  Office of Chemical Safety and Pollution Prevention
  (7101)
                                                               EPA 712-C-16-003
                                                                   October 2016

Ecological Effects
Test Guidelines

OCSPP 850.1730:

Fish Bioconcentration Factor (BCF)

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  NOTICE
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  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.
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  	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.
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  	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.

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, et seq.) 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 E1022, 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 LC50 for acute toxicity testing refers to the toxicant concentration at which the LC50 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)(11)).
      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.
      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 (L·Kg[-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 (k2), 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, k2 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 "k2"; however, recent literature uses k2 to describe only the gill elimination rate constant.  The total depuration rate constant, "kT" includes the gill elimination rate constant k2, and also the metabolic transformation rate constant kM, the fecal egestion rate constant kE, and the growth dilution rate constant kG.  kT 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, "k2" will be used as synonymous of "kT" 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 (log10 KOW) stands for the logarithm to the base 10 of the octanol-water partition coefficient.
      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 (k1), 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, k1 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[-1]·day[-1]).
(d) General considerations

      (1) Summary of the test.  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. During the exposure phase determination of the concentration of test substance (and major metabolites) in whole fish (or specified tissues) are made periodically.  The exposure phase lasts until steady state is achieved or to 28 days, whichever comes first; however, this period may be extended for certain test substances until steady-state is reached appropriately, or 60 days, whichever comes first.  At termination of the uptake phase, the groups of remaining fish are transferred to a medium free of the test substance for the 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.  The test is designed 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 fish. This guideline is based on the aqueous exposure route, which is preferred for evaluating the bioconcentration potential of a test substance in fish.  However, for pesticides, in some cases, when it has been adequately demonstrated that quantitation of the test substance cannot be conducted, or when the test substance concentration cannot be kept constant, or when there are other analytical difficulties, in consultation with the Agency, there may be other options to evaluate bioconcentration/bioaccumulation potential in fish.

      (2) General test guidance.  The general guidance in OCSPP 850.1000 applies to this guideline except as specifically noted herein.
      (3) Preliminary information.  OCSPP 850.1000 lists the type of information that should be known about the test substance before testing, and discusses methods for preparation of test solutions.  In addition, the following data on the test substance should be known to help design the range-finding and definitive BCF tests.
            (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 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 (Cf) against time becomes nearly parallel to the 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 (Cf) 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, k2, 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 k2 is summarized in Table 1.  For nonionic organic test substances, estimates of k2 may be readily made using an empirical relationship between k2 and the n-octanol-water partition coefficient (KOW) or between k2 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 (k1) and depuration (k2) rate constants (see paragraphs (j)(5) and (j)(12) 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 Cf in exponential form is shown in Equation 2.  As steady-state is approached (i.e., as time (t) approaches infinity), Equation 2 reduces to the form in Equation 3 where Cf,s 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 Cf to Cf,s) when k2 is known (Equation 5).
		Equation 1
                  where:
                  k1 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);
                  	k2 is the rate constant for the depuration (loss) of test substance in units of day[-1];
                  Cf is the concentration of the test substance in whole fish in milligrams per kilogram (mg/Kg) wet weight;
                  	Cw is the soluble water concentration of the test substance in milligrams per liter (mg/L);
                  Cf s 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.
		Equation 2
		Equation 3
		Equation 4
		Equation 5
                  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 (Cf) plotted against linear time (t) to reach 1.6/k2 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 k2 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., Cf/Cf,S = 0.80).  Equation 6 is then rearranged to provide a solution or estimate of the time to achieve 80% of steady-state (t80 in days) given a known k2.
		Equation 6
		Equation 7
                  Similarly the formula for estimating 95% of steady-state (t95 in days) is provided in Equation 8.
		Equation 8
	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
                           Depuration Rate Constant,
                                 k2 (day[-1])
Time (in days) to reach 50, 80, 90 and 95% of steady-state residue levels or of depuration

                              t50 = 0.693/k2 (a)
                              t80 = 1.6/k2 [(b)]
                               t90 = 2.3/k2 (c)
                               t95 = 3.0/k2 (d)
1.689
0.41
0.95
1.4
1.8
0.652
1.1
2.5
3.5
4.6
0.251
2.8
6.4
9.2
12
0.097
7.1
17
24
31
0.037
19
43
62
80
[(a)] Equivalent to time to reach 50% of steady state or depuration (Cf/Cf,0 = 0.50).
[(b][)] Equivalent to time to reach 80% of steady state or depuration (Cf/Cf,0 = 0.20).
[(c][)] Equivalent to time to reach 90% of steady state or depuration (Cf/Cf,0 = 0.10).
[(d][)] Equivalent to time to reach 95% of steady state or depuration (Cf/Cf,0 = 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 (teq ≈ 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 k2. A k2 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 k2 (day[-1]) for fish may be obtained from an empirical relationship with log10 KOW for organic test substances with log10 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 k2 see reference in paragraph (j)(18) of this guideline.  Estimates of k2 for log10 KOW using Equation 10 are listed in Table 3.
		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, described in Equation 11 (see reference in paragraph (j)(7) of this guideline).  These relationships apply only to chemicals with log10 KOW values between 2 and 6.5 (see reference in paragraph (j)(15) of this guideline).  Estimates of log10 KOW values from aqueous solubility using Equation 11 are listed in Table 3.
	log10 KOW = (0.862) (log10 s) + 0.710  (r[2] = 0.994)	Equation 11
	Table 3. -- Estimates of KOW from Aqueous Solubility and k2 from Empirical Relationship with KOW
                       Aqueous solubility (moles/liter)
                         Log10 KOW (from Equation 11)
                       k2 (days[-1]) (from Equation 10)
                                 2.20 x 10[-3]
                                      3.0
1.689
                                 1.53 x 10[-4]
                                      4.0
0.652
                                 1.05 x 10[-5]
                                      5.0
0.251
                                 7.30 x 10[-7]
                                      6.0
0.097
                                 1.92 x 10[-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, k2, assuming a linear uptake, first-order kinetic model.  Estimates of k2 may be used from existing data for a phylogenetically similar species of fish  or may readily be made using an empirical relationship between k2 and KOW  or k2 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 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 Cf,0 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 Cf to Cf,0) when k2 is known (Equation 14).
		Equation 12
		Equation 13
		Equation 14
                  The half-life of the test substance in fish tissue (time to 50% depuration, t50) based on Equation 14, is represented in Equation 15.  This equation can be rearranged into the form in Equation 16 to solve for t50, when k2 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 k2 values are provided in Table 1.
		Equation 15
		Equation 16
                  Similarly the formula for estimating 95% depuration, t95 (i.e. 5% of initial residue remains, Cf/Cf,0 = 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, Cf /Cf,0 = 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 k2 is summarized in Table 1.
		Equation 17
	(3) Test organism
            (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
                                    ([o]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 beryllina); shiner perch (Cymatogaster aggregata); 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 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;
                  (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)(1) 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,  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 (HCl) 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 EC10 or less than one-tenth the LC50 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 LC50, or below the chronic NOAEC.  A chronic NOAEC for an untested species may be estimated by dividing its acute 96-h LC50 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 [[14]C] 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.

      (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:
 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.
 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 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.
            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 (um) 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.
            (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 to1080 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 >= 10[4]).  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)(11) of this guideline in such instances (i.e., see Equation A2 in the referenced paragraph).  At 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[o]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.
            (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 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)(7) and (j)(18) 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)
                                Sampling Period
                                     Water
                                     Fish

                                    Minimum
                                   frequency
                                  Additional
                                  Sampling[1]
                                    Minimum
                                   frequency
                                  Additional
                                  Sampling[1]
                                      -1
                                       2

                                 Uptake phase
                                     2[2]

                                Add 40-80 fish
                                     1[st]
                                       2
                                      (2)
                                       4
                                      (4)
                                     2[nd]
                                       2
                                      (2)
                                       4
                                      (4)
                                     3[rd]
                                       2
                                      (2)
                                       4
                                      (4)
                                     4[th]
                                       2
                                      (2)
                                       4
                                      (4)
                                     5[th]
                                       2

                                       6

                               Depuration phase

                            Transfer fish to water
                             free of test chemical
                                     6[th]
                                       2
                                      (0)
                                       4
                                      (4)
                                     7[th]
                                       2
                                      (0)
                                       4
                                      (4)
                                     8[th]
                                       2
                                      (0)
                                       4
                                      (4)
                                     9[th]
                                       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 log10 KOW Values Between 3.0 and 6.8 (with No Pooling of Fish)
Parameter
                                   Log10 KOW

                                      3.0
                                     3.13
                                      4.0
                                      5.0
                                      5.5
                                      6.0
                                      6.4
                                      6.6
                                    >=6.8
k2 (day[-1])[a]
                                     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
Exposure[a]
                                      S95
                                      S95
                                      S95
                                      S95
                                      S95
                                  S90[d] = 24
                                   S85 = 29
                                  S90 ≈ 42
                                  S85 ≈ 42
Post-exposure[a]
                                      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
Additional[b]
                                      ...
                                      ...
                                      ...
                                      ...
                                      14
                                      18
                                      23
                                      21
                                      34
SX
                                      1.8
                                      2.0
                                      4.6
                                      12
                                      19
                                     24[d]
                                     31[d]
                                     41[d]
                                     46[d]
SX + 2 or 7[d]
                                      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
                                     28[d]
                                     45[d]
                                     55[d]
                                     60[d]
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
Additional[b]
                                      ...
                                      ...
                                      ...
                                      ...
                                      ...
                                      39
                                      61
                                      75
                                      81
UX + SX/2
                                      6.7
                                      7.0
                                      11
                                      22
                                      32
                                      44
                                      67
                                      82
                                      88
Additional[b]
                                      ...
                                      ...
                                      ...
                                      ...
                                      ...
                                      ...
                                      73
                                      89
                                      95
UX + SX/1.3
                                      7.2
                                      7.5
                                      12
                                      25
                                      37
                                      51
                                      79
                                      96
                                      102
Additional[b]
                                      ...
                                      ...
                                      ...
                                      ...
                                      ...
                                      ...
                                      84
                                      103
                                      109
UX + SX
                                      7.6
                                      8.0
                                      13
                                      28
                                      42
                                      59
                                      90
                                      110
                                    116[e]
[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 S80 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 log10 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 (Cf,L) 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 (BCFL) can be calculated using the lipid normalized concentration of the test substance in fish, Cf,L (Equation 19).
	  Cf,L= CfLfish	Equation 18
	  BCFL= Cf,LCw	Equation 19
      where:
      Cf,L = 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
      BCFL = 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= (wi)(xi)wi	Equation 20
      where:
            TWAC is the time weighted average concentration, the carried weight wi is time tj  -  tj-1, the number of hours or days at the concentration xi; and
      xi is the average concentration (Cj + Cj-1)/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 (k1) and depuration (k2) rate constants.  The preferred method for obtaining BCFK and the rate constants, k1 and k2, is to use nonlinear parameter estimation methods (see paragraph (j)(18) 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), (j)(12), (j)(15), (j)(16), (j)(17), (j)(24), and (j)(26) of this guideline).
            (i) Graphical method for determination of depuration (loss) rate constant k2 and k1
                  (A) Determination of k2.  Derivatizing Equation 13 using the natural log yields Equation 21.  The concentration of the test substance found in each 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 -k2.  Alternatively, k2 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)).
ln Cf dep   =  ln Cf, 0  -  k2  tdep                                      Equation 21
		Equation 22
            Where the subscript dep stands for depuration;
                  Cf dep is the concentration of the test substance in fish at time tdep during the depuration phase;
            Cf, 0 is the concentration in fish at the start of the depuration phase; and
                  Cf 1 and Cf 2 are measured concentrations of the test substance in fish, at different times t1 and t2 during the depuration, respectively.
                  (B) Determination of k1.  Rearrangement of Equation 2 yields Equation 23.  Given k2, calculate k1 using Equation 23 of this guideline.

  k1= Cf, up  k2Cw [1-exp-k2  tup]	Equation 23

            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);
                  k2 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 Cf up is measured.
            (ii) Method for calculation of uptake and depuration (loss) rate constants.
                  (A) The preferred means for obtaining the bioconcentration factor and k1 and k2 rate constants is to use nonlinear parameter estimation methods with the aid of a computer.  These programs find values for k1 and k2 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 k1 and k2.
	Cf= Cwx k1k2 1-e-k2t;     0<t<tU	Equation 24
	Cf= Cwx k1k2 e-k2(t-tU)-e-k2t;  t>tU	Equation 25
            where:
            tU = time at the end of the uptake phase.
                  (B) As k2 in most cases can be estimated from the depuration curve with relatively high precision, and because a strong correlation exists between the two parameters k1 and k2 if estimated simultaneously, it may be advisable first to calculate k2 from the depuration data only, and subsequently calculate k1 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).
            (i) BCFSS
                  (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).
		Equation 26
            where:
                  \s \* MERGEFORMAT = mean test substance concentration in fish (or specified tissue) on a wet weight basis at steady-state; and
                  \s \* MERGEFORMAT = 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 (k1) to the depuration rate constant (k2) 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.
		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.

	  BCFK,G= k1k2- kG 	Equation 28
            where BCFK,G is the kinetic bioconcentration factor corrected for growth dilution, and kG is the growth rate constant expressed in units per day (day[-1]).  The experimental calculation of the growth rate is based on the assumption that fish growth is exponential with time.  For the purpose of this experiment, the fish weight is strongly preferred, but the fish length may be used as a surrogate measure if necessary.  Plot the natural logarithm of 1/fish weight against time (day) or the natural logarithm of 1/fish length against time (day).  Individual measured fish weights (or lengths) should be plotted as opposed to mean values.  The growth rate constant is the slope of the line obtained.  A value of kG should be calculated for each treatment concentration and the controls, and any statistically significant difference described and explained, if possible.
      (5) Determination of the metabolism rate constant, kM.  Based on the results of the study, for chemicals with significant metabolism (e.g., their bioconcentration factors are smaller than predicted by KOW alone), the metabolism rate constant, kM should be calculated.  For details about how to conduct these calculations see paragraphs (j)(3) and (j)(11) of this guideline.
      (6) Model discrimination.  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 parameters are calculated from the model that best describes the measured concentrations of test substance in fish and water.  Most bioconcentration data have been assumed to be reasonably well described by a simple two-compartment or two-parameter model, as indicated by the rectilinear curve which approximates to the points for concentrations in fish, during the depuration phase.  Where these points cannot be described by a rectilinear curve then more complex models should be employed, see paragraph (j)(26) of this guideline.
(g) Tabular summary of test conditions.  Table 7 lists the important conditions that should prevail during this test.  Meeting these test conditions will greatly increase the likelihood that the completed test will be acceptable or valid.
	Table 7. -- Summary of Test Conditions for Fish BCF Test
Test type
Renewal or flow-through (preferred)
Test species
See Table 4 and paragraphs (e)(3)(i) of this guideline
Test duration
Uptake phase: until steady state (maximum 60 days)
Depuration phase:  duration of time to reach 95% elimination of the test substance, or until the concentration in tissue is less than 10% of the steady-state concentration or below the detection limit in tissue (maximum 56 days)
Temperature
Species specific, see Table 4 of this guideline; temperature should be constant within +-2°C during the test
Light quality
Ambient laboratory illumination
Light intensity
50-100 ft-c around 540-1080 lux
Photoperiod
Regime selected from 12 h light:12 h dark to 16 h light:8 h dark
pH
Between 6.0 and 8.5; constant during test within +- 0.5 pH unit
Water hardness (as CaCO3)
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 of fish (wet weight) per liter per day (0.1 - 1.0 g of fish (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, k1, k2 (when appropriate, BCFK,G, kG, kM)

(h) Test validity elements.  This test would be considered unacceptable or invalid if one or more of the conditions in Table 8 occurred.  This list should not be misconstrued as limiting the reason(s) that a test could be found unacceptable or invalid.  Except for the conditions listed in Table 8 and in OCSPP 850.1000, it is unlikely that a study will be rejected when there are slight variations from guideline environmental conditions and study design unless the control organisms are affected, the precision of the test is reduced, the power of a test to detect differences is reduced, and/or significant biases are introduced in defining the magnitude of effect on measurement endpoints as compared to guideline conditions.  Before departing significantly from this guideline, the investigator should contact the Agency to discuss the reason for the departure and the effect the change(s) will have on test acceptability.  In the test report, all departures from the guideline should be identified, reasons for these changes given, and any resulting effects on test endpoints noted and discussed.
	Table 8. -- Test Validity Elements for the Fish BCF Test
--------------------------------------------------------------------------------
1. Treatments were not randomly or indiscriminately assigned to individual test vessel locations or individual test organisms were not impartially or randomly assigned to test vessels.
2. A dilution water control [or a solvent (vehicle) control, when a solvent was used] was not included in the test.
3. More than 10% of the organisms in the control group [dilution water or solvent control] showed mortality or sublethal effects.  Death or other adverse effects were greater than 5% per month or exceeded 30% in all for a test extended over several weeks or months.
4.  The uptake phase was terminated before 28 days and steady-state was not reached.
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)(1) 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 LC50 and a NOAEC and LOAEC from a chronic study (i.e., an early life stage test or a full life cycle test).
	(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.
		(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.
            (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; Cf 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 k1 and k2, 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.
(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). DOI: 10.1520/E1022-94R07.
      (2) Arnot, J.A., and F.A.P.C. Gobas (2004) A food web bioaccumulation 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(1) 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).
      (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)  Könemann, 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.
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