United States       Prevention, Pesticides     EPA712-C-96-137
          Environmental Protection    and Toxic Substances     April 1996
          Agency         (7101)
&EPA   Ecological Effects Test
          OPPTS 850.1045
          Penaeid Acute Toxicity
                 'Public Draft"

     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

     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:

     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,  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

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,

     (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-

     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

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

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

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-

    (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

    (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.

     (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

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-

    (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

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]