[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: 40CFR797.1930]
[Page 132-136]
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. 797.1930 Mysid shrimp acute toxicity test.
(a) Purpose. This guideline 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 guideline prescribes a test using mysid
shrimp as test organisms to develop data on the acute toxicity of
chemicals. The United States Environmental Protection Agency (EPA) will
use data from these tests in assessing the hazard of a chemical to the
aquatic environment.
(b) Definitions. The definitions in section 3 of the Toxic
Substances Control Act (TSCA) and in part 792--Good Laboratory Practice
Standards of this chapter, apply to this test guideline. The following
definitions also apply to this guideline.
(1) ``Death'' means the lack of reaction of a test organism to
gentle prodding.
(2) ``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.
(3) ``LC50'' means that experimentally derived
concentration of test substance that is calculated to kill 50 percent of
a test population during continuous exposure over a specified period of
time.
(4) ``Loading'' means the ratio of test organisms biomass (grams,
wet weight) to the volume (liters) of test solution in a test chamber.
(5) ``Retention chamber'' means a structure within a flow-through
test chamber which confines the test organisms, facilitating observation
of test organisms and eliminating loss of organisms in outflow water.
(6) ``Static system'' means a test chamber in which the test
solution is not renewed during the period of the test.
(c) Test procedures--(1) Summary of the test. In preparation for the
test, 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. The test substance
is introduced into each test chamber. In a flow-through test, the rate
at which the test substance is added is adjusted to establish and
maintain the desired concentration of test substance in each test
chamber. The test is started by randomly introducing mysids acclimated
in accordance with the test design into the test chambers. Mysids in the
test chambers are observed periodically during the test, the dead mysids
removed and the findings recorded. Dissolved oxygen
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concentration, pH, temperature, salinity, the concentration of test
substance, and other water quality characteristics are measured at
specified intervals in test chambers. Data collected during the test are
used to develop concentration-response curves and LC50 values
for the test substance.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to determine:
(A) Which life stage (juvenile or young adult) is to be utilized in
the definitive test.
(B) The test solution concentrations for the definitive test.
(ii) The mysids should be exposed to a series of widely spaced
concentrations of test substance (e.g., 1, 10, 100 mg/l, etc.), usually
under static conditions.
(iii) This test should be conducted with both newly hatched juvenile
(< 24 hours old) and young adult (5 to 6 days old) mysids. For each age
class (juvenile or young adult), a minimum of 10 mysids should be
exposed to each concentration of test substance for up to 96 hours. The
exposure period may be shortened if data suitable for the purpose of the
range-finding test can be obtained in less time. The age class which is
most sensitive to the test substance in the range-finding test shall be
utilized in the definitive test. When no apparent difference in
sensitivity of the two life stages is found, juveniles shall be utilized
in the definitive test. No replicates are required, and nominal
concentrations of the chemical are acceptable.
(4) Definitive test. (i) The purpose of the definitive test is to
determine the concentration-response curves and the 48- and 96-hour
LC50 values with the minimum amount of testing beyond the
range-finding test.
(ii) The definitive test shall be conducted on the mysid life stage
(juveniles or young adults) which is most sensitive to the test
substance being evaluated.
(iii) A minimum of 20 mysids per concentration shall be exposed to
five or more concentrations of the chemical chosen 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). An equal number of mysids shall be placed in two or more
replicates. If solvents, solubilizing agents or emulsifiers have to be
used, they shall be commonly used carriers and shall not possess a
synergistic or antagonistic effect on the toxicity of the test
substance. The concentration of solvent shall not exceed 0.1 ml/1. The
concentration ranges shall be selected to determine the concentration-
response curves and LC50 values at 48 and 96 hours.
(iv) Every test shall include controls consisting of the same
dilution water, conditions, procedures, and mysids from the same
population or culture container, except that none of the chemical is
added.
(v) The dissolved oxygen concentration temperature, salinity, and pH
shall be measured at the beginning and end of the test in each chamber.
(vi) The test duration is 96 hours. The test is unacceptable if more
than 10 percent of the control organisms die or exhibit abnormal
behavior during the 96 hour test period. Each test chamber should be
checked for dead mysids at 24, 48, 72, and 96 hours after the beginning
of the test. Concentration-response curves and 24-, 48-, 72- and 96-hour
LC50 values should be determined along with their 95 percent
confidence limits.
(vii) In addition to death, any abnormal behavior or appearance
shall also be reported.
(viii) Test organisms shall be impartially distributed among test
chambers in such a manner that test results show no significant bias
from the distributions. In addition, test chambers within the testing
area shall be positioned in a random manner or in a way in which
appropriated statistical analyses can be used to determine the variation
due to placement.
(ix) The concentration of the test substance in the chambers should
be measured as often as is feasible during the test. At a minimum,
during static tests the concentration of test substance shall be
measured at each concentration at the beginning and at the end of the
test. During the flow-through test, the concentration of test substance
should be measured at the beginning and end of the test and in at
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least one appropriate chamber whenever a malfunction is detected in any
part of the test substance delivery system. Equal aliquots of test
solution may be removed from each replicate chamber and pooled for
analysis. Among replicate test chambers of a treatment concentration,
the measured concentration of the test substance should not vary more
than 20 percent.
(5) [Reserved]
(6) Analytical measurements--(i) Test chemical. Deionized water
should be used in making stock solutions of the test substance. Standard
analytical methods should be used whenever available in performing the
analyses. The analytical method used to measure the amount of test
substance in a sample shall be validated before beginning the test by
appropriate laboratory practices. An analytical method is not acceptable
if likely degradation products of the test substance, such as hydrolysis
and oxidation products, give positive or negative interferences which
cannot be systematically identified and corrected mathematically.
(ii) Numerical. The number of dead mysids shall be counted during
each definitive test. Appropriate statistical analyses should provide a
goodness-of-fit determination for the concentration-response curves. A
48- and 96-hour LC50 and corresponding 95 percent interval
shall be calculated.
(d) Test conditions--(1) Test species--(i) Selection. (A) The mysid
shrimp, Mysidopsis bahia, is the organism specified for these tests.
Either juvenile (<24 hours old) or young adult (5 to 6 days old) mysids
are to be used to start the test.
(B) Mysids to be used in chronic toxicity tests should originate
from laboratory cultures in order to ensure the individuals are of
similar age and experimental history. Mysids used for establishing
laboratory cultures may be purchased commercially or collected from
appropriate natural areas. Because of similarities with other mysids
species, taxonomic verification should be obtained from the commercial
supplier by experienced laboratory personnel or by an outside expert.
(C) Mysids used in a particular test shall be of similar age and be
of normal size and appearance for their age. Mysids shall not be used
for a test if they exhibit abnormal behavior or if they have been used
in a previous test, either in a treatment or in a control group.
(ii) Acclimation. (A) Any change in the temperature and chemistry of
the dilution water used for holding or culturing the test organisms to
those of the test shall be gradual. Within a 24-hour period, changes in
water temperature shall not exceed 1 [deg]C, while salinity changes
shall not exceed 5 percent.
(B) During acclimation mysids should be maintained in facilities
with background colors and light intensities similar to those of the
testing areas.
(iii) Care and handling. Methods for the care and handling of mysids
such as those described in paragraph (f)(1) of this section can be used
during holding, culturing and testing periods.
(iv) Feeding. Mysids should be fed during testing. Any food utilized
should support survival, growth and reproduction of the mysids. A
recommended food is live Artemia spp. (48-hour-old nauplii).
(2) Facilities--(i) Apparatus. (A) Facilities which may be needed to
perform this test include: (1) flow-through or recirculating tanks for
holding and acclimating mysids; (2) a mechanism for controlling and
maintaining the water temperature during the holding, acclimation and
test periods; (3) apparatus for straining particulate matter, removing
gas bubbles, or aerating the water, as necessary; and (4) an apparatus
for providing a 14-hour light and 10-hour dark photoperiod with a 15 to
30 minute transition period. In addition, for flow-through tests, flow-
through chambers and a test substance delivery system are required.
Furthermore, it is recommended that mysids be held in retention chambers
within test chambers to facilitate observations and eliminate loss of
test organisms through outflow water. For static tests, suitable
chambers for exposing test mysids to the test substance are required.
Facilities should be well ventilated and free of fumes and disturbances
that may affect the test organisms.
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(B) Test chambers shall be loosely covered to reduce the loss of
test solution or dilution water due to evaporation and to minimize the
entry of dust or other particulates into the solutions.
(ii) Cleaning. Test substance delivery systems and test chambers
shall be cleaned before each test following standard laboratory
practices.
(iii) Construction materials. (A) Materials and equipment that
contact test solutions should be chosen to minimize sorption of test
chemicals from dilution water and should not contain substances that can
be leached into aqueous solution in quantities that can affect test
results.
(B) For use in the flow-through test, retention chambers utilized
for confinement of test organisms can be constructed with netting
material of appropriate mesh size.
(iv) Dilution water. (A) Natural or artificial seawater is
acceptable as dilution water if mysids will survive and successfully
reproduce in it for the duration of the holding, acclimating and testing
periods without showing signs of stress, such as reduced growth and
fecundity. Mysids shall be cultured and tested in dilution water from
the same origin.
(B) Natural seawater shall be filtered through a filter with a pore
size of <20 microns prior to use in a test.
(C) Artificial seawater can be prepared by adding commercially
available formulations or by adding specific amounts of reagent-grade
chemicals to deionized water. Deionized water with a conductivity less
than 1 [micro]ohm/cm at 12 [deg]C is acceptable for making artificial
seawater. When deionized water is prepared from a ground or surface
water source, conductivity and total organic carbon (or chemical oxygen
demand) shall be measured on each batch.
(v) Test substance delivery system. In flow-through tests,
proportional diluters, metering pumps, or other suitable systems should
be used to deliver test substance to the test chambers. The system used
shall be calibrated before each test. Calibration includes determining
the flow rate through each chamber and the concentration of the test
substance in each chamber. The general operation of the test substance
delivery system should be checked twice daily during a test. The 24-hour
flow through a test chamber shall be equal to at least 5 times the
volume of the test chamber. During a test, the flow rates should not
vary more than 10 percent among test chambers or across time.
(3) Test parameters. Environmental parameters of the water contained
in test chambers shall be maintained as specified below:
(i) The test temperature shall be 25[deg]C. Excursions from the test
temperature shall be not greater than 2[deg]C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation. Aeration, if needed to achieve this level, shall be done
before the addition of the test substance. All treatment and control
chambers shall be given the same aeration treatment.
(iii) The number of mysids placed in a test solution shall not be so
great as to affect results of the test. Loading shall not exceed 30
mysids per liter for a static test. Loading requirements for the flow-
through test will vary depending on the flow rate of dilution water. The
loading shall not cause the dissolved oxygen concentration to fall below
the recommended levels.
(iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15
to 30 minute transition period.
(v) Salinity of 20 parts per thousand 3
percent.
(e) Reporting. The sponsor shall submit to the EPA all data
developed during the test that are suggestive or predictive of acute
toxicity and all concomitant toxicologic manifestations. In addition to
the general reporting requirements prescribed in part 792--Good
Laboratory Practice Standards of this chapter, the reporting of test
data shall include the following:
(1) The source of the dilution water, its chemical characteristics
(e.g., salinity, pH, etc.) and a description of any pretreatment.
(2) Detailed information about the test organisms, including the
scientific name and method of verification, age, source, history,
abnormal behavior, acclimation procedures and food used.
(3) A description of the test chambers, the depth and volume of
solution in the chamber, the way the test was
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begun (e.g., conditioning, test substance additions, etc.), the number
of organisms per treatment, the number of replicates, the loading, the
lighting, the test substance delivery system and the flow rate expressed
as volume additions per 24 hours.
(4) The measured concentration of test substance in test chambers at
the times designated.
(5) The number and percentage of organisms that died or showed any
other adverse effects in the control and in each treatment at each
observation period.
(6) Concentration-response curves shall be fitted to mortality data
collected at 24, 48, 72, and 96 hours. A statistical test of goodness-
of-fit shall be performed and the results reported.
(7) The 96-hour LC50 and when sufficient data have been
generated, the 24-, 48-, and 72-hour LC50's and the
corresponding 95-percent confidence limits and the methods used to
calculate the values. These calculations shall be made using the average
measured concentration of the test substance.
(8) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
(9) The data records of the holding, acclimation and test
temperature and salinity.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) U.S. Environmental Protection Agency, ``Bioassay Procedures for
the Ocean Disposal Permit Program,'' EPA Report No. 600-9-78-010 (Gulf
Breeze, Florida, 1978).
(2) [Reserved]
[50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19068, May 20, 1987;
52 FR 26150, July 13, 1987]