United States       Prevention, Pesticides     EPA 712-C-96-115
          Environmental Protection    and Toxic Substances     April 1996
          Agency        (7101)
&EPA    Ecological Effects Test
          OPPTS 850.1025
          Oyster Acute Topxicity
          Test (Shell Deposition)
                '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 850.1025  Oyster acute toxicity test (shell deposition).
     (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 797.1800 Oyster Acute Tox-
icity 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 to be  used to develop
data on the  acute toxicity of chemical  substances and mixtures  ("chemi-
cals") to  Eastern oysters, Crassostrea virginica (Gmelin). The Environ-
mental Protection Agency will use data from these tests in assessing the
hazard of a chemical to the environment.

     (c) Definitions. The definitions in section  3 of the Toxic Substances
Control Act (TSCA) and the  definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

     Acute toxicity is  the discernible adverse effects induced in an orga-
nism within a short period of time (days) of exposure to a chemical. For
aquatic animals this usually refers to continuous exposure to the chemical
in water for a period of up  to 4 days. The effects (lethal or sublethal)
occurring  may  usually be  observed within  the period of  exposure with
aquatic organisms. In this  test guideline, shell deposition  is used as the
measure of toxicity.

     EC50 is that experimentally derived concentration of a chemical in
water that is calculated to induce shell  deposition 50 percent  less  than
that of the controls in a test batch of organisms during continuous exposure
within a particular exposure period which should be stated.

     Shell deposition  is the measured length of shell growth  that  occurs
between the time the  shell is  ground at test initiation and test termination
96 h later.

     Umbo means the  narrow  end (apex) of the oyster shell.

     Valve height means the greatest  linear dimension of the  oyster  as
measured  from the  umbo to  the ventral  edge  of the valves (the farthest
distance from the umbo).

     (d) Test procedures—(1)  Summary of the test, (i) The water solu-
bility and the vapor pressure  of the test chemical should be known. Prior
to testing, the structural formula of the test chemical, its purity, stability

in water and light, w-octanol/water  partition coefficient, and pKa values
should be known prior to testing.  The results of a biodegradability test
and the method of analysis for the quantification of the chemical in water
should also be known.

     (ii) For chemicals with limited solubility under the test conditions,
it may not be possible to determine an EC50.  If it is observed  that the
stability or homogeneity of the test chemical cannot be maintained,  then
care should be taken in the interpretation of the results and a note made
that these results may not be reproducible.

     (iii) Test chambers are filled with appropriate volumes of  dilution
water.  The  flow  of dilution water through each chamber  is adjusted to
the rate desired.  The test chemical  is introduced into  each test chamber
and the flow-rate adjusted to establish and maintain the desired concentra-
tion  in each test  chamber. Test oysters, which have been acclimated and
prepared by grinding away a portion of the shell periphery, are randomly
introduced into the test and control chambers. Oysters in the test and  con-
trol chambers are observed daily during the test for evidence of feeding
or unusual conditions, such as shell gaping, excessive mucus production
or formation of fungal growths in the test chambers. The observations are
recorded and dead oysters removed. At the end of 96 h the increments
of new shell growth are  measured in all oysters.  The concentration-re-
sponse curve and EC50 value for  the test  chemical are developed from
these data.

     (2) Range-finding test. A range-finding test should be conducted to
establish test chemical concentrations for the definitive  test. The test is
conducted in the same way as the  definitive test except  a widely spaced
chemical concentration series (i.e. log-interval) is used.

     (3) Definitive test, (i)  Oysters which meet condition  criteria (age,
size, reproductive status, health) and which have been acclimated to test
conditions should have approximately 3 to  5 mm of the shell periphery,
at the rounded (ventral) end, ground away with a small electric disc grinder
or other appropriate device, taking care to remove the shell  rim uniformly
to produce a smooth,  rounded, blunt profile. The oyster's  valves should
be held together  tightly during grinding to  avoid vibrating the shell and
injuring the adductor muscle.  Oysters from which so  much of the shell
rim has been removed that an opening into the shell cavity is visible should
not be used.

     (ii) It is desirable to have shell growth values for the low and  high
concentrations relatively close to, but different from, 0 and 100  percent.
Therefore, the range of concentrations to which the  oysters are  exposed
should be such that in  96 h relative to the controls, very little shell growth
occurs in oysters  exposed to the highest concentration and shell growth
is  slightly less than controls  at the lowest  concentration. Oysters in the

remaining concentrations should have increments of shell growth such that
the concentration producing 50 percent shell growth relative to the growth
is bracketed with at least one concentration above and one below it.

     (iii) The test should be carried out without adjustment of pH unless
there is evidence of marked change in the pH of the solution.  In this  case,
it is advised that the test be repeated with pH adjustment to that of the
dilution water and the results reported.

     (iv) The test begins when at least 20 prepared oysters are placed in
each of the test chambers containing the appropriate concentrations of test
substance and controls. The  steady-state flows and test chemical concentra-
tions should be documented.  At  least five test chemical concentrations
should be used.  The dilution factor between concentrations should not ex-
ceed 1.8.

     (v) Test oysters should  be impartially distributed  among test chambers
in such a manner that test results show no significant  bias from the dis-
tributions. The oysters should be spread out equidistantly from one another
so that the entire test chamber is used. The oysters should also be placed
with the left (cupped) valve  down and the open, unhinged ends all oriented
in the same direction facing  the incoming flow of test  solution.

     (vi) The oysters are inspected at least after 24,  48, 72, and 96 h.
Oysters are  considered  dead if touching of the gaping shell produces no
reaction. Dead oysters are removed when observed and mortalities  are re-
corded. Observations at  3 h and 6 h are also desirable.

     (vii)  Shell growth  is the primary criterion used  in this test guideline
to evaluate the toxicity of the test chemical. Shell growth increments in
all  oysters should be measured after 96-h exposure.  Record the  length
of the longest "finger"  of new shell growth to the nearest 0.1 mm. Oysters
should be handled very gently  at this stage to prevent damage to the new
shell growth.

     (viii) Records should be kept of visible abnormalities such as loss
of feeding activity (failure to  deposit feces), excessive mucus production
(stringy material floating  suspended  from oysters),  spawning, or appear-
ance of shell (closure or gaping).

     (ix) The criteria for a valid definitive test are:

     (A) The mortality  in  the  controls should not exceed 10 percent at
the end of the test.

     (B) The dissolved oxygen concentration should be  at least 60 percent
of air saturation throughout the test.

     (C) If evidence of  spawning is observed, the test should  be repeated.

     (D) There should be evidence that the concentration of the substance
being tested has been satisfactorily maintained over the test period.  The
concentration of the test substance should be measured:

     (7) In each chamber at time 0-h.

     (2) In each chamber at 96-h; and
        In at least one  appropriate chamber whenever a malfunction is
detected in any part of the test chemical delivery system.

     (E) Dissolved oxygen, temperature,  salinity, and  pH measurements
should be made at the beginning and end of the test in each chamber.

     (F) A minimum of 2 mm of new shell  growth should be observed
in control oysters (solvent and dilution water).

     (4) Test results, (i)  At the end of the test, appropriate statistical anal-
ysis  should be conducted on the oyster  shell deposition test data.  The
probit transformation should  then be applied to the response variable and
then regressed, using least squares regression, on  dose or log-dose.  An
F Test for linearity should be conducted to determine whether the chosen
regression technique adequately describes the experimental data.

     (ii) Calculate the ratio of the  mean  shell growth  for each group  of
test oysters  (exposed to  each of the test  chemical  concentrations) to  the
mean shell growth of the group  of control oysters. From these  data  the
concentration-response curve is  drawn  and an EC50  along  with  the
95 percent confidence limits on the value  are determined from the curves.
The mean measured concentration of test  chemical should be used to cal-
culate the EC50 and to plot the concentration-response curve.

     (e) Test conditions — (1) Test species — (i) Selection. (A) The Eastern
oyster, Crassostrea virginica, should be used as the test organism.

     (B) Oysters used in the same  test should be 30 to 50 mm  in valve
height  and should be as  similar in age and/or size as possible to reduce
variability. The standard  deviation of the valve height should be less than
20 percent of the mean.

     (C) Oysters used in the same test should be from the  same source
and from the same holding and acclimation tanks.

     (D) Oysters should  be in a prespawn condition of gonadal develop-
ment prior to and during the test as determined by direct or histological
observation of the gonadal tissue for the presence of gametes.

     (ii) Acquisition. Oysters may be cultured in the laboratory, purchased
from culture facilities or commercial harvesters, or collected from a natural
population in an unpolluted area free from  epizootic disease.

     (iii) Acclimation.  (A) Oysters  should  be attended to immediately
upon arrival. Oyster shells should be brushed clean of fouling organisms
and the transfer of the oysters to the holding water should be gradual to
reduce stress  caused by differences  in water  quality characteristics and
temperature. Oysters should be held for at least 12 to 15 days before test-
ing. All oysters should be maintained in dilution water at the test tempera-
ture for at least 2 days before they are used.

     (B) During holding, the oysters  should not be crowded, and the dis-
solved oxygen concentration should be above  60 percent  saturation. The
temperature of the holding water should be the same as that used for test-
ing. Holding tanks should be kept clean and free of debris. Cultured algae
may be added to dilution water sparingly, as  necessary to support life and
growth and such that test results are not affected as confirmed by previous

     (C) Oysters should be handled as little as possible. When handling
is necessary, it should be done as gently, carefully, and quickly as possible.

     (D) A batch of oysters is  acceptable for testing if the percentage mor-
tality over the 7-day period prior to  testing is less than 5 percent. If the
mortality  is between 5 and 10 percent, acclimation  should continue for
7 additional days. If the  mortality is greater than 10 percent,  the entire
batch of oysters should be rejected. Oysters which appear diseased or oth-
erwise stressed or which  have cracked,  chipped,  bored, or  gaping shells
should not be used. Oysters infested with mudworms (Polydora sp.)  or
boring sponges (Cilona cellata) should not be used.

     (2) Test facilities—(i) Apparatus. (A) In addition to normal labora-
tory  equipment,  an oxygen meter, equipment  for delivering the test chemi-
cal, adequate  apparatus for temperature control, and test  tanks made  of
chemically inert material are needed.

     (B) Constant conditions in the test facilities should be maintained as
much as possible throughout the test. The preparation and storage of the
test material, the holding of the oysters and all  operations and tests should
be carried out in an environment free  from harmful concentrations of dust,
vapors and gases and in such a way as to avoid cross-contamination. Any
disturbances that may change the behavior of the oysters should be avoid-

     (ii) Dilution water. A constant supply of good quality unfiltered sea-
water should be available  throughout  the holding, acclimation, and testing
periods. Natural seawater is  recommended, although  artificial seawater
with food  added may be  used. In either case, to ensure each oyster is
provided equal amounts of food, the water should come from a thoroughly
mixed common source  and should be delivered at a flowrate  of at least
1 and preferably 5 L/h per oyster. The flowrate should be ±10  percent
of the nominal flow. A dilution water is  acceptable if oysters will survive

and grow normally for 14 days without exhibiting signs of stress; i.e.  ex-
cessive  mucus production (stringy material floating  suspended from oys-
ters), lack of feeding, shell gaping, poor shell closing in response to prod-
ding, or excessive mortality. The dilution water should have a salinity in
excess of 12 ppt,  and should be similar to that in the  environment from
which the test oysters originated. A natural seawater  should have a weekly
range in salinity of less  than 10 ppt and a monthly range in pH of less
than 0.8 unit. Artificial seawater salinity should not  vary more than 2  ppt
nor more than 0.5  pH unit. Oysters should be tested in dilution water from
the same origin.

    (3) Test parameters—(i) Carriers. Stock solutions of substances of
low aqueous solubility may be prepared by ultrasonic dispersion or, if nec-
essary, by use of organic solvents, emulsifiers or dispersants of low tox-
icity to oysters.  When such carriers  are used the control oysters  should
be exposed  to the  same concentration of  the carrier as that used in  the
highest  concentration of the test substance. The concentration of such car-
riers should  not exceed 0.1 mL/L.

    (ii) Dissolved oxygen. The  dissolved oxygen concentrations  should
be at least 60 percent of the saturation value and should be recorded daily.

    (iii) Loading. The loading rate  should not crowd oysters and should
permit adequate  circulation of water while avoiding physical agitation of
oysters by water current.

    (iv) Temperature. The test temperature should  be  20 °C. Temporary
fluctuations  (less than 8 h) within ±5 °C are  permissible. Temperature
should be recorded continuously.

    (v) pH. The  pH should be measured at the beginning  and end of
the test in each test chamber.

    (f) Reporting. In addition to the reporting  requirements as specified
under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart
J, the following specific information should be reported:

    (1) The source of the dilution water, the  mean, standard deviation
and range of the salinity, pH, temperature, and dissolved oxygen during
the test period.

    (2) A description of the  test procedures used (e.g. the flow-through
system, test  chambers, chemical delivery system, aeration, etc.).

    (3) Detailed information about the oysters used,  including  the  age
and/or  size  (i.e.  height),  source,  history,  method  of confirmation  of
prespawn condition, acclimation procedures, and food used.

    (4)  The  number of  organisms tested,  the  loading rate,  and  the

     (5) The methods of preparation of stock and test solutions, and the
test chemical concentrations used.

     (6) The number of dead and live test  organisms, the percentage of
organisms that died,  and  the  number that showed any abnormal effects
in the control and in each test chamber at each observation period.

     (7) The 96-h shell growth measurements of each oyster;  the mean,
standard deviation and range of the measured shell growth at 96 h of oys-
ters in each concentration of test substance and control.

     (8) The calculated 96-h  EC50 and  its  95 percent  confidence limits
and the  statistical methods used to calculate these values.

     (9) When observed, the 96-h observed no-effect concentration (the
highest  concentration tested at which there were no mortalities, abnormal
behavioral or physiological effects and at which shell growth did not differ
from controls).

     (10) A  graph  of  the concentration-response curve  based on  the
96-h chemical concentration and shell growth measurements upon which
the EC50 was calculated.

     (11) Methods and data records of all chemical analyses of water qual-
ity parameters and test substance concentrations, including method valida-
tions and reagent blanks.

     (12) Any incidents in the course of the test which might have influ-
enced the results.

     (13) A statement that the test was carried out in agreement with the
prescriptions of the  test guideline given  above (otherwise a description
of any deviations occurring).