 M3,
I 144,
1213,
1303,
2321.
TEST
1117,
1211 1,
1242,
2231 ,
2423.
MHO,
1141,
1214,
1234,
2224,
2502.
II 14,
1 146,
12110,
2101,
SYSTEM
1 1 18,
121 12,
1244,
2241,
HIM,
1142,
1216,
1236,
2312,
1121,
1 148,
121 13,
2102,
NUMBER
IIII2,
1226,
1245,
2322,
III 12,
1145,
1217,
1237,
2313,
1 131,
1 149,
1221,
2211,
1 1113,
1227,
1246,
2323,
1122,
1 147,
1219,
1241,
2324,
1132,
141 10,
1222,
2212,
1153,
1228,
1304,
2331,
1134,
11410,
1223,
1243,
2332,
1 133,
M56,
1224,
2213,
1154,
1231,
2215,
2333,
1135,
1151,
1225,
1305,
2411,
1136,
1211,
1301,
2221,
84
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
ENDPOIIMTS TEST SYSTEM NUMBER
Adrenal function 1131.
Airway resistance 1145, 1147.
Alveolar macrophage - bacterial activity 1137.
Alveolar macron .age - cytotoxicity I 1410.
Alveolar macrophage - enzymatic profile I 1410.
Alveolar macrophage - morphology I 1410.
Arterial blood Pnn I 148.
t,oz
Arterial blood Pn I 148.
U2
ATP, ADP, AMP tissue levels 1113, 1116, illl I, I I I 12,
1156, 1306.
ATP release from platelets 1115.
Bicarbonate concentration in the blood 1148.
Biochemical development of the nervous 1156, 1306.
system
Blood pH I 148.
Brain neurochemistry 1152, 1156.
Breathing frequency 1145, 1147.
cAMP 1138.
Cardio-vascular response 1147.
Cel I number 1119, Mill.
cGMP 1138.
(conti nued)
185
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
Kontinued)
ENDPOINTS
TEST SYSTEM NUMBER
Ci Iiary beating
Clin icaI symptoms
Colony formation
Comparative metabolism
Creative phosphokinase
Cyto logicaI
Cytochrome P-450
CMS Function
Deposit ion
Dose-response curve
Electrocardiograms (EGG)
Enzyme activity
Fetal anomalies
FetaI ma I format i on
Fetal toxicity
Growth of neonatal rats
Heart-rate
Hematology
Histology
Hydrolytic enzyme activity
Immune response and host resistance
(continued)
186
I I 14.
1121, 1122.
I I I 10, I I I 13.
1133, 1134, 1135.
1302.
1114, 11410.
1136, 1156, 1306.
1151, 1152, 1156.
1114, 1144.
1122, 1141, 1142, 1143,
I 145.
1147.
Mil, IM2, 1132, 1133,
1142, 1145, 11410.
1301, 1303, 1304, 1305,
1306.
1301, 1303.
1301, 1302, 1303, 1304,
1305, 1306.
1304.
I 147.
1152.
1114, 1141, 1142.
II110.
I 152.
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
(continued)
ENDPOINTS
TEST SYSTEM NUMBER
In{activity model
Isozyme prof i les
LC50
LD50
Learning behavior in rats
Learning in primates
Lethality
Li ver enzymes
Locomotor activity
Lung clearance
Lung compliance
Lymphocyte cytotoxicity
Mamma Ii an teratoIogy
Maximum tolerated dose (MTD)
1/2 MTD
I/4MTD
MetabolItes
Memory in primates
Minute volumes
Morphology
Motor and sensory activity
1149.
1131, 1302.
1141, 1142, 1143.
1121, I 122.
1151, 1152.
1151, 1)53, 1154.
1141, 1142, 1143, 1149,
1152, 1301, 1303, 1304,
1305, 1306.
I 136.
1151, 1152, 1153, 1304.
1144.
I 145, I 147.
1117.
1301 .
I 136.
I 136.
1136.
MM, MI2, MI3, 1133,
1134, 1135, M36, 1142.
1151, 1153, 1154.
1145, 1147.
1119, Mill, IIII2.
1151, 1152, 1153, 1154.
(continued)
187
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
(continued)
ENDPOINTS
TEST SYSTEM NUMBER
Mutation frequency
NADH
NADPH
Neutrophil phagocytosis
Oxidatant production in alveolar macrophages
Phagocytic index
Platelet function
Post natal development
Protein determinations
Pulmonary mechanics
Residual lung volumes
Righting reflex
Sensory modality
Sequencing of behavior
Serum constituents
Serum isoenzyme patterns
Sleep-time
Somite development
Static compliance curves
Task discrimination
Teratogenicity
Thyroid function
I I I 13.
1113, 1156.
1113, 1156.
I I 18.
I 137.
I I 18. I I I I I.
1115, 1116.
1302, 1304, 1305, 1306.
1119, 1305.
1145, 1146, 1147.
1145, 1146, 1147.
1132.
1151, 1152, 1153.
1151, 1152, 1153.
1131.
1131, 1302.
I 132.
1305.
1145, 1146, 1147.
1151, 1154.
1301, 1302, 1303, 1304,
1305, 1306.
1131.
(conti nued)
188
-------
INDEX IX: ENDPOINTS OF THE GENERAL AND PERINATAL TOXICITY TEST SYSTEMS
(continued)
ENDPOINTS TEST SYSTEM NUMBER
Tidal volume I 145, I 147.
Total eel I protein 1119, I I I I I, I I I 12.
Urinary constituents 1131.
Viability of neonatal rats 1304, 1306.
189
-------
INDEX X: ENDPOINT DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS
ENDPOINTS
IN-VITRO
TEST TYPE
"IN-VIVO
OTHER
Carcinogenesi s
Mutagenesis
Chromosomal
aberrations
1231
1232
1233
1234
1235
1236
1225
TEST SYSTEM NUMBER*
121 13
1225
Point mutation 1211
1212
1214
1216
1217
1218
1219
Primary DNA Damage 1215
1218
1219
1221
1222
1224
1227
1229
1213
1214
121 10
121 I 1
12112
1242
1245
1223 1245
1226
1228
1242
*An underlined number indicates that the test system has more than one
endpoi nt.
190
-------
INDEX XI: EMDPOINT DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS
ENDPOINT
TEST SYSTEM NUMBER*
Bioconcentration Factors (BCF)
Effect Concentration (EC)
5Q% Effect Concentration (EC50)
5Q% Lethal Concentration (LC50)
Maximum Acceptable Toxicant
Concentration (MATC)
2501, 2502.
2101, 2102, 2212. 2214,
2215, 2223, 2224, 2231,
2312, 2313, 2322, 2331,
2332, 2333, 2411, 2412,
2421, 2422, 2423.
2221, 2222, 2321.
221 I , 2221, 2222, 2223,
2224, 2311, 2512. 2321.
2323, 2324.
2212, 2213, 224It, 2312.
*An underlined number indicates that the test system has more than one
endpoint.
tThis endpoint is based on change within the ecosystem processes.
191
-------
INDEX XII: APPLICATION DISTRIBUTION TABLE
APPLICATION: AIR
TEST SYSTEM
NUMBER:
1 1 14
1 1 15
1 119
UNO
1 1 1 1 1
1 1 1 13
1 138
1 141
1 142
145
146
147
148
149
1410
WATER SOIL
1 1 15
1 1 16
1 1 18
1 1 19
1 1 MO
1 1 1 1 1
II 1 13
1 138
1 146
1 148
1 152
1 155
(conti nued )
FOOD MULTIMEDIA
1 II
1 12
1 13
1115
II 17
1 1 12
121
122
131
132
133
134
135
136
137
1 1 38 1 38
1 143
1 144
1 146
1 147
1 148
151
153
1 154
192
-------
INDEX XII: APPLICATION DISTRIBUTION TABLE (continued)
APPLICATION: AIR WATER SOIL FOOD MULTIMEDIA
TEST SYSTEM
NUMBER: 1156
121 I
1212
1213
1214
1215
1216
1217
1218
1219
12110
121 II 121 I I
12112 12112 12112
121 13
1221
1222
1223
1224
1225
1226
1227
1228
1229 1229
1231
1232
1233
1234
1235
1236
1237
1241
1242
1243
1244
1245 1245
1246 1246
1301
1302
1303
1304 1304 1304
1305
1306
2101
2102
2211
(continued)
193
-------
INDEX XII: APPLICATION DISTRIBUTION TABLE (continued)
APPLICATION: AIR WATER SOIL FOOD MULTIMEDIA
TEST SYSTEM
NUMBER: 2212
2213
2214
2215
2221
2222
2223
2224
2231
2241
2311
2312
2313
2321
2322 2322
2323
2324
2331
2332
2333
241 I
2412
2421 2421 2421
2422
2423
2501
2502
194
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL
TEST SYSTEMS
SAMPLES TEST SYSTEM NUMBER
Aerosols 1141.
Air 1113, 1114, 1115, 1141,
1142, 1145, 1146, 1147,
1148, 1149, I 1410.
Alkylating agents I 1113.
Aromatic ami nes I I I 13.
Asbestos 1146.
AWT effluent I I 19.
Benzene I 152.
Cadmium MM, 1112, 1114, MIM,
1149, 11410.
Chlorinated aliphatic hydrocarbons 1133, M34.
Chlorinated aromatic hydrocarbons 1133, 1134.
Coal dust M46, I 148.
Copper Mil.
Diesel fuel MM, MI3, M45, 1146,
M47, 1148.
Dtoxin M52, 1301.
Dolomite II I 10.
Drugs 1135.
(conti nued)
195
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Energy related pollutants
Ethers
Fly ash
Gases
Gasoline exhaust
Heavy meta t s
Hydrocarbons
Industrial pollutants
I norgani cs
Manganese
Mercury
Nickel
Nitrogen oxides
Nitroso amines
NO
Non-ionizing radiation
1113, 1114, 1115, 1117,
I I I 10, I I I I I, I 132, I 138,
1145, 1146, 1149, 11410,
1155, 1156, 1306.
I 152.
1119, I I I 10, I II I I.
MM, 1145, 1149, 11410.
1113, IM4, 1115, M46.
MM, I! 19, Ml 13, I 146,
1151, 1154, 1303, 1306.
1116, 1118. IIII3.
IM3, MI4, MIS, 1117,
II 19, II 110, MMI, M32,
1138, 1141, M42, 1146,
1148, 1149, M4IO, 1155,
1156, 1306.
1119, II!12, 1301, 1302,
1303, 1304, 1306.
MM, I I Ml, I 1410.
I I I I, II12.
I M4, 11111,1 149, I 1410.
MM, 1138, 1145.
II I 13.
I 138.
MM, IM2, M37, 1138,
1145, 1149, 11410.
151.
(continued)
196
-------
INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
Organics
Oxidants
Ozone
Paraquat
Part ' cu 1 ates
Pesticides
Pheny Is
Platium
Radioactive aerosols
Rock dust
Si 1 icic acid
so2
Stack gases
Su I fates
Sulfuric acid
Sul fur oxides
Technical grade materials
TEST SYSTEM NUMBER
1 1 16,
1122,
1301,
1305,
II 12,
1 1 12,
1 148,
1 145,
1 1 1 10,
1 149,
1 122,
1151,
1303,
1 152.
II 1 II.
1 144.
1148.
MHO.
1 1 1 1,
! 148.
1146,
1 145,
1 1 14,
II 1 1,
1122,
1 1 19, 1 1112, 1121,
1131, 1145, 1155,
1302, 1303, 1304,
1306.
1146, 1148.
1137, 1138, 1145,
1149, 11410.
1 148.
1137, 1141, 1145,
1 1410.
1135, 1141, 1142
1152, 1301, 1302,
1304, 1305.
1112, 1145, 1147,
1 148.
1146, 1148, 1149.
1 145.
1 145.
1141, 1142, 1143.
(continued)
197
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INDEX XIII: SAMPLE DISTRIBUTION OF THE GENERAL AND PERINATAL TOXICITY
TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Toxic substances
Trace metals
Transportation related pollutants
Tri halomethanes
Vaporizable Iiquids
Water
Xenob iotics
Zinc
I I 10, I 121,
132, 1135,
143, 1145,
I 122,
I 141,
I 151,
131,
142,
152,
301, 1302, 1303, 1304,
305.
Ill, 1119, 11111,1148,
1410, 1151, 1155, 1303,
306.
113, 1114, 1115, 1117,
138, 1145, 1146, 1147,
148, 1149, I 1410, 1155,
156, 1306.
I 152.
1142.
1115, 1116.
1135, 1136.
I I I I.
198
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS*t
SAMPLES
ALKYLATING AGENTS
Aldehydes
Alkane sulfonic esters
Alkyl and a 1 kane ha 1 ides
Alkylsulfates
Ary 1 d i a 1 ky 1 1 r i azenes
Az i ri d ines
Azoxy and hydrazo alkanes
Diazoalkanes
Epoxides
Lactones
Nitrogen, sulfur,
and oxide mustards
Phosphoric acid esters
1211,
1219,
121 1,
1219,
1223,
1211,
1219,
1211,
1219,
1212,
121 1 ,
1219,
1223.
1211,
121 1,
1219,
121 1,
1219,
1223.
121 1,
1219,
1211,
1219,
1224.
1211,
121 10,
TEST
1212,
121 10.
1212,
12110,
1224.
1212,
12110,
1212,
121 10,
121 10,
1212,
121 10,
121 10,
1212,
121 10.
1212,
121 10,
1212,
121 10,
1212,
121 10,
1212,
121 1 1,
SYSTEM
1213,
1213,
1211 1,
1213,
12111,
1213,
1221,
12111.
1213,
121 1 1,
1224.
1213,
1213,
121 1 1,
1213,
1221 ,
1213,
121 13,
1216,
1221 ,
NUMBER
1216,
1216,
12)13,
1216,
1221,
1216,
1222,
1216,
121 13,
1216,
1216,
121 13,
1216,
1222,
1216,
1221,
1217,
1222,
1217,
1217,
1221,
1217,
1222,
1217,
1223.
1217,
1221,
1217,
1217,
1221 ,
1217,
1223,
1217,
1222,
1218,
1223.
1218,
1218,
1222,
1218,
1223.
1218,
1218,
1222,
1218,
1218,
1222,
1218,
1224,
1218,
1223,
1219,
SuI tones
1211, 1212, 1213, 1221, 1222, 1223,
1224.
(conti nued)
*The data base on test systems 1214, 1215, and 121 12 is not yet available,
tThe scope of this index extends beyond the test system texts.
99
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
ALKYLATING AGENTS (continued)
Triazines
AROMATIC AMIDES
AZO DYES
HALOGENATED ETHERS AND
HALOHYDRINS
HALOGENATED HYDROCARBONS
AND RELATED DERIVATIVES
Fluorocarbons
Halogenated aromatics
VinyI and vinyIidine
derivatives
HETEROCYCLICS
Acridines and quinicrines
Benzimidazoles
Cyclodienes
Di benzo-p-d i oxi ns
Dicarboximides
FIuorenones
Fuorocoumarins
Phenothiazines
Th ioxanthi nes
Other
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 121 I I, 12113, 1224.
1211, 1212, 1213, 1216, 1217, I2|8,
1219, 12110, 121 I I, 12113, 1224.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 1224.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 1211 I.
1211, 1212, 1213, 12110.
121 I, 1212, 12110, 121 I I.
1211, 1212, 1216, 1218, 1219, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 1221, 1222, 1223, 1224.
1211, 1212, 12110.
1211, 1212.
1211, 1212.
1211, 1212.
1211, 1212, 1216, 1217, 1218, (219,
12110, 1221, 1222, 1223, 1224.
1211, 1212.
1211, 1212.
1211, 1212, 1213, 1216, 1217, 1218,
1219.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III.
(continued)
200
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
HYDRAZINES, HYDROXYLAMINES
CARBAMATES, HYDRAZIDES, AND UREAS
Carbamates
Hydraz ides
Hydrazines
Hydro-.•/ [amines
Ureas and thfoureas
INORGANIC DERI,ATIVES
Halogens and derivatives
Metal and metal fold
derivatives
Ozone
Sulfur and nitrogen oxides
NATURAL PRODUCTS
Antibotics
Mycotoxins
Aflatoxin
Other
Pyrrol izidine alkaloids
Steroids
Xanthines
NITRO DERIVATIVES
1211, 1212, 1213, 1216, 1217,
1219, 12110, 121 I I , 1221 , 1222,
1211, 1212, I2III.
1211, 1212, 12110.
121
1212, 1213, 1216, 1217,
1219, 12110, 121 I I , 1221 , 1222,
1218,
1223.
1218,
1223.
1212, 1213, 1216, 1217, 1218,
1219, 12110, 12]I I .
1211,
12110.
1212, 1216, 1217, 1218, 1219,
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III, 1221, 1222, 1223.
1212, 12110, 121 I I.
1211, 1212, 1213, 12110, 1211 I, 1221,
1222, 1223.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 1221, 1222, 1223, 1224.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 1221, 1222, 1223, 1224.
1211, 1212, 12110, 1221, 1222, 1223,
1224.
12110, 1221, 1222, 1223.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 12111, 1221, 1222, 1223,
1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, 121 I I.
(continued)
201
-------
INDEX XIV: SAMPLE DISTRIBUTION OF THE GENOTOXICITY TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
NITRO DERIVATIVES (continued)
Nitrofurans
N itroimidazoles
Nitroquinolines and
compounds
N-NITROSO COMPOUNDS
Nitrosamides
N itrosamines
N itrosoureas
NUCLEIC ACID BASES AND ANALOGS
ORGANIC PEROXIDES
ORGANO-METALLICS
Organo lead derivatives
Organo mercury derivatives
Other
POLYNUCLEAR AROMATICS
OTHER
Esters and anhydrides
N-oxides
Quaternary ammonium
compounds
Qu inones
Sulfites
1211, 1212, 1216, 12(7, 1218, 1219,
12110, 1221, 1222, 1223.
1212, 12110.
1211, 1212, 1213, 12110, 1221, 1222,
1223, 1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III, 1221, 1222, 1223,
1224.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III, 1221, 1222, 1223,
1224.
1211, 1212, 1213, 1216, 1217, 1218,
12)9, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110, I2III, 12113.
1211, 1212, 12110.
1221, 1222, 1223.
1211, 1212, I2III, 1221, 1222, 1223.
1211, 1212, 12110, 1221, 1222, 1223.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 1221, 1222, 1223, 1224.
1211, 1212, 1213, 12110, 121II.
1211, 1212, 1213, 12110.
1211, 1212, 1213, 1216, 1217, 1218,
1219, 12110.
1211, 1212, 1216, 1217, 1218, 1219,
12110, 121II.
1212, 121.3, 12110, I2III.
?02
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS
SAMPLES
Ac i d ra i n
Air pol 1 utants
Ambient
Ambient ai r
Chloramines
Chlorinated hydrocarbons
Ch lor ine
CO
Coa 1 storage
Complex wastes
Detergents
Dissolved gases
Dredge sediment
Drt I I ing muds
TEST SYSTEM NUMBER
2411 .
2411, 2421.
2101, 2102, 2411, 2423.
241 1 .
2102.
2501.
2102.
241 1 .
2101 .
2322.
2101 .
2215.
2102, 2322.
2211, 2221, 2311, 2321,
Energy related
Gaseous pollutants
Heavy metals
2333.
2101, 2211, 2212, 2215,
2221, 2224, 2231, 2311,
2321, 2331, 2332, 2333,
2411, 2423.
2411, 2421.
2101, 2102, 2211, 2221,
2222, 2223, 2311, 2321,
2322, 2332, 2412, 2421,
2422, 2423, 2501.
(cont inued)
203
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Herb ic ides
Industrial
Industrial air pollutants
Industrial effluents
Industrial sludge
Industrial waste discharges
Industrial waste effluents
Inorgan ics
Insecticides
Leachates
Meta I s
Multi chemicals
Monoch lorinated organ ics
N0
NO
Nutrients
2101, 2211, 2221, 2311,
2312, 2313. 2321.
2101, 2102, 2211, 2212,
2214, 2215, 2221, 2224,
2231, 2311, 2321, 2331,
2332, 2411, 2412, 242!,
2422, 2423, 2502.
2411.
2211, 2221, 2311, 2321,
2331, 2332, 2421.
2412.
2101.
2422.
2212, 2241, 2502.
2101, 2211, 2221, 2311,
2312, 2313. 2321, 2501.
2101.
2213, 2215, 2224, 2231,
2324.
2214.
2102.
2101.
2101.
2101.
241 I.
2101, 2102.
(continued)
204
-------
INDEX XV: SAMPLE DISTRIBUTION OF THE ECOLOGICAL TEST SYSTEMS (continued)
SAMPLES
TEST SYSTEM NUMBER
Oi Is
Organ ics
Ortho-P
Other
Oxygen
Ozone
PCB'r,
PsnTachlorophenol
Pesticides
Radiolabeled material
Receiving waters
so2
Tox-P
Toxic organ ics
Transportation related
Waste oi I
2211, 2221, 2311, 2321,
2331, 2332.
2212, 2215, 2224, 2231,
2241, 2331, 2332, 2502.
2101 .
2101 .
2215.
241 I .
2102, 2211, 2221, 2311,
2312, 2321, 2501.
2312.
2101, 2213, 2222, 2323,
2324, 2331, 2332, 2333,
2421, 2422, 2423.
2501.
2101, 21 02.
241 I .
2101.
2331, 2332.
2411, 2421.
2213.
205
-------
TECHNICAL REPORT DATA
read Inttnictions on the reverse before completing)
1. REPORT NO.
EPA-600/9-78-037
2.
4. TITLE AND SUBTITLE
SHORT-TERM TESTS FOR HEALTH AND ECOLOGICAL
EFFECTS. Part I: Program Overview.
Part II: Directory of Tests
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
November 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Biochemistry Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Trianglp Parl^ M.r. 77711
10. PROGRAM ELEMENT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
TVianglo Pat-L- M C 97711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
gl
TE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is the proceedings of an Office of Health and Ecological Effects
(OHEE), U.S. Environmental Protection Agency workshop held at the Research
Triangle Park, North Carolina, in January of 1978.
The proceedings consists of eight papers. The first paper is the keynote
address; the other seven papers overview the work being done in short-term testing
for health and ecological effects by the various U.S. Environmental Protection
Agency, Office of Health and Ecological Effects Laboratories.
Included with the proceedings in the Directory of Short-Term Tests for Health
and Ecological Effects, which is also published separately as EPA-600/1-78-052.
The directory, which was compiled as a result of the workshop, provides basic
information about the individual short-term tests for health and ecological effects.
The test systems are cross-indexed.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
environmental tests
laboratories
biological laboratories
directories
indexes (documentation)
short term tests
06 F, T
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
IIij[SLASSIEIEB___
20VSECUHTTV CLASS (Till.
21. NO. OF PAGES
215
20V'StCU'RYTY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Farm 2220-1 (9-73)
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