[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR797.1050]
[Page 101-105]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 797_ENVIRONMENTAL EFFECTS TESTING GUIDELINES--Table of Contents
Subpart B_Aquatic Guidelines
Sec. 797.1050 Algal acute toxicity test.
Subpart A [Reserved]
Subpart B_Aquatic Guidelines
Sec.
797.1050 Algal acute toxicity test.
797.1300 Daphnid acute toxicity test.
797.1330 Daphnid chronic toxicity test.
797.1400 Fish acute toxicity test.
797.1600 Fish early life stage toxicity test.
797.1930 Mysid shrimp acute toxicity test.
797.1950 Mysid shrimp chronic toxicity test.
Authority: 15 U.S.C. 2603.
Source: 50 FR 39321, Sept. 27, 1985, unless otherwise noted.
Subpart A [Reserved]
(a) Purpose. The guideline in this section is intended for use in
developing data on the acute toxicity of chemical substances and
mixtures (``chemicals'') subject to environmental effects test
regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-
469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This
[[Page 102]]
guideline prescribes test procedures and conditions using freshwater and
marine algae to develop data on the phytotoxicity of chemicals. The
United States Environmental Protection Agency (U.S. EPA) will use data
from these tests in assessing the hazard of a chemical to the
environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and the definitions in part 792--Good
Laboratory Practice Standards of this chapter 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 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 container in which the test solution
is not renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. (i) 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.
(ii) At the end of 96 hours enumerate the algal cells in all
containers to determine inhibition or stimulation of growth in test
containers compared to controls. Use data to define the concentration-
response curve, and calculate the EC10, EC50, and
EC90 values.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to determine:
(A) If definitive testing is necessary.
(B) Test chemical concentrations for the definitive test.
(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 contain equal
volumes of test solution and approximately 1x10\4\ Selenastrum cells/ml
or 7.7x10\4\ 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/l)
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 EC10's,
EC50's, and EC90's for algal growth for each
species 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, 8, 16, 32, and 64 mg/l). Algae shall 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
contain equal volumes of test solution and approximately 1x10\4\
Selenastrum cells/ml or 7.7x10\4\ 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 highest concentrations
of test substance compared to controls.
[[Page 103]]
(iii) Every test shall 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 any of the test chambers, a separate carrier control is
required.
(iv) The test begins when algae from 5- to 10-day-old stock cultures
are placed in the test chambers containing test solutions having the
appropriate concentrations of the test substance. Algal growth in
controls should reach the logarithmic growth phase by 96 hours. If
logarithmic growth cannot be demonstrated, the test shall be repeated.
At the end of 24, 48, 72, and 96 hours the algal growth response (number
or weight of algal cells/ml) in all test containers and controls shall
be determined by an indirect (spectrophotometry, electronic cell
counters, dry weight, etc.) or a direct (actual microscopic cell count)
method. Indirect methods shall be calibrated by a direct microscopic
count. The percentage inhibition or stimulation of growth for each
concentration, EC10, EC50, EC90 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 shall be performed:
(A) 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,
flocculations, adherence of algae to test containers, or aggregation of
algal cells.
(B) In test concentrations where growth is maximally inhibited,
algistatic effects may be differentiated from algicidal effects by the
following two methods for Skeletonema and by the second method for
Selenastrum.
(1) Add 0.5 ml of a 0.1 percent solution (weight/volume) of Evans
blue stain to a 1 milliliter aliquot of algae from a control container
and to a 1 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 10 to
30 minutes, examine microscopically, and determine the percent of the
cells which stain blue (indicating cell mortality). A staining control
shall be performed concurrently using heat-killed or formaldehyde-
preserved algal cells; 100 percent of these cells shall stain blue.
(2) Remove 0.5 ml aliquots of test solution containing growth-
inhibited algae from each replicate test container having the
concentration of test substance evaluated in paragraph (c)(4)(v)(B)(1)
of this section. 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 9 days, and observe for algal growth to
determine if the algistatic effect 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 shall be used in the preparation of the nutrient medium.
The pH of the test solution shall be measured in the control and test
containers at the beginning and at the end of the definitive test. The
concentration of test chemical in the test containers shall be
determined at the 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
[[Page 104]]
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 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 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 capricornutum, 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 shall 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 2 subcultures lasting 7 days each prior to the
start of the definitive test. All algae used for a particular test shall
be from the same source and the same stock culture. Test algae shall not
have been used in a previous test, either in a treatment or a control.
(2) Facilities--(i) General. (A) Facilities needed to perform this
test include: 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 spent
glassware, algae and test solutions at the end of the test and any bench
covering, lab clothing, or other contaminated materials.
(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 shall 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 shall be cleaned and sterilized prior to use according to
standard good laboratory practices.
(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 appropriate 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 are to be included in
the nutrient medium used for test 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 shall be 7.5 (0.1) for Selenastrum and 8.1 (0.1)
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 shall be used in making stock
solutions of
[[Page 105]]
the test chemical. If a carrier other than nutrient medium is absolutely
necessary to dissolve the chemical, the volume used shall not exceed the
minimum volume necessary to dissolve or suspend the chemical in the test
solution.
(3) Test parameters. (i) The test temperature shall be 24 [deg]C for
Selenastrum and 20 [deg]C for Skeletonema. Excursions from the test
temperature shall be no greater than 2 [deg]C.
Temperature should be recorded hourly during the test.
(ii) Test chambers containing Selenastrum shall be illuminated
continuously and those containing Skeletonema shall be provided a 14-
hour light and 10-hour dark photoperiod with a 30 minute transition
period under fluorescent lamps providing 300 25
uEin/m\2\ sec (approximately 400 ft-c) measured adjacent to the test
chambers at the level of test solution.
(iii) Stock algal cultures should be shaken twice daily by hand.
Test containers shall be placed on a rotary shaking apparatus and
oscillated at approximately 100 cycles/minute for Selenastrum and at
approximately 60 cycles/minute for Skeletonema during the test. The rate
of oscillation should be determined at least once daily during testing.
(iv) The pH of nutrient medium in which algae are subcultured shall
be 7.5 (0.1) for Selenastrum and 8.1 (0.1) for Skeletonema, and is not adjusted after the
addition of the algae. The pH of all test solutions shall be measured at
the beginning and end of the test.
(v) Light intensity shall be monitored at least daily during the
test at the level of the test solution.
(e) Reporting. The sponsor shall 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 of this
Chapter, the following shall be reported:
(1) Detailed information about the test organisms, including the
scientific name, method of verification, and source.
(2) 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.
(3) 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.
(4) The number of algal cells per milliliter in each treatment and
control and the method used to derive these values at the beginning, 24,
48, and 72 hours, and end of the test; the percentage of inhibition or
stimulation of growth relative to controls; and other adverse effect in
the control and in each treatment.
(5) The 96-hour EC10, EC50, and
EC90 values, and when sufficient data have been generated,
the 24, 48, and 72 hour LC50's and 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.
(6) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
(7) 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.
(8) If the range-finding test showed that the highest concentration
of the chemical tested (not less than 1000 mg/l 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.
(9) 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.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987]