United States Prevention, Pesticides EPA712-C-96-137
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1045
Penaeid 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 850.1045 Penaeid 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 797.1970 Penaid 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 tests using penaeid shrimp as
test organisms to develop data on the acute toxicity of chemicals. The
Environmental Protection Agency will use data from these tests in assess-
ing the hazard of a chemical to the aquatic environment.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice
Standards apply to this test guideline. The following definitions also apply
to this guideline:
Concentration-response curve is the curve produced from toxicity test
data when percent response (e.g. mortality) values are plotted against con-
centration of test substance for a given length of exposure.
Death is the lack of reaction of a test organism to gentle prodding.
Flow-through is a continuous passage of test solution or dilution
water through a test chamber, holding, or acclimation tank with no recy-
cling.
LC50 is the experimentally derived concentration of test substance
that is calculated to have killed 50 percent of a test population during
continuous exposure over a specified period of time.
Loading is the ratio of test organism 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
the 95 percent level), and below which no tested concentration caused such
an occurrence.
ppt is parts per thousand (salinity units).
(d) Test procedures—(1) Summary of the test. Prior to testing, the
bottoms of the test chambers are covered with 2 to 3 cm of sand and
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then filled with appropriate volumes of dilution water. The flow is adjusted
to the rate desired to achieve loading requirements. Penaeids are intro-
duced into the test chambers according to the experimental design. The
shrimp are acclimated by maintaining them in the test chambers for a pe-
riod of 3 to 7 days prior to the beginning of the test. The test begins
when the test substance is introduced into the test chambers. The rate of
flow is adjusted to maintain the desired test substance concentration in
each chamber. The shrimp are observed during the test; dead shrimp are
counted, removed, and the findings recorded. Dissolved oxygen concentra-
tion (DOC), pH, temperature, salinity, test substance concentration, and
other water quality characteristics are measured at specified intervals in
selected test chambers. The concentration of test substances with low water
solubility may have to be determined with more frequency. 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 the test substance concentrations to be used for the definitive
test. Substances which have low solubility and/or unusual adsorbance char-
acteristics may require special handling procedures (physical procedures
or the use of carrier substances) and attention to the type of materials
used in the testing chambers to enhance solubility or decrease adsorption.
(ii) The shrimp should be exposed to a series of widely spaced con-
centrations of test substance (e.g. 1, 10, 100 mg/L, etc.).
(iii) A minimum of five penaeid shrimp should be exposed to each
concentration of test substance for up to 96 h. No replicates are required
and nominal concentrations of the chemical are acceptable.
(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.
(ii) A minimum of 20 shrimp per concentration should 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 shrimp 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 requested concentration-response curves and LC50 values.
The concentration of test substance in test solutions should be determined
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prior to use and at designated times. Abnormal or unexpected observations
should trigger chemical analysis of the test water. If a specific test chamber
seems to be affected, its water should be analyzed.
(iii) Every test should include controls consisting of the same dilution
water, conditions, procedures, and shrimp from the same population or
culture container, except that none of the chemical is added. If carriers
are used, a separate carrier control should also be included.
(iv) The DOC, temperature, salinity, and pH should be measured at
the beginning of the test and at 24, 48, 72, and 96 h in each test chamber.
(v) The test duration is 96 h. The test is unacceptable if more than
10 percent of the control organisms die or appear to be stressed or diseased
during the 96-h test period. Each test chamber should be checked for dead
shrimp at 3, 6, 12, 24, 48, 72, and 96 h after the beginning of the test.
Concentration-response curves and 48- and 96-h LC50 values should be
determined along with their 95 percent confidence limits.
(vi) In addition to death, any abnormal behavior or appearance should
also be reported.
(vii) Distribution of shrimp among test chambers should be random-
ized. In addition, test chambers within the testing area should be positioned
in a random manner or in a way in which appropriate statistical analyses
can be used to determine the variation due to placement.
(viii) The concentration of dissolved test substance (that which passes
through a 0.45 (im filter) in the test chambers should be measured as often
as is feasible during the test. The concentration of test substance should
be measured:
(A) In each chamber at the beginning of the test and at 48 and
96 h after the start of the test.
(B) In at least one chamber containing the next to the lowest test
substance concentration at least once every 24 h during the test.
(C) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system. Among replicate
test chambers of a treatment concentration, the measured concentration of
the test substance should not vary more than 20 percent.
(ix) Observations on compound solubility should be recorded. The
investigator should report the appearance of surface slicks, precipitates,
or material adhering to the sides of the test chambers.
(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
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analyses. The analytical method used to measure the amount of test sub-
stance in a sample should be validated before beginning the test by appro-
priate laboratory practices. 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 corrected mathematically.
(ii) Numerical. The number of dead shrimp 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 intervals 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. This test should
be conducted using one of three species of penaeid: Penaeus aztecus
(brown shrimp), Penaeus duorarum (pink shrimp), or Penaeus setiferus
(white shrimp). Post-larval juvenile shrimp should be utilized. Shrimp may
be reared from eggs in the laboratory or obtained directly as juveniles
or adults. Shrimp used in a particular test should have been obtained from
the same source, be of similar age, and be of normal size and appearance.
Shrimp 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 control
group.
(ii) Acclimation. During acclimation, shrimp should be maintained
in facilities with background colors and light intensities similar to those
of the testing areas. In addition, any change in the temperature and chem-
istry of the dilution water used for holding and acclimating the test orga-
nisms 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 2 percent.
(iii) Care and handling. Upon arrival at the test facility, the shrimp
should be transferred to water closely matching the temperature and salin-
ity of the transporting medium. Shrimp should be held in glass tanks of
30 L capacity or larger. No more than 22 to 24 shrimp should be placed
in a 30 L tank unless the flow-through apparatus can maintain dissolved
oxygen levels above 60 percent of saturation. With species of the genus
Penaeus, a minimum flow rate of 7.5 L/g body weight day should be
provided. Larger flows, up to 22 L/g body weight day, may be desirable
to ensure dissolve oxygen concentrations above 60 percent of saturation
and the removal of metabolic products. The period of acclimation to ambi-
ent laboratory conditions should be at least 4 to 7 days.
(iv) Feeding. Penaeid shrimp should not be fed during testing. Every
2 or 3 days during the acclimation period, shrimp should be fed fish pieces
approximately 1 cm2. Uneaten food should be removed daily.
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(2) Facilities—(i) Apparatus. (A) Facilities which may be needed
to perform this test include: Flow-through tanks for holding and
acclimating penaeid shrimp; a mechanism for controlling and maintaining
the water temperature and salinity during the holding period; apparatus
for straining particulate matter, removing air bubbles, or aerating water
when necessitated by water quality requirements; and an apparatus provid-
ing a 12-h light and 12-h dark photoperiod with a 15- to 30-min transi-
tion period. Facilities should be well ventilated, free of fumes, and free
of all other disturbances that may affect test organisms.
(B) Acid-washed sand, free of excess organic matter, should be placed
in the bottom of test chambers to a depth of 2-3 cm.
(C) Test chambers should be loosely covered to reduce the loss of
test solution or dilution water due to evaporation, minimize entry of dust
and other particles, and prevent escape of the shrimp.
(ii) Cleaning. Test substance delivery systems and test chambers
should be cleaned before each test following standard laboratory practices.
(iii) Construction materials. 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.
(iv) Dilution water. (A) Natural or artificial seawater is acceptable
as dilution water if shrimp will survive in it without signs of stress, such
as unusual behavior or discoloration. Shrimp should be acclimated and
tested in dilution water from the same origin.
(B) Natural seawater should be filtered through a 5 (im filter with
a pore size < 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
0.1 mS/m 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. Proportional diluters, metering
pumps, or other suitable systems should be used to deliver 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 in each chamber. The general
operation of the test substance delivery system should be checked twice
daily during a test. The 94-h flow through a test chamber should be equal
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to a 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 as specified below:
(i) Temperature of 23 + 1 °C.
(ii) DOC between 60 and 105 percent saturation. Aeration, if needed
to achieve this level, should be done before the addition of the test sub-
stance. All treatment and control chambers should be given the same aer-
ation treatment.
(iii) The number of shrimp placed in a test solution should not be
so great as to affect results of the test. Loading requirements will vary
depending on the flow rate of dilution water. The loading should not cause
the DOC to fall below the recommended levels.
(iv) Photoperiod of 12-h light and 12-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 by the test that are suggestive or predictive of acute toxicity and
all other toxicological manifestations. In addition to the general reporting
requirements prescribed under Good Laboratory Practice Standards, 40
CFR part 792, subpart J, the reporting of test data should include the fol-
lowing:
(1) The nature of the test, laboratory, name of the investigator, test
substance, and dates of the 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, 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
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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 DOC 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
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, FL 1978).
(2) [Reserved]
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