United States Prevention, Pesticides EPA712-C-96-136
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1035
Mysid Acute Toxicity
Test
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1035 Mysid acute toxicity test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1930 Mysid Shrimp Acute
Toxicity Test and OPP 72-3 Acute Toxicity Test for Estuarine and Marine
Organisms (Pesticide Assessment Guidelines, Subdivision E—Hazard
Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024,
1982.
(b) Purpose. This guideline prescribes a test using mysids as test or-
ganisms to develop data on the acute toxicity of chemicals. The Environ-
mental Protection Agency will use data from these tests in assessing the
hazard of a chemical to the aquatic environment.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and in 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. The following definitions also apply
to this test guideline.
Concentration-response curve is the curve produced from toxicity
tests when percent response (e.g. mortality) values are plotted against con-
centration of test substance for a given length of exposure.
Death means the lack of reaction of a test organism to gentle prod-
ding.
Flow-through means a continuous or an intermittent passage of test
solution or dilution water through a test chamber or a holding or acclima-
tion tank, with no recycling.
LC50 means the experimentally derived concentration of test sub-
stance that is calculated to kill 50 percent of a test population during con-
tinuous exposure over a specified period of time.
Loading means the ratio of test organisms biomass (grams, wet
weight) to the volume (liters) of test solution in a test chamber.
No observed effect concentration (NOEC) is the highest tested con-
centration in an acceptable toxicity test which did not cause the occurrence
of any specified adverse effect (statistically different from the control at
95 percent level), and below which no tested concentration caused such
an occurrence.
Retention chamber means a structure within a flow-through test cham-
ber which confines the test organisms, facilitating observation of test orga-
nisms, and eliminating loss of organisms in outflow water.
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Static system means a test chamber in which the test solution is not
renewed during the period of the test.
(d) 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, dead mysids are removed, and the
findings recorded. Dissolved oxygen concentration, pH, temperature, salin-
ity, the concentration of test substance, and other water quality characteris-
tics 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) 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 con-
centrations 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 h 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 h. 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 should be utilized in the defini-
tive test. When no apparent difference in sensitivity of the two life stages
is found, juveniles should be utilized in the definitive test. No replicates
are required, and nominal concentrations of the test chemical are accept-
able.
(3) Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration-response curves and the 48- and 96-h LC50 values
with the minimum amount of testing beyond the range-finding test.
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(ii) The definitive test should 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 should be exposed
to five or more concentrations of the test 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 are introduced into the test and
control chambers by stratified random assignment and should be placed
in two or more replicates. If solvents, solubilizing agents, or emulsifiers
have to be used, they should be commonly used carriers and should not
possess a synergistic or antagonistic effect on the toxicity of the test sub-
stance. Preferred carriers are dimethyl formamide, triethylene glycol, ace-
tone, or ethanol. Use of carriers should be avoided, if possible, as they
may serve as a carbon source for bacteria. The concentration of solvent
should not exceed 0.1 mL/L. The concentration ranges should be selected
to determine the concentration-response curves and LC50 values at 48 and
96 h.
(iv) Every test should include controls consisting of the same dilution
water, conditions, and procedures, and mysids from the same population
or culture container, except that none of the test chemical is added.
(v) The dissolved oxygen concentration, temperature, salinity, and pH
should be measured at the beginning and end of the test in each chamber.
(vi) The test duration is 96 h. The test is unacceptable if more than
10 percent of the control organisms die or exhibit abnormal behavior dur-
ing the 96-h test period. Each test chamber should be checked for dead
mysids at 24, 48, 72, and 96 h after the beginning of the test. Concentra-
tion-response curves and 24-, 48-, 72- and 96-h LC50 values should be
determined along with their 95 percent confidence limits.
(vii) In addition to death, any abnormal behavior or appearance
should also be reported.
(viii) Test organisms should 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 should
be positioned in a random manner or in a way in which appropriate statis-
tical 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. During static tests,
the concentration of test substance should be measured at a minimum at
the beginning and at the end of the tests. During the flow-through test,
the concentration of test substance should be measured at the beginning
and end of the test and in at least one appropriate chamber whenever a
malfunction is detected in any part of the test substance delivery system.
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Equal aliquots of test solution may be removed from each replicate cham-
ber 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.
(4) 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 sub-
stance in a sample should be validated by appropriate laboratory practices
before beginning the test. An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis and oxida-
tion products, give positive or negative interferences which cannot be sys-
tematically identified and mathematically corrected.
(ii) Numerical. The number of dead mysids should be counted during
each definitive test. Appropriate statistical analyses should provide a good-
ness-of-fit determination for the concentration-response curves. A 48- and
96-h LC50 and corresponding 95 percent interval should be calculated.
An NOEC and the slope of the dose-response curve should also be deter-
mined.
(e) Test conditions—(1) Test species—(i) Selection. (A) The mysid,
Mysidopsis bahia, is the organism specified for these tests. Either juvenile
(<24 h old) or young adult (5 to 6 days old) mysids are to be used to
start the test. It has recently been proposed, under paragraph (g)(2) of this
guideline, to place this species in a new genus, Americamysis.
(B) Mysids to be used in acute 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 verifica-
tion should be obtained from the commercial supplier by experienced lab-
oratory personnel or by an outside expert.
(C) Mysids used in a particular test should be of similar age and
be of normal size and appearance for their age. Mysids should 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 should be gradual. Within a 24-h period, changes in water
temperature should not exceed 1 °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.
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(iii) Care and handling. Methods for the care and handling of mysids
such as those described under paragraph (g)(l) of this guideline can be
used during holding, culturing, and testing periods.
(iv) Feeding. Mysids should be fed daily during testing. Any food
utilized should support survival, growth, and reproduction of the mysids.
A recommended food is live Anemia spp. (48-h-old nauplii).
(2) Facilities—(i) Apparatus. (A) Facilities which may be needed
to perform this test include:
(7) Flow-through or recirculating tanks for holding and acclimating
mysids.
(2) A mechanism for controlling and maintaining the water tempera-
ture during the holding, acclimation, and test periods.
(3) Apparatus for straining particulate matter, removing gas bubbles,
or aerating the water, as necessary.
(4) An apparatus for providing a 14-h light and 10-h dark
photoperiod with a 15 to 30 min 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 reten-
tion 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. Fa-
cilities should be well ventilated and free of fumes and disturbances that
may affect the test organisms.
(B) Test chambers should 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
should be cleaned before each test following standard laboratory practices.
(iii) Construction materials. (A) Materials and equipment that con-
tact 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
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showing signs of stress, such as reduced growth and fecundity. Mysids
should be cultured and tested in dilution water from the same origin.
(B) Natural seawater should be filtered through a filter with a pore
size of < 20 (im prior to use in a test.
(C) Artificial seawater can be prepared by adding commercially avail-
able formulations or specific amounts of reagent-grade chemicals to
deionized water. Deionized water with a conductivity less than
1 (lohm/cm at 12 °C is acceptable for making artificial seawater. When
deionized water is prepared from a ground or surface water source, con-
ductivity and total organic carbon (or chemical oxygen demand) should
be measured on each batch.
(v) Test substance delivery system. In flow-through tests, propor-
tional diluters, metering pumps, or other suitable systems should be used
to deliver test substance to the test chambers. The system to be used should
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-h flow through a
test chamber should be equal to at least 5x 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 con-
tained in test chambers should be maintained as specified below:
(i) The test temperature should be 25 °C. Excursions from the test
temperature should be not greater than ±2 °C.
(ii) Dissolved oxygen concentration between 60 and 105 percent satu-
ration. Aeration, if needed to achieve this level, should be done before
the addition of the test substance. All treatment and control chambers
should be given the same aeration treatment.
(iii) The number of mysids placed in a test solution should not be
so great as to affect results of the test. Loading should 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
should not cause the dissolved oxygen concentration to fall below the rec-
ommended levels.
(iv) Photoperiod of 14 h light and 10 h darkness, with a 15 to
30 min transition period.
(v) Salinity of 20 ±3 ppt.
(f) Reporting. The sponsor should submit to the EPA all data devel-
oped during the test that are suggestive or predictive of acute toxicity and
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all concomitant toxicologic manifestations. In addition to the reporting re-
quirements as specified under Good Laboratory Practice Standards, 40
CFR part 792, subpart J, the following specific information should be re-
ported:
(1) The nature of the test, laboratory, name of the investigator, test
substance, and dates of test should be supplied.
(2) A detailed description of the test substances should be provided.
This information should include the source, lot number, composition, phys-
ical and chemical properties, shelf life and storage conditions, and any
carrier or additives used.
(3) Detailed information about the shrimp should be provided: Com-
mon and scientific names, source of supply, age, history, weight, acclima-
tion procedure, and feeding history should be reported.
(4) A description of the experimental design including the number
of test solution concentrations, number of replicates, and number of shrimp
per replicate should be provided.
(5) The source of the dilution water, its chemical characteristics (e.g.
salinity), and a description of any pretreatment.
(6) A description of the test chambers, the depth and volume of solu-
tion in the chamber, the number of organisms per treatment, the number
of replicates, the loading, the lighting, the test substance delivery system
and flow rate expressed as volume additions per 24 h.
(7) The concentration of the test substance in each test chamber be-
fore the start of the test and at the end.
(8) The number of dead shrimp and measurements of water tempera-
ture, salinity, and dissolved oxygen concentration in each test chamber
should be recorded at the protocol-designated times.
(9) Methods and data records of all chemical analyses of water quality
and test substance concentrations, including method validations and rea-
gent blanks.
(10) Recorded data for the holding and acclimation period (tempera-
ture, salinity, etc.).
(11) Concentration-response curves should be fitted to mortality data
collected at 24, 48, 72, and 96 h. A statistical test of goodness-of-fit should
be performed.
(12) For each set of mortality data, the 48- and 96-h LC50 and 95
percent confidence limits should be calculated on the basis of the average
measured concentration of the test substance. When data permits, LC50
values with 95 percent confidence limits should be computed for 24- and
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72-h observations. The NOEC and slope of the dose-response curves
should also be calculated.
(13) The methods used in calculating the concentration-response
curves and the LC50 values should be fully described.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Environmental Protection Agency, Bioassay Procedures for the
Ocean Disposal Permit Program, EPA Report No. 600-9-78-010 (Gulf
Breeze, Florida, 1978).
(2) Price, E.W. et al. Observations on the genus Mysidopsis Sars,
1864 with the designation of a new genus, Americamysis, and the descrip-
tions of Americamysis alleni and A. stucki (Pericarda: Mysidacea:
Mysidae), from the Gulf of Mexico. Proceedings of the Biological Society
of Washington 107:680-698 (1994).
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