EPA-660/3-75-009
APRIL 1975
Ecological Research Series
Methods for Acute Toxicity Tests with
Fish, Macroinvertebrates,
and Amphibians
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
series. This series describes research on the effects of pollution
on humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investigations
include formation, transport, and pathway studies to determine the
fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living
organisms in the aquatic, terrestrial and atmospheric environments.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
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not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.
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EPA-660/3-75-009
April 1975
METHODS FOR ACUTE TOXICITY TESTS WITH FISH, MACROINVERTEBRATES,
AND AMPHIBIANS
Environmental Protection Agency
Library Systems Branch, Room 2903
401 M Street, S.W.
Washington, D.C. 20460
by
The Committee on Methods for Toxicity Tests
with Aquatic Organisms
Program Element 1BA021
Project Officer
Charles E. Stephan
National Water Quality Laboratory
National Environmental Research Center
6201 Congdon Boulevard
Duluth, Minnesota 55804
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For Sale by the National Technical Information Service
U.S. Department of Commerce, Springfield, VA 22151
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ABSTRACT
Four detailed methods for conducting acute toxicity tests with freshwater,
estuarine, and marine fish, macroinvertebrates, and amphibians are presented
in an integrated format. Nomenclature is consistent with that used in
other branches of toxicology. Concepts incorporated into the methods are
applicable to toxicity tests with most aquatic organisms.
This report was prepared by the Committee on Methods for Toxicity Tests
with Aquatic Organisms under the partial sponsorship of the U. S.
Environmental Protection Agency. Work was completed as of December,
1974.
ii
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CONTENTS
Sections Page
I Conclusions 1
II Recommendations 2
III Introduction 3
A. Background 3
B. Nomenclature 5
IV Methods 9
A. Preface 9
B. Equipment 9
C. Dilution water 14
D. Test organisms 20
E. Test procedure 30
F. Reports 47
V Discussion 50
VI References 57
iii
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TABLES
No. Page
1 Recommended Reconstituted Fresh Waters 17
2 Recommended Procedure for Preparing 18
Reconstituted Sea Water
3 Recommended Species and Test Temperatures 21
4 Recommended Prophylactic and Therapeutic 26
Treatments for Freshwater Fish
5 Percentage of Ammonia that is Un-ionized 44
in Distilled Water at Different Temperatures
and pH's
6 Hypothetical Sets of Data and Calculated 48
Results
iv
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ACKNOWLEDGMENTS
The committee wishes to acknowledge the congenial and cooperative spirit
of the participating organizations, which made possible the writing of
these methods. The committee also wishes to express its appreciation to
the many people who offered suggestions and reviewed the manuscript,
especially Mrs. Bea Smith and Mr. Paul H. Eschmeyer.
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SECTION I
CONCLUSIONS
1. A diverse group of scientists interested in aquatic toxicology can
reach, agreement concerning detailed methods for conducting toxicity
tests with aquatic animals.
2. Many of the methods written in the past for conducting toxicity
tests with aquatic organisms were not as detailed or as consistent
as desirable.
3. Most acute toxicity tests, can he conducted according to uniform,
detailed methods.
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SECTION IT
RECOMMENDATIONS
1. Toxicity tests with aquatic organisms should be conducted according
to uniform, detailed methods whenever possible to maximize the number
of reliable comparisons that can be made concerning relative
toxlcity and relative sensitivity.
2. Whenever toxiclty tests are conducted with aquatic organisms, the
methods presented herein should be followed as closely as possible.
3. Reports of toxicity tests should contain all information necessary
to allow correct use of the results.
4. More effort should be expended toward writing a consistent set of
detailed methods for toxicity tests with aquatic organisms by a
procedure that allows input by all interested persons from academic,
industrial, contract, regulatory, and other organizations.
5. Additional research on methodology is needed to identify improvements
that should be made in the present methods. In particular, research
should be directed toward identifying means of determining the
overall quality or "healthiness" of aquatic animals used for toxicity
tests.
6. To obtain the most meaningful data from a toxicity test with aquatic
organisms, the investigator should consult with an aquatic biologist,
analytical chemist, biometrician, and aquatic toxlcologist before
the test.
7. Aquatic toxicologlsts should regularly exchange information
concerning methodology with each other and with other toxicologists
in the United States and in other countries and should cooperate to
avoid proliferation of committees writing methods.
2
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SECTION III
INTRODUCTION
A. BACKGROUND
The Committee on Methods for Toxicity Tests with Aquatic Organisms was
organized in November, 1971, at the suggestion of Mr. Andrew J. Culver,
Jr., to foster cooperation, uniformity, and excellence in the field of
aquatic toxicology. The members of the committee and the organizations
with which they were affiliated are:
John G. Eaton
National Water Quality Laboratory
U. S. Environmental Protection Agency
Duluth, Minnesota
Eugene E. Kenaga
National Agricultural Chemicals Assoc.
Dow Chemical USA
Midland, Michigan
Richard A. Kimerle, Ph.D.
Soap and Detergent Association
Monsanto Industrial Chemicals Company
St. Louis, Missouri
Ernest C. Ladd
Manufacturing Chemists Association
FMC Corporation
Philadelphia, Pennsylvania
Kenneth J. Macek, Ph.D.
Bionomics, EG&G, Inc.
Wareham, Massachusetts
Leif L. Marking
Fish Control Laboratory
U. S. Department of the Interior
La Crosse, Wisconsin
Foster L. Mayer, Ph.D.
Fish-Pesticide Research Laboratory
U. S. Department of the Interior
Columbia, Missouri
John A. McCann
Technical Services Division
Office of Pesticide Programs
U. S. Environmental Protection Agency
Beltsville, Maryland
Patrick R. Parrish
Gulf Breeze Environmental Research Lab.
U. S. Environmental Protection Agency
Gulf Breeze, Florida
Charles E. Stephan
Newtown Fish Toxicology Station
U. S. Environmental Protection Agency
Cincinnati, Ohio
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Nelson E. Stewart
National Marine Water Quality Lab.
U. S. Environmental Protection Agency
Narragansett, Rhode Island
Patrick Parrish is now affiliated with Bionomics, EG&G, Inc., Pensacola,
Florida; Charles Stephan with the National Water Quality Laboratory,
U. S. Environmental Protection Agency, Duluth, Minnesota; and Nelson
Stewart with the Oregon Fish Commission, Newport, Oregon.
To help the committee in its work, a Subcommittee on Effluent Tests was
organized with the following members:
Howard Alexander Ernest C. Ladd
Dow Chemical Company FMC Corporation
Midland, Michigan Philadelphia, Pennsylvania
Dale L. Bacon
3M Company
St. Paul, Minnesota
Russell 0. Blosser
National Council for Air and
Stream Improvement
New York, New York
Thomas E. Braidech
National Field Investigations
Center - Cincinnati
U. S. Environmental Protection Agency
Cincinnati, Ohio
Kenneth L. Dickson, Ph.D.
Center for Environmental Studies
Virginia Polytechnic Institute and
State University
Blacksburg, Virginia
Carlos M. Fetterolf, Jr.
Michigan Department of Natural
Resources
Lansing, Michigan
Kenneth J. Macek, Ph.D.
Bionomics, EG&G, Inc.
Wareham, Massachusetts
William H. Peltier
Region IV
U. S. Environmental Protection Agency
Athens, Georgia
Ronald Preston
Region III
U. S. Environmental Protection Agency
Wheeling, West Virginia
Anne Spacie
Tarrytown Technical Center
Union Carbide Corporation
Tarrytown, New York
Charles E. Stephan
National Water Quality Laboratory
U. S. Environmental Protection Agency
Duluth, Minnesota
Anne Spacie is now affiliated with Purdue University, West Lafayette, Indiana.
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In addition, many other people were contacted in the organizations of
the members of the committee and subcommittee and through a mailing list
of about 150 names and all known existing methods were reviewed.
Although the methods presented herein were written mainly by and for
aquatic toxicologists in the United States, to encourage international
cooperation comments were solicited and received from scientists in
other countries, particularly Canada and Great Britain. Although the
committee gratefully acknowledges helpful advice from many people, it
accepts full responsibility for the methods contained herein.
The committee felt that an immediate need existed for detailed methods for
conducting toxicity tests with aquatic organisms. Therefore it undertook
the task of writing some such methods in order to make recommendations
for conducting such tests to give guidance to those who desire it, to
discourage the use of methods considered unacceptable, and to encourage
uniformity in methodology and nomenclature so that the results of
toxicity tests with aquatic organisms would be more useful. Nomenclature
consistent with that used in other branches of toxicology was used
whenever possible to facilitate communication between aquatic and other
toxicologists.
Because of the formation of sections dealing with aquatic toxicology
within Committee E-35 on Pesticides and Committee D-19 on Water of the
American Society for Testing and Materials (ASTM), the Committee on
Methods for Toxicity Tests with Aquatic Organisms is dissolving with the
publication of this document to help prevent a proliferation of
competing committees and conflicting methods.
B. NOMENCLATURE
In a toxicity test two or more treatments are used to study the effect of
a toxic agent on test organisms which are usually all of the same
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species. Although toxicity tests with aquatic organisms can be
conducted by applying the toxic agent directly to the test organisms,
such as by injection or in food, most tests are conducted by exposing the
test organisms to test solutions containing various levels of a toxic
agent. One or more control treatments are used to provide a measure of
the acceptability of the test by giving some indication of the healthiness
of the test organisms and the suitability of the dilution water, test
conditions, handling procedures, etc. A control treatment is an exposure
of the test organisms to dilution water with no toxic agent added. The
other treatments are exposures of the test organisms to dilution water
with toxic agent added. The toxic agent can be one or more pure
chemicals or a complex mixture such as a formulation or an effluent.
Sometimes the test solutions are not true solutions because they contain
undissolved toxic agent. Test solutions are often prepared by dissolving
a toxicant in a solvent, preferably water, to form a stable stock
solution, and then adding a portion of the stock solution to dilution
water. Generally the most important data obtained from a toxicity
test are the percentages of test organisms that are affected in a
specified way by each of the treatments. The result derived from these
data is a measure of the toxicity of the toxic agent to the test organisms
under the conditions of the test or, in other words, a measure of the
susceptibility of the test organisms to the toxic agent.
Acute toxicity tests are generally used to determine the level of toxic
agent that produces an adverse effect on a specified percentage of the
test organisms in a short period of time. Because death is normally an
easily detected and obviously important adverse effect, the most common
acute toxicity test is the acute mortality test. Experimentally, 50%
effect is the most reproducible measure of the toxicity of a toxic agent
to a group of test organisms, and 96 hours is often a convenient,
reasonably useful exposure duration. Therefore, the measure of acute
toxicity most often used with fish, macroinvertebrates-, and amphibians
6
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is the 96-hour median lethal concentration (96-hr LC50). Thus the result
of an acute mortality test is the statistically derived best estimate
of the LC50, which is the concentration of toxicant in dilution water
that is lethal to exactly 50% of the test organisms during continuous
exposure for a specified period of time, based on data from one experiment.
However, the measure of acute toxicity most often used with daphnids
and midge larvae is the 48-hour median effective concentration (48—hr
EC50) based on immobilization. The terms median lethal concentration
(LC50) and median effective concentration (EC50) are consistent with
the widely used terms median lethal dose (LD50) and median effective dose
(ED50), respectively. However, whereas "concentration" refers to the
concentration of toxicant in the test solution, "dose" refers to the
amount of toxicant that enters the test organism. For toxic agents or
tests to which neither concentration nor dose applies, such as tests
with temperature, the terms median lethal level (LL50) and median
effective level (EL50) should be used.
Acute toxicity tests in which test organisms are exposed to test solutions
containing a toxic agent can be conducted by at least four techniques:
1. In the static technique test solutions and test organisms are placed
in test chambers and kept there for the duration of the test.
2. The recirculation technique is like the static technique except that
each test solution is continuously circulated through an apparatus
to maintain water quality by such means as filtration, aeration,
and sterilization and then returned to the test chamber.
3. The renewal technique is like the static technique except that the
test organisms are periodically exposed to fresh test solution of
the same composition, usually once every 24 hours, either by
transferring the test organisms from one test chamber to another or
by replacing the test solution.
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4. In the flow-through technique test solutions flow into and out of
the test chambers on a once through basis for the duration of the
test. Two procedures can be used. In the first large volumes of
the test solutions are prepared before the beginning of the test
and these flow through the test chambers. In the second and more
common procedure fresh test solutions are prepared continuously or
every few minutes in a toxicant delivery system.
With any of these techniques a pump or stirrer can be used to create a
current in the test chambers to accommodate particular test organisms,
but this will often increase aeration and volatilization.
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SECTION IV
METHODS
A. PREFACE
The BASIC STATIC ACUTE TOXICITY TEST METHOD, BASIC FLOW-THROUGH ACUTE
TOXICITY TEST METHOD, EFFLUENT STATIC ACUTE TOXICITY TEST METHOD, and
EFFLUENT FLOW-THROUGH ACUTE TOXICITY TEST METHOD are presented herein in
a format that eliminates repetition and does not require referencing
from one method to another. Items that do not apply to all four methods
are clearly labelled according to the methods to which they do apply.
The BASIC test methods are the ones that aquatic toxicologists will
normally use when conducting acute toxicity tests. When special situations
arise, the BASIC test methods can be modified to meet special needs.
The most important special need at this time is effluent testing and so
two EFFLUENT test methods were written by making appropriate
modifications in the BASIC test methods.
B. EQUIPMENT
1. Facilities
The facilities should include tanks for holding and acclimating test
organisms, and a constant-temperature area or recirculating water bath
for the test chambers. For STATIC tests there should be a dilution-
water tank that may be used to prepare reconstituted water and is
sometimes elevated so dilution water can flow by gravity into holding
and acclimation tanks and test chambers. For FLOW-THROUGH tests there
should be an elevated headbox so dilution water can flow by gravity into
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holding and acclimation tanks and the toxicant delivery system. For
FLOW-THROUGH tests ceilings should be at least 10 feet high to
accommodate proportional diluters, and strainers and air traps should be
included in the water supply system. Holding, acclimation, and dilution-
water tanks and headboxes should be equipped for temperature control and
aeration. Air used for aeration must be free of oil and fumes; filters to
remove oil and water are desirable. During holding, acclimation, and
testing, test organisms should be shielded from disturbances. The test
facility must be well ventilated and free of fumes. For BASIC tests, a
16-hour light and 8-hour dark photoperiod should be provided with a 15-
to 30-minute transition period controlled by a system such as that
described by Drummond and Dawson (1970).
Organisms requiring special conditions must be accommodated during holding,
acclimation, and testing. For example, burrowing mayfly nymphs should be
provided a burrowing substrate, such as that described by Fremling and
Schoening (1973) and Fremling (1974); immature stream insects should
always be in a current in a system such as that described by Nebeker and
Lemke (1968); and penaeid shrimp and bottom-dwelling fish should be
provided a silica sand substrate. Since cannibalism can occur among
many species of decapods, the claws of crabs and crayfish should be
banded or the individuals should be physically isolated, by such means as
screened compartments.
2. Construction Materials
Construction materials and commercially purchased equipment that may
contact any water into which test organisms are placed should not
contain any substances that can be leached or dissolved by the water.
In addition, materials and equipment that contact stock solutions or test
solutions should be chosen to minimize sorption of toxicants from water.
To minimize leaching, dissolution, and sorption, glass, #316 stainless
10
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steel, and perfluorocarbon plastics must be used whenever possible.
Unplasticized plastics can be used for holding and acclimation tanks
and in the water supply system. "Rubber, copper, brass, and lead must
not come in contact with dilution water, stock solutions, effluent
samples, or test solutions.
3. Toxicant Delivery Systems for FLOW-THROUGH Tests
Although many toxicant delivery systems can be used (Xowe, 1964; Sprague,
1969; Freeman, 1971; Cline and Post, 1972; Grarrmo and Kollberg, -1972;
Bengtsson, 1972; Lichatowich et _al., 1973; Shumway and Palensky, 1973;
Abram, 1973; Schimmel, Hansen, and Forester, 1974; D. DeFoe, National
Water Quality Laboratory, Duluth, Minnesota, personal communication; R.
Carton, Western Fish Toxicology Station, Corvallis, Oregon, personal
communication), the proportional diluter CMount and Brungs, 1967) is
probably the best for routine use. It is accurate over extended periods
of time, is nearly trouble—free, and has fail-safe provisions. A small
chamber to promote mixing of toxicant-bearing water and dilution water
should be used between the diluter and the test chambers for each
concentration. If duplicate test chambers are used, separate delivery
tubes must be run from this mixing chamber to each duplicate. Alterations
in the design of the proportional diluter, such as the use of six or
more concentrations, have been useful in some situations (Esvelt and
Connors, 1971; McAllister, Mauck, and Mayer, 1972; Benoit and Puglisi,
1973; Chandler, Sanders, and Walsh, 1974; D. Allison, National Water
Quality Laboratory, Duluth, Minnesota, personal communication; S.
Schimmel et al., Gulf Breeze Environmental Research Laboratory, Gulf
Breeze, Florida, personal communication; V. Snarski and F. Puglisi,
National Water Quality Laboratory, Duluth, Minnesota, personal communication)
The flow rates through the test chambers must be five water volumes per
24 hours for daphnids, and must be at least five water volumes per 24
hours for all other animals. It is usually desirable to construct the
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toxicant delivery system so that it can provide at least ten water
volumes per 24 hours. The flow rates through the test chambers should
not vary by more than 10% from any one test chamber to any other or
from one time to another within a test.
For high concentrations of effluent approaching 100%, it might be
desirable to use paired flows (Jackson and Brungs, 1967; American Public
Health Association, 1971) but pumps are sometimes unreliable and small
tubes and openings tend to clog, especially in effluent testing.
Alternatively, the proportional diluter (Mount and Brungs, 1967) can be
modified by eliminating the W-l cell and the M-l cell and introducing
100% effluent directly into the C-2 cell.
The calibration of the toxicant delivery system should be checked
carefully before and after each test. This should include determining
the flow rate through each test chamber and measuring either the
concentration of toxicant in each test chamber or the volume of solution
used in each portion of the toxicant delivery system. The general operation
of the toxicant delivery system should be checked daily during the test.
4. Test Chambers
Test chambers can be made by welding, not soldering, stainless
steel or by gluing double-strength or stronger window glass with
clear silicon adhesive. Silicon adhesive sorbs some organochlorine
and organophosphorus pesticides which are then difficult to remove.
Therefore, as little of the adhesive as possible should be in contact
with water; extra beads of adhesive should be on the outside of chambers
rather than on the inside. For larger organisms (over 0.5 g each) the
test solution should be between 15 and 30 cm deep.
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For STATIC tests larger organisms are often exposed in 19.6-liter (5-
gallon) wide-mouth soft-glass bottles containing 15 liters of solution
(Hesselberg and Burress, 1967) or in 30 cm X 60 cm X 30 cm deep all-
glass test chambers. Smaller organisms are often exposed in 3.9-liter
(1-gallon) wide-mouth soft-glass bottles or battery jars containing
2 to 3 liters of solution. Daphnids and midge larvae are often exposed
in loosely covered 250-ml beakers containing 200 ml of solution.
For FLOW-THROUGH tests test chambers can be made by modifying glass
bottles, battery jars, or beakers to provide screened overflow, holes or
V-notches. Larger organisms are often exposed in 30 liters of solution
in a 30 cm X 60 cm X 30 cm deep all-glass test chamber in BASIC tests
and in 15 to 30 liters of solution in EFFLUENT tests. Smaller organisms
are often exposed in 2 to 4 liters of solution.
5. Cleaning
Toxicant delivery systems and test chambers must be cleaned before
use. New ones must be washed with detergent and rinsed with 100%
acetone, water, acid (such as 5% concentrated nitric acid), and
twice with tap or other clean water. At the end of every BASIC
test, if the toxicant delivery system or test chambers are to be used
again, they should be (a) emptied, (b) rinsed with water, (c) cleaned by
a. procedure appropriate for removing the toxicant tested (e.g., acid to
remove metals and bases; detergent, organic solvent, or activated
carbon to remove organic compounds), and (d) rinsed twice with water.
At the end of every EFFLUENT test, if the toxicant delivery system or
test chambers are to be used again, they must be cleaned the same as
new ones. Acid is useful for removing mineral deposits, and 200 mg of
hypochlorite/liter is useful for removing organic matter and for disinfection.
However, acid and hypochlorite must not be used together. Test chambers
and toxicant delivery systems must be rinsed with dilution water just
before -use.
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C. DILUTION WATER
1. General Requirements
An adequate supply of a dilution water that is acceptable to the test
organisms and the purpose of the test must be available. For acute
toxicity tests a minimal criterion for an acceptable dilution water is
that healthy test organisms will survive in it for the duration of
acclimation and testing without showing signs of stress, such as
discoloration or unusual behavior. Because daphnids are more sensitive
to many toxicants than most other freshwater aquatic animals, a more
realistic criterion for an acceptable freshwater dilution water is that
first instar daphnids will survive in it for 48 hours without food. A
more stringent criterion for an acceptable dilution water is that test
organisms will survive, grow, and reproduce satisfactorily in it. Water
in which daphnids will survive and reproduce satisfactorily should be an
acceptable dilution water for most tests with freshwater animals.
For BASIC tests, the dilution water should be intensively aerated by such
means as air stones, surface aerators, and screen tubes (Rucker and
Hodgeboom, 1953; Wm. Spoor, National Water Quality Laboratory, Duluth,
Minnesota, personal communication) prior to the introduction of the
toxicant. Adequate aeration will bring the pH and the concentration of
dissolved oxygen and other gases into equilibrium with air, and minimize
oxygen demand and the concentration of volatiles. For BASIC tests, the
concentration of dissolved oxygen in the dilution water should be between
90% and 100% saturation, and water that may be contaminated with undesirable
microorganisms should be passed through a properly maintained ultraviolet
sterilizer equipped with an intensity meter.
2. Reconstituted Water
The recommended reconstituted waters (Tables 1 and 2) should be used as
the dilution water for as many BASIC tests as possible to maximize the
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number of reliable comparisons that can be made concerning relative
toxicity and relative sensitivity. Reconstituted water is prepared by
adding known amounts of specified reagent—grade chemicals to water which
meets the following specifications:
Specific conductance <1 micromho/cm
Total organic carbon (TOG) or <1 mg/1
chemical oxygen demand CCOD) <2 mg/1
Boron, fluoride <100 yg/1 each
Un-ionized ammonia <20 yg/1
Aluminum, ars eni c, chromium,
cobalt, copper, iron, lead,
nickel, zinc <1 yg/1 each
Residual chlorine <3 yg/1
Cadmium, mercury, silver <100 ng/1 each
Total organophosphorus pesticides <50 ng/1
Total organochlorine pesticides plus
polychlorinated biphenyls (PCBrs) <50 ng/1
Distilled water and carbon-filtered deionized water are generally
acceptable, but the specific conductance must be measured on each batch
from which reconstituted water is to be prepared, and the other
characteristics must be measured at least twice a year and whenever
significant changes in these characteristics are expected. If the water
is prepared from a surface water, TOG, or COD must be measured on each
batch. If the water is prepared from a chlorinated water, residual
chlorine must be measured on each batch or it must be shown that first
instar daphnids can survive in each batch of reconstituted water for
48 hours without food.
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Whenever possible, the soft reconstituted fresh water (Table la) should
be used for BASIC tests with freshwater animals. The other reconstituted
fresh waters (Tables la and Ib) should be used for studying the effects
of water quality on the results of toxicity tests. However, the buffers
used in Table Ib may react chemically with some toxicants.
Whenever possible, reconstituted sea water (Table 2) of 34 g/kg (ppt,
°/oo) salinity and pH 8.0 should be used for BASIC tests with true
marine stenohaline species, and 25 g/kg salinity and pH 8.0 with
euryhaline species. Other salinities can be used for studying the
effects of water quality on the results of toxicity tests. The initial
salinity of the reconstituted sea water given in Table 2 is 34+0.5
g/kg, and the desired test salinity is attained at time of use by dilution
with water that meets the specifications listed above.
All reconstituted waters should be intensively aerated prior to use,
except that the buffered soft fresh waters listed in Table Ib should be
aerated before but not after the addition of the buffer chemicals listed
therein.
3. Alternative Dilution Waters
Use of a reconstituted water for FLOW-THROUGH tests is generally
impractical. Alternative dilution waters should be uncontaminated and
constant quality and should meet the following specifications:
Suspended solids <20 mg/1
TOG <10 mg/1
Un—ionized ammonia <20 yg/1
Residual chlorine <3 pg/1
Total organophosphorus pesticides <50 ng/1
Total organochlorine pesticides plus PCB's <50 ng/1
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Table 1. RECOMMENDED RECONSTITUTED FRESH WATERS3
Table la. Quantities of reagent-grade chemicals required to prepare recommended
reconstituted fresh waters and the resulting water qualities.
Name
Very soft
Soft
Hard
Very hard
Salts required (mg/1)
NaHC03
12
48
192
384
CaS04-2H20
7.5
30.0
120.0
240.0
MgSOt,
7.5
30.0
120.0
240.0
KC1
0.5
2.0
8.0
16.0
PH"
6.4-6.8
7.2-7.6
7.6-8.0
8.0-8.4
Hardness0
10-13
40-48
160-180
280-320
Alkalinity0
10-13
30-35
110-120
225-245
Table Ib. Quantities of reagent-grade chemicals to be added to aerated soft
reconstituted fresh water for buffering pH. The solutions should
not be aerated after addition of these chemicals.
PHd
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
Milliliters of solution for 15. liters of water
1.0 N NaOH
1.3
5.0
19.0
19.0
6.5
8.8
11.0
16.0
1.0 M KH2P04
80.0
30.0
30.0
20.0
0.5 M H3B03
40.0
30.0
20.0
18.0
aFrom Marking and Dawson (1973).
^Approximate equilibrium pH after aeration and with fish in water.
cExpressed in mg/1 as CaC03.
"Approximate equilibrium pH with fish in water.
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Table 2. RECOMMENDED PROCEDURE FOR PREPARING RECONSTITUTED SEA WATER*
Add the following reagent-grade chemicals in the amounts and order listed
to 890 ml water. Each chemical must be dissolved before another is added.
Chemical
NaF
SrCl2-6H20
H3B03
KBr
KC1
CaCl2'2H20
Na2S04
MgCl2-6H20
NaCl
Na2Si03«9H20
Na4EDTAb
NaHC03
Amount
3 mg
20 mg
30 mg
100 mg
700 mg
1.47 g
4.00 g
10.78 g
23.50 g
20 mg
1 mg
200 mg
If the resulting solution is diluted to 1 liter, the salinity should be
34 + 0.5 g/kg and the pK 8.0 + 0.2. The desired test salinity is
attained by dilution at time of use.
aFrom Kester et_ al. (1967), Zaroogian e_t al. (1969), and Zillioux ^t al.
(1973).
^Tetrasodium ethylenediaminetetraacetate. This should be omitted when
toxicity tests are conducted with metals. When tests are conducted
with plankton or larvae, the EDTA should be omitted and the medium
should be stripped of trace metals (Davey et al_., 1970).
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A freshwater dilution water is considered to be constant quality if the
monthly ranges of the hardness, alkalinity, and specific conductance are
less than 10% of their respective averages and if the monthly range of
pH is less than 0.4 unit. A brackish or marine dilution water is
considered to be constant quality if the weekly range of the salinity
is less than 6 g/kg and if the monthly range of pH is less than 0.8 unit.
Alternative freshwater dilution waters should be obtained from an
uncontaminated well or spring if possible; only as a last resort should a
dechlorinated water be used. If a dechlorinated water is used, at the
beginning of STATIC tests and daily during FLOW-THROUGH tests either it
must be shown that first instar daphnids can survive in it for 48 hours
without food or residual chlorine must be measured. When possible, an
alternative dilution water with a hardness of 44 mg/1 as CaCOs should be
used for BASIC tests with freshwater animals, with a salinity of 34 g/kg
for BASIC tests with true marine stenohaline species, and with a salinity
of 25 g/kg for BASIC tests with euryhaline species.
4. Effluent Tests
For EFFLUENT tests, the dilution water must be a representative sample of
the receiving water obtained as close to the point of discharge as
possible, but upstream of or outside the zone of influence of the effluent,
The sample of the receiving water must not be aerated or altered in any
way except that it may be filtered through a sieve or screen with 2 mm
or larger holes. For STATIC EFFLUENT tests the dilution water should be
obtained from the receiving water as close to the start of the test as
possible, but never more than 96 hours prior to the beginning of the
test. For FLOW-THROUGH EFFLUENT tests the dilution water may be obtained
either by continuous sampling from the receiving water during the test
or as one or more batches; if obtained in batches, the dilution water
should not be obtained from the receiving water more than 96 hours prior
19
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to the beginning of the test. If an acceptable dilution water cannot be
obtained from the receiving water, aerated receiving water, or an
uncontaminated, well-aerated surface, ground, or reconstituted water
with hardness, alkalinity, and specific conductance within 25% and pH
within 0.2 unit of those of the receiving water at the time of the test
may be used. If a dechlorinated water is used, at the beginning of
STATIC tests and daily during FLOW-THROUGH tests either it must be
shown that first instar daphnids can survive in it for 48 hours without
food or residual chlorine must be measured.
D. TEST ORGANISMS
1. Species
Whenever possible, BASIC tests should be conducted with the species
listed in Table 3. If a recommended species is not available, organisms
of the recommended genus should be used. The scientific name of the
species used must be verified.
Whenever possible, EFFLUENT tests should be conducted with the most
sensitive important species indigenous to or regularly stocked into the
receiving water in the vicinity of the discharge. However, this species
will depend on the receiving water, the composition of the effluent, etc.,
and is therefore generally difficult to identify without conducting
tests with a variety of species. Therefore, tests are usually conducted
with a readily available, commercially, or recreationally important
species. Because centrarchids and salmonids are among the more sensitive
species to many toxicants, are often available, are easy to handle, and
are important in many receiving waters, they are often acceptable. The
scientific name of the species used must be verified.
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Table 3. RECOMMENDED SPECIES AND TEST TEMPERATURES
Recommended test
Recommended species3 temperature (°C)
Freshwater
Vertebrates
Coho salmon, Oncorhynchus kisutch 12
Rainbow trout, Salmo gairdneri 12
Brook trout, Salvelinus fontinalis 12
Goldfish, Carassius auratus 22
Fathead minnow, Pimephales promelas 22
Channel catfish, Ictalurus punctatus 22
Bluegill, Lepomis macrochirus 22
Invertebrates^
Daphnids, Daphnia magna or I), pulex 17
Amphipods, Gammarus lacustris, G_. fasciatus, or 17
Q. pseudolimnaeus 17
Crayfish, Orconectes sp., Cambarus sp., Procambarus 22
sp., or Pacifastacus leniusculus 22
Stoneflies, Pteronarcys sp. 12
Mayflies, Baetis sp. or Ephemerella sp. 17
Mayflies, Hexagenia limbata or fl. bilinata 22
Midges, Chironomus sp. 22
Marine and estuarine
Vertebrates
Sheepshead minnow, Cyprinodon variegatus 22
Mummichog, Fundulus heteroclitus 22
Longnose killifish, Fundulus similis 22
Silverside, Menidia sp. 22
Threespine stickleback, Casterosteus aculeatus 22
Pinfish, Lagodon rhomboides 22
Spot, Leiostomus xanthurus 22
Shiner perch, Cymatogaster aggregata 12
Pacific staghorn sculpin, Leptocottus armatus 12
Sanddab,. Citharichthys stigmaeus 12
Flounder, Paralichthys dentatus, P_. lethostigma 22
English sole, Parophrys vetulus 12
21
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Table 3 (continued). RECOMMENDED SPECIES AND TEST TEMPERATURES
Recommended test
Recommended species3 temperature (°C)
Marine and estuarine
Invertebrates*5
Shrimp, Penaeus setiferus, P_. duorarun, or 22
P_. aztecus 22
Grass shrimp, Palaemonetes sp. 22
Shrimp, Crangon sp. 22
Oceanic shrimp, Pandalus jordani 12
Blue crab, Callinectes sapidus 22
Dungeness crab, Cancer magister 12
aThe scientific name must be verified.
"Freshwater amphipods, daphnids, and midge larvae should be cultured and
tested at the test temperature. Other invertebrates should be held and
tested within 5° C of the temperature of the water from which they were
obtained. They should be tested at the recommended test temperature if
it is within this range; otherwise they should be tested at the temperature
from the series 7, 12, 17, 22, and 27° C that is closest to the recommended
test temperature and is within the allowed range.
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2. Source
Usual sources of freshwater fish, are private, state, and federal hatcheries,
Whenever trout are to be used, certified disease-free fish (free of
infectious pancreatic necrosis, furunculosis, kidney disease, and
whirling disease) should be obtained if possible. Freshwater amphipods,
daphnids, and midge larvae should be reared in the testing facility
from laboratory cultures. Daphnids from cultures in which ephippia are
being produced should not be used. The other suggested species are
usually obtained directly from wild populations in relatively unpolluted
areas. However, collecting permits may be required by local and state
agencies. Organisms captured by electroshoeking should not be used. All
organisms in a test should be from the same source and as healthy and
uniform in size and age as possible.
For BASIC tests analysis of organisms for pesticides, PCB's, phthalates,
mercury, and the toxicant being tested is desirable. Organisms should
not be used if the total concentration of organochlorine pesticides plus
PCB's exceeds 0.3 ug/g (wet weight).
Although EFFLUENT tests should be conducted with a species that is
indigenous to or stocked into the receiving water, the test organisms
themselves do not have to be taken from the receiving water. Often it
is difficult to obtain organisms in good condition from the receiving
water and sometimes collecting permits are difficult to obtain. In
addition, it is often difficult to determine whether or not motile
organisms have been exposed to the effluent.
3. Size
a. Fish Very young (not yet actively feeding), spawning or recently
spent fish should not be used. The use of fish that weigh between 0.5
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and 5.0 g each is usually desirable. Embryos and newly hatched fish are
sometimes more sensitive than older stages and can be tested if appropriate
precautions are taken. In any single test all fish should be from the same
year class, and the standard length (tip of snout to end of caudal peduncle)
of the longest fish should be no more than twice that of the shortest
fish.
b. Invertebrates Immature organisms should be used whenever possible.
Among freshwater organisms, daphnids should be in the first instar;
amphipods, stoneflies, and mayflies in an early instar; and midges in the
second or third instar.
c. Amphibians Young larvae should be used whenever possible.
4. Care and Handling
To avoid unnecessary stress, organisms should not be subjected to rapid
changes in temperature or water quality. In general, aquatic organisms
should not be subjected to more than a 3° C change in water temperature
in any 12-hour period. Holding and acclimation tanks should be sterilized
with an iodophor or with 200 mg of hypochlorite/liter for 1 hour,
scrubbed well once during the hour, and then rinsed well between groups
of test organisms. When organisms are first brought into the facility,
they should be quarantined at least until they appear to be disease-free.
To maintain organisms in good condition during holding and acclimation,
crowding should be avoided and the dissolved oxygen concentration must
be maintained between 60% and 100% saturation; gentle aeration may be
used if necessary. Organisms should be fed at least once a day and
tanks scrubbed at least twice a week. Organisms should be observed
carefully during holding and acclimation for signs of disease, stress,
physical damage, and mortality. Dead and abnormal individuals must be
discarded.
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Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as
possible, so that the organisms are not unnecessarily stressed. Organisms
that touch dry surfaces or are dropped or injured during handling must
be discarded. Small dipnets are best for handling larger organisms.
Such nets are commercially available, or can be made from small-mesh
nylon netting, nylon or silk bolting cloth, plankton netting, or similar
material. Nets coated with urethane resin are best for handling catfish.
Smooth glass tubes with rubber bulbs should be used for transferring
smaller organisms such as daphnids and midge larvae. Equipment used to
handle aquatic organisms should be sterilized between uses with an
iodophor, 200 mg of hypochlorite/liter, or 30% formalin plus 1% benzalkonium
chloride. Hands should be washed or sterilized before handling or
feeding test organisms.
5. Disease Treatment
Freshwater fish may be chemically treated to cure or prevent diseases by
using the treatments recommended in Table 4, but if they are severely
diseased, it is often better to destroy the entire lot. Until acceptable
treatments have been proven effective, all other diseased animals should
be discarded. Generally organisms should not be treated during the
first 16 hours after they arrive at the facility because they are probably
stressed due to collection or transportation and some are treated during
transit. However, immediate treatment is necessary in some situations,
such as treatment of bluegills for columnaris during hot weather. Tests
must not be begun with treated organisms for at least 10 days after
treatment for BASIC tests and at least 4 days after treatment for EFFLUENT
tests. Tanks and test chambers which may be contaminated with
undesirable microorganisms should be sterilized for 1 hour with an iodophor
or with 200 mg of hypochlorlte/liter.
25
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Table 4. RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR FRESHWATER
FISHa
Disease
External
bacteria
Monogenetic
trematodes ,
fungi, and
external
protozoa^
Parasitic
copepods
Chemical
Benzalkonium chloride
(Hyamine 1622R)
Nitrofurazone (water mix)
Neomycin sulfate
Oxytetracycline hydrochloride
(water soluble)
Formalin plus zinc-free
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
DexonR (35% AI)
Trichlorfon
(MasotenR)
Concen tration
(mg/D
1-2 AIb
3-5 AI
25
25 AI
25
0.1
150-250
2-6
15000-30000
2000-4000
20
0.25 AI
Application
30-60 minc
30-60 minc
30-60 minc
30-60 minc
1-2 hours0
30-60 minc
30-60 minc
5-10 min dip
e,c
30-60 minc
f
aThese recommendations do not imply that these treatments have been cleared or
registered for these uses. Appropriate state and federal regulatory agencies
should be consulted to determine if the treatment in question can be used and
under what conditions the uses are permitted. These treatments should be used
only on fish intended for research. They have been found dependable, but
efficacy against diseases and toxicity to fish may be altered by temperature
or water quality. Researchers are cautioned to test treatments on small lots
of fish before making large-scale applications. Prevention of disease is
preferred, and newly acquired fish should be treated with the formalin-
malachite green combination on three alternate days if possible. However,
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Table 4 (continued). RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR
FRESHWATER FISH
generally fish should not be treated on the first day they are in the facility.
This table is merely an attempt to indicate the order of preference of
treatments that have been reported to be effective. Before a treatment is used,
additional information should be obtained from sources such as Davis (1953),
Hoffman and Meyer (1974), Reichenbach-Klinke and Elkan (1965), Snieszko (1970),
and van Duijn (1973).
"AI - active ingredient.
GTreatment may be accomplished by (1) transferring the fish to a static
treatment tank and back to a holding tank; (2) temporarily stopping the
flow in a flow-through system, treating the fish in a static manner,
and then resuming the flow to flush out the chemical; or (3) continuously
adding a stock solution of the chemical to a flow-through system by means
of a metered flow or the technique of Brungs and Mount (1967).
dOne treatment is usually sufficient except for "Ich," which must be treated
daily or every other day until no sign of the protozoan remains. This may
take 4-5 weeks at 5-10° C and 11-13 days at 15-21° C. A temperature of 32° C
is lethal to Ich in one week.
Minimum of 24 hours but may be continued indefinitely.
^Continuous treatment should be employed in static or flow-through systems
until no copepods remain, except that treatment should not be continued
for over 4 weeks and should not be used above 27° C.
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6. Holding
After collection or transportation, Invertebrates and amphibians must have
been in holding or acclimation tanks for at least 10 days and fish for
at least 14 days before they are used for BASIC tests; all test organisms
must have been in holding or acclimation tanks for at least 2 days before
they are used for EFFLUENT tests. They should be held under stable
conditions of temperature and water quality in uncontaminated, constant-
quality water in a flow-through system with a flow rate of at least two
water volumes per day or, for EFFLUENT tests only, in a recirculating
system in which the water flows through a carbon filter and an
ultraviolet sterilizer. Water from a well or spring should be used for
freshwater organisms whenever possible. Only as a last resort should a
dechlorinated water be used. For BASIC tests water that may be contaminated
with undesirable microorganisms should be passed through an ultraviolet
sterilizer, and the un-ionized ammonia concentration in the holding tanks
should be less than 20 yg/1. When possible, the organisms should be
held in dilution water and at the temperature at which they are to be
tested. During long holding periods, however, it is generally easier and
safer to hold fish at lower temperatures rather than at higher temperatures
because the metabolic rate and the number and severity of disease out-
breaks are reduced. However, the recommended test temperatures listed in
Table 3 are generally good temperatures at which to hold the respective
organisms. Aquatic invertebrates should be held within 5° C of the
temperature of the water from which they were obtained.
7- Acclimation
Freshwater amphipods, daphnids, and midge larvae should be acclimated to
water quality and temperature by rearing them in the dilution water at
the test temperature.
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Other organisms can be acclimated (in a flow-through system with a flow
rate of at least two water volumes per day for FLOW-THROUGH tests)
simultaneously to the dilution water and test temperature after transferring
an appropriate number of similar-length individuals from a holding tank
to an acclimation tank. They should be acclimated to the dilution water
by gradually changing the water in the acclimation tank from 100% holding
water to 100% dilution water over a period of 2 pr more days for BASIC
tests and 24 or more hours for EFFLUENT tests. All organisms must remain
in 100% dilution water for at least 2 days for BASIC tests and for at
least 24 hours for EFFLUENT tests before they are used for tests. For
BASIC tests water that may be contaminated with undesirable microorganisms
should be passed through an ultraviolet sterilizer, and the un—ionized
ammonia concentration in the acclimation tanks should be less than 20
Ug/1. They should be acclimated to the test temperature by changing the
water temperature at a rate not to exceed 3° C within 72 hours for BASIC
tests and not to exceed 3° C within 24 hours for EFFLUENT tests until the
allowable test temperature range is reached. They must be maintained for
at least 2 days for BASIC tests and 24 hours for EFFLUENT tests at the
allowable test temperature range before tests are begun with them.
Longer acclimation times are generally desirable.
A group of organisms must not be used for a test if the individuals appear
to be diseased or otherwise stressed or if more than 3% for BASIC tests
or 5% for EFFLUENT tests die during the 48 hours Immediately prior to
the beginning of the test. If a group fails to meet these criteria, all
individuals must be either discarded or treated, held an additional 10
days for BASIC test or 4 days for EFFLUENT tests, and reacclimated if
necessary.
Young amphibian larvae and fish that have been actively feeding for less
than about 20 days, amphipods, daphnids, and midge larvae must be fed,
and all other insects may be fed, up to the beginning of the test. For
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BASIC tests all other amphibian larvae and fish over 0.5 g each must
not be fed for 96 hours, and all other invertebrates over 0.5 g each
must not be fed for 48 hours, before the beginning of the test. For
EFFLUENT tests all other amphibian larvae, fish, and invertebrates over
0.5 g each must not be fed for 48 hours before the beginning of the test.
E. TEST PROCEDURE
1. Experimental Design
For BASIC and EFFLUENT STATIC tests at least 10 organisms, and for EFFLUENT
FLOW-THROUGH tests at least 20 organisms, must be exposed to each
treatment, but they may be divided between two or more test chambers.
The use of more organisms and replicate test chambers for each treatment
is desirable. For BASIC FLOW-THROUGH tests at least 30 organisms must
be exposed to each treatment, except that if replicate test chambers are
used separately in the statistical analysis, at least 20 organisms must
be exposed to each treatment. If replicates are used, they must be true
replicates with no water connections between the replicate test chambers.
Randomization of the treatments is desirable; if replicates are used,
random assignment of one test chamber for each treatment in a row,
followed by random assignment of a second test chamber for each
treatment in another or an extension of the same row, is recommended
rather than total randomization. A representative sample of the test
organisms should be impartially distributed to the test chambers, either
by adding one (if there are to be less than 11 organisms per container)
or two (if there are to be more than 11 organisms per container) test
organisms to each chamber, and then adding one or two more, and repeating
the process until each test chamber has the desired number of test
organisms in it. Alternatively, the organisms can be assigned either
by random assignment of one organism to each test chamber, random
assignment of a second organism to each test chamber, etc., or by total
30
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randomization. It is often convenient to assign organisms to other
containers and then add them to the test chambers all at once.
Every test requires a control which consists of the same dilution water,
conditions, procedures, and organisms as are used in the remainder of
the test. If any additive is present in any of the test chambers, an
additive control is also required. This additive control is treated
the same as the regular control except that the highest amount of additive
present in any other test chamber is added to this test chamber. If the
toxicant is a mixture or formulation, none of the ingredients of the
mixture or formulation is considered an additive. A test is not acceptable
if more than 10% (5% for BASIC PLOW-THROUGH tests) of the organisms die
in any control in a test determining an LC50 or show the effect in a test
determining an EC50.
It is desirable to repeat the test at a later time to obtain information
on the reproducibility of the results of the test.
2. Dissolved Oxygen Concentration
Test solutions must not be aerated in the test chambers or in the toxicant
delivery system. For BASIC STATIC tests the dissolved oxygen concentration
in each test chamber must be between 60% and 100% saturation during the
first 48 hours of the test and must be between 40% and 100% saturation
after 48 hours. For BASIC FLOW-THROUGH tests the dissolved oxygen
concentration in each test chamber must be between 60% and 100%
saturation at all times during the test.
3. Test Temperature
For BASIC tests the test temperature must be selected from the series
7, 12, 17, 22, and 27° C. The actual test temperature must not deviate
from the selected test temperature by more than 1° C at any time during
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the test. The temperatures recommended in Table 3 should be used as the
selected test temperatures for the species listed therein whenever
possible. Other temperatures from the series can be used for studying the
effect of temperature on the results of toxicity tests. For aquatic
invertebrates the selected test temperature should be within 5° C of the
temperature of the water from which they were obtained. They should be
tested at the recommended test temperature if it is within this range;
otherwise they should be tested at the temperature from the series 7, 12,
17, 22, and 27° C that is closest to the recommended test temperature
and is within the allowed range.
For EFFLUENT STATIC tests the selected test temperature should be the
temperature of the receiving water measured just outside the zone of
influence of the effluent at noon (local time) on the day before
acclimation begins, because the temperature at noon usually approximates
the average temperature for the day. For aquatic invertebrates the
selected test temperature should be within 5° C of the temperature of the
water from which they were obtained, and as close to the measured noon
temperature of the receiving water as possible. The actual test
temperature must not deviate from the selected test temperature by more
than 2° C at any time during the test.
The effect of temperature on the results of EFFLUENT STATIC tests can be
determined by conducting additional toxicity tests with different test
temperatures. When special tests are conducted to collect additional
information about an effluent, it must be remembered that the
characteristics of the effluent and the receiving water may vary
significantly within short periods of time. Thus tests to determine the
effect of temperature should all be conducted at the same time on the
same samples of the effluent and the receiving water.
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For EFFLUENT FLOW-THROUGH tests the. actual test temperature must always
be between the daily low and the dally high temperature of the receiving
water measured at the time of the test just outside the zone of influence
of the effluent. For aquatic invertebrates the test temperature should
always be within 5° C of the temperature of the water from which they
were obtained and should always be between the daily low and the daily
high temperature of the receiving water measured at the time of the
test just outside the zone of influence of the effluent if possible. The
actual test temperature may be constant throughout the test or may
fluctuate within the allowable test temperature range with the temperature
of the receiving water.
4. Loading
The grams of organism per liter of solution in the test chambers must
not be so high that it affects the results of the test. Therefore the
loading must be limited to insure that the concentration of dissolved
oxygen and toxicant is not decreased below acceptable levels, that the
concentration of metabolic products does not increase above acceptable
levels, and that the organisms are not stressed due to crowding. For
STATIC tests with the species listed in Table 3 the loading in the test
chambers must not exceed 0.8 g/liter at or below the temperatures
specified as the recommended test temperatures and 0.4 g/liter at higher
temperatures. For FLOW-THROUGH tests with the species listed in Table 3
the loading in the test chambers must not exceed 2 g per liter of test
solution passing through the test chamber in 24 hours and must not exceed
20 g/liter of test solution in the test chamber at any time at or below
the temperatures specified as the recommended test temperatures; at
higher temperatures the loading must not exceed 1 g/Cliter/day) or 10 g/liter.
(The recommended test temperatures listed in Table 3 have no bearing on
the temperatures at which EFFLUENT tests should be conducted.)
33
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For BASIC STATIC tests lower loadings must be used if the concentration
of dissolved oxygen does not remain above 60% saturation for the first
48 hours of the test and above 40% saturation after 48 hours.
For BASIC FLOW-THROUGH tests lower loadings must be used if necessary to
meet the following three criteria at all times during the test in each
test chamber:
a. the concentration of dissolved oxygen must not fall below 60%
saturation;
b. the concentration of un—ionized ammonia must not exceed 20 vg/1; and
c. the concentration of toxicant must not be lowered by more than 20%
because of uptake by the test organisms.
For EFFLUENT tests, when the concentration of dissolved oxygen in the
dilution water is less than 60% saturation at the beginning of the test,
lower loadings than those specified above should be used. If the
dissolved oxygen concentration is less than 60% saturation in any test
chamber at any time during an EFFLUENT test, it may also be desirable to
conduct an additional toxicity test by a modified procedure by slowly
bubbling air or oxygen through the solutions in the test chambers during
the test. When tests are conducted by such modified methods, the exact
methodology must be described in detail in all reports of the results
of the test. In order to determine the effect of the test organisms on
the dissolved oxygen concentration during EFFLUENT tests, the dissolved
oxygen concentration should be measured in duplicate test chambers that
do not contain test organisms.
Comparable loadings should be used for other species.
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5. Toxicant
For BASIC tests the toxicant should be added to the dilution water (in
the toxicant delivery system for FLOW-THROUGH tests) without the use of
any solvents or other additives, except water, if possible. If additives
other than water are necessary, the amount used must be kept to a
minimum. Hydrochloric acid, nitric acid, potassium hydroxide, sodium
hydroxide, and sulfuric acid may be used to prepare aqueous stock solutions,
but they may affect the pH of the test solutions appreciably. Acetone,
dimethylformamide (EMF)» ethanol, methanol, and trlethylene glycol may
be used to prepare stock solutions, but the concentration of solvent in
any test solution must not exceed 0.5 ml/liter in BASIC STATIC tests and
0.1 ml/liter in BASIC FLOW-THROUGH tests. The stability of the toxicant
in the stock solution should be determined.
For EFFLUENT tests the toxicant is a sample of an effluent. The sample of
the effluent must not be aerated or altered In any way except that it
may be filtered through a sieve or screen with 2 mm or larger holes.
Samples must be covered at all times and violent agitation must be avoided.
Undissolved materials must be uniformly dispersed by gentle agitation
Immediately before any aliquot of the sample is taken for use, especially
in the headbox for EFFLUENT FLOW-THROUGH tests. The timing of the test
and the collection of samples should be based on an understanding of the
short- and long-term operations and schedules of the discharger if
possible.
For EFFLUENT STATIC tests separate tests generally should be conducted on
at least two grab samples and more tests may often be desirable, especially
if there are known sources of variability such as process changes. Tests
on composite samples may be desirable In some cases. Tests should be
begun as soon as possible, but must be begun within 8 hours, after the
sample Is-obtained. The temperature of the sample should be adjusted to
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the test temperature (+2° C) and maintained at that temperature until
portions are added to the dilution water. Often it is convenient to
store the sample in the constant temperature water bath or area in
which the test chambers are placed during the test.
For EFFLUENT FLOW-THROUGH tests on effluents that are discharged
continuously for over 96 hours, the sample of the effluent must be taken
continuously from the. discharge line and introduced directly into a small
effluent hjeadbox that feeds the toxicant delivery system. If the discharge
rate is not reasonably constant, flow—proportional continuous sampling
may be desirable. For effluents that are only discharged in batches, a
grab sample must be used and the test must begin within 8 hours after
the sample is obtained. The temperature of the sample should be adjusted
to be within the allowable test temperature range before it is added
to the dilution water.
Special EFFLUENT tests may be conducted on altered or treated samples of
the effluent or on other samples to obtain additional information
concerning the toxicity of the effluent. For example, a special
EFFLUENT STATIC test can be conducted by mixing effluent with dilution
water and letting the solutions age for a period of time, such as 24
hours, before adding the test organisms to determine if toxicity increases
or decreases with time. When special tests are conducted, the exact
methodology must be described in all reports of the results of the tests.
6. Beginning the Test
STATIC tests are begun either by Ca) adding toxicant to the test chambers
18 to 24 hours after the test organisms are added or (b) adding test
organisms to the test chambers within 30 minutes after the toxicant is
added to the dilution water. The first alternative (a) allows the test
organisms to partially acclimate to the test chambers and precludes loss
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of toxicant due to hydrolysis, sorption, or evaporation prior to
exposure of the test organisms. The second alternative (b) conserves
dissolved oxygen and prevents the exposure of test organisms to the
toxicant before it is evenly dispersed; this alternative must be used when
tests are conducted on aged solutions of a toxicant in dilution water and
when tests are conducted with daphnids or midge larvae. A gentle swirl
with a glass rod is usually sufficient to disperse the toxicant.
FLOW-THROUGH tests are begun either by (a) placing test organisms in
the test chambers after the test solutions have been flowing through the
test chambers long enough so that the toxicant concentrations are
constant or (b) activating the toxicant metering device in the toxicant
delivery system several days after the test organisms were placed in
test chambers that had dilution water flowing through them. The first
alternative (a) allows the investigator to study the behavior of the
toxicant and the toxicant delivery system immediately prior to the
beginning of the test, whereas the second alternative (b) allows the test
organisms to partially acclimate to the test chambers before the
beginning of the test.
7. Feeding
The test organisms must not be fed while in the test chambers.
8. Duration
A test begins when the test organisms are first exposed to the toxicant.
Daphnids and midge larvae must be exposed to the toxicant for at least
48 hours. All other organisms must be exposed for 96 hours in BASIC
STATIC tests, for 48 to 96 hours in EFFLUENT STATIC tests, and for at
least 96 hours in all FLOW-THROUGH tests. When BASIC FLOW-THROUGH tests
are conducted with larger organisms (over 0.5 g each), it is usually
desirable to determine the shape of the toxicity curve, i.e., LC50 or
EC50 vs. time, throughout an 8-day exposure.
37
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9. Biological Data
The number of dead or affected organisms in each test chamber must be
counted every 24 hours after the beginning of the test. More
observations are desirable, especially near the beginning of the test.
For BASIC FLOW-THROUGH tests the number should be counted at enough
appropriate times during the test to define the shape of the toxicity
curve. A suggested schedule is to count the number of dead or affected
organisms in each test chamber 3, 6, 12, and 24 hours after the beginning
of the test and twice a day thereafter to the end of the test. It is
more important to obtain data that define the shape of the toxicity
curve than it is to obtain data at prespecified times other than every
24 hours.
Dead organisms must be removed as soon as they are observed in STATIC
tests and at least once every 24 hours in FLOW-THROUGH tests.
Death is the adverse effect most often used to study acute toxicity with
aquatic organisms. The criteria for death are usually the lack of
movement, especially the absence of gill movement in fish, and the lack
of reaction to gentle prodding. However, because death is not easily
determined for some invertebrates, an EC50 is often determined rather than
an LC50. The effect generally used for determining an EC50 with daphnids
and midge larvae is immobilization, which is defined as the lack of
movement except for minor activity of appendages. The effects generally
used for determining an EC50 with crabs, crayfish, and shrimp are
immobilization and loss of equilibrium. Other effects can be used for
determining an EC50, but the effect and its definition must always be
38 .
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reported. General observations on such things as erratic swimming, loss
of reflex, discoloration, changes in behavior, excessive mucus production,
hyperventilation, opaque eyes, curved spine, hemorrhaging, molting, and
cannibalism should be reported.
The weights and standard lengths of the test organisms should be determined
by measuring representative organisms before the test or the control
organisms after the test. Organisms that are to be used in a test must
not be weighed or measured after acclimation has begun.
10. Chemical and Physical Data
a. BASIC STATIC tests
If a freshwater dilution water is used, its hardness, alkalinity, pH, and
specific conductance must be measured. If a brackish or marine dilution
water is used, its salinity and pH must be measured. Measurement of
suspended solids and TOG or COD is desirable. The dissolved oxygen
concentration must be measured at the beginning of the test and every 48
hours thereafter to the end of the test in the control and the high,
medium, and low toxicant concentrations as long as test organisms are
present. The pH should be measured at the beginning and end of the test
in the control and the high, medium, and low toxicant concentrations. If
possible, the concentration of toxicant should be measured at the
beginning and end of the test in all test chambers. Measurement of
degradation products of the toxicant is desirable.
b. BASIC FLOW-THROUGH tests
(1) Analytical method for toxicant analysis When the identity of the
toxicant is known, the concentration of toxicant in the test chambers
39
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must be measured. When the concentration is not measured, the usefulness
of the flow-through technique may be greatly diminished. The analytical
method for measuring the concentration of toxicant must be validated
before the beginning of the test. At a minimum, a measure of the accuracy
of the method must be obtained by using the method of known additions
with dilution water from a tank containing test organisms; on each of two
separate days three samples must be analyzed at the next to the lowest
toxicant concentration that will be used in the toxicity test. It is
also desirable to study the accuracy and precision of the analytical
method by use of reference or split samples or interlaboratory studies
and by comparison with alternative, preferably reference or corroborative,
methods of analysis. The accuracy of standard solutions should be
checked against other standard solutions whenever possible.
An analytical method is not acceptable if likely degradation products of
the toxicant, such as hydrolysis and oxidation products, give positive or
negative interferences, unless it is shown that such degradation products
are not present in the test chambers during the test. In general,
atomic absorption spectrophotometric methods for metals and gas
chromatographic methods for organic compounds are preferable to
colorimetric methods.
In addition to measuring the total concentration of toxicant in the test
chambers, it is usually desirable to make measurements of either the "dissolved"
or the "undissolved" fraction of the toxicant. Especially for inorganic
substances the "dissolved" fraction is usually defined and determined as
that which passes through a 0.45 micron membrane filter. Glass filter
holders are best for organic toxicants, but plastic holders are best for
metals. Filters and their holders must always be prewashed by filtering
distilled water and then filtering a portion of the solution of interest
before the final filtration is performed. The final portion of the
filtered distilled water should be analyzed for toxicant to make sure
40
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that the filter Is not contaminated with toxicant. The sample must be
filtered within 30 minutes after it is taken from the test chamber.
Whenever samples from a toxicity test are analyzed, at least one reagent
blank must also be analyzed, if appropriate. Also, at least one sample
for the method of known additions must be prepared by adding toxicant to
water from a control test chamber to match the next to the lowest
toxicant concentration used in the toxicity test.
(2) Behavior of the toxicant Data should be available or should be
generated to show that under the conditions of the test at the flow rate
used (a) no more than 20% of the toxicant at the next to the lowest
concentration will degrade in or volatilize from the test chambers or
both; and (b) at the loading used, no less than 80% of the toxicant that
would be present in the next to the lowest concentration without test
organisms in the test chamber will be present with test organisms in the
test chamber. The latter problem can be alleviated by reducing the
loading, and both problems by using a faster flow rate.
(3) Toxicant measurements during the test It is desirable to measure
the concentration of toxicant in the test chambers as often as practical
during the test. At a minimum the concentration of toxicant must be
measured in (a) each test chamber at least once during the test; (b) at
least one test chamber at the next to the lowest toxicant concentration
at least once every 24 hours during the test; and (c) at least one
appropriate test chamber whenever a malfunction is detected in any part
of the toxicant delivery system. For replicate test chambers at the same
toxicant concentration, the highest measured concentration divided by
the lowest measured concentration must be less than 1.2. If it is not,
the toxicant delivery sys-tem should be checked, and additional samples
from the proper test chambers should be analyzed .to determine if the
41
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sampling or analytical methods are not precise enough. In addition, the
measured concentration of toxicant in any test chamber must be no more
than 30% higher or lower than the concentration calculated from the
composition of the stock solution and the calibration of the toxicant
delivery system. If the difference is more than 30%, measuring the
concentration of toxicant in the test solution flowing into the test
chamber will indicate whether the problem is in the toxicant delivery
system or in the test chamber. Measurement of degradation products of the
toxicant is desirable.
(4) Analyses of water quality-—Certain measurements must be performed
at least once every 30 days or at the beginning of the test, if data are
available to show that the quality of the dilution water is constant, and
.daily if such data are not available. For a freshwater dilution water
hardness, alkalinity, pH, specific conductance, TOG or COD, and suspended
solids must be measured, and for a brackish or marine dilution water
salinity, pH, TOC or COD, and suspended solids must be measured. For
fresh waters it is also desirable to measure the concentration of calcium,
magnesium, sodium, potassium, chloride, and sulfate. The dissolved oxygen
concentration must be measured at the beginning of the test and every
48 hours thereafter to the end of the test in the control and the high,
medium, and low toxicant concentrations as long as test organisms are
present. The pH should be measured at least once in the control and the
high, medium, and low toxicant concentration.
c. EFFLUENT STATIC tests
The dissolved oxygen concentration must be measured at the beginning of
the test and every 48 hours thereafter to the end of the test in the
control and the high, medium, and low effluent concentrations as long as
test organisms are present. The pH and specific conductance must be
42
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measured at the beginning of the teat In the. control and the high,
medium, and low effluent concentrations.
d. EFFLUENT FLOW-THROUGH tests
The dissolved oxygen concentration, pH, and specific conductance must be
measured at the beginning of the test and every 24 hours thereafter to
the end of the test in the control and the high, medium, and low effluent
concentrations as long as test organisms are present.
e. All Tests
Temperature must be recorded at least hourly throughout acclimation and
throughout the test in at least one test chamber. Additional measurements
on dilution water, test solutions, and effluent samples are often
desirable. When water samples are taken from test chambers, they must be
taken midway between the top, bottom, and sides of the test chambers and
should not include any surface scum or material stirred up from the
bottom or sides.
Methods used for analysis of water quality must be those specified in the
latest edition of the Annual Book of Standards, Part 31 (American Society
for Testing and Materials, 1974) or Methods for Chemical Analysis of Water
and Wastes (U. S. Environmental Protection Agency, 1974). Residual
chlorine can be measured to 3 yg/1 using a modified amperometric method
CAndrew and Glass, 1974). The concentration of un—ionized ammonia can be
calculated from the concentration of total ammonia, pH, and temperature
according to Table 5. Salinity should he measured with a conductivity
salinometer or by a chlorinlty titration.
11. Range-finding Test
Unless the approximate toxlctty of the toxicant Is already known, it is
usually desirable to conduct a range-finding test to determine the toxicant
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Table 5. PERCENTAGE OF AMMONIA THAT IS UN-IONIZED IN DISTILLED WATER
AT DIFFERENT TEMPERATURES AND pH'Sa
Temperature
(°C)
7
12
17
22
27
PH
6.0
0.01
0.02
0.03
0.04
0.06
6.5
0.05
0.07
0.10
0.14
0.21
7.0
0.15
0.22
0.32
0.45
0.65
7.5
0.46
0.68
1.00
1.43
2.03
8.0
1.45
2.13
3.08
4.39
6.15
8.5
4.44
6.44
9.14
12.7
17.2
9.0
12.8
17.9
24.1
31.5
39.6
9.5
31.7
40.8
50.2
59.2
67.4
10.0
59.5
68.5
76.1
82.1
86.8
aFrom Thurston, Russo, and Emerson (1974).
44
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concentrations that should be used in the definitive test. Generally
groups of five organisms are exposed to three to five widely spaced
toxicant concentrations and a control for 24 to 96 hours using the static
or flow-through techniques. The greater the similarity between the
range-finding test and the definitive test, the more useful the results
of'the range-finding test will be.
Meaningful range-finding tests may often be difficult to conduct for
EFFLUENT tests because the characteristics of the effluent and the
receiving water may vary significantly within short periods of time.
However, many nonchlorinated effluents have an LC50 between 2% and 100%.
If a range-finding test is to be conducted with the same grab sample of
the effluent with which a definitive EFFLUENT test is to be conducted,
the range-finding test can last 8 hours at the most.
12. Definitive Test
For the determination of an LC50 or an EC50, a control and at least five
concentrations of toxicant in a geometric series should be used. More
treatments are desirable to insure the acceptability of the test and to
provide additional data for various lengths of exposure. A definitive test
must meet both of the following criteria so that the LC50 or EC50 can be
calculated with reasonable accuracy:
a. Except for the controls, the concentration of toxicant in each
treatment must be at least 60% of the next higher one for BASIC tests and
at least 50% of the next higher one for EFFLUENT tests.
b. One treatment other than the control must have killed or affected
less than 35% of the organisms exposed to it, and one treatment must have
killed or affected more than 65% of the organisms. This requirement
does not apply to EFFLUENT tests If 100% effluent does not kill or affect
more than 65% of the organisms exposed to it.
45
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If an LC or EC near the extremes of toxicity is to be calculated, such
as an LC10 or an EC90, at least one treatment must have killed or affected
a percentage of test organisms, other than 0% and 100%, near the
percentage for which the LC or EC is to be calculated. This requirement
might be met in a test to determine an LC50 or an EC50, but special tests
with appropriate toxicant concentrations will often be necessary.
Other ways of providing information concerning the extremes of toxicity
are to report the highest concentration of toxicant that actually killed
or affected no greater a percentage of the test organisms than did the
control treatment in the toxicity test or to report the lowest
concentration of toxicant that actually killed or affected all of the test
organisms exposed to it. These alternatives are normally more informative
than reporting a result such as an LC2 or an EC98 unless several
partial kills or effects are obtained close to 2% or 98%.
13. Calculations
For each set of data the LC50 or EC50 and its 95% confidence limits must
be calculated on the basis of (a) the measured initial concentrations of
toxicant, if available, or the calculated initial concentrations for
BASIC STATIC tests, (b) the average measured concentrations of toxicant,
if available, or the calculated average concentrations for BASIC FLOW-
THROUGH tests, (c) the calculated initial volume per cent of the effluent
in the test solutions for EFFLUENT STATIC tests, and (d) the calculated
average volume per cent of the effluent in the test solutions for
EFFLUENT FLOW-THROUGH tests. The "volume per cent" equals "(100 X volume
of effluent)/(volume of effluent plus volume of dilution water)0" If
other LC or EC values are calculated, their 95% confidence limits must
also be calculated. A variety of methods can be used to calculate an LC
or EC (Finney, 1964, 1971), but the most widely used are the probit, logit,
moving average, and Litchfield-Wilcoxon (1949) methods. The percentage
46
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of the test organisms that die or show the effect in the control treatment
must not be used in the calculation of the results.
To be sure that the calculations are being performed correctly, results
calculated for the hypothetical sets of data in Table 6, assuming ten test
organisms per treatment, should fall within the ranges of acceptable values
given therein.
F. REPORTS
A report of the results of a test must include the following:
1. name of method, investigator, and laboratory, and date test was
conducted;
2. for BASIC tests a detailed description of the toxicant, including its
source, composition, known physical and chemical properties, and any
additives used, and for EFFLUENT tests a detailed description of the
effluent, including its source, date, and time of collection,
composition, known physical and chemical properties, and variability;
3. the source of the dilution water, its chemical characteristics, and
a description of any pretreatment, and for EFFLUENT tests the date
and time of collection;
4. detailed information about the test organisms, including scientific
name, standard length, weight, age, life stage, source, history,
observed diseases, treatments, and acclimation procedure used;
5. a description of the experimental design and the test chambers, the
depth and volume of solution in the test chambers, the way the test
was begun, the number of organisms per treatment, the loading, the
lighting, and for FLOW-THROUGH tests a description of the toxicant
delivery system and the flow rate as the average number of water
volumes of test solution passing through each test chamber in 24 hours;
6. definition of the criterion used to determine the effect and a summary
of general observations on other effects or symptoms;
7. percentage of organisms that died or showed the effect in the control
treatment;
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Table 6, HYPOTHETICAL SETS OF DATA AND CALCULATED RESULTS
Table 6a. Percentage mortality
Data
set
A
B
C
D
E
Concentrations (ug/1)
100
100
100
100
100
100
60
100
100
100
100
100
36
100
100
40
70
30
22
10
70
10
20
20
13
0
0
0
0
0
7.8
0
0
0
0
0
Control
0
0
0
0
0
Table 6b. Range of acceptable values
Data
set
A
B
C
D
E
LC50
25.5-27.5
19.0-21.5
35.5-37.2
29.4-30.0
35.4-40.5
95% Confidence limits
Lower
21.1-24.0
14.7-19.4
26.1-30.7
23.5-23.9
28.1-30.8
Upper
28.6-30.8
22.9-24.5
43.4-45.3
36.3-37.4
44.5-46.0
48
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8. for daphntds and midge larvae, the 24- and 48-hour, and for all other
organisms, the 24^-, 48-, and 96-hour LC50 or EC50 values and their
95% confidence limits and the method used to calculate them (If 100%
effluent does not kill or affect more than 65% of the test organisms,
report the percentage of test organisms killed or affected by
various concentrations of the effluent), and for BASIC FLOW-THROUGH
tests enough, other LC50 or EC50 values to define the shape of the
toxicity curve;
9. methods used for, and the results of, all chemical analyses of water
quality and toxicant concentration, including validation studies and
reagent blanks;
10. the average and range of the acclimation temperature and the test
temperature;
11. anything unusual about the test;
12. any deviation from these methods; and
13. any other relevant information.
49
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SECTION V
DISCUSSION
These methods concern acute toxicity tests with fish, macroinvertebrates,
and amphibians, but many of the considerations dealt with apply directly
to other kinds of toxicity tests and aquatic organisms. Whenever toxicity
tests are conducted with aquatic organisms, the methods presented here
should be followed as closely as possible. Of necessity, some of the
recommendations are based on a professional consensus rather than on
scientific data. Therefore, the committee encourages additional research
on methodology and hopes that the present methods will serve as the starting
point for such work.
Many reasons exist for conducting toxicity tests with aquatic organisms
and various organisms, endpoints, and techniques are commonly used. Since
no detailed method can satisfy all needs, four different methods have
been written, identified by titles that clearly specify a definite set of
procedures. Use of these methods for special purposes may require
modification or specification of additional details, such as choosing one
particular species.
Since not all details are covered in these methods, the successful
execution of these tests will require some training or experience in
aquatic toxicology or aquatic biology or both as well as a familiarity
with the material in many of the references, especially American Public
Health Association (1971) and Sprague (1969, 1973). It is essential to
50
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conduct tests so that they meet specific needs, but the present methods
should cover most situations. To obtain the most meaningful data, the
investigator should consult with an aquatic biologist, analytical
chemist, biometrician, and aquatic toxicologist before the test.
The static technique provides the easiest measure of toxicity and is often
the only practical means of estimating the influence of variables such
as temperature and water quality on the results of toxicity tests. The
static technique should not be used for exposures lasting longer than 96
hours. Flow-through tests can last longer than 96 hours because this technique
provides for continual addition of test solution to the test chambers,
maintenance of the dissolved oxygen and toxicant concentrations and pH
at desired levels, and removal of degradation and metabolic products.
The flow-through technique should be used when other than the static is
desired, although the use of the recirculation and renewal techniques may
be justified in some cases.
Probably the most important consideration in the preparation for toxicity
tests with aquatic organisms is obtaining an adequate supply of water of
acceptable quality. Reconstituted water can be prepared from almost any
water, but the cost of preparing it from low quality water can be very
high. Continuous treatment of water for flow-through tests can be very
costly. For tests with freshwater organisms municipal water supplies
often contain unacceptable concentrations of copper, lead, zinc, fluoride,
j'
and chlorine or chloramines. Metals can be removed with chelating resins.
Sodium bisulfite should be better for dechlorinating water than sodium
sulfite; both are much more reliable than carbon filters, especially
for removing chloramines. For acute tests with freshwater animals it
is always desirable to continuously biomonitor the quality of the dilution
water by determining daily if first instar daphnids can survive in it for
48 hours without food. The results of tests conducted in freshwater
51
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dilution water prepared from chlorinated water must almost always be
suspect unless the acceptability of the water has been proven with first
instar daphnids. Systems have been developed to provide acceptable sea
water from surface water sources (Clark and Clark, 1964; Wood, 1965;
Tenore and Huguenin, 1973; White jit aL_., 1973).
The recommended reconstituted waters have been shown to be acceptable to
aquatic organisms. Daphnids can survive and reproduce satisfactorily in
the soft and the hard reconstituted fresh waters (H. Sanders, Fish-
Pesticide Research Laboratory, Columbia, Missouri, personal communication;
R. Winner, Miami University, Oxford, Ohio, personal communication). The
recommended sea water is acceptable for artemia (Zillioux et al., 1973)
and oyster larvae (Zaroogian et al., 1969; Calabrese _e_t al., 1973).
Although some general information on the care and handling of aquatic
animals is available (Brauhn and Schoettger, 1974; National Academy of
Sciences, 1973a, 1973b, 1974a, 1974b), a continuing problem in aquatic
toxicology is the lack of detailed information concerning quality control
of test organisms. This deficiency stems from a lack of information on
the effects of nutrition, diseases, acclimation, and other stresses on
aquatic organisms. Most investigators would rather not use organisms that
have been treated to prevent or cure diseases; they usually find, however,
that it is impractical to try to use only untreated organisms, partly
because most organisms are treated during transportation. In spite of
probable limitations, some investigators favor the use of a reference
toxicant as a measure of the quality of test organisms (Marking, 1966).
Not enough information is available to recommend any one compound,
although antimycin has been found useful for detecting stressed freshwater
fish (Hunn, Schoettger, and Whealdon, 1968; L. Marking, Fish Control
Laboratory, La Crosse, Wisconsin, personal communication). Complete
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acclimation may take considerably longer than even that specified for
basic tests; therefore, acclimation times longer than the minimum specified
should be used when possible.
The recommended species were selected on the basis of availability,
importance, past use, and ease of handling in the laboratory, and were
chosen primarily to encourage uniformity so that much Information becomes
available about a few species rather than a little information about many
species. Much work needs to be done to identify species that are important
in aquatic environments over wide geographical regions, are readily
available, and can be used easily in toxicity tests. Some organisms,
especially oyster embryos, are not listed because their use in toxicity
tests requires special methodology (Woelke, 1972; Calabrese et_ al., 1973).
Test organisms should not be fed during acute toxicity tests and should
not be fed for a time before tests when possible because fecal matter and
uneaten food can affect the biological activity of some toxic agents and
increase biological oxygen demand. These problems are most severe with
the static technique, but can be important even with the flow-through
technique. Because daphnids and midge larvae usually cannot live much
longer than- 48 hours without food, these organisms should be fed up to the
beginning of the test. Withholding food for up to 12 days from healthy
fish and macroinvertebrates weighing over 0.5 g each should not affect
the results of toxicity tests.
Among the many factors taken into account in the selection of the
recommended test temperatures were the acceptable temperatures for holding
individual species in the laboratory; the solubility of oxygen in water;
the rates of metabolism, degradation, and volatilization; the consumption
of dissolved oxygen; the effect of temperature on the results of toxicity
tests; the prevelance of diseases; past practice; and the relationship of
53
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test temperature to room temperature. The series 7, 12, 17, 22, and 27° C
was chosen because it better suits more organisms than does any other
series. Although the recommended test temperatures were deemed to be very
acceptable to the organisms, they should also be convenient for use by
most investigators. Optimum temperatures for growth were not a major
consideration in the selection of recommended test temperatures because
organisms in nature spend very little of their time at the optimum
temperature, it would be necessary to know the optimum temperature for
each life stage of each organism used in tests, organisms can function well
over a range of temperatures, diseases are often much more prev»lant at
optimum temperatures than at lower acceptable temperatures, and the use of
optimum temperatures as test temperatures would be very inconvenient for
many investigators. An additional consideration for freshwater fish is
that they function well at temperatures 5° C higher and lower than the
recommended test temperatures so the effect of temperature on the results
of toxicity tests can be studied at 7, 12, and 17° C for cold water
freshwater fish and at 17, 22, and 27° C for warm water freshwater fish.
Other temperatures have been recommended for other kinds of tests with
some of these organisms (National Academy of Sciences, 1973a).
The methods are applicable to freshwater, estuarine, and marine animals.
The large amount of information available on freshwater organisms and the
small amount on estuarine and marine organisms are a direct reflection of
the amount of research that has been done with the various kinds of aquatic
life. It is hoped that gaps in available information will be filled soon.
The biological testing of aqueous effluents has some advantages and some
disadvantages when compared with chemical testing. Therefore, the various
methods should be used to complement one another. In biological tests
aquatic organisms integrate the synergistic and antagonistic effects of
all components over the duration of the exposure. Even though one biological
54
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test normally costs more than one chemical test, reasonable coverage with
biological tests may not cost more than reasonably complete coverage with
chemical tests when all levels of all potentially harmful toxic agents
are taken into accqunt. All tests have their limitations and no amount of
biological and chemical testing can provide enough data to guarantee
complete protection of the receiving water as a natural resource. However,
a reasonable amount of testing can provide a basis for a plausible
amount of protection.
Usually a flow-through test involving continuous sampling of both the
effluent and the receiving water is considered the best way to conduct
toxicity tests on aqueous effluents because in this way the test"organisms
are exposed to all occurring levels of all toxic agents. The flow-through
technique is particularly advantageous over the static technique for
effluents that are variable, have a high biological oxygen demand, or
contain degradable or volatile toxicants. All three of these problems
tend to be less important with treated effluents than with untreated ones.
However, the static technique is often used for effluent testing because
it generally provides useful data at a lower cost than does the flow-through
technique.
Some effluent testing must be tailored to meet the specific requirements
of regulatory agencies. In such cases the requirements of the regulatory
agency should be followed regardless of any other recommendations or an
agreement should be reached before the tests are started.
An attempt was made to balance scientific considerations against practical
considerations and the reliability of the results. The major consideration
was that the common uses of the results do not require or justify stricter
requirements than those set forth herein. The requirements for acceptable
static tests are lenient because of the inherent limitations of the
55
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technique. Special considerations for effluent tests include the
variability of effluents and receiving waters, the fact that the tests
will often be conducted in field situations and in mobile laboratories,
and the uses to which the results will be put. Although any of the tests
can be improved by using more test organisms, etc., the committee feels
that the requirements as presented will meet the needs of most situations.
The methods are designed to insure that the results will be accurate and
precise enough for the majority of situations requiring acute toxicity
tests. A decision to conduct more precise tests will usually be based on
personal preference rather than on a practical need for more precise results.
The 95% confidence limits must be reported because they provide a good
indication of the precision of a test. The ranges of acceptable values
given in Table 6 were derived by comparing the values calculated by
various people using a variety of methods of calculation. Ranges are
given because there are no single correct values. When valid methods of
calculation are correctly used, values within the ranges given should
be obtained.
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SECTION VI
REFERENCES
Abram, F. S. H. 1973. Apparatus for control of poison concentration in
toxicity studies with fish. Water Res. 7:1875-1879.
American Puhlic Health Association. 1971. Standard Methods for the
Examination of Water and Wastewater. 13th ed. New York. 874 p.
American Society for Testing and Materials. 1974. Annual Book of
Standards, Part 31. Philadelphia. 844 p.
Andrew, R. W. and G. E. Glass. 1974. Amperometric methods for determining
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61
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EBA-660/3-75-009
4. TITLE AND SUBTITLE
Methods for Acute Toxicity Tests with Fish,
invertebrates, and Amphibians.
3. RECIPIENT'S ACCESSION>NO.
5. REPORT DATE
12/74 (Completion)
Ma^iO- g PERFORMING ORGANIZATION CODE
7.AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.
Committee on Methods for Toxicity Tests with Aquatic
Organisms.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
National Water Quality Laboratory
6201 Congdon Blvd.
Duluth, MN 55804
12. SPONSORING AGENCY NAME AND ADDRESS
National Water Quality Laboratory
6201 Congdon Blvd.
Duluth, MN 55804
10. PROGRAM ELEMENT NO.
1BA021
11. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Four detailed methods for conducting acute toxicity tests with freshwater,
estuarine, and marine fish, macro invertebrates , and amphibians are presented
in an integrated format. Nomenclature is consistent with that used in
other branches of toxicology. Concepts incorporated into the methods are
applicable to toxicity tests with most aquatic organisms.
This report was prepared by the Committee on Methods for Toxicity Tests
with Aquatic Organisms under the partial sponsorship of the U. S.
Environmental Protection Agency. Work was completed as of December,
1974.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Water pollution Fish
Amphibians Methodology
Aquatic animals Invertebrates
Bioassay Test procedures
Diseases Toxicity
Effluents
18. DISTRIBUTION STATEMENT
Release unlimited
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Acute
Median lethal concentration
Macro invertebrates
Toxicity tests
19. SECURITY CLASS (This Report) 21. NO. OF PAGES
67
20. SECURITY CLASS (This page} 22. PRICE
EPA Form 2220-1 (9-73)
ft U.S. GOVERNMENT PRINTING OFFICE: 1975-698-400/133 REGION 10
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