United States       Prevention, Pesticides     EPA712-C-96-127
           Environmental Protection    and Toxic Substances     April 1996
           Agency         (7101)
&EPA    Ecological Effects Test
           OPPTS 850.1710
           Oyster BCF
                 '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 850.1710  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-

     (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-

     (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.

     (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

     (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.

     (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

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

     (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-

     (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 
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)


     S = 3.0/antilog(-0.414 log P + 0.122)


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



                 Table—Time to Steady-State in Days—Continued

Test Period




  1 Hours
  2 Additional sampling times may be needed to confirm that steady-state  has been

           (v) The following  criteria should be met in order for the test to be

           (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.

     (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-

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-

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.


     (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.

       (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-

       (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


             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.


     (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

     (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).

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