BG-8
August, 1982
ALGAL ACUTE TOXICITY TEST
OFFICE OF TOXIC SUBSTANCES
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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Office of Toxic Substances EG-8
Guideline for Testing Chemicals August, 1982
ALGAL ACUTE TOXICITY TEST
(a) Purpose. The guideline in this 'section'is intended for
use in developing data on the acute toxicity of chemical
substances and mixtur'es ("chemicals") subject to environmental
effects test regulations under the Toxic Substances Control Act
(TSCA) (P.L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.).
This guideline prescribes test procedures and conditions using
freshwater and marine algae to develop data on the phy to toxicity"
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of chemicals. The United States Environmental Protection Agency
(USEPA) will use data from these tests in assessing the hazard of
a chemical to the environment.
(b). Def initions . The definitions in Section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in Part 792—
Good Laboratory' Practice Standards apply to this test
guideline. The following definitions also apply to this
guideline:
(1) "Algicidal" means having the property of killing algae.
(2) "Algistatic" means having the property of inhibiting
algal growth.
(3) "ECx" means the experimentally derived chemical
concentration that is calculated to effect X percent of the test
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August, 1982
criterion.
(4) "Growth" means a relative measure of the viability of an
algal population based on the number and/or weight of algal cells
per volume of nutrient medium or test solution in a specified
period of time.
(5) "Static system" means a test contai er in which the test
solution is not renewed during the period oL the test.
. (.c) Test procedures — (1) -Summary of the test. (A) In
preparation for the test, fill test containers with appropriate
volumes of nutrient medium and/or test solution. Start the test
by introducing algae into the test and control containers in the
growth chambers. Environmental conditions within the growth
chambers are established at predetermined limits.
(3) At the end of 96 hours enumerate the algal cells in all
containers to determine inhibition or stimulation of growth in
t'.st containers compared to controls. Use data to define the
concentration-response curve, and calculate the EC-10, EC-50, and
EC-90 values.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be1
conducted to determine if:
(A) definitive testing is necessary
(B) test chemical concentrations for the definitive test.
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August, 1982
(ii) Algae are exposed to a widely spaced (e.g., log
interval) chemical concentration series. The lowest value in the
series, exclusive of controls, should be at the chemical's
detection limit. The upper value, for water soluble compounds,
should be the saturation concentration. No replicates are
required; and nominal concentrations of the chemical are
acceptable unless definitive testing is not required.
(iii) The test is performed once for each of the recommended
algal species or selected alternates. Test chambers should
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contain equal volumes of test solution and 'approximately 1 x 104
Selenastrum cells/ml or 7.7 x 104 Skeletonema cells/ml of test
solution. The algae should be exposed to each concentration of
test chemical for up to 96 hours. The exposure period may be
shortened if data suitable for the purposes of the range-finding
test can be obtained in less time.
(iv) Definitive testing is not necessary if the highest
chemical concentration tested (water saturation concentration or
1000 mg/1) results in less than a 50 percent reduction in growth
or if the lowest concentration tested (analytical detection
limit) results in greater than a 50 percent reduction in growth.
(4) Definitive test. (i) The purpose of the definitive
test is to determine the concentration response curves, the EC-
10's, EC-50's, and Ec-90's for algal growth for each species
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tested, with a minimum amount of testing beyond the range-finding
test.
(ii) Algae should be exposed to five or more concentrations
of the test chemical in a geometric series in which the ratio is
between 1.5 and 2.0 (e.g., 2, 4, 6, 8, 16, 32 and 64 mg/1).
Algae should be placed in a minimum of three replicate test
containers for each concentration of test chemical and control.
More than three replicates may be required to provide sufficient
quantities of test solution for .determination of test substance
concentration at the end of the test. ''Each test chamber should
contai-n equal volumes of test solution and approximately 1 x 104
Selenastrum cells ml"1 or 7.7 x 104 Skeletonema cells/ml of test
solution. The chemical concentrations should result in greater
than 90 percent of algal growth being inhibited or stimulated at
the lowest concentrations of test substance compared to controls.
(iii) Every test should include a control consisting of the
same nutrient medium, conditions, procedures, and algae from the
same culture, except that none of the test substance is added.
If a carrier is present in a-ny of the test chambers, a separate
carrier control is required.
(iv) The test begins when algae from seven to ten-day-old
stock cultures are placed in the test chambers containing test
solutions having the appropriate concentrations of the test
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August, 1982
substance. Algal growth in controls should reach the logarithmic
growth phase by 96 hours (at which time the number of algal cells
should be approximately 1.5 x 106/ml for Skeletonema or 3.5 x
106/ml for Selenastrum). If growth in controls does not reach
this logarithmic phase within this 96-hour period, the test is
invalidated and should be repeated. At the end of 96 hours the
algal growth response (number or weight of algal cells/ml) in all
test containers and controls should be determined by an indirect
(spectrophotometry, electronic cell counters, dry weight, etc.)
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or a direct (actual microscopic cell count) method. Indirect
methods should be calibrated by a direct microscopic count. The
percentage inhibition or stimulation of growth for each
concentration, EC-10, EC-50, EC-90 and the concentration-response
curves are determined from these counts.
(v) At the end of the definitive test, the following
additional analyses of algal growth response should be performed:
(1) Determine whether the altered growth response between
controls and test algae was due to a change in relative cell
numbers, cell sizes or both. Also note any unusual cell shapes,
color differences, f locculations, adherence of algae to test
containers, or aggregation of algal cells.
(2) In test concentrations where growth is maximally
inhibited, algistatic effects may be differentiated from
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algicidal effects by the following two methods:
(A) Add 0.5 ml of a 0.1 percent solution (weight/volume) of
Evans blue stain to a one milliliter aliquot of algae from a
control container and to a one milliliter aliquot of algae from
the test container having the lowest concentration of test
chemical which completely inhibited algal growth (if algal growth
was not completely inhibited, select an aliquot of algae for
staining from the test container having the highest concentration
of test chemical which inhibited algal growth). Wait ten to
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thirty minutes, examine microscopically, and determine the
percent of the cells-which stain blue (indicating cell
mortality). A staining control should be performed concurrently
using heat-killed or formaldehyde-preserved algal cells; 100
percent of these cells should stain blue.
(B) Remove 0.5 ml aliquots of test solution containing
g rowt h-i nh ib i ted algae from each replicate test container having
the concentration of test substance evaluated in (2) (I) above.
Combine these aliquots into a new test container and add a
sufficient volume of fresh nutrient medium-to dilute the test
chemical to a concentration which does not affect growth.
Incubate this subculture under the environmental conditions used
in the definitive test for a period of up to nine days, and
observe for algal growth to determine if the algistatic effect
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noted after the 96-hour test is reversible. This subculture test
may be discontinued as soon as growth occurs.
(5) [Reserved]
(6) Analytical measurements — (i) Chemical. (A) Glass
distilled or deionized water should be used in the preparation of
the nutrient medium. The pH of the test solution should be
measured in the control and test containers at the beginning and
at the end. of the definitive test1. The concentration of test
chemical in-the test containers should be determined at the
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beginning and end of the definitive test by standard analytical
methods which have been validated prior to the test. An
analytical method is unacceptable if likely degradation products
of the chemical, such as hydrolysis and oxidation products, give
positive or negative interference.
(B) At the end of the test and after aliquots have been
removed for algal growth-response determinations, microscopic
examination, mortal staining, or subculturing, the replicate test
containers for each chemical concentration may be pooled into one
sample. An aliquot of the pooled sample may then be taken and
the concentration of test chemical determined. In addition, the
concentration of test chemical associated with the algae alone
should be determined. Separate and concentrate the algal cells
from the test solution by centrifuging or filtering the remaining
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pooled sample and measure the test substance concentration in the
algal-cell concentrate.
(ii) Numerical. Algal growth response (as percent of
inhibition or stimulation in the test solutions compared to the
controls) is calculated at the end of the test. Mean and
standard deviation should be calculated and plotted for each
treatment and control. Appropriate statistical analyses should
provide a goodness-of-fit determination for the concentration
response curves. The concentration response curves are plotted
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using the mean measured test solution concentrations obtained at
the end of the test. . -
(d) Test conditions—(1) Test species . Species of algae
recommended as test organisms for this test are the freshwater
green alga, Selenastrum capr icornu turn, and the marine diatom,
Skeletonema costatum. Algae to be used in acute toxicity tests
may be initially obtained from commercial sources and
subsequently cultured using sterile technique. Toxicity testing
should not be performed until algal cultures are shown to be
actively growing (i.e. capable of logarithmic growth within the
test period) in at least two subcultures lasting seven days each
prior to the start of the definitive test. All algae used for a
particular test should be from the same source and the same stock
culture. Test algae should not have been used in a previous
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test, either in a treatment or a control.
(2) Facilities — (i) General. (A) Facilities needed to
perform this test includes: a growth chamber or a controlled
environment room that can hold the test containers and will
maintain the air temperature, lighting intensity and photoperiod
specified in this test guideline; apparatus for culturing and
enumerating algae; a source of distilled and/or deionized water;
and apparatus for carrying out analyses of the test chemical.
(B) Disposal facilities should be adequate to accommodate
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spent glassware, algae and' test solutions at the end of the test
and any bench covering, lab clothing, or other contaminated
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ma terials.
(ii) Test containers. Erlenmeyer flasks should be used for
test containers. The flasks may be of any volume between 125 and
500 ml as long as the same size is used throughout a test and the
test solution volume does not exceed 50 percent of the flask
volume .
(iii) Cleaning and sterilization. New test containers may
contain substances which inhibit growth of algae. They should
therefore be cleaned thoroughly and used several times to culture
algae before being used in toxicity testing. All glassware used
in algal culturing or testing should be cleaned and sterilized
prior to use'according to standard good laboratory practices.
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(iv) Conditioning. Test containers should be conditioned by
a rinse with the appropriate test solutions prior to the start of
the test. Decant and add fresh test solutions after an appro-
priate conditioning period for the test chemical.
(v) Nutrient medium. (A) Formulation and sterilization of
nutrient medium used for algal culture and preparation of test
solutions should conform to those currently recommended by the
U.S. EPA for freshwater and marine algal bioassays. No chelating
agents should be included in the nutrient medium used for test
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solution preparation. Nutrient medium should be freshly prepared
for algal testing, and may be dispensed in appropriate volumes in
test containers and sterilized by autoclaving or filtration. The
pH of the nutrient medium should be 7.5 for Selenastrum and 8.1
for Skeletonema at the start of the test and may be adjusted
prior to test chemical addition with 0.1N NaOH or HC1.
(B) Dilution water used for preparation of nutrient medium
and test solutions should be filtered, deionized or glass
distilled. Saltwater for marine algal nutrient medium and test
solutions should be prepared by adding a commercial, synthetic,
sea salt formulation or a modified synthetic seawater formulation
to distilled/deionized water to a concentration of 30 parts per
thousand.
(vi) Carriers . Nutrient medium should be used in making
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stock solutions of the test chemical. If a carrier other than
nutrient medium is absolutely necessary to dissolve the chemical,
the volume used should not exceed the minimum volume necessary to
dissolve or suspend the chemical in the test solution.
(3) Test parameters. (A) The test temperature should be
maintained at 24°±l°c for Selenastrum and 20°±1°C for
Skeletonema. Temperature should be recorded hourly during the
test. ' •
(B) Test chambers containing Selenastrum should be
illuminated continuously and those containing Skeletonema should
be provided a 14-hour light and 10-hour dark photoperipd with a
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30 minute transition period under fluorescent lamps providing 300
±25 uEin/m^ sec (approximately 400 ft-c) measured adjacent to
the test chambers at the level of test solution.
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(C) Stock algal cultures should be shaken twice daily by
hand. Test containers should be placed on a rotary shaking
apparatus and oscillated at approximately 100 cycles/min for
Selenastrum and at approximately 60 cycles/min for Skeletonema
during the test. The rate of oscillation should be determined at
least once daily during testing.
(D) The pH of nutrient medium in which algae are subcultured
should be 7.5 for Selenastrum and 8.1 for Skeletonema, and is not
adjusted after the addition' of the algae. The pH of all test
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solutions and controls should be measured at the beginning and
end of the test.
(E)' Light intensity should be monitored at least daily
during the test at the level of the test solution.
(e) Reporting . The sponsor should submit to the EPA all
data developed by the test that are suggestive or predictive of
acute phytotoxicity. In addition to the general reporting
requirements prescribed in Part 792—Good Laboratory Practice
Standards , the following should be reported:
(i) Detailed 'information about the test organisms, 'including'
the scientific name, method of verification, and source; . -
(ii) A description of the test chambers and containers , the
volumes of solution in the containers, the way the test was begun
(e.g. conditioning, test substance additions, etc.), the number
of replicates, the temperature, the lighting, and method of
incubation, oscillation rates, and type of apparatus;
(iii) The concentration of the test chemical in the control
and in each treatment at the end of the test and the pH of the
solutions;
( iv) The number of algal cells in.each treatment and control
and the method used to derive these values at the beginning and
end of the test; the percentage of inhibition or stimulation of
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growth, relative to controls; and other adverse effect .in the
control and in each treatment;
(v) The 96-hour EC-10, EC-50 and EO90 values and -their 95-
percent confidence limits, the methods used to derive these
values, the data used to define the shape of the concentration-
response curve and the goodness-of-fit determination;
(vi) Methods and data records of all chemical analyses of
water quality and test substance concentrations, including method
validations and reagent blanks;
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(vii) The results of any optional analyses such as: '
microscopic appearance of algae, size or color changes, percent
mortality of cells and the fate of subcultured cells, the
concentration of test substance associated with algae and test
solution supernate or filtrate;
(viii) If the range-finding test showed that the highest
concentration of the chemical tested (not less than 1000 mg/1 or
saturation concentration) had no effect on the algae, report the
results and concentration and a statement that the chemical is of
minimum phytotoxic concern;
(ix) If the range-finding test showed greater than a 50
percent inhibition of algal growth at a test concentration below
the analytical detection limit, report the results,
concentration, and a statement that the chemical is phytotoxic
below the analytical detection limit.
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