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

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     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

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     •   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

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 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

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     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

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     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

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      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

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                        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

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                                                    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

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      •   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

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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

                                       33

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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.
                                      34

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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.
                                      35

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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,
                                       37

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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.
                                      38

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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.
                                      42

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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.
                                       45

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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

                                      46

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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.
                                       47

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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.
                                      51

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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
            Endpoint Pistribution.of the  GenotoKtcfvty'vo
              Test Systems	
       Endpoint DIstrfbOtlon bf':lhe  Eco'togfeat"Test Systems^'!' 1 "^
       Appl jcation Distribution Tabiei•:£  c;-;i i©'••'.bcr.. •tgrtw-.-.r.,
 .'.M
, •-"'•«•
      Sample Distribution  of the Genera,!  and.Perinatal  Toxici-tvr -  ^
      •• Test-Systems.  ;  ;  ;  ;  ;  ?™: '.°:!i:.c'.: 'T' ^. V 'f;"     '  -"Ji"
                                                            ***•••
      Sample Distri but fort  of'the'Genotoxfcfiy "test Systems/.
      Samp Ie DIstrIbut Tori  6f'the ' Edofog fcaI  f^st Systems...
                                                                                145
                                                                               OQOj
 154
 160
 168
 172
 175
 176
 ISO
 184

 185

 190
000 £
 191
 192

 195
 199
203
                                       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

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     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

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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.

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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

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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

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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

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                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

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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

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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

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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.

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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

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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.

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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

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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

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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

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 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

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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

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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

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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

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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

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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

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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

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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

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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.

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INDICES
   153

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                    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

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            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

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         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

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           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

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          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

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          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

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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

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     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

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 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

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 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

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 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

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        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

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                                    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)

-------