United States Prevention, Pesticides EPA712-C-96-127
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1710
Oyster BCF
'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 850.1710 Oyster BCF.
(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 materials used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1830 Oyster
Bioconcentration Test and OPP 72-6 Aquatic Organism Accumulation
Tests (Pesticide Assessment Guidelines, Subdivision E—Hazard Evalua-
tion; Wildlife and Aquatic Organisms) EPA report 540/09-82-024, 1982.
(b) Purpose. This guideline is to be used for assessing the propensity
of chemical substances to bioconcentrate in tissues of estuarine and marine
molluscs. This guideline describes a bioconcentration test procedure for
the continuous exposure of Eastern oysters (Crassostrea virginicd) to a
test substance in a flow-through system. EPA will use data from this test
in assessing the hazard a chemical or pesticide may present to the environ-
ment.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and in 40 CFR Part 792—Good Laboratory Practice
Standards are applicable to this test guideline. The following definitions
also apply:
(1) Acclimation is the physiological compensation by test organisms
to new environmental conditions (e.g. temperature, salinity, pH).
(2) Bioconcentration is the net accumulation of a chemical directly
from water into and onto aquatic organisms.
(3) Bioconcentration factor (BCF) is the quotient of the concentration
of a test chemical in tissues of aquatic organisms at or over a discrete
time period of exposure divided by the concentration of test chemical in
the test water at or during the same time period.
(4) Depuration is the elimination of a test chemical from a test orga-
nism.
(5) Depuration phase is the portion of a bioconcentration test after
the uptake phase during which the organisms are in flowing water to which
no test chemical is added.
(6) 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 period of exposure (which should be stated).
(7) Loading is the ratio of the number of oysters to the volume (liters)
of test solution passing through the test chamber per hour.
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(8) Organic chlorine is the chlorine associated with all chlorine-con-
taining compounds that elute just before lindane to just after mirex during
gas chromatographic analysis using a halogen detector.
(9) Organochlorine pesticides are those pesticides which contain car-
bon and chlorine, such as aldrin, DDD, DDE, DDT, dieldrin, endrin, and
heptachlor.
(10) Steady-state is the time period during which the amounts of test
chemical being taken up and depurated by the test oysters are equal, i.e.
equilibrium.
(11) Steady-state bioconcentration factor is the mean concentration
of the test chemical in test organisms during steady-state divided by the
mean concentration of the test chemical in the test solution during the
same period.
(12) Stock solution is the concentrated solution of the test substance
which is dissolved and introduced into the dilution water.
(13) Test chamber is the container in which the test oysters are main-
tained during the test period.
(14) Test solution is dilution water containing the dissolved test sub-
stance to which test organisms are exposed.
(15) Umbo is the narrow end (apex) of the oyster shell.
(16) Uptake is the sorption of a test chemical into and onto aquatic
organisms during exposure.
(17) Uptake phase is the initial portion of a bioconcentration test dur-
ing which the organisms are exposed to the test solution.
(18) Valve height is 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. Oysters are continu-
ously exposed to a minimum of one constant, sublethal concentration of
a test chemical under flow-through conditions for a maximum of 28 days.
During this time, test solution and oysters are periodically sampled and
analyzed using appropriate methods to quantify the test chemical con-
centration. If, prior to day 28, the tissue concentrations of the chemical
sampled over three consecutive sampling periods have been shown to be
statistically similar (i.e. steady-state has been reached), the uptake phase
of the test is terminated, and the remaining oysters are transferred to un-
treated flowing water until 95 percent of the accumulated chemical resi-
dues have been eliminated, or for a maximum depuration period of 14
days. The mean test chemical concentration in the oysters at steady-state
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is divided by the mean test solution concentration at the same time to
determine the bioconcentration factor (BCF). If steady-state is not reached
during 28 days of uptake, the steady-state BCF should be calculated using
non-linear parameter estimation methods.
(2) [Reserved]
(3) Range-finding test. The oyster acute toxicity test is used to deter-
mine the concentration levels to be used in the oyster bioconcentration
test.
(4) Definitive test, (i) The following data on the test chemical should
be known prior to testing:
(A) Solubility in water.
(B) Stability in water.
(C) Octanol-water partition coefficient.
(D) Acute toxicity (e.g. propensity to inhibit shell deposition) to oys-
ters.
(E) The validity, accuracy, minimum detection, and minimum quan-
tification limits of selected analytical methods.
(ii) At least two concentrations should be tested to assess the propen-
sity of the compound to bioconcentrate. The concentrations selected should
not stress or adversely affect the oysters and should be less than one-
tenth the EC50 or
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estimate should also be used to designate a sampling schedule. The uptake
phase should continue until steady-state has been reached. The uptake
phase should continue for at least 4 days, but need not be longer than
28 days.
(A) The time to steady-state (S in hours) can be estimated from the
water solubility of the octanol-water partition coefficient for chemicals
whose uptake and depuration follow a two-compartment, two-parameter
model (ASTM, 1986, under paragraph (g)(l) of this guideline). The fol-
lowing equations were developed from data on fish but are considered
useful in this test as well:
S = 3.0/antilog(0.431 log W - 2.11)
or
S = 3.0/antilog(-0.414 log P + 0.122)
where
W = water solubility (mg/L)
P = octanol-water partition coefffient
For example, S for a chemical of log P 4.0 would be estimated as
3.0/antilog(-0.414(4.0) + 0.122) = 3.0/0.029 = 103.4 h.
Bioconcentration kinetic studies have also been performed specifically for
molluscs, e.g. as investigated by Hawker and Connell (under paragraph
(g)(2) of this guideline) and these may also be consulted.
(B) The depuration phase should continue until at least 95 percent
of the accumulated test substance and metabolites have been eliminated,
but no longer than 14 days.
(C) Based on the estimate of the time to steady-state, one of the fol-
lowing sampling schemes may be used to generate the appropriate data.
Table—Time to Steady-State in Days
Test Period
Exposure2
S<4
Sampling
days
11
61
1
2
3
4
S>4<14
Sampling
days
41
1
3
7
10
12
14
S>15<21
Sampling
days
1
3
7
10
14
18
22
S>21
Sampling
days
1
3
7
10
14
21
28
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Table—Time to Steady-State in Days—Continued
Test Period
Depuration2
S<4
Sampling
days
11
61
121
1
S>4<14
Sampling
days
1
2
4
6
S>15<21
Sampling
days
1
3
7
10
S>21
Sampling
days
1
3
7
10
14
1 Hours
2 Additional sampling times may be needed to confirm that steady-state has been
attained
(v) The following criteria should be met in order for the test to be
valid:
(A) If it is observed that the stability or homogeneity of the test chem-
ical cannot be maintained in the test solution, care should be taken in
the interpretation of the results and a note should be made that these results
may not be reproducible.
(B) The mortality in the controls should not exceed 10 percent at
the end of the test.
(C) The dissolved oxygen concentration should be >60 percent of
saturation throughout the test.
(D) There should be evidence (using measured test chemical con-
centrations) that the concentration of the chemical being tested has been
satisfactorily maintained over the test period.
(E) If evidence of spawning is observed, the test should be discon-
tinued and later repeated.
(F) Temperature variations from 20 °C should be held to a minimum,
preferably ±2 °C.
(vi) The following methodology should be followed:
(A) The test should not be started until the test chemical delivery
system has been observed to be functioning properly and the test chemical
concentrations have equilibrated (i.e. the concentration does not vary more
than 20 percent). Analyses of two sets of test solution samples taken prior
to test initiation should document this equilibrium. At initiation (time 0),
test solution samples should be collected immediately prior to the addition
of oysters to the test chambers.
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(B) The appropriate number of oysters (see paragraph (d)(4)(vii)(A)
of this guideline) should be brushed clean and should be impartially dis-
tributed among test chambers in such a manner that test results show no
significant bias from the distributions. The number of oysters used in ths
test will depend on the length of the test, number of replicate test chambers
used, and if, in addition to a nonsolvent control, a solvent-control is used.
Also important are the size of each oyster and the size of the test chamber.
For example, in a 28-day test, a minimum of 28 oysters in the uptake
(exposure) phase and an additional 20 oysters in the depuration phase per
test chemical concentration would be needed. These oysters could be dis-
tributed among two or more replicates at each concentration. A minimum
of 48 oysters would be required for each control. The oysters should be
spread out equidistant from one another and placed with the left (cupped)
valve down and the unhinged ends (opposite from umbo) all oriented in
the same direction facing the incoming flow.
(C) Oysters should be exposed to the test chemical during the uptake
phase until steady state has been reached or for a maximum of 28 days.
The uptake phase should be a minimum of at least 4 days. Then the re-
maining oysters should be transferred to untreated flowing water and sam-
pled periodically to determine if depuration of the test chemical occurs.
Every test should include a control consisting of the same dilution water,
conditions, procedures, and oysters from the same group used in the test,
except that none of the test chemical is added. If a carrier is present in
the test chamber, a separate carrier control is required.
(D) Oysters should be observed (and data recorded) at least daily for
feeding activity (deposition of feces) or any unusual conditions such as
excessive mucus production (stringy material floating suspended from oys-
ters), spawning, or appearance of shell (closure or gaping). If gaping is
noted, the oyster(s) should be prodded. Oysters which fail to make any
shell movements when prodded are to be considered dead, and should be
removed promptly with as little disturbance as possible to the test
chamber(s) and remaining live oysters.
(E) For oysters sampled, careful examination of all the tissues should
be made at the time of shucking for any unusual conditions, such as a
watery appearance or differences in color from the controls.
(F) Observations on compound solubility should also be recorded.
These include the appearance of surface slicks, precipitates, or material
adsorbing to the test chamber.
(vii) Sampling. (A) At each of the designated sampling times, trip-
licate water samples and enough oysters should be collected from the test
chamber(s) to allow for tissue analyses of at least four oysters. The con-
centration of test chemical should be determined in a minimum of four
oysters analyzed individually at each sampling period. If individual analy-
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sis is not possible, due to limitations of the sensitivity of the analytical
methods, then pairs, triplicates or more oysters may be pooled to constitute
a sample for measurement. A similar number of control oysters should
also be collected at each sample point, but only those collected at the
first sampling period and weekly thereafter, should be analyzed. Triplicate
control water samples should be collected at the time of test initiation
and weekly thereafter. Test solution samples should be removed from the
approximate center of the water column.
(B) At each sampling period the appropriate numbers of oysters are
removed and treated as follows:
(7) The valve height of each oyster should be measured.
(2) Oysters should be shucked as soon as practical after removal and
should never be refrigerated or frozen in the shell. The shell should be
opened at the hinge, the adductor muscle severed and the top valve re-
moved. The remaining adductor muscle should be severed where it at-
taches to the lower valve and the entire oyster removed.
(3) The shucked oysters should then be drained 3 min, blotted dry,
weighed and analyzed immediately for the test chemical. If analyses are
delayed, the shucked oysters should be wrapped individually in aluminum
foil (for organic analysis) or placed in plastic or glass containers (for metal
analysis) and frozen.
(C) If a radiolabeled test compound is used, a sufficient number of
oysters should also be sampled at termination to permit identification and
quantitation of any major (greater than 10 percent of parent) metabolites
present. It is crucial to determine how much of the activity present in
the oyster is directly attributable to the parent compound, and to correct
the bioconcentration factor appropriately.
(5) Test results (i) Steady-state has been reached when the mean
concentrations of test chemical in whole oyster tissue for three consecutive
sampling periods are statistically similar (F test, P = 0.05). A BCF is then
calculated by dividing the mean tissue residue concentration during steady-
state by the mean test solution concentration during the same period. A
95 percent confidence interval should also be derived from the BCF. This
should be done by calculating the mean oyster tissue concentration at
steady-state (X0 ) and its 97.5 percent confidence interval X0 + t (S.E.)
where t is the t statistic at P = 0.025 and S.E. is the one standard error
of the mean. This calculation would yield lower and upper confidence lim-
its (Lo and Uo). The same procedure should be used to calculate the mean
and 97.5 percent confidence interval for the test solution concentrations
at steady-state, XS + t (S.E.), and the resulting upper and lower confidence
limits (Ls and Us). The 95 percent confidence interval of the BCF would
then be between Lo/Us and Uo/Ls. If steady-state was not reached during
the maximum 28-day uptake period, the maximum BCF should be cal-
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culated using the mean tissue concentration from that and all the previous
sampling days. An uptake rate constant should then be calculated using
appropriate techniques. This rate constant is used to estimate the steady-
state BCF and the time to steady-state.
(ii) If 95 percent elimination has not been observed after 14 days
depuration then a depuration rate constant should also be calculated. This
rate constant should be based on the elimination of the parent compound.
(iii) Oysters used in the same test should be 30 to 50 mm in valve
height and should be as similar in age/size as possible to reduce variability.
The standard deviation of the height should be less than 20 percent of
the mean (N = 30).
(6) Analytical measurements, (i) All samples should be analyzed
using EPA methods and guidelines whenever feasible. The specific meth-
odology used should be validated before the test is initiated. The accuracy
of the method should be measured by the method of known additions.
This involves adding a known amount of the test chemical to three water
samples taken from an aquarium containing dilution water and a number
of oysters equal to that to be used in the test. The nominal concentration
of these samples should be the same as the concentration to be used in
the test. Samples taken on two separate days should be analyzed. The accu-
racy and precision of the analytical method should be checked using ref-
erence or split samples or suitable corroborative methods of analysis. The
accuracy of standard solutions should be checked against other standard
solutions whenever possible.
(ii) An analytical method should not be used if likely degradation
products of the test chemical, such as hydrolysis and oxidation products,
give positive or negative interferences, unless it is shown that such deg-
radation products are not present in the test chambers during the test.
Atomic absorption spectrophotometric methods for metal and gas
chromatographic methods for organic compounds are preferable to colori-
metric methods. Spectrophotometry is also acceptable provided Beer's law
is followed and an acceptable extinction coefficient can be determined.
(iii) In addition to analyzing samples of test solution at least one rea-
gent blank should also be analyzed when a reagent is used in the analysis.
(iv) When radiolabelled test compounds are used, total radioactivity
should be measured in all samples. At the end of the uptake phase, water
and tissue samples should be analyzed using appropriate methodology to
identify and estimate the amount of any major (at least 10 percent of the
parent compound) degradation products or metabolites that may be present.
(e) Test conditions—(1) Test species, (i) The Eastern oyster,
Crassostrea virginica, should be used as the test organism.
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(ii) 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.
(iii) Oysters used in the same test should be from the same source
and from the same holding and acclimation tank(s).
(iv) 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.
(v) 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.
(vi) The holding and acclimation of the oysters should be as follows:
(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 days before testing. All oysters should be
maintained in dilution water at the test temperature 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 waters should be the same as that used for
testing. 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, such that test results are not affected, as confirmed by
previous testing. Oysters should be handled as little as possible. When
handling is necessary, it should be done as gently, carefully, and quickly
as possible.
(C) 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, bared, or gaping shells
should not be used. Oysters infested with mudworms (Polydora sp.) or
boring sponges (Cilona cellata) should not be used.
(2) Facilities—(i) Apparatus. (A) An oxygen meter, dosing equip-
ment for delivering the test chemical, adequate apparatus for temperature
control, test tanks made of chemically inert material and other normal lab-
oratory equipment are needed.
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(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-
ed.
(C) The test chambers should be made from materials that will not
absorb the test substance. Delivery systems and test chambers should be
cleaned before and after each use. If absorption of the test substance oc-
curs, those applicable parts of the delivery system should be discarded.
(D) The test substance delivery system used should accommodate the
physical and chemical properties of the test substance and the selected
exposure concentrations. The apparatus used should accurately and pre-
cisely deliver the appropriate amount of stock solution and dilution (sea)
water to the test chambers. The introduction of the test substance should
be done in such a way as to maximize the homogeneous distribution of
the test substance throughout the test chamber.
(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 (algae) added may be used. In either case, to ensure each oyster
is provided equal amounts of food, the water should come from a thor-
oughly mixed common source and should be delivered at a flow rate of
at least one, 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 over the period in which the test is con-
ducted without exhibiting signs of stress, i.e. excessive mucus production
(stringy material floating suspended from oysters), lack of feeding, shell
gaping, poor shell closing in response to prodding, or excessive mortality.
The dilution water should have a salinity in excess of 12 ppt, and should
be similiar to that in the environment from which the test oysters origi-
nated. 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 units. Artificial
seawater should not vary more than 2 ppt nor more than 0.5 pH units.
Oysters should be tested in dilution water from the same origin. If natural
sea water is used, it should meet the following specifications, measured
at least twice a year.
Substance Concentration
Suspended solids <20 mg/L
Un-ionized ammonia <20 mg/L
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Substance Concentration
Residual chlorine <3 jig/L
Total organophosphorus pesticides <50 jig/L
Total organophosphorus pesticides <50 jig/L
plus RGB's
(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 (100 mg/L).
(ii) Dissolved oxygen. This dissolved oxygen concentration should
be at least 60 percent of the air saturation value and should be measured
daily in each chamber.
(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
excursions (less than 8 h) within + 5 °C are permissible. Temperature
should be recorded continually.
(v) pH. The pH should be measured daily in each test chamber.
(vi) The amount of total organic carbon (TOC) in the dilution water
can affect the bioavailability of some chemicals. Thus, TOC should be
measured daily.
(f) Reporting. In addition to the reporting requirements prescribed
in 40 CFR Part 792—Good Laboratory Practice Standards, the report
should contain the following:
(1) The source of the dilution water, the mean, standard deviation
and range of the salinity, pH, TOC, 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 age, size
(i.e. height), weight (blotted dry), source, history, method of confirmation
of prespawn condition, acclimation procedures, and food used.
11
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(4) The number of organisms tested, sampling schedule, loading rate
and flowrate.
(5) The methods of preparation of stock and test solutions and the
test chemical concentrations used.
(6) The number of dead and live organisms, the percentage of oysters
that died and the number that showed any abnormal effects in the control
and in each test chamber at each observation period.
(7) Methods and data records of all chemical analyses of water quality
parameters and test chemical concentrations, including method validations
and reagent blanks.
(8) Description of sampling, sample storage (if required) and analyt-
ical methods of water and tissue analyses for the test chemical.
(9) The mean, standard deviation and range of the concentration of
test chemical in the test solution and oyster tissue at each sampling period.
(10) The time to steady-state.
(11) The steady-state or maximum BCF and the 95 percent confidence
limits.
(12) The time to 95 percent elimination of accumulated residues of
the test chemical from test oysters.
(13) Any incidents in the course of the test which might have influ-
enced the results.
(14) If the test was not done in accordance with the prescribed condi-
tions and procedures, all deviations should be described in full.
(g) References.
(1) American Society for Testing and Materials. ASTM E 1022-84.
Standard practice for conducting bioconcentration tests with fishes and
saltwater bivalve molluscs. In 1986 Annual Book of ASTM Standards,
vol. 11.04: Pesticides; resource recovery; hazardous substances and oil
spill response; waste disposal; biological effects, pp. 702-725 (1986).
(2) Hawker, D.W. and D.W. Connell, Bioconcentration of lipophilic
compounds by some aquatic organisms, Ecotoxicology and Environmental
Safety 11:184-197(1986).
(3) Schimmel, S.C. and R.L. Garnas, Interlaboratory comparison of
the ASTM bioconcentration test method using the eastern oyster, pp. 277-
287. In R.C. Banner and R.T. Hansen (eds.), Aquatic Toxicology and Haz-
ard Assessment: Eighth Symposium, ASTM STP 891, American Society
for Testing and Materials, Philadelphia, PA (1985).
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