EPA
United States
Environmental
Protection
Agency
Office of
Solid Waste and
Emergency Response
Publication 9345.0-051
March 1994
ECO Update
Office of Emergency Remedial Response
Hazardous Site Evaluation Division (5204G)
Intermittent Bulletin
Volume 2, Number 2
Catalogue of Standard Toxicity Tests for
Ecological Risk Assessment
This Bulletin, which serves as a companion to "Using
Toxicity Tests in Ecological Risk Assessments" (ECO
Update Vol. 1, No. 1), consists of an annotated list of
standardized aquatic, sediment, terrestrial, and microbial
toxicity tests currently in use at Superfund sites. Future
Bulletins will address new approaches to measuring
toxicity, as they become available.
In addition to the standardized approaches described in
this Bulletin, the literature describes many other toxicity
tests. In some cases, an investigator may identify a non-
standard test that appears more relevant to a site and its
contaminant picture than the standardized tests. Before
deciding to use the non-standard test, the investigator
should ascertain that the test is both repeatable and logical.
The BTAG can assist the investigator in making a sound
decision.
Most of the terms used in this document are defined in
the companion Bulletin. Reference numbers in the text
indicate source documents (listed at the end of this
Bulletin) that more fully describe test protocols. In the
case of acute aquatic toxicity tests, the catalogue also
directs the reader to sources describing additional
standardized tests.
IN THIS BULLETIN
Aquatic Toxicity Test 1
Sediment Toxicity Tests 3
Terrestrial Toxicity Tests 3
Microbial Toxicity Tests 4
References 4
Aquatic Toxicity Tests
The aquatic toxicity tests described here are the most
commonly used tests and are highly standardized.
Investigators1 should have little difficulty identifying
laboratories capable of performing them.
While these tests have the advantage of wide acceptance
and well-established protocols, other tests can contribute to the
ecological risk assessment of Superfund sites. The references
at the end of this Bulletin include sources of information about
many such tests. The Regional Biological Technical
Assistance Group (BTAG)2 also is a source of information
about toxicity tests that are especially well suited for use at a
particular site.
Acute Freshwater Toxicity Tests
The following two tests measure the lethality of water
samples to freshwater organisms, indicating the toxicity of
water samples. They are described in Reference 5.
1 The term "investigator" refers to the individual charged with
responsibility for designing and/or carrying out any part of an ecological risk
assessment. Investigators can include government scientists, contractors, or
university scientists. However, the site manager (remedial project manager or
on-site coordinator) retains ultimate responsibility for the quality of the
ecological risk assessment.
2 These groups are sometimes known by different names, depending on
the Region, and not all Regions have established BTAGs. Readers should
check with the appropriate Superfund manager for the name of the BTAG
coordinator or other sources of technical assistance in their Region. A more
complete description of BTAG structure and function is available in "The
Role of BTAGs in Ecological Assessment" (ECO Update Vol. 1, No. 1).
ECO Update is a Bulletin series on ecological risk assessment of Superfund sites. These Bulletins serve as supplements to Risk Assessment Guidance for
Superfund, Volume II: Environmental Evaluation Manual (EPA/540-1-89/001). The information presented is intended to provide technical information to EPA and
other government employees. It does not constitute rulemaking by the Agency, and may not be relied on to create a substantive or procedural right enforceable by any
other person. The Government may take action that is at variance with these Bulletins.
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The Daphnia pulex or Daphnia magna acute toxicity test
evaluates acute toxicity of a sample to a water flea belonging
to the genus Daphnia. The test uses a static or static-renewal
design and lasts 24, 48, or 96 hours. Observations at 24, 48,
and 96 hours permit the calculation of 24-, 48-, and 96-hour
LC values.
Two documents describe additional acute freshwater
toxicity tests. Methods for Measuring the Acute Toxicity of
Effluents and Receiving Waters to Freshwater and Marine
Organisms (Reference 6) describes acute toxicity tests with
three species of freshwater fish and three invertebrate species.
The test organisms include both warm-water and cold-water
species. The guide includes range finding and definite tests.
The second document, Annual Book ofASTM Standards:
Water and Environmental Technology (Reference 1), contains
guidelines for acute toxicity tests and sediment elutriate tests
using freshwater fish, macroinvertebrates, and amphibians.
The guidelines describe static, static-renewal, and flow-
through techniques and recommend that investigators consider
EC50s along with lethality.
Chronic Freshwater Toxicity Tests
These tests measure both lethal and sublethal effects over
the life cycle or partial life cycle of freshwater organisms,
providing information useful in assessing the potential long-
term effects of contamination. Originally developed for the
NPDES permitting program, the tests have since been used in
assessing the toxicity of water associated with waste sites. All
of these tests are described in Reference 8.
The Ceriodaphnia dubia survival and reproduction test
estimates chronic toxicity of a sample to Ceriodaphnia dubia,
which is a water flea. The test uses the static-renewal design
and lasts for seven days, monitoring both the survival of test
organisms and the number of offspring they produce.
The fathead minnow (Pimephales promelas) larval
survival and growth test uses the static-renewal design and
lasts for seven days, tracking the survival of test organisms
and their increase in weight.
The fathead minnow (Pimephales promelas) embryo-
larval survival and teratogenicity test assesses the chronic
toxicity of a sample to minnows, beginning as embryos and
extending to the larval stage. The test uses the static-renewal
design and lasts for seven days, noting both the survival of the
fish and the induction of terata.
The algal (Selenastrum capricornutum) growth test
identifies both biostimulatory3 and chronic toxic effects of a
sample to a one-celled freshwater alga. The test uses the static
design and lasts 96 hours, most commonly monitoring cell
density (cells per mL). Alternative measures include biomass
3 Biostimulation refers to excessive growth of algae, a condition not likely
to occur at Superfund sites.
(weight of living matter), chlorophyll content, or light
absorbance.
Acute Marine Toxicity Tests
These tests measure short-term lethality to marine and
estuarine organisms. Protocols differ little from acute toxicity
tests for freshwater organisms, with the marine/estuarine tests
incorporating the appropriate species substitutions and test
condition adjustments.
The static acute toxicity test using larvae of bivalve
mollusks evaluates the acute toxicity of test media to one of
four species of bivalve mollusks (invertebrates such as clams,
with two piece shells). The test lasts 48 hours and notes
abnormal shell development. (See Reference 1.)
The static acute toxicity test using silversides (Menidia
species) or sheepshead minnow (Cyprinodon variegatus)
evaluates the acute toxicity of test media to these fish species.
The screening test lasts 24 hours and the definitive test 48
hours. Both record mortality as a lack of movement. (See
Reference 6.)
Two documents describe additional acute marine toxicity
tests. The Annual Book of ASTM Standards: Water and
Environmental Technology (Reference 1) contains guidelines
for tests using marine and estuarine fish and
macroinvertebrates. This volume presents a variety of tests
including static, static-renewal, and flow-through designs.
Test durations vary from two to eight days, depending on the
species selected.
The second document, Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms (Reference 6) provides protocols for tests
using four species of marine and estuarine fish and one
invertebrate species. The test organisms include both warm-
water and cold-water organisms. Range finding and definitive
tests are represented in the guide. Duration and test conditions
vary with the species and specific test selected.
Chronic Marine Toxicity Tests
The following tests measure both lethal and sublethal
effects over the life cycle or partial life cycle of marine and
estuarine organisms, providing information about the potential
long-term effects of contamination. The tests using fish
resemble the survival, growth, and teratogenicity tests for
freshwater fish, with the appropriate adjustments of test
conditions and species substitutions. All of these tests are
described in Reference 7.
The inland silverside (Menidia beryllina) larval survival
and growth test is a static-renewal test that lasts for seven
days and measures the survival and increase in weight of
inland silverside larvae.
The sheepshead minnow (Cyprinodon variegatus) larval
survival and growth test uses the static-renewal design and
lasts for seven days, monitoring the survival of test organisms
and their increase in weight.
March 1994 • Vol. 2, No. 2
ECO Update
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The sheepshead minnow (Cyprinodon variegatus)
embryo-larval survival and teratogenicity test assesses the
chronic toxicity of a sample to minnows, beginning as
embryos and extending to the larval stage. The test uses the
static-renewal design and lasts for nine days, recording both
the survival of the fish and any development of terata.
The mysid (Mysidopsis bahia) survival, growth, and
fecundity test evaluates the chronic toxicity of a sample to
shrimp, beginning as juveniles and extending through their
sexual maturation. The test uses the static-renewal design and
lasts for seven days, monitoring survival, weight gain, and egg
production.
The sea urchin (Arbacia punctulata) fertilization test
evaluates toxicity to the eggs and sperm of Arbacia
punctulata. The test exposes dilute sperm suspensions to a
water sample for one hour. Then eggs are added, and 20
minutes later the test terminates. The technician then
calculates the percent fertilization.
The algal (Champia parvula) sexual reproduction test uses
a static design and lasts five to seven days, exposing a mixture
of male and female algae to the sample for two days and then
transferring them to a nontoxic medium. At the end of the
test, the test organisms are scored for their production of
cystocarps, the structures that result from fertilization.
Sediment Toxicity Tests
Because of the paucity of data concerning sediment
toxicity, tests designed specifically to evaluate the toxicity of
sediments are in their early stages of development. Currently,
protocols exist for chronic toxicity tests only. As research
continues, new tests should become available to investigators.
For additional information on sediment toxicity tests,
consult ASTM's Standard Guide for Conducting Sediment
Toxicity Tests with Freshwater Invertebrates (Reference 4).
In addition, Protocols for Short Term Toxicity Screening of
Hazardous Waste Sites by Greene et al. (Reference 5)
describes a lettuce seed germination assay and a lettuce root
elongation assay that can be used to measure the toxicity of
elutriates.
Chronic Freshwater Sediment Toxicity
Tests
The following tests assess the chronic toxicity of sediment
samples to three freshwater invertebrates. For these tests a
layer of water overlies the sediment sample. The design may
either be static or involve flow-through replacement of the
overlying water. Test duration varies with the objectives of
the study but generally does not extend beyond 30 days.
These tests are described in Reference 2.
Hyalella azteca sediment toxicity tests evaluate sediment
toxicity to Hyalella azteca, an amphipod that swims in the
water column and burrows in the sediment surface. Short-
term tests last 10 days or fewer and evaluate the survival,
growth, and development of the test organisms. Longer tests
can last up to 30 days allowing the evaluation of the effect of
the sediment on reproduction behavior, sexual development,
egg production, and the development of offspring.
Chironomus tentans and Chironomus riparius sediment
toxicity tests evaluate sediment toxicity to midge larvae. The
larvae burrow into sediment to build a casing within which
they mature. Tests lasting 10-14 days evaluate the effect of
exposure on survival and growth. Longer tests assess the
effects of toxicity on development and reproduction.
Chronic Marine Sediment Toxicity Test
The ten-day static sediment toxicity test using marine
and estuarine amphipods measures the acute toxicity of
marine sediments to amphipods that burrow in the sediment.
This test also can evaluate sublethal effects, such as
emergence from a highly toxic sediment and the inability to
re-burrow into clean sediment at the termination of the assay.
(See Reference 3.)
Terrestrial Toxicity Tests
Compared with aquatic toxicity tests, few protocols exist
for evaluating the toxicity of soils. However, several
techniques currently in development should soon be
standardized, increasing the number of options available.
Some investigators have tried to overcome the lack of
standardized terrestrial toxicity tests by preparing elutriates
and analyzing these by means of aquatic toxicity tests.
However, this approach does not account for the toxicity of
contaminants that remain sorbed to soil particles. On the other
hand, analyzing elutriates with aquatic toxicity tests can prove
useful when exploring the mobility of contaminants.
These tests are described in Reference 5.
The earthworm (Eisenia foetida) survival assay
estimates toxicity of soil or solid waste to earthworms. The
test uses the static design and lasts 14 days, monitoring the
survival of the test organisms. This assay usually involves the
use of soil samples, but can be conducted in sediment diluted
with artificial soil. An alternate test design—exposing
earthworms to artificial soil mixed with water samples or
elutriate dilutions—makes this assay useful in assessing water
samples or elutriates.
The lettuce (Latuca saliva) seed germination assay
employs a static design and lasts, as specified in the protocol,
120 hours (5 days). As in the earthworm test, the test can use
sample soil or sediment diluted with artificial soil, or artificial
soil wetted with a water sample or an elutriate of soil or
sediment.
The lettuce (Latuca saliva) root elongation assay also
uses a static design and lasts, as specified in the protocol, 120
hours (5 days). This test monitors both seed germination and
seedling length. In this test, the technician places the seeds on
pieces of filter paper wetted with either a water sample, an
elutriate sample, or a sample dilution.
March 1994 • Vol. 2, No. 2
ECO Update
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Microbial Toxicity Tests
Microbial toxicity tests assess toxic effects on the
microbial community and can serve as cost-effective and rapid
screening indicators. They are described in Reference 4.
The ATP-TOX system test measures a sample's effect on
bacterial growth. For this test, bacteria are suspended in a
water sample or soil or sediment elutriate. After several life
cycles, a technician estimates the density of bacterial growth
using a method that gives the test its name: since each
bacterium has a fairly constant concentration of the chemical
compound adenosine triphosphate (ATP), measuring the ATP
content of a suspension of bacteria provides a reliable estimate
of the bacterial population.
The Microtek test measures the toxicity of water samples
or elutriates to Photobacterium phosphoreum, a species of
bioluminescent marine bacteria. Some contaminants inhibit
the bacteria's metabolism, decreasing the intensity of light
emitted. Other contaminants stimulate the bacteria and cause
an increase in luminescence.
Conclusion
As discussed in the companion Bulletin to this document,
toxicity tests represent one set of tools that can be used to
evaluate possible adverse ecological effects at Superfund sites.
To be effective, these tests must be planned and evaluated
carefully, in the context of an overall ecological risk
assessment designed to meet specific objectives. Site
managers are urged to consult with their Regional BTAGs to
ensure that tests appropriate to specific circumstances are
selected and that the tests are conducted in such a manner as to
be useful in supporting remedial decisions.
References
1. American Society for Testing and Materials (ASTM).
1992. Annual Book of ASTM Standards: Water and
Environmental Technology, Vol. 11.04. American Society
for Testing and Materials, Philadelphia, PA. 1426 pp.
2. American Society for Testing and Materials (ASTM).
1992. Standard Guide for Conducting Sediment Toxicity
Tests with Freshwater Invertebrates. American Society
for Testing and Materials, Philadelphia, PA. 23 pp.
3. American Society for Testing and Materials (ASTM).
1992. Standard Guide for Conducting 10-day Static
Sediment Toxicity Tests with Marine and Estuarine
Amphipods. American Society for Testing and Materials,
Philadelphia, PA. 24 pp.
4. Bitten, G., B.J. Dutka, and C.W. Hendricks. 1989.
Chapter 6, Section 4 in Warren-Hicks, W., B.R. Parkhurst
and S.S. Baker Jr., eds. Ecological Assessment of
Hazardous Waste Sites: A Field and Laboratory
Reference. EPA/600/3-89/013. Environmental Research
Laboratory, Office of Research Development,
Environmental Protection Agency, Corvallis, OR.
U.S.
5. Greene, J.C., Bartels, W.J. Warren-Hicks, B.R. Parkhurst,
G.L. Linder, S.A. Peterson, and W.E. Miller. 1989.
Protocols for Short-Term Toxicity Screening of
Hazardous Waste Sites. EPA/600/3-88/029.
Environmental Research Laboratory, Office of Research
and Development, U.S. Environmental Protection
Agency, Corvallis, OR.
6. Weber, C.I. 1991. Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater
and Marine Organisms. 4th edition. EPA/600/4-90/027.
Environmental Monitoring Systems Laboratory, Office of
Research and Development, U.S. Environmental
Protection Agency, Cincinnati, OH.
7. Weber, C.I., W.I. Horning, D.J. Claim, T.W. Neiheisel,
P. A. Lewis, E.L. Robinson, J. Menkedick, and F. Kessler.
1988. Short-Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Marine and
Estuarine Organisms. EPA/600/4-87/028. Environmental
Monitoring and Support Laboratory, Office of Research
and Development, U.S. Environmental Protection
Agency, Cincinnati, OH.
8. Weber, C.I., W.H. Peltier, TJ. Norberg-King, W.B.
Horning II, F. Kessler, and J. Menkedick. 1989. Short-
term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Freshwater Organisms.
2nd edition. EPA/600/4-89/001. Environmental
Monitoring Systems Laboratory, Office of Research and
Development, U.S. Environmental Protection Agency,
Cincinnati, OH.
March 1994 • Vol. 2, No. 2
ECO Update
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