United States	Office of Chemical Safety	EPA 712-C-16-003
Environmental Protection and Pollution Prevention	October 2016
ImhI M % Agency	(7101)
Ecological Effects
Test Guidelines
OCSPP 850.1730:
Fish Bioconcentration
Factor (BCF)

-------
NOTICE
This guideline is one of a series of test guidelines established by the United States Environmental
Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing
pesticides and chemical substances to develop data for submission to the Agency under the Toxic
Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and
Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of
Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines
issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS
as part of the guideline's number. Any test guidelines developed after April 22, 2010 will use the new
acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions under
TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also
used by EPA, the public, and the companies that are subject to data submission requirements under
TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on either
EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant
and without prior notice. At places in this guidance, the Agency uses the word "should." In this
guidance, the use of "should" with regard to an action means that the action is recommended rather
than mandatory. The procedures contained in this guideline are strongly recommended for generating
the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in
specific situations. You may propose alternatives to the recommendations described in these guidelines,
and the Agency will assess them for appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www .epa.gov/ocspp and select "Test Methods & Guidelines" on the
navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by Series
under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-
OPPT-2009-0576.
i

-------
OCSPP 850.1730: Fish Bioconcentration Factor (BCF)
(a)	Scope
(1)	Applicability. This guideline is intended for use in meeting testing requirements of
the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.)
and/or the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.). It describes
procedures that, if followed, would result in data that would generally be of scientific
merit for the purposes described in paragraph (b) of this guideline.
(2)	Background. The source materials used in developing this harmonized OPPTS test
guideline are 40 CFR 797.1520 Fish Bioconcentration Test; 72-6 Aquatic Organism
Accumulation Tests (Pesticide Assessment Guidelines, Subdivision E Hazard Evaluation:
Wildlife and Aquatic Organisms); 165-4 Laboratory Studies of Pesticide Accumulation in
Fish (Pesticide Assessment Guidelines, Subdivision N Chemistry: Environmental Fate);
the Organisation for Economic Cooperation and Development (OECD) Guideline 305
Bioaccumulation in Fish: Aqueous and Dietary Exposure; and the American Society for
Testing and Material ASTM International El022, Standard Guide for Conducting
Bioconcentration Tests with Fishes and Saltwater Bivalve Mollusks. Other sources used,
beyond what is referenced in subsequent paragraphs, are referenced in paragraph (j)(6),
(j)(10), and (j)(20) of this guideline.
(b)	Purpose. This guideline is intended for use in developing data on the propensity of chemical
substances and mixtures ("test chemicals" or "test substances") to bioconcentrate in the tissues of
fish. The purpose of the study is to determine uptake and depuration rate constants and
bioconcentration factors (BCFs) for fish exposed to a test substance in aqueous solution under
flow-through systems. For BCFs equal to or greater than e.g. 500, an additional purpose is to
identify if the test substance accumulates as the parent compound and/or its major metabolic
products and to identify and quantify the accumulation of these major metabolic products at
steady state. BCFs may be used to help assess risks to fish and to other organisms above them in
the food chain (including humans). The Environmental Protection Agency will use data from
this test in assessing the hazard and risks a test substance may present in the aquatic
environment.
(c)	Definitions. The definitions in OCSPP 850.1000 apply to this test guideline. The following
more specific definitions also apply:
Asymptotic LCso for acute toxicity testing refers to the toxicant concentration at which the
LCso becomes a constant for a prolonged exposure time.
Bioaccumulation refers to the net uptake of a pesticide from the environment by all
possible routes (e.g., respiration, diet, dermal) from any source (e.g., water, sediment, and
other organisms) (see paragraph (j)(l 1)).
Bioconcentration refers to the net accumulation of a test substance by the fish as a result
of uptake directly from aqueous solution, through gill membranes or other external body
surfaces.
1

-------
Bioconcentration factor (BCF), is the ratio, at any time during the bioconcentration test,
of the concentration of test substance in the whole fish or specified tissues thereof
(milligrams per kilogram (mg/Kg) wet weight) at that time, to the aqueous concentration
of the test substance (milligrams per liter (mg/L)). The BCF is expressed in units of test
solution volume per mass of fish (or tissue thereof), defined here as liters of test solution
per kilogram of fish (LKg"1). See bioconcentration factor, kinetic (BCFk) and
bioconcentration factor, steady-state (BCFss). Note: The term "in the whole fish"
includes any test substance sorbed onto external surface areas of the fish.
Bioconcentration factor, kinetic (BCFk) is the BCF calculated directly from the kinetic
uptake and depuration rate constants determined from the study. At steady state, the
BCFk equals the BCFss assuming first order uptake and elimination kinetics applies.
Bioconcentration factor, steady-state (BCFss) refers to the BCF that exists when uptake
and elimination are equal (balance of the flux of test substance into and out of the
organism). Operationally defined, in this study, as where the BCF does not change
significantly in the uptake phase over a period of two to four days under uniform or
constant aqueous test substance concentration.
Depuration is the elimination (or loss) of accumulated test substance from a test
organism as a result of any active (i.e., metabolic breakdown) or passive process (i.e.,
respiration or fecal elimination). The term applies in an aqueous environment without the
presence of the test substance.
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 substance is added.
Depuration rate constant (ki), is the numerical value defining the rate of reduction (or
loss) in the concentration of the test substance by the test fish (or specified tissues
thereof). For first-order kinetics, ki is expressed as the fraction of tissue residue lost per
unit of time, defined here per day (day"1). Historically, the depuration rate constant has
been described by the term "fo however, recent literature uses k-2 to describe only the
gill elimination rate constant. The total depuration rate constant, 'W includes the gill
elimination rate constant fo, and also the metabolic transformation rate constant ku, the
fecal egestion rate constant fe, and the growth dilution rate constant ko. k\ is then the
sum total of all four processes, in units per day (day"1) (see paragraph (j)(2) and (j)(3) of
this guideline). In this guideline, "fo" will be used as synonymous of 'W in order to
keep the document consistent with previous OPPTS and OECD and ASTM guidance
regarding bioconcentration in fish.
Elimination is the general term for the loss of a substance from an organism that occurs
by any active or passive means. The term is applicable in an environment with or
without the presence of the test substance.
Octanol-water partition coefficient (Kow) is the ratio of the concentration of a chemical in
n-octanol (or 1-octanol) to its unionized concentration in the aqueous phase in an
equilibrated two-phase octanol-water system. The abbreviation log Kow (logio Kow)
stands for the logarithm to the base 10 of the octanol-water partition coefficient.
2

-------
Steady-state or steady-state plateau refers to a situation when the total flux of test
substance into an organism equals the total flux out with no net change in mass or
concentration of the test substance. Steady-state differs from equilibrium in that it is
achieved as a result of a balance of transport and transformation processes acting upon
the test substance, whereas equilibrium is the end result of a physical-chemical
partitioning process.
Uptake is the acquisition of a substance from the environment by an organism as a result
of active or passive processes. In this study, uptake is primarily through the gills during
respiration.
Uptake rate constant (ki), is the numerical value defining the rate of increase in the
concentration of test substance in a test fish (or specified tissues thereof) following the
exposure of test fish to a medium containing the aqueous test substance. For first-order
kinetics, ki is expressed in units of volume of test solution per mass of tissue per time,
defined here as liters of test solution per kilogram of tissue per day (L Kg^ day"1).
(d) General considerations
(1)	Summary of the test. The test consists of two phases—the exposure {uptake) and
post-exposure (
-------
(i)	Test substance solubility in test water (e.g. seawater for estuarine and marine
fish) and test environmental conditions.
(ii)	Octanol-water partition coefficient (Kow), which is useful in estimating the
expected steady-state BCF.
(iii)	The negative logarithm to the base 10 of the acid dissociation constant (pKa).
(iv)	Toxicity of the test substance to the species of fish to be tested. This is
necessary for the establishment of test concentrations (to levels where adverse
effects are expected).
(4)	Range-finding test. It may be useful to conduct preliminary experiments in order to
optimize the test conditions of the definitive test, e.g. selection of test substance
concentrations, duration of the uptake and depuration phases. Pretest method
development and experimental design should be conducted to minimize results reported
as "not detected at the limit of detection", 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.
(5)	Definitive test. The goal of the definitive test is to determine the steady-state
bioconcentration factor (BCFss) as well as the kinetic bioconcentration factor (BCFk) for
the test substance (and its major metabolites) for the test organism. The test consists of
two phases—the exposure (uptake) and post-exposure (depuration) phases. During the
uptake phase, separate groups of fish of one species are exposed to at least two aqueous
concentrations of the test substance until steady state is achieved and documented
(minimum of 4 days) or to 28 days, whichever comes first. If the steady-state has not
been reached after 28 days of exposure, typically for test substances with log Kow >5
(Table 2), the uptake phase may be extended taking further measurements until steady-
state is reached or 60 days, whichever comes first. During the exposure phase
determination of the concentration of test substance (and major metabolites) in whole fish
(or specified tissues) and exposure water are made periodically. Steady-state is
operationally considered reached when a plot of the concentration of test substance in
whole fish against time becomes parallel to the time axis and three successive analyses of
the concentration in whole fish made on samples taken at intervals of at least 2-7 days are
within plus or minus (±) 20 percent (%) of each other. When pooled fish samples are
analyzed at least four successive analyses should be made to achieve steady state. For
test substances which are taken up slowly, the intervals would more appropriately be 7
days. Subsequent to reaching steady state, the groups of remaining fish are then
transferred to a medium free of the test substance for a post-exposure (depuration) phase.
During the depuration phase determination of the concentration of the test substance (and
major metabolites) in whole fish (or specified tissues) are made periodically. The
depuration phase lasts until 95% of the mass is depurated or for a maximum of 56 days,
whichever comes first. Because it is important to maintain stable concentrations of the
test substance in water, it is recommended that this test be conducted using a flow-
through exposure regime, although a static renewal technique may also be used if stable
exposure and environmental conditions can be maintained throughout the duration of the
4
-------
study. BCFs based on total radiolabeled residues in fish tissue and exposure water can be
used to help determine whether major metabolic products or degradates should be
identified and quantified. If the BCF in terms of total radiolabeled residues is greater
than or equal to 500, an attempt should be made to identify and quantify test substance
metabolic products or degradates representing greater than or equal to 10% of total
residues in fish tissues at steady state. Further, residues of toxicological concern should
also be quantified, even if they are less than 10% of the total residues in fish tissues at
steady state. The elements of an acceptable test are described in Table 8 of this guideline.
(e) Test standards
(1)	Test substance. Whether radiolabeled or not, the chemical purity of the test
substance tested should be as high as practical. For pesticides, the substance to be tested
should be technical grade unless the test is designed to test a specific formulation,
mixture, or end-use product. For pesticides, if more than one active ingredient constitutes
a technical product, then the technical grade of each active ingredient should be tested
separately, in addition to the combination, if applicable. If radiolabeled, the radiopurity
should be known. Radiolabeled test substances are used in an effort to simplify analyses
of test solutions and test organisms. However the following issues should be considered
when radiolabeled test substances are used:
(i)	Many radiolabeled substances contain more than 1% radiolabeled impurities,
which can greatly affect the apparent BCF if the impurity has a high BCF. For
this reason, if radiolabeled, the test substance should be of the highest purity
possible (i.e. greater than 95%, but greater than 98% is preferred); and
(ii)	If the BCF is equal to or greater than 500, verification is strongly
recommended to determine whether the radioactivity is associated with the parent
chemical or with metabolites.
(2)	Test duration. The duration of the test is chemical specific. To determine the
duration of this test, an estimation of the uptake phase should be made prior to testing
based upon either previous experience with the same test material in a different species, a
test with a similar material, the results of a preliminary range-finding test, or from the
water solubility or octanol-water partition coefficient (Kow) of the test material, as
described in paragraph (e)(2)(i) of this guideline for estimating the uptake phase and in
paragraph (e)(2)(ii) of this guideline for the depuration phase. It is important to note that
these estimates are based on the assumption that uptake and depuration patterns will
follow first order kinetics. If first order kinetics are obviously not obeyed or expected not
be obeyed based on practical experience, more complex models should be employed to
estimate the duration of each phase of the test (e.g paragraph (j)(19) of this guideline).
(i) Uptake (exposure) phase duration. Generally, the uptake phase should be
run until steady-state is reached or to 28 days, whichever comes first; however, if
the steady-state has not been reached by 28 days, typically for test substances with
log Kow >5 (Table 2), the uptake phase may be extended taking further
measurements until steady-state is reached or 60 days, whichever comes first.
Steady-state is operationally considered achieved when a plot of the concentration
of test substance in whole fish (C/) against time becomes nearly parallel to the
5
-------
time axis, as demonstrated by three consecutive analyses of tissue concentrations,
taken over appropriate intervals (e.g., 2 days for test substances that
bioconcentrate rapidly to 7 days for test substances that bioconcentrate slowly),
being within ±20% of each other. In cases where samples are within 20% of each
other, but the concentration of test substance in whole fish (C/) shows an
increasing trend during the sampling period, additional sampling and
measurements are recommended to ensure that steady state has been reached.
When fish tissue is pooled for analysis, at least four successive analyses should be
made for documenting that steady-state has been achieved. Before performing the
test, an estimate of the time to reach steady-state may be obtained from an
estimate of the depuration rate, fo, and using a linear uptake, first-order kinetic
model (see paragraph (e)(2)(i)(A) of this guideline) or from an empirical
relationship observed with partition coefficients (see paragraph (e)(2)(i)(B) of this
guideline). The relationship between the estimated duration of the uptake phase
(time to steady-state) and ki is summarized in Table 1. For nonionic organic test
substances, estimates of ki may be readily made using an empirical relationship
between ki and the n-octanol-water partition coefficient (Kow) or between ki and
aqueous solubility (see paragraph (e)(2)(i)(C) of this guideline).
(A) Estimate of time to steady-state assuming first-order kinetics. The
time for the test substance to reach some percentage of steady-state may
be obtained by use of the general first-order kinetic equation (Equation 1)
containing the uptake (k\) and depuration (ki) rate constants (see
paragraphs (j)(5) and (j )( l 2) of this guideline). If the concentration of the
test substance in exposure water (Cw) is held constant then an appropriate
solution to the differential first-order equation for C/in exponential form is
shown in Equation 2. As steady-state is approached (i.e., as time (/)
approaches infinity), Equation 2 reduces to the form in Equation 3 where
Cfs is the concentration of the test substance in the fish at "steady-state".
Equation 2 can then be rewritten in terms of the "steady-state"
concentration in fish (Equation 4), which can be further rearranged to
provide an estimate of time to reach a given fraction of "steady-state" (i.e.,
ratio of C/to Q,) when k'2 is known (Equation 5).
dCf
—— = klCw-k2Cf	Equation 1
dt
where:
ki is the rate constant for the uptake of test substance in units of
L Kg"1 • day"1 (liter of water per kilogram of fish per day);
is the rate constant for the depuration (loss) of test substance in units of
day"1;
C/is the concentration of the test substance in whole fish in milligrams per
kilogram (mg/Kg) wet weight;
6
-------
Cw is the soluble water concentration of the test substance in milligrams
per liter (mg/L);
Cfs is the concentration of the test substance in whole fish at steady state,
in milligrams per kilogram (mg/Kg) wet weight; and
t is time in units of days.
C/ =
'O
k
v 2 y
(cj(l-e"*2t)	Equation 2
Cf,=
h
V^2 J
(cj	Equation 3
Cy =(c/jiS)(\-e kl')	Equation 4
v
c
r =l-e^	Equation 5
c/.
As a guide, the statistically optimal duration of the uptake phase for the
production of a statistically acceptable BCFk is that period which is
required for the curve of the logarithm of the concentration of the test
substance in fish (C/) plotted against linear time (!) to reach I.6/&2 or 80%
of steady-state, but not more than 3.0/k2 or 95% of steady-state (see
paragraph (j)(24) of this guideline). Estimated time to 50%, 80%, 90%
and 95% of steady-state using the first-order kinetic model relationship
with ki is summarized in Table 1.
Using Equation 5, the fraction of "steady-state" of interest in Equation 6 is
set to achieve 80% of steady-state (i.e., C/Cfs = 0.80). Equation 6 is then
rearranged to provide a solution or estimate of the time to achieve 80% of
steady-state (tso in days) given a known k}.
0.80 = 1 —e	Equation 6
- 0.80)) 1.6	_	,
fg0 = ——	- =	Equation 7
2	2
Similarly the formula for estimating 95% of steady-state (Y95 in days) is
provided in Equation 8.
,m =^(1-0.95)^10	Equat.on8
k2	k2
Table 1.—Estimated Time to Obtain 50, 80, 90 and 95% of Steady-State Residue
Levels or Depuration, Using First-Order Kinetics Model Based on a k2 Value
7
-------
Depuration Rate
Constant,
k2 (day1)
Time (in days) to reach 50, 80, 90 and 95% of steady-state residue levels or
of depuration
tso = 0.693/1(2(a)
t80= 1.6/1(2 (b)
f9o = 2.3//c2(c)
= 3.0//C2  Equivalent to time to reach 50% of steady state or depuration (Cf/Cf,o = 0.50).
(b)	Equivalent to time to reach 80% of steady state or depuration (Cf/Cf:o = 0.20).
(c)	Equivalent to time to reach 90% of steady state or depuration (Q/Qo = 0.10).
(d)	Equivalent to time to reach 95% of steady state or depuration (Cf/Cf o = 0.05).
(B) Estimate of time to steady-state (partition coefficient model). A
model based on the partition coefficient of lipophilic compounds on
bioconcentration kinetics with fish (Equation 9), described in paragraph
(e)(2)(i)(A) of this guideline may be used (see reference in paragraph
(j)(15) of this guideline) as an alternative to the linear first-order kinetic
model. The units of teq in this relationship are in hours, not days.
Estimated time to steady-state (leq ~ tss) using the partition coefficient
empirical model is summarized in Table 2.
teq (hours) = (6.54 x 10"3) (Kow) + 55.31	Equation 9
Table 2.—Estimated Duration of Uptake (Exposure) Phase to Obtain Steady-State
Residue Levels Using the Partition Coefficient Empirical Relationship
Log Kow
Time to Steady-State,
teq (hours)
Time to Steady-State,
teq (days)
3.0
61.9
2.6
4.0
121
5.0
5.0
709
30
6.0
6596
275
6.5
20,737
864
(C) Estimation methods for l<2 A k} value for the test substance from a
phylogenetically similar species of fish, if available, may be used to make
a rough estimate of the time to steady-state. Alternatively an estimate of
k'2 (day"1) for fish may be obtained from an empirical relationship with
logio Kow for organic test substances with logio Kow values between 2 and
6.5 described in Equation 10 (see reference in paragraph (j)(26) of this
guideline). For other alternative relationships to derive an estimate of k-2
see reference in paragraph (j)(18) of this guideline. Estimates of ki for
logio Kow using Equation 10 are listed in Table 3.
logio^2 =(-°-414Xlogio Kow)+\Al (r2 = 0.95)	Equation 10
If the partition coefficient (Kow) is not known, an estimate can be made
from a knowledge of the aqueous solubility of the test substance using the
empirical relationship with solubility, which is in units of moles per liter,
8
-------
described in Equation 11 (see reference in paragraph (j)(7) of this
guideline). These relationships apply only to chemicals with logio Kow
values between 2 and 6.5 (see reference in paragraph (j)(l 5) of this
guideline). Estimates of logio Kow values from aqueous solubility using
Equation 11 are listed in Table 3.
logioKow= (0.862) (logio s) + 0.710 (r2 = 0.994)	Equation 11
Table 3.—Estimates of Kow from Aqueous Solubility and kz from Empirical
Relationship with Kow
Aqueous solubility (moles/liter)
Logio Kow (from Equation 11)
to (days-1) (from Equation 10)
2.20 x10"3
3.0
1.689
1.53 x10"4
4.0
0.652
1.05 x10"5
5.0
0.251
7.30 x10"7
6.0
0.097
1.92 x10"7
6.5
0.060
Model not applicable to predict Kow
7.0
0.037
(ii) Post-exposure (Depuration) phase duration. 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 done unless uptake of the
test substance during the uptake phase has been insignificant (e.g. the BCF is less
than 10). A period of half the duration of the uptake phase is usually sufficient
for an appropriate (e.g. 95%) reduction in the body burden of the test substance to
occur. If the time required to reach 95% loss is impractically long, exceeding for
example twice the 28 day duration bound 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% of steady-state concentration). However, for test substances
having more complex patterns of uptake and depuration than are represented by a
one-compartment fish model, yielding first order kinetics, allow longer depuration
phases for determination of loss rate constants. More complex models should be
employed in this case (e.g. paragraph (j)(19) of this guideline). The depuration
phase period may, however, be governed by the period over which the
concentration of test substance in the fish remains above the analytical detection
limit.
(A) Prediction of depuration phase duration. Before performing the
test, a prediction of the duration of the depuration phase may be made
from an estimate of the depuration rate, fo, assuming a linear uptake, first-
order kinetic model. Estimates of may be used from existing data for a
phylogenetically similar species of fish or may readily be made using an
empirical relationship between and Kow or k-2 and aqueous solubility
(see paragraph (e)(2)(i)(C) of this guideline).
A prediction of the time needed to reduce the body burden to some
percentage of the initial concentration may be obtained from the general
first-order uptake and depuration equation, Equation 1 (see references in
paragraphs (j)(16) and (j)(26) of this guideline). For the depuration phase
9
-------
the soluble concentration of the test substance in water, Cw is set to zero
{i.e., depuration phase is conducted in dilution water without test
substance present) and Equation 1 reduces to Equation 12. Then an
appropriate solution in exponential form to the differential first-order
equation (Equation 12) where the concentration of the test substance in the
fish at the start of the depuration period is rewritten as C/,o is Equation 13.
Rearranging Equation 13 provides a form to estimate the time to depurate
a percentage of the initial starting tissue concentration {i.e., ratio of C/to
Cfo) when k-/ is known (Equation 14).
dC
= -k2Cf	Equation 12
dt
C, =(cf,,V")
Equation 13
cr
—— = e 2	Equation 14
C
The half-life of the test substance in fish tissue (time to 50% depuration,
tso) based on Equation 14, is represented in Equation 15. This equation
can be rearranged into the form in Equation 16 to solve for tso, when ki is
known; the time is the same as estimated to reach 50% of steady state for
the uptake exposure phase. Half-life predictions based on this first-order
kinetic model applying various values are provided in Table 1.
0.50 = efc2'50	Equation 15
Ln{1/0.50) 0.693
t50 =—		- =		Equation 16
k2	k2
Similarly the formula for estimating 95% depuration, t95 {i.e. 5% of initial
residue remains, CflCfo = 0.05), in days is provided in Equation 17 and
Table 1. The time to 95% depuration is equivalent to the time to reach
95%) of steady state (compare Equations 8 and 17 of this guideline). The
time to reach 80% depuration {i.e., 20% of initial residue remains, CflCfo
= 0.20) is equivalent to the time to reach 80% of steady state (see Equation
7 of this guideline). Estimated time to 50%, 80%, 90% and 95% of
depuration using the first-order kinetic model relationship with ki is
summarized in Table 1.
Ln{ll 0.05) 3.0	^	_
t95=—		- = —	Equation 17
k2	k2
(3) Test organism
10
-------
(i) Species. Important considerations 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 selecting 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 Table 4 of this guideline. The bluegill sunfish, Lepomis macrochirus, has
traditionally been used in studies submitted to the Office of Pesticides Program
(OPP) to fulfill guideline requirements under the Federal Insecticide, Fungicide
and Rodenticide Act (FIFRA). 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 species and the experimental method should be reported in this
case.
Table 4.—Fish Species and Size, and Temperature Recommendations
Species
Common
Name
Test
temperature
(°C)
Total length
of test animal
(cm)
Danio rerio (Teleostei, Cyprinidae)
Zebra-fish
20-25
3.0 ±0.5
Pimephales promelas (Teleostei, Cyprinidae)
Fathead
minnow
20-25
5.0 ±2.0
Cyprinus carpio (Teleostei, Cyprinidae)
Common carp
20-25
5.0 ± 3.0
Oryzias latipes (Teleostei, Poeciliidae)
Ricefish
20-25
4.0 ± 1.0
Poecilia reticulata (Teleostei, Poeciliidae)
Guppy
20-25
3.0 ± 1.0
Lepomis macrochirus (Teleostei, Centrarchidae)
Bluegill
20-25
5.0 ±2.0
Oncorhynchus mykiss (Teleostei, Salmonidae
Rainbow trout
13-17
8.0 ±4.0
Gasterosteus aculeatus (Teleostei, Gasterosteidae)
Three-spined
stickleback
18-20
3.0 ± 1.0
Various estuarine and marine species have been used in different countries, for
example: Spot (Leiostomus xanthurus)., sheepshead minnow (Cyprinodon
variegatus); silverside (Menidia heryllina)\ shiner perch (Cymatogaster
aggregala); English sole (Parophrys vetulus); staghorn sculpin (Leptocottus
armatus); the euryhaline species three-spined stickleback (Gasterosteus
aculeatus); sea bass (Dicentracus labrax); bleak (Alburnus alburnus).
The freshwater fish listed are easy to rear and/or are widely available throughout
the year. They are capable 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. The availability of marine and estuarine species is partially
confined to the respective countries.
Fish used in the same test should be from the same source and from the same
year-class and from the same holding and acclimation tank(s). Juvenile fish
(preferred), post-larval or older, actively feeding, may be tested. Recommended
test species and size of test organisms are listed in Table 4. Weight should be
relatively constant for the duration of the study (see paragraph (e)(3)(v) of this
guideline). In any one test, select fish of similar weight such that the smallest is
11
-------
no smaller than two-thirds of the weight of the largest. Since weight and age of a
fish sometimes appears to have a significant effect on BCF values (see paragraph
(j)(8) of this guideline), these details should be accurately provided. It is
recommended that a sub-sample of the stock of fish is weighed and length
measured before the test in order to estimate the mean weight and length (see
paragraph (j)(5) of this guideline).
If adult fish are used, report whether male or female, or both are used in the
experiment. They should not be in a spawning state or recently spent (just after
spawning/resorption of eggs) either before or during the test. If both sexes are
used, differences in lipid content between sexes should be documented to be non-
significant before the start of the exposure and at test termination. This is
important because male and female fish may need to be pooled to obtain a
sufficient size sample to analyze and detect parent material and potentially its
major metabolites.
(ii)	Holding and acclimation. Fish brought into the laboratory should be held for
a minimum of 14 days prior to use. A minimum of 7 days of this period are used
for acclimation to environmental conditions (e.g., temperature, light intensity,
temperature, dilution water) similar to those used in the test. To maintain
organisms in good condition and avoid unnecessary stress, they should not be
crowded or subjected to rapid changes in temperature or water quality.
Acclimation water should be from the same dilution water source as used in the
test; if not, acclimation to the dilution water should be done gradually over a 48-
hour settling-in period. Within a 24-hour period, changes in water temperature
during holding or acclimation should not exceed 3 degrees Celsius (°C), and for
saltwater species, changes in salinity change should not exceed 2 parts per
thousand (ppt).
Following a 48-hour settling-in period, mortalities should be recorded, and the
following guidelines should be applied:
(A)	Mortalities of greater than 10% of the population in the 7 days of
acclimation: rejection of entire batch;
(B)	Mortalities of between 5 and 10% of the population during the 7 days
of acclimation: acclimation continued for additional 7 days;
(C)	Mortalities of less than 5% of the population during the 7 days of
acclimation: acceptance of batch.
(iii)	Health status and condition. Fish should not receive treatment for disease
during a test. Fish should not be used for a test:
(A) If more than 5% of the culture or acclimating group dies or shows
signs of stress (e.g., disease, physical damage, or abnormalities) during the
48 hours preceding the test;
12
-------
(B)	If they have been used in a previous test, either in a treatment or in a
control group;
(C)	If more than 10% die during the 7 days preceding the test; and
(D)	If they were administered treatment for disease within two weeks
preceding the test.
(iv)	Care and handling. Organisms should be handled as little as possible, but
when necessary, it should be done as carefully and quickly as possible. Any
disturbance which might change the behavior of the test fish should be avoided.
Detailed instructions for the care and handling of fish, such as those described
under paragraph (j)(l) of this guideline can be followed during the culturing,
holding and testing periods.
(v)	Diet and feeding. During the acclimation and test periods, feed an
appropriate 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% of body weight per day; this keeps the lipid concentration in most species of
fish at a relatively constant level during the test.
During the test, the amount of feed should be recalculated, for example, once per
week, in order to maintain consistent body weight and lipid content. For this
calculation, the weight of the fish in each test vessel can be estimated from the
weight of the fish sampled most recently in that test vessel. Do not weigh the fish
remaining in the test vessel. Siphon uneaten food and feces daily from the test
vessels shortly after feeding (30 minutes to 1 hour). Keep the test vessels as clean
as possible throughout the test so that the concentration of organic matter is kept
as low as possible, since the presence of organic carbon may limit the
bioavailability of the test substance (paragraph (j)(8) of this guideline).
Since many feeds are derived from fishmeal, feed should be analyzed periodically
to identify background contaminants such as heavy metals (e.g., arsenic,
cadmium, lead, mercury, and selenium) and persistent pesticides, especially
chlorinated insecticides. An analysis of the nutrient composition of the diet should
be included in the report. Many commercial feed companies provide both the
analysis of the lipid and protein content and the list of supplements.
(4) Administration of test substance
(i) Preparation of test solutions. Preparation of test solutions depends on the
solubility and stability of the test substance. The preparation of test solutions is
described in OCSPP 850.1000. Dilution water source and quality used in the test
are described in OCSPP 850.1000 and paragraph (e)(7)(vi) of this guideline.
Radiolabeled test substances can facilitate the analysis of water and fish samples,
13
-------
and should be used when conducting metabolite or degradate identification and
quantification.
The concentration of vehicle solvent should not exceed 0.1 milliliters per liter
(mL/L) and should ideally be the same in all test vessels. Its contribution (together
with the test substance) to the overall content of organic carbon in the test water
should be known.
The pH of stock solutions may be adjusted to match the pH of dilution water or to
a neutral pH if pH change does not affect the stability of the test substance in water.
The pH of test solutions may be adjusted after the addition of the test substance or
stock solution into the dilution water. However, all pH adjustments need to be made
prior to the addition of test organisms. Hydrochloric acid (HC1) and sodium
hydroxide (NaOH) may be used for this adjustment if warranted.
(ii)	Exposure technique. Because it is important to maintain stable
concentrations of the test material in water, it is recommended that this test be
conducted using the flow-through exposure technique, but where this is not
possible (e.g. when the test organisms are adversely affected) a semi-static
technique may be used provided that test elements are met (see Table 8 of this
guideline). Guidance on the exposure techniques is provided in OCSPP
850.1000.
(iii)	Treatment concentrations. To document that the potential to
bioconcentrate is independent of the concentration of the test substance,
bioconcentration values for the test substance should be determined using at least
two concentrations during the exposure phase which are a factor of 10 apart, plus
the appropriate control(s). Preliminary toxicity tests or a range-finding test can be
used to establish the appropriate test solution concentrations for the definitive test.
The two concentrations selected should not stress or adversely affect the fish.
These concentrations should be selected based on an appropriate toxicity
endpoint, e.g., less than the ECio or less than one-tenth the LCso determined in
either a range finding test or a 96-h definitive acute mortality test (see OCSPP
850.1075), or select the higher (or highest) concentration of the test substance to
be about of 1% of the acute asymptotic LCso, or below the chronic NOAEC. A
chronic NOAEC for an untested species may be estimated by dividing its acute
96-h LCso by an appropriate available acute-to-chronic ratio (e.g., ratios for some
chemicals are about 3, but a few are above 100). The highest test concentration
should be less than the solubility limit of the test material in water under the test
conditions; recommend at most that the highest concentration be no more than
one-half the solubility of the test substance under test conditions. If possible,
choose the other test concentration such that it differs from the highest
concentration by a factor of 10. If this is not feasible because of the analytical
detection limit, a lower factor than 10 between concentrations can be used or the
use of carbon-14 [14C] labeled test substance should be considered. The limiting
factor of how low one can test is based on the detection and quantitation limits of
the analytical methods. The concentration of the test material in the test solution
should be at least 10 times greater than the detection limit in water.
14
-------
(5)	Controls. Every test includes a dilution water control. However, if a vehicle
(solvent) is used, tests include a solvent control and the dilution water control is optional.
If a vehicle (solvent) control is used, care should be taken to maintain the vehicle
(solvent) concentration consistent across treatment concentrations (i.e., the two test
substance concentrations should have the same solvent (vehicle) concentration). Controls
consist of the same dilution water (and solvent (vehicle), if applicable), conditions,
procedures, and test population as the test solutions, except that no test substance is
added. The controls are used to relate possible adverse effects observed in the
bioconcentration test to a matching control group.
A test is considered unacceptable if:
(i)	For tests of standard durations, more than 10% of the organisms in
any control showed signs of disease, stress (e.g., abnormal
behavior, excessive mucus), and/or death.
(ii)	For tests that are extended by several weeks or months, if death or
other adverse effects were greater than 5% per month or exceeded
30% in the dilution or solvent control treatment.
(6)	Number of test organisms and replicates. The number of fish used in this test will
depend on the duration of the test and the number of replicate test vessels used. Also
important are the size of each fish and the size of the test chamber. The numbers of fish
per test concentration should be selected such that a minimum of four fish per sample are
available at each sampling interval. An example of sampling frequency and number of
test fish samples is provided in Table 5 of this guideline. The fish may be distributed
among two or more replicates at each treatment. The number of replicates should be
selected to account for variability. Estimates of the variability of test material in fish
tissue may be determined from a range-finding test or from other residue studies with test
substances with similar Kow values. These estimates of variability and a power analysis
may be used to help determine the number of replicates to use and whether compositing
fish would help reduce variability (see paragraph (j) of this guideline). If greater
statistical power (see OCSPP 850.1000) is needed to meet the objective of the study, the
number of test fish should be increased. Each replicate test vessel should contain an
equal volume of test solution and equal numbers of test fish. Replicate test vessels
should be physically separated, since the test vessel is the experimental unit.
(i) Loading. High water-to-fish ratios should be used in order to minimize the
reduction in the concentration of the pesticide in the water (Cw) caused by the
addition of the fish at the start of the test and to avoid decreases in dissolved
oxygen concentration. It is also important that the loading rate is appropriate for
the test species and size of fish used. In any case, a loading rate of less than or
equal to 1.0 g of fish (wet weight) per liter of water per day is recommended.
Higher loading rates can be used if it is shown that the concentration of test
substance is stable in the presence of the fish (i.e. can be maintained within plus
or minus (±) 20%), and that the concentration of dissolved oxygen does not fall
below 60% saturation. Rather than increasing the loading, extra replicates can be
15
-------
used if needed to obtain sufficient biomass for analysis {i.e., pool across two
replicates). In choosing appropriate loading regimes, the normal habitat of the fish
species should be considered. For example, bottom-dwelling fish may demand a
larger bottom area of the aquarium for the same volume of water than pelagic fish
species.
(ii) Introduction of test organisms. The test should not be started until the test
substance delivery system has been observed to be functioning properly and the
test substance concentrations have equilibrated (see OCSPP 850.1000). Test
vessels for treatment levels should be randomly or indiscriminately located within
the test area, and test organisms should be randomly or indiscriminately
distributed among test vessels. Further guidance is provided in OCSPP 850.1000.
(7) Facilities, apparatus and supplies. Normal laboratory equipment should be used,
especially the following:
(i)	Facilities. Facilities for culturing, holding, acclimating, and testing fish that are
well ventilated and free of fumes and disturbances which may affect the test
organisms. There should also be flow-through or recirculation tanks for culturing
and acclimating fish. Equipment for culturing and/or handling food sources for fish.
(ii)	Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature and lighting during the culturing, holding,
acclimation, and test periods. Apparatus for aerating dilution water and removing
gas bubbles as necessary. For flow-through tests, apparatus for aerating the dilution
water in the head box before mixing with the test substance or delivery to test
vessels. An apparatus providing a 30-minute lighting transition period may be
needed.
(iii)	Water quality testing instruments. Equipment for determination of water
quality characteristics (pH, hardness, temperature, etc)
(iv)	Cleaning of test system. Test substance delivery systems and test vessels
should be cleaned before each test. See OCSPP 850.1000 for further information.
(v)	Test containers and delivery system. Construction materials and equipment
that may contact the stock solution, test solution, or dilution water should not
contain substances that can be leached or dissolved into aqueous solutions in
quantities that can affect the test results. Construction materials and equipment that
contact stock or test solutions should be chosen to minimize sorption of test
substances. Refer to OCSPP 850.1000 for additional information on appropriate
construction materials. Test vessels, which should be constructed of chemically
inert material, should be of a capacity to maintain the loading rate and
environmental conditions. Test vessels should be loosely covered to reduce the loss
of test solution or dilution water due to evaporation, to minimize the entry of dust
or other particulates into solutions, and to prevent loss of test fish. A flow-through
system, if used, should contain an appropriate test substance delivery system.
16
-------
Many different sizes of test vessels have been used successfully. The size, shape,
and depth of the test vessel is appropriate if the specified flow rate and loading
requirements can be achieved.
(vi) Dilution water. Clean surface water, ground water, reconstituted water, or
natural or artificial seawater (for saltwater species) are acceptable as dilution
water if the test species will survive in it for the duration of the culturing, holding,
acclimation, and testing periods without showing signs of stress.
Natural seawater should be filtered through a filter with a pore size of <20
micrometers (|im) prior to use in a test. Artificial seawater can be prepared by
adding commercially available formulations or specific amounts of reagent-grade
chemicals to reagent water (deionized, distilled, or reverse osmosis water),
surface water, or ground water. For saltwater species, a salinity should be selected
from a range of 15 and 25 ppt. For artificial seawater or natural seawater that is
diluted with freshwater, salinity should be maintainable within a weekly range of
2 ppt.
Dechlorinated tap water is not recommended (either as the freshwater source,
preparation of artificial seawater, or dilution of natural seawater) because some
forms of chlorination are difficult to remove adequately. If dechlorinated tap
water is used, recommended maximum chlorine levels as well as other ways to
demonstrate suitability as a dilution water source are in OCSPP 850.1000.
Dissolved oxygen in the dilution water (prior to use in a test) should be between
90 and 100% saturation. If necessary, the dilution water can be aerated before the
addition of the test substance.
For freshwater testing, hardness, alkalinity, and conductivity should be measured
in the dilution water at the beginning of the test. For saltwater testing, salinity
should be measured in the dilution water at the beginning of the test.
Measurement of total organic carbon (TOC) or chemical oxygen demand (COD)
in the dilution water at the beginning of the test is recommended, but at a
minimum, TOC and COD should be analyzed periodically in the dilution water
source to document and characterize their magnitude and variability. For tests
with cationic substances, TOC or COD should be measured at the beginning of
the test.
(8) Environmental conditions. Environmental parameters during the test should be
maintained as specified below. The number and frequency of measurements
recommended for documenting and confirming the magnitude and variability of water
quality parameters (e.g., temperature, dissolved oxygen, pH, and salinity) in test solutions
during the test are described in detail in OCSPP 850.1000.
17
-------
(i)	Temperature. Test temperatures are provided in Table 4 of this guideline for
recommended test species. During a given test, the selected temperature should be
constant within plus or minus (±) 2 °C.
(ii)	pH and salinity. The pH should be between 6.0 and 8.5 for freshwater species
and between 7.5 and 8.5 for saltwater species and should vary less than 1 pH unit
during the test within a test vessel and between test concentrations (including
control). During a given test, the salinity (selected from a range of 15 to 25 ppt)
should be constant within ± 2 ppt.
(iii)	Lighting and photoperiod. A photoperiod should be selected from regimes
of 12 hours light: 12 hours dark to 16 hours light: 8 hours dark. For any given test,
the light regime should be constant. Light intensity should range from 540 tol080
lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition
period between light and dark is recommended.
(iv)	Dissolved oxygen. The dissolved oxygen concentration should be between
60 and 100% saturation during the test. If aeration is needed to achieve an
appropriate dissolved oxygen level, it should be done before addition of the test
substance. For flow-through exposures, the dilution water may be aerated
vigorously prior to delivery to the test vessels (e.g., in the diluter head box) such
that the dissolved oxygen concentration is at or near 90 to 100% saturation. If the
water is heated, precautions should be taken to ensure that supersaturation of
dissolved gases is avoided. Aeration of the test solutions during the test is not
recommended. Gentle aeration of test vessels during the exposure period is
permitted only in cases where the dissolved oxygen levels are in danger of
dropping below 60% saturation. In such cases, assurances should be made that the
use of aeration does not stress the test organisms; test substance concentrations
should be measured during the test to ensure that they are not affected by the use
of aeration; and all treatment and control vessels should be given the same
aeration treatment.
(v)	Total and dissolved organic carbon. The amount of TOC and dissolved
organic carbon (DOC) in the dilution water can affect the biovailability of some
test materials. Thus, they should be monitored routinely during both the uptake
and depuration phases. The contribution to the organic carbon content in water
from the test fish (excreta) 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 material and, if used, the solubilizing agent by more than 20%.
Monitoring the TOC and DOC, including the solubilizing agent (vehicle) if used,
is especially important and should occur more often when the logarithm base ten
of the octanol-water partition coefficient is greater than or equal to 4 (i.e., Kow>
104). In such instances, the apparent bioconcentration factor should be corrected
if necessary, to reflect the effect of TOC and DOC in the dilution water. The
scientist is advised to consult the open literature and paragraph (j)(l 1) of this
guideline in such instances (i.e., see Equation A2 in the referenced paragraph). At
18
-------
a minimum particulate matter should be measured weekly in the test chambers
during the test.
(vi) Flow in a flow-through system. During a test, the flow rates should not vary
more than 10% between any one replicate and another. The minimum number of
test vessel volume replacements should be five per 24-hour period. It is
recommended that diluter systems be monitored for proper adjustment and
operation at least twice daily throughout the test period to better ensure that the
target test concentrations are achieved and maintained. The flow rate to each test
vessel should be measured at the beginning and end of the test.
(9) Observations
(i) Measurement of test substance. The analytical methods used to measure the
amount of dissolved test substance in a water sample and the concentration in fish
tissue should be validated before beginning the test, as described in OCSPP
850.1000, and the relevant method detection limit(s) and limit(s) of quantification
should be reported. The fish and water samples should be handled throughout the
test in such a manner as to minimize contamination and loss (e.g. resulting from
adsorption by the sampling device). For BCF calculation, the concentration of the
test substance in the water (Cw) should be determined after the water is filtered
and/or centrifuged. Analytical confirmation of test concentrations and method
validation are discussed in 850.1000.
When radiolabeled test substance is used, total radioactivity should be measured
in all samples. At the end of the uptake phase, if the bioconcentration factor is
>500, water and tissue samples should be analyzed using appropriate
methodology to identify and estimate the amount of any major (i.e. greater than
10% of the parent compound) or toxicologically relevant degradation products or
metabolites that may be present. A sufficient number of fish should be sampled at
termination of the uptake phase to permit identification and quantitation of any
major (greater than 10% percent of parent) or toxicologically relevant degradates
or metabolites present. It should be determined how much of the activity present
in the fish is directly attributable to the parent compound, and the
bioconcentration factor should be corrected appropriately.
It is preferable to analyze fish and water immediately after sampling in order to
prevent degradation or other losses and to calculate approximate uptake and
depuration rates as the test proceeds. Immediate analysis also avoids delay in
determining when a plateau has been reached. Failing immediate analysis, the
samples should be stored by an appropriate method. Information on the proper
method of storage for the particular test material should be obtained before the
beginning of the study—for example, deep-freezing, holding at 4°C, duration of
storage, extraction, etc. If analyses are delayed, fish should be wrapped
individually in e.g. aluminum foil (for organic analysis) or placed in plastic or
glass containers (for metal analysis), and appropriately frozen. If the samples are
stored for extended periods of time, the storage stability of the parent and major
metabolites and/or degradates should be demonstrated.
19
-------
(ii)	Water Samples. Water should be sampled from the test vessels for the
determination of test substance before addition of fish and during both uptake and
depuration phases. Water samples should be collected during the uptake phase in
order to document the exposure concentration and stability during the exposure
phase. More frequent sampling may be appropriate to document exposure and
stability. The frequency of sampling should be sufficient to document test
substance stability (see Table 8). At a minimum, water should be sampled at the
same time as the fish and before feeding (see example sampling schemes in
Tables 5 to 6 of this guideline). At initiation (time 0), water samples should be
collected immediately prior to the addition of fish to the test vessels.
(iii)	Fish Samples.
(A)	Sampling methodology. Fish samples should be obtained for
analysis by removing an appropriate number of fish (normally a minimum
of four) from the test vessels at each sampling time. Fish samples should
be rinsed quickly with water, euthanized immediately, using the most
appropriate and humane method, and blotted dry.
If fish are sexually mature, the gender should be determined and recorded.
Each individual fish should be weighed (wet weight) and length measured
immediately after the fish is euthanized. If a fish is parsed into smaller
fractions, the wet weight of each component (e.g., edible, nonedible, liver)
for each individual fish should also be measured.
(B)	Analysis of fish samples - Individual fish versus pooling. The
concentration of the test substance should be determined for each weighed
individual fish. The BCF is expressed as a function of the total wet weight
of the fish. For special purposes, BCF calculations for specified tissues or
organs (e.g. muscle, liver) may be included if the fish are sufficiently
large, or the fish may be divided into edible (fillet) and nonedible (viscera)
fractions (i.e., for evaluating human health exposure). If analysis of each
individual fish is not possible, due to limitations of the sensitivity of the
analytical methods, then pairs, triplicates or more fish may be pooled to
constitute a sample for measurement. The same number of fish should be
pooled to constitute a sample at each sampling point. A similar number of
control fish should also be collected at each sample point, but only those
collected at the first sampling period and weekly thereafter, may need to
be analyzed. Pooling restricts 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 used
for the test design. See paragraphs (j)(9) and (j)(13) of this guideline for
an introduction to relevant pooling procedures.
(C)	Lipid content. BCF values for organic test substances should also be
expressed both as a function of total wet weight and as a function of total
lipid content in the fish. The total lipid content should be determined for
20
-------
fish on each sampling occasion, including fish exposed to the test material
and fish from the control. Suitable methods should be used for
determination of the total lipid content (see paragraphs (j)(V) and (j )( l 8) of
this guideline). The chloroform/methanol extraction technique has been
recommended as a standard method (see paragraph (j)(14) of this
guideline). The various methods do not give identical values (see
paragraphs (j)(23) and (j)(25) of this guideline), so it is important to
provide details of the method used. When possible, the analysis for total
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 mean total
lipid content of the fish samples (as mg/Kg wet weight) at the end of the
experiment should not differ from that at the start by more than ± 25%.
(D)	Determination of solids. The tissue percent solids should also be
determined to allow conversion of lipid concentration from a wet to a dry
basis. This can be accomplished by measuring the percent solids from fish
sampled from the control vessels.
(E)	Sampling frequency. Control fish should be sampled at the
beginning and end of uptake phase and at the end of the depuration phase.
Fish exposed to the test substance should be collected on at least five
occasions during the uptake phase and at least on four occasions during
the depuration phase. Since in some instances it will be difficult to obtain
a reasonably precise estimate of the BCF value based on this low number
of samples (especially when other than simple first-order depuration
kinetics are indicated), it may be advisable to take samples at a higher
frequency in both periods. Table 6 of this guideline gives a theoretical
sample schedule for chemicals with various log Kow values. Table 5
provides an example of a theoretical sample schedule with higher-
frequency that can be useful when other than simple first order is
followed.
(iv) Theoretical fish and water sampling scheme. An example of an acceptable
water and fish sampling schedule showing number of water samples and fish
samples to collect is given in Table 5 of this guideline. Based on the estimates of
the duration of the uptake and depuration phases discussed in paragraphs (e)(2)
and the recommended sampling rate in paragraphs (e)(9) of this guideline
theoretical sampling schemes for test chemicals with log Kow from 3.0 through
6.8 are shown in Table 6.
Table 5.—Theoretical Example of Fish and Water Sampling Program for
Bioconcentration Tests (With No Pooling of Fish)

Water
Fish

Minimum
Additional
Minimum
Additional
Sampling Period
frequency
Sampling1
frequency
Sampling1
-1
2



Uptake phase
22

Add 40-80 fish
1st
2
(2)
4
(4)
21
-------

Water
Fish

Minimum
Additional
Minimum
Additional
Sampling Period
frequency
Sampling1
frequency
Sampling1
2nd
2
(2)
4
(4)
3rd
2
(2)
4
(4)
4th
2
(2)
4
(4)
5th
2

6




Transfer fish to water
Depuration phase


free of test chemical
6th
2
(0)
4
(4)
7th
2
(0)
4
(4)
8th
2
(0)
4
(4)
9th
2
(0)
4
(4)
1	Values in parentheses are numbers of samples (water, fish) to be taken if additional sampling is carried
out.
2	Sample water after minimum of 3 "test vessel-volumes" have been delivered to allow time to reach a
steady state concentration.
Table 6.—Examples of Estimated Minimum Sampling Schedules for Non-Ionic Test
Substances with logio Kow Values Between 3.0 and 6.8 (with No Pooling of Fish)
Parameter
Logio Kow
3.0
3.13
4.0
5.0
5.5
6.0
6.4
6.6
>6.8
fe (day-1)3
1.689
1.493
0.652
0.251
0.156
0.097
0.066
0.055
-0.045
S95 (days)
1.8
2.0
4.6
12
19
31
45
55
-66
Sx value used in calculations for estimating duration and sampling times of designated phases
Exposure3
S95
S95
S95
S95
S95
S90d = 24
Sss = 29
S90 = 42
Sss =42
Post-exposure3
S95
S95
S95
S95
S95
S95
S95 = 45
S95 = 55
S93 = 59
Fish Sampling Schedule
Exposure (Uptake) phase (days from test initiation)
Test initiation
0
0
0
0
0
0
0
0
0
Sx/16
0.1
0.1
0.3
1
1
1
2
4
3
Sx/ 8
0.2
0.3
0.6
2
2
3
4
5
6
Sx/4
0.4
0.5
1.2
3
5
6
8
10
11
Sx/2
0.9
1.0
2.3
6
9
12
15
20
23
Additional13




14
18
23
21
34
Sx
1.8
2.0
4.6
12
19
24d
31d
41d
46d
Sx + 2 or 7d
3.8
4.0
6.6
14
21
26
38
48
53
Sx + 4 or 14 = Ux
5.8
6.0
8.6
16
23
28d
45d
55d
60d
Post-exposure (depuration) phase (days from test initiation)
Ux+Sx/ 8





31
51
62
67
Ux+ Sx/4
6.3
6.5
9.8
19
27
34
56
69
74
Additional13





39
61
75
81
Ux + Sx/2
6.7
7.0
11
22
32
44
67
82
88
Additional13






73
89
95
Ux + Sx/1.3
7.2
7.5
12
25
37
51
79
96
102
Additional13






84
103
109
22
-------
Parameter
Logio Kow
3.0
3.13
4.0
5.0
5.5
6.0
6.4
6.6
>6.8
Ux + Sx
7.6
8.0
13
28
42
59
90
110
116e
a Estimated number of days to 95% of steady-state (in these examples, S95 is estimated from depuration
rate constant, k2, and Log Kow; see Equation 10, of this guideline).
b Additional midterm sampling point so that no two consecutive points are more than ~7 days apart.
c Ux = length of uptake phase. If pooled fish tissue is used for analysis, an additional consecutive analysis
should be made Sx + 5 or 21 = Ux
d Duration of uptake phase may be terminated after documenting steady-state has been reached or 28
days (Sx greater than or equal to Sso is preferred); for test substances with a slow rate of uptake, the
duration of the uptake phase period may be increased for up to 60 days.
e Duration of depuration phase is to estimated 95% depuration or 56 days, whichever comes first.
(v)	Test solution appearance. Observations on test solution appearance and test
substance solubility should be made daily and at the beginning and end of the test.
The appearance of surface slicks, precipitates, or material adhering to the sides of
the test vessels or in any part of the mixing and delivery system should be
recorded at a minimum at the beginning and end of the test and during the test
when the test solution appearance changes.
(vi)	Mortality, appearance and behavior. Mortality, abnormal behavior,
feeding activity (deposition of feces), or appearance, and the number of test fish
exhibiting these characteristics should be recorded daily. For sampled fish,
careful examination of all the tissues should be made at the time of sampling for
any unusual conditions, such as a watery appearance or differences in color from
the controls.
(f) Treatment of results
(1) Lipid normalization of tissue concentrations. Since, for many organic substances,
there is a clear relationship between the potential for bioconcentration and lipophilicity,
there is also a corresponding relation between the lipid content of the test fish and the
observed bioconcentration of such substances. Thus, to reduce this source of variability
in test results for those substances with high lipophilicity (i.e., with logio Kow greater
than or equal to 3), bioconcentration should be expressed in relation to lipid content in
addition to whole body wet weight. Each individual test substance concentration in fish
(mg/Kg wet weight) should be adjusted to its lipid normalized value (Cfi) by dividing
this value by the lipid content (fraction; Lfish) of the fish using Equation 18. The lipid
content should be determined on the same biological material as is used to determine the
concentration of the test substance in whole fish or a specific tissue (e.g., edible, non-
edible tissue). The test substance tissue concentration is normalized to the lipid content.
Furthermore, a lipid normalized bioconcentration factor (BCF/,) can be calculated using
the lipid normalized concentration of the test substance in fish, Cfi (Equation 19).
cf
CfL= 		Equation 18
Lfish
BCFl =	Equation 19
23
-------
where:
Cfh = lipid normalized concentration of test substance in fish (mg/Kg wet weight);
Lfish = lipid content in fish based on a wet weight basis, expressed as a fraction; and
BCFz, = is the lipid normalized bioconcentration factor.
(2)	Descriptive statistics
(i)	Environmental conditions. Descriptive statistics (time-weighted mean,
standard deviation, coefficient of variation, minimum, maximum) should be
calculated by treatment level and aquaria for temperature, pH, dissolved oxygen,
and salinity (if relevant).
(ii)	Aqueous test material concentration. Descriptive statistics (time-weighted
average [TWA] concentration and standard deviation, minimum, maximum,
coefficient of variation) should be calculated by treatment level of the test
material concentration for the exposure period. The bioconcentration factors
should be typically expressed based upon the time-weighted average
concentration, which may be calculated following the methods such as described
in the references in paragraph (j)(21), or other similar methods. See example in
Equation 20. An explanation of the rationale and method used should be provided
with the study report.
Cw = TWAC =	Equation 20
Lwi
where:
TWAc is the time weighted average concentration, the carried weight w', is time tj
- tj-i, the number of hours or days at the concentration x,\ and
Xi is the average concentration (Cj + Cj-i)/2.
(3)	Determination of uptake and depuration rate constants. Concentrations of the test
substance in fish (or specified tissues) and water as a function of time throughout the
uptake and depuration phases are used to determine the uptake (ki) and depuration (ki)
rate constants. The preferred method for obtaining BCFk and the rate constants, ki and
fo, is to use nonlinear parameter estimation methods (see paragraph (j)(l 8) of this
guideline). See paragraph (f)(3)(i)(A), below, for a description of these methods. If the
depuration curve is obviously not first-order, then more complex models should be
employed (see paragraphs (j)(4), (j)(5), 
-------
sample of fish (or specified fraction or tissue) during the depuration phase
should be plotted against sampling time on semi-log paper. The slope of
that line, calculated using Equation 21 of this guideline, is -fo.
Alternatively, k-j may be calculated from two points in the graph using
Equation 22. 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
kinetics (see paragraph (f)(3)(ii)).
Ill Cydep 111 Cy 0 ~ tdep	Equation 21
C
k2 = A f\	Equation 22
Ln
(h -0
Where the subscript dep stands for depuration;
Cfdep is the concentration of the test substance in fish at time tdep during the
depuration phase;
C/o is the concentration in fish at the start of the depuration phase; and
C/i and C/2 are measured concentrations of the test substance in fish, at
different times t\ and h during the depuration, respectively.
(B) Determination of ki. Rearrangement of Equation 2 yields Equation
23. Given fo, calculate ki using Equation 23 of this guideline.
= —		—		r-	Equation 23
[1 — exp(—^2 tWpJ]
where:
the subscript up stands for uptake;
Cf,up = the concentration in the fish (or specified fraction or tissue) at time
of uptake tup, read from the midpoint of the smooth uptake curve produced
by the data when log concentration is plotted versus time (on an
arithmetical scale);
is obtained from the slope of the plot derived from Equations 21 or 22;
and
tup is the time, in days, where the midpoint of the uptake curve occurs and
the same time at which Cfup is measured.
25
-------
(ii) Method for calculation of uptake and depuration (loss) rate constants.
(A) The preferred means for obtaining the bioconcentration factor and ki
and k2 rate constants is to use nonlinear parameter estimation methods
with the aid of a computer. These programs find values for ki and ki given
a set of sequential time concentration data and the model conditions
described in Equations 24 and 25. This approach provides standard
deviation estimates and confidence estimates for ki and ki.
where:
tu = time at the end of the uptake phase.
(B) As in most cases can be estimated from the depuration curve with
relatively high precision, and because a strong correlation exists between
the two parameters ki and ki if estimated simultaneously, it may be
advisable first to calculate k-/ from the depuration data only, and
subsequently calculate ki from the uptake data using nonlinear regression.
(iii) Interpretation when concentrations are near detection limit. 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 indication of good quality bioconcentration
data. The difference between the uptake/depuration constants calculated at two
test concentrations should be less than 20%. 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%.
(4) Determination of steady-state and kinetic BCF values. Both the BCFss and BCFk
should be calculated. At a minimum, both the BCFss and BCFk should be computed for
the whole fish. Whenever possible, they should also be calculated for edible (fillet) and
nonedible (viscera) fractions. For special purposes BCF values for specified tissues or
organs (e.g. muscle, liver) may be determined if the fish are sufficiently large. BCF
determinations should always be based on concentrations of the test substance in fish
tissue and exposure water, and not on total radiolabeled residues. The whole fish BCF
values are expressed as a function of the total wet weight of the fish and BCF values for a
given tissue or fraction are based on the total wet weight of that respective tissue or
fraction. Bioconcentration values should be expressed in relation to lipid content in
addition to whole body weight (i.e., Equation 19).
Cf = C„ x | (1 - e-K'ty,
0 < t < tjj
Equation 24
Equation 25
(i) BCFss
26
-------
(A) Steady-state reached during uptake phase. The uptake curve of the
test substance is calculated by plotting its concentration in 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, the BCFss should be calculated using Equation 26 of this
guideline. The BCFss should be related to both the total weight and lipid
content of the fish {i.e., BCFss corrected for lipid content or BCFss, l =
BCFss/L).
BCFSS =
(n \
l f,s
y C w
Equation 26
where:
Cf,s = mean test substance concentration in fish (or specified tissue) on a
wet weight basis at steady-state; and
Cw = mean aqueous test substance concentration during the exposure
(uptake) phase.
(B) Steady-state not reached during uptake phase. When no steady
state is reached, it may be possible to calculate a BCFss of 80-95% of
steady-state of equilibrium. If steady-state was not reached during the
maximum 60-day uptake period, the maximum BCFss should be
calculated using the mean tissue concentration from that and all the
previous sampling days. An uptake rate constant should then be
calculated using appropriate techniques (i.e. by fitting an equation to the
data). This rate constant is used to estimate the BCFss and the time to
steady-state. If 95% elimination has not been observed after 56 days
depuration then a depuration rate constant should also be calculated. This
rate constant should be based on the elimination of the parent compound
(i.e. using Equations 21 and 22).
(ii) BCFk. The BCFk is calculated as the ratio of the uptake rate constant (k/) to
the depuration rate constant (ki) assuming first-order kinetics (Equation 27 of this
guideline). The rate constants are obtained as described in paragraphs (f)(3)(i)(A)
and (f)(3)(i)(B) of this guideline.
bcfk =
k
\ 2 y
Equation 27
(iii) BCFk,g. Assuming that fecal egestion and metabolism are negligible, if the
test is conducted for an extended period of time (e.g., greater than 28 days), a
kinetic bioconcentration factor, corrected for growth dilution, should be
calculated using Equation 28 of this guideline.
27
-------
BCFKig — —~—	Equation 28
k2 — kg
where BCI
-------
Water hardness (as CaCCb)
Should generally range between 40 and 180 mg/L for freshwater
species; for testing with metals, 40 - 50 mg/L
TOC (dilution water)
< 2 mg/L
Dissolved oxygen
> 60% of saturation
Age of test organisms
Juveniles preferred; see Table 4 for fish sizes
Number of organisms per
concentration
Sufficient to provide 4 organisms per treatment on each sampling day
Number of replicate test
vessels per concentration
Use an appropriate number of vessels, consistent with loading rate
Test vessel size/volume
Use standard rectangular or cylindrical vessels of a suitable capacity in
compliance with loading rate
Loading
< 1.0 g offish (wet weight) per liter per day (0.1 -1.0 g offish (wet
weight per liter per day is normally recommended
Feeding regime
Sufficient for maintenance of healthy condition, 1 - 2% of body weight
per day
Test vessel aeration
Not recommended; gentle aeration of test vessels may only be used in
cases where the dissolved oxygen levels are in danger of dropping
below 60% saturation. In such cases, assurances should be made that
the use of aeration does not stress the test organisms; test substance
concentrations should be measured during the test; and all treatment
and control vessels should be given the same aeration treatment.
Test concentrations
At least two concentrations, plus appropriate control should be used.
Vehicle concentration, if
vehicle used
< 0.1 mL/L, not recommended to be used if at all possible
Measures of effect or
Measurement endpoints
BCFss and BCFk (at a minimum for whole fish, optionally for special
purposes also on edible and/or non-edible tissues) on a wet weight and
on a lipid normalized basis, ki, fe (when appropriate, BCFkg, /
-------
5.	A surfactant or dispersant was used in the preparation of a stock or test solution. (However, adjuvants
may be used when testing pesticide typical end-use products.)
6.	Fish in the test treatments showed evidence of adverse effects (e.g., abnormal behavior (erratic
swimming, lying on bottom of test vessel), lack of feeding, or excessive mortality, such that chemical
uptake and depuration was likely impacted.
(i) Reporting.
(1)	Background information. Paragraph (k)(l) of OCSPP 850.1000 describes the
minimum background information to be supplied in the report.
(2)	Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences that
may have influenced the results of the test, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(3)	Test substance.
(i)	Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e., for pesticides, the identity and
concentration of active ingredient(s)) should be provided.
(ii)	The following physical/chemical properties should be included: solubility in
water and/ or saltwater, Kow, hydrolysis half-life
(iii)	If the test substance is radiolabeled the precise position of the labeled atoms,
the radiopurity, and the percentage of radioactivity associated with impurities
should be identified.
(iv)	Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(v)	Methods of preparation of the test substance and the treatment concentrations
used in the range-finding and definitive test, or limit test.
(vi)	If a vehicle (solvent) is used to prepare stock or test substance, the following
should be provided: the name and source of the vehicle, the nominal
concentration(s) of the test substance in the vehicle in stock solutions or mixtures,
and the vehicle concentration(s) used in the treatments and solvent control. A
description of the solvents, concentration, and effect on solubility of those that
were tried prior to initiation of the final study should be included.
(vii)	The toxicity of the chemical to fish (ideally to the test species) should be
provided. The toxicity should be reported as an acute 96-h LCso and a NOAEC
and LOAEC from a chronic study (i.e., an early life stage test or a full life cycle
test).
30
-------
(4)	Test organism.
(i)	The scientific and common name.
(ii)	Method and person verifying the species.
(iii)	Age and life stage of test organisms at test initiation and method of
verification.
(iv)	Information about the fish used, including strain, source, culture practices,
including media used, feeding history, any pretreatment, acclimation, and health
status.
(v)	The mean and range of the weight (wet, blotted dry) and length of the fish at
test initiation. If a growth rate constant should be calculated, provide the
individual fish weights and lengths.
(vi)	The fish lipid content for the control and test fish at study initiation, at the end
of the uptake, and at the end of the depuration period.
(5)	Test system and conditions. Provide a description of the test system and conditions
used in the bioconcentration study should be provided.
(i)	Description of the test container should be provided, including size, type,
material, fill volume.
(ii)	Description of the exposure technique used: static renewal or flow-through. If
static renewal describe the frequency of test solution renewal and if flow-through
a description of the flow-through system, including flow rates and test vessel
turnover rate. For closed systems include a description of the closed system
design.
(iii)	Description of the dilution water and any water pretreatment: source/type;
temperature; salinity (saltwater); pH; hardness and alkalinity (freshwater);
dissolved oxygen; total organic carbon or chemical oxygen demand; particulate
matter; conductivity; metals, pesticides, and residual chlorine concentrations
(mean, standard deviation, range). Describe the frequency and sample date(s) for
documenting dilution water quality and consistency.
(iv)	Use of aeration, if any, and location within exposure system of aeration (e.g.,
test solution or dilution water prior to test substance addition).
(v)	Number of test organisms added to each test vessel at test initiation.
(vi)	Number of test vessels (replicates) per treatment level and control(s).
(vii)	Methods used for treatment randomization and assignment of test organisms
to test vessels.
(viii)	Date of introduction of test organism to test solutions and test duration.
31
-------
(ix)	Loading rate.
(x)	Photoperiod and light source.
(xi)	Methods and frequency of environmental monitoring performed during the
definitive or limit test for test solution temperature, dissolved oxygen, pH, salinity
(if applicable), and light intensity.
(xii)	Methods and frequency of measuring the dissolved test substance to verify
exposure concentrations.
(xiii)	Methods and frequency of counting number of dead test organisms and
measuring any other toxic symptoms.
(xiv)	For the definitive and limit tests, description of all analytical procedures,
accuracy of the method, method detection limit, and limit of quantification.
(xv)	Detailed description of the diet: source, composition, and a nutrient analysis
(at least lipid and protein content), amount given and frequency. Feed should be
analyzed periodically to identify background contaminants such as heavy metals
(e.g., arsenic, cadmium, lead, mercury, and selenium) and persistent pesticides,
especially chlorinated insecticides.
(xvi)	Method used for determining amount to feed to fish.
(xvii)	Sampling schedule for tissue and water samples and a description of the
tissue and water samples analyzed.
(xviii)	Methods used to obtain, prepare and store tissue and water samples.
(xix)	Methods used for measurements of lipids in tissue. The accuracy of the
method, method detection limit, and limit of quantification should be given.
(xx)	The number of test substance and control treatments and the nominal solution
concentrations for each treatment.
(6) Results.
(i) Provide the results of any preliminary or supplementary studies performed.
32
-------
(ii)	The percentage of fish that died or showed any abnormal or adverse effects in
the control and in each test vessel and whether fish were sexually mature.
(iii)	Environmental monitoring data results (e.g., test solution temperature,
dissolved oxygen, light intensity, pH) in tabular form (raw data should be
provided for measurements not made on a continuous basis), and descriptive
statistics (e.g., mean, standard deviation, minimum, maximum).
(iv)	Results of lipid measurements for whole fish or specific tissues (e.g. edible,
viscera, liver), if applicable, reported on a wet weight basis.
(v)	Mean and range of the weight (wet, blotted dry) and length of the fish at each
test interval of the uptake and depuration phase for the control and test groups. In
order to calculate the growth rate, if applicable, provide in a table format the
individual fish weights and lengths for the control fish and the treated fish, and
calculate the growth rate constant. Additionally, provide a plot of fish weight or
log transformed fish weight against time.
(vi)	Length of uptake and depuration phases and the rationale behind them.
(vii)	Tabular summary of concentrations of parent compound in fish tissue and
exposure water by sampling time (raw data). Tabular representation of data; C/
and Cw (with mean, standard deviation and range, if appropriate) for all sampling
times (Cf expressed in mg/Kg of wet weight (ppm) of whole body or specified
tissues thereof (e.g. lipid), and Cw expressed in mg/L (ppm). Cw values for the
control series.
(viii)	Graphical representations of uptake and depuration curves.
(ix)	Uptake and depuration rate constants with 95% confidence limits.
(x)	The steady state and kinetic BCF values (both expressed in relation to the
whole body and the total lipid content, if measured, of the animal or specified
tissues thereof), confidence limits and standard deviation (as available).
(xi)	The time to steady state and the time to 95% elimination of accumulated
residues of the test substance from test fish.
(xii)	Description of statistical method(s) used for calculation of the ki and fo,
including software package, and the basis for the choice of method. Results of
any goodness-of-fit tests should be provided.
(xiii)	Where radiolabeled test substances are used and the bioconcentration factor
was greater than e.g. 500, the identities and quantities of any major metabolites or
metabolites of toxicological concern at the time of steady state or at the end of the
uptake phase should be provided.
(xiv)	Provide a table with a summary of all results from the aqueous exposure
test, including the bioconcentration factor(s), rate constants, and key results.
33
-------
(j) References.
(1)	American Society for Testing and Materials. ASTM E1022-94 (Reapproved 2007).
Standard Guide for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve
Mollusks. Current edition approved October 1, 2007. Published October 2007. Originally
approved in 1984. Last previous edition approved in 2002 as E1022 - 94(2002). DO I:
10.1520/E1022-94R07.
(2)	Arnot, J.A., and F A.P C. Gobas (2004) A food web bioaccuniulation model for
organic chemicals in aquatic ecosystems. Environ. Toxicol. & Chem. 23(10): 2343-2355.
(3)	Arnot, J.A., D. Macay, and M. Bonnell (2008) Estimating metabolic
biotransformation rates in fish from laboratory data. Environmental Toxicology and
Chemistry, 27(2): 341-351.
(4)	Bintein, S., J. Devillers and W. Karcher (1993) Nonlinear dependance of fish
bioconcentration on n-octanol/water partition coefficient. SAR and QSAR in
Environmental Research 1:29-39.
(5)	Branson, D.R., G.E. Blau, H.C. Alexander and W.B. Neely (1975) Bioconcentration
of 2,2',4,4'-tetrachlorobiphenyl in rainbow trout as measured by an accelerated test.
Transactions of the American Fisheries Society 104:785-792.
(6)	Chiou, C.T. and D.W. Schmedding (1982) Partitioning of organic compounds in
octanol-water systems. Environmental Science and Technology 16:4-10.
(7)	Compaan, H. (1980) Chapter 2.3, Part II in The determination of the possible effects
of chemicals and wastes on the aquatic environment: degradation, toxicity,
bioaccumulation. Government Publishing Office, The Hague, The Netherlands (1980).
(8)	Connell, D.W. (1988) Bioaccumulation behavior of persistent chemicals with aquatic
organisms. Reviews of Environmental Contaminant Toxicology 102:117-156.
(9)	Environmental Protection Agency. 1974. 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 Triangle Park, NC 27711 (1974).
(10)	Environmental Protection Agency. 1994. Document ID 822-R-94-002. Great Lake
Water Quality Initiative Technical Support Document for the Procedure to Determine
Bioaccumulation Factors (1994).
(11)	Environmental Protection Agency. 2009. User's Guide and Technical
Documentation, KABAM version 1.0, (Kow (based) Aquatic BioAccumulation Model).
Environmental Fate and Effects Division. Office of Pesticides Program. April 7, 2009.
(12)	Ernst W. (1985) 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).
34
-------
(13)	Food and Drug Administration. 1975. Pesticide analytical manual. Vol. 1. 5600
Fisher's Lane, Rockville, MD 20852, (1975).
(14)	Gardner, W.S., W.A. Frez and E.A. Cichocki (1985) Micromethod for lipids in
aquatic invertebrates. Limnology and Oceanography 30:1099-1105.
(15)	Hawker, D.W. and D.W. Connell (1988) Influence of partition coefficient of
lipophilic compounds on bioconcentration kinetics with fish. Water Research 22: 701-
707.
(16)	Konemann, H. and K. Van Leeuwen (1980) Toxicokinetics in Fish: Accumulation
and Elimination of Six Chlorobenzenes by Guppies. Chemosphere 9:3-19.
(17)	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.
(18)	Kristensen, P. and N. Nyholm. (1987) CEC. Bioaccumulation of chemical
substances in fish: the flow-through method—Ring Test Programme, 1984-1985 Final
report, March 1987.
(19)	Organization for Economic Cooperation and Development. (1993) Guidelines for
testing of chemicals. Paris (1993).
(20)	OECD, Paris (1995). Direct Phototransformation of chemicals in water. Guidance
Document. February 1996.
(21)	OECD Guidelines for Testing of Chemicals 211. 1998. Daphnia magna
Reproduction Test. Annex 6. Calculation of a Time-Weighted Mean.
(22)	OECD Guidelines for Testing of Chemicals 305. 2012. Bioaccumulation in Fish:
Aqueous and Dietary Exposure. Adopted October 2, 2012. 72 pp.
(23)	Randall R.C., H. Lee, R.J. Ozretich, J.L. Lake and R.J. Pruell (1991) Evaluation of
selected lipid methods for normalizing pollutant bioaccumulation. Environ. Toxicol.
Chem. Vol.10, pp. 1431-1436.
(24)	Reilly P.M., R. Bajramovic, G.E. Blau, D.R. Branson, and M.W. Sauerhoff (1977).
Guidelines for the optimal design of experiments to estimate parameters in first order
kinetic models. The Canadian Journal of Chemical Engineering 55:614-622.
(25)	Schlechtriem, C., A. Fliedner, and C. Schafers (2012) Determination of lipid content
in fish samples from bioaccumulation studies: contributions to the revision of guideline
OECD 305. Environmental Sciences Europe 2012, 24:13.
(26)	Spacie, A. and J.L. Hamelink (1982) Alternative models for describing the
bioconcentration of organics in fish. Environmental Toxicology and Chemistry 1:309-
320.
35
-------