8010 Introduction

8010 INTRODUCTION*
8010 A. General Discussion
1. Uses of Toxicity Tests
Toxicity tests are desirable in water quality evaluations be-
cause chemical and physical tests alone are not sufficient to
assess potential effects on aquatic biota.
1–3
For example, the
effects of chemical interactions and the influence of complex
matrices on toxicity cannot be determined from chemical tests
alone. Different species of aquatic organisms are not equally
susceptible to the same toxic substances nor are organisms
equally susceptible throughout the life cycle. Even previous
exposure to toxicants can alter susceptibility. In addition, organ-
isms of the same species can respond differently to the same
level of a toxicant from time to time, even when all other
variables are held constant.
Toxicity tests are useful for a variety of purposes, which
include determining: (a) suitability of environmental conditions
for aquatic life, (b) favorable and unfavorable environmental
factors, such as dissolved oxygen (DO), pH, temperature, salin-
ity, or turbidity, (c) effect of environmental factors on waste
toxicity, (d) toxicity of wastes to a test species, (e) relative
sensitivity of aquatic organisms to an effluent or toxicant, (f)
amount and type of waste treatment needed to meet water
pollution control requirements, (g) effectiveness of waste treat-
ment methods, (h) permissible effluent discharge rates, and (i)
compliance with water quality standards, effluent requirements,
and discharge permits. In such regulatory assessments, use tox-
icity test data with receiving-water and site-specific discharge
data on volumes, dilution rates, and exposure times and concen-
trations.
2. Test Procedures
There is a need to use correct terminology (see 8010B, Ter-
minology), and environmentally relevant test procedures to meet
regulatory, legal, and research objectives.
3– 8
The procedures given below allow measurement of biological
responses to known and unknown concentrations of materials in
both fresh and saline waters. These toxicity tests are applicable
to routine monitoring requirements as well as research needs.
Refer to Part 9000 for microbiological methods and Part 10000
for field and other types of biological laboratory methods for
water quality evaluations. Refer to Section 10900 for identifica-
tion aids for aquatic organisms.
Reasonable uniformity of procedures and of data presentation
is essential. The use of standardized methods described below
will ensure adequate uniformity, reproducibility, and general
usefulness of results without interfering unduly with the adapt-
ability of the tests to local circumstances.
Quality assurance practices for toxicity test methods include
all aspects of the test that affect the quality of the data. These
include sampling and handling, source and condition of test
organisms, performance of reference toxicant tests, and the test
procedures themselves. Quality assurance/quality control guide-
lines are available for single compound testing and general
laboratory practices
9
and for effluent evaluations in technical
guidance manuals for conducting acute and short-term chronic
toxicity tests with effluents.
10 –12
Other publications covering aspects of toxicity test procedures
are available.
8,13–15
Also, see 8010I.
3. References
1. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1991. Technical Support
Document for Water Quality-Based Control. EPA-505/2-90-001
(PB91-127415), Off. Water, U.S. Environmental Protection
Agency, Washington, D.C.
2. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1987. Permit Writer’s
Guide to Water Quality-Based Permitting for Toxic Pollutants. Off.
Water, U.S. Environmental Protection Agency, Washington, D.C.
3. GROTHE, D.R., K.L. DICKSON & D.K. REED-JUDKINS, eds. 1996.
Whole Effluent Toxicity Testing: An Evaluation of Methods and
Prediction of Receiving System Impacts. SETAC Pellston Work-
shop on Whole Effluent Toxicity, Sept. 16 –25, 1995, Pellston,
Mich. SETAC Press, Pensacola, Fla.
4. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Annual Book
of ASTM Standards, Section 11, Water and Environmental Tech-
nology. Volume 11.05 Pesticides; Resources Recovery; Hazardous
Substances and Oil Spill Responses; Waste Disposal; Biological
Effects. American Soc. Testing & Materials, W. Conshohocken, Pa.
5. ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT. 1981.
OECD Guidelines for Testing of Chemicals. Organization for Eco-
nomic Cooperation and Development, Paris, France.
6. BERGMAN, H., R. KIMERLE & A.W. MAKI, eds. 1985. Environmental
Hazard Assessment of Effluents. Pergamon Press, Inc., Elmsford,
N.Y.
7. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Standard
guide for conducting acute toxicity tests on aqueous ambient sam-
ples and effluents with fishes, macroinvertebrates, and amphibians.
E1192-97 (2003), Annual Book of ASTM Standards, Vol. 11.06.
American Soc. Testing & Materials, W. Conshohocken, Pa.
8. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Standard
guide for conducting acute toxicity tests on test materials with
fishes, macroinvertebrates, and amphibians. E729-96 (2002), An-
nual Book of ASTM Standards, Vol. 11.06. American Soc. Testing
& Materials, W. Conshohocken, Pa.
9. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1987. Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice
Standards. Proposed Rule. 40 CFR Part 160; Federal Register
52:48920.
10. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Methods for Mea-
suring the Acute Toxicity of Effluents and Receiving Waters to
Freshwater and Marine Organisms, 5th ed. EPA-821-R-02-012, Off.
Water, U.S. Environmental Protection Agency, Washington, D.C.
11. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Short-Term Meth-
ods for Estimating the Chronic Toxicity of Effluents and Receiving
* Approved by Standard Methods Committee, 1997. Editorial revisions, 2009.
Joint Task Group: 20th Edition—Wayne L. McCulloch (chair), Jeffrey A. Black,
Dennis T. Burton, William T. Goodfellow, Donald R. Grothe, James J. Pletl,
William J.Rue.
1

Waters to Marine and Estuarine Organisms, 3rd ed., EPA-821-R-
02-014. Off. Water, U.S. Environmental Protection Agency, Wash-
ington, D.C.
12. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Short-Term Meth-
ods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms, 4th ed., EPA-821-R-02-013. Off.
Water, U.S. Environmental Protection Agency, Washington, D.C.
13. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Standard
practice for statistical analysis of toxicity tests conducted under
ASTM guidelines. E1847-96 (2003), Annual Book of ASTM Stan-
dards, Vol. 11.06. American Soc. Testing & Materials, W. Consho-
hocken, Pa.
14. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Standard
guide for selection of resident species as test organisms for aquatic
and sediment toxicity tests. E1850-04, Annual Book of ASTM
Standards, Vol. 11.06. American Soc. Testing & Materials,
W. Conshohocken, Pa.
15. ENVIRONMENT CANADA. 1999. Guidance Document on Application
and Interpretation of Single-Species Tests in Environmental Toxi-
cology. Report EPS 1/RM/34, Environment Canada, Ottawa, Ont.
4. Bibliography
KLAASON, C.D., M.O. AMDUR &J.DOULL, eds. 1986. Casarett and
Doull’s Toxicology, 3rd ed. Macmillan, New York, N.Y.
RAND, G.M. ed. 1995. Fundamentals of Aquatic Toxicology, 2nd ed.
Taylor and Francis, Washington, D.C.
8010 B. Terminology
An aquatic toxicity test is a procedure in which the responses
of aquatic organisms are used to detect or measure the presence
or effect of one or more substances, wastes, or environmental
factors, alone or in combination.
1. General Terms
Acclimate—to accustom test organisms to different environmen-
tal conditions, such as temperature, light, and water quality.
Control—treatment in a toxicity test that duplicates all the con-
ditions of the exposure treatment but contains no test material.
Definitive test—toxicity test designed to establish concentration
at which a particular endpoint occurs. Exposures for these
tests are longer than for screening or range-finding tests,
incorporating multiple concentrations at closer intervals and
multiple replicates.
Range-finding test—preliminary test designed to establish ap-
proximate toxicity of a solution. Test design incorporates
multiple, widely spaced concentrations with single replicates;
exposure is usually 8 to 24 h.
Response—the measured biological effect of the variable tested.
In acute toxicity tests, the response usually is death or immo-
bilization. In plant toxicity tests, the response can be death,
growth inhibition, or reproductive inhibition. In biostimula-
tion tests, the response is biomass increase.
Screening test—toxicity test to determine if an effect is likely to
be observed; test design incorporates one concentration, mul-
tiple replicates, exposure 24 to 96 h.
2. Toxicity Terms
Acute toxicity—relatively short-term lethal or other effect, usu-
ally defined as occurring within 4 d for fish and macroinver-
tebrates and shorter times (2 d) for organisms with shorter life
spans.
Asymptotic LC
50
—toxicant concentration at which LC
50
ap-
proaches a constant for a prolonged exposure time.
Chronic toxicity—toxicity involving a stimulus that lingers or
continues for a relatively long period of time, often one-tenth
of the life span or more. “Chronic” should be considered a
relative term depending on the life span of an organism. A
chronic toxic effect can be measured in terms of reduced
growth, reduced reproduction, etc., in addition to lethality.
Dose—amount of toxicant that enters the organism. Dose and
concentration are not interchangeable.
Toxicity—potential or capacity of a test material to cause adverse
effects on living organisms, generally a poison or mixture of
poisons. Toxicity is a result of dose or exposure concentration
and exposure time, modified by such variables as temperature,
chemical form, and availability.
Effective concentration (ECP)—toxicant concentration estimated to
cause a specified effect in a designated proportion (P) of test
organisms. The effect is usually sublethal (e.g., a change in
respiration rate or loss of equilibrium). The exposure time also
is specified; for example, the 96-h EC
50
for loss of equilibrium
is the effective concentration for 50% of the test organisms in
96 h, for this kind of effect.
Exposure time—time test organism is exposed to test solution.
Inhibition concentration (ICP)—toxicant concentration estimated
to cause a specified percentage (P) inhibition or impairment in
a qualitative biological function. For example, an IC
25
could
be the concentration estimated to cause a 25% reduction in
growth of larval fish, relative to the control. Use this term with
any toxicological test that measures a change in rate (respira-
tion, number of progeny, decrease in number of algal cells,
etc.).
Lethal concentration (LCP)—toxicant concentration estimated
to produce death in a specified proportion (P) of test organ-
isms. Usually defined as median (50%) lethal concentration,
LC
50
, i.e., concentration killing 50% of exposed organisms at
a specific time of observation (e.g., 96-h LC
50
).
Lowest-observed-effect concentration (LOEC)—in a full- or partial-
life-cycle test, the lowest toxicant concentration in which the
values for the measured response are statistically significantly
different from those in the control.
Median tolerance limit (TLm)—test material concentration at
which 50% of test organisms survive for a specified exposure
time. This term has been superseded by median lethal con-
centration (LC
50
) and median effective concentration (EC
50
).
No-observed-effect concentration (NOEC)—in a full- or partial-
life-cycle test, the highest toxicant concentration in which the
INTRODUCTION (8010)/Terminology
2
INTRODUCTION (8010)/Terminology

values for the measured response are not statistically signifi-
cantly different from those in the control.
3. Biostimulation Terms
Limiting nutrient—among the required nutrients, the one that is
inadequate in quantity for growth (while others remain suffi-
cient).
Maximum standing crop—maximum weight of organisms during a
test, specified as wet or dry weight.
Nutrient—specific substance required for organism growth.
4. Solution Renewal Terms
Flow-through test—test in which solution is replaced continu-
ously in test chambers throughout the test duration.
Renewal test—tests in which organisms are exposed to solutions
of the same composition that are renewed periodically during
the test period (with renewals usually at 24-h intervals). This
is accomplished by transferring test organisms or replacing
test solution.
Static test—test in which solutions and test organisms are placed
in test chambers and kept there for the duration of the test.
5. Evaluation of Results
Acute-to-chronic ratio—numerical relationship between acute and
chronic toxicity that is applied to acute toxicity test values to
estimate toxicant concentration that is safe for chronic or long-
term exposure of a test organism.
Chronic value (ChV)— geometric mean of the NOEC and LOEC
from partial- and full-life-cycle tests and early-life-stage tests.
Maximum allowable toxicant concentration (MATC)—toxicant
concentration that may be present in a receiving water without
causing significant harm to productivity or other uses. MATC
is determined by long-term tests of either partial life cycle
with sensitive life stages or a full life cycle of the test organism.
6. Bibliography
STEPHAN, C.E., D.I. MOUNT, D.J. HANSEN, J.H. GENTILE, G.A. CHAPMAN &
W.A. BRUNGS. 1985. Guidelines for Deriving Numerical National
Water Quality Criteria for the Protection of Aquatic Organisms and
Their Uses. NTIS PB85-227049, U.S. Environmental Research
Laboratories, Duluth, Minn.; Narragansett, R.I.; and Corvallis, Ore.
U.S. ENVIRONMENTAL PROTECTION AGENCY. 1991. Technical Support Doc-
ument for Water Quality-Based Control, EPA-505/2-90-001
(PB91-127415). Off. Water, U.S. Environmental Protection
Agency, Washington, D.C.
RAND, G.M., ed. 1995. Fundamentals of Aquatic Toxicology, 2nd ed.
Taylor and Francis, Washington, D.C.
U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Methods for Measuring
the Acute Toxicity of Effluents and Receiving Waters to Freshwater
and Marine Organisms, 5th ed., EPA-821-R-02-012. Off. Water,
U.S. Environmental Protection Agency, Washington, D.C.
U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Short-Term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms, 4th ed., EPA-821-R-02-013. Off.
Water, U.S. Environmental Protection Agency, Washington, D.C.
8010 C. Basic Requirements
1. General Requirements
The basic requirements and desirable conditions for toxicity
tests are: (a) an abundant supply of water of desired quality (see
8010E.4b), (b) an adequate and effective flowing water system
constructed of nonpolluting or absorbing materials (see
8010F.1a), (c) adequate space and well-planned holding, cultur-
ing, and testing equipment and facilities (see 8010E.3), (d) an
adequate source of healthy experimental organisms (see
8010E.4), and (e) appropriate lighting facilities for plant toxicity
tests. Much valuable information and advice regarding general
requirements and desirable conditions for toxicity testing are
available.
1–9
2. Requirements for Specific Test Purposes
The facilities, equipment, and water supplies needed for ef-
fective tests depend on the type of tests and their objectives.
6
For
effluent and monitoring compliance tests requiring receiving
water as the dilution water, use water immediately upstream
and outside the waste’s zone of influence. When studies
require the use of laboratory-grade water, use a water supply
free from pollution and one that provides for acceptable
survival, growth, and reproduction of the aquatic test organ-
isms to be studied. The most important requirements for
designing a toxicity testing program are defining the study
objective and establishing quality control practices to ensure
that the data are of sufficient quality to address the objectives
and ensure credibility.
3. References
1. U.S. DEPARTMENT OF COMMERCE. 1970. Aquarium Design Criteria,
special ed. National Fisheries Center Aquarium.
2. CLARK, J.R. & R.L. CLARK, eds. 1964. Sea Water Systems for Ex-
perimental Aquariums, U.S. Fish & Wildl. Serv. Bur. Sports Fish &
Wildl. Res. Rep. 63. U.S. Government Printing Off., Washington,
D.C.
3. SPOTTE, S. 1973. Marine Aquarium Keeping—The Science, the An-
imals, the Art. Wiley Interscience Publ., New York, N.Y.
4. LASKER, R. & L.L. VLYMER. 1969. Experimental Seawater Aquarium,
U.S. Fish & Wildl. Serv. Bur. Commercial Fish. Circ. 334. U.S.
Government Printing Off., Washington, D.C.
5. STEPHAN, C.E., D.I. MOUNT, D.J. JANSEN, J.H. GENTILE, G.A CHAPMAN
& W.A. BRUNGS. 1985. Guidelines for Deriving Numerical National
Water Quality Criteria for the Protection of Aquatic Organisms and
Their Uses, NTIS PB85-227049. U.S. Environmental Research Lab-
oratory, Duluth, Minn.
INTRODUCTION (8010)/Basic Requirements
3
INTRODUCTION (8010)/Basic Requirements

6. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2002. Methods for
Measuring the Acute Toxicity of Effluents and Receiving Waters
to Freshwater and Marine Organisms, 5th ed., EPA-821-R-02-
012. Off. Water, U.S. Environmental Protection Agency, Wash-
ington, D.C.
7. DENNY, J.S. 1987. Guidelines for The Culture of Fathead Minnows
Pimephales promelas for Use in Toxicity Tests, EPA-600/3-87-001.
Environmental Research Lab., U.S. Environmental Protection
Agency, Duluth, Minn.
8. U.S. ENVIRONMENTAL PROTECTION AGENCY/U.S. ARMY CORPS OF ENGI-
NEERS. 1998. Evaluation of Dredged Material Proposed for Discharge
in Waters of the U.S.—Testing Manual (Inland Testing Manual),
USEPA 823-B-98-004. Washington, D.C.
9. AMERICAN SOCIETY FOR TESTING AND MATERIALS. 2007. Annual Book
of ASTM Standards, Section 11 Water and Environmental Technol-
ogy. Volume 11.05 Pesticides; Resources Recovery; Hazardous Sub-
stances and Oil Spill Responses; Waste Disposal; Biological Effects.
American Soc. Testing & Materials, W. Conshohocken, Pa.
8010 D. Conducting Toxicity Tests
1. Types of Toxicity Tests: Their Uses, Advantages, and
Disadvantages
Toxicity tests are classified according to (a) duration—short-
term, intermediate, and/or long-term, (b) method of adding test
solutions—static, renewal, or flow-through, and (c) purpose—
effluent quality monitoring, single compound testing, relative
toxicity, relative sensitivity, taste or odor, growth rate, etc.
Short-term toxicity tests are used for routine monitoring suit-
able for effluent discharge permit requirements and for explor-
atory tests. They may use endpoints other than mortality. Acute
definitive tests typically use mortality as an endpoint or other
discrete observations to determine effects due to the toxicant
(i.e., LC
50
or EC
50
values). These tests also may be used to
indicate a suitable range of toxicant concentrations for interme-
diate and long-term tests. Short-term tests, rather than longer-
duration tests, are used to obtain toxicity data as rapidly and
inexpensively as possible. They are valuable for estimating over-
all toxicity, for screening test solutions or materials for which
toxicity data do not exist, for assessing relative toxicity of
different toxicants or wastes to selected test organisms, or for
relative sensitivity of different organisms to different conditions
of such variables as temperature and pH. The results of these
tests can be used to calculate acceptable concentrations for very
short exposures, such as those that might occur as organisms
pass through an effluent zone of initial dilution or a mixing zone.
Toxicity tests of intermediate duration typically are used when
longer exposure durations are necessary to determine the effect
of the toxicant on various life stages of long-life-cycle organ-
isms, and to indicate toxicant concentrations for life-cycle tests.
Long-term toxicity tests are generally used for estimating
chronic toxicity. Long-term testing may include early-life-stage,
partial-life-cycle, or full-life-cycle testing. Exposures may be as
short as7dtoexpose specific portions of an organism’s life
cycle, 21 to 28 d to several months or longer for traditional
partial-life-cycle and full-life-cycle tests with fish.
To establish a successful testing program, consider the fol-
lowing: Use caution when static tests are used to evaluate solu-
tions with high BOD and/or COD levels or high bacterial
populations. These tests can be conducted successfully with
incorporation of rigorous DO monitoring and acceptable aera-
tion. Volatile or unstable toxicants may decrease in concentra-
tion during the test, resulting in an underestimation of the expo-
sure concentration affecting the test organisms. Metabolic prod-
ucts, such as ammonia, may increase to undesirably high
concentrations, resulting in stress or death of test organisms and
overestimation of the concentration that causes a toxic response.
Toxicant concentration may be reduced by sorption on sedi-
ments, test chamber walls, by the food provided for the test
organisms, or by combination with the mucus or metabolic
products of the test organisms and in their bodies.
Flow-through toxicity tests are desirable for high-BOD or
COD samples and for those that contain unstable or volatile
substances. Organisms with high metabolic rates are difficult to
maintain under static exposure conditions, while flow-through
tests provide well-oxygenated test solutions and continuous re-
moval of metabolic wastes. Use flow-through toxicity tests
whenever there is evidence or expectation of rapid degradation
of the test solution. Such a change is indicated when the survival
time of test animals in a fresh solution is significantly shorter
than in a corresponding 2-d-old solution (provided that adequate
DO is present throughout both tests). Flow-through toxicity tests
are also desirable for industrial effluents and chemicals that are
removed appreciably from solution by precipitation, by test
organisms, or by other means.
The LC
50
values may be useful measures of acute toxicity but
they do not represent concentrations that are safe or harmless in
aquatic habitats. Concentrations of wastes that are not demon-
strably toxic in 96 h may be toxic at longer exposure periods in
a receiving water. Thus the 96-h LC
50
may represent only a
fraction of long-term toxicity. When estimating safe discharge
rates or dilution ratios for effluents or other pollutants on the
basis of acute toxicity evaluations, use acute-to-chronic ratios
determined primarily from life-cycle tests; however, NOEC val-
ues determined from shorter-duration chronic toxicity tests can
be used. Even the provision of an apparently ample margin of
safety can fail to accomplish its purpose when there is cumula-
tive toxicity that cannot be predicted from acute toxicity results.
No single, simple acute-to-chronic ratio is valid for all wastes
or toxicants. However, research on effluents has shown that
acute-to-chronic ratios for whole effluents often are around 10.
An acute-to-chronic ratio of 20 commonly has been used for
nonpersistent chemicals, while a factor of 100 has been used for
persistent chemicals. The constituents of a complex waste re-
sponsible for acute toxicity may be, but are not necessarily, the
constituents responsible for chronic or cumulative toxicity de-
monstrable in diluted waste that is no longer acutely toxic. The
chronic toxicity may be lethal after a long exposure period or it
may only cause impairment of function. Knowledge of a waste’s
acute toxicity often can be helpful in predicting and preventing
INTRODUCTION (8010)/Conducting Toxicity Tests
4
INTRODUCTION (8010)/Conducting Toxicity Tests

acute damage to aquatic life in receiving waters, as well as in
regulating toxic waste discharges.
2. Short-Term Toxicity Tests
a. Range-finding toxicity tests: For effluents or materials of
unknown toxicity, conduct short-term (usually 24-h or 48-h),
small-scale range-finding or exploratory tests to determine ap-
proximate concentration range to be included in definitive short-
term tests. For effluents with low or slow-acting toxicity, 48- or
96-h tests may be necessary. Expose test organisms to a wide
range of concentrations of the test substance, usually in a loga-
rithmic ratio (e.g., 0.01, 0.1, 1, 10, and 100% of the sample).
Attempt to include concentrations that will kill all organisms and
others that will kill few or no organisms. For short-term, defin-
itive tests, select a geometrically spaced series of concentrations
between the highest concentration that killed no, or only a few,
test organisms and the lowest concentration that killed most or
all test organisms.
Prepare test concentrations as described in 8010F.2b.
b. Short-term definitive tests: Because death is an important,
easily detected adverse effect, the most commonly used tests are
for acute lethality. These tests are most appropriate for routine
monitoring and checking conformity with NPDES require-
ments.
1
If it is not possible to perform a range-finding toxicity
test before a definitive acute toxicity test, using a concentration
series with a 0.5 (100, 50, 25, 12.5, 6.25%) or 0.3 (100, 30, 10,
3, 1%) dilution factor may be appropriate. Short-term tests may
be static, renewal, or flow-through. Exposure periods for these
tests usually are 48 or 96 h. Static or renewal tests often are used
when the test organisms are phyto- or zooplankton because these
organisms are easily washed out in flow-through tests. Static and
renewal tests are considerably less expensive to perform than
flow-through tests. Overnight express mail shipments of samples
often make static and renewal tests the method of choice for
regulatory compliance testing.
Test solutions may be renewed daily if required because of
oxygen demand, if the toxicant is unstable or volatile, or (in the
case of whole effluents), daily variation in the composition of the
effluent. Renewals also may be less frequent. If the test material
has high BOD and/or COD level or is relatively unstable, use test
vessels with maximum surface area-to-volume ratio, or use the
renewal or flow-through technique.
Test duration is determined by the toxicant and the test ob-
jectives and usually is the same for different groups of organ-
isms. For short-life-cycle organisms (e.g., phytoplankton), the
usual exposure time can cover many generations. Determine test
duration, in part, by the length of the life cycle. Generally,
expose fish and large invertebrates in static and static renewal
tests for 96 h and in flow-through tests for an equal period unless
the toxicant’s composition is variable. In this case, longer expo-
sure may be useful to assess effects of toxicant variability.
Expose Daphnia and Ceriodaphnia for 48 h. Short-term tests
have been limited arbitrarily to 96 h, but longer tests sometimes
are desirable because death does not always occur within the 48-
or 96-h period. When some test animals, though still alive, are
dying or evidently affected after 96-h exposure, prolong the test
or express the results of the test as a 48- or 96-h EC
50
, defining
the observed effect. If tests are continued for longer periods, the
test organisms may need to be fed.
Feed test organisms as directed in specific sections of Part
8000. Record feeding and ensure that it is equivalent in each
container.
Special tests may be conducted on altered or treated samples
of effluent to obtain additional toxicity information. For exam-
ple, effluent dilution water mixtures may be aged 24 to 48 h
before adding the test organisms, to determine changes in tox-
icity. When special tests are conducted, describe methods in
detail.
3. Intermediate-Term Toxicity Tests
No sharp time separation exists between short- and interme-
diate-term or between intermediate- and long-term tests. Usually
tests lasting 10 d or less are considered short-term while inter-
mediate tests may last from 11 to 90 d. The length of the test
organism’s life cycle helps to determine what is short-term,
intermediate, or long-term for that species. Intermediate-length
tests may be static, renewal, or flow-through, but flow-through
tests are recommended for most situations. For conduct of tests,
see 8010F.3a.
4. Long-Term, Partial- or Complete-Life-Cycle Toxicity
Tests
With few exceptions, use flow-through tests with exposure
extending over as much of the life cycle as possible. Continue
tests from egg to egg or beyond, or for several life cycles for
smaller forms. Determine the maximum concentrations of toxi-
cant not producing harmful effects with continuous exposure.
The overall objective of this type of test is to determine NOECs
or chronic value (ChV) of effluents, toxicants, or wastes. Use
life-cycle tests whenever possible to determine acute-to-chronic
ratios and the effects on growth, reproduction, development of
sex products, maturation, spawning, success of spawning and
hatching, survival of larvae or fry, growth and survival of dif-
ferent life stages, deformities, behavior, and bioaccumulation,
although bioaccumulation (or bioconcentration) often is deter-
mined with more mature animals in specially designed tests.
2
In life-cycle or partial-life-cycle tests, ensure that water qual-
ity factors (e.g., temperature, pH, salinity, and DO) follow the
natural seasonal cycle unless the test objective is to study one of
these factors. It may be essential that the natural annual cycle be
duplicated if the development of sex products, spawning, and
development of eggs and larvae are to be normal. Whenever
possible, do not let toxicant concentrations vary by more than
⫾15% from the selected concentration because of uptake by test
organisms, absorption, precipitation, or other factors.
In these tests, select five or more concentrations on the basis
of short- or intermediate-term tests and set up the exposure
chambers at least in duplicate. Vary exposure chambers, spawn-
ing chambers, and other equipment to meet the needs of the
different organisms. (See Sections 8111 through 8910.) Other
apparatus, water supplies, and analytical determinations are
listed in 8010E.
5. Short-Term Tests for Estimating Chronic Toxicity
Tests are available to estimate long-term effects of a toxicant
or effluent after a relatively short (7 d) exposure. Endpoints for
INTRODUCTION (8010)/Conducting Toxicity Tests
5
INTRODUCTION (8010)/Conducting Toxicity Tests