[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: 40CFR797.1950]
[Page 136-141]
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. 797.1950 Mysid shrimp chronic toxicity test.
(a) Purpose. This guideline is intended for use in developing data
on the chronic 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 using mysids as
test organisms to develop data on the chronic 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) ``Chronic toxicity test'' means a method used to determine the
concentration of a substance that produces an adverse effect from
prolonged exposure of an organism to that substance. In this test,
mortality, number of young per female and growth are used as measures of
chronic toxicity.
(2) ``Death'' means the lack of reaction of a test organism to
gentle prodding.
(3) ``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.
(4) ``G1 (Generation 1)'' means those mysids which are used to begin
the test, also referred to as adults; G2 (Generation 2) are the young
produced by G1.
(5) ``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.
(6) ``Loading'' means the ratio of test organism biomass (gram, wet
weight) to the volume (liters) of test solution in a test chamber.
(7) ``MATC'' (Maximum Acceptable Toxicant Concentration) means the
maximum concentration at which a chemical can be present and not be
toxic to the test organism.
(8) ``Retention chamber'' means a structure within a flow-through
test chamber which confines the test organisms, facilitating observation
of test organisms and eliminating washout from test chambers.
(c) Test procedures--(1) Summary of the test. (i) In preparation for
the test, the flow of test solution through each
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chamber is adjusted to the rate desired. The test substance is
introduced into each test chamber. 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
retention chambers within the test and the control chambers. Mysids in
the test and control chambers are observed periodically during the test,
the dead mysids removed and the findings reported.
(ii) Dissolved oxygen concentration, pH, temperature, salinity, the
concentration of test substance and other water quality characteristics
are measured at specified intervals in selected test chambers.
(iii) Data collected during the test are used to develop a MATC
(Maximum Acceptable Toxicant Concentration) and quantify effects on
specific chronic parameters.
(2) [Reserved]
(3) Range-finding test. (i) A range-finding test should be conducted
to establish test solution concentrations for the definitive test.
(ii) The mysids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually
under static conditions.
(iii) A minimum of 10 mysids should be exposed to each concentration
of test substance for a period of time which allows estimation of
appropriate chronic test concentrations. 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 concentration-response curves, LC50 values, and
effects of a chemical on growth and reproduction during chronic
exposure.
(ii) A minimum of 40 mysids per concentration shall be exposed to
four 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/1). 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 should not exceed 0.1 ml/1. The
concentration ranges should be selected to determine the concentration
response curves, LC50 values and MATC. Concentration of test
substance in test solutions should be analyzed prior to use.
(iii) Every test should 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.
(iv) The dissolved oxygen concentration, temperature, salinity, and
pH shall be measured weekly in each chamber.
(v) The test duration is 28 days. The test is unacceptable if more
than 20 percent of the control organisms die, appear stressed or are
diseased during the test. The number of dead mysids in each chamber
shall be recorded on days 7, 14, 21, and 28 of the test. At the time
when sexual characteristics are discernible in the mysids (approximately
10 to 12 days in controls; possible delays may occur in mysids exposed
to test substances), the number of males and females (identified by
ventral brood pouch) in each chamber shall be recorded. Body length (as
measured by total midline body length, from the anterior tip of the
carapace to the posterior margin of the uropod) shall be recorded for
males and females at the time when sex can be determined simultaneously
for all mysids in control and treatment groups. This time cannot be
specified because of possible delays in sexual maturation of mysids
exposed to test substances. A second observation of male and female body
lengths shall be conducted on day 28 of the test. To reduce stress on
the mysids, body lengths can be recorded by photography through a
stereomicroscope with appropriate scaling information. As offspring are
produced by the G1 mysids (approximately 13 to 16 days in controls), the
young shall be counted and separated into retention chambers at the same
test substance concentration as the chambers where they originated. If
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available prior to termination of the test, observations on the
mortality, number of males and females and male and female body length
shall be recorded for the G2 mysids. Concentration-response curves,
LC50 values and associated 95 percent confidence limits for
the number of dead mysids (G1) shall be determined for days 7, 14, 21,
and 28. An MATC shall be determined for the most sensitive test criteria
measured (cumulative mortality of adult mysids, number of young per
female, and body lengths of adult males and females).
(vi) In addition to death, any abnormal behavior or appearance shall
also be reported.
(vii) 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
appropriate statistical analyses can be used to determined the variation
due to placement.
(viii) The concentration of the test substance in the chambers
should be measured as often as is feasible during the test. The
concentration of test substance shall be measured:
(A) At each test concentration at the beginning of the test and on
days 7, 14, 21, and 28; and
(B) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
Equal aliquots of test solutions may be removed from each test 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 employed 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. (A) The number of dead mysids, cumulative young per
female, and body lengths of male and female mysids shall be recorded
during each definitive test. Appropriate statistical analyses shall
provide a goodness-of-fit determination for the day 7, 14, 21 and 28
adult (Gl) death concentration-response curves.
(B) A 7-, 14-, 21- and 28-day LC50, based on adult (Gl)
death, and corresponding 95 percent confidence intervals shall be
calculated. Appropriate statistical tests (e.g., analysis of variance,
mean separation test) should be used to test for significant chemical
effects on chronic test criteria (cumulative mortality of adults,
cumulative number of young per female and body lengths of adult male and
females) on designated days. An MATC shall be calculated using these
chronic tests criteria.
(d) Test conditions--(1) Test species--(i) Selection. (A) The mysid
shrimp, Mysidopsis bahia, is the organism specified for these tests.
Juvenile mysids, <=24 hours old, 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 mysid
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.
(D) 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 water used for holding or culturing the
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test organisms to those of the test should be gradual. Within a 24-hour
period, changes in water temperature should not exceed 1 [deg]C, while
salinity changes should 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. nauplii (approximately 48 hours
old).
(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, flow-through chambers and a
test substance delivery system are required. It is recommended that
mysids be held in retention chambers within test chambers to facilitate
observations and eliminate loss through outflow water.
(B) Facilities should be well ventilated and free of fumes and
disturbances that may affect test organisms.
(C) 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 use 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 the dilution water and should not contain substances that
can be leached into aqueous solution in quantities that can affect the
test results.
(B) 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 or glass-distilled water. Deionized water with a
conductivity less than 1 [micro]ohm/cm at 12 [deg]C is acceptable as the
diluent 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. 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 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 rate through a chamber shall be
equal to at least 5 times the volume of the chamber. The flow rates
should not vary more than 10 percent among 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 no greater than 2
[deg]C.
(ii) Dissolved oxygen concentration between 60 and 105 percent
saturation.
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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 requirements for the
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-
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 by the test that are suggestive or predictive of chronic
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, average length, age, source,
history, observed diseases, treatments, 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 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 first time (day) that sexual characteristics can be observed
in controls and in each test substance concentration.
(6) The length of time for the appearance of the first brood for
each concentration.
(7) The means (average of replicates) and respective 95 percent
confidence intervals for:
(i) Body length of males and females at the first observation day
(depending on time of sexual maturation) and on day 28.
(ii) Cumulative number of young produced per female on day 28.
(iii) Cumulative number of dead adults on day 7, 14, 21 and 28.
(iv) If available prior to test termination (day 28), effects on G2
mysids (number of males and females, body length of males and females
and cumulative mortality).
(8) The MATC is calculated as the geometric mean between the lowest
measured test substance concentration that had a significant (P<0.05)
effect and the highest measured test substance concentration that had no
significant (P<0.05) effect in the chronic test. The most sensitive of
the test criteria for adult (Gl) mysids (cumulative number of dead
mysids, body lengths of males and females or the number of young per
female) is used to calculate the MATC. The criterion selected for MATC
computation is the one which exhibits an effect (a statistically
significant difference between treatment and control groups; P<0.05) at
the lowest test substance concentration for the shortest period of
exposure. Appropriate statistical tests (analysis of variance, mean
separation test) should be used to test for significant chemical
effects. The statistical tests employed and the results of these tests
shall be reported.
(9) Concentration-response curves shall be fitted to the cumulative
number of adult dead for days 7, 14, 21, and 28. A statistical test of
goodness-of-fit shall be performed and the results reported.
(10) An LC50 value based on the number of dead adults
with corresponding 95 percent confidence intervals for days 7, 14, 21
and 28. These calculations shall be made using the average measured
concentration of the test substance.
(11) Methods and data records of all chemical analyses of water
quality and test substance concentrations, including method validations
and reagent blanks.
[[Page 141]]
(12) 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 19069, May 20, 1987]