United States Prevention, Pesticides EPA712-C-96-129
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1730
Fish 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 850.1730 Fish BCF
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 797.1520 Fish Bioconcentration
Test; OPP guideline 72-6 Aquatic Organism Bioavailability/
Biomagnification/Toxicity Tests (Pesticide Assessment Guidelines, Sub-
division E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA re-
port 540/09-82-024, 1982 and OPP 165-4 Laboratory Studies of Pesticide
Accumulation in Fish (Pesticide Assessment Guidelines, Subdivision N—
Environmental Fate) EPA report 540/09-82-031, 1982; and OECD 305E
Bioaccumulation: Flow-Through Fish Test.
(b) Introduction—(1) Purpose. The purpose of the study is to deter-
mine uptake and depuration rate constants and bioconcentration factors
(BCFs) for fish exposed to a test chemical in aqueous solution. Another
purpose is to identify and quantify major degradates at steady state. BCF
values for the test chemical should always be based on concentrations of
the chemical in fish tissue and exposure water, and not on total
radiolabeled residues. BCFs may be used to help assess risks to the fish
and to nontarget organisms (including humans) above them in the food
chain.
(2) Criteria for performing test. The test is most commonly required
for chemicals that are relatively persistent (stable) in water and have a
relatively high potential for bioaccumulation as indicated by log Pow (log
of the octanol/water partition coefficient) values less than or equal to 1.0.
(3) Criteria for degradate characterization. BCFs based on total
radiolabeled residues in fish tissue and exposure water can be used to help
determine whether major degradates should be identified and quantified.
If the BCF in terms of total radiolabeled residues is greater than or equal
to 1,000, OPP requires that an attempt be made to identify and quantify
pesticide degradates representing greater than or equal to 10 percent of
total residues in fish tissues at steady state. If degradates representing
greater than or equal to 10 percent of total radiolabeled residues in the
fish tissue are identified and quantified, then degradates in the test water
should also be identified and quantified.
(4) Desired information on the test chemical. To determine whether
a BCF test is warranted (see paragraph (b)(2) of this guideline), it is nec-
essary to know aqueous fate characteristics of the test chemical that deter-
mine its persistence in water and its octanol/water partition coefficient.
Aqueous fate characteristics include rates of abiotic hydrolysis,
biodegradation, direct photolysis in natural sunlight, and volatilization
from water. Henry's law constant (approximated by the ratio of the chemi-
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cal's vapor pressure to its solubility in water) is a good indicator of vola-
tilization potential. It is necessary to know the test chemical's solubility
in water to ensure that exposure concentrations do not exceed it. It is also
necessary to know the toxicity of the chemical to test fish to ensure expo-
sure concentrations do not adversely effect them (see paragraph (g)(2) of
this guideline). The purity of the test chemical should be known as well
as its radiopurity if radiolabeled. The structure and radiolabeled positions
should be known. An appropriate analytical method, of known accuracy,
precision, and sensitivity, for the quantification of the substance in the
test solutions and in biological material must be available, together with
details of sample preparation and storage. Analytical detection limit of test
substance in both water and fish tissues should also be known.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
Bioconcentration/bioaccumulation is the increase in concentration of
the test substance in or on an organism (specified tissues thereof) relative
to the concentration of test substance in the surrounding medium.
The bioconcentration factor (BCF or KB) at any time during the up-
take phase of this accumulation test is the concentration of test substance
(expressed in milligrams per gram or parts per million) in/on the fish or
specified tissues thereof, divided by the concentration of the chemical in
the surrounding medium (BCF = Cf/Cw).
The depuration (loss) rate constant (k2) is the numerical value defin-
ing the rate of reduction in the concentration of the test substance in the
test fish (or specified tissues thereof) following the transfer of the test
fish from a medium containing the test substance to a medium free of
that substance (k2 is expressed in day1).
The exposure or uptake phase is the time during which fish are ex-
posed to the test chemical.
Kinetic concentration factors (BCFK) are bioconcentration factors
calculated directly from kinetic rate constants (ki/k2).
The octanol-water partition coefficient (Pow) is the ratio of the solu-
bility of a chemical in w-octanol and water at equilibrium and can also
be expressed as Kow. Log Pow is used as an indication of a chemical's
potential for bioconcentration by aquatic organisms.
A plateau or steady-state is reached when the the plot of yhe con-
centration of test substance in fish (Cf) against time becomes parallel to
the time axis and three successive analyses of Cf made on samples taken
at intervals of at least 2 days are within + 20 percent of each other, and
there are no significant differences among the three sampling periods. At
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least four successive analyses are required when pooled samples are ana-
lyzed. For test substances which are taken up slowly, the intervals would
more appropriately be 7 days.
The postexposure or depuration (loss) phase is the time, following
the transfer of the test fish from a medium containing test substance to
a medium free of that substance, during which the depuration (or the net
loss) of the substance from the test fish (or specified tissue thereof) is
studied.
The steady state bioconcentration factor is found when the BCF does
not change significantly over a prolonged period of time, the concentration
of the test substance in the surrounding medium being constant during
this period of time.
The uptake rate constant (k\) is the numerical value defining the rate
of increase in the concentration of test substance in/on test fish (or speci-
fied tissues thereof) when the fish are exposed to that chemical (ki is
expressed in day *).
(d) Principle of test—(1) Uptake and depuration phase. The test
consists of two phases—the exposure (uptake) and postexposure
(depuration) phases. During the uptake phase, separate groups of fish of
one species are exposed to at least two concentrations of the test substance
until steady state is achieved or to a maximum of 28-60 days (see para-
graph (g)(3) of this guideline). They are then transferred to a medium free
of the test substance for a depuration phase of adequate duration (see para-
graph (g)(4) of this guideline). The concentration of the test substance in/
on the fish (or specified tissue thereof) and in water is followed through
both phases of the test.
(2) Determination of rate constants and BCFs. (i) Concentrations
of the test chemical in fish tissue and water as a function of time through-
out the uptake and depuration phases are used to determine the uptake
(ki) and depuration (k2) rate constants (see paragraph (i)(l) of this guide-
line.
(ii) Both the steady state and kinetic bioconcentration factors should
be calculated (see paragraph (i)(2) of this guideline). The steady state
bioconcentration factor (BCFs) is calculated as the ratio of the concentra-
tion in the fish (Cf) and to that in the water (Cw) at apparent steady-state.
The kinetic bioconcentration factor (BCFK) is calculated as the ratio of
the uptake rate constant (ki) to the depuration rate constant (k2) assuming
first-order kinetics
(iii) At a minimum, BCFs should be computed for the whole fish.
Whenever possible, they should also be calculated for edible and nonedible
tissue. BCFs should be related to both the weight and lipid content of
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the fish. If first-order kinetics are obviously not obeyed, more complex
models should be employed under paragraph (d)(2)(iv) of this guideline.
(iv) Model discrimination. Most bioconcentration data have been as-
sumed to be reasonably well described by a simple two-compartment/two-
parameter model, as indicated by the rectilinear curve which approximates
to the points for concentrations in fish, during the depuration phase, when
these are plotted on semilog paper. (Where these points cannot be de-
scribed by a rectilinear curve then more complex models should be em-
ployed, see paragraph (k)(20) of this guideline.)
(A) Graphical method for determination of depuration (loss) rate con-
stant k2.
Plot the concentration of the test substance found in each sample of
fish against sampling time on semilog paper. The slope of that line is
k2.
k2 = ln(Cn/Cf2)/(t2-ti)
Note that deviations from a straight line may indicate a more complex
depuration pattern than first order kinetics. A graphical method may be
applied for resolving types of depuration deviating from first order kinet-
ics.
(B) Graphical method for determination of uptake rate constant ki.
Given k2, calculate ki as follows:
Equation 1
ki = Cfk2/Cw Tlx (1 - e-k2t)
The value of Cf is read from the midpoint of the smooth uptake curve
produced by the data when log concentration is plotted versus time (on
an arithmetical scale).
(C) Computer method for calculation of uptake and depuration (loss)
rate constants.
The preferred means for obtaining the bioconcentration factor and ki
and k2 rate constants is to use nonlinear parameter estimation methods
on a computer. These programs find values for ki and k2 given a set of
sequential time concentration data and the model:
Equation 2
Cf = Cw x ki/k2 x (1 - e-k2t) 0 < t < tc
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Equation 3
Cf = Cw x ki/k2 x (e-k2(t -tc) - ek-2t t > tc
where tc = time at the end of the uptake phase.
This approach provides standard deviation estimates of ki and k2.
(D) As k2 in most cases can be estimated from the depuration curve
with relatively high precision, and because a strong correlation exists be-
tween the two parameters ki and k2 if estimated simultaneously, it may
be advisable first to calculate k2 from the depuration data only, and subse-
quently calculate ki from the uptake data using nonlinear regression.
(e) Materials—(1) Exposure tanks and tubes. Care should be taken
to avoid the use of materials, for all parts of the equipment, that can dis-
solve, sorb or leach and have an adverse effect on the fish. Standard rec-
tangular or cylindrical tanks, made of chemically inert material and of a
suitable capacity in compliance with loading rate (see paragraph (e)(7) of
this guideline), can be used. The use of soft plastic tubing should be mini-
mized. Use Teflon, stainless steel and/or glass tubing. Experience has
shown that for substances with high adsorption coefficients, such as the
synthetic pyrethroids, silanized glass may be required. In these situations
the equipment will have to be discarded after use.
(2) Diluter. For flow-through tests, a system which continuously dis-
penses and dilutes a stock solution of the test substance (e.g. metering
pump, proportional diluter, saturator system) is required to deliver the test
concentrations to the test chambers. Preferably allow at least five volume
replacements through each test chamber per day. The flow rates of stock
solutions and dilution water should be checked both 48 hours before and
then at least daily during the test. Include in this check the determination
of the flow-rate through each test chamber and ensure that it does not
vary by more than 20 percent either within or between chambers. The
flow-through mode is to be preferred, but where this is not possible (e.g.
when the test organisms are adversely affected) a semi-static technique
may be used provided that the validity criteria are satisfied (see paragraph
(g)(ll) of this guideline).
(3) Dilution water, (i) Natural water is generally used in the test
and should be obtained from uncontaminated and uniform quality source.
The dilution water must be of a quality that will allow the survival of
the chosen fish species for the duration of the acclimation and test periods
without them showing any abnormal appearance or behavior. Ideally, it
should be demonstrated that the test species can survive, grow and repro-
duce in the dilution water (e.g. in laboratory culture or a life-cycle toxicity
test). The water should be characterized at least by pH, hardness, total
solids, total organic carbon and, preferably also ammonium, nitrite and
alkalinity and, for marine species, salinity. Although the parameters which
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are important for optimal fish well-being are not fully known, the follow-
ing Table 1. gives recommended maximum concentrations of a number
of parameters for fresh and marine test waters.
Table 1.—Some chemical characteristics of an acceptable dilution water
Substance
Limit con-
centration
Participate matter 5 mg/L
Total organic carbon 2 mg/L
Un-ionized ammonia 1 mg/L
Residual chlorine 10 mg/L
Total organophosphorus pesticides 50 ng/L
Total organochlorine pesticides 50 ng/L
plus polychlorinated biphenyls 25 ng/L
Total organic chlorine 1 jig/L
Aluminium 1 jig/L
Arsenic 1 jig/L
Chromium 1 jig/L
Cobalt 1 ng/L
Copper 1 |ig/L
Iron 1 jig/L
Lead 1 jig/L
Nickel 1 jig/L
Zinc 1 jig/L
Cadmium 100 ng/L
Mercury 100 ng/L
Silver 100 ng/L
(ii) The water should be of constant quality during the period of a
test. The pH value should be within the range 6.0 to 8.5, but during a
given test it should be within a range of ±0.5 pH units. In order to ensure
that the dilution water will not unduly influence the test result (for exam-
ple, by complexation of the test substance) or adversely affect the perform-
ance of the stock of fish, samples should be taken at intervals for analysis.
Determination of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions
and cations (e.g. Ca, Mg, Na, K, Cl, SCU), pesticides (e.g. total
organophosphorus and total organochlorine pesticides), total organic car-
bon and suspended solids should be made, for example, every 3 months
where a dilution water is known to be relatively constant in quality. If
water quality has been demonstrated to be constant over at least 1 year,
determinations can be less frequent and intervals extended (e.g. every 6
months).
(iii) The natural particle content as well as the total organic carbon
(TOC) of the dilution water should be as low as possible to avoid adsorp-
tion of the test substance to organic matter which may reduce its
bioavailability. The maximum acceptable value is 5 mg/L for particulate
matter (dry matter, not passing a 0.45 (im filter) and 2 mg/L for total
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organic carbon. If necessary, the water should be filtered before use. The
contribution to the organic carbon content in water from the test fish (ex-
creta) and from the food residues should be as low as possible. Throughout
the test, the concentration of organic carbon in the test vessels should not
exceed the concentration of organic carbon originating from the test sub-
stance and, if used, the solubilizing agent by more than 10 mg/L
(±20 percent).
(4) Test chemical. Whether radiolabeled or not, the chemical purity
of the test chemical should be as high as practical (preferably greater than
or equal to 98 percent). If radiolabeled, the radiopurity should be greater
than or equal to 95 percent.
(5) Test chemical stock solutions. Prepare a stock solution of the
test substance at a suitable concentration. The stock solution should pref-
erably be prepared by simply mixing or agitating the test substance in
the dilution water. The use of solvents or dispersant (solubilizing agents)
is not recommended; however this may occur in some cases in order to
produce a suitably concentrated stock solution. Solvents which may be
used are, ethanol, methanol, ethylene glycol monomethyl ether, ethylene
glycol dimethyl ether, dimethylformamide and triethylene glycol. Dispers-
ant which may be used are Cremophor RH40, Tween 80, methylcellulose
0.01 percent and HCO-40. Care should be taken when using readily bio-
degradable agents as these can cause problems with bacterial growth in
flow-through tests.
(6) Test species, (i) Important criteria in the selection of species are
that they are readily available, can be obtained in convenient sizes and
can be satisfactorily maintained in the laboratory. Other criteria for select-
ing fish species include recreational, commercial, ecological importance
as well as comparable sensitivity, past successful use etc. Recommended
test species and test conditions are given in the following Table 2. Other
species may be used but the test procedure may have to be adapted to
provide suitable test conditions. The rationale for the selection of the spe-
cies and the experimental method should be reported in this case.
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Table 2.—Fish species recommended for testing
Species
Danio rerio 1 (Teleostei, Cyprinidae) (Hamilton-Bu-
chanan) Zebra-fish
Pimephales promelas (Teleostei, Cyprinidae)
(Rafinesque) Fathead minnow
Cyprinus carpio (Teleostei, Cyprinidae) (Linnaeus) Com-
mon carp
Oryzias latipes (Teleostei, Poecilliidae) (Temminck and
Schlegel) Ricefish
Poecilia reticulata (Teleostei, Poeciliidae) (Peters) Guppy
Lepomis macrochirus (Teleostei Centrarchidae)
(Rafinesque) Bluegill
Oncorhynchus mykiss (Teleostei Salmonidae (Walbaum)
Rainbow trout
Gasterosteus aculeatus (Teleostei, (Gasterosteidae) (Lin-
naeus) Three-seined stickleback
Test tem-
perature
(°C)
20-25
20-25
20-25
20-25
20-25
20-25
13-17
18-20
Total length
of test ani-
mal
(cm)
3.0±0.5
5.0±2.0
5.0±3.0
4.0±1.0
3.0±1.0
5.0±2.0
8.0±4.0
3.0+1.0
1 Meyer A. and G. Orti. Proceedings of the Royal Society of London 252 (Series
B):231 (1993).
(ii) Various estuarine and marine species have been used in different
countries, for example: Spot (Leiostomus xanthurus}; Sheepshead minnow
(Cyprinodon variegatus); Silverside (Menidia beryllind); Shiner perch
(Cymatogaster aggregata); English sole (Parophrys vetulus); Staghorn
sculpin (Leptocottus armatus); Three-spined stickleback (Gasterosteus
aculeatus}; Sea bass (Dicentracus labrax); Bleak (Alburnus alburnus)
(iii) The fresh water fish listed are easy to rear and/or are widely
available throughout the year, whereas the availability of marine and estua-
rine species is partially confined to the respective countries. They are capa-
ble of being bred and cultivated either in fish farms or in the laboratory,
under disease-and parasite-controlled conditions, so that the test animal
will be healthy and of known parentage. These fish are available in many
parts of the world.
(7) Reference chemicals. The use of reference compounds of known
bioconcentration potential would be useful in checking the experimental
procedure, when required. However, specific substances cannot yet be rec-
ommended.
(f) Fish care and health—(1) Acclimation. Acclimate the stock pop-
ulation of fish for at least 2 weeks in water at the test temperature and
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feed throughout on a sufficient diet (see paragraph (f)(3) of this guideline)
and of the same type to be used during the test.
(2) Pretest mortality and health, (i) Following a 48-h settling-in
period (during acclimation), mortalities are recorded and the following cri-
teria applied:
(A) Mortalities of greater than 10 percent of population in 7 days,
reject the entire batch.
(B) Mortalities of between 5 and 10 percent of population in 7 days,
acclimate for 7 additional days.
(C) Mortalities of less than 5 percent of population in 7 days, accept
the batch. If more than 5 percent mortality during the second 7 days, reject
the entire batch.
(ii) Ensure that fish used in tests are free from observable diseases
and abnormalities. Discard any diseased fish. Fish should not receive treat-
ment for disease in the two weeks preceding the test, or during the test.
(3) Feeding, (i) During the acclimation and test periods, feed an ap-
propriate diet of known lipid and total protein content to the fish in an
amount sufficient to keep them in a healthy condition and to maintain
body weight. Feed daily throughout the acclimation and test periods at
a level of approximately 1 to 2 percent of body weight per day; this keeps
the lipid concentration in most species of fish at a relatively constant level
during the test. The amount of feed should be recalculated, for example,
once per week, in order to maintain consistent body weight and lipid con-
tent. For this calculation, the weight of the fish in each test chamber can
be estimated from the weight of the fish sampled most recently in that
chamber. Do not weigh the fish remaining in the chamber.
(ii) Siphon uneaten food and faeces daily from the test chambers
shortly after feeding (30 min to 1 h). Keep the chambers as clean as pos-
sible throughout the test so that the concentration of organic matter is kept
as low as possible (see paragraph (e)(3), since the presence of organic
carbon may limit the bioavailability of the test substance under paragraph
(k)(6) of this guideline.
(iii) Since many feeds are derived from fishmeal, the feed should be
analyzed for the test substance. It is also desirable to analyze the feed
for pesticides and heavy metals.
(g) Exposure conditions during test—(1) Optional preliminary
test to determine optimal conditions. It may be useful to conduct a pre-
liminary experiment in order to optimize the test conditions of the defini-
tive test, e.g. selection of test substance concentrations, duration of the
uptake and depuration phases.
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(2) Exposure concentrations of test chemical, (i) During the uptake
phase, expose fish under flow-through conditions to at least two concentra-
tions of the test substance in water. Normally, select the higher (or highest)
concentration of the test substance to be about 1 percent of its acute as-
ymptotic LC50, and to be at least tenfold higher than its detection limit
in water by the analytical method used. The highest test concentration can
also be determined by dividing the acute 96-h LC50 by an appropriate
acute/chronic ratio (e.g. appropriate ratios for some chemicals are about
3, but a few are above 100). If possible, choose the other concentrations
such that it differs from the one above by a factor of 10. If this is not
possible because of the 1 percent of LC50 criterion and the analytical limit,
a lower factor than 10 can be used or the use of 14C labeled test substance
should be considered.
(ii) No exposure concentration used should be above the solubility
in water of the test substance.
(iii) Where a solubilizing agent is used in the stock solution, its di-
luted concentration in the exposure water should not be greater than
0.1 mL/L and should be the same in all test vessels. Its contribution (to-
gether with the test substance) to the overall content of organic carbon
in the test water should be known. However, every effort should be made
to avoid the use of such materials.
(iv) Minimize results reported as ' 'not detected at the limit of detec-
tion' ' by pretest method development and experimental design, since such
results cannot be used for rate constant calculations. Pretest results can
be used to determine the exposure concentrations necessary to ensure that
concentrations in fish tissue are generally above method detection limits.
(3) Duration of uptake phase, (i) A prediction of the duration of
the uptake phase and time required to reach steady state can be obtained
from practical experience (e.g. from a previous study or an accumulation
study on a structurally related chemical) or from certain empirical relation-
ships utilizing knowledge of either the solubility in water or the octanol/
water partition coefficient of the test substance (see paragraph (g)(5) of
this guideline).
(ii) The uptake phase should be run for 28 days unless it can be
demonstrated that equilibrium has been reached earlier. If the steady-state
has not been reached by 28 days, the uptake phase should be extended,
taking further measurements, until steady-state is reached or 60 days,
whichever is shorter. The depuration phase is then begun.
(4) Duration of depuration phase, (i) The depuration period is
begun by transferring the fish to the same medium but without the test
substance in another clean vessel. A depuration phase is always necessary
unless uptake of the substance during the uptake phase has been insignifi-
cant (e.g. the BCF is less than 10).
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(ii) A period of half the duration of the uptake phase is usually suffi-
cient for an appropriate (e.g. 95 percent) reduction in the body burden
of the substance to occur (see paragraph (g)(5) of this guideline for an
explanation of the estimation). If the time required to reach 95 percent
loss is unpractically long, exceeding for example twice the normal duration
of the uptake phase (i.e. more than 56 days) a shorter period may be used
(e.g. until the concentration of test substance is less than 10 percent of
steady-state concentration). However, for substances having more complex
patterns of uptake and depuration than are represented by a one-compart-
ment fish model, yielding first order kinetics, allow longer depuration
phases for determination of loss rate constants. The period may, however,
be governed by the period over which the concentration of test substance
in the fish remains above the analytical detection limit.
(5) Prediction of the duration of the uptake and depuration
phases — (i) Prediction of the duration of the uptake phase. (A) Before
performing the test, an estimate of k2 and hence some percentage of the
time needed to reach steady-state may be obtained from empirical relation-
ships between k2 and the w-octanol/water partition coefficient (Pow) or k2
and the aqueous solubilities.
(B) (7) An estimate of k2 (day-1) may be obtained from the following
empirical relationship (see paragraph (k)(20) of this guideline):
Equation 1
log k2 = -0.414 log Pow + 1.47(r2 = 0.95)
For other relationships see see paragraph (k)(14) of this guideline.
(2) If the partition coefficient (Pow) is not known, an estimate can
be made (see paragraph (k)(4) of this guideline) from a knowledge of the
aqueous solubility (s) of the substance using:
Equation 2
log Pow = 0.862 log(s) + 0.710(r2 = 0.994)
where s = solubility expressed as moles per liter: (n=36)
(3) These relationships apply only to chemicals with log Pow values
between 2 and 6.5 (see paragraph (k)(12) of this guideline).
The time to reach some percentage of steady-state may be obtained
by applying the k2-estimate, from the general kinetic equation describing
uptake and depuration (first-order kinetics):
dCf/dt = kiCw - k2Cf
or, if Cw is constant:
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Equation 3
Cf=k1/k2-Cw(l-(exp)k2t
When steady-state is approached (as t approaches infinity), equation
3 may be reduced (see paragraphs (k)(3) and (k)(9) of this guideline) to:
Cf = ki/k2Cw
or
Cf/Cw = ki/k2 = BCF
Then ki/k2-Cw is an approach to the concentration in the fish at steady-
state (Cf,s). Equation 3 may be transcribed to:
Cf=Cf,s(l-(exp)-k2t
or
Equation 4
Cf/Cf,s = 1 - e-k2t
Applying equation 4, the time to reach some percentage of steady-state
may be predicted when k2 is preestimated using equation 1 or 2.
As a guideline, the statistically optimal duration of the uptake phase
for the production of statistically acceptable data (BCFK) is that period
which is required for the curve of the logarithm of the concentration of
the test substance in fish plotted against linear time to reach its midpoint,
or 1.6/k2, or 80 percent of steady-state but not more than 3.0/k2 or 95
percent of steady-state (see paragraph (k)(19) of this guideline).
The time to reach 80 percent of steady-state is (equation 4):
0.8 = 1 - e k2t
or
Equation 5
tso = 1.6/k2
Similarly 95 percent of steady-state is:
Equation 6
t9s - 3.0/k2
For example, the duration of the uptake phase (up) for a test substance
with log POW = 4 would be (using equations 1, 5, and 6):
12
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Iogk2 = -0.414.(4)+ 1.47
k2 = 0.652 days-1
up (80 pct)= 1.6/0.652 i.e. 2.45 days (59 h)
or
up (95 pct)= 3.0/0.652 i.e. 4.60 days (110 h)
Similarly, for a test substance with s = 10 5 mol/L, (log(s) = - 5.0),
the duration of up would be (using equations 1, 2 and 5, 6):
log (Pow) = -0.862 (-5.0) + 0.710 = 5.02
log k2 =-0.414 (5.02)+ 1.47
k2 = 0.246 days !
up (80 pet) = 1.6/0.246, i.e. 6.5 days (156 hours)
or
up (95 pet) = 3.0/0.246, i.e. 12.2 days (293 hours)
Alternatively, the expression:
teq = 6.54 x 10 3POW + 55.31 (hours)
may be used to calculate the time for effective steady-state to be reached
(see paragraph (k)(12) of this guideline).
(ii) Prediction of the duration of the depuration phase. (A) A pre-
diction of the time needed to reduce the body burden to some percentage
of the initial concentration may also be obtained from the general equation
describing uptake and depuration (first order kinetics) (see paragraphs
(k)(13) and (k)(20) of this guideline.
For the depuration phase, Cw is assumed to be zero. The equation
may then be reduced to:
dCf/dt = -k2Cf
or
Cf=Cf,o(exp)-k2t
where Cf=o is the concentration at the start of the depuration period.
50 percent depuration will then be reached at the time (tso):
or
Similarly 95 percent depuration will be reached at:
13
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t95 = 3.0/k2
If 80 percent uptake is used for the first period (1.6/k2) and 95 percent
loss in the depuration phase (3.0/k2), then depuration phase is approxi-
mately twice the duration of the uptake phase.
It is important to note, however, that the estimations are based on
the assumption that uptake and depuration patterns will follow first order
kinetics. If first order kinetics are obviously not obeyed, more complex
models should be employed (e.g. paragraph (k)(16) of this guideline).
(6) Numbers and characteristics of test fish, (i) Select the numbers
of fish per test concentration such that a minimum of four fish per sample
are available at each sampling. If greater statistical power is required, more
fish per sample will be necessary.
(ii) If adult fish are used, report whether male or female, or both
are used in the experiment. If both sexes are used, differences in lipid
content between sexes should be documented to be nonsignificant before
the start of the exposure; pooling all male and all female fish may be
necessary.
(iii) In any one test, select fish of similar weight such that the smallest
are no smaller than two-thirds of the weight of the largest. All should
be of the same year-class and come from the same source. Since weight
and age of a fish appear sometimes to have a significant effect on BCF
values (see paragraph (k)(6) of this guideline) record these details accu-
rately. It is recommended that a sub-sample of the stock of fish is weighed
before the test in order to estimate the mean weight (see paragraph (h)(2)
of this guideline).
(7) Loading of fish, (i) Use high water-to-fish ratios in order to mini-
mize the reduction in Cw caused by the addition of the fish at the start
of the test and also to avoid decreases in dissolved oxygen concentration.
It is important that the loading rate is appropriate for the test species used.
In any case, a loading rate of 0.1-1.0 g of fish (wet weight) per liter
of water per day is normally recommended. High loading rates can be
used if it is shown that the required concentration of test substance can
be maintained within ±20 percent limits, and that the concentration of
dissolved oxygen does not fall below 60 percent saturation.
(ii) In choosing appropriate loading regimes, take account of the nor-
mal habitat of the fish species. For example, bottom-living fish may de-
mand a larger bottom area of the aquarium for the same volume of water
than pelagic fish species.
(8) Light and temperature. The photoperiod is usually 12 to 16 h
and the temperature (±2 °C) should be appropriate for the test species
(see Table 3. under paragraph (e)(6)(i) of this guideline). The type and
14
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characteristics of illumination should be known. Caution should be given
to the possible phototransformation of the test substance under the irradia-
tion conditions of the study. Appropriate illumination should be used
avoiding exposure of fish to unnatural photoproducts. In some cases it
may be appropriate to use a filter to screen out UV irradiation below
290 nm.
(9) Water quality measurements. During the test, dissolved oxygen,
TOC, pH and temperature should be measured in all vessels. Total hard-
ness and salinity (if relevant) should be measured in the controls and one
vessel at the higher (or highest) concentration. As a minimum, dissolved
oxygen and salinity (if relevant) should be measured 3 times—at the begin-
ning, around the middle, and end of the uptake period—and once a week
in the depuration period. TOC should be measured at the beginning of
the test (24 h and 48 h prior to test initiation of uptake phase) before
addition of the fish and, at least once a week, during both uptake and
depuration phases. Temperature should be measured daily, pH at the begin-
ning and end of each period and hardness once each test. Temperature
should preferably be monitored continuously in at least one vessel.
(10) Controls. In addition to the two test concentrations, a control
group of fish is held under identical conditions except for the absence
of the test substance, to relate possible adverse effects observed in the
bioconcentration test to a matching control group and to obtain background
concentrations of test substance. One dilution water control and if relevant,
one control containing the solubilizing agent should be run.
(11) Validity of test. For a test to be valid the following conditions
apply:
(i) The temperature variation is less than ±2 °C.
(ii) The concentration of dissolved oxygen does not fall below
60 percent saturation.
(iii) The concentration of the test substance in the chambers is main-
tained within + 20 percent of the mean of the measured values during the
uptake phase.
(iv) The mortality or other adverse effects/disease in both control and
treated fish is less than 10 percent at the end of the test; where the test
is extended over several weeks or months, death or other adverse effects
in both sets of fish should be less than 5 percent per month and not exceed
30 percent in all.
(h) Sampling and analysis of fish and water—(1) Fish and water
sampling schedule, (i) Sample water from the test chambers for the deter-
mination of test substance concentration before addition of the fish and
during both uptake and depuration phases. As a minimum, sample the
15
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water at the same time as the fish and before feeding. During the uptake
phase, the concentrations of test substance are determined in order to check
compliance with the validity criteria (see paragraph (g)(ll) of this guide-
line).
(ii) Sample fish on at least five occasions during the uptake phase
and at least on four occasions during the depuration phase. Since on some
occasions it will be difficult to calculate a reasonably precise estimate of
the BCF value based on this number of samples (especially when other
than simple first-order depuration kinetics are indicated), it may be advis-
able to take samples at a higher frequency in both periods (see the follow-
ing Table 3.) Store the extra samples as described in paragraph (h)(3) and
analyze them only if the results of the first round of analyses prove inad-
equate for the calculation of the BCF with the desired precision.
(iii) An example of an acceptable sampling schedule is given in the
following Table 3.
Table 3.—Theoretical example of sampling schedule for bioconcentration tests of substances
with log Pow = 4
Fish Sampling
Uptake phase
1st
2nd
3rd
4th
5th
Depuration phase
6th
7th
8th
9th
Sample tim
Minimal re-
quired fre-
quency
(days)
-1
0
03
0.3
0.6
1.2
24
47
5.0
59
93
14.0
e schedule
Additional
sampling
(days)
0.9
1 7
33
5.3
70
11 2
17.5
No. of water
samples**
2*
2
2
(2)
2
(2)
2
(2)
2
(2)
2
No. of fish per sample**
Add 45-80 fish
4
(4)
4
(4)
4
(4)
4
(4)
6
Transfer fish to water
free of test chemical
4
(4)
4
(4)
4
(4)
6
(4)
*Sample water after minimum of 3 "chamber-volumes" have been deliv-
ered.
**Values in parentheses are numbers of samples (water, fish) to be taken
if additional sampling is carried out.
16
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Note: Pretest estimate of k2 for log Pow of 4.0 is 0.652 days-1. The total
duration of the experiment is set to 3 x up = 3 x 4.6 days =14 days.
For the estimation of up see paragraph (g)(5) of this guideline.
Other schedules can readily be calculated using other assumed values of
POW to calculate the exposure time for 95 percent uptake.
(iv) Continue sampling during the uptake phase until a steady-state
has been established or for 28 days, whichever is the shorter. If the steady-
state has not been reached within 28 days continue until a steady-state
has been attained or 60 days, whichever is shorter. Before beginning the
depuration phase transfer the fish to clean tanks.
(2) Sampling methodology, (i) Obtain water samples for analysis by
siphoning through inert tubing from a central point in the test chamber.
Since neither filtration nor centrifuging appears always to separate the
nonbioavailable fraction of the test substance from that which is
bioavailable (especially for superlipophilic chemicals, those chemicals with
a log POW greater than or equal to 5) (see paragraphs (k)(6) and (k)(8)
of this guideline), samples may not be subjected to those treatments. In-
stead, measures should be taken to keep the tanks as clean as possible
and the content of total organic carbon should be monitored during both
the uptake and depuration phases (see paragraph (g)(9) of this guideline).
(ii) Remove an appropriate number of fish (normally a minimum of
four) from the test chambers at each sampling time. Rinse the sampled
fish quickly with water, blot dry, kill instantly, using the most appropriate
and humane method, and then weigh.
(3) Sample storage, (i) It is preferable to analyze fish and water im-
mediately after sampling in order to prevent degradation or other losses
and to calculate approximate uptake and depuration rates as the test pro-
ceeds. Immediate analysis also avoids delay in determining when a plateau
has been reached.
(ii) Failing immediate analysis, store the samples by an appropriate
method. Obtain information on the proper method of storage for the par-
ticular test substance before the beginning of the study—for example,
deep-freezing, holding at 4 °C, duration of storage, extraction, etc.
(4) Analysis of fish samples, (i) Radiolabeled test substances can
facilitate the analysis of water and fish samples, and may be used to deter-
mine whether degradate identification and quantification should be made.
BFCs based on total radiolabeled residues (e.g. by combustion or tissue
solubilization) can serve as one of the criteria for determining if degrades
identification and quantification is necessary. However, BCF determina-
tions for the parent compound should be based upon the concentration
of the parent compound in fish and water, not upon total radiolabeled resi-
dues.
17
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(ii) If the BCF in terms of total radiolabeled residues is greater than
or equal to 1,000, it may be advisable, and for certain categories of chemi-
cals such as pesticides strongly recommended, to identify and quantify
degradates representing greater than or equal to 10 percent of total residues
in fish tissues at steady state. If degradates representing greater than or
equal to 10 percent of total radiolabeled residues in the fish tissue are
identified and quantified, then it is also recommended to identify and quan-
tify degradates in the test water. The major metabolites may be character-
ized at steady-state or at the end of the uptake phase, whichever is the
sooner. It is possible to combine a fish metabolism study with a
bioconcentration study to identify and quantify residues in tissues.
(iii) The concentration of the test substance should usually be deter-
mined for each weighed individual fish. If this is not possible, pooling
of the samples on each sampling occasion may be done but pooling does
restrict the statistical procedures which can be applied to the data. If a
specific statistical procedure and power are important considerations, then
an adequate number of fish to accommodate the desired pooling, procedure
and power, should be included in the test. See paragraphs (k)(7) and
(k)(10) of this guideline for an introduction to relevant pooling procedures.
(5) Determination of lipid content. BCF should be expressed both
as a function of total wet weight and, for high lipophilic substances, as
a function of the lipid content. Determine the lipid content of the fish
on each sampling occasion if possible. Suitable methods should be used
for determination of lipid content (see paragraphs (k)(5) and (k)(15) of
this guideline). Chloroform/methanol extraction technique may be rec-
ommended as standard method (see paragraph (k)(ll) of this guideline).
The various methods do not give identical values (see paragraph (k)(18)
of this guideline), so it is important to give details of the method used.
When possible, the analysis for lipid should be made on the same extract
as that produced for analysis for the test substance, since the lipids often
have to be removed from the extract before it can be analyzed
chromatographically. The lipid content of the fish (as mg/kg wet weight)
at the end of the experiment should not differ from that at the start by
more ±25 percent. The tissue percent solids should also be reported to
allow conversion of lipid concentration from a wet to a dry basis.
(6) Quality of analytical method. Since the whole procedure is gov-
erned essentially by the accuracy, precision, and sensitivity of the analyt-
ical method used for the test substance, check the precision and reproduc-
ibility of the chemical analysis experimentally, as well as recovery of the
test substance from both water and fish to ensure that they are satisfactory
for the particular method. Also, check that the test substance is not detect-
able in the dilution water used. If necessary, correct the values of Cw and
Cf obtained from the test for the recoveries and background values of con-
trols. Handle the fish and water samples throughout in such a manner as
18
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to minimize contamination and loss (e.g. resulting from adsorption by the
sampling device).
(i) Data analysis—(1) Determination of uptake and depuration
rate constants, (i) Obtain the uptake and depuration curves of the test
substance by plotting its concentration in/on fish (or specified tissues) in
the uptake and in the depuration phase against time on arithmetic scales.
The depuration rate constant (k2) is usually determined from the depuration
curve (i.e. a plot of the decrease in test substance concentration in the
fish with time). The uptake rate constant (ki) is then calculated given k2
and a value of Cf which is derived from the uptake curve. See paragraph
(d)(2)(iv) of this guideline for a description of these methods. The pre-
ferred method for obtaining BCFK and the rate constants, ki and k2, is
to use nonlinear parameter estimation methods on a computer (see para-
graph (k)(15) of this guideline). Otherwise, graphical methods may be used
to calculate ki and k2. If the depuration curve is obviously not first-order,
then more complex models should be employed (see paragraphs (k)(3),
(k)(4), (k)(9), (k)(12), (k)(13), (k)(14), (k)(19, and (k)(20) of this guide-
line) and advice sought from a biostatistician.
(ii) The uptake rate constant, the depuration (loss) rate constant (or
constants, where more complex models are involved), the bioconcentration
factor, and where possible, the confidence limits of each of these param-
eters are calculated from the model that best describes the measured con-
centrations of test substance in fish and water.
(iii) The results should be interpreted with caution where measured
concentrations of test solutions occur at levels near the detection limit of
the analytical method. Clearly defined uptake and loss curves are an indi-
cation of good quality bioconcentration data. The variation in uptake/
depuration constants between the two test concentrations should be less
than 20 percent. Observed significant differences in uptake/depuration
rates between the two applied test concentrations should be recorded and
possible explanations given. Generally the confidence limit of BCFs from
well-designed studies approach + 20 percent.
(2) Determination of the steady state and kinetic BCFs. (i) Obtain
the uptake curve of the test substance by plotting its concentration in/on
fish (or specified tissues) in the uptake phase against time on arithmetic
scales. If the curve has reached a plateau, that is, become approximately
asymptotic to the time axis, calculate the steady state BCFs from the fol-
lowing relationship:
Cf at steady state (mean)/Cw at steady state (mean)
(ii) When no steady state is reached, it may be possible to calculate
a BCFs of sufficient precision for hazard assessment from a steady-state
at 80 percent (1.6/k2) or 95 percent (3.0/k2) of equilibrium.
19
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(iii) Determine the concentration factor (BCFK) as the ratio ki/k2,
the two first-order kinetic constants.
(iv) The BCF is expressed as a function of the total wet weight of
the fish. However, for special purposes, specified tissues or organs (e.g.
muscle, liver), may be used if the fish are sufficiently large or the fish
may be divided into edible (fillet) and nonedible (viscera) fractions. Since,
for many organic substances, there is a clear relationship between the po-
tential for bioconcentration and lipophilicity, there is also a corresponding
relationship between the lipid content of the test fish and the observed
bioconcentration of such substances. Thus, to reduce this source of varia-
bility in test results for those substances with high lipophilicity (i.e. with
log POW greater than or equal to 3), bioconcentration should be expressed
in relation to lipid content in addition to whole body weight. The lipid
content should be determined on the same biological material as is used
to determine the concentration of the test substance, when feasible.
(j) Test report. The test report must include the following informa-
tion.
(1) Summary. Test chemical and test species, uptake and depuration
rate constants, and steady state and kinetic BCFs
(2) Materials, (i) Exposure tanks and tubes—material and size of
tanks.
(ii) Diluter-type and description.
(iii) Dilution water. Source, description of any pretreatment, and
water characteristics including pH, hardness, temperature, dissolved oxy-
gen concentration, residual chlorine levels (if measured), total organic car-
bon, suspended solids, salinity of the test medium (if appropriate) and any
other measurements made.
(iv) Test substance. Physical nature and, where relevant, physico-
chemical properties; chemical identification data (including the organic
carbon content, if appropriate); if radio-labeled, the precise position of the
labeled atoms and the percentage of radioactivity associated with impuri-
ties.
(v) Stock solutions. Method of preparation of stock solutions and fre-
quency of renewal (the solubilizing agent, its concentration and its con-
tribution to the organic carbon content of test water must be given, when
used).
(vi) Test species. Scientific name, strain, source, any pretreatment,
age, size-range, etc.
20
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(vii) Care of fish. Acclimation, pretest mortality and health, feeding
(e.g. type of foods, source, composition—at least lipid and protein content
if possible, amount given and frequency).
(3) Test conditions— (i) Test design. Number and size of test cham-
bers, water volume replacement rate, number of replicates, number of fish
per replicate tank, number of test concentrations, controls.
(ii) Exposure concentrations. The nominal concentrations, the means
of the measured values and their standard deviations in the test vessels.
(iii) Length of uptake and depuration phases. Give the lengths of
the uptake and depuration phases and the rationale behind them
(iv) Light. Type and characteristics of illumination used and
photoperiods.
(v) Water quality within test vessels. pH, hardness, TOC, tempera-
ture and dissolved oxygen concentration.
(4) Sampling and analysis, (i) Sampling frequency for fish and water
samples.
(ii) Sample storage.
(iii) Sample extraction and analysis.
(iv) Detection and quantification limits.
(v) Accuracy and precision—results of spike and replicate analyses
(5) Results, (i) Data obtained in any preliminary test.
(ii) Validity of the test. Fish mortality and/or abnormal behavior for
exposed and control, variations in exposure concentrations, variations in
temperature, and minimum dissolved oxygen with respect to test validity
criteria.
(iii) Lipid content of the test fish.
(iv) Uptake and depuration curves of the test chemical in fish; graphi-
cal representation of data.
(v) Concentrations of parent in fish tissue and exposure water. Tab-
ular representation of data; Cf and Cw (with standard deviation and range,
if appropriate) for all sampling times (Cf expressed in milligrams per gam
of wet weight (parts per million) of whole body or specified tissues thereof
e.g. lipid, and Cw expressed in milligrams per gam of wet weight (parts
per million). Cw values for the control series (background should also be
reported).
21
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(vi) Uptake and depuration rate constants. Give values and 95 percent
confidence limits for the uptake and depuration (loss) rate constants, de-
scribe the computation.
(vii) Steady state and kinetic BCFs. The BCFs and the BCFK (both
expressed in relation to the whole body and the total lipid content, if meas-
ured, of the animal or specified tissues thereof), confidence limits and
standard deviation (as available).
(viii) Degradate concentrations. Where radiolabeled substances are
used, and when required, the accumulation of any major metabolites at
steady state or at the end of the uptake phase.
(ix) Deviations and/or unusual observations. Report anything unusual
about the test, any deviation from these procedures, and any other relevant
information.
(k) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) American Society for Testing and Materials. ASTM E-1022-84.
Standard Practice for conducting Bioconcentration Tests with Fishes and
Saltwater Bivalve Molluscs (1988).
(2) Bintein, S. et al. Nonlinear dependance of fish bioconcentration
on w-octanol/water partition coefficient. Environmental Research 1:29-390
(1993).
(3) Branson, D.R. et al. Transactions of the American Fisheries Soci-
ety 104:785-792 (1975).
(4) Chiou, C.T. and Schmedding D.W. Partitioning of organic com-
pounds in octanol-water systems. Environmental Science and Technology
16:4-10 (1982).
(5) Compaan, H. Chapter 2.3, Part II in The determination of the
possible effects of chemicals and wastes on the aquatic environment: deg-
radation, toxicity, bioaccumulation. Government Publishing Office, The
Hague, The Netherlands (1980).
(6) Connell, D.W. Bioaccumulation behavior of persistent chemicals
with aquatic organisms. Reviews of Environmental Contaminant Toxi-
cology 102:117-156(1988).
(7) Environmental Protection Agency. Section 5, A(l) Analysis of
Human or Animal Adipose Tissue in Analysis of Pesticide Residues in
Human and Environmental Samples. Thompson J.F. (ed). Research Tri-
angle Park, NC 27711 (1974).
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(8) Environmental Protection Agency. 822-R-94-002. Great Lake
Water Quality Initiative Technical Support Document for the Procedure
to Determine Bioaccumulation Factors (1994).
(9) Ernst W. Accumulation in Aquatic Organisms. In: Appraisal of
tests to predict the environmental behavior of chemicals. Ed. by Sheehman
P., Korte F., Klein W. and Bourdeau P.H. Part 4.4 pp 243-255. 1985
SCOPE, John Wiley & Sons Ltd., New York (1985).
(10) Food and Drug Administration. Pesticide analytical manual. Vol.
1. 5600 Fisher's Lane, Rockville, MD 20852, (1975).
(11) Gardner et al. Limnology and Oceanography 30:1099-1105
(1995).
(12) Hawker, D.W. and D.W. Connell D.W. Influence of partition
coefficient of lipophilic compounds on bioconcentration kinetics with fish.
Water Research 22: 701-707.
(13) Konemann, H. and K. Van Leeuwen Toxicokinetics in Fish: Ac-
cumulation and Elimination of Six Chlorobenzenes by Guppies. Chemo-
sphere 9:3-19 (1980).
(14) Kristensen P. (1991) Bioconcentration in fish: comparison of
bioconcentration factors derived from OECD and ASTM testing methods;
influence of particulate organic matter to the bioavailability of chemicals.
Water Quality Institute, Denmark.
(15) Kristensen, P. and N. Nyholm. CEC. Bioaccumulation of chemi-
cal substances in fish: the flow-through method—Ring Test Programme,
1984-1985 Final report, March 1987.
(16) Organization for Economic Cooperation and Development.
Guidelines for testing of chemicals. Paris (1993).
(17) OECD, Paris (1995). Direct Phototransformation of chemicals
in water. Guidance Document. February 1996.
(18) Randall R.C., Lee H., Ozretich R.J., Lake J.L. and Pruell
R.J.(1991). Evaluation of selected lipid methods for normalizing pollutant
bioaccumulation. Environ. Toxicol. Chem. Vol.10, pp. 1431-1436.
(19) Reilly P.M. et al. Guidelines for the optimal design of experi-
ments to estimate parameters in first order kinetic models. Canadian Jour-
nal of Chemical Engineering 55:614-622 (1977).
(20) Spacie, A. and J.L. Hamelink Alternative models for describing
the bioconcentration of organics in fish. Environmental Toxicology and
Chemistry 1:309-320 (1982).
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