[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: 40CFR797.1400]
[Page 115-121]
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. 797.1400 Fish acute toxicity test.
(a) Purpose. This guideline may be used to develop 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 guideline prescribes tests to be used to develop data on the
acute toxicity of chemicals to fish. The United States Environmental
Protection Agency (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
[[Page 116]]
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) Acclimation means the physiological compensation by test
organisms to new environmental conditions (e.g., temperature, hardness,
pH).
(2) Acute toxicity test means a method used to determine the
concentration of a substance that produces a toxic effect on a specified
percentage of test organisms in a short period of time (e.g., 96 hours).
In this guideline, death is used as the measure of toxicity.
(3) Carrier means a solvent used to dissolve a test substance prior
to delivery to the test chamber.
(4) Conditioning means the exposure of construction materials, test
chambers, and testing apparatus to dilution water or to test solutions
prior to the start of a test in order to minimize the sorption of the
test substance onto the test facilities or the leaching of substances
from the test facilities into the dilution water or test solution.
(5) Death means the lack of opercular movement by a test fish.
(6) Flow-through means a continuous or an intermittent passage of
test solution or dilution water through a test chamber, or a holding or
acclimation tank with no recycling.
(7) Incipient LC50 means that test substance
concentration, calculated from experimentally-derived mortality data,
that is lethal to 50 percent of a test population when exposure to the
test substance is continued until the mean increase in mortality does
not exceed 10 percent in any concentration over a 24-hour period.
(8) LC50 means that test substance concentration,
calculated from experimentally-derived mortality data, that is lethal to
50 percent of a test population during continuous exposure over a
specified period of time.
(9) Loading means the ratio of fish biomass (grams, wet weight) to
the volume (liters) of test solution in a test chamber or passing
through it in a 24-hour period.
(10) Static means the test solution is not renewed during the period
of the test.
(11) Test solution means the test substance and the dilution water
in which the test substance is dissolved or suspended.
(c) Test procedures--(1) Summary of the test. (i) Test chambers are
filled with appropriate volumes of dilution water. If a flow-through
test is performed, the flow of dilution water through each chamber is
adjusted to the rate desired.
(ii) The test substance is introduced into each test chamber. In a
flow-through test, the amount of test substance which is added to the
dilution water is adjusted to establish and maintain the desired
concentration of test substance in each test chamber.
(iii) Test fish which have been acclimated in accordance with the
test design are introduced into the test and control chambers by
stratified random assignment.
(iv) Fish in the test and control chambers are observed periodically
during the test; dead fish are removed at least twice each day and the
findings are recorded.
(v) The dissolved oxygen concentration, pH, temperature and the
concentration of test substance are measured at intervals in selected
test chambers.
(vi) Concentration-response curves and LC50 values for
the test substance are developed from the mortality data collected
during the test.
(2) [Reserved]
(3) Range finding test. If the toxicity of the test substance is not
already known, a range finding test should be performed to determine the
range of concentrations to be used in the definitive test. The highest
concentration of test substance for use in the range finding test should
not exceed its solubility in water or the permissible amount of the
carrier used.
(4) Definitive test. (i) A minimum of 20 fish should be exposed to
each of five or more test substance concentrations. The range of
concentrations to which the fish are exposed should be such that in 96
hours there are at least two partial mortality exposures bracketing 50
percent survival.
(ii) For exposure to each concentration of a test substance, an
equal number of test fish shall be placed in two or more replicate test
chambers. Test fish
[[Page 117]]
shall be impartially distributed among test chambers in such a manner
that test results show no significant bias from the distributions.
(iii) Every test shall include a control consisting of the same
dilution water, conditions, procedures, and fish from the same group
used in the test, except that none of the test substance is added.
(iv) Mortality data collected during the test are used to calculate
a 96-hour LC50. The 24-, 48-, and 72-hour values should be
calculated whenever there is sufficient mortality data to determine such
values. If the 96-hour LC50 is less than 50 percent of the
estimated 48-hour LC50 in a flow-through test, the test shall
be continued until the mean increase in mortality at any test
concentration does not exceed 10 percent over a 24-hour period or until
14 days.
(v) Test fish shall not be fed while they are being exposed to the
test substance under static conditions or during the first 96 hours of
flow-through testing. If the test continues past 96 hours, the fish
should be fed a suitable food at a maintenance level every other day
beginning on test day 5. Any excess food and the fecal material should
be removed when observed.
(5) Test results. (i) Death is the primary criterion used in this
test guideline to evaluate the toxicity of the test substance.
(ii) In addition to death, any abnormal behavior such as, but not
limited to, erratic swimming, loss of reflex, increased excitability,
lethargy, or any changes in appearance or physiology such as
discoloration, excessive mucous production, hyperventilation, opaque
eyes, curved spine, or hemorrhaging shall be recorded.
(iii) Observations on compound solubility shall be recorded. The
investigator shall report the appearance of surface slicks,
precipitates, or material adhering to the sides of the test chamber.
(iv) Each test and control chamber shall be checked for dead fish
and observations recorded at 24, 48, 72, and 96 hours after the
beginning of the test or within one hour of the designated times. If the
test is continued past 96 hours, additional observations shall be made
every 24 hours until termination.
(v) The mortality data is used to calculate LC50's and
their 95 percent confidence limits, and to plot concentration-response
curves for each time interval whenever sufficient data exists. The
methods recommended for use in calculating LC50's include
probit, logit, binomial, and moving average angle.
(vi) A test is unacceptable if more than 10 percent of the control
fish die or exhibit abnormal behavior during a 96-hour test. If a flow-
through test is continued past 96 hours, the maximum allowable
additional mortality is 10 percent.
(6) Analytical measurements--(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and
particulate matter of the dilution water should be measured at the
beginning of each static test and at the beginning and end of each flow-
through test. The month to month variation of the above values should be
less than 10 percent and the pH should vary less than 0.4 units.
(B) During static tests, the dissolved oxygen concentration,
temperature, and pH shall be measured in each test chamber at the
beginning and end of the test. The test solution volume shall not be
reduced by more than 10 percent as a result of these measurements.
(C) During flow-through tests, dissolved oxygen, temperature and pH
measurements shall be made in each chamber at the beginning and end of
the test.
(ii) Collection of samples for measurement of test substance. Test
solution samples to be analyzed for the test substance should be taken
midway between the top, bottom, and sides of the test chamber. These
samples should not include any surface scum or material dislodged from
the bottom or sides. Samples should be analyzed immediately or handled
and stored in a manner which minimizes loss of test substance through
microbial degradation, photodegradation, chemical reaction,
volatilization, or sorption.
(iii) Measurement of test substance. (A) For static tests, the
concentration of the test substance shall be measured at a minimum in
each test chamber at each test concentration at the beginning (0-hour,
before fish are added) and at the end of the test. During flow-
[[Page 118]]
through tests, the concentration of test substance shall be measured as
follows:
(1) In at least the chamber of each test concentration at 0-hour.
(2) In at least the chamber of each test concentration at 96-hours
and every 4 days thereafter, as long as the test is continued.
(3) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
(4) Equal aliquots of test solution may be removed from each
replicate chamber and pooled for analysis.
(B) Filters and their holders used for determining the dissolved
test substance concentrations should be prewashed with several volumes
of distilled water and undergo a final rinse with test solution. Glass
or stainless steel filter holders are best for organic test substances,
while plastic holders are best for metals. The sample should be filtered
within 30 minutes after it is taken from the test chamber.
(C) The analytical methods used to measure the amount of test
substance in a sample shall be validated before beginning the test. The
accuracy of a method should be verified by a method such as using known
additions. This involves adding a known amount of the test substance to
three water samples taken from a chamber containing dilution water and
the same number and species of fish as are used in the test. The nominal
concentration of the test substance in those samples should span the
concentration range to be used in the test.
(D) An analytical method is not acceptable if likely degradation
products of the test substance give positive or negative interferences,
unless it is shown that such degradation products are not present in the
test chambers during the test.
(E) In addition to analyzing samples of test solution, at least one
reagent blank, containing all reagents used, should also be analyzed.
(F) If the measured concentrations of dissolved test substance are
considerably lower (e.g., <50 percent) than the nominal concentrations,
the total test substance concentration should be measured in the highest
test concentration.
(G) Among replicate test chambers, the measured concentrations shall
not vary more than 20 percent. The measured concentration of the test
substance in any chamber during the test should not vary more than 30
percent from the measured concentration at time 0.
(H) The mean measured concentration of test substance shall be used
to calculate all LC60's and to plot all concentration-
response curves.
(d) Test conditions--(1) Test species--(i) Selection. The test
species for this test are the rainbow trout (Salmo gairdneri), bluegill
(Lepomis macrochirus) and fathead minnow (Pimephales promelas). The
particular species of fish to be used will be prescribed in the test
rule.
(ii) Age and condition of fish. (A) Juvenile fish shall be used.
Fish used in a particular test shall be the same age and be of normal
size and appearance for their age. The longest fish shall not be more
than twice the length of the shortest.
(B) All newly acquired fish should be quarantined and observed for
at least 14 days prior to use in a test.
(C) Fish shall not be used for a test if they appear stressed or if
more than five percent die during the 48 hours immediately prior to the
test.
(iii) Acclimation of test fish. (A) If the holding water is not from
the same source as the test dilution water, acclimation to the dilution
water should be done gradually over a 48-hour period. The fish should
then be held an additional 14 days in the dilution water prior to
testing. Any changes in water temperature should not exceed 3 [deg]C per
day. Fish should be held for a minimum of 7 days at the test temperature
prior to testing.
(B) During the final 48-hours of acclimation, fish should be
maintained in facilities with background colors and light intensities
similar to those of the testing area and should not be fed.
(2) Facilities--(i) General. Facilities needed to perform this test
include:
(A) Flow-through tanks for holding and acclimating fish.
(B) A mechanism for controlling and maintaining the water
temperature
[[Page 119]]
during the holding, acclimation and test periods.
(C) Apparatus for straining particulate matter, removing gas
bubbles, or insufficient dissolved oxygen, respectively.
(D) Apparatus for providing a 16-hour light and 8-hour dark
photoperiod with a 15- to 30-minute transition period.
(E) Chambers for exposing test fish to the test substance.
(F) A test substance delivery system for flow-through tests.
(ii) Construction materials. Construction materials and commercially
purchased equipment that may contact the stock solution, test solution,
or dilution water should not contain substances that can be leached or
dissolved into aqueous solutions in quantities that can alter the test
results. Materials and equipment that contact stock or test solutions
should be chosen to minimize sorption of test chemicals. Glass,
stainless steel, and perfluorocarbon plastic should be used whenever
possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for
holding tanks, acclimation tanks, and water supply systems, but they
should be used to remove rust particles. Rubber, copper, brass,
galvanized metal, epoxy glues, and lead should not come in contact with
the dilution water, stock solution, or test solution.
(iii) Test substance delivery system. In flow-through tests,
diluters, metering pump systems, or other suitable devices should be
used to deliver the test substance to the test chambers. The system used
should be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
substance delivered to each chamber. The general operation of the test
substance delivery system should be checked twice daily during a test.
The 24-hour flow rate through a test chamber should be a minimum of 6
tank volumes. During a test, the flow rates should not vary more than 10
percent from one test chamber to another.
(iv) Test chambers. Test chambers made of stainless steel should be
welded, not soldered. Test chambers made of glass should be fused or
bonded using clear silicone adhesive. As little adhesive as possible
should be left exposed in the interior of the chamber.
(v) Cleaning of test system. Test substance delivery systems and
test chambers should be cleaned before each test. They should be washed
with detergent and then rinsed in sequence with clean water, pesticide-
free acetone, clean water, and 5 percent nitric acid, followed by two or
more changes of dilution water.
(vi) Dilution water. (A) Clean surface or ground water reconstituted
water, or dechlorinated tap water is acceptable as dilution water if the
test fish will survive in it for the duration of the holding,
acclimating, and testing periods without showing signs of stress, such
as discoloration, hemorrhaging, disorientation or other unusual
behavior. The quality of the dilution water should be constant and
should meet the following specifications measured at least twice a year:
------------------------------------------------------------------------
Substance Maximum
------------------------------------------------------------------------
Particulate matter........................ 20 mg/liter.
Total organic carbon or................... 2 mg/liter.
chemical oxygen demand.............. 5 mg/liter.
Un-ionized ammonia........................ 1 [micro]g/liter.
Residual chlorine......................... 1 [micro]g/liter.
Total organochloring pesticides........... 50 [micro]g/liter.
Total organocholorine pesticides plus 50 [micro]g/liter.
polychlorinated biphenyls (PCBs).
or organic chlorine................. 25 [micro]g/liter.
------------------------------------------------------------------------
(B) The concentration of dissolved oxygen in the dilution water
should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/l for
tests with trout, and 8.0 to 8.9 mg/l for tests with bluegill or fathead
minnow at sea level. If necessary, the dilution water can be aerated
before the addition of the test substance. All reconstituted water
should be aerated before use. Buffered soft water should be aerated
before but not after the addition of buffers.
(C) If disease organisms are present in the dilution water in
sufficient numbers to cause infection, they should be killed or removed
by suitable equipment.
(D) Glass distilled or carbon filtered deionized water with a
conductivity less than 1 micromho/cm is acceptable for use in making
reconstituted water. If the reconstituted water is prepared from a
ground or surface water source, conductivity, and total organic carbon
(TOC) or chemical oxygen demand
[[Page 120]]
(COD) should be measured on each batch.
(vii) Carriers. (A) Distilled water should be used in making stock
solutions of the test substance. If the stock volume however is more
than 10 percent of the test solution volume, dilution water should be
used. If a carrier is absolutely necessary to dissolve the test
substance, the volume used should not exceed the minimum volume
necessary to dissolve or suspend the test substance in the test
solution. If the test substance is a mixture, formulation, or commercial
product, none of the ingredients is considered a carrier unless an extra
amount is used to prepare the stock solution.
(B) Triethylene glycol and dimethyl formamide are the prefered
carriers, but acetone may also be used. The concentration of triethylene
glycol in the test solution should not exceed 80 mg/1. The concentration
of dimethyl formamide or acetone in the test solution should not exceed
5.0 mg/1.
(3) Test parameters--(i) Loading. The number of fish placed in a
test chamber should not be so great as to affect the results of the
test. The loading should not be so great that the test substance
concentrations are decreased by more than 20 percent due to uptake by
the fish. In static tests, loading should not exceed 0.5 grams of fish
per liter of solution in the test chamber at any one time. In flow-
through tests loading should not exceed 0.5 grams of fish per liter of
test solution passing through the chamber in 24 hours. These loading
rates should be sufficient to maintain the dissolved oxygen
concentration above the recommended levels and the ammonia concentration
below 20 [micro]g/l.
(ii) Dissolved oxygen concentration. (A) During static tests with
rainbow trout the dissolved oxygen in each test chamber shall be greater
than 5.5 mg/1. In tests with bluegill and fathead minnows, the DO shall
be maintained above 4.5 mg/1.
(B) During flow-through tests the dissolved oxygen concentration
shall be maintained above 8.2 mg/1 in tests with trout and above 6.6 mg/
l in tests with bluegills or fathead minnows.
(iii) Temperature. The test temperature shall be 22 [deg]C for
bluegill and fathead minnow and 12 [deg]C for rainbow trout. Excursions
from the test temperature shall be no greater than 2 [deg]C. The temperature shall be measured at least
hourly in one test chamber.
(iv) Light. A 16-hour light and 8-hour dark photoperiod should be
maintained.
(e) Reporting. The sponsor shall submit to the EPA all data
developed by the test that are suggestive or predictive of toxicity. In
addition to the reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reported test data
shall include the following:
(1) The source of the dilution water, a description of any
pretreatment, and the measured hardness, acidity, alkalinity, pH,
conductivity, TOC or COD and particulate matter.
(2) A description of the test chambers, the depth and volume of
solution in the chamber, the specific way the test was begun (e.g.,
conditioning, test substance additions), and for flow-through tests, a
description of the test substance delivery system.
(3) Detailed information about the test fish, including the
scientific name and method of verification, average weight (grams, wet
weight), standard length, age, source, history, observed diseases,
treatments, and mortalities, acclimation procedures, and food used.
(4) The number of replicates used, the number of organisms per
replicate, the loading rate, and the flow rate for flow-through tests.
(5) The measured DO, pH and temperature and the lighting regime.
(6) The solvent used, the test substance concentration in the stock
solution, the highest solvent concentration in the test solution and a
description of the solubility determinations in water and solvents if
used.
(7) The concentrations of the test substance at each test
concentration just before the start of the test and at all subsequent
sampling periods.
(8) The number of dead and live tests organisms, the percentage of
organisms that died, and the number that showed any abnormal effects in
the control and in each test chamber at each observation period.
(9) The 96-hour LC50, and when sufficient data have been
generated, the 24-, 48-, 72-, and incipient LC50 values,
[[Page 121]]
their 95 percent confidence limits, and the methods used to calculate
the LC50 values and their confidence limits.
(10) When observed, the observed no effect concentration (the
highest concentration tested at which there were no mortalities or
abnormal behavioral or physiological effects).
(11) The concentration-response curve at each observation period for
which a LC50 was calculated.
(12) Methods and data records of all chemical analyses of water
quality parameters and test substance concentrations, including method
validations and reagent blanks.
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19062, May 20, 1987;
54 FR 29715, July 14, 1989; 54 FR 33148, Aug. 11, 1989]