United States
Environmental Protection
Agency
Office of Emergency and Office of
Remedial Response Research and Development
Washington DC 20460 Cincinnati, OH 4626$
Superfund
EPA/540/S-95/501
Engineering Bulletin
Biological Toxicity Testing
Purpose
Section 121(b) of the Comprehensive Environmental
Response, Compensation, and Liability Act (CERCLA) man-
dates the U.S. Environmental Protection Agency (EPA) to
select remedies that "utilize permanent solutions and alter-
native technologies or resource recovery technologies to
the maximum extent practicable" and to prefer remedial
actions in which treatment "permanently and significantly
reduces the volume, toxicity, or mobility of hazardous
substances, pollutants, and contaminants as a principal
element." The Engineering Bulletins comprise a series of
documents that summarize the latest information available
on selected treatment and site remediation technologies
and related issues. They provide summaries of and refer-
ences for the latest information to help remedial project
managers (RPMs), on-scene coordinators (OSCs), contrac-
tors, and other site cleanup managers understand the type
of data and site characteristics needed to evaluate a tech-
nology or other remedial tool for potential applicability to
their Superfund or other hazardous waste site. Those
documents that describe individual treatment technologies
focus on remedial investigation scoping needs. Addenda
will be issued periodically to update the original Bulletins.
Abstract
Biological toxicity testing is an important tool in per-
forming ecological assessments at Superfund sites. Site
managers* legislatively mandated to protect the environ-
ment can use biological toxicity testing to support deci-
sions made at any stage of the remedial process. Providing
information that chemical-specific testing alone cannot
supply, these tests evaluate the aggregate toxic effects of all
contaminants in a medium. They also can be an important
indicator of increased toxicity caused by incomplete treat-
ment of contaminated media.
This Engineering Bulletin is intended to provide site
managers with information on ecological assessment and
biological toxicity testing, applicability of biological toxic-
ity testing, planning effective biological toxicity assess-
ments, descriptions of test methods, limitations, current
trends, and sources of additional information. Additional
emphasis has been placed on terminology and references
for biological toxicity test methods in order to provide a
basic understanding from which to seek additional informa-
tion as needed. This Bulletin is not intended to be a
comprehensive review of toxicity test methods. RPMs and
OSCs are encouraged to contact the Biological Technical
Assistance Group representative for their region for addi-
tional information.
Ecological Assessment and Biological
Toxicity Testing
CERCLA and the National Oil and Hazardous Sub-
stances Pollution Contingency Plan (NCP) require that
remedial actions at hazardous waste sites protect human
health and the environment. Site managers are also re-
sponsible for compliance with all applicable or relevant and
appropriate requirements (ARARs), including numerous stat-
utes and regulations enacted to protect natural resources.
In response to these mandates, the Office of Emergency and
Remedial Response (OERR) and the Office of Waste Pro-
grams Enforcement (OWPE) issued a joint memorandum in
December 1988 directing Regional Offices to perform "thor-
ough and consistent" ecological assessments at all Super-
fund sites. EPA followed up the memorandum by publish-
ing the Risk Assessment Guidance for Superfund. Volume II.
Environmental Evaluation Manual [1, pp. 1-57]**. The
manual defines an environmental evaluation, or more pre-
cisely an ecological assessment, as a "qualitative and/or
quantitative appraisal of the actual or potential effects of a
hazardous waste site on plants and animals other than
people and domesticated species." Ecological assessments
at Superfund sites should supply site managers with the
information necessary to determine potential and actual
threats to the natural environment caused by current con-
* For brevity, the term "site managers" will be used to denote RPMs, OSCs, potentially responsible party (PRP) contractors, and other site
remediation professionals. In cases where the information presented only applies to one or more of these groups, the included groups
will be identified.
** [reference number, page number]
-------
United States
Environmental Protection
Agency
Office of Emergency and Office of
Remedial Response Research and Development
Washington, DC 20460 Cincinnati, OH 4S26&
Superfund
EPA/540/S-95/501
Engineering Bulletin
Biological Toxicity Testing
Purpose
Section 121(b) of the Comprehensive Environmental
Response, Compensation, and Liability Act (CERCLA) man-
dates the U.S. Environmental Protection Agency (EPA) to
select remedies that "utilize permanent solutions and alter-
native technologies or resource recovery technologies to
the maximum extent practicable" and to prefer remedial
actions in which treatment "permanently and significantly
reduces the volume, toxicity, or mobility of hazardous
substances, pollutants, and contaminants as a principal
element." The Engineering Bulletins comprise a series of
documents that summarize the latest information available
on selected treatment and site remediation technologies
and related issues. They provide summaries of and refer-
ences for the latest information to help remedial project
managers (RPMs), on-scene coordinators (OSCs), contrac-
tors, and other site cleanup managers understand the type
of data and site characteristics needed to evaluate a tech-
nology or other remedial tool for potential applicability to
their Superfund or other hazardous waste site. Those
documents that describe individual treatment technologies
focus on remedial investigation scoping needs. Addenda
will be issued periodically to update the original Bulletins.
Abstract
Biological toxicity testing is an important tool in per-
forming ecological assessments at Superfund sites. Site
managers* legislatively mandated to protect the environ-
ment can use biological toxicity testing to support deci-
sions made at any stage of the remedial process. Providing
information that chemical-specific testing alone cannot
supply, these tests evaluate the aggregate toxic effects of all
contaminants in a medium. They also can be an important
indicator of increased toxicity caused by incomplete treat-
ment of contaminated media.
This Engineering Bulletin is intended to provide site
managers with information on ecological assessment and
biological toxicity testing, applicability of biological toxic-
ity testing, planning effective biological toxicity assess-
ments, descriptions of test methods, limitations, current
trends, and sources of additional information. Additional
emphasis has been placed on terminology and references
for biological toxicity test methods in order to provide a
basic understanding from which to seek additional informa-
tion as needed. This Bulletin is not intended to be a
comprehensive review of toxicity test methods. RPMs and
OSCs are encouraged to contact the Biological Technical
Assistance Croup representative for their region for addi-
tional information.
Ecological Assessment and Biological
Toxicity Testing
CERCLA and the National Oil and Hazardous Sub-
stances Pollution Contingency Plan (NCP) require that
remedial actions at hazardous waste sites protect human
health and the environment. Site managers are also re-
sponsible for compliance with all applicable or relevant and
appropriate requirements (ARARs), including numerous stat-
utes and regulations enacted to protect natural resources.
In response to these mandates, the Office of Emergency and
Remedial Response (OERR) and the Office of Waste Pro-
grams Enforcement (OWPE) issued a joint memorandum in
December 1988 directing Regional Offices to perform "thor-
ough and consistent" ecological assessments at all Super-
fund sites. EPA followed up the memorandum by publish-
ing the Risk Assessment Guidance for Superfund. Volume II.
Environmental Evaluation Manual [1, pp. 1-57]**. The
manual defines an environmental evaluation, or more pre-
cisely an ecological assessment, as a "qualitative and/or
quantitative appraisal of the actual or potential effects of a
hazardous waste site on plants and animals other than
people and domesticated species." Ecological assessments
at Superfund sites should supply site managers with the
information necessary to determine potential and actual
threats to the natural environment caused by current con-
* For brevity, the term "site managers" will be used to denote RPMs, OSCs, potentially responsible party (PRP) contractors, and other site
remediation professionals. In cases where the information presented only applies to one or more of these groups, the included groups
will be identified.
** [reference number, page number]
-------
ditions, remedial actions, and contaminants remaining at a
remediated Superfund site. An ecological assessment gen-
erally is composed of four interconnected activities, which
are described in the following subsections: problem formu-
lation; exposure assessment; ecological effects assessment;
and risk characterization [2, p. 3]. Figure 1 depicts these
activities and their components. As shown in Figure 1 and
discussed below, toxicity testing is an integral component
of the ecological effects assessment activity.
Problem Formulation
Problem formulation includes development of assess-
ment objectives and assessment endpoints. Assessment
objectives are usually qualitative statements identifying the
environmental values to be investigated. A typical assess-
ment objective would be to determine if areas of a river
flowing through the site have reduced populations of game
fish, possibly due to contamination of the river water. To be
PROBLEM FORMULATION
Qualitatively evaluate contaminant release, migration, and fate
Identify:
- Contaminants of ecological concern - Exposure pathways
- Receptors - Known effects
Select endpoints of concern
Specify objectives and scope
EXPOSURE ASSESSMENT
Quantify release, migration, and fate
Characterize receptors
Measure or estimate exposure point
concentrations
f COLOCICAL EFFECTS ASSESSMENT
Literature
Toxicity testing
Field studies
RISK CHARACTERIZATION
Current adverse effects
Future adverse effects
Uncertainty analysis
Ecological significance
REMEDIAL OBJECTIVES
ANALYSIS OF REMEDIAL
ALTERNATIVES
REMEDY SELECTION
RECORD OF DECISION
REMEDIAL DESIGN
REMEDIAL ACTION
Figure 1. Ecological assessment of Superfund sites: Overview. Source: ( 2, p.3)
Engineering Bulletin: Biological Toxicity Testing
-------
r
r
useful in guiding ecological assessments, assessment objec-
tives must be translated into quantifiable assessment end-
points. Identification of potentially affected areas of the
river where the concentration of contaminants in the sur-
face water could result in a greater than 10 percent reduc-
tion in bass populations could be an assessment endpoint
for the example assessment objective.
Exposure Assessment
Exposure assessment quantifies the magnitude and
routes of contaminant exposure to which ecological recep-
tors are subjected. Using the above example, an exposure
assessment would determine the concentration of contami-
nants in the surface water, sediment, and food sources for
bass populations upstream, within the boundaries, and
downstream of the site.
Ecological Effects Assessment
Toxicity testing is conducted within the ecological
effects assessment. Toxicity tests expose test organisms to
soil, water, or sediment and evaluate the effects of the
medium on the survival, growth, reproduction, behavior,
or other attributes of the test organisms [3, p. 1]. These
attributes are usually referred to as indicators. The quanti-
tative expression of an indicator (i.e., the results of a
biological toxicity test) is called a measurement endpoint.
Common measurement endpoints used in toxicity testing
are listed in Table 1 [4, pp. 9-10]. Measurement endpoints
for an assessment should be relevant to the assessment
objectives. Use of toxicity tests with measurement end-
points such as LC50 and NOEC for fathead minnows (stan-
dardized tests for fish toxicity testing) and LC50 for prey
species that spend a portion of their lives in river sediment
would be appropriate for the cited example.
Measurement endpoints are combined with other com-
ponents of the risk assessment to evaluate the assessment
endpoints. Given the potential complexity of ecological
interactions at Superfund sites, multiple measurement end-
points often will be required to evaluate a single assessment
endpoint. The most useful assessment endpoints are those
for which there are well-developed measurement end-
points, test methods, field measurement techniques, and
predictive models [5, p.24]. Using the above example, the
relationship of indicators, measurement endpoints, assess-
ment endpoints, and assessment objectives is shown in
Figure 2.
Risk Characterization
Risk characterization involves a direct comparison of
the results of the ecological effects assessment with the
results of the exposure assessment, drawing conclusions in
support of the assessment objectives. The data collected in
the exposure assessment for areas upstream, within the
boundaries, and downstream of the site are compared to
determine the distribution of contaminants. The relative
effects of these contaminants, determined by the ecologi-
Table 1. Common Measurement Endpoints Used in
Toxicity Testing
NOEC
LOEC
MATC
EC
50
LC
so
No-Observed-Effect Concentration.
(The highest concentration of a
contaminated medium at which no
statistically significant effect relative to
negative controls was observed in test
organisms.)
Lowest-Observed-Effect Concentration.
(The lowest concentration of a contaminated
medium at which a statistically significant
effect relative to negative controls was
observed in test organisms.)
Maximum Acceptable Toxicant
Concentration. (The maximum
concentration at which a contaminated
medium can be present and not be toxic to
the test organism. The MATC is normally
calculated using the geometric mean of the
lowest concentration for which an adverse
effect was observed [LOEC] and the highest
concentration that did not yield any adverse
effects [NOEC].)
Median Effective Concentration.
(The concentration of a contaminated
medium that produces a designated effect on
50 percent of the test organisms.)
Median Lethal Concentration.
(The concentration of a contaminated
medium that produces mortality in 50
percent of the test organisms.)
cal effects assessment, are then overlaid upon the contami-
nants distribution. This process generates a risk description
that includes conclusions on the ecological risks, uncertain-
ties associated with the conclusions, and interpretations of
the ecological significance of the observed effects. For the
cited example, the risk characterization could conclude
that game fish populations are probably being affected by
contaminants in three areas of the site, as evidenced by
greater than 10 percent reductions in bass populations; and
although sport fishing may be affected, the abundance of
these fish in other areas of the site should preclude overall
degradation of that part of the river ecosystem flowing
through the site. More detailed information on performing
ecological assessments can be obtained by consulting the
"Sources of Additional Information" section of this Bulletin.
RPMs and OSCs are especially encouraged to utilize their
Regional contacts for the Biological Technical Assistance
Croup (BTAG) listed in that section.
Engineering Bulletin: Biological Toxicity Testing
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ASSESSMENT OBJECTIVE
ASSESSMENT ENDPOINTS
* Determine which areas of a river flowing
through the site have reduced populations
of game fish, possibly due to contamina-
tion of the river water.
MEASUREMENT ENDPOINTS
INDICATORS
Areas with
potential for >10%
reduction in
bass populations
Fathead
Minnow LC50,
NOEC
Sediment
toxicity to fish prey
Surface water
toxicity to fish prey
Surface water
toxicity to fish
Figure 2. Relationship of indicators, endpoints, and objectives in the ecological assessment process.
Applicability of Biological Toxicity
Testing
Biological toxicity testing is an important tool that is
potentially applicable to any stage of the site remediation
process It can be applied to the initial stages of site
prioritization, used in waste and site characterization, em-
ployed in the establishment of cleanup standards, used in
the selection of treatment technologies, and finally, utilized
in site monitoring during and after remediation. Figure 3
lists these applications and their corresponding stages in
the remedial process [6, p. 8]. This section discusses these
and other applications of biological toxicity testing.
Site Prioritization
When determining which contaminated sites should
be addressed first, collection of information that allows the
ranking of sites according to relative risk is an important
process. Although ARARs and chemical analysis are typi-
cally used to prioritize sites, toxicity tests can identify sites
that are impacted by contaminants that preliminary chemi-
cal screening may not identify. For example, pentachlo-
rophenol (PCP) is a common contaminant at wood-preserv-
ing sites. Analytical quantification of PCP is difficult, as
evidenced by an estimated quantitation limit in water of 50
parts per billion (ppb) [7, p. 8280A-32]. The presence of
interfering contaminants can potentially raise the limit by
an order of magnitude. Also, the acceptable spike recovery
limit under the EPA Contract Laboratory Program is 9 to 103
percent [8]. When chemical testing is used alone, the
reported low concentrations of PCP may indicate that the
site is of lower priority. In contrast, the Microtox bioassay
is highly sensitive to PCP, showing an EC50 at concentra-
tions as low as 80 ppb. The addition of biological toxicity
testing therefore could indicate that the site actually is of
high priority from an ecological risk perspective.
Toxicity tests can be employed to classify the type of
toxic effects produced by the mixture of contaminants at a
site They can be designed to provide information on acute
or chronic toxicity of contaminants associated with media
at a site. Acute, toxicity tests generally indicate the test
organisms' rapid response to contaminants, using a test
indicator of survival. These tests can provide rapid screen-
ing information useful in site prioritization. Chronic toxic-
ity tests generally measure the test organisms' longer-term
response to contaminants including survival, changes in
growth rates, reproductive capacity, biochemical and physi-
ological functions, behavior, incidence of genetic muta-
tions, tumors, and cancer [9, p. 6]. These tests can identify
sites that did not display acute toxicity but should be further
investigated due to their chronic toxicity. Use of toxicity
tests in conjunction with chemical analyses in support of
ARARs facilitates a more accurate site discovery and notifi-
cation process.
Engineering Bulletin: Biological Toxicity Testing
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Figure 3. Potential role of biological toxicity testing in the NCP site remediation process. Source: (6, p.8)
r
Waste Characterization
Toxicity tests evaluate the aggregate toxic effects of all
contaminants in the medium of concern. Identification and
quantification of individual compounds in a complex mix-
ture of contaminants can be prohibitively expensive and
provide no information on additive, synergistic, or com-
petitive interactions of the compounds. Biological toxicity
tests, such as fish bioassays, have been used since the 1960s
to determine the toxicity of complex aqueous wastes,
including municipal and industrial effluents [10, p. 331]. At
Superfund sites that formerly produced chemicals, little
scientific literature may be available on the toxicity of the
chemicals produced that are now contaminating the site.
Toxicity tests can evaluate the toxicity of substances whose
biological effects have not been well characterized. Over
10 million chemicals had been documented in the Ameri-
can Chemical Society's Chemical Abstracts Service as of
April 1990. The "environmental" toxicity of only a small
portion of these compounds (mostly pesticides and herbi-
cides) is known [11, p. 266]. Various modeling approaches,
such as structural-activity models, may be used to predict
the availability and toxicity of compounds, but toxicity test
results are needed to verify the toxic effects of compounds
under specific environmental conditions. Information on
the biological effects of wastes at a site can be important to
the preliminary assessment and site inspection (PA/SI),
remedial action prioritization (RAP), and remedial investi-
gation and feasibility study (RI/FS) stages of the remedial
process.
Site Characterization
Toxicity tests can be a cost-effective approach to char-
acterizing the distribution of contaminants potentially af-
fecting resident organisms throughout a site. Determining
the distribution of site contaminants at a Superfund site by
chemical analysis is very expensive and may not show the
spatial relationship of contaminants and biosensitive areas.
Many of the standard acute toxicity tests are comparable in
cost to comprehensive chemical analyses [12]. More so-
phisticated chronic toxicity tests provide additional infor-
mation useful in site characterization, but can be substan-
tially more expensive. Biological toxicity testing used in
support of site characterization provides information useful
to the PA/SI, RAP, and RI/FS stages of the remediation
process.
Cleanup Standards
Toxicity test results can be used to help ensure that
cleanup standards established for a site will be protective of
the environment. Cleanup standards that are based upon
human risk assessments or technology performance may
not be protective of all organisms at a site. The use of
toxicity tests as a component in the cleanup standard-
setting process provides a more complete picture of the
overall protection derived from remediating site contami-
nation to a specific level.
Toxicity tests can be used, in some cases, to determine
which compounds are contributing to the observed effects
on organisms at a site. Using a toxicity identification
evaluations (TIE) approach, investigators can manipulate
test conditions to selectively affect certain compounds and
compare these results to results from unmanipulated tests.
For example, a chelating agent can be added to one set of
water tests and the results compared to results from tests
where the agent was not added to the medium. If per-
formed with the proper controls, a reduction in toxicity in
Engineering Bulletin: Biological Toxicity Testing
-------
the chelated tests would indicate a likelihood that the toxic
effect is at least partially due to compounds that can be
removed by chelation (e.g., certain metals). This type of
information would allow establishment of cleanup stan-
dards for specific groups of compounds based upon toxicity
tests. EPA has developed a three-phase approach to TIE that
progressively narrows the focus of the evaluation from
toxicity characterization, through toxicity identification, to
toxicity confirmation. This approach currently can be used
for water and sediment samples. For further information,
the reader is referred to the corresponding EPA documents
Toxicity tests can help determine the potential for a
remediated site to support a viable ecological community.
CERCLA and the NCP define natural resources to include
biota and their supporting resources and designate natural
resource trustees charged with their protection. CERCLA
Section 104(b)(2) requires EPA to promptly notify the
appropriate natural resource trustees of the potential for
natural resource injuries resulting from releases under in-
vestigation. If natural resources are damaged, the trustees
are allowed to file for monetary compensation. Toxicity
testing that indicates the potential for restoration of natural
resources provides site managers with valuable information
for negotiating with natural resource trustees concerning
the potential for natural resource damage assessments.
Selection of remedial alternatives that protect and restore
natural resources often reduces the chances of costly and
time-consuming natural resource damage proceedings that
may delay negotiated settlements [18, p. 1-9]. Toxicity
tests used in support of the development of cleanup stan-
dards provide valuable information for the RI/FS stage of
remediation.
Treatment Selection
Toxicity tests can be an important tool in the evaluation
of different treatment technologies investigated through
treatability studies. Remedy screening treatability studies
are generally performed to determine the potential feasibil-
ity of several remedial technologies. Remedy selection
treatability studies are usually employed to develop perfor-
mance and cost data on a smaller group of treatment
technologies [19, pp. 8-10]. Toxicity tests performed on
the medium of interest before and after remedy screening
treatability studies can identify which technologies are
potentially effective at reducing the toxic properties of
contaminated soil, sediment, and water. When used in the
evaluation of remedy selection studies, toxicity testing can
help determine the degree of treatment required to reduce
toxic effects to an acceptable level. Cost of treatment
estimates can then be generated.
Toxicity tests have been used to evaluate bench-scale
treatment technologies for several mine drainage remedia-
tion projects. Acute toxicity tests have been used to
augment bench-scale treatment investigations of mine drain-
age from a drainage tunnel near Leadville, Colorado. Forty-
eight-hour water flea and minnow tests were used to
evaluate five different chemical/physical treatment tech-
niques. Results from these tests were used to determine
which of the techniques cost-effectively maximized the
removal of metal toxicity. Based upon the toxicity tests and
other chemistry and engineering information, a treatment
system was recommended and constructed.
Similar tests were used to evaluate the effectiveness of
seven different artificially constructed wetlands in reducing
toxicity of effluents from mine drainage in Idaho Springs,
Colorado. Toxicity testing also was used to evaluate the
effectiveness of modifications to the pretreatment of drain-
age prior to entering these pilot-scale wetlands. Biological
toxicity tests used in the treatment selection process sup-
port decisions made during the RI/FS and remedial action
design and construction processes.
Site Monitoring
Toxicity tests can be an integral component of reme-
dial and post-remedial monitoring. Remedial actions, such
as excavation, can increase soil loading to adjacent water
bodies. Dredging of contaminated sediment can resus-
pend particles, potentially impacting downstream organ-
isms. In order to monitor the effects of these activities,
biological toxicity tests can be employed. Some remedia-
tion technologies have the potential to produce com-
pounds that are more toxic than the original contaminants
(e.g., intermediate metabolites during bioremediation).
Biological toxicity testing can be used to monitor for these
concerns [10, p. 336][20, pp. 105-112]. The long-term
effectiveness of remedial actions can be monitored through
periodic toxicity testing. Depending on the cleanup goals
established, either acute or chronic tests can be employed.
If the remediation was not completely successful, it is
important to identify problems and implement corrective
actions as soon as possible [6, p. 11]. Toxicity testing can
also be incorporated into long-term monitoring of reme-
diation residuals that remain on site (e.g., solidified or
thermally treated soil).
Toxicity testing was used at a site in Michigan to
evaluate the removal of organic contaminants from soil
after treatment by thermal desorption. Using an earth-
worm test, it was determined that the soil was as toxic after
treatment as before. Further investigation showed that,
while the toxicity attributed to organic contaminants was
removed, the treatment had increased the bioavailability of
manganese through the removal of organic matter to
which it had previously been bound. Both aquatic and
terrestrial toxicity tests have been used at several sites to
evaluate the effectiveness of mine tailings remediation
projects. At one site, aquatic tests were used to pin-point
instream impacts from tailings and to evaluate the effec-
tiveness of isolating tailings drainage and runoff from a
creek. At another site, earthworm tests were used to
determine the degree of residual toxicity remaining after
mine tailings and roaster piles were removed.
These examples emphasize the need to consider all
relevant data when determining whether cleanup goals
have been achieved. A decision that cleanup goals have
been met based upon only one type of test may lead to an
Engineering Bulletin: Biological Toxicity Testing
-------
incorrect conclusion. Toxicity tests used in support of site
monitoring can help ensure a thorough evaluation of reme-
J"*'- dial action design, construction, implementation, and suc-
cess.
Planning Effective Biological Toxicity
Assessments
Site managers charged with planning or reviewing
biological toxicity testing should be aware of the elements
that comprise effective biological toxicity assessments and
the ideal characteristics of toxicity tests. Important ele-
ments discussed in the following subsections include: the
objective, the reference site, the medium analyzed, the test
organisms, the test methodology, the test site, the statisti-
cal analysis to be used to interpret the results, and the
quality assurance/quality control (QA/QC) standards nec-
essary to ensure the collection of valid data [3, p. 3]. These
elements are interrelated; changes in one area affect the
other areas. Site managers should review all elements if
changes are made to any one.
onsite or offsite contamination. Upstream locations are
often appropriate for surface water and sediment toxicity
tests. Upwind, upgradient areas can be appropriate refer-
ence sites for terrestrial tests. Site managers should con-
sider factors such as sediment and soil particle size, vegeta-
tion, slope, previous usage, the presence of fill material, and
unrelated sources of contamination when choosing a refer-
ence site. Careful evaluation of these factors will allow
investigators to match site characteristics as closely as
possible, reducing the effect of noncontaminant differ-
ences on data comparisons.
In some cases, negative controls (i.e., a medium that is
known to be nontoxic to the test organisms and is geochemi-
cally similar to the test medium) are used instead of a
reference site. This approach provides a reasonable worst-
case comparison by eliminating most of the non-contami-
nant factors that affect organisms at a reference site. Con-
sequently, the difference in organism response between
the test medium and the negative control may be greater
than the difference in organism response between the test
medium and the reference site.
The Objective
The development of clear, attainable objectives is the
most critical element in a toxicity assessment. Objectives
need to reflect the type and level of information required
from the study. Each of the applications shown in Figure 3
may require a different set of objectives. For example, the
objective of toxicity tests used in site characterization may
be to identify the areas with the highest toxicity to organ-
isms in order to prioritize remediation of the site. The other
elements of the assessment would reflect this objective
(e.g., the testing may evaluate both soil and water media,
using acute toxicity test methodologies). If the assessment
objective is to set toxicity-based cleanup standards for soil
at the site, the other elements would focus on the evalua-
tion of soil and the use of chronic toxicity test methodolo-
gies.
An important component of establishing assessment
objectives is the development of data quality objectives
(DQOs). DQOs are qualitative and quantitative statements
specifying the quality of data needed to support test con-
clusions. They are developed in accordance with the
intended end use of the data to be collected. The three
stages of DQO development are: identify decision types,
identify data uses/needs, and design the data collection
program. For further information on development of DQOs,
consult Data Quality Objectives for Remedial Response
Activities. Development Process [21].
The Reference Site
Toxicity tests usually compare the response of one or
more test species exposed to contaminated media with the
species' response to media from an area unaffected by the
site or other sources of contamination. This unaffected
area, or reference site, should be situated as close to the
Superfund site as possible without being impacted by
The Medium
Toxicity tests most often evaluate the effects of con-
taminants in surface water, sediment, or soil. For the latter
two, samples may be tested as bulk samples or processed
first, using water as an extraction fluid to remove sub-
stances adsorbed to the solid particles, and the water
extract used for the test. Bulk soil or sediment tests evaluate
the toxicity of the medium itself, while water extract tests
provide information about the potential toxicity of runoff,
leachate, or water associated with sediment disturbances,
such as dredging. Obtaining information on the medium
being tested is important for characterizing the medium
and in some instances, estimating contaminant availability.
For water, important factors to consider include alkalinity,
hardness, pH, temperature, biochemical oxygen demand,
total dissolved oxygen, total dissolved solids, total organic
carbon, nitrogen, and phosphorus. For sediment and soil,
important factors include grain size distribution, bulk den-
sity, humic content, percent moisture, pH, and total or-
ganic carbon. These factors also must be considered when
establishing control and reference site samples. The effect
of any adjustments to the medium (e.g., increasing the
total dissolved oxygen content or diluting samples to deter-
mine ranges of toxicity) must also be considered and should
be kept to a minimum whenever possible.
Test Organisms
Information on specific test organisms is presented in
the "Descriptions of Biological Toxicity Test Methods"
section of this Bulletin. In most cases, use of standard
organisms will be sufficient to meet assessment objectives.
Organisms typically used for toxicity tests include: bacte-
ria; algae; seeds; young plants; aquatic macroinvertebrates,
such as amphipods, chironomids, and sediment worms;
mollusks; sea urchins; aquatic vertebrates, such as amphib-
ians and fish; and terrestrial invertebrates, such as earth-
Engineering Bulletin: Biological Toxicity Testing
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worms [22, pp. 1-4]. When choosing standard organisms,
it is important to consider species that are representative of
resident organisms, sensitive to site contaminants, appli-
cable to assessment endpoints, consistent with DQOs, and
supportive of assessment goals.
The use of nonstandard organisms, such as contami-
nant sensitive, resident invertebrates or fish species, may
provide better representation of the actual effects of site
contaminants. Additional factors that are standardized
when using designated test species, such as test conditions,
organism age, positive identification, and physiological
condition, must be examined more closely when using
nonstandard organisms. When considering resident spe-
cies for toxicity tests, species-specific factors should be
considered. These factors include the species'potential for
exposure, relative sensitivity to contaminants, role in the
ecological functions of the site, potential for wildlife and
human consumption, time spent on site, characteristics
that contribute to the ease or difficulty of conducting the
test, appropriateness as a surrogate for other species, and
recognized value (e.g., importance as a game fish) [23, p.
3]. Furthermore, collection of some organisms may be
regulated by Federal, State, and local regulations (e.g.,
threatened and endangered species regulations) that must
be followed when gathering test species.
In order to accomplish most biological toxicity assess-
ment objectives, it is usually necessary to use more than one
test organism. For soil tests, one plant and one animal
species can be used. When evaluating aqueous phases, it is
recommended that at least one fish and one invertebrate
species be used, unless the site is known to have only one
contaminant and one of the groups of organisms is known
to be insensitive to that contaminant [3, p. 8]. For sediment
tests, a battery of tests has been recommended [24, p. 556].
In instances where this number of tests is not feasible,
sediment evaluations should include at least two inverte-
brate species, including one that spends a portion of its life
in sediment (e.g., the amphipod, Hyalella azteca ).
Test Method
Information on specific test methods is presented in
the "Descriptions of Biological Toxicity Test Methods"
section of this Bulletin. Standard toxicity test methods have
been developed for the evaluation of contaminated soil,
sediment, and water by several organizations, including
The American Society for Testing and Materials (ASTM),
EPA, and private companies [4][25][26][27][28], Biological
toxicity test methods should supply the following informa-
tion: scope and application; summary of method; sample
collection, preservation, and handling; interferences; equip-
ment (including test organisms); reagents; procedures;
calculations; quality assurance/quality control measures;
data validation and reporting (including statistical presen-
tation); and health and safety considerations. The method
should specify the use of one or more negative controls. A
medium from an identified reference site is sometimes used
for this purpose [25, p. 6].
Table 2 presents the characteristics of an ideal biologi-
cal toxicity test [24, p. 543]. Depending on the objectives
Table 2. Characteristics of an Ideal Biological Toxicity Test
- Rapid
- Simple
- Replicable
- Inexpensive
- Standardized
- Sensitive
- Discriminatory
- Ecologically relevant
- Relatable to field effects
- Useful in developing, and relatable
to, regulatory standards
Source: (24, p.543)
and level of effort required for the assessment, the test may
only meet a portion of these characteristics. For example,
toxicity tests designed to determine the chronic effects of
sediment contaminants on sediment dwelling invertebrates
will meet many of the characteristics, especially sensitivity,
ecological relevance, and usefulness for regulatory stan-
dards. This type of test is not rapid or inexpensive com-
pared to tests focusing on acute effects. Conversely, if the
objective is to identify areas of the site with the highest
sediment toxicity to aquatic macroinvertebrates, an acute
toxicity test that is rapid (e.g., 48 hours), simple, and
relatively inexpensive is more appropriate. The results of
the acute test, however, may be less useful in developing
regulatory or site-specific standards than the results of the
chronic tests.
Test Site
The majority of toxicity tests are performed at labora-
tories on samples of media collected and shipped from the
site of interest. Some companies have mobile laboratory
facilities that can be set up onsite, reducing the time
between sample collection and testing. The advantages of
laboratory testing include constant conditions, standard-
ized protocols, and readily available equipment [3, p. 8].
In situ toxicity tests allow the test organisms to be in
constant exposure to the medium of concern under actual
site conditions. This type of test may provide a more
realistic evaluation of contaminant toxicity, and if using
species present on the site, can generate data that are
directly applicable to the ecological risk assessment. Addi-
tionally, in situ tests do not invoke the regulatory issues or
disposal requirements raised by offsite shipment of con-
taminated media. However, use of in situ toxicity tests
provides little control over changing test conditions. For
example, heavy rainfall during a toxicity test may increase
stream flow, changing the chemical conditions to which
test organisms are exposed. Also, testing designed to
simulate a reasonable worst-case scenario may be disrupted
by changing site conditions, calling into question the strin-
gency of the test. For these reasons, site conditions during
the test should be closely monitored.
Statistical Analysis
The type of statistical analysis used to evaluate toxicity
test results depends on the test objectives and the measure-
ment endpoint used. Measurements that estimate the
effects from specific dilutions (e.g., LC50 and EC50) can be
8
Engineering Bulletin: Biological Toxicity Testing
-------
r
subjected to regression models that assume the greater the
dilution of contaminants, the lesser the effect. Coefficients
of variation can be calculated for these types of point
estimates.
Measurements that compare test dilutions with con-
trols and evaluate whether differences are significant (e.g.,
NOEC and LOEC) make use of hypothesis testing [3, p. 8].
Using the null hypothesis that there is no difference be-
tween a test dilution and the control, the test data should
result in acceptance or rejection of the hypothesis at a
confidence level determined by project DQOs or other
requirements. A thorough discussion on the statistical
analysis of toxicity test results is presented in other EPA
documents [29][30].
QA/QC Standards
In order for test results to be defensible, it is necessary
to have the appropriate level of supporting QA/QC. As
stated earlier, toxicity test methods should specify QA/QC
measures to be followed, starting from sample collection
and concluding with report preparation. These measures
should be consistent with the DQOs for the project. Bio-
logical toxicity assessment plans should have a separate
section on project QA/QC. Large-scale efforts may need to
have separate quality assurance project plans (QAPPs). The
level of QA/QC effort is dictated by the end use of the data.^
EPA has divided data collection projects into four categories
based upon data usage. Category I projects require the
most rigorous and extensive QA; Category IV projects
require the least. Most biological toxicity testing efforts fit
within Category II or III, producing results that complement
other inputs to a decision process, or producing results used
to evaluate and select basic options, respectively. Two
documents useful in establishing appropriate QA are Prepa-
ration Aids for the Development of Category II Quality
Assurance Project Plans and the complementary document
for the development of Category III QAPPs [31][32].
Descriptions of Biological Toxicity Test
Methods
Biological toxicity tests should be chosen to accom-
plish the stated objectives of the study. Depending on
those objectives, tests that differ in measurement end-
points, range of media concentrations, contaminant deliv-
ery scenarios, and organism selection may be chosen. The
following subsections discuss these differences.
Measurement Endpoints
Acute toxicity tests measure an organism's short-term
response (typically 1 to 5 days) to contaminants. The
measurement endpoint for acute toxicity tests usually re-
lates to survival of the test organisms [3, p. 2]. Chronic
toxicity tests generally are more sensitive than acute tests
and commonly expose test organisms to lower contami-
nant concentrations. They also typically require more
financial resources and time to perform. (Chronic tests
presented later in this Bulletin use exposure durations
ranging from 2 to 90 days.) Measurement endpoints for
chronic tests usually include survival, growth, reproductive
impairment, nerve impairment, reduced or abnormal mo-
tility, development of structural abnormalities (teratoge-
nicity), development of chromosomal abnormalities
(genotoxicity and mutagenicity), and behavioral changes
[3, p. 3]. Given the amount and types of data generated by
chronic tests, their expense and duration are often justified.
When designed properly, both acute and chronic toxicity
tests provide valuable, statistically defensible results. Com-
mon measurement endpoints for acute and chronic toxicity
tests were presented in Table 1.
Range of Media Concentrations
Biological toxicity tests can be divided into three cat-
egories based upon the range of media concentrations used
in the tests. These categories are screening, range-finding,
and definitive tests.
Screening tests generally examine the acute and chronic
effects of undiluted samples on the test organisms. These
tests can be useful for site prioritization and site character-
ization by distinguishing between areas of high toxicity and
low/no toxicity. Since these tests are performed at one
concentration (undiluted samples), they are generally less
expensive than range-finding and definitive tests. Signifi-
cant results from screening tests point out the potential
need for definitive tests.
Range-finding tests generally examine the test organ-
isms' acute response to a broad range of media dilutions.
They commonly utilize three or more media dilutions and
do not usually require replicate tests [27, pp. 5-7, 13-15].
Consequently, range-finding tests are usually more expen-
sive than screening tests, but less expensive than definitive
tests. Range-finding tests help identify appropriate dilu-
tions for definitive tests.
Definitive tests establish concentration-response rela-
tionships or NOECs between media concentrations and the
responses of test organisms. These tests typically use a
range of concentrations established by the range-finding
tests. Replicate test units are used when employing defini-
tive tests. These tests can be useful in waste characteriza-
tion, development of cleanup standards, and site monitor-
ing [3, p. 8].
It should be noted that most sediment and soil tests are
currently conducted using undiluted samples only. This
limitation is necessitated by the lack of established tech-
niques for performing sediment and soil dilutions.
Contaminant Delivery Scenarios
There are three contaminant delivery scenarios used for
aqueous-phase toxicity tests. Static tests utilize the same
contaminated medium, with no additions of the medium
throughout the test duration. Static-renewal tests deliver
new test solution to the test organisms by replacing all or a
portion of the aqueous phase at specified times during the
Engineering Bulletin: Biological Toxicity Testing
-------
test. The flow-through method continuously delivers new
aqueous phase to the test organisms, minimizing the abi-
otic loss of contaminants. Of the three approaches, flow-
through systems require the most complex equipment and
incur the greatest expense [3, pp. 5, 8].
Toxicity tests on soil are most commonly of the static
design. Depending on the objectives of the study, how-
ever, aqueous extracts of the soil or the waste can be
introduced to the soil and static-removal or flow-through
tests can be employed. For example, the effects on earth-
worms of leachate from a solidified waste that is to remain
at a site can be examined by pouring the leachate into the
test chamber once (static), intermittently (static-renewal),
or continuously (flow-through) depending on the exposure
scenario of interest. A modification used for sediment
testing is to periodically renew the overlying water while
not renewing the sediment.
Organism Selection
Table 3 lists commonly used biological toxicity tests
that support remedial activities at contaminated sites. The
tests are identified by the organisms employed in the
evaluation. The same species are often used for both acute
and chronic testing, with conditions and duration being
modified to differentiate between the two types of tests.
Table 3 is not intended to be a comprehensive list of
available tests. The focus of the table is on tests that allow
estimations of the effects of contaminated media on popu-
lations (groups of the same species) and, to a lesser extent,
communities (populations of different species) at a site.
Effects at the ecosystem level (as evaluated in microcosm
experiments) and effects of contaminated media on higher
organisms (e.g., birds and mammals) are not covered in the
table or this Bulletin. For more information on these aspects
of biological toxicity testing, refer to Compendium of
Ecological Risk Assessment Tools [33], and consult the
appropriate Regional BTAG member listed in the "Sources
of Additional Information" section of this Bulletin.
General Guidance
When choosing biological toxicity test methods, the
objectives of the monitoring program and available data on
contaminant concentrations should be considered. Acute
tests are usually conducted when concentrations of con-
taminants are in the part per million (ppm) range; chronic
test are usually conducted when contaminants are in the
ppb range. The appropriateness of methods, however, can
be greatly influenced by the characteristics of the medium
being investigated. As previously discussed, water quality
conditions, such as hardness, alkalinity, pH, and/or organic
content will affect the toxicity of both organic and metal
contaminants. For example, high organic content, hard-
ness, or alkalinity will reduce copper toxicity, while high
organic content can make certain organic contaminants
more soluble and therefore, more bioavailable.
Of the tests listed in Table 3, both the minnow and
water flea acute and chronic tests are commonly used for
evaluating the toxicity of streams, lakes, leachates, and
effluents. Acute tests using these organisms are recom-
mended for use in support of feasibility studies where
previous toxicity tests and extensive chemical analyses have
not been performed. If no statistically significant mortality
is observed in the acute tests, chronic tests using these
organisms then should be considered. This approach will
indicate whether acute or chronic risks may need to be
examined in the subsequent stages of the remediation
process.
This approach also should be used to evaluate other
types of contaminated media as well. For marine and
estuarine water, shrimp and fish (silverside or sheepshead
minnow) acute and chronic tests are commonly used. For
whole sediment testing, current EPA methods list amphi-
pods and midge larvae for fresh water sediment and amphi-
pods for marine and estuarine sediment [29][30]. For soil
testing, acute earthworm and 4-day seed germination tests
can be conducted when soil contaminants are in the ppm
range. Genotoxicity, root elongation, and earthworm
reproductive tests are appropriate when soil contamination
is present in ppb levels.
Limitations
Biological toxicity testing should be recognized as one
tool in the overall process of environmental assessment.
When properly utilized it provides information on the
potential effects of contaminated media on the ecological
conditions of the site of interest. Toxicity tests are not
intended to be absolute proof of environmental damage [1,
p. 1]. In order to properly use the information obtained
from toxicity tests, it is important to understand their
limitations.
Biological toxicity tests generally do not differentiate
between the individual contaminants contributing to
the response of the test organism. A review of perti-
nent literature, however, may indicate a test organism's
relative sensitivity to a specific type of contaminant.
For example, a well executed literature search would
reveal that green algae, such as Selenastrum, are more
sensitive to low concentrations of some heavy metals
than higher level organisms, such as fish [24, p. 548].
By using several test organisms, and changing test
conditions (i.e., using the TIE approach), it may be
possible to determine which contaminants are produc-
ing the effects of concern.
Most tests require the identification of a reference site
that is presumably unaffected by the contaminants of
concern. Incorrect selection of the reference site may
cause underestimation of the toxic effects of the me-
dium. This limitation may be addressed through the
use of appropriate laboratory negative controls and the
use of multiple reference sites.
Biological toxicity testing may not be necessary for all
sites. Sites which have contaminant concentrations
below levels associated with documented acute or
chronic effects may not need to be tested. Review of
pertinent literature and site chemical data may prevent
the performance of unnecessary toxicity tests.
10
Engineering Bulletin: Biological Toxicity Testing
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Different organisms display widely different responses
to the same contaminant; this often necessitates the
gathering of prior information on site contaminants
and species sensitivity. The performance of multiple
tests using different species may address this limita-
tion.
Ancillary test conditions (e.g., alkalinity of the test
water) can adversely affect test results. Nonstandard
test conditions also make the comparison of results
between sites and over time difficult to interpret. Test
conditions specified in standardized test methods, there-
fore, should be followed where possible. Also, addi-
tional negative controls should be used to address this
limitation.
In many cases, single toxicity tests will not provide
sufficient information to assist site managers in evalu-
ating the environmental conditions of the site. Several
tests using different species are usually required for
surface water testing [3, p. 9]. Choosing tests that use
site-appropriate organisms may reduce the number of
tests required.
Toxicity test results, when not evaluated in light of
additional information (e.g., taxonomic surveys, chemi-
cal analyses, and computerized toxicity modeling),
present only a partial, and possibly incorrect, picture of
environmental conditions at a site [53, p. 827]. Inter-
pretation of results can be difficult if supporting infor-
mation is unavailable. Since biological toxicity testing
in support of site remediation is a developing field, site
managers may not have adequate experience in results
interpretation. The use of all available resources, in-
cluding BTAG contacts, will help ensure proper site
evaluation.
The collection, shipment, and biological toxicity test-
ing of media contaminated with hazardous waste is
regulated under the treatability study samples require-
ments of 40 CFR 261.4(e-f). (These regulations allow
the collection, shipment, and testing of up to 10,000
kilograms (kg) of each medium contaminated with
non-acute hazardous waste and up to 2,500 kg of each
medium contaminated with acute hazardous waste to
proceed under reduced regulation. These regulations
do specify packing, shipping, storage, and notification
requirements for sample collectors and testing facili-
ties.) [54, pp. 1 3, 38-40] [55, pp. 8365-8366]. Onsite
and in situ testing may reduce regulatory require-
ments.
Current Trends in Biological Toxicity
Testing
The use of biological toxicity testing in conjunction
with predictive modeling has gained considerable interest
in recent years. Models, such as EPA's LC50, are being used
to support field and laboratory toxicity testing by providing
site managers with tools for estimating the biological ef-
fects of contaminated media [33, p. 243]. Additional
information on models is presented in the "Sources of
Additional Information" section of this Bulletin.
Refinement, standardization, and validation of addi-
tional biological toxicity test methods is an ongoing task at
EPA, academic, and private laboratories. The Compendium
of Ecological Risk Assessment Tools, produced in Septem-
ber 1993, list 56 laboratory study methods [33]. ASTM also
continues to update its guides for conducting toxicity tests,
incorporating new species and test procedures [25][26].
Sources of Additional Information
Given the complexity of many Superfund sites, the
difficulty in interpreting toxicity data, and the relatively
recent emphasis on quantitative ecological risk assessment,
site managers are strongly encouraged to make use of
biological toxicity testing resources. For RPMs and OSCs,
the Regional BTAG contacts listed in Table 4 are valuable
sources of information on biological resource issues. An
updated list of contacts is periodically published in ECO
Update, an EPA bulletin described in Table 5. The BTAG
member should be contacted early in the remedial process.
Following initial review of site data, the BTAG member can
make recommendations on the need for biological toxicity
testing. The BTAG contact also should be consulted when
test plans, QAPPs, interim reports, and data summaries are
delivered. BTAG comments on these documents can save
time and money by pointing to the need for additional or
fewer tests [56, p. 2].
RPMs and OSCs are also encouraged to utilize the ^H
Center for Technical Assistance on Ecological Assessment of
Superfund and RCRA Sites. The Center is part of the
Ecological Monitoring Research Division of the National
Exposure Research Laboratory (NERL) in Cincinnati, OH.
The Center supports the Regions by providing technical
reviews, conducting aquatic and terrestrial ecological as-
sessment studies, and performing ecotoxicity testing. Avail-
able assistance with ecological assessments includes collec-
tion and assessment of aquatic and terrestrial biological
systems, assessment of physical habitat, and performance
of ecotoxicity assessments. Assistance with ecotoxicity
assessments includes toxicity testing of water, sediments,
and soils with vertebrates, invertebrates, and plants. The
Center has both in-field and laboratory toxicity testing
capabilities. Most of the tests listed in Table 3 can be
performed in support of Regional efforts. Additionally, the
Center has constructed 12 artificial streams that can be
modified to simulate a variety of site conditions. For further
information on the Center, contact Dr. James Lazorchak at
(513)569-7076.
Non-governmental site managers are encouraged to
consult experts in the academic and private sector, and
work closely with the overseeing regulatory agency.
Table 5 lists additional sources of information on bio-
logical toxicity testing. Many of these sources are available
to both EPA and the general public. jtf
12
Engineering Bulletin: Biological Toxicity Testing
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Table 4. U.S. EPA Regional BTAG Coordinators/Contacts
r
EPA Headquarters
Steve Ells
OWPE
USEPA (OS-510)
401 M Street, S.W.
Washington, DC 20460
(703) 603-8934
(703)603-6724 FAX
Joseph Tieger
USEPA(OS-SIOW)
401 M Street, S.W.
Washington, DC 20460
(202)260-3104
David Charters
Mark Sprenger
ERT
USEPA(MS-IOI)
2890 Wood bridge Ave.
Building 18
Edison, NJ 08837-3679
(908) 906-6825 - David
(908) 906-6826 - Mark
(908) 321-6724 FAX
Region 1
Susan Svirsky
Waste Management Division
USEPA Region 1 (HSS-CAN7)
JFK Federal Building
Boston, MA 02203
(617) 573-9649
(617) 573-9662 FAX
Region 2
Shari Stevens
Environmental Services Division
USEPA Region 2 (MS-220)
2890 Woodbridge Ave.
Building 209
Edison, NJ 08837
(908) 906-6994
(908) 321-6616 FAX
Region 3
Robert Davis
Technical Support Section
USEPA Region 3 (3HW13)
841 Chestnut St.
Philadelphia, PA 19107
(215)597-3155
(215) 597-9890 FAX
Region 4
Lynn Wellman
USEPA Region 4 (4WD-OHA)
345 Courtland St., N.E.
Atlanta, GA 30365
(404) 347-3555 x6366
(404) 347-0076 FAX
Region 5
Brenda Jones
USEPA Region 5 (5HSRLT-5J)
77 W. Jackson Blvd.
Chicago, IL 60604-1602
(312)886-7188
(312) 886-0753 FAX
Region 6
Jon Rauscher
Susan Swenson Roddy
USEPA Region 6 (6H-SR)
First Interstate Tower
1445 Ross Ave.
Dallas, TX 75202-2733
(214)665-8513
(214) 665-6762 FAX
Region 7
Bob Koke
USEPA Region 7 (SPFD-REML)
726 Minnesota Ave.
Kansas City, KS 66101
(913)551-7468
(913) 551-7063 FAX
Region 8
Gerry Henningsen
USEPA Region 8 (8HWM-SM)
999 18th Street, Suite 500
Denver, CO 80202-2466
(303) 294-7656
(303) 293-1230 FAX
Region 9
Clarence Callahan
USEPA Region 9
75 Hawthorne St. (H93)
San Francisco, CA 94105-3901
(415)744-2314
(415) 744-1916 FAX
Region 10
Julius Nwosu
USEPA Region 10 (ES-098)
1200 6th Ave.
Seattle, WA 98101
(206)553-8086
(206) 55 3-0119 FAX
EPA Contact
Technical questions regarding this Bulletin may be
directed to:
Dr. James Lazorchak
U.S. Environmental Protection Agency
National Exposure Research Laboratory
26 W. Martin Luther King Drive
Cincinnati, Ohio 45268
(513)569-7076
Acknowledgments
This Bulletin was prepared for the U.S. Environmental
Protection Agency, Office of Research and Development
(ORD), National Risk Management Research Laboratory
(NRMRL), Cincinnati, Ohio, by Science Applications Inter-
national Corporation ( SAIC) under Contract No. 68-CO-
0048. Dr. Steven Safferman, formerly of EPA, initiated the
project, Dr. James Lazorchak served as the EPA Technical
Project Monitor. Mr. Jim Rawe served as SAIC's Work
Assignment Manager. This Bulletin was authored by Mr.
Kurt Whitford of SAIC.
The following additional Agency personnel have con-
tributed their time and expertise by reviewing and com-
menting on the document: Mr. Edward Bates, Mr. Paul de
Percin, and Mr. Mark Meckes of NRMRL; Dr. Gerald Ankley
of NERL in Duluth, MN; Dr. William (Skip) Nelson of NERL
in Narragansett, Rl; Mr. Thomas Taccone of Region 2; Mr.
Ron Preston of Region 4; Mr. James Hahnenberg of Region
5; and Dr. Clarence Callahan of Region 9. Additional SAIC
personnel contributing to development of this document
include Mr. Clyde Dial, Dr. Robert Hoke, Ms. Jo-Ann
Hockemeier, Ms. Lisa Kulujian, and Ms. Debbie Seibel.
Engineering Bulletin: Biological Toxicity Testing
13
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Table 5. Additional Sources of Information on Biological Toxicity Testing
Sample
Collection
Characterization of Hazardous Waste Sites-A Methods Manual: Volume II. Available Sampling Methods,
Second Edition. EPA-600/4-84/076, December 1984.
Compendium of Ecological Risk Assessment Tools. September 28, 1993.
Macroinvertebrate Field and Laboratory Methods for Evaluating the Biological Integrity of Surface Waters.
EPA/600/4-90/030, November 1990.
Fish Field and Laboratory Methods for Evaluating the Biological Integrity of Surface Waters. EPA/600/R-
92/111, March 1993.
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisims, EPA/600/4-91/002. December 1994.
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and
Estuarine Organisims. EPA/600/4-91/003, December 1994.
Predictive
Models
LCS0. Model estimates LC50 values for species based upon experimental data. Contact the Center for
Exposure Assessment Modeling, Environmental Research Laboratory, USEPA, Athens, GA 30613-0801.
(706)546-3130.
FGETS (Food and Gill Exchange of Toxic Substances.) Model predicts bioaccumulation and survival of
several types of fish exposed to pollutants. Contact the Center for Exposure Assessment Modeling,
Environmental Research Laboratory, USEPA, Athens, GA 30613-0801. (706) 546-3130.
Databases
AQUIRE (AQUatic toxicity Information REtrieval.) Database containing toxicity information from reports
published in the open literature. Contact the Scientific Outreach Program, Environmental Research
Laboratory, USEPA, Duluth, MN 55804. (218) 720-5602.
ASTER (Assessment Tool for the Evaluation of Risk.) Toxicological database and predictive model
containing effects data for pollutants in aquatic ecosystems. Contact the Scientific Outreach Program,
Environmental Research Laboratory, USEPA, Duluth, MN 55804. (218) 720-5602.
Publications
ECO Update (Publication 9345.0-051) and BTAG Forum (Publication 9200.3251). Intermittent bulletins
providing information on toxicity testing and other ecological assessment topics. Contact BTAG Forum,
USEPA, 303 Methodist Building, 11th and Chapline Streets, Wheeling, WV 26003. (304) 234-0245.
REFERENCES
1. Risk Assessment Guidance for Superfund, Volume II, En-
vironmental Evaluation Manual. EPA/540/1-89/001,
U.S. Environmental Protection Agency, March 1989.
2. Ecological Assessment of Superfund Sites: An Overview.
ECO Update, Vol. 1, No. 2. Publication 9345.0-051,
U.S. Environmental Protection Agency, December 1991.
3. Using Toxicity Tests in Ecological Risk Assessment. ECO
Update, Vol. 2, No. 1 Publication 9345.0-051. U.S. Envi-
ronmental Protection Agency, September 1994.
4. Evaluation of Terrestrial Indicators for Use in Ecological
Assessments at Hazardous Waste Sites. EPA/600/R-92/
183, U.S. Environmental Protection Agency, September
1992.
5. Suter, G.W. Ecological Risk Assessment. Lewis Publish-
ers, 121 South Main Street, Chelsea, Ml 48118, 1992.
6. Protocols for Short Term Toxicity Screening of Hazard-
ous Waste Sites. EPA/600/3-88/029, U.S. Environmental
Protection Agency, July 1988.
7. Test Methods for Evaluating Solid Waste. SW-846,
Third Edition, U.S. Environmental Protection Agency,
1986.
8. USEPA Contract Laboratory Program, Statement of
Work for Organics Analysis. U.S. Environmental Protec-
tion Agency, February 1988.
9. National Research Council. Testing for Effects of Chemi-
14
Engineering Bulletin: Biological Toxicity Testing
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r
cals on Ecosystems, A Report by the Committee to Re-
view Methods for Ecotoxicology. National Academy
Press, 2101 Constitution Avenue N.W., Washington
D.C. 20418, 1981.
10. Marty, G.D., et al. Fish-based Biomonitoring to Deter-
mine Toxic Characteristics of Complex Chemical Mix-
tures: Documentation of Bioremediation at a Pesticide
Disposal Site. Aquatic Toxicology, Vol. 19, pp. 329-340,
1991.
11. Baudo, R., j.P. Giesy, and H. Muntau eds. Sediments:
Chemistry and Toxicity of In-Place Pollutants. Lewis
Publishers, 121 South Main Street, Chelsea, Ml 48118,
1990.
12. Personal communications between Deana Jenigen of
SAIC and several commercial laboratories. July 1994.
13. Methods for Aquatic Toxicity Identification Evaluations:
Phase I Toxicity Characterization Procedures, 2nd Edi-
tion. EPA/600/6-91/003, U.S. Environmental Protection
Agency, February 1991.
14. Toxicity Identification Evaluation: Characteri-zation of
Chronically Toxic Effluents, Phase I. EPA/600/6-91/
005F, U.S. Environmental Protection Agency, May 1992.
15. Methods for Aquatic Toxicity Identification Evaluations:
Phase II Toxicity Identification Procedures for Samples
Exhibiting Acute and Chronic Toxicity. EPA/600/R-92/
080, U.S. Environmental Protection Agency, September
1993.
16. Methods for Aquatic Toxicity Identification Evaluations:
Phase III Toxicity Confirmation Procedures for Samples
Exhibiting Acute and Chronic Toxicity. EPA/600/R-92/
081, U.S. Environmental Protection Agency, September
1993.
17. Sediment Toxicity Identification Evaluation: Phase I
(Characterization), Phase II (Identification), and Phase III
(Confirmation) Modifications of Effluent Procedures.
Prepared by Environmental Research Laboratory, 6201
Congdon Blvd., Duluth, MN 55804. U.S. Environmen-
tal Protection Agency, 1991.
18. The Role of Natural Resource Trustees in the Superfund
Process. ECO Update, Vol. 1, No. 3. Publication
9345.0-051. U.S. Environmental Protection Agency,
March 1992.
19. Guide for Conducting Treatability Studies Under
CERCLA. EPA/540/R-92/071a, U.S. Environmental Pro-
tection Agency, October 1992.
20. Nelson, W.G., and D.|. Hansen. Development and Use
of Site-Specific Chemical and Biological Criteria for As-
sessing New Bedford Harbor Pilot Dredging Project. En-
vironmental Management, Vol. 15, pp. 105-112, 1991.
21. Data Quality Objectives for Remedial Response Activi-
ties, Development Process. EPA/540/G-87/003, U.S.
Environmental Protection Agency, March 1987.
22. Catalogue of Standard Toxicity Tests for Ecological Risk
Assessment. ECO Update, Vol. 2, No. 2. Publication
9345.0-051, U.S. Environmental Protection Agency, Sep-
tember 1994.
23. Field Studies for Ecological Risk Assessment (Draft).
ECO Update, Vol. 2, No. 3 Publication 9345.0-051, U.S.
Environmental Protection Agency, October 1993. EPA
Contact: Ron Preston, (304) 234-0245.
24. Giesy, J.P. and R.A. Hoke. Freshwater Sediment Toxicity
Bioassessment: Rationale for Species Selection and Test
Design. Journal of Great Lakes Research, Vol. 15, pp.
539-569, 1989.
25. Standard Guide for Conducting Sediment Toxicity Tests
with Freshwater Invertebrates. E 1383-94, American
Society for Testing and Materials, April 1994.
26. Standard Guide for Conducting 10-day Static Sediment
Toxicity Tests with Marine and Estuarine Amphipods.
E 1 367-92, American Society for Testing and Materials,
January 1993.
27. Compendium of ERT Toxicity Testing Procedures. EPA/
540/P-91/009, U.S. Environmental Protection Agency,
January 1991.
28. Microtox Product Literature. Microbics Corporation,
Carlsbad, CA, 1994.
29. Methods for Measuring the Toxicity and
Bioaccumulation of Sediment-associated Contaminants
with Freshwater Invertebrates. EPA/600/R-94/024, U.S.
Environmental Protection Agency, June 1994.
30. Methods for Assessing the Toxicity of Sediment-associ-
ated Contaminants with Estuarine and Marine Amphi-
pods. EPA/600/R-94/025, U.S. Environmental
Protection Agency, June 1994.
31. Preparation Aids for the Development of Category II
Quality Assurance Project Plans. EPA/600/8-91/004,
U.S. Environmental Protection Agency, February 1991.
32. Preparation Aids for the Development of Category III
Quality Assurance Project Plans. EPA/600/8-91/005,
U.S. Environmental Protection Agency, February 1991.
33. Compendium of Ecological Risk Assessment Tools
(Draft). Prepared by Eastern Research Group, 110
Hartwell Avenue, Lexington, MA 02173-3198. U.S. En-
vironmental Protection Agency, September 1993.
34. Baud-Grasset, S., et al. Reduction of Genotoxicity of a
Creosote-Contaminated Soil after Fungal Treatment De-
termined by the Tradescantia-Micronucleus Test. Muta-
tion Research, Vol. 303, pp. 77-82, 1993.
35. Application of Tradescantia Micronucleus Assay for In
Situ Evaluation^ Potential Genetic Hazards from Expo-
sure to Chemicals at a Wood Preserving Site. EPA/600/
J-92/282, U.S. Environmental Protection Agency, 1991.
36. Application of a Plant Test System in the Identification
of Potential Genetic Hazards at Chemical Waste Sites.
EPA/600/D-91/275, U.S. Environmental Protection
Agency, 1991.
37. Kaiser, K.L.E. and V.S. Palabrica. Photobacterium
Phosphoreum Toxicity Data Index. Water Pollution Re-
search Journal of Canada, Vol 26, pp. 361-431, 1991.
38. Middaugh, D.P., et al. Detoxification of Pentachlo-
rophenol and Creosote Contaminated Groundwater by
Physical Extraction: Chemical and Biological Assess-
ment. Archives of Environmental Contamination and
Engineering Bulletin: Biological Toxicity Testing
15
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