&ER&
United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
EPA-600/2-79-122
June 1979
Research and Development
Terrestrial Ecology
Protocols for
Environmental
Assessment Programs:
Workshop Proceedings
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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 report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
EPA REVIEW NOTICE
This report has been reviewed by the U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents necessarily
reflect the views and policy of the Agency, nor does mention of trade names or
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-79-122
June 1979
Terrestrial Ecology Protocols
for Environmental Assessment Programs:
Workshop Proceedings
R.L Waterland, Compiler
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
Contract No. 68-02-2611
Task No. 43
Program Element No. INE624
EPA Project Officer: Raymond G. Merrill
Industrial Environmental Research Laboratory
Office of Energy, Minerals, and Industry
Research Triangle Park, NC 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Washington, DC 20460
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ABSTRACT
Through combined efforts of EPA's Industrial Environmental Research
Laboratory (IERL-RTP) and Corvallis Environmental Research Laboratory
(CERL), a workshop was held in Coryallis, Oregon, during November 1978 to
discuss potential tests for inclusion in, and make recommendatons for a
terrestrial ecology bioassay testing protocol for use in IERL
Environmental Assessment programs. Workshop participants included both
government and private researchers in the fields of plant physiology, soil
microbiology, and entomology. Specific issues addressed at the workshop
included: what tests should be included in a Level 1 protcol, what should
Level 1 to Level 2 decision criteria be, and what kinds of tests would be
appropriate at Level 2.
This report serves as the proceedings of the workshop. It
summaries key points of discussion and presents the results, conclusions,
and recommendations reached in addressing stated workshop issues.
Recommended Level 1 plant, soil, and animal assays are discussed, and
suggested kinds of Level 2 procedures, based on Level 1 findings, are
presented.
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TABLE OF CONTENTS
Section Page
1 INTRODUCTION 1
2 GENERAL DISCUSSION 7
2.1 Developing State of Terrestrial Ecology 7
2.2 Features of Appropriate Tests 7
2.3 Difficulties of Gas Sample Testing 9
2.4 Decision Criteria 9
2.5 Quality Assurance 11
2.6 Intra-EPA Protocol Comparability 11
3 PLANT BIOASSAYS 13
3.1 Candidate Tests 13
3.2 Recommended Level 1 Protocol 15
3.3 Suggested Level 2 Procedures 16
4 SOIL ASSAYS 17
4.1 Candidate Tests 17
4.2 Recommended Level 1 Protocol 19
4.3 Suggested Level 2 Procedures 21
5 ANIMAL ASSAYS 22
5.1 Candidate Tests 22
5.2 Recommended Level 1 Protocol 23
5.3 Suggested Level 2 Procedures 23
6 SUMMARY AND CONCLUSIONS 24
APPENDICES — CONSULTANTS' REPORTS 28
APPENDIX A — D. Tingey, EPA/CERL 29
APPENDIX B — S. Sandhu, EPA/HERL-RTP 32 ,
APPENDIX C ~ J. Bromenshenk, University of Montana . . 37
APPENDIX D — K. Duke, Battelle-Columbus 48
APPENDIX E -- D. McCune, Boyce Thompson Institute ... 55
APPENDIX F — R. Rogers, University of Nevada 66
APPENDIX G -- D. Shriner, ORNL 71
APPENDIX H — T. Tibbitts, University of Wisconsin . . 81
ill
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SECTION 1
INTRODUCTION
In 1975 EPA's Industrial Environmental Research Laboratory,
Research Triangle Park (IERL-RTP) initiated a series of Environmental
Assessment (EA) programs. These programs were designed to:
t Systematically evaluate the physical, chemical, and biological
characteristics of all effluent streams from an energy
conversion or industrial process
• Predict the probable effects of those streams on the environment
• Rank those streams relative to their individual hazard potential
t Identify and define .necessary control technology development
programs to reduce the potential hazard presented by those
streams
To satisfy these aims, key aspects of an EA are a characterization of the
total pollution potential presented by waste streams from a process and a
comparison of the nature of the potential environmental insult to existing
standards or defined environmental goals.
Types of EA programs currently underway include assessments of low,
medium, and high Btu gasification, fluidized bed combustion, stationary
conventional combustion, coal cleaning, and coal liquification processes.
In performing these EA's, IERL is satisfying one of its roles in
supporting the regulatory and enforcement offices of EPA by anticipating
future control technology needs and developing the data bases needed to
support standards development.
To support the EA programs a tiered source sampling, chemical
analysis, and bioassay approach has been defined to provide the data
needed to evaluate potential environmental impact. This tiered approach
incorporates three levels of sampling and analysis comprehensiveness and
detail:
• Level 1: Screening ~ structured to identify potential problem
effluents and pollutants
• Level 2: Confirmation -- structured to confirm the existence
of problem effluents and pollutants
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• Level 3: Risk Assessment ~ structured to quantify the extent
of environmental impact from problem
effluents
The tiered approach was adopted because it offered potential cost
savings over a direct approach in which all streams would be carefully
sampled and completely analyzed in one pass. In the tiered approach
effluent streams are first surveyed, or screened, using simplified,
generalized sampling and analysis methods (Level 1) which permit ranking
streams on a more hazardous to less hazardous basis. Detailed sampling
and analysis (Level 2) would then be performed on those priority streams
identified at Level 1. Level 2 would thus confirm screening results and
provide better quantitative information on potential environmental hazard.
In each level of the tiered approach, both chemical and biological
characterizations of an effluent stream are performed. The chemical
characterization provides a quantitative, engineering type numerical
evaluation of a stream's potential hazard, along with control technology
development input. The biological characterization provides a direct
measure of a stream's potential hazard in terms of a biological response.
In addition, the biological testing aids in identifying toxicant
synergisms and antagonisms. Thus the dual chemical and biological
characterizations are designed to supplement each other.
To date, through efforts coordinated by the Process Measurements
Branch of IERL-RTP, Level 1 sampling and chemical analysis procedures have
been defined (References 1, 2), and Level 2 chemical analysis procedures
are being defined. However, since IERL expertise is mainly in the fields
of chemistry and engineering, a Bioassay Subcommittee of the overall IERL
Environmental Assessment Steering Committee was formed to advise and
coordinate EPA inhouse and contractor efforts in developing appropriate
bioassay testing protocols to parallel the chemical analysis procedures.
The subcommittee draws representation from five Office of Health and
Ecological Effects laboratories. As such, it represents expertise in the
fields of pollutant effects on human health, aquatic ecology, terrestrial
ecology, and carcinogenicity and mutagenicity.
Initial subcommittee efforts resulted in defining a preliminary
Level 1 bioassay protocol (Reference 3). The prescribed protocol
specified testing of whole effluent samples for:
• Mutagenicity (one test)
• Human health effects (two in vitro tests, one in vivo test)
• Aquatic ecology effects (three fresh water tests, three marine
tests)
• Terrestrial ecology effects (two tests)
Since little experience with complex effluent testing using any of
the tests specified existed, a series of pilot studies were initiated upon
publication of this preliminary Level 2 protocol. These pilot studies
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were designed in part to validate the protocol and to evaluate the utility
of the data obtainable. Results of the pilot studies are still being
evaluated. However, based in part on these results and other criticism
advanced since publication of the preliminary protocol, concerns have been
raised over the propriety of including the terrestrial ecology effects
tests specified. Questions concerning ease of sample collection,
transport, and analysis, and of ultimate data interpretation have led to
concerns over whether other terrestrial ecology tests now being developed
might be more appropriate than the ones specified.
In response to these concerns IERL and the Corvallis Environmental
Research Laboratory (CERL), organized a workshop to specifically address a
recommended terrestrial ecology testing protocol for use in the EA tiered
approach. Specific issues to be addressed at this workshop were:
e What should the specific terrestrial ecology test protocols be
for Level 1 bioassay testing?
• What should the decision criteria be for specifying Level 2
terrestrial ecology test needs based on Level 1 results?
• What are potential terrestrial ecology protocols for Level 2
bioassay testing?
Specific questions, relating to these issues, needing answers included:
• What specific tests should be used for testing the variety of
gaseous, liquid, and solid samples to be encountered?
• What are sample size, integrity, age, etc. requirements for
candidate tests?
t Can candidate tests accommodate the currently defined sampling
procedures?
t Should Level 2 testing include applying Level 1 test procedures
to fractionated effluent samples?
• Are chronic effects tests feasible for Level 2 testing?
The workshop was held on November 14-15, 1978 in Coryallis,
Oregon. Technical participants in the workshop are listed in Table 1.
The agenda for the workshop included an introductory session, where the
background on EA program needs and the ground rules for discussion were
established, three working sessions on potential plant, soil, and animal
assays respectively, and a summary session.
In the introductory session it was noted that, in developing
recommendations for potential test protocols, six criteria were to be
considered for candidate tests:
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TABLE 1. TERRESTRIAL ECOLOGY BIOASSAY WORKSHOP PARTICIPANTS
Participant
Affiliation
Dr. J. Bremenshenk
Dr. K. Duke
Dr. R. Eagar
Dr. J. Gillett
Chairman, Soil and Animal
Assay Sessions
Dr. A. Goloff
Dr. B. Lighthart
Dr. D. McCune
Dr. R. Merrill
Workshop Chairman
Dr. R. Rogers
Entomologist/Ecologist
University of Montana
Missoula, Montana
Associate Manager
Ecology and Ecosystem Analysis
Section
Battelle - Columbus Laboratories
Columbus, Ohio
Soil Microbiologist
Union Carbide Corporation
Tarrytown, New York
Research Ecologist
U.S. EPA
Corvallis Environmental Research
Laboratory
Corvallis, Oregon
Plant Physiologist
Union Carbide Corporation
Tarrytown, New York
Soil Microbiologist
U.S. EPA
Corvallis Environmental Research
Laboratory
Corvallis, Oregon
Plant Physiologist
Boyce Thompson Institute for
Plant Research
Ithica, New York
Research Chemist
U.S. EPA
Industrial Environmental Research
Laboratory
Research Triangle Park, N.C.
Soil Microbiologist
University of Nevada
Las Vegas, Nevada
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TABLE 1. Concluded
Participant
Affiliation
Dr. W. Rosen
Dr. S. Sandhu
Dr. D. Shriner
Dr. T. Tibbitts
Dr. D. Tingey
Chairman, Plant Assay
Session
Dr. L. Water land
Meeting Coordinator
Plant Physiologist
U.S. EPA
Office of Toxic Substances
Washington, D.C.
Research Biologist
U.S. EPA
Health Effects Research Laboratory
Research Triangle Park, N.C.
Research Ecologist
Oak Ridge National Laboratory
Oak Ridge, Tennessee
Professor of Horticulture
University of Wisconsin
Madison, Wisconsin
Plant Physiologist
U.S. EPA
Corvallis Environmental Research
Laboratory
Corvallis, Oregon
Leader, Process Analysis Section
Acurex Corporation
Mountain View, California
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• Cost
• Sample requirements (form and quantity)
• Relevance to terrestrial ecology and biology
• Availability (existence of accepted test procedures and a
validation data base)
• Comparability (among different laboratories performing the test)
• Response (easily measured and sensitive)
Pertaining to these criteria it was noted that, for Level 1, tests
must cost no more than $500 to $700 and must take no more than 2 to 3
months to complete from receipt of samples to test report completion. In
addition, it was noted that, based on current Level 1 sampling procedures,
available sample quantities for liquid and solid effluent streams would be
essentially unlimited (within reason), but that gas stream particulate and
organic species (sorbent extract) samples would probably be limited to 250
to 300 mg quantities. Based on these constraints, discussion in each of
the working sessions focused on the above issues and criteria.
This report serves as the proceedings of the workshop and presents
the results, conclusions, and recommendations reached in defining a Level
1 terrestrial ecology bioassay protocol, suggesting potential Level 2
protocols, and outlining decision criteria for specifying Level 2 tests
based on Level 1 results. As such, the remainder of the report is
organized as follows. Section 2 presents a summary of general discussion
and overall concerns raised at the workshop which do not specifically
relate to a given (plant, soil, animal) session topic area. Sections 3
through 5 summarize discussion specific to each topic area: plant, soil,
and animal assays respectively. Section 6 summarizes overall workshop
conclusions and recommendations. Finally, several of the technical
participants listed in Table 1 served as EPA consultants. Their reports
on conclusions and recommendations reached are reproduced in the
Appendices.
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SECTION 2
GENERAL DISCUSSION
Throughout the workshop, even into the summary sessions, several
issues or concerns of a general nature were raised, issues not strictly
focused on the discussion of a given class (plant, soil, animal) of
candidate assays for incorporation into the EA phased analysis approach.
This general discussion is summarized in this section.
2.1 DEVELOPING STATE OF TERRESTRIAL ECOLOGY
The first of these general points of discussion concerned the
current relatively undeveloped state of the terrestrial bioassays.
Specifically it was noted that research in the field of toxicant effects
on terrestrial ecology lags that in the areas of carcinogenesis/mutagenesis,
human health effects, and aquatic ecology effects by several years.
Comprounding this is the fact that, although several government groups
want to employ terrestrial bioassay testing, few are funding developmental
work and coordination of ongoing efforts is poor. Thus, virtually all
existent terrestrial bioassay test procedures must be considered
developmental. Few terrestrial assays have been validated, and
experimentation with other than pure compounds has not been performed. Of
course it should be noted that experience with complex effluents testing
was largely lacking for all tests proposed in the preliminary Level 1
bioassay protocol prior to initiating the EA pilot studies. But this fact
must be underscored for terrestrial ecology tests, and supplemented by
noting that the degree of pure compound testing in terrestrial ecology '
protocols has been limited. Therefore, it must be emphasized that any set
of test procedures recommended now for inclusion in the EA methodology
should be taken only as the best available at present, given the
constraints that a test must be inexpensive, simple, and reliable.
However, as other test procedures develop, and interpretation of test data
becomes more discriminating, other protocols may become more appropriate.
2.2 FEATURES OF APPROPRIATE TESTS
The above led into the next general area of discussion: what
generic kinds of tests would be appropriate for incorporation into the
tiered approach? For example, is it necessary for a test just to give a
response when subjected to an effluent sample, or should we expect the
response to be indicative of some real environmental impact, such as crop
loss? This kind of question relates closely to the above discussed state
of development of terrestrial bioassays. Several test procedures are
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currently being developed which give responses other than lethality to
known toxicants. However, quantitatively relating these responses to
effects on an organism's growth, development, and reproduction is often
quite difficult. Furthermore, the ties between these specific effects on
an organism and more global impacts on an ecosystem are even more tenuous.
With this in mind, it was decided to focus attention on tests that
have broad applicability and integrate organism responses. Responses
measured should, where possible, be correctable to effects on the test
organism's growth, development, and/or reproduction. In addition,
recommended protocols should include, as a minimum, a representative test
for each of the producer, consumer, and decomposer (recycler) organism
classes. In this regard, though, discussion should focus on bioassays
that measure toxicity and not mutagenicity.
Another concern in this area related to the question of the what
are acceptable levels of false positive and false negative responses in a
given test. Clearly, what is desired in any candidate test is a minimum
of both. However, this is not generally possible. Thus, it was decided
that for Level 1 screening tests, a minimum of false negative responses
was key, with a reasonable level of false positive responses acceptable.
Level 2 testing would be designed to remove the false positives. Of
course, it must be noted here that, for most terrestrial ecology assays,
little is currently known about the incidence of false positive or
negative responses. Thus, this information must await future test
development and validation.
Finally, discussion focused on the eventual need to include chronic
effects testing. Here, the needs of anTA program, and the desire to
identify chronic toxic effects are somewhat at odds. An EA requires rapid
test procedures, especially at the Level 1 stage, but at Level 2 as well.
But chronic effects testing, by its very nature, requires lengthy test
times, which currently fit well only in Level 3. There is concern,
though, that by delaying chronic effects testing to Level 3, many
potential effluent streams would have been screened out at Levels 1
and 2. In fact, IERL has requested that the bioassay subcommittee
consider specifying a chronic effects or life cycle test for optional use
on Level 1 samples. Though this would be considered a Level 2 test,
testing of Level 1 samples would be considered when indicated. What is
required is an inexpensive, simple, short term chronic effects test.
Although, none is currently available, such a test may become available in
the future. For example, the Arabidopsis bioassay being developed at the
Corvallis Environmental Research Laboratory is a short term life cycle
test which satisfies some of the needs of a chronic effects test. Thus it
was decided that, of necessity, chronic effects testing may not be
addressed at Level 1, but some form of life cycle testing should be
incorporated into the Level 2 protocol when it becomes definitely
established. Hopefully, some life cycle tests, such as the Arabidopsis
bioassay, will be well developed in time for inclusion into a detailed
Level 2 protocol.
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2.3 DIFFICULTIES OF GAS SAMPLE TESTING
The next general area of discussion concerned the difficulty of gas
sample testing. Although gas fumigation techniques are standard
procedures and can be adapted to virtually all terrestrial assays, most
current bioassay procedures require very large volumes of sample. This
problem is compounded if flow through testing is required instead of
static gas sample testing. In this regard, flow through testing is the
preferred approach since it is often difficult to make a valid assessment
of dose-response under static exposure conditions.
Large gas samples are difficult to collect in the field, transport
to the laboratory, and store until use. Compounding this problem is the
fact that collecting large gas samples requires using some form of plastic
(e.g., Tedlar) bag. Sample component loss through the bag, chemical
reactions with the bag material, and absorption to the bag walls have been
experienced (Reference 4), leading to a very real concern about sample
integrity by the time it is assayed.
These problems can be circumvented by performing tests onsite in
the field. But this approach introduces a different set of problems.
Besides it would be inordinately expensive, and not feasible at some test
sites. Gas sample compression or cooling would reduce the volume
requirement for sample transportation and storage, but this approach also
suffers from maintenance of sample integrity questions.
The best solution to the gas sample testing problem would involve
decreasing the sample size requirement of a given test procedure; in other
words, miniaturizing the test. However, this must be considered more of a
long term solution. For example, work is currently proceeding toward
miniaturizing the stress ethylene protocol, but results are not expected
for at least 24 months. Therefore, it was decided that, for the present,
gas stream testing be performed in the laboratory, using static procedures
to minimize, to the extent possible, sample size requirements, and hope
that the sample tested remained sufficiently representative of the gas
stream sampled to give trustworthy results. In this respect, it was noted
that, although some sample loss and chemical reactions might occur, a gas
sample would probably maintain its, phytotoxicity for some reasonable time
period. Furthermore, given the screening nature of Level 1, the loss of
some component or some chemical transformation might not seriously
invalidate test conclusions at Level 1. Moreover, if Level 1 tests were
positive on these potentially degraded gas samples, one might conclude
that the sample contained highly toxic and stable compounds. For the
future, though, the development of "miniature" test assays should be
strongly encouraged, and the use of flow through gas sample testing
pursued over static testing.
2.4 DECISION CRITERIA
The propriety of specifying Level 1 to Level 2 decision criteria as
a workshop output was also raised as an issue. After discussing the most
recently proposed decision criteria for the other (mutagenesis, health
effects, aquatic effects) tests in the Level 1 protocol, and noting the
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tentative nature of these criteria, it was decided to leave specifying
detailed criteria to future Bioassay Subcommittee efforts. This was
deemed appropriate since the subcommittee is in the best position to
assure consistent decision criteria among all mutagenesis, health effects,
aquatic, and terrestrial tests. However, the workshop would offer
guidance as to what kinds of Level 2 testing would be indicated based on
Level 1 results.
In general, it was agreed that:
• Level 2 terrestrial ecology testing emphasize using Level 1
test procedures on effluent sample fractions if terrestrial
ecology tests give positive, or high toxicity responses
• Level 2 terrestrial ecology testing emphasize testing
unfractionated samples with other species and other responses,
and life cycle testing if Level 1 terrestrial ecology tests
give negative, or low toxicity responses, but other Level 1
tests (health effects, aquatic effects) give positive, or high
toxicity responses.
These recommendations are summarized in more detail in the test assay
sections (Sections 3 to 5) of this report. It should be noted that these
recommendations conform to the objectives of Level 2: to confirm Level 1
results and to isolate toxic species through fractionation. High toxicity
streams should not need confirmation and could go directly to
fractionation. Lower toxicity streams should need confirmation.
Tempering the above recommendation was the observation that the
most care in describing Level 1 to Level 2 decision criteria is required
in cases where samples are of moderate toxicity. Level 2 would definitely
be indicated for samples which gave high toxic responses in all biotests
and triggered high in the Level 1 chemical analyses. Conversely, Level 2
testing would be of low priority for samples which elicited no response in
any bioassay and triggered low in the chemical analyses. It is in the
middle ground, where samples elicit low to moderate toxicity in several
tests, or the range of responses varies from not detectable to high
toxicity over several tests, that the most difficulty in making a Level 2
decision will be encountered. One possible approach might include the
following:
• A high toxicity response in any test should automatically raise
a stream's priority
t A moderate toxicity reponse both in one health test and one
ecological test should raise a stream's priority
t A low toxicity response in one health test, one aquatic ecology
test, and one terrestrial ecology test should raise a stream's
priority
With respect to reporting Level 1 results, the method of assigning
relative toxicity values (not detectable, low toxicity, moderate toxicity,
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high toxicity) based on the maximum applicable dose in a test and the
LDso, LCso, or EC50 response of a test, as reported in Reference 5,
was deemed appropriate.
In a related vein, it was noted that some form of pre-Level 1
decision criteria should also be specified, based on a stream's chemical
characterization. For example, a stream with very low pH, or with
significant quantities of arsenic present would probably not need to be
subjected to bioassay.
2.5 QUALITY ASSURANCE
The next area of general discussion revolved around specifying
quality control and quality assurance procedures. In other words, what
must be done to assure that good data giving interpretable results come
from a given procedure. Three general areas must be addressed h'ere:
• The mechanics of sample and data handling including sample
chain of custody records and prevention of data transcription
errors
• Individual test procedure quality assurance including
provisions for negative control samples (blanks), positive
control samples, and audit samples.
• Test results reproducibility and interpretation.
It was agreed that the burden for several aspects of specifying a
QA procedure should logically fall to IERL and the Bioassay Subcommittee.
Specifically, outlining chain of custody and data transcription checking
procedures and providing laboratory audit samples fall in this category.
However, specifying positive control compounds will be the responsibility
of the individual drafting the detailed procedures document for each
test. Thus, appropriate positive control species are discussed below in
each test assay section of this report. In addition, it was decided that
to ensure test validity and reproducibility, each effluent tested should
be assayed at a minimum of three concentration levels, in addition to
controls, and that four replicate tests be performed.
2.6 INTRA-EPA PROTOCOL COMPARABILITY
The final general area of concern related to assuring that the
bioassay protocol specified for use in IERL EA programs be comparable,
where possible, to protocols being developed and employed by other EPA
organizations. Currently, due to mandates under the Clean Air Act
Amendments, the Clean Water Act Amendments, the Toxic Substances Control
Act (TOSCA), the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA), and the Resource Conservation and Recovery Act (RCRA), several
EPA offices including the Office of Toxic Substances (OTS), the Office of
Solid Waste (OSW), the Office of Pesticide Programs (OPP), and the Office
of Water Planning and Standards (OWPS) are. developing bioassay protocols
to assess the impact of various process, product, and waste streams.
These are in addition to the subject IERL protocols. Thus striving for
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comparability, to the degree possible, among these protocols, adopting
standard procedures, standard controls, standard data interpretation
procedures, etc., would seem to be quite important. Of course the needs
of an EA program may not quite mesh with those of other offices, but where
possible a consistent set of assays should be adopted.
It was noted that IERL is indeed attempting to perform this
activity. A representative from OTS was a workshop participant, in part
to relate recent OTS efforts in developing protocols for use in TOSCA
mandated assays. A document summarizing the specific procedures
recommended for use in implementing TOSCA is scheduled for release in
early 1979. Findings of this study, and others like it, should be
incorporated as appropriate into the IERL protocol development.
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SECTION 3
PLANT BIOASSAYS
This section summarizes workshop discussion focused on identifying,
weighing, and selecting plant test procedures for inclusion in Level 1 and
Level 2 bioassay protocols. As noted in Section 2.2, it was generally
agreed that effects on photosynthetic systems (producers) needed to be
addressed in the protocol, and that the current aquatic algal tests were
not sufficient. Thus, defining specific terrestrial plant tests for use
with solid, liquid, and gaseous samples was addressed in this session.
3.1 CANDIDATE TESTS
The discussion opened by listing candidate tests for consideration
without regard to applicability (solid, liquid, or gas samples). Tests
suggested were:
• Stress ethylene
t Foliar injury
• Seed germination
• Seedling growth
• Tradescantia (mutagenesis)
• Pollen viability
t Arabidopsis life cycle
• Pea tendril coiling
• Tomato petiole angle
a Cucumber leaf enlargement
• Bean hook opening
• Turgor changes
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However, preliminary discussion quickly removed from consideration
the last five in the list as being too undeveloped with respect to other
tests to be considered at present.
The stress ethylene test is currently included in the preliminary
Level 1 protocol for gas sample testing (in fact it is the only test
presently specified for whole gas sample testing). However, the test can
be used to assay liquid and liquid extracts of solid samples as well
(administration by foliar spray or root irrigation). It is one of the
more developed of the plant tests, beinq extensively used by the Corvallis
Environmental Research Laboratory (CERL) amonq others. The test gives a
general, integrated, reproducible response. [CERL results have been
corroborated by the Battelie-Columbus Laboratories). It is a quite
sensitive test; no false negative or false positive responses have been
detected. In addition, increased ethylene production has been correlated
with decreased growth, establishing the desired link between test response
and physiological function. The test cost is about $800 per test series
(blank, positive control, three sample concentrations, four replicates).
A static gas fumigation procedure has been developed, though a large
sample quantity (1300 £.) is still required. To overcome this drawback,
work to miniaturize the procedure is currently underway, though the
miniature procedure will not be available for at least 24 months.
The foliar injury test is also relatively well developed and can be
used to assay extracts of solid, liquid, and gas samples through
fumigation, foliar spray, or root irrigation. It is a less sensitive test
than the stress ethylene procedure, though it has been shown to be dose
responsive and reliable. The potential for more false negative responses
exists than with the stress ethylene test, however, the incidence of false
positive responses should be low.
The seed germination and root elongation assays can probably be
considered the most developed assays for liquid samples, though extracts
of solids and possibly solids can be tested. Well documented procedures
for performing these tests exist (References 6, 7). The root elongation
test is attractive because it provides an evaluation of morphological
development of plant systems. It is a more sensitive measure of effects
than seed germination, which is actually a measure of lethal effect,
though both tests are attractive for EA needs. A variety of seeds can be
used to detect species response variations.
The Tradescantia assay is a quite sensitive mutagenesis test.
However, S02 is highly toxic to the test organism, thus the presence of
S0£ in the test sample can mask any mutagenic response. For this reason
(S02 will almost always be encountered in gas samples from an energy
conversion process) and the fact that the test is only in the early stages
of development, it is not as attractive an alternative for current EA
needs. Moreover, since the workshop focused on identifying candidate
tests to measure toxicity, not mutagenicity, the Tradescantia assay seemed
inappropriate.
Similarly for the pollen viability assay. In spite of the fact
that this test would require small samples for gas testing and is quite
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sensitive to SC>2 and inorganic compounds, it hasn't been tested widely
and is therefore not sufficiently developed for current needs.
The Arabidopsis test offers the potential for being a rapid,
simple, inexpensive life cycle test. But it also is too undeveloped to be
used at present.
3.2 RECOMMENDED LEVEL 1 PROTOCOL
Based on the above discussion, the recommended plant assays for
incorporation into the Level 1 protocol are:
• The stress ethylene test coupled with the foliar injury assay
• The root elongation test coupled with the seed germination
assay
The stress ethylene/foliar injury test is proposed for use with gas
samples, with optional use on liquid, solid leachate, and possibly solid
samples. The seed germination/root elongation assay is proposed for use
on liquid and solid extract samples.
The combination stress ethylene/foliar injury procedure was chosen
because it provides the sensitivity of the stress ethylene test with a
measure of response severity provided by the foliar injury test. Although
the stress ethylene protocol is less well developed for use with liquid
and solid extract samples, enough experience with it exists to justify
suggesting it for these samples. A static fumigation procedure was
recommended for gas sample testing and root irrigation for liquid samples
and extracts of solid samples. Some concern was expressed over the
organic content of the plant growth medium buffering the toxicity of
samples tested. However, the concensus opinion was that this can be
avoided with care and proper medium choice. Use of a low organic medium
was suggested.
The combination seed germi nation/root elongation test was proposed
for liquid and solid extract sample testing. Incorporation into the
germination/growth medium was proposed for liquid and solid leachate
sample testing. The choice of species to be used was deferred until
actual protocol drafting. Current OTS thought suggests using six species.
Positive control compounds suggested for both tests were ozone or
chlorine" for gas samples, and some choice of compounds from the reference
list of 10 being used by CERL in stress ethylene test work for liquid and
solid extract tests (Reference 7). The need to rigorously define plant
culture conditions in eventual protocol drafting was emphasized.
The concern that neither of the suggested procedures does life
cycle testing was expressed. But, as noted in Section 2.2, it was decided
to defer life cycle testing to Level 2, or until the Arabidopsis assay
becomes sufficiently developed.
15
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3.3 SUGGESTED LEVEL 2 PROCEDURES
Suggestions for appropriate kinds of tests for Level 2 included:
t Testing for the same (Level 1) responses with other species
t Testing for other responses with the same (Level 1) species,
testing responses indicative of effects on a different stage of
life, or full life cycle testing
• Testing fractionated effluent samples using the Level 1 test
procedures.
Which of these kinds of tests would be called for would depend on Level 1
results obtained. If the Level 1 plant tests gave positive (toxic)
responses, then Level 2 would emphasize sample fraction testing using the
same Level 1 procedures. If the Level 1 plant tests gave negative or low
toxicity results, but other bioassay tests triggered positive, then Level
2 plant testing would emphasize testing with other species, and testing
other responses, or other life cycle stages with Level 1 species. It was
recommended that both sample fractionation and effect confirmation were
important facets of Level 2, though the focus on one of these should be
determined by what was found at Level 1.
16
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SECTION 4
SOIL ASSAYS
As noted in Section 2.2, a responsive bioassay protocol for
terrestrial ecology effects should include representative tests for
effects on each of producer, consumer, and decomposer organisms. Plant
assays discussed in Section 3 focused on producers; soil assays discussed
in this section emphasize decomposers.
4.1 CANDIDATE TESTS
As in the plant session, the soil assay session opened by listing
candidate test procedures for consideration. Those advanced were:
• Soil core microcosm
• Endogenous respiration
• Specific microbial tests in culture, or in homogenized soil,
measuring:
— Specific respiration/substrate degradation
Starch
Cellulose
Pectin
Protein
— Nitrogen fixation
— Nitrification
— Sulfate reduction
-- Hydrogen (tritium) oxidation
t Sludge testing
Sludge testing was rather quickly eliminated from further consideration
because these are somewhat poorly defined tests with no standard
17
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procedures established. Furthermore interpreting data from these tests is
relatively unclear.
The use of cultured bacterial colony testing was also rejected.
Interesting organisms tend to be difficult to culture and single strain
testing lacks the broad sensitivity and integrated response aspects
desired of a Level 1 test.
Soil testing provides the desired integrated responses. However,
it must be remembered that soil testing can mask potential effects; soil
samples can be resistant to many toxic compounds and soil can rebound from
an initial insult. Thus cultured organisms can be highly sensitive to
toxic effects, whereas soil cultures would show less sensitivity.
However, this may not be a detriment; such behavior is more indicative of
real world impacts. Besides, the rebounding characteristics of soil
colonies allows differentiating between reversible and irreversible
effects.
The soil core microcosm test is currently specified in the
preliminary Level 1 protocol. However, its use as a Level 1 screen is
probably not warranted. The test is quite information-rich, but
interpretation of all the data provided is not a straightforward task at
present. Thus, as it stands the test can be considered too sophisticated
for use in Level 1. Besides, the test is too expensive ($1300 to 2500)
and too lengthy (8 weeks) for Level 1 needs.
Many of the objections to using the microcosm test would be
overcome if only a single response was measured. However, the choice of
an appropriate response is not simple. Calcium efflux (specified in the
current Level 1 protocol) is the easiest to measure and has a very low
coefficient of variation. Phosphate, sulfate, and nitrogen (ammonia,
nitrite, or nitrate) export are also potential responses. However, these
are all species likely to be found in text effluent samples, so test
results are liable to be difficult to interpret. Carbon dioxide release
(also specified in the current Level 1 protocol) as a measure of
respiration is also a possibility, but again, data interpretation would be
difficult. As testimony to the above, what little work has been done with
the soil microcosm test on complex effluents is seemingly contradictory
and difficult to interpret.
It was agreed, then, that use of the soil core microcosm test not
be recommended for use in a Level 1 protocol, but perhaps be further
developed for use in Level 2 assays. For current Level 1 needs it was
agreed that homogenized soil testing offered the best approach. Thus, the
choice of appropriate response to measure remained. Both specific
respiration and hydrogen oxidation were deemed too undeveloped at
present. Hydrogen oxidation as measured by tritium conversion to water
may prove, in the future, to be a very rapid, inexpensive, simple
response, perfect for Level 1 needs. However, current experience at the
University of Nevada, Las Vegas, and the EPA Environmental Monitoring and
Support Laboratory, Las Vegas, has only been developed with $03,
cadmium, and mercury.
18
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The most promising of the remaining responses were concluded to be
endogenous respiration (as measured by C02 release), nitrogen fixation
(as measured by acetylene reduction), and nitrification.
Endogenous respiration is a stable function, though not
particularly sensitive. However, much experience with measuring this
response exists. Work at CERL has shown a very low coefficient of
variation can be obtained (1.6 percent) in the rate of 0)3 released
versus time after the initial rapid release with test sample addition has
occurred. However, COg must be monitored for at least 30 days to allow
soil reequilibration, before irreversible toxic effects can be identified.
Both nitrification and nitrogen fixation are quite sensitive
functions. But nitrogen fixation is perhaps the more developed of the two
tests.
4.2 RECOMMENDED LEVEL I PROTOCOL
Based on the above discussion, the recommended soil assay for
incorporation into the Level 1 protocol is a homogenized soil test, where
endogenous respiration (C02 release), and nitrogen fixation (acetylene
reduction) are monitored. These two functions represent a combination of
a stable, though less sensitive, response (endogenous respiration), and a
highly sensitive response (nitrogen fixation), capable of identifying
irreversible toxic effects
Table 2 shows required test time, estimated test costs (for blank,
positive control, and three sample concentrations testing, four replicate
tests), and potential positive controls for each response. The test time
requirement is in addition to the initial 14 days equilibration period
before test sample addition. It was agreed that 30-day monitoring of
C02 release was needed to allow the mitigating properties of the soil to
take effect. A shorter, perhaps 14 day, test could be specified, but this
would also capture reversible effects and the rebounding properties of
soil would not be observed. Of course it could be argued that assaying
for immediate responses is appropriate for Level 1 screening, with system
resiliancy being an appropriate response to look for at Level 2. But, it
was noted that if the endogenous respiration assay was limited to short
term effects, the incidence of false positive responses might be
unacceptably high and much of the screening nature of the test might be
lost. Thus the longer term endogenous respiration test was recommended.
The recommended soil assay was proposed for use on all solid,
liquid, and gaseous samples. Liquid samples and leachate testing would be
introduced by soil irrigation and solid samples by mixing with the test
soil. Static gas sample testing would be performed by placing a test
atmosphere over the soil sample. Although there has been very little
experience with testing gas samples using this technique, it was deemed
straightforward enough to warrant inclusion.
In actual test implementation it would be wise to attempt to define
a standard soil to be used for all testing. A soil of a certain type,
obtained from a well-defined geographical location would suffice. A
19
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TABLE 2. RECOMMENDED LEVEL 1 SOIL ASSAYS
Test
Response
Time
Required
Estimated
Cost
Candidate Positive
Control Compounds
Endogenous Respiration 30 days $500-1000
Nitrogen Fixation
(C2H2 reduction)
24 hours $500-1000
Gas: Ethylene oxide
Liquid: 2,4 dinitrophenol,
Solid: Silver or cadmium
compound
Gas: Ethylene oxide
Liquid: Sodium azide
Solid: Silver or cadmium
compound
20
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synthetic soil would meet the standard soil needs, but work to develop a
good synthetic soil has been singularly unsuccessful. Homogenization
procedures should also be standardized. Final soil particle size should
be less than 2 mm.
For the future it was emphasized that other responses may become
more appropriate for Level 1 needs as they become developed. Hydrogen
oxidation and nitrification seem particularly promising in this respect.
In fact, hydrogen oxidation shows such good promise that its active
development deserves encouragement. It was suggested that pilot testing,
not only of the suggested Level 1 tests, but also the hydrogen oxidation
and nitrification assay procedures be considered.
4.3 SUGGESTED LEVEL 2 PROCEDURES
Analogous to what was suggested for appropriate Level 2 tests in
the plant assay discussion, the kinds of soil assays suggested for
consideration at Level 2 included testing effluent fractions using the
Level 1 procedures, and testing whole effluents using different tests or
monitoring different responses. Specifically, the soil core microcosm
test, and homogenized soil assays measuring specific respiration of a
variety of substrates were proposed. The use of a nitrification assay as
a backup was also suggested.
Again, as for plant assays, the emphasis given to fractionation or
confirmation in Level 2 would be based on what was found at Level 1. If
the Level 1 soil assay tests indicated significant toxicity, then effluent
fraction testing would be emphasized. However, if the Level 1 soil assay
tests gave negative responses, but other tests in the Level 1 protocol
triggered positive, then soil core microcosm and specific respiration
testing would be emphasized.
21
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SECTION 5
ANIMAL ASSAYS
With plant assays satisfying the need to test producer organisms
and soil assays satisfying decomposer needs, the third workshop session
treated animal assays which focus on consumer organisms. The need for
fast, simple assays which are inexpensive, require minimum test organism
maintenance costs, and which require small sample size, caused discussion
to focus immediately to insect assays. In addition, a higher animal test
using rats is already specified in the Level 1 health effects protocol.
Thus, only insect assays are discussed below.
5.1 CANDIDATE TESTS
As in the plant and soil sessions, discussion in the animal assay
session opened by listing candidate test procedures. Candidate test
species suggested were honeybees (Apis me!1 ifera), fruit flies (Drosophila
melanogaster), houseflies, and mosquito larvae, as much single compound
assay work has been done with all these species in the past. Appropriate
responses to measure included lethality (acute LCso), bioaccumulation,
enzyme activity, and behavioral alteration. However, it was quickly
decided that, for Level 1 needs, lethality was the simplest response to
measure, though the life span shortening endpoint deserves some
consideration (test time required increases to 3 weeks). Assaying gaseous
(fumigation), liquid, and solid samples (ingestion) using these insects
would be straightforward.
Honeybee testing is widely used, with standardized procedures being
employed by many laboratories throughout the country. Honeybees have the
advantage of possessing a clean genetic line; all bees in a colony are
derived from a single queen which lives an average of 8 years. Thus bees
don't easily develop resistance to toxic compounds. In addition,
honeybees are often more sensitive organisms than Drosophila. Using bees
as a test organism would also allow a logical sequence of test procedures
to be defined through Level 2 to Level 3, where community and social
studies could be performed. An auxiliary advantage bees have over other
insects is the fact that they are a beneficial insect, commercially
important as honey producers and pollenators.
Drosgphila testing is also quite widely used, thus this insect, and
toxicant effects on it, are also well understood. Drosophila colonies are
also easier to manage in a laboratory setting, and Drosophila are smaller
organisms than honeybees, thus a smaller sample would be required for
22
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assay needs. Houseflies and mosquito larvae have no clear advantages over
Drosophi'la as test insects. The fact that they are pests tends to act
subtly to their disadvantage.
5.2 RECOMMENDED LEVEL 1 PROTOCOL
Based on the above discussion the recommended animal assays for use
in Level 1 testing are the use of honeybees and Drpsophila to assay
gaseous (fumigation), liquid, and solid (ingestion) samples. Acute
("inhalation", or oral) LCso was the suggested biological response.
Estimated assay costs are in the $300 to $500 range. Caged insect assays
were proposed. The use of methyl parathione, Sevin^ or other carbamate
insecticides, or monosodium methane arsenate as positive control compounds
was suggested.
Specific procedures will await detailed protocol drafting. To aid
in this, the results of a recent workshop organized by EPA's Office of
Pesticide Programs (Dr. A. Vaughan, Coordinator) will become available in
the near future. The workshop, held in Washington, D.C. on November 8-9,
1978, and entitled "Conference to Develop Test Methods for Determining
Pesticide Effects on Bees," was organized to specifically draft procedures
for short term caged assays as well as chronic, 60-day, outdoor tests.
Results from this workshop should be available in early 1979.
5.3 SUGGESTED LEVEL 2 PROCEDURES
As with the plant and soil assays discussed above, suggested Level
2 animal tests will include effluent fraction testing and whole effluent
testing using different species (houseflies and mosquito larvae were
proposed) and measuring different responses (bioaccumulation, behavior
modification, and enzyme activity were recommended). Emphasis on
fractionation or confirmation testing will be applied based on Level 1
results, as discussed in Sections 3.3 and 4.3. The potential for
including a Drosophila mutagenicity assay at Level 2 also exists.
23
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SECTION 6
SUMMARY AND CONCLUSIONS
The recommended terrestrial ecology tests for incorporation into
the Level 1 bioassay protocol are listed in Table 3, which also indicates
the suggested method of sample administration for each potential sample
type (gas, liquid, solid). Specifying the specific details of each
individual procedure is deferred until actual protocols are drafted. The
responsibility for drafting individual protocols will be assigned by the
Bioassay Subcommittee of the IERL Environmental Assessment Steering
Committee.
The fact that none of the tests recommended for Level 1 can really
be considered adequately developed deserves emphasis. For all the tests,
experience with complex effluent testing is nonexistent, and for several
of the tests pure compound experience is limited. In addition, experience
with gas sample testing using the soil assay procedures, with liquid and
solid sample testing using the stress ethylene test, and with solid (not
leachate) sample testing using all the plant and soil tests is deficient.
For these reasons, pilot testing of all the proposed procedures is
strongly recommended before they become included in a definite Level 1
protocol. In addition, it must be emphasized that any set of test
procedures recommended now for inclusion in the EA methodology should be
taken only as the best available at present, given the constraints that a
test must be inexpensive, simple, and reliable. As other test procedures
develop, and interpretation of test data becomes more discriminating,
other protocols may become more appropriate.
Suggested kinds of procedures for Level 2 tests include:
• Effluent sample fraction testing using Level 1 procedures
• Testing for Level 1 responses in other test species (e.g.,
houseflies and mosquito larvae in the animal assays)
• Testing for different responses or testing for effects on
different life cycle stages with Level 1 tests species (e.g.,
specific respiration assays with a variety of substrates in the
soil assay; bioaccumulation, enzyme activity, and behavior
modification in the insect assays)
In addition the soil core microcosm test is recommended as a Level 2 soil
assay.
24
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TABLE 3. RECOMMENDED LEVEL 1 BIOASSAY PROTOCOL FOR TERRESTRIAL ECOLOGY
Plant Tests
Soil Tests
Animal Tests
ro
en
1. Stress ethyl ene and
foliar injury:
• Gas: Fumigation
• Liquid: Root
irrigation
(optional)
• Solid: Medium
incorporation
(optional)
2. Seed germination and
root elongation
1. Homogenized soil assay
measuring:
-- • Endogenous respiration
release)
• Liquid:
• Solid:
Irrigation
Leachate
irrigation
1. Honeybee acute
™ Nitrogen fixation
reduction)
• Gas: Fumigation
t Liquid: Irrigation
t Solid: Soil incorporation
and leachate
irrigation
• Gas: Fumigation
• Liquid: Ingestion
• Solid and Solid
Leachate: Ingestion
2. Drosophila acute
• Gas: Fumigation
• Liquid: Ingestion
• Solid and Solid
Leachate: Ingestion
T-1866
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Specific criteria for deciding Level 2 test needs based on Level 1
results should be outlined by the Bioassay Subcommittee as a whole.
However, it is proposed that:
• Level 2 terrestrial ecology testing emphasize using Level 1
test procedures on effluent sample fractions if terrestrial
ecology tests give positive, or high toxicity responses
• Level 2 terrestrial ecology testing emphasize testing
unfractionated samples with other species and other responses
if Level 1 terrestrial ecology tests give negative, or low
toxicity responses, but other Level 1 tests (health effects,
aquatic effects) give positive or high toxicity responses
In addition to the above protocol recommendations, several other
points deserve emphasis. These include:
• The development of other test procedures and other test
responses for potential inclusion into the phased analysis
should be encouraged. Specific tests/test responses showing
promise for meeting Level 1-2 needs include:
-- The Tradescantia assay
-- Miniaturization of the stress ethylene procedure
-- The Arabidopsis assay as a potential life cycle test
-- Monitoring nitrification in a soil assay
-- Monitoring hydrogen oxidation in a soil assay
t A rapid simple chronic effects assay is sorely needed. The
Arabidopsis procedure shows some promise here
• The need to ensure comparability between the IERL protocol and
those being defined by other EPA offices is of critical
importance
26
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REFERENCES
1. Hammersma, J. W., S. L. Reynolds, and R. F. Maddalone, "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment,"
EPA-600/2-76-160a, June 1976.
2. Lentzen, D. E., et £[., "IERL-RTP Procedures Manual: Level 1
Environmental Assessment (Second Edition)," EPA-600/7-78-201,
October 1978.
3. Duke, K. M., M. E. Davis, and A. J. Dennis, "IERL-RTP Procedures
Manual: Level 1 Environmental Assessment Biological Tests for
Pilot Studies," EPA-600/7-77-043, August 1977.
4. "Gas Sample Storage," Draft Report, Arthur D. Little, Cambridge,
Massachusetts, EPA Contract 68-02-2156, March 1979.
5. "Level 1 Bioassay Data Reporting Format," Litton Bionetics,
Kensington, Maryland, EPA Contract 68-02-2681, November 1978.
6. Rubinstein, R., et al.. "Test Methods for Assessing the Effects of
Chemicals on Plants," EPA-560/5-75-008, June 1975.
7. Ratsch, H. C., et al_., "Root Elongation, A Bioassay for Determining
the Phytotoxicity of Chemicals," Corvallis Environmental Research
Laboratory Draft Report, July 1978.
27
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APPENDICES
CONSULTANTS' REPORTS
28
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APPENDIX A
D. Tingey
EPA/IERL
29
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Corvallis Environmental Research Laboratory
SUBJECT: Bioassay Workshop held in Corvallis November 14 & 15 DATE: November 27, 1978
FROM. David T. Tingey
TO: File
The bioassay workshop meeting opened with a discussion presented by Dr.
Ray Merrill from IERL, RTP, who presented an overview of the environ-
mental assessment program and the role of the bioassays in the program,
sample constraints, and sample limitations. Also he defined level 1
bioassays as screening bioassays and level 2 bioassays as confirmatory
bioassays.
Specific discussions were held to select the bioassays for plants,
soils, and animals, with the constraints that the bioassays selected
must be already developed but not necessarily validated. Also there was
no money available for any new level 1 bioassay development.
Level 1 bioassays selected for plants were: Phytotoxicity to include
stress ethylene and foliar injury, and a test on plant growth probably
root elongation similar to the test we are developing for OTS. The soil
bioassays would incude a time for the soil to equilibrate and then it
would be stressed and for short term changes in respiration, and in a
specific process, such as nitrogen fixiation would be measured. As a
level 1 bioassay, there was also expressed interest in the tritium
oxidation system developed by Las Vegas, but there was no money available
to develop the protocol. Level 1 bioassays proposed for the animal
system were to determine LC50 for honeybee and for drosophila.
Level 2 bioassays for terrestrial systems would be initated under either
of two criteria. One the terrestrial test level 1 bioassays showed high
toxicity. If this were the case then Level 2 bioassays would be run of
fractioned samples using the same tests as Level 1. However, if level 1
bioassays for the terrestrial component did not show high toxicity but
either the aquatic or the human health tests show high toxicity, level 2
bioassays would be run on unfractioned samples and would include addi-
tional species in the same tests.
The specific level 2 protocols for plants would include phytotoxicity
tests, a root elongation test as proposed in level 1 and would also
include a full life cycle test, most likely Arabidopsis. Level 2 soil
tests would include a soil microcosm and specific substrate respiration
using C-14 labeled compounds such as starch, pectin, cellulose, and
protein. Level 2 animal bioassays would include behavioral changes in
addition to LC50 data.
30
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The two proposed grants to be funded in FY 79 from the IERL money were
discussed. The proposal of Lyle Craker to miniaturize the stress ethylene
system was well received by the workshop participants. They felt this
was a valuable addition to the bioassay protocol systems. However, the
grant proposal of Ken Williamson from OSU did not receive a good recep-
tion from the woshop participants.
Protocols have been proposed for plants soils and animals for both level
1 and level 2 as a result of the workshop. It is now necessary to
determine if all of the proposed protocols will be implemented in a
Level I sampling program. For the tests selected at levels 1 and 2
protocols will need to be prepared and these protocols will need to be
validated during pilot studies. Discussions need to be held with IERL
concerning the availability of resources to have the protocols written
and validated during pilot testing.
31
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APPENDIX B
S. Sandhu
EPA/HERL - RTP
32
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
DATE: December 14, 1978
SUBJECT: Summary of Terrestrial Bloassay Workshop Held at Corvallis, Oregon,
November 14-15, 1978
FROM-. Shahbeg Sandhu
Biologist, HERL (MD 68)
TO: James Dorsey
Chief, Process Measurement Br., IERL (MD 62)
Dr. Ray Merrill of PMB, IERL-RTP presented the general philosophy for
the biotesting of industrial effluents and emissions. He outlined the
objectives and approaches for the phased approach for environmental
assessment program.
The workshop participants expressed concern for the lack of bioassays in
Level I matrix to measure chronic effects. They felt that'all the
bioassays except Ames test measure the acute toxic effects and that
acute effects may not be reliable indicators of chronic effects.
It was emphasized by the workshop participants that although health
effects and aquatic bioassays proved to be adequate in pilot studies,
there is a definite need for biotesting of industrial wastes using green
plants and soil microflora. It was recommended that terrestrial bioassays
representing three basic processes in ecosystems i.e. photosynthesis,
reproduction and consumption should be included in Level I biotest
matrix.
The workshop participants were briefed on the difficulties experienced
in the pilot studies in applying terrestrial bioassays, especially thos
designed to measure the biological effects of gases.
The following bioassays were considered for Level I testing:
y
Plants
1. Stress ethyl ens production and fclisr nu
2. Tradescantia micronuclei and pollen tube elongation
3. Seed germination and seedling growth
4. Pea tendril coiling
5. Tomato petiole angle measurement
6. Bean hook opening
7. Chromosomal breaks in beans
8. Stomatal movements
9. Pollen germination
33
EPA FORM 1320-6 (REV. 3-76)
-------
The problem with most of these bioassays except numbers 2 and 3 listed
above, is that they have never been used systematically for evaluating
the toxidty of complex mixtures. Two among these bioassays were selected
for further consideration. These were: 1) seed germination and seedling
growth; and 2) stress ethylene production and foliar injury.
The stress ethylene bioassay designed primarily to measure the effects
of gases 1s still under development. The previously approved stress
ethylene production protocol included in IERL-RTP Level 1 Bioassay
Manual, required the transportation of large volume of gases into labora-
tory. The pilot studies performed by IERL-RTP have clearly shown the
transportation of large volume of gases to be impractical. In addition,
there are serious reservations in the scientific community on the reli-
ability of tills bioassay. A new version of this bioassay is under
development at the University of Massachusetts. This research effort
sponsored by EPA, If sucessful, will eliminate the necessity for trans-
porting large volume of gases from Industrial sources into laboratory.
However the development and implementation of this bioassay will take
two to three years. I do agree that foliar Injury test has some merit
but its sensitivity to low concentrations of gases has to be verified.
The seed germination and seedling growth bioassay has been used by
biologists for the past thirty years to measure the genetic damage
caused by radiation and chemicals. It 1s en inexpensive, rapid, simple
and reliable bioassay. However it suffers from the basic limitation of
need for transporting large volume of gases Into laboratory for seed
treatment. For testing the liquid and solid samples, this bioassay
appears to be very useful. However, some developmental work on this
bioassay is needed, which, I do not believe should be too expensive or
should take too long to accomplish. The following areas need to be
addressed relevant to .this bioassay:
1. Growth medium; I.e. peatmoss, sand, etc.
2. Climatic variables; .i.e. temperature, humidity, light intensity, etc.
3> Plant species; i.e. lettuce, tomato, grasses, raddish, etc.
One of plant species which may be of great use in biotesting is Arabidopsis.
It has very small seeds and short life cycle. I understand that Dr.
David Tingey is already looking into the possibility of utilizing this
species.
It is unfortunate that Tradescantia micronucleus assay was not given a
serious consideration. This bioassay measures the genetic damage induced
by environmental pollutants in the germ cells. The preliminary studies
performed by Dr. Te S. Ma of the Southern Illinois University, on the
diesel exhaust has shown Tradescantia test to be very sensitive for in-
situ measuring the effect of gases.
34
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Soil Bloassays
The use of soil microflora for testing the biological effects of Industrial
wastes is fretted with a lot of problems. The numerous types of soils
and variables associated with soil makes it very difficult to implement
the biotests based on the use of soil.
After considerable discussion the following soil bioassays were recommended
for consideration for Level I biotesting.
1. Seed germination and seedling growth
2. Respiration
3. Nitrogen fixation
The major limitation for these biotests is that although these methods
have been known to biologists for a long time, these have never been
used for testing the complex environmental mixtures. The appropriate
protocol for each of these bioassays has to be developed and validated
before these could be used for routine biotesting.
The workshop participants recommended that the nethods of extracting and
leaching toxicants from soil should be further examined.
Animal Bioassays
Two bioassays based on the use of honeybees and Drosophlla were recommended
for consideration for Level I testing. Both tests looked very promising.
Drosophila has been used quite extensively for genetic and behavioral
studies. Honeybees although somewhat difficult to manage appear to be
useful experimental organisms.
Level I to Level II Trigger Criteria
It was suggested that the data from terrestrial bioassays may be expressed
in terms of degree of toxicity i.e. not detectable, low, medium, and
high. Those samples showing high toxicity should be tested in Level II
bioassays. The samples showing less than high toxic response in terrestrial
bioassays and showing consistent toxic response in health effects or
aquatic bioassays, should be retested using Level I terrestrial bioassays
employing additional species of plants and insects. If the effects
initially detected are confirmed in other representative species, then
Level II tests should be performed on fractionated samples.
Level II, testing will be performed on fractionated samples uisng the
same bioassays as in Level I. In addition, the life cycle studies in
plants and insects were recommended.
35
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It was recommended that soil microcosms should be included in Level II
test matrix.
Dr. David Tingey assumed the responsibility of writing the protocols for
the proposed bioassays. It was recommended that the reliability of
these bioassays should be evaluated in pilot studies.
In summary the following bioassays were recommended for Level I pilot
studies to evaluate the terrestrial effects.
Plants
Soil
1.
2.
1.
2.
Animal
1.
2.
Stress ethylene and foliar injury
Seed germination and seedling growth
Nitrogen Fixation
Respiration
Drosphila melanogaster
Honeybees
For Level II the use of plant life, cycles and soil microcosms were
recoi-anended. Among all the bioassays included in Level I, terrestrial
bioassays appear to be least developed. After pilot studies, no more
than three terrestrial tests should be selected for Level I.
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APPENDIX C
J. Bromenshenk
University of Montana
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Reference: Acurex Project 7347
Contract No. 68-02-2611
Subcontract RB82542A
SUMMARY AND DISCUSSION OF THE TERRESTRIAL BIOASSAY WORKSHOP
DEFINITION OF TERRESTRIAL ECOLOGY BIOASSAY PROTOCOL
FOR ENVIRONMENTAL ASSESSMENT PROGRAMS
by
Jerry J. Bromenshenk, Ph.D.
Ecologist/Entomologist/Consultant
733 West Sussex, No. 3
Missoula, Montana 59801
November 25, 1978
INTRODUCTION
The Terrestrial Bioassay Workshop held November 14 and 15, 1978, in
Gorvallis, Oregon, reviewed the status of~~the IERL-RTP Environmental Assessment
(EA) Methodologies with specific reference to Terrestrial Biotests. The
stated objectives were to specify a battery of terrestrial ecology bioassay
tests for the Level 1 protocol, to formulate Level 1 to Level 2 decision
criteria, and to propose Level 2 terrestrial bioassay protocols. The parti-
cipants were asked to address the critical question of whether the current
protocols proposed for the IERL-RTP terrestrial ecology tests were appropriate
and if there were other tests available for use which would be better than
those proposed or which also should be included.
The following viewpoints were expressed by workshop participants and are
relevant to a summary and discussion of the outcome of the meeting:
(1) The workshop organizers hoped to "cast in concrete" a Level 1
terrestrial ecology bioassay protocol (TEBP) which could then be
presented for program review.
(2) While acknowledging the need for specifying a Level 1 TEBP, the
attendees stressed that any bioassays proposed during the workshop
must be defined as those thought to be the best tests currently
available but not necessarily the best tests which could be
performed—a major consideration for the tests proposed was that
results be reproducible by different laboratories.
(3) The Level 1 biotests, which were recommended, demonstrate only acute
effects. But there is no evidence that acute effects are indicative
of chronic effects in terms of biological responses. Chronic effects
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may be considerably different from acute effects. Also, the biotests
proposed for use do not consider latency. Thus, it must be recog-
nized and stated in the IERL-RTP program that these responses cannot
be addressed because of a lack of suitable, well-developed, repeat-
able bioassays which also can meet the stated Level 1 cost and time
constraints. The conclusion should be that at present these tests
cannot be performed, not that they don't need to be done.
(4) Ideally the decision criteria for specifying the needs for Level 2
terrestrial ecology tests based on Level 1 results would involve
unequivocal triggers to determine the necessity of Level 2 testing.
However, the consensus of the workshop participants seemed to be
that limitations of currently available bioassays and a lack of
knowledge concerning the complex ecological effects of individual
pollutants, not to mention multimedia, multipollutant streams,
requires that a step by step, case by case, decision process by
qualified experts be utilized.
(5) The workshop committee felt that they could not set forth a set of
perfect Level 1 bioassay tests, nor could they establish, in many
cases, easy to follow, clearly-delineated decision criteria for
proceeding from Level 1 to Level 2. Despite these problems, the
committee supported the inclusion of terrestrial ecology bioassays
in the IERL-RTP program. Bioassays give the actual reactions of
individual organisms or populations to pollutants, they respond to
synergistic, additive, and antagonistic properties of mixtures of
pollutants, and they integrate responses through time. Chemical
and physical characterization of pollutant streams can attempt only
to predict a very limited number of biological responses to rela-
tively few substances for which there is an existing data base
concerning effects such as toxicity or mutagenicity. Also, this
usually only can be done for individual chemicals rather than
mixtures.
WORKSHOP SUMMARY AND CONCLUSIONS
The participants of the Terrestrial Bioassay Workshop recommended a set
of specific tests to be used in a terrestrial ecology bioassay protocol for
the Level 1 Environmental Assessment Program. Also, they indicated appropriate
categories of tests for a Level 2 protocol. The bioassays for Level 1 were
chosen keeping in mind the need to evaluate pollutant streams which involves
a chemical test matrix of liquid, solid, and gaseous samples. The recomen-
dations for the protocols for Level 1 and Level 2 bioassays were that:
(1) Plant bioassays for Level 1 focus on phytotoxicity and be comprised
of: (a) the stress ethylene plant response tests coupled with
determinations of foliar injury, and (b) seed germination tests
linked to seedling growth observations. Level 2 testing would
incorporate fractionation of chemical samples, tests of the responses
of additional plant species, and tests which would include full life
cycles of plants.
(2) Soil bioassays for Level 1 concentrate on endogenous soil respiration
but also include tests of ethylene reduction (an indication of
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nitrogen fixation). Possible inclusion of a test of effects on
hydrogen-oxidizing microorganisms using the tritium-labeled hydrogen
(tritiated water) procedure developed by EPA-Las Vegas was suggested.
Level 2 testing would utilize soil microcosms, specific substrate
respiration, and presumably fractionation of chemical samples for
further testing.
(3) Animal bioassays for Level 1 examine zootoxicity and encompass
in vivo toxicity tests using invertebrates in addition to the
mammalian bioassays to be performed under the health effects testing
program (IERL-RTP Procedures Manual: Level 1, EPA-600/4-77-043,
April, 1977). Honeybees (Apis mellife-pa) and/or fruit flies
(.Dvosaphita melanogaster) were proposed as the test organisms.
Level 2 testing would take into account fractionated chemical
samples, additional invertebrate species, and other biological
responses such as evidenced by behavior, physiology, reproduction,
and full life cycles.
The decision criteria for specifying Level 2 terrestrial ecology test
needs based on Level 1 results was one of the most difficult issues addressed
at the workshop. However, there were several points of general agreement:
(1) It should not be necessary to perform Level 1 bioassays nor to make
decisions concerning Level 2 for chemical streams containing
substances known to be extremely hazardous or containing unacceptable
levels of these substances. For example,-a chemical stream with a
pH of 2 would be expected to kill just about anything in it, while
streams containing quantities of arsenic, cyanide, or mercury would
be subject to existing regulations and standards concerning accept-
able levels of release of the contaminants and the control of their
release into the environment. It is important that the final
procedures manual contain a statement about not performing needless
bioassays on chemical streams of this sort and provide guidelines
towards making this determination.
(2) Ideally, Level 1 results should provide easy to interpret warning
flags, yes-no choices, or triggers which would decide both the need
for Level 2 testing as well as specify the types of tests to be
performed. In some cases, this will probably occur. For example,
if the results of all the Level 1 chemical and biological tests
indicate no hazard, the decision is fairly straightforward; there is
no need to proceed to Level 2. On the other hand, if all the
chemical and biological tests indicated that the degree of hazard
was high, then one would definitely proceed to Level 2. However, if
only some of the chemical and biological tests indicated a possible
hazard, if some indicated a high degree of hazard and others low or
none, or if most suggested that the degree of hazard was moderate,
then the decision criteria would become much more complex and would
not be simple or clearly defined.
(3) Obviously, there is a need to prioritize or rank chemical streams in
terms of harmful biological (health, ecological) effects based on
the Level 1 results. Ranking within a single biotest such as
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assigning toxicity values (no detectable toxicity, low toxicity,
moderate toxicity, and high toxicity) based on Lethal Dose 50% of
specified concentrations to rats (as in Figure 4, page 11, of the
Draft document entitled Bioassay Procedures for Screening Complex
Effluent Samples) can be easily standardized. But prioritizing and
ranking the results of a battery of bioassays is much more difficult
and is dependent on a clear definition of goals and an understanding
of what the tests results mean.
(4) Limitations of knowledge concerning problems such as substances for
which there is little or no data on toxicity or mutagenicity to
organisms, synergisms and antagonisms of chemicals which may alter
harmful effects, the adequacy and the representativeness of the
specified Level 1 tests in terms of the biological effects not only
to a few specific systems but also in a more holistic sense, and the
weighting of Level 1 bioassay results will necessitate, in most
cases, an ad hoc decision process which must be conducted by quali-
fied experts.
During the final session of the workshop, participants expressed concern
for quality controls at all stages or levels of the test program from the
initial sample collection (including the planning of its collection) through
the final data interpretation and reporting. The proposed controls for quality
assurance included the use of standard control samples, reference controls,
single blind controls, and splits of the samples and tests between laboratories
performing the assays. Reference compounds for the toxicity tests should
include materials representative of inorganic, organic, and organo-metallic
pollutants. Workshop participants from the Corvallis Environmental Research
Laboratory had participated in the development of the stress ethylene plant
response test and indicated that they had a list of phytotoxic chemicals that
could be used as a data base from which to pick toxicity references. For the
other Level 1 bioassays, 2-4-nitrophenol was suggested for the soil respiration
inhibition test, while chemicals from the pesticide testing programs were
suggested for use in the invertebrate tests and included monosodium methane
arsenate, an organophosphate insecticide (methyl parathion, preferably Penncap
M ^y), and a carbamate insecticide (Sevin Qy).
The need to completely describe the procedures for each of the proposed
Level 1 bioassays was recognized, but it was felt that this could not be
accomplished at the workshop. There was some concern that the bioassays that
had appeared in the previous IERL-RTP procedures manual were not precise
enough concerning many of the procedural details.
GENERAL DISCUSSION
Some points concerning the discussions at the Terrestrial Bioassay
Workshop deserve further mention.
Levels 1, 2, and 3 were defined and for the most part follow the expla-
nation given in the background information documents provided at the meeting.
However, according to at least one of the background information documents,
chronic sublethal effects would be relegated to Level 3. I do not believe
that the workshop participants were aware of this. There was discussion as to
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whether chronic sublethal tests belonged in Level 2. Some felt that inclusion
of these tests at Level 1 was desirable, although probably not feasible.
It should be mentioned that in choosing Level 1 bioassays, the workshop
group took into account many factors such as chemical matrix (gas, solid,
liquid), biological processes (photosynthesis, decomposition, consumption,
nitrogen fixation, etc.) and responses (growth, development, reproduction),
and procedures (cost, sample type, relevance, availability, comparability).
The stress ethylene plant response test was thought by some members of
the group to be overly general, while others were concerned about the ability
to quantify foliar injury, since it may be expressed in many different ways
(lesions, chlorosis, mottle, necrosis). The idea of coupling the two should
offset some of the uncertainties of each taken alone. There was some mention
that not all investigators who have performed the stress ethylene test were
satisfied with it, but no one at the meeting explained what specific problems
had been encountered. Apparently, space was a problem, and a miniaturized
version of the test using plants grown on agar in test tubes is being
developed. I should think that such a highly artificial system would have to
be very carefully evaluated against full size controls in order to determine
the effects of miniaturization. Also, there was no mention made at the meeting
that foliar injury linked with histopathological examinations of the damaged
area can provide considerable information concerning the nature of the damage
and may provide a useful "fingerprint" for specific chemicals or chemical
classes.
Seed germination and seedling growth were chosen because these provide
information concerning two important biological processes—reproduction and
growth. There exists an extensive body of knowledge concerning this type of
test, and it was surprising that germination and growth tests had not been
included in the original bioassay protocol.
Pollen tube elongation and pollen viability were favored by some members
of the committee but were excluded because of problems concerning pollen
viability during storage and the interpretation of the results.
Tradeseantia was suggested as a potentially valuable test organism for
mutagenicity in higher organisms (eukaryotes as compared with the prokaryotes
used in the Ames test). The Tradesoantia assay involves a color change of
cells of the stamens or filaments of the flowers. Although it was not brought
out at the meeting, the main problem with this method may be that large
numbers of cells must be counted to give significant results, especially if
the chemical dosages are very low (a background incidence of the color change
must be taken into account).
Several other plant bioassays were proposed and discussed, but none
seemed to offer as much potential as those chosen, or the tests were not as
well developed.
Since the soil microcosm test was the only Level 1 test above a species
level, it received considerable attention. However, the sensitivity of the
system was considered to be low, and the test appeared to be data rich but
difficult to interpret. The test probably would be difficult to standardize
because of chemicals already present in the soil cores. There is a question
of which soil types to use and a problem of how to obtain a standard core.
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On the other hand, procedures such as soil respiration have had many
years of usage, and these procedures have been standardized and are considered
to be reliable. Several alternative or additional tests were advocated,
including a Mason jar soil CC>2 test, nitrogen fixation, nitrification, bio-
degradation profiles (starch, cellulose, protein, pectin), and hydrogen-
oxidation (tritiated water). Many of these seemed to have merit, and it was
suggested that they be examined for possible inclusion at a later date. Soil
microcosm testing was moved to Level 2, since it tends to be data rich. It
was felt that this data richness might tend to send everything to Level 2,
whenever the test was performed.
During the discussion of the animal bioassays, concern was expressed that
many major groups of animals, such as birds and invertebrates, were not repre-
sented. No one at the meeting was prepared or qualified to indicate an appro-
priate bird test. However, insects were deemed to offer considerable potential
as bioassays. There appeared to be mutual agreement that insects should be
included, and the debate turned to which species to use. Honeybees and fruit
flies were given priority. I shall discuss this topic more completely in the
specific discussion section of this paper.
\
Quality assurance was recognized as an essential aspect of the bioassay
program. Care must be taken to insure that quality assurance is built into
the protocols. Problems such as the effects of sample integrity and age,
effects of collection procedures, the form in which chemicals should be tested,
the need for the testing of chemical fractions in Level 2, whether Level 2
requires new or additional chemical samples, and the need to concentrate
samples for testing were discussed on a case by case basis. However, quality
assurance and these other problems require that qualified experts be involved
not only in the development of the procedures manual but also during the
testing. There must be cooperation and involvement by biologists and chemists
during all stages of the program to insure its validity and success.
SPECIFIC DISCUSSION
In this section, I shall confine my remarks to my personal expertise,
i.e., environmental entomology.
There are several programs that already require or will soon require
testing of pesticides and other toxic substances on honeybees because there
have been substantial losses of bees in recent years attributable to the use
or misuse of insecticides. This in turn affects the products and services
provided by bees—honey, wax, and pollination. In addition, honeybees have
been tested and rated according to their sensitivity to hundreds of chemicals.
Toxicity testing procedures for bees are well-defined. Also, honeybees appear
to be more susceptible to harm from many toxic chemicals than other animals
including man. There is a chance, although slight, that chemical toxins may
pass to human food chains through honey.
Since a queen honeybee may live for several years and is the only repro-
ductively mature member of a colony, resistance to hazardous substances is
unlikely to evolve, although resistance to pesticides, for example, has
evolved rapidly in many other insect species. Since all worker bees in a
colony are haploid offspring of an individual queen, genetic variability can
be limited, and controlled breeding can be used to convert bee colonies to the
"same" genetic composition.
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Finally, honeybees not only could be used in a variety of ways in a
Level 1 and a Level 2 protocol, but they could be used in a Level 3 protocol.
In fact, a few monitoring programs in the United States and Europe presently
are using honeybees in a manner representative of the projected Level 3
protocol.
Conversely, Drosophila melanogasteip is a good test organism. Extensive
genetic research has been conducted with it. This insect also has been used
in bioassays of pollutants. It would be difficult to say whether there is a
more extensive body of information concerning Drosaphila than Apis mellifera
but suffice to say that both have been studied over a very long period of years.
Drosophila, at first glance, would appear to be somewhat easier to rear
and maintain than honeybees. But once an apiary has been established, honeybee
colonies are more or less self-sustaining and require very little care.
Honeybees tend to be more vigorous if the colonies are kept outdoors and the
bees are free to fly. They can be brought into the laboratory for testing,
and also they can be tested in the field (field testing using Drosophila would
be less convenient). In order to be able to conduct bioassays all year round,
a laboratory doing honeybee bioassays probably would have to be located in the
south or in a coastal area of the United States—areas with warm winters.
The preceeding summarizes comments regarding insects made at the Corvallis
workshop. The following comments reflect ray own opinions and may not represent
those of the other members of the workshop committee.
Chemicals other than pesticides have been shown to have significant
effects on many terrestrial insect systems. These include gaseous, liquid,
and solid chemical forms and include smog, ozone, hydrocarbons, nitrous oxides,
fluorocarbons, sulfur oxides, dusts, major and trace elements, radionuclides,
and acid mists. Routes of entry into the body of an insect may be by inhala-
tion, injestion, or penetration through the cuticle. Biomagnification may
occur as toxic materials are passed along insect food chains.
Biologically active pollutants have been shown to affect the biochemistry,
physiology, and behavior of insects. Toxins may produce both lethal and sub-
lethal effects such as shortened life spans, reduced hatchability, lowered
fecundity, genetic alterations (mutations, teratorlogies), toxicant avoidance,
disorientation, memory loss, and temporary and/or permanent behavioral modifi-
cations. Thus, effects may occur at any level of organization from the
biochemical to the ecosystem.
I have attached to this report two papers (References 1 and 2) which
reference my comments and provide a more detailed discussion of pollutant-
insect interactions.
It should be noted that honeybees and fruit flies are not the only insects
that have been studied as regards hazardous chemicals. There exists a large
quantity of information concerning insect pests of forests and agriculture,
butterflies (industrial melanism), ground-dwelling beetles (predators and sapro-
phages), mosquitoes, and soil arthropods.
The workshop left open the decision of whether to use honeybees or fruit
flies or both. Each species has its own unique advantages and disadvantages
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for use in a bioassay program. It may well be that each may be used but for
different tests, either in Level 1 or especially in Level 2.
During post-workshop inquiries, I found that bioassay protocol using
honeybees is being put together for EPA's Pesticide Hazard Evaluation Division.
A workshop was held about three weeks before the Terrestrial Bioassay Workshop.
The outcome of that workshop was the framework for a screening protocol
concerning the effects of pesticides on honeybees. This protocol should be
available for comment by February, 1979. Information concerning this program
can be obtained from Allen Vaughn, OPP, EEB, HED, Room E 107, TS 769, 401 M
Street SW, Washington, D.C. 20460, telephone (202) 426-0224.
Briefly, the proposed pesticide protocol involves acute toxicity, dose-
response , linear regression curve determinations using caged bees in the
laboratory or possibly colonies in the field. The procedures have been
developed and tested by E.L. Atkins and associates at the University of
California, Riverside, and by R.J. Barker and associates at the USDA-ARS Bee
Research Laboratory,.Tucson, Arizona.
A second set of tests to be used will examine sublethal or chronic
effects which may be disruptive to the social organization of bee colonies
and which could be far more important in terms of harm than mortality per se.
The projected tests would consist of 60-day tests, probably carried out in a
screened greenhouse. All experiments would start with clean equipment. The
choice of the number of bees and the type of hive used would be left, within
certain guidelines, to the investigator. At 30 days and at 60 days, the
amount of brood would be determined by measuring the comb area covered by
brood. At the end of 60 days, determinations such as the weight of bees
(indicates number) and the weight of the product (honey) could be made.
During the run period, observations could be made concerning rates of mortality
(as measured by dead bee traps), abnormal adult behavior, morphogenic changes,
etc. Controls and standards would be utilized where appropriate.
Although this protocol is being developed with reference to pesticides,
it should be easy to adapt to other hazardous chemicals. Liquids and solids
could be administered in controlled dosages in water or food (honey, pollen)
or could be misted or dusted onto the bees. Gases would require some type of
fumigation apparatus similar to that needed for the plant bioassays.
Drosophila melanogastep is a very useful organism for genetic and life
cycle studies, although possible evolution of resistance would have to be
continuously monitored. Drosophila's life span is relatively short. Rearing
procedures are standardized. A fumigation apparatus using half pint milk
bottles (the standard rearing containers) has been developed by M.E. Ginevan,
Argonne National Laboratory, Argonne, Illinois 60439, and D. Lane, Department
of Civil Engineering, University of Kansas, Lawrence, Kansas 66045. These
investigators have conducted effects tests of sulfur dioxide over the life
cycle of the flies and on specific life stages (egg, larvae, adult). They
carried out experiments over periods from 4 to 20 days using continuous, "low"
level (0.4-0.7 ppm) sulfur dioxide fumigation. The types of responses that
they quantified included increases in development time, decreases in survival,
genetically-controlled differential treatment response, and reduced activity
levels (torpid, fed sporadically).
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The workshop committee recommended that the Level 1 insect bioassays
concentrate on in vivo toxicity. However, since both Drosophila melanogaster
and Apis mellifera have short life cycles, it is feasible to incorporate
determinations of effects such as shortening of life spans simply by contin-
uing the toxicity evaluations for a period of three weeks. Drosophila has a
generation time of about ten days, while honeybee queens develop from egg to
adult in approximately 15 days, workers in 21 days, and drones in 24 days.
The queen mates 5 days after the imaginal molt, and she begins to lay eggs
2 days later. Shortening of lifespans of honeybees affects honey production
since it is the oldest bees that do most of the foraging.
Either in Level 1 or Level 2 if a sufficient number of graded dosages
were administered, one could perform linear regressions or probit analyses of
dose-response. This information could be applied towards making predictions
(restricted to insects) about the possible consequences (toxic effects) of
releasing specified amounts of the chemicals of concern into the environment.
Suggestions made at the workshop for Level 2 insect bioassays included
fractionation of chemicals for toxicity and other types of evaluations, the
use of additional insect species, and the assessment of effects on full life
cycles, reproduction, and behavior.
Drawing from experience in pesticide testing, additional insect species
might include mosquitoes, aphids, crickets, or grasshoppers. Mosquitoes are
very sensitive to many toxins, often more so than even honeybees or fruit
flies. Crickets are convenient test animals and some species have a pest -
status. Grasshoppers and aphids are two major pests that insecticides are
designed to control. Any number of other major pest insects such as those of
forests, croplands, orchards, or gardens could be used. The insecticide
industry conducts tremendous numbers of tests concerning the efficiency and
mode of action of their products on target organisms.
I should think that a mutagenicity assay using Drosophila would be appro-
priate for Level 2. Like the proposed Tradescant-ia mutagenic test, this would
provide information specifically concerning higher (eukaryotic) organisms,
unlike the Ames tests that focuses on bacteria (prokaryotes).
Another type of test which might be considered for Level 2 is a morpho-
genic test. For example, E.L. Atkins and associates at the University of
California, Riverside, have been investigating morphogenic responses of
honeybees to pesticides. In this test, the chemical(s) is inserted with a
micro-pipette into brood at different concentrations and at different stages
of the insects' development. As the adults emerge, the number dead, deformed,
underweight, etc. are recorded. The results are then correlated with dosages
and stage at which the chemical was introduced. Thus, the test provides an
assessment of interactions with growth and development.
Insect behavior could be a very sensitive and very rapid bioassay and
would provide information not given by any test other than the aquatic effects
assay using Dapknia. Abnormal web spinning by spiders (Arachnids) has been
used by pharmaceutical companies in evaluations of the effects of certain
drugs. The rate of singing or chirping by crickets is altered by chemicals
and other environmental stresses. Honeybees demonstrate disorientation,
losses of memory, or impaired locomotor activity in response to sublethal
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dosages of insecticides and chemicals such as carbon dioxide and nitrous
oxides. Pence and Lomax (Biodynamics of the Excised Honey Bee Abdomen.
Insect World Digest 1(1):16-24, 1973) reported that the movements of excised
abdomens of bees can be used as signatures for specific pollutants. They have
performed the procedure for odors, gases, polluted water, and pesticides in
soils.
Effects on reproduction such as altered fecundity and offspring viability
seem to me to be part of life cycle observations and not a separate category.
The Level 2 tests that I have indicated are meant to be illustrative and
not an attempt at a comprehensive treatment of the subject. Any number of
potentially useful and informative bioassays of chemical streams could be
performed using insects or other invertebrates. I believe that insects offer
many advantages as test organisms in environmental assessment protocols
because of their abundance in species and numbers, small size, short life
spans, importance to ecosystems, sensitivity to environmental perturbations,
and history of use in evaluations of chemicals in pollution monitoring and in
ecological studies.
REFERENCES
1. Bromenshenk, J. J., and C. E. Carlson, "Impact on Insect Pollinators,"
in Air Pollution and Metropolitan Woody Vegetation, W. H. Smith and
L. W. Dochinger, eds., Yale University Printing Service, 1975.
2. Bromenshenk, J. J., "Yet Another Job for Busy Bees," The Sciences,
Vol. 18, No. 6, p. 12, July 1978.
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APPENDIX D
K. Duke
Battelle-Columbus Laboratories
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SUMMARY OF
EPA TERRESTRIAL BIOASSAY WORKSHOP. NOVEMBER 14, 15. 1978
Summary
The EPA Terrestrial Bioassay Workshop was held in Corvallis,
Oregon November 14 and 15, 1978, under the direction of Acurex Corporation.
The workshop focused on the phased approach to biological testing of
complex effluents from energy and industrial processes and had three
objectives:
1. Develop recommendations for Level 1 terrestrial bioassays
2. Develop Level 1 to Level 2 decision criteria
3. Suggest potential Level 2 testing procedures.
Participants included professional researchers familiar with
terrestrial bioassay procedures and/or EPA's phased approach to testing.
The summary presented here is taken from the author's notes and
represents his understanding of the conclusions reached at the workshop.
The author's comments on these conclusions is presented in a separate
section of this report.
Level 1 Test Procedures
Level 1 testing is the initial screening level of the phased
approach. Two terrestrial tests, plant stress ethylene and soil
microcoms, had been originally proposed for the bioassay protocol. However,
EPA pilot studies had revealed some problems with these tests. One
objective of this workshop was to develop recommendations for Level 1
terrestrial tests which would meet Level 1 criteria and the
acceptance of the EPA Bioassay Subcommittee and other technical experts.
In developing the recommendations at least six criteria were considered
for each candidate test: (1) cost, (2) sample requirements (form and
quantity), (3) relevance to terrestrial ecology and biology, (4) availability
(existence of accepted test procedures and a data base developed from
previous use of test), (5) comparability (among different labs performing
the test), and (6) response (easily measured and sensitive).
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Four candidate tests were suggested for the plant (photosynthetic)
aspect of the terrestrial environment. They were foliar injury/stress
ethylene, Tradescantia mutogenicity/toxicity, seed germination rate
and seedling growth and dvelopment (several specific tests). Three
of the four tests (Tradescantia was dropped) passed the screening and were
recommended for Level 1 tests.
A second aspect of the terrestrial environment is the process
of decomposition in the soil. This process is important because it is
key to nutrient recycling in ecosystems. Numerous suggestions for suit-
able tests were made including several bacterial tests, nigrogen fixation,
sludge testing, and the soil microcom tests. Two tests, ethylene
reduction and total endogenous soil respiration, were finally recommended
for Level 1.
Animal (consumer) tests suggested for consideration were
primarily acute toxicity tests using mosquito larvae, Drosophila, honey
bee, and others. The honey bee/Drosophila actue toxicity tests were
finally recommended.
Level 1 to 2 Decision Criteria
While some time was spent discussing this topic, no definitive
recommendation was made. The results from Level 1 testing—biological and
chemical—are needed to priortize the waste stream tested. Those high
in priority are subjected to Level 2 first. Medium priority streams are
tested later if time, money, sample quantity, etc. permit. Lowest
priority streams, while not eliminated out of hand from further testing,
are not likely to proceed to Level 2. It was expressed at workshop
that streams obviously toxic in both chemistry and biology and those which
showed no toxicity could be easily prioritized. It was the ones with
indeterminant results that would provide the most difficulty in accurately
assigning priorities. However, no recommendation was developed for
these "grey" area streams.
Level 2 Test Procedures
Recommendations for potential Level 2 plant tests were of two
types. The first recommendation was to retain the same tests as used for
Level 1 but to use different species. New tests which could be used at
Level 2 were the Tradescantia mutogenicity/toxicity test and numerous
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growth and developmental tests such as pea seedling growth, bean hook
opening, and others. A terminal bud genetics test was also suggested.
All tests suggested will require further developmental work to be suit-
able for Level 2 use.
Level 2 tests suggested for the decomposition process included
the soil microcosm and specific substrate respiration tests. Again,
developmental work will be necessary before they can be actually implemented.
The suggested animal tests for Level 2 focused on life cycle
and behavioral tests using various different species. These types of
tests are directed at long term effects (life cycle) and subtle
subacute (behavioral) responses which are often very sensitive. Some
developmental work would be required for these tests before incorporation
into Level 2.
Comments
Level 1 Test Procedures
In the author's opinion those tests recommended for Level 1
comprise an adequate protocol capable of meeting the objectives of the
phased approach. This protocol is definitely superior to previous Level 1
terrestrial 'scheme. Some of the problems remaining with the newly
recommended protocdl include the use of tedlar or teflon bags (needed
because of the large volume required) to collect the gas sample for
the stress ethylene test. Some of the constituents of the gas are known
to either adhere to or pass through the walls of the bag altering the nature
of the sample that will be tested. The constituents so lost may or may
not be toxic ones of interest. In spite of these sample problems, stress
ethylene test is still of use in Level 1 because of its high sensitivity
and relatively linear dose response curve. -The sample problem is partially
offset by the data obtained through on-site gas chromatography for Level 1
chemistry and the decomposition and animal tests on gases which, because
of the small quantity needed, can be collected in glass containers (where
adherence and permeation are not a problem).
Another aspect involving the Level 1 animal tests deserve some comment.
No final decision was made as to which of the two insect species, honey
bee or Drosophila, should be used in the acute test. At present, 1 would
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recommend Drosophila in spite of the compelling argument for the honey
bee based on its great economic value. The Drosophila are smaller
requiring smaller sample quantities (expecially critical for particulate
and gaseous samples). They are easier to handle because they don't sting.
They can be maintained year round in laboratories under constant environmental
conditions. Honey bees are more often reared outside and become less
active in the winter which can alter their response in acute bioassays.
The endogenous soil respiration test is comparatively less
sensitive and more variable than some of the other bioassays recommended.
This variability problem must be recognized when using the test results.
This test still remains an important and useful one in that it involves
a critical component of terrestrial ecosystems for which data are needed
at Level 1 to aid in effluent prioritization.
Performing Level 1 bioassays on-site especially for those such
as the stress et^ylene test where sample quantity and quality are a
problem has been suggested. I believe the additional cost, quality'
control, and logistics problems of on-site testing far outweigh any
advantages obtained by such testing. This is particularly true at
Level 1 where the tests need to be cost effective, quick screens for
toxicity rather than expensive, more definitive procedures. Off-site
laboratory tests are fully adequate for Level 1 testing and probably
Level 2. Only Level 3 may need on-site tests.
One final area of comment for Level 1 is the need for further
test development. While the tests suggested are adequate for Level 1,
there are many others which show promise of being even better (cheaper,
more reliable, more replicable, smaller sample requirements, etc.) but
need some additional developmental work to make them acceptable. This
developmental work involves both refinement of test procedures and
validation of the test in actual routine usage. Such developmental
work will be slow or lacking altoghther unless the interest in this work
is organized into some type of test development program. Since the
development of these tests has value to both IERL-RTP, other EPA ORD
groups, and the EPA program offices responsible for implementing
various environmental laws (e.g., TSCA, RCRA), some type of coordinated
jointly funded effort should be possible and certainly desirable.
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Level 1 to 2 Decision Criteria
The author believes this to be one of the most important
difficulties to be solved in the phased approach. There is little
experience in synthesizing both Level 1 chemical and biological data to
obtain a realistic ranking of waste streams. Streams showing high
toxicity or no detectable toxicity in Level 1 biological tests and
revealing significant quantities of many known toxic substances or none
in Level 1 chemistry are easily prioritized. It is the test results in
between these two extremes that present interpretation problems, i.e.,
samples containing no significant quantities (detectable with Level 1
^
chemical procedures) of known toxicants which give toxic responses in
several bioassays or samples giving toxic responses in some biological
tests and nontoxic responses in others. This problem is compounded by
the fact that there is a potential for false negative test results and
that the tests have varying sensitivities. Varying sensitivity means that
if higher priority is placed, for example, on a relatively insensitive
health test (because it is indicative of effects on a man) rather than a
very sensitive terrestrial test, it is possible that a waste stream
containing a toxic chemical may be given a lower priority than it
deserves. Until our knowledge about the correlation between laboratory
test results and effects on man is improved, the weighting of one piece
of Level 1 information over another should be done with care. Some
system to use all Level 1 data is needed. Suggestions for using such
data include:
1. A "high" (as per the Litton evaluation scheme) in any test
should automatically raise a stream's priority
2. A "medium" both in one health test and one ecological test
should raise a stream's priority
3. A "low" in one health test, one aquative ecology test,
and one terrestrial ecology test should raise a stream's
priority.
As the test results for Level 1 bioassays accumulate.for different kinds
of samples, it may become apparent that some of the tests are particularly
sensitive and reliable and others are insensitive or variable. At that
time, it may be appropriate to either revamp the test protocol or weight
the sensitive, reliable test results higher.
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In interpretation of test results, the use of the "degree of
hazard" method and the MATE or MEG values should be approached with
caution. Level 1 chemistry does not provide data fully compatible with
MEG and MATE values leading to misleading rankings of waste streams. This
was demonstrated by the Level 1 pilot study results.
Level 2 Test Procedures
Level 2 recommendations resulting from the workshop carry the
necessity for further test development. My comments on this need have
already been given in my Level 1 comments. Coordinated developmental
efforts are needed if effective Level 2 procedures are to become available.
A final suggestion for Level 2 concerns the philosophy and
the procedures to be used. Level 2 has two objectives: (1) to confirm
Level 1 results and (2) to isolate the toxic chemical(s) through
fractionation ari retesting. High priority streams receiving bad marks
in several Level 1 chemical and biological tests should not need confirm-
ation. It should be possible to go directly to fractionation. Medium
priority streams should need confirmation. This confirmation may be
either using Level 1 tests on different test species, using completely
new tests, or both. Once toxicity has been confirmed, chemical fraction-
ation and testing should follow. Level 2 biological tests on the fractions
could well be the same as used in Level 1 since the objective of testing
the fractions is similar to Level 1, i.e., to determine which of the
fractions is toxic. Some refinement in Level 1 procedures (e.g.,
altering the range or number of doses, etc.) may be appropriate to better
identify the toxic response. However, it is unlikely that major test
changes would be needed for most areas of biological testing.
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APPENDIX E
D. McCune
Boyce Thompson Institute for Plant Research
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REPORT TO ACUREX CORPORATION ON
TERRESTRIAL BIOASSAY WORKSHOP IN
CORVALLIS, OREGON ON 14-15 NOVEMBER 1978
1. Summary
The general conclusions reached by the workshop were that
there are now suitable candidates for bioassays in Level 1 screening
for plant, soil, and animal components of the terrestrial system.
It was also concluded that other sets of protocols can be recom-
mended for further investigation and possible development as Level 2
bioassays.
A consideration of the problems involved in biological as well
as screening protocols led to the conclusion that decision-criteria
for transition from Level 1 to Level 2 screening in terrestrial
systems are not independent of but contingent on the results of
sampling, chemical analyses, and bioassays for toxicity in aquatic
and mammalian systems.
Throughout the discussions, it was evident that many lacunae
are present in Level 1 protocols as regards their description,
relevance of results to intended use, and quality of data derived
from screening procedures.
2. Protocols for Level 1 Bioassays
The following protocols were recommended for bioassays in the
terrestrial ecology systems on the basis of three kinds of criteria.
Firstly, they represented significant components (receptors) of the
terrestrial system and their responses had relevance to possible
adverse effects. Secondly, the bioassays generally were appropriate
to stream-classifications, i.e. gas, liquid, or solid, with respect
to the material to be assayed. Thirdly, the bioassays were among
the most practical in terms of availability, cost, efficiency, etc.
2.1. Animal Bioassays
Two test systems were recommended and differ mainly in the organism
to be used: honeybee (Apis mellifera) and fruit fly (Drosophila
melanogaster).
2.1.1. Protocols
Specifics of these test protocols were not formulated at the
workshop, but were to be formalized later.
2.1.2. Rationale for Selection
The following kinds of considerations entered into the selection
of these bioassay systems.
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20 November 1978
page 2
(a) Cost. Well within appropriate range.
(b) Availability. Stock cultures of organisms are easily and
economically maintained and numerous sources are available.
Moreover, protocols for the exposure of the organisms have
been developed, and extensive background data of their
genetics, tolerances, and behaviour are available.
(c) Sample suitability. The particular stream-phases to which
this test would be applied were not specified. But it
appeared that gas-, liquid-, or solid-phase materials, in
the amounts to be obtained, could be accomodated because
the materials could be administered topically or by
ingestion.
(d) Relevance of responses. Definite endpoints of each system
could be achieved in terms of morbidity, aberrant behaviour,
mortality, or bio-accumulation. Further, these responses
would be relevant to environmental effects.
(e) Comparability. Suitable standards are available for quality
control and assurance. In the honeybee system, methyl
parathione and monosodium methyl arsenate were suggested
as standards.
2.2. Soil Bioassays
Two systems were recommended: endogenous respiration and
acetylene (C2H2> reduction. Both are process-oriented and use the
same general test system but differ chiefly in the response measured.
Of the two processes, the former is a generalized index of activity
whereas the latter is a surrogate measure of nitrogen-fixation.
2.2.1. Protocols
Specific protocols were not recommended at this time but details
can be made available. It was proposed that three concentrations
with four replicates together with appropriate controls and standards
for quality control and assurance are accomodated by the procedure.
One difficulty will be in the selection.of a standard soil or the
kinds of soils to be used.
2.2.2. Rationale for Selection
The following factors entered into the selection of these
systems:
(a) Cost. The cost per test would probably be no greater than
S500 per stream-sample.
(b) Availability. The procedures are off-the-self and a data
base is available that allows judgements as to the sensitivity,
specificity, and precision of measurements.
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(c) Sample. Sample requirements appeared to be non-critical
for liquid or solid streams. Also, sample integrity appeared
to present no problem.
(d) Relevance of Response. Sample induced changes in the time-
course of endogenous respiration would indicate a general
change in the biotic processes of the soil, which could
be indicative of an adverse effect on any of many possible
organisms or on their interaction. The acetylene reduction
would be much more specific as to target and process
affected because of its use as a surrogate measure for
nitrogen-fixation. Both false positives and false negatives
may be high in endogenous respiration and of unknown
frequency in the acetylene reduction system.
(e) Comparability. It was noted that the endogenous respiration
test is relatively precise with coefficients of variation
of about 1.6%. It was also noted that sources of acetylene
must be screened before acceptance for use in the test.
Suitable standards are available for quality control and
assurance: aqueous solutions of silver or cadmium salts, sodium
aziue, or 2,4-dinitrophenol or gaseous ethylene oxide in the
endogenous respiration; sodium azi.de in the acetylene reduction.
2.3. Plant Bioassays
It was agreed that algae cannot serve as surrogates for
terrestrial plants and therefore suitable bioassay system that
employs higher plants was needed. The stress ethylene test was
recommended for use subject to clarification and refinement of the
description published in EPA-600/7-77-043 and some caveats concerning
its utility and relevance.
The plant bioassay system poses problems, of which some are
local and others global with reference to the phased approach.
2.3.1. Sample
It seems to me that the greatest problems in the utility of
this system arise in the matter of the sample's integrity, amount,
and relevance to control of process streams . From these standpoints
the plant bioassay system may be subject to false negatives. "
(a) Integrity. The principal problems lie in the possible pro-
cesses in the sample once it has been obtained; its sorption on or
reaction with the walls of the container; gas phase or heterogeneous
reactions or condensation in the sample itself owing to the continued
concentration of materials and lower temperatures during the interval
between sampling and bioassay. Thus it is entirely possible that
the bioassay will be conducted with a toxicant of composition different
from what is obtained by chemical analyses. It is worth noting that
the absence of some processes, such as photochemical transformations,
in the atmosphere, may also bias the results of,the bioassay.
At present, only two approaches seem to be available to check
on the integrity of samples. Firstly, gaseous samples of known
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page 4
phytotoxicity could be run. through the sampling system as standards
for quality control and assurance. Secondly, Level 1 chemical
analyses could be used to determine the possibility of those pro-
cesses that would adversely affect the integrity of the sample or
possibly to resynthesize the effluent at the point of bioassay.
Some discussion was devoted to the possibility of development of a
field-bioassay system. However, the scale and need for precise
environmental controls in the stress-ethylene test renders it
unsuitable for field use.
(b) Amount. The amount of sample available appeared to be
almost as limiting a factor as integrity. Aside from bioassay in
the field, the only other possibilities were to increase the size
of the sample by increasing the volume of the container or by physical
concentration of the sample and then revolatilization. The last
possibility would gravely affect the integrity of the sample. At
present the only answer appeared to be to obtain as large a sample
as was practical and economical.
(c) Relevance. Even if sufficient sample of unquestioned
integrity were obtained, there would still be the question of reference
of the gaseous process-streams to their possible environmental effects
for two reasons.
Firstly, stream classification, sampling, and physical and
chemical analyses are process- and not receptor-oriented. This
results in the problem that one cannot always have a direct mapping
of a stream or its components on the possible receptors . For
example: the classification of streams as gas, liquid, or solid
apparently does not account for the possibility that the atmosphere
will deliver components derived from liquid streams, through evaporation
of ponds, or from solid streams, by re-entrainment of particles,
to the terrestrial system as though they were originally in the
gaseous streams.
Secondly, it would appear that whereas the Source Assessment
Sampling System (SASS) is suited to the sampling needs and physical
and chemical analyses of Level 1, it is more refined than is
warranted by the bioassay (on plants) at Level 1. That is, it is
possible that coarse and fine particles by themselves or interactively
with the gaseous components could be active in the plant bioassay
and should be relevant to any environmental assessment.
2.3.2. Protocol
Two principal points of discussion, which were somewhat related
to the protocol for this bioassay, were whether this test was
sufficiently robust to be useful and whether alternatives were more
suitable. With reference to the latter point, it was concluded
that other bioassays are available but not of off-the-shelf availability
and might be better suited to Level 2 procedures. With reference
to the former point, the test appeared to be robust because similar
results were obtained by Battelle-Columbus and Corvallis Environmental
Research Laboratories. Nevertheless, it seems as though the protocol
could be more specific in its descriptions of some details and con-
sider the cost-effectiveness of the experimental designs.
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20 November 1978
page 5
(a) Culture and maintainance of plants. 'Dare' soybean should
be used, and in selection for uniformity and size a phenological
as well as a chronological age should be specified so that tissues
present represent the same physiological range of ages and thereby
the same potential range of susceptibilities. It was also suggested
that due emphasis be placed on the necessity for uniform and repro-
ducible conditions under which the plants are to be grown. This
includes the medium or artificial soil in pots and supply of mineral
nutrients and water as well as temperature, humidity, light intensity
and quality, and photoperiod. In short, one needs uniform plants
that are not subjected to environmental stresses for precise and
sensitive bioassays.
(b) Measures of response. It was recommended that the occurrence
(with respect to kind,frequency, and severity) of foliar injury
be a co-equal measure of response with ethylene evolution. This
was recommended not only because necrosis biases the ethylene evolution
but also because the occurrence of foliar symptoms is the most
documented response of plants to air pollutants and has some
relationship (but not an invariant one) to growth and yield. Thus
it can be used as a measure of relative toxicity and potential effect.
(c) Dose-Response. There is some doubt in my mind as to how
useful the dose-response or range-finding approach will prove in
this or the soil bioassay system -- except to test two hypotheses:
(1) montonicity of response; (2) greater than (synergistic) or less
than (antagonistic) additive affects of the components of the sample.
In the protocols for aquatic or other systems this facet of the
dose-response situation does not seem to be addressed, and if it
isn't, calculations of EC^Q'S may not be correct and indeed irrelevant.
It seems as though some caveat should be given.
2.3.3. Cost and Comparability
It appears that for this bioassay the fixed costs would be
relatively great owing to the need for certain facilities in which
to grow and test the plants as well as the need for personnel to
acquire the expertise required. Once a laboratory has the capability
for this bioassay, marginal costs should be relatively slight if
plants can be produced on a routine basis and not started and stopped
in response to irregular requests for bioassays or shipments of
samples. That is, economies of scale can be achieved only within
a laboratory unless other facilities have soybeans under routine
production for other purposes. Unlike tests for mutagenicity or
mammalian toxicity, the plant bioassay system is not widespread.
Thus, if samples are to be screened routinely by (for example) two
laboratories, the use of a third as an occasional referee may involve
extraordinarily high costs.
It would follow that the costs of Level 1 screening and quality
control and assurance would become more favorable the more concurrence
is achieved within the Offices of EPA as to the need for and nature
of a suitable plant bioassay.
As was stated above, a standard material such as chlorine (C12)
to be used in quality assurance, could be used alone but also as
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20 November 1978
page 6
an added component (spike) to a sample as it is being taken as a
test of sample integrity and the ability of the sample-bioassay
system to respond to it.
Given the nature of the sample vis a vis the problem of
environmental assessment, one would expect false regatives to be
much more frequent than false positives.
3. Possible Protocols for Level 2 Bioassays
A considerable body of discussion developed in response to the
problem of what criteria should be used in the selection.of tests
and what tests best fit these criteria. It also appeared that
the tests to be used at Level 2 cannot be arrived at independently
of what criteria will be used to decide to go to Level 2-testing.
Certain tests were proposed for Level 2 in that their possible
development and suitability be explored. Other tests were mentioned
as possibilities, as yet more remote, for the terrestrial system.
3.1. Animal Systems
Although the protocols to be used were not specified, it was
felt that the fruit fly and honeybee systems could be used for this
phase of biological screening. A system with mosquito larvae was
also suggested. Both systems could be reduced in scale, if necessary,
to what would be necessary for the reduced sizes of samples that
would be coming from the Level 2 chemical fractionation of effluent
streams. No measures of response were decided upon apart from the
general desire to have exposures and measures encompass the life-cycle
and monitor behavioural characteristics.
3.2. Soil Systems
Three test systems were proposed as possibilities for Level 2
bioassays. The soil-core microcosm and substrate respiration were
suggested as major tests and nitrification was suggested as a possible
back-up bioassay.
One theme, which was often implied but never stated explicitly
throughout the discussions, was apparent in points raised for dis-
cussion of soil-tests: concurrence of bioassays in Levels 1, 2, or
3 with those mandated or to be mandated in connection with the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) or the
Toxic Substance Control Act (TOSCA). This point was not resolved
but a general impression was that some degree of concurrence would
be worthwhile from the standpoints of both efficiency of bioassay
systems and planning for possible regulatory actions.
The major criteria that led to the choice of these bioassay
systems were: (1) the tests would give a composite or integral
measure of effects on the three major functional components of soil,
i.e. producers, consumers and decomposers; (2) the tests would give
more specific profiles as to the effects of toxicants on soil-processes;
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page 7
(3) the techniques, protocols, and baseline data are available and
minim-urn amount of work need be done to bring the techniques "off the
shelf".
3.2.1. Soil microcosm
Three major considerations that favored the use of this bioassay
at Level 2 instead of Level 1 appeared to be cost, specificity, and
time required (> 6 weeks). The cost was uncertain but estimated
to be in the range of $1300 to $2500 per test. The specificity of
the test would suffer if the effluent stream contained the same
materials, such as calcium (Ca), sulfate (804), phosphate (P04>,
dissolved organic carbon (DOC), or bicarbonate, as those whose
mobility from the soil was to be measured, and the occurrence of
false positives and false negatives was unknown. Thus, the test
seemed better suited for Level 2 where chemical fractionation is
present and the time and cost of the tests are more comensurate with
the specifity and richness of the data obtained.
It should be noted that more needs to be specified as to what
soil(s) and the composition(s) of simulated rain are to be used in
this test.
3.2.2. Substrate respiration
In this bioassay system more specific information would be
obtained on biotic processes and thereby it appeared to complement
the soil microcosm wherein physical and chemical characteristics,
mobility of toxicant, and general effects on abiotic and biotic
processes were measured.
Within this bioassay, the effects of a toxicant on respiration
would be measured (on a time-course?) with cellulose, starch, pectic
substances, or protein as substrate. It was estimated that the
test would cost about $500 per substrate and require about 15 days
for completion. The occurrences of false positives and false
negatives were unknown. The kind(s) of soil to be used was not
recommended insofar as its physical, chemical, and biological
characteristics were concerned.
3.2.3. Other bioassays
Inasmuch as certain key functions of soil were to be used as
measures of response (endogenous respiration and a surrogate of
N-fixation of Level 1, and substrate respiration at Level 2), it
was felt that nitrification should also be considered, at least at
Level 2.
Another consideration, with reference to Level 2 where more
would be known of the chemical composition of toxicants, was that
degradation or fate of effluents might be considered as part of a
bioassay system.
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page 8
3.3. Plant systems
Several criteria were explored with respect to the kinds of
bioassays that could be suitable for Level 2 screening. Perhaps
the three major considerations were that: (1) the test be an
efficient yet rich source of information as to probable environmental
effects; (2) the test exploit what was found in Level 1 sampling
and physical and chemical screening; (3) the test be commensurate
with other Level 2 procedures in yielding data that could be used
in planning, regulation, and control. Three kinds of, bioassays
were proposed.
3.3.1. Primary
One recommendation appeared to be that the same bioassays be
performed at Level 2 as at Level 1 but that they be expanded in
scope in two different ways: chemically and botanically.
Firstly, the Level 2 test should be performed on fractions of
the sample, which chemical analyses and extrinsic information may
show to have the greater potential toxicities. Secondly, the
tests should be performed with additional species of plants that
were subjected to the toxicant for the full life-cycle (possibly
a system with Arabidopsis could be developed for this purpose) .
The desire to broaden the range of test species was based on the
knowledge that a considerable variability in tolerance to any compound
exists between and even within species. The extension of tests over
a larger time period was based on the knowledge that chronic as
well as acute effects are possible and that in any plant, certain
stages of growth or reproduction are more sensitive to pollutant-
induced effects. There was also the consideration that a definite
and meaningful end-point could be achieved if the yield of biomass
or seed were used as a measure of response.
It should be noted here that whereas temporal patterns of
occurrence are placed in Level 3 sampling and analysis, these data
are of great relevance to whatever plant bioassays are to be performed
and certainly should be in hand to plan Level 3 bioassays. When the
plant is a receptor, the variables describing duration of exposure,
number of exposures that occur, and intervals between successive
exposures are almost co-equal to concentration of pollutant in the
prediction of possible or potential effects.
3.3.2. Secondary
Two tests with higher plants were proposed as other candidates
for Level 2 screening: seedling growth and seed germination.
Several reasons favored the use of these bioassay systems.
(a) Availability: a considerable body of literature and data
is available and procedures have been described in Test Methods for
Assessing the Effects of Chemicals on Plants", EPA-68-01-2249, Final
Report, Office of Toxic Substances (OTS), 30 June 1975.
(b) Costs. These bioassays are relatively small in scale, require
easily acquired facilities, and are relatively inexpensive.
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(c) Comparability. Considerable data is available for quality
assurance and to determine relative toxicity. Inasmuch as OTS may
recommend these tests, a degree of concurrence will be achieved.
(d) Relevance. Firstly, the tests could be developed to
accomodate gaseous, liquid, or solid samples and thereby be more
compound-oriented (as would be consistent with Level 2 fractionation)
and more independent of the phase of the feedstock- or effluent-
stream. Secondly, two critical phases in the growth and development
of higher plants would be studied. Also, the responses have meaningful
end-points. Thirdly, the combination of compound-orientation and the
germination of growth of seedlings would be significant in terms of
the potential effects of environmental accumulation of materials,
especially in the soils.
3.3.3. Tertiary
Some discussion was given to the suitability of other tests,
e.g. chromosome breakage in buds and protoplasmic streaming in
stamen hairs of Tradescantia; pollen germination, pollen-tube or
root-hair growth, and other tests. These were relegated to the
area of possibilities to be explored later owing to the use of a
more standard test for mutagenicity, difficulty of interpretation,
and the more holistic characteristics of the tests above.
4. Decision-Criteria-. Level 1
The discussion as to what decision-criteria should be specified
for the transition from Level 1 to Level 2 screening in terrestrial
systems left this question unanswered and also raised the question
of what relevance one level had to another in terrestrial systems.
The general consensus appeared to be that: (1) multiple decisions
are involved; (2) results for any terrestrial bioassay system
cannot be used independently from those of the other bioassays or
sampling and chemical analysis; (3) an ad hoc decision may be
needed for each stream or each chemical based upon its characteristics,
once analyses have been made.
In my opinion, the decision-criteria for terrestrial systems
cannot be formulated until certain problems are resolved and the
roles played by these bioassays are made more explicit in the phased
approach , especially in view of the unknown frequencies of false
positives and negatives.
4.1. Hierarchy
It would appear that more systematic and extensive exposition
of decisions that are involved in the phased approach would be worth-
while. The results of any bioassay, such as the stress ethylene,
must answer at least two major questions -- "What rank should be
assigned to this stream as a control priority?" "Should Level 2
bioassay be done?" However, there are other decisions that should
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be made first, such as, "Should another sample be taken and re-te^i-pH
at Level 1?" and "Is this.a valid result?"/ The orde? in which these
decisions should be made is probably the inverse of the order in
which they are listed. In view of the role played by integrity of
sample in the plant-bioassay, the latter two questions would most
likely be asked with respect to negative results.
It should be noted that all except the first question can be
answered in the metric of yes or no. Therefore, it would probably
be better to phrase the first question as a compound, e.g. "Should
this stream receive priority, if so at what rank?"
4.2. Contingency
One general conclusion, except for the mutagenesis test, was
that decisions for Level 2 are contingent upon the results of
chemical screening and other bioassays. With respect to the plant
bioassay, it appeared that a highly positive (toxic) response in
itself or that slight toxic response and high mammalian toxicity
indicated fractionation and Level 2 bioassay were adviseable.
4.3. Validity
It appeared that means for the validation of decisional models
or criteria for decisions were not developed to any extent. Never-
theless, two kinds of validity checks are more or less implied in
the phased approach with respect to prioritization of streams.
Firstly, the entire battery of bioassays at a level appear to
be a check of the validity of the quantitative measure — degree of
hazard (DOH). However, those weighting coefficients (MAC's or TLV's),
which enter .into the computation of the DOH, may have been derived
from the same bioassay systems. Thus the bioassays offer an inde-
pendent check on the suitability of a linear additive model to derive
a measure of DOH mainly with respect to: (1) possible synergism of
components of a stream; or, (2) possible effects of one or more
unknown but highly toxic components.
Secondly, the utility of the bioassays themselves seems to be
compromised by the manifold of different results that may be obtained.
If one assumes that twelve bioassays (three each for mammalian, fresh
water, salt water, and terrestrial systems) are run with results
measured on the scale of no, low, moderate, or high toxicity, then
there are 412 possible outcomes. Obviously because of commonalities
in the assays, not all outcomes are equiprobable. Nevertheless, it
would seem adviseable to drive the biological tests numerically with
data on hand (from the numerous, known compounds already screened
independently through the bioassay-system) to determine the kinds of
events (sets of outcomes) that can occur, the probability of certain
events, and their significance with respect to the decisions to
validate whatever criteria are chosen.
D.C. McCune
20 November 1978
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APPENDIX F
R. Rogers
University of Nevada, Las Vegas
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A report submitted to Larry R. Waterland, Leader of the
Process Analysis Section of the ACUREX Corporation in
Fulfillment of Subcontract RB 82544A.
Part I. General Summary of ACUREX Workshop
In this section of the report I will discuss in a general way the outlined
needs IERL has for an EA program and the plant-animal bioassays. Since my
expertise is soil microbiology, I will reserve specific comments on the
soil bioassays for the next section of the report
I inferred from Ray Merrill's presentation that there is a very urgent need
for usable bioassays to compliment existing chemical tests in order to
characterize the hazardous of effluent streams. As was explained, rapid,
cost effective terrestrial bioassays are required for level I screening of
effluent streams with a somewhat more complex and time-consumming assays being
used to confirm or refute the findings of level I.
Tables 6 and 7 of document E were presented to show the correlation between
chemical and biological testing. It appreared to me that the correlation
was not as predictive as would be hoped for. This is especially evident in
Table 7 data where DOH's do not always compare with biological response.
I assume that these data can be used to emphasize the necessity of a biological
screen. On the otherhand, however, the discrepancy might be that either the
wrong chemical evaluation index is being used or more important, the
bioassays are not responsive to hazardness of the stream. I would suggest
that these possibilities be explored.
Other information was given which underscored the difficulty of preserving
the integrity of gas samples. Discussion of this subject was raised several
times throughout the meeting. Two possible methods of solving this problem
were expressed. One was to improve upon the method and containers used for
sampling and the other was to push for the use of methods in the bioassay
area which could use smaller volumes of gas. It was suggested that the
development of miniaturized bioassays systems be persued for gas evaluation
studies. Such a system would use less gas hence the problems associated
with gas handling would be reduced.
I was personally very pleased that terrestrial bioassays are being considered.
There has been a great deal of effort both in industry and government to
influence policymakers to only require aquatic assays. Any document written
on terrestrial bioassays will certainly bring welcome emphasis to a neglected
subject.
The discussion on plant bioassays seemed to be centered on whether or not the
stress ethylene plant test was the most acceptable system for level I testing.
During the insuing discussion several plant bioassays were purposed. It was
finally decided that given the constraints of cost, sample type, relevance
to need, availability of test materials, comparability of data output, and
response that the stress ethylene procedure was the logical method to use.
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-2-
It was also understood that terrestrial plants not aquatic plants would
be used. The stress ethylene test appears to lend itself very well
to gaseous assays. Apparently there is a fair amount of data which has been
obtained from at least two different laboratories who have used the pro-
cedure .
The final list of tests for the level I plant bioassays were:
(1) for gas streams - stress ethylene, and foliar injury
(2) for liquid streams - seed germination, and seedling growth
(3) for solids - seedling growth
Some concern was voiced over the media in which the seedlings were going to be
grown. For example, if vermiculite is used something should be done to
ameliorate its toxic properties.
Level II plant bioassays should consist of more long-range growth, development
and reproduction testing. Such testing could include the use of microcosms.
Animal bioassays centered on the use of honey bees, drosophila, and mosquito.
The use of these assays seems to be very straight-forward. Much data has
been generated with the systems and they lend themselves to the testing of
all three phases of streams. These tests can be used for both levels I and II.
Part II. Discussion of Soil Bioassays
The hopes of the soil bioassays was apparently being based on using soil
core microcosms for both level I and level II testing. Such testing uses
respiration and nutrient export as markers for toxic insult. I am not in
favor of using this assay for level I testing because the assay requires too much
time for completion (some 8 weeks) to be considered as a rapid screen. Another
criticism of the system is that the effect of complex toxic materials has not
been determined. For example, if Ca export is being used as an index of
perturbation would Ca in the stream being applied to the system cause erroneous
results. Also, no data has been generated which will allow for a determination
of the applicability of using gaseous and liquid streams. In my opinion this
system is still in the developmental stage and can not be considered as an
"available" bioassay. This is not to say that the system does not hold promise
of being a worthwhile assay. With development, its use as a level II test could
be most useful.
Other tests were purposed which would rely on specific microbial functions.
Such tests included nitrogen fixation, nitrification, hydrogen oxidation,
the degradation of certain key compounds such as starch, cellulose, pectin, and
protein, and lastly microbial respiration. Of these methods, endogenous C02
production (a measure of respiration), and nitrogen fixation (as measured by C2H2
reduction) were chosen.
There are some good reasons why these methods should be used. Both tests are
rapid and uncomplicated. A change in CO2 production reflects an initial change
in the metabolism in some or all of the heteratrophic microbiological population.
However, it has some problem in that given time unaffected members of the
population can cause the C02 flow to return to the pretreatment level. On the
otherhand, such a deficiency is balanced by using another test which determines
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-3-
stream affects on a specific group of organisms i.e. dinitrogen fixers. I would
certainly urge the concurrent development of tests which use other specific
groups of organisms since different toxic materials can affect different
microbes in a variety of ways.
Since I have used the hydrogen oxidation assay on different gaseous and solid
toxic materials I would suggest that this method be further developed and
evaluated. There are several references available on the ease of use, rapidity
of the test, ubiquity of oxidizing organisms, and the effect of toxicants on the
oxidation process (ref 1 through 7).
Three reference chemicals were suggested for use with the soil bioassays. These
were silver compounds (I would suggest AgNO3>, sodium azide, and 2-4 dinitro-
phenyl-ethyleneoxide.
In summary, I feel that the suggested soil bioassay test have promise of providing
useful data. However, more time will be required before the two level I tests
and the level II soil core microcosm systems can be validated. Since this test
will require further data base development I strongly urge that the hydrogen
oxidation test also be evaluated at the same time.
ROBERT D. ROGERS, PH.D.
Soil Microbiologist
University of Nevada-Las Vegas
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References
1. Rogers, R.D., D.V. Bradley, and J.C./Farlane. 1978. The role of a
hydrogen-oxidizing microorganism, Alcaligenes paradoxus, in environmental
tritium oxidation. To be published in J. Soil Sci. Soc. Amer.
2. McFarlane, J.C., R.D. Rogers, and D.V. Bradley. 1978. Environmental
tritium oxidation in surface soil. Environ. Sci. Tech. 12:590-593.
3. McFarlane, J.D., R.D. Rogers, and D. V. Bradley. 1978. The effect of
SC>2 on soil microorganism activity. The bioenvironmental impact of a
coal-fired power plant. Fourth Interim Report, Colstrip, Montana. To
be published by USEPA.
4. Bradley, D. V., R.D. Rogers, J. C. McFarlane. 1978. Physiological and
morphological studies on a tritium oxidizing soil microorganism, Alcaligenes
paradoxus, and its possible use as a biological monitor. To be published
by USEPA.
5. McFarlane, J.D., R.D. Rogers . and D.V. Bradley. 1978. Tritium oxidation
in surface soils - A survey of soils near five nuclear fuel reprocessing
plants. To be published by Environ. Sci. Tech.
6. McFarlane, J.C., R.D. Rogers, and D.V. Bradley. 1978. Elemental tritium
analysis by bio-oxidation. To be published in Health. Phys.
7. Wiersma, G.B.,R.D. Rogers, J.C. McFarlane, and D.V. Bradley. 1978.
Biological monitoring techniques for assessing exposure. 1978. To be
published in Proceedings of the biological monitoring workshop held at
Raleigh, N.C. March 21-22, 1978.
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APPENDIX 6
D. Shriner
Oak Ridge National Laboratory
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Terrestrial Bioassay Workshop
Key Points of Discussion
David S. Shriner
Research Ecologist
Environmental Sciences Division
Oak Ridge National Laboratory
A. General Summary
Ray Merrill began the workshop by presenting an overview of the
phased approach to Environmental Assessment being developed by EPA/IERL,
and outlined for us the specific requirements for a bioassay protocol.
From my point of view, the most important aspects of this discussion
with regard to our task for the remainder of the workshop centered around
the need for the level one bioassays to be true screening tests aimed
at a preliminary ranking or prioritization of process effluents. Perhaps
the most important point from the standpoint of limitations on bioassay
systems is the limited sample size likely to be available under current
protocol. I feel that sample size should be given some serious thought
in relationship to bioassay requirements. Is it conceivable that modifi-
cation of the sampling protocols to accommodate the bioassay protocols
would be more effective and easier-in the long run than trying to work
with the bioassay systems under limiting conditions of sample size?
Other questions of a general nature which recurred throughout the
course of our two-day discussion had to do with sample collection, handling
and storage. For liquid and solid samples, these problems are relatively
straightforward, and do not seem to me to pose any significant barrier to
use in bioassay protocols. Gaseous sample collection, handling, and
storage, however, represent what I perceive to be a significant problem
which must be dealt with before any widespread use of the gaseous phase
testing can be promoted. Of course, the more reactive the species in
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the sample, the greater the danger of significant transformations occurring
during transport or storage, and it is conceivable that some of these trans-
formations might be missed under routine sampling conditions. For example,
consider a sample of tail gas from a sulfur recovery plant. The sample
will contain a mixture of sulfur species, probably including H2S, COS,
CS2, and S02. Our on-line monitor will record total S concentration at
the time of sample collection, and a second analysis for total S at the
time of use of the sample would likely confirm the presence of similar
quantities of total S in the sample. However, speciation may have changed
dramatically between the two analyses, resulting in a significantly differ-
ent response of plants to the sample in, for example, the stress ethylene
test. Our discussions did not resolve this point to my satisfaction, and
I feel it is an area, based on my personal experience with transport of
gas samples, that still requires some research effort.
I was interested in the brief discussion of on-site vs. off-site
bioassays. My feeling is that while some important advantages might be
gained by on-site bioassay testing, by far the most desirable circumstances
in virtually all of the bioassay tests - especially from the aspect of
minimizing operational problems and maximizing quality control and quality
assurance measures, will be to conduct the bioassay tests at a permanent,
off-site location.
We discussed a number of attributes of bioassay test systems which
are desirable and/or mandatory (cost, sample type, relevance, availability,
comparability, and response).
Of the above, cost, while important in the overall picture, can
probably be eliminated as a factor in bioassay test selection since most of
the tests meeting the remainder of the criteria will likely fall within
a relatively narrow range of costs anyway.
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Relevance is an important point in test selection. It is very impor-
tant that the test selection be geared to likely pathways of exposure in
natural systems (i.e., if contamination of soils is suspected, a test
measuring foliar injury of vegetation would probably be inappropriate - even
though foliar injury might result in some cases. Obviously a test known
to be consistently sensitive to stress on root systems might be expected
to be more sensitive, and we are left with seed germination, seedling
growth, and stress ethylene tests as more likely candidates.
We reviewed a number (10 by my count) of potential phytotoxicity
bioassays and came to a general agreement that stress ethylene and seed
germination tests are the two best of the currently available options. A
large data base exists for seed germination testing, and a growing data
base exists for the stress ethylene test. I will make additional comments
*
in the area of phytotoxicity testing in Part B, specific comments in my
area of expertise.
The discussion on soil bioassays included, again, a number of available
tests. Perhaps the first accomplishment of this discussion session was to
thoroughly discuss the merits of the proposed soil-core microcosm test.
I felt satisfied that our discussion of the method and its potential strengths
and weaknesses left little room for doubt that this method is unsuitable -
at least at the present time - as a level one assessment tool. I see a
couple of major problems which were fundamental in the arrival at this con-
clusion: 1) Most of the proposed measurement parameters, involving nutrient
efflux (Ca++, P04S, S04=, NH4-N, NOg-N) are potential constituents in effluent
streams. Furthermore, in many energy-related process and waste streams,
Ca and/or 50^= are present in relatively significant quantities. In
such cases, measurement of calcium efflux from a soil core microcosm would
present more problems in interpretation than I would consider acceptable
for a level I bioassay; and 2) My impression from our discussion and the
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comments of those more familiar with this test than I, is that there is a
great deal of ambiguity associated with interpretation of the results.
Furthermore, there are not sufficient data yet available to make any real
assessment of the frequency of false positives or false negatives. I feel
that it is a key point that not only must these bioassays be rapid, inexpen-
sive and reproducible, but they must lend themselves to very clear-cut,
straightforward interpretation of results. A "yes" or "no" answer is
required, and every effort should be made to minimize the frequency of
"maybe" results, since each such questionable response must be treated
as a positive.
There appeared to be general agreement that endogenous soil respira-
tion measurements are one indication of soil microbial activity for which
adequate data exist to establish this type of test as reasonably repli-
cable. A 30-day test was recommended to permit stabilization of the
system after manipulation.
Endogenous respiration measurement does not appear to be adequate to
stand alone as a soil bioassay. It was therefore suggested that a second
bioassay be included in the protocol, with nitrogen fixation and nitrifi-
cation assays, because of their broad use in agricultural work - an estab-
lished data base - and their relative sensitivity, being recommended. Of
the two, consensus was for the acetylene reduction assay for nitrogenase
activity, a 24-hour test. Each of these tests were thought to be applicable
to liquid, solid, or gas samples.
In addition, it was agreed that the hydrogen oxidation test discussed
by Rogers should be given development priority.
All of the soil tests selected necessarily call for the evaluation of
basically site-specific soils and soil microbial populations. For the
purposes of IERL this may not constitute a significant problem if the tests
are to be conducted in-house, at a limited number of sites. However, should
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others wish to reproduce the tests, it may well be worth specifying that
soil chemical and physical parameters be as fully characterized as possible.
Some degree of standardization of loam, sand, and clay fractions, exchange
capacity, and organic matter content could help. Textural variations alone
could influence moisture status at micro-sites of biological activity within
the soil matrix, and contribute to unacceptable variability in response
data.
We discussed synthetic soil preparations which might be standardized,
but there was strong feeling that this was an approach which had been
unsuccessfully explored in the past, and was probably not worth additional
effort. It would seem to me, however, that soil microbial populations might
be able to be standardized. This could conceivably be accomplished by soil
sterilization - perhaps by gamma irradiation, and a subsequent standard
reinoculation of soil microflora and fauna. Such standardization might
make replicability, reproducibility, and data interpretation easier.
Animal tests were discussed briefly. Because of the large volumes of
data existing on both honeybee and drosophila as test organisms, it was
generally agreed after discussion, that acute oral IC™ tests for liquid
and solid samples would be appropriate, and that the same organisms could
also be used in exposure to gaseous samples as well.
A discussion occurred on what should be the decision criteria for
specifying Level 2 tests on the basis of Level 1 results. I was favorably
impressed with the use of the Maximum Applicable Dose concept where it is
appropriate. Once the Level one response has been categorized as either
high or low, it was suggested that two different types of Level two assess-
ments might be appropriate depending upon the type of Level 1 response
observed. If level one testing established a high toxicity level in a
particular sample, fractionation of the sample (sample size permitting) and
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re-running of the Level one tests with each of the fractions would be
an appropriate Level two response. If, however, a given sample tested
high in only one test, or moderate in several tests, the appropriate Level
two response for those tests lower in toxicity might be to rerun the test
with additional species to confirm the potential hazard before going to
the expense of fractionation.
Using the above scheme, stress ethylene and foliar injury would be
utilized as both Level one and Level two tests as would seed germination.
Based on Level one, or other Level two data, full life cycle studies were
also regarded as a Level two test for plants.
Endogenous soil respiration and acetylene reduction were Level one
soil tests, with soil core microcosm and specific substrate respiration
suggested as Level two tests for soils.
Animal testing should employ the acute LC50 tests with honey bee and
Drosophila as both Level one and Level two test, with complete life cycle,
bioaccumulation, and behavioral tests being included as potential Level
two tests if appropriate.
There was discussion on the need for inclusion of blanks, reference
samples, and positive controls in the testing scheme for all tests. The
need for these steps cannot be overemphasized in my opinion. We discussed on
numerous occasions the variability and frequency of false hits with each of
these tests. Any meaningful interpretation of data from such a testing
protocol will have to utilize positive controls and appropriate reference
chemicals to establish standards for comparison. I would recommend, in
the case of phytotoxicity in seed germination and seedling growth tests, the
adoption of the 10 chemicals currently being used by Tingey in the round-
robin experiment as standard reference chemicals. For soils, 2,4-D, silver,
cadmium, and sodium azide were suggested as appropriate reference chemicals.
And for animals, parathion and Monosodium metharsenate (MSMA) were suggested.
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For standard reference gases, ozone, clorine, hydrogen chloride, and
ethylene oxide were suggested as references. I would comment here that
chlorine gas and HC1 gas behave somewhat similarly in terms of their
effects on plants, and would, in my opinion, represent an unnecessary
duplication.
I would like to now offer comments on some of the specific questions
(on page 4 of our agenda handout from the workshop) which have not been
addressed elsewhere:
- Effects of sample integrity - although we discussed this problem
on several occasions, I don't feel that it was ever resolved in
a totally satisfactory manner. The samples of most critical concern
from the standpoint of sample integrity will be, in my opinion, the
gaseous samples. My opinion, from frequent discussions with chemical
engineers on our staff about this exact problem, is that the solution
is an engineering problem. Given the resources, it is a solvable
problem, albeit a costly one.
- Effects of sample treatment procedures - also a real problem. I
would certainly opt for sample treatment procedures which would opti-
mize the utility of the samples for bioassay purposes. The introduc-
tion and subsequent removal of toxic solvents from a sample could
seriously alter the sample's integrity.
- Can Level one tests accommodate samples from Level 1 sampling? Given
the choice, I would certainly prefer to see separate samples collected
for bioassay purposes. One of the biggest reasons for this is the
limitation of sample size. A SASS train sample is not large enough
in most cases to permit Level I and II testing from the same sample,
especially if fractionation were required.
- Should solids be tested directly...? Both solids and leachates, or
extracts of the solids should be tested, in my opinion. A toxic
response to a solid could be attributed to a high concentration of
a heavy metal, by analysis. However, the heavy metal might not
have been in a soluble form, and the toxic response could have actually
been due to highly soluble sulfate, for example.
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- Should samples be concentrated? If an octanol/water partition
coefficient were to suggest potential for bioaccumulation, and
especially if mutagenic or teratogenic activity were suspected,
concentration would be called for. For simple toxicity tests,
however, we can expect chemicals in the environment to generally
undergo dilution during environmental transport, making concentra-
tion of the samples a probably unnecessary step.
- Does Level 2 testing require new (fresh) samples?- It is probably
unrealistic to expect samples to maintain their integrity from
collection through to the completed assessment of the Level 1 test
in a state acceptable for Level II testing. However, if fresh
samples were to be collected for Level II testing, I would recommend
-simultaneous repeats of the Level I tests on the fresh sample. The
*i
costs would be minimal, and since you have already flagged the
material as potentially problematic, it would seem to me you couldn't
afford not to rerun the Level I tests concurrent with the Level II
tests.
B. Phytotoxicity Assays
I wish to make only a few additional comments to the ones I have
already made under "general comments".
First of all, I would encourage IERL to be aware of the potential
impact of their planning and protocol development exercise. Even though
the tests are presently planned for in-house use at EPA labs, because of
their applicability to current requirements of enforcement arms of EPA
under TSCA and RCRA, these tests could easily find themselves being used
by hundreds of private commercial testing labs with varying standards of
quality assurance. For this reason, every possible effort should be
made to make the protocols as clear-cut and unambiguous as possible.
Along this line, perhaps the most critical step in getting reliable,
reproducible results from seedling growth and stress-ethylene testing will
be plant culture conditions. I would strongly urge reliance upon the
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materials developed for this purpose by Ted Tibbitts and his subcommittee
associates of the American Horticultural Society.
Three recent current versions of the seed germination test have
been tested by various labs. I have no experience with the Franklin
Institute version currently favored by TSCA, but can speak directly to
the versions being used at Corvallis and Oak Ridge. My opinion is that
there are no significant differences in principle between the two versions.
They should be cross-calibrated with one another, but I would not antici-
pate any significant differences in response.
One final comment - I am personally troubled by the proposed static
exposure conditions for gaseous sample testing of plant response. I feel
«
that it is extremely difficult to make valid assessment of dose-response
under static exposure conditions, and I would urge at least further con-
sideration of flow-through exposure conditions for the implementation of
stress-ethylene, foliar injury, and seedling growth tests.
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APPENDIX H
T. Tibbitts
University of Wisconsin
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Report on Workshop
Terrestrial Ecology Bioassay Protocol
for Environmental Assessments Program
T. W. Tibbitts
University of Wisconsin - Madison
Summary of Meeting
A. Level 1 Protocol
The participants at the meeting concurred in the following outline
of protocol for the Level 1 bioassays.
Plants Soil Microorganisms Animal
Rate of ethylene production Respiration of soil Honey bee survival
population
Foliar injury Drosophila survival
Acetylene reduction of '
Seed Germination percentage soil
Seedling growth
These protocol were accepted because all have been reasonably well defined
and have been utilized either in other bioassay programs or evaluated in more
than one laboratory as useful bioassays for the Environmental Assessments Program.
It was apparent that the group were more comfortable with recommending
the animal tests because of the large amount of use and standardization that
has been developed for these assays in pesticide evaluation. (This should
be documented, however)
There were greater reservations for recommending the plant and soil micro-
organism tests because none of the assays have been adequately standardized for
bioassay use and/or evaluated for use with mixed 'stream' effluents.
A summarization of the group response to each of these bioassays is
as follows:
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Plants
Rate of ethylene production - This should be an effective bioassay
for it provides a non-specific response of plants that monitors many different
types of stress reactions in the plant. It is known to occur with water stress,
pressure, chemical toxicity and any tissue injury. It also monitors a very
relevant response of plants,ie non-normal stress or injury to the tissues.
The test has been duplicated successfully in two different laboratories.
Foliar Injury
Has been utilized effectively in many types of pesticide testing and could
be utilized in conjunction with the ethylene production assay to reduce greatly
the cost of this assay. The fact that the Office of Toxic Substances was to
include this bioassay in their recommended procedures encouraged its inclusion in
this protocol.
Seedling Germination Percentage
Procedure for this have been described in the EPA 560/5-75-008 report of Test
Methods for Assessing the Effects of Chemicals on Plants. This could provide a
test requiring a minimum amount of space and minimum amount of environmental
control. It was felt that this could be utilized with gaseous toxicants even
though germination would have to be on a moist substrate.
Seedling Growth
The group encouraged this test to provide evaluation of morphological develop-
ment of plant systems. It also could be conducted in a small system and requires
a minimum of environmental control for the first level of assaying. It could
be conducted without light. It was indicated that this test could be combined
with the seedling germination test to minimize cost.
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Soil Microorganisms
Respiration of the soil community - The group supported this bioassay because
it was a very basic response for providing energy in all living systems and for
the decomposition of organic matter. It also was a rather non-specific response
that might be altered by toxicity to one of several different metabolic systems
within organisms. It was obvious, though, that the sys*tem might be overly sensi-
tive and respond to nearly any alteration of the physical or chemical soil system
even though the alteration was noc a distinct chemical toxicity. The group also
expressed the concern for false negatives,for respiration might be stimulated
by the addition of organic substrate in the stream effluents.
Acetylene reduction of Soils
The group supported this assay because of its basic relevance to nitrogen
fertilization for plants and because there has been considerable development of
this procedure for the extensive assay work in soil nitrification.
Honey bee survival
The group supported this assay because of the very high value that society
places upon honey bees and because careful testing procedures have been developed
for evaluation of pesticide toxicity. There was some concern expressed for the
problems of maintaining active colonies at all times, but this did not seem to
be a serious limitation. Insect tests are useful animal tests because of the
large populations that can be evaluated with very small samples. They are parti-
cularly useful, thus, for gas samples.
Drosophila survival
The group supported this bioassay because of the very detailed research data
available on drosophila and its response to different types of toxicants. There
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also is good information for genetic response evaluations. The very small size
of this organism also is of considerate advantage. The lack of significant
relevance to life on this planet is the biggest disadvantage of this test.
B. Level 2 Protocol Recommendations
Plants Soil " Animals
Full Life Cycle Soil core microcosm Full Life Cycle
tests
Behavioral
Substrate Respiration
There was no attempt to carefully detail Level 2 studies but recommendations
were made to provide a basis for encouraging investigation of acceptable
standardized procedures for each.
Plant Full Life Cycle
It was proposed that there was a need to follow plants from seed to seed to
determine toxicity to plant responses not studied in the seedling plants or
foliar injury studies. Responses that should be studied include reproductive
initiation, flowering phase initiation, sexual tissue development, and ferti-
lization. It could also permit study of genetic changes. Arapadopsis was sug-
gested as a useful plant species for it has been grown from seed to seed in
test tubes in a minimum of space.
Soil Core Microcosm
This test was proposed for Level 2 testing instead of Level 1 because no
effective means of standardizing the soil to be used in each test was able to be
recommended and because the response tests involve determinations for chemicals
that may often be present in the stream placed on the microcosm. Thus, the results
obtained may not be definitive enough to establish toxicity effects.
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Substrate Respiration
Recommended for Level 2 because each substrate utilized had a rather
restricted relevance to the functioning of the whole organism, and therefore,
a large number of substrates, involving excessive expense, would be required to
get meaningful results.
Animal Full Life Cycle
No particular animal studies were proposed although bee, drosophila, flies
and mosquitos should be investigated because standardized procedures are available
for all.
Behavioral
No particular animal studies were proposed, however, comments indicated support
for using bees for these studies.
The following proposed studies were not included in this list for the following
reasons:
Hydrogen oxidation
This soil microorganism test appears to have usefulness for bioassays but
required additional evaluation and standardization to make useful. It is a very
specific test of unknown relevancy but would likely have little interference from
materials in the stream.
Plant tests
Plant responses including pea epicotyl growth, bean hypocotyl opening, pea
tendril, tomato epinasty, turgor swelling, pollen growth, and cucumber leaf enlarge-
ment were not recommended because these tests either were very specific response
test, i.e. hormone response, or were too difficult to standardize for this bioassay
testing.
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Tradescantia stamenal hair alterations.
This bioassay procedure was not encouraged because of the complexity of
maintaining flowering plants in an available test form over the entire year.
Soil nitrification
This test was excluded because it was a rather specific test involving nitrogen
availability to plants and thus, closely paralleled the actylene reduction test
that was included. It might be considered as an additional Phase II test.
Mosquito and house fly survival
This test was excluded because it closely paralleled the drosophila test
and thus, was an unnecessary duplication of effort.
The following additional recommendations were made by the committee:
Level 2 testing would be undertaken when Level 1 bioassays indicated toxicity
however, there would have to be some judgement involved in each specific case to
determine what Level 2 bioassays should be undertaken.
Types of Level 2 Testing Recommended.
1. Fractionation of streams for the purpose of identifying the specific toxicants
in the mixtures of Level 1 testing.
2. Longer term testing to establish chronic effects of toxicants.
3. Test for toxic effects on all stages of plant growth, development, and repro-
duction.
4. Determine if toxicity occurs in several different species.
Samples for Inclusion in Bioassay.
1. Sample at least three concentrations of stream effluent.
2. Control sample without stream effluent.
87
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3. Control sample with a reference toxicant.
4. Control sample with a blind toxicant.
E. Bioassays are required for the following reasons:
1. Confirm the expected degree of toxicity for chemicals known to be in
the stream.
2. Establish toxicity from unrecognized chemicals in the stream.
3. Determine unrecognized toxicities from interacting levels of 2 or more
chemicals in the stream.
F. Specific Comments in My Area of Expertise.
1. Level 1 Plant Experiments
a. There is a need of carefully detailing the environmental conditions
and growing procedures for all plant experiments. This should include
the following factors: (See attachment for examples of standardized
growing conditions.)
Seed
Seed supplier and storage conditions
Regular germination tests to insure seed vitality
Selection of a self-pollinated plant or hybrid to reduce genetic
variability
Cultural Procedures
Media composition
Compaction of media
Seed sowing depth
Nutrition of media
Watering procedures
Rotation of plants in chamber
88
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Environmental conditions and instruments for measurement
Temperature of air
Radiation intensity
Relative humidity
Fresh air supply for CO control
Temperature of soil
b. For ethylene and leaf injury tests plants should be grown to a
particular stage described by leaf size and leaf number with a pre-
scribed acceptable variation. Growing time should not deviate +
1 day from seeding to test time.
c. A procedure for quantitative estimation of leaf injury should be
agreed upon to eliminate individual differences and inconsistencies
in visual estimates of leaf injury.
d. The germination test could be easily modified to obtain data on time
for emergence as well as percentage of germination. This is a more
sensitive measure of toxicity than percentage of germination.
e. All plant experiments may be subject to undesirable ethylene build-up
in the growing container or chambers if inadequate amounts of fresh
air are not directed through the growing system.
2. Level 2 Plant Experiments
The life cycle test proposed for Arabadopsis within small tubes should
be carefully evaluated to establish if plants growing under the very slow
rates of dry matter accumulation in small tubes have similar sensitivity
to toxicants as plants grown under normal growing environments.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-79-122
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE Terrestrial Ecology Protocols for
Environmental Assessment Programs: Workshop
Proceedings
5. REPORT DATE
June 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
R.L. Waterland, Compiler
8. PERFORMING ORGANIZATION REPORT NO,
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Acurex Corporation
485 Clyde Avenue
Mountain View, California 94042
10. PROGRAM ELEMENT NO.
INE624
11. CONTRACT/GRANT NO.
68-02-2611, Task 43
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Proceedings; 10/78 - 1/79
14. SPONSORING AGENCY CODE
EPA/600/13
. SUPPLEMENTARY NOTESIERL_RTp project officer & Raymond G. Merrill, MD-62, 919/
541-2557.
16. ABSTRACT
repOrf jg f^g proceedings of a workshop held in Corvallis, Oregon,
during November 1978, to discuss potential tests for inclusion in, and make recom-
mendations for, a terrestrial ecology bioassay testing protocol for use in EPA/
lERL-RTP's environmental assessment programs. The workshop, sponsored
jointly by EPA's IERL-RTP and ERL-Corvallis , included participants representing
both government and private researchers in the fields of plant physiology, soil micro-
biology, and entomology. Questions addressed included: What tests should be included
in a Level 1 protocol ? What should Level 1 to Level 2 decision criteria be ? and
What kinds of tests would be appropriate at Level 2 ? The report summarizes key
points of discussion and presents the results, conclusions, and recommendations
reached in addressing stated workshop questions. Recommended Level 1 plant, soil,
and animal assays are discussed, and Level 2 procedures are suggested, based on
Level 1 findings.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Pollution
Assessments
Testing
Ecology
Bioassay
Plant Physiology
Soil Microbiology
Entomology
Pollution Control
Stationary Sources
Terrestrial Ecology
13 B
14B
06F
06A
06C
06M
13. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. Or PAGES
93
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
90
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