Section

[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

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.

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.

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(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]