
  Office of Chemical Safety and Pollution Prevention
  (7101)
                                                               EPA 712-C-16-004
                                                                   October 2016

Ecological Effects
Test Guidelines



OCSPP 850.1710:

Oyster 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.
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OPPTS 850.1710:  Oyster 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 OCSPP test guideline are 40 CFR 797.1830 Oyster Bioconcentration Test; 72-6 Aquatic Organism Accumulation Tests (Pesticide Assessment Guidelines, Subdivision E Hazard Evaluation: Wildlife and Aquatic Organisms); ASTM 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)(15), (j)(18), 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 estuarine and marine mollusks.  The purpose of the study is to determine uptake and depuration rate constants and bioconcentration factors (BCFs) for an estuarine and marine mollusk species exposed to a test substance in aqueous solution under flow-through systems.  For BCFs equal to or greater than 500, an additional purpose is to identify if the test substance accumulates as the parent compound and/or its major metabolic products and/or degradates and to identify and quantify the accumulation of these major metabolic products and/or degradates at steady state.  This guideline describes a bioconcentration test procedure for Eastern oysters (Crassostrea virginica).  BCFs may be used to help assess risks to mollusks 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.  In addition, the following more specific definitions apply:
      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)(21)).
      Bioconcentration refers to the net accumulation of a test substance by the oyster 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 mollusk (excluding the shell) or specified tissues thereof (milligrams per kilogram (mg/Kg) wet weight) at that time, to the aqueous test substance concentration (milligrams per liter (mg/L)).  The BCF is expressed in units of test solution volume per mass of the mollusk (or tissue thereof), defined here as liters of test solution per kilogram of tissue (L·Kg[-1]).  See also bioconcentration factor, kinetic (BCFK) and bioconcentration factor, steady-state (BCFSS).
      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 three consecutive sampling periods (taken at appropriate intervals) 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 (e.g., metabolic breakdown) or passive process.  The term applies in an 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) of the test substance from oyster tissue.  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 "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 the sum total of all four processes, in units per day (day[-1]) (see paragraph (j)(2) of this guideline).  In this guideline, "k2" will be used as synonymous of "kT" in order to keep the document consistent with previous OCSPP (OPPTS), and current OECD and ASTM guidance regarding bioconcentration in oyster and 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 (1-octanol) to its unionized concentration in the aqueous phase in an equilibrated two-phase octanol-water system.  The abbreviation log KOW stands for the logarithm to the base 10 of the octanol-water partition coefficient (log10 KOW).
      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.
	Umbo is the narrow end (apex) of the oyster shell.
      Uptake is the sorption of a substance from the environment into and onto an organism as a result of active or passive processes.  In this study, uptake is primarily through the gills during respiration; however, due to the nature of the study, it appears likely that a small amount of uptake could occur from consumption of food containing the test substance.
      Uptake rate constant (k1), is the numerical value defining the rate of increase in the concentration of test substance in tissue following the exposure of test animals 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]).
      Valve height is the greatest linear dimension of the oyster as measured from the umbo to the ventral edge of the valves (the farthest distance from the umbo).
(d) 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 oysters are exposed continuously to at least two aqueous, sublethal, concentrations of the test substance under flow-through conditions.  During the exposure phase, test solution and oysters are periodically sampled and analyzed using appropriate methods to quantify the test substance concentration (and major degradates and/ or metabolites, if appropriate).  The exposure phase generally lasts for 28 days.  If, prior to day 28, the tissue concentrations of the test material sampled over three consecutive sampling periods (taken at appropriate intervals) have been shown to be statistically similar (i.e. steady-state has been reached for a minimum of 4 days), the uptake phase of the test may be terminated.  At termination of the uptake phase, the groups of remaining oysters are transferred to a medium free of the test substance for the post-exposure (depuration) phase.  During the depuration phase, test solution and oysters are periodically sampled and analyzed using appropriate methods to quantify the test substance concentration (and major degradates and/ or metabolites, if appropriate).  The depuration phase lasts until 95% of the mass is depurated, or generally for a maximum of 14 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 oyster. If steady-state is not reached during 28 days of uptake, the BCFSS should be calculated using non-linear parameter estimation methods.
      (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.  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 (i.e. saltwater or seawater) and test environmental conditions.
            (ii) Octanol-water partition coefficient (KOW), which may be useful in estimating the expected steady-state BCF.
            (iii) The negative logarithm to the base 10 of the acid dissociation constant (pKa), when applicable.
            (iv) Toxicity of the test substance to oysters (e.g. propensity to inhibit shell deposition or toxicity to embryo-larval stages) to ensure exposure concentrations do not adversely affect them.
      (4) Range-finding test.  It may be useful to conduct a preliminary experiment 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, firstly to minimize results reported as "not detected at the limit of detection", since such results cannot be used for rate constant calculations.  Furthermore, pretest results can be used to determine the exposure concentrations necessary to ensure that concentrations in oysters 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) of the test substance (and its major degradates and/or metabolites, if appropriate) for the test organism.  The test consists of two phases--the exposure (uptake) and post-exposure (depuration) phases.  During the uptake phase, oysters are exposed to aqueous concentrations of the test substance until steady state is reached and documented (minimum of 4 days), or generally for a maximum of 28 days, whichever comes first.  During the exposure phase determination of the concentration of test substance (and major degradates and/or metabolites, if appropriate) in soft oyster tissue and exposure water are made periodically.  Steady-state is operationally considered reached when a plot of the concentration of test substance in oyster tissue against time becomes parallel to the time axis and three successive analyses of the concentration in soft oyster tissue made on samples taken at intervals of at least 2 days are within plus or minus (+-) 20 percent (%) of each other.  When pooled oyster samples are analyzed, at least four successive analyses should be made to document that steady-state has been achieved.  For test substances which are taken up slowly, the intervals would more appropriately be longer than 2 days (e.g. 7 days).  Subsequent to reaching steady state, the groups of remaining oysters are 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 degradates and/or metabolites, if appropriate) in oyster (or specified tissues if applicable) are made periodically.  The depuration phase lasts until 95% of the mass is depurated, or generally for a maximum of 14 days, whichever comes first.  If a radiolabeled test substance is used, BCFs based on total radiolabeled residues in oyster tissue and exposure water can be used to help determine whether major degradates or metabolites 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 representing greater than or equal to 10% of total residues in oyster 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 oyster tissue at steady state.  The elements of an acceptable test are described in Table 4 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 (preferably greater than 98%) unless the test is designed to test a specific formulation, mixture, or end-use product.  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 using radiolabeled test substances:
            (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 and/ or degradation products.
      (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 the estimates using water solubility and the KOW are based on the assumption that uptake and depuration patterns will follow first order kinetics.  This estimate should also be used to design a sampling schedule.  The uptake phase should continue until steady-state has been reached.  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)(13) of this guideline).  
            (i) Uptake (exposure) phase duration.  The uptake phase should be run until steady-state is reached or generally to 28 days, whichever comes first.  The minimum duration is 4 days -- corresponding to the minimum amount of time to document steady-state has been reached for a test substance that theoretically reaches steady-state in a day or less.  Steady-state is operationally considered achieved when a plot of the concentration of test substance in oyster (Coy) against time becomes nearly parallel to the time axis, as demonstrated by three consecutive analysis 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 oyster (Coy) shows an increasing trend during the sampling period, additional sampling and measurements are recommended to ensure that steady state has been reached.  When oyster 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 constant, k2, and using a linear uptake, first-order kinetic model, and water solubility or partition coefficients.  
            The time to reach 95% of steady state (t95 in days, Equation 1) for test substances whose uptake and depuration follow first order kinetics, may be estimated using the octanol-water partition coefficient (KOW) of the test substance using Equations 1 and 2, the water solubility of the test substance (Equations 1, 2 and 3 if the solubility is expressed in moles/L or Equation 4 if the solubility is expressed in mg/L).  These equations were developed from data on fish but are considered useful in this test as well (see paragraph (j)(1) of this guideline).  These relationships apply only to chemicals with log10 KOW values between 2 and 6.5 (see reference in paragraph (j)(19) of this guideline).  For other alternative relationships to derive an estimate of k2 see reference in paragraph (j)(11) of this guideline.  Bioconcentration kinetic studies have also been performed specifically for mollusks, e.g. as investigated by Hawker and Connell (paragraph (j)(10) of this guideline) and these may also be consulted.
                  
		Equation 1
		Equation 2
	log10 KOW = (0.862) (log10 M) + 0.710  (r[2] = 0.994)	Equation 3
		Equation 4
            where:
	t95 is the time to 95% of steady-state in days (hours for Equation 4);
            k2 is the rate constant for the depuration (loss) of test substance in units per time (day[-1] in Equations 1 or hour[-1] in Equation 4);
            	M is the solubility of the test substance expressed in units of moles per liter (mol/L);
            	s is the solubility of the test substance expressed in units of milligrams per liter (mg/L); and
            KOW is the octanol-water partition coefficient.
            For example, t95 for a test substance with a log KOW of 4.0 would be estimated as 4.6 days using Equations 1 and 2 (as shown in Equation 5).
                              
t95= 3.010[-0.4144.0+ 1.47]=4.6 days	Equation 5

            (ii) Post-exposure (Depuration) phase duration.  The depuration period is begun by transferring the oysters to the same medium but without the test substance into clean test vessels.  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).  The depuration phase should continue until at least 95% of the accumulated test substance and metabolites have been eliminated, but generally no longer than 14 days.  Assuming a first-order kinetic model, the time to 95% loss or depuration of the test substance is the same as the time estimated to reach 95% steady-state in the uptake phase, t95 (see Table 1 and paragraphs (e)(2)(ii) in OCSPP 850.1730).  However, for test substances having more complex patterns of uptake and depuration than are represented by a one-compartment oyster 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)(13) of this guideline).  The depuration phase period, may be governed by the period over which the concentration of test substance in the oyster remains above the analytical detection limit.
      (3) Test organism
            (i) Species.  The Eastern oyster, Crassostrea virginica, should be used as the test organism.
            Oysters may be cultured in the laboratory, purchased from culture facilities or commercial harvesters, or collected from a natural population in an unpolluted area free from epizootic disease. Oysters used in the same test should be from the same source and from the same holding and acclimation tank(s).  
            Oysters used in the test should be 30 to 50 mm in valve height and should be as similar in age and/ or size as possible to reduce variability.  The standard deviation of the valve height should be less than 20% of the mean. Oysters should be in a prespawn condition of gonadal development prior to and during the test.  This may be determined by direct or histological observation of the gonadal tissue for the presence of gametes or may be inferred based upon size and condition.
            (ii) Holding and acclimation.  Oysters should be attended to immediately upon arrival at the test facility. Oyster shells should be brushed clean of fouling organisms. Oysters should be held and acclimated in dilution water from the same origin as that used for testing. If the organisms arrive in water, the transfer of the oysters to the holding water should be gradual to reduce stress caused by differences in water quality characteristics and temperature. Oysters should be held for a period of time long enough to demonstrate that they are not diseased or stressed. Holding times of 10 to 12 days before testing have been used. During holding and acclimation, the oysters should not be crowded, and the dissolved oxygen concentration should be above 60% saturation. Holding tanks should be kept clean and free of debris. All oysters should be maintained in dilution water at the test temperature for at least 48 hours before they are used. 
            Mortalities should be recorded, and the following recommendations should be applied:
                  (A) Mortalities of greater than 10% of the population in the 7 days directly preceding the test: rejection of entire batch;
                  (B) Mortalities of between 5 and 10% of the population during the 7 days directly preceding the test: acclimation/holding continued for additional 7 days;
                  (C) Mortalities of less than 5% of the population during the 7 days directly preceding the test: acceptance of batch.
                  
            (iii) Health status and condition.  Oysters should not receive treatment for disease during a test.  Oysters should not be used for a test:
                  (A) If they appear diseased or otherwise stressed, or if they have cracked, chipped, bored, or gaping shells, or abnormalities;
                  (B) If they are infested with mudworms (Polydora sp.) or boring sponges (Cliona cellata);
                  (C) If they have been used in a previous test, either in a treatment or a control group;
            (iv) Care and handling.  Organisms should be handled as little as possible but when handling is necessary it should be done as gently, carefully and quickly as possible.  Any disturbance which might change the behavior of the test oysters should be avoided.  
            (v) Diet and feeding.  Oysters should be provided enough food to support survival and growth during holding, acclimation, and testing. Holding and acclimating oysters in natural seawater that is not expected to contain disease-causing organisms at adverse concentrations and contains as much natural phytoplankton as possible is advantageous. Cultured algae may be added to the water as necessary to support oyster survival and growth. If natural seawater that is not supplemented with an additional food source is used, it should not be passed through an ultraviolet sterilizer or a filter of <20 micrometers (um). If unsterilized and unfiltered natural seawater is used without adding algae, at least 1 liter per hour per individual (L/h/individual) is usually the minimum flow rate for mollusks of the size 40-50 mm (umbo to distal valve edge) to provide an adequate food supply that supports the desired growth rate. If the presence of disease-causing organisms is suspected in natural seawater, then filtration through a smaller sized filter and addition of a supplemental algal source to the test system may be necessary.
	(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 oyster tissue samples, and should be used when conducting degradate and/or metabolite 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 and ensure there is an adequate food supply, this test should be conducted using the flow-through exposure technique. Additional guidance on flow-through exposures 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 also be used to establish the appropriate test solution concentrations for the definitive test.  The two concentrations selected should not stress or adversely affect the oysters and should be less than one-tenth the IC50 or <IC10 determined in either the range-finding or 96-h definitive test under the OCSPP 850.1025 (Oyster Acute Toxicity, Shell Deposition) guideline or less than one-tenth the EC50 or <EC10 determined in the 48-h definitive test under the OCSPP 850.1055 (Bivalve Acute Toxicity, Embryo-Larval Development) guideline.  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, the other concentration should be chosen 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 may 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 not acceptable 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 total number of oysters 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 oyster and the size of the test chamber.  The concentration of test material should be determined in a minimum of four oyster samples, at each sampling period.  For example, in a 28-day test, a minimum of 28 oysters in the uptake (exposure) phase (four oysters sampled at each of seven sampling days) and an additional 20 oysters in the depuration phase (four oysters sampled at each of five sampling days) per test material concentration and control would be needed.  Examples of sampling frequency and number of samples are provided in Tables 1 and 2 of this guideline, respectively.  If analysis of individual oysters is not possible, due to limitations of the sensitivity of the analytical methods, then pairs, triplicates or more oysters may be pooled to constitute a sample for measurement.  These oysters should be distributed among replicates for each treatment.  Estimates of the variability of test material in oyster 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 if the number of replicates used should be adjusted (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 oysters should be increased.  Each replicate test vessel should contain an equal volume of test solution and equal numbers of oysters.  Replicate test vessels should be physically separated, since the test vessel is the experimental unit.
            (i) Loading.  The loading rate should not crowd oysters and should permit adequate circulation of water around each shell while avoiding physical agitation of the oysters by water currents. Flow rates should be sufficient to promote adequate oyster shell growth and maintain environmental conditions. A flow rate of 1 L/h/individual has been demonstrated to allow for adequate shell growth and is recommended as the minimum flow rate if using unfiltered natural seawater that is not supplemented with additional algae
            

            (ii) Introduction of test organisms.  The test should be started by introducing oysters that have been acclimated to test conditions into the test vessels after the test system has equilibrated (see OCSPP 850.1000). Within a test vessel, the oysters should be spread out equidistant from one another so that the entire test vessel is used. The oysters should all be placed with the left (cupped) valve down and the open, unhinged ends all oriented in the same direction facing the incoming flow of test solution. 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 oysters that are well ventilated and free of fumes and disturbances which may affect the test organisms. There should be flow-through tanks for holding and acclimating oysters and a system for culturing algae, if prepared on-site.
            (ii) Environmental control equipment.  Mechanisms for controlling and maintaining the water temperature, lighting, salinity, and flow during the culturing, holding, acclimation and test periods. Apparatus for aerating dilution water and removing gas bubbles as necessary. 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 (e.g., pH, salinity, 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 guidance. 
            (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 and to minimize entry of dust and other particles into the solutions. The flow-through system should contain an appropriate test substance delivery system.  
            (vi) Dilution water.  A dilution water is acceptable if oysters will survive in it for the duration of the culturing, holding, acclimation, and testing periods without showing signs of stress. Clean natural unfiltered seawater may be used; such water should come from a thoroughly mixed common source to ensure each oyster is provided equal amounts of food. Clean artificial seawater or filtered natural seawater with food (algae) added may be used especially if the presence of disease organisms is suspected in natural seawater. Natural seawater should be filtered through a filter with a pore size of <20 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 well water. Dechlorinated tap water is not recommended for 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.
            The dilution water should have a salinity in excess of 12 parts per thousand (ppt), and should be similar to that in the environment from which the test oysters originated.  For unfiltered natural seawater that is not diluted with freshwater to reduce salinity, salinity of >12 ppt is recommended with a weekly range in salinity of <5 ppt. For artificial seawater or natural seawater that is diluted with freshwater, salinity should be maintainable within a weekly range of 2 ppt.
            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. 
            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. 
            Specifications for dilution water quality and constancy are described in OCSPP 850.1000.
      (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.  The recommended water temperature is 20 °C. During a given test, the temperature should be constant within plus or minus (+-) 2 °C. However, if unfiltered natural seawater that has not been previously held is used, temporary fluctuations (less than 8 hours) of +-5 °C may occur and be tolerated by oysters (i.e., not affect control performance) due to their adaptations to fluctuating tidal habitats.
            (ii) pH and salinity. The pH should be between 7.5 and 8.5 and vary less than 1 pH unit during the test within a test vessel and between test concentrations (including control). For unfiltered natural seawater that is not diluted with freshwater to reduce salinity, a salinity of >12 ppt, with a range of <5 ppt, is recommended. For artificial seawater or natural seawater that is diluted with freshwater, salinity should be 20 ppt and constant within +-2 ppt during the test. 
            (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 to 1080 lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition period between light and dark is suggested.
            (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. 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 oysters (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)(21) 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 flow rate should be sufficient to promote adequate oyster shell growth and maintain environmental conditions. A flow rate of 1 L/h/individual has been shown to provide adequate environmental conditions. If unfiltered natural seawater that is not supplemented with additional algae is used, then the recommended flow rate is 1 L/hour/individual. 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 weekly and 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 oyster 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 oyster 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 oysters 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 oyster is directly attributable to the parent compound, and the bioconcentration factor should be corrected appropriately.

            It is preferable to analyze oysters 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.  Oysters should never be refrigerated or frozen in the shell.  If analyses are delayed, shucked oysters 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. Triplicate control water samples should be collected at the time of test initiation and at least weekly thereafter.  Triplicate water samples should be collected from the test substance exposure vessels before addition of oysters and periodically during both uptake and depuration phases.  Water samples are collected during the uptake phase in order to document the exposure concentration and stability of the test substance during the exposure phase.  Frequency of sampling should be sufficient to document test substance stability (see Table 2) and at a minimum be collected at the same time as the oysters (see example sampling schemes in Tables 1 and 2 of this guideline).  At initiation (time 0), water samples are collected immediately prior to the addition of oysters to the test vessels.
            (iii) Oyster samples.
                  (A) Sampling methodology.  Oyster samples should be obtained for analysis by removing an appropriate number of oysters from the test vessels (see paragraph (e)(6)) at each sampling time and treated as follows:
                  The valve height of each oyster should be measured prior to shucking.  Oysters should be shucked as soon as practical after removal and should never be refrigerated or frozen in the shell.  The shell should be opened at the hinge, the adductor muscle severed and the top valve removed.  The remaining adductor muscle should be severed where it attaches to the lower valve and the entire oyster tissue removed. The shucked oysters should be drained for approximately 3 minutes, blotted dry, weighed (wet) and analyzed immediately for the test material or stored if needed as described in paragraph (e)(9)(i) of this guideline.  
                  (B) Analysis of oyster samples--Individual oyster versus pooling.  The concentration of the test substance should usually be determined for each individual oyster.  If analysis of each individual oyster is not possible, due to limitations of the sensitivity of the analytical methods, then pairs, triplicates or more oysters may be pooled to constitute a sample for measurement.  The same number of oysters should be pooled to constitute a sample at each sampling point.  A similar number of control oysters should also be collected at each sample point. 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 oysters to accommodate the desired pooling, procedure and power, should be used for the test design.  See paragraphs (j)(6) and (j)(8) of this guideline for an introduction to relevant pooling procedures.
                  (C) Lipid content.  BCF values for organic test substances should be expressed both as a function of total wet weight and as a function of lipid content in the oysters (Equations 6 and 7).  The total lipid content should be determined for oysters on each sampling occasion if possible.  Suitable methods should be used for determination of the total lipid content (see paragraph (j)(5)).  The chloroform/ methanol extraction technique is recommended as a standard method (see paragraph (j)(9) of this guideline).  The various methods do not give identical values (see paragraph (j)(16) of this guideline); it is important to give details of the method used in the report.  When possible, the analysis for lipid should be made on the same extract as that produced for analysis for the test substance, since the lipids often have to be removed from the extract before it can be analyzed chromatographically.
                  (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 oysters sampled from the control vessels.
                  (E) Sampling frequency.  Based on the estimate of the time to steady-state and deuration, one of the examples of sampling schemes shown in Table 1 may be used to generate the appropriate data.  A similar number of control oysters should also be collected at each sampling interval, but only those collected at the first sampling period and weekly thereafter, may need to be be analyzed.  Since in some circumstances it will be difficult to calculate a reasonably precise estimate of the BCF value based on this number of samples (especially when other than simple first-order depuration kinetics are indicated), it may be advisable to take samples at a higher frequency in the uptake and depuration periods.  Table 2 of this guideline shows a general sampling program for an oyster BCF study.  The table also provides an example of a sampling program with higher frequency that may be useful when other than simple first order uptake and depuration kinetics are observed.  The additional samples should be stored as described in paragraph (e)(9)(i) and analyzed if the results of the first round of analyses prove inadequate for the calculation of the BCF with the desired precision. 
Table 1. --  Example Sampling Schedules Based on Estimated Time to Steady-State
Test Period
Sampling Day (except as noted) for Test Substance with
an Estimated Time to Steady-State (t95) of:

t95 < 4 days
4 < t95 < 14 days
15 < t95 < 21 days
t95 > 28 days
Exposure Phase[2]
1[1]
4[1]
1
1

6[1]
1
3
3

1
3
7
7

2
7
10
10

3
10
14
14

4
12
18
21

--
14
22
28
Depuration Phase[2]
1[1]
1
1
1

6[1]
2
3
3

12[1]
4
7
7

1
6
10
10

--
--
--
14
[1] hours
[2] Additional sampling times may be needed to confirm that steady-state has been attained.

	Table 2. -- Example of Oyster and Water Sampling Program for Bioconcentration Tests (With No Pooling of Oysters)
Sampling Period
Water
Fish 

Minimum
frequency
Additional
Sampling[1]
Minimum
frequency
Additional
Sampling[1]
-1
3



Uptake phase
3

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

6

Depuration phase


Transfer oysters to water free of test chemical
6[th]
3
(0)
4
(4)
7[th]
3
(0)
4
(4)
8[th]
3
(0)
4
(4)
9[th]
3
(0)
6
(6)
[1]Values in parentheses are numbers of samples (water, oyster) 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.

            (iv) 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.
            (v) Mortality, appearance and behavior.  Oysters should be observed (and data recorded) at least daily for feeding activity (deposition of feces) or any unusual conditions such as excessive mucus production (stringy material floating suspended from oysters), spawning, or appearance of shell (closure or gaping).  If gaping is noted, the oyster(s) should be prodded. Oysters which fail to make any shell movements when prodded are to be considered dead, and should be removed promptly with as little disturbance as possible to the test chamber(s) and remaining live oysters.  For oysters sampled, careful examination of all the tissues should be made at the time of shucking for any unusual conditions, such as a watery appearance or differences in color from the controls.
(f) 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 oysters and the observed bioconcentration of such substances.  Thus, to reduce this source of variability in test results for those substances with high lipophility (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 wet weight test substance concentration in oyster tissue (mg/kg wet weight) should be adjusted to its lipid normalized value using Equation 6.  The lipid content should be determined on the same biological material as is used to determine the concentration of the test substance (e.g., individual oyster, pooled oysters).  The bioconcentration factor should be calculated using the lipid normalized concentration of test substance in oyster using Equation 7.
	  Coy,L= CoyL	Equation 6
	  BCFL= Coy,LCw	Equation 7
      where:
      Coy,L is the lipid normalized concentration of test substance in oyster (mg/kg wet weight);
      L is the lipid content in oyster based on a wet weight basis, expressed as a fraction;
      BCFL is the lipid normalized bioconcentration factor; and
      Cw is the concentration of the test substance in the filtered and/ or centrifuged water.
      (2) Descriptive statistics.
            (i) Environmental conditions.  Descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) should be calculated by treatment level and aquaria for temperature, pH, dissolved oxygen, and salinity.
            (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 paragraph (j)(14), or other similar methods (see example in Equation 8).  An explanation of the rationale and method used should be provided with the study report.
	  TWAC= (wi)(xi)wi	Equation 8
      where:
            TWAC is the test substance time-weighted average concentration, the weight wi is the period of time tj  - tj-1, or 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 oysters and water as a function of time throughout the uptake and depuration phases should be 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)(12) of this guideline).  Otherwise, graphical methods may be used to calculate k1 and k2.  See paragraph (f)(3)(i)(A) to (f)(3)(ii), 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)(3), (j)(4), (j)(7), (j)(17), and (j)(19) of this guideline).  
            (i) Graphical method for determination of depuration (loss) rate constant k2 and k1.
                  (A) Determination of k2.  Plot the concentration of the test substance found in each oyster, during the depuration phase, against sampling time on semilog paper.  The slope of the line, calculated using Equation 9 of this guideline, is -k2.  Alternatively, k2 may be calculated from two points in the graph using Equation 10.  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)(B)).
	lnCoy dep=lnCoy, 0-k2xtdep	Equation 9
		Equation 10
            where:
                  Coy dep is the concentration of the test substance in oyster at time of depuration tdep;
            Coy 0 is the concentration in oyster at the start of the depuration phase; and
                  Coy 1 and Coy 2 are measured concentrations of the test substance in oyster at different times t1 and t2 of the depuration phase, respectively
                  (B) Determination of k1.  Given k2, calculate k1 using Equation 11 of this guideline.  
                    
  k1= Coy up  k2Cw [1-exp-k2  tup]	Equation 11

            where:
                  Coy up  = the mean concentration in oyster 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 Equation 9 or 10; and
                  tup is the time, in days, where the midpoint of the uptake curve occurs, and the same time at which Coy 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 12a and 12b.  This approach provides standard deviation estimates and confidence estimates for k1 and k2.
	Coy= Cwx k1k2 1-e-k2t; 0<t<tU	Equation 12(a)
	Coy= Cwx k1k2 e-k2(t-tU)-e-k2t; t>tU	Equation 12(b)
            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 depuration curves are an indication of good quality bioconcentration data.  The difference between the uptake/ depuration constants calculated at the 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.  BCF determinations should always be based on concentrations of the test substance in oyster tissue and exposure water, and not on total radiolabeled residues.  The oyster BCF values are expressed as a function of the total wet weight of the oyster.  Bioconcentration values should be expressed in relation to lipid content in addition to whole body weight (Equation 7).  
      (i) BCFSS.
                  (A) Steady-state reached during uptake phase.  The uptake curve of the test substance should be obtained by plotting its concentration in oyster in the uptake phase against time on arithmetic scales.  If the curve has reached a plateau, that is, become approximately asymptotic to the time axis, calculate the BCFSS using Equation 13 of this guideline.  The BCFSS should be related to both the total wet weight and lipid content of the oyster.  A 95% confidence interval (CI) should also be derived for the BCFSS.  This should be done using Equation 14 of this guideline to yield the mean and lower and upper confidence limits of the concentration of the test substance in oyster at steady state at 97.5% CI.  The same procedure should be used to calculate the mean and lower and upper confidence limits at 97.5% CI for the test solution concentrations at steady-state using equation 15.  The 95% CI of the BCF would then be between Loy/Uw and Uoy/Lw.
		Equation 13
            where:
                  \s \* MERGEFORMAT = mean test substance concentration in oyster on a wet weight basis at steady-state; and
                  \s \* MERGEFORMAT = mean water test substance concentration during the exposure (uptake) phase.
	97.5% CI CoyLoy , Uoy= Coy, S+- (tn-1; 0.025)(SEoy) 	Equation 14
                   where:
                   	\s \* MERGEFORMAT is the test substance concentration in oyster at steady state;
                   Loy and Uoy are the lower and upper limits of Coy, respectively;
                   tn-1; 0.025 is the student's t value for n-1 degrees of freedom at p = 0.025;
                  SEoy is the standard error of the mean of the oyster test substance concentration at steady state.
	97.5% CI CwLw , Uw= Cw+- (tn-1;  0.025)(SEw) 	  Equation 15
            where:
                  \s \* MERGEFORMAT is the time weighted average water test substance concentration during the exposure phase;
            Lw and Uw are the lower and upper limits of Cw, respectively;
                  	tn-1, 0.025 is the student's t value for n-1 degrees of freedom at p = 0.025;
                  SEw is the standard error of the mean of the water 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 28-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 14 days depuration then a depuration rate constant should also be calculated. This rate constant should be based on the elimination of the parent compound (i.e. using Equations 9 and 10).
            (ii) BCFK..  The BCFK should be calculated as the ratio of the uptake rate constant (k1) to the depuration rate constant (k2) assuming first-order kinetics (Equation 16 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 16
      (5) 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 should be calculated from the model that best describes the measured concentrations of test substance in oyster 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 oyster, during the depuration phase, when these are plotted on semilog paper.  Where these points cannot be described by a rectilinear curve then more complex models should be employed (see paragraph (j)(19) of this guideline).
(g) Tabular summary of test conditions.  Table 3 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 3. -- Summary of Test Conditions for the Oyster Test
Test type
Flow-through
Test species
Eastern oyster (Crassostrea virginica)
Test duration
Uptake phase: until steady state is reached (minimum of 4 days) or generally 28 days, whichever comes first.
Depuration phase: until 95% of the test substance is eliminated, or if this takes longer, then until the concentration in tissue is less than 10% of the steady-state concentration or below the detection limit in tissue (generally a maximum of 14 days). 
Temperature
20 °C (constant during test within +-2 °C) 
However, if unfiltered natural seawater that has not been previously held is used, temporary fluctuations (less than 8 hours) of +-5 °C may occur and be tolerated by oysters (i.e., not affect control performance) due to their adaptations to fluctuating tidal habitats.
Light quality
Ambient laboratory illumination.
Light intensity
540-1080 lux (approximately 50-100 ft-c)
Photoperiod
Selected from among 12 hours light:12 hours dark to 16 hours light:8 hours dark schemes
Salinity
Artificial or natural seawater that is diluted with freshwater: 20 ppt (range of +-2 ppt during test);
Unfiltered natural seawater that is not diluted with freshwater to reduce salinity: >12 ppt (range of <5 ppt during test).
pH
The monthly range of pH is less than 0.8 pH units.
TOC (dilution water)
<= 2 mg/L.
Dissolved oxygen
Minimum of 60% saturation, aeration not recommended.
Size of test organisms 
30 to 50 mm in valve height; similar in age and/or size.  Standard deviation of valve height should< 20 percent of the mean.
Number of organisms per concentration
Sufficient to provide 4 oysters per treatment on each sampling period.  For example, for a 28-day test, 48 organisms is the minimum number of oysters.
Number of replicate test vessels per concentration
Use an appropriate number of vessels, consistent with loading rate
Test vessel size/volume
Sufficient to completely submerge the oysters throughout the test; may use standard rectangular or cylindrical vessels of a suitable capacity in compliance with loading rate.
Loading
Flow rate is adequate to promote adequate shell growth and maintain environmental conditions. Flow rate of 1 L/h/individual has been shown to be adequate when using unfiltered natural seawater that is not supplemented with additional algae
Feeding regime
Phytoplankton naturally occurring in the dilution water (if using unfiltered, unsterilized natural seawater) or supplemented (needed if using artificial seawater).
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 on a wet weight, and on a lipid normalized basis, k1 and k2.

(h) Test validity elements.  This test would be considered unacceptable or invalid if one or more of the conditions in Table 4 occurred.  This list should not be misconstrued as limiting the reason(s) that a test could be found unacceptable or invalid.  However, except for the conditions listed in Table 4 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 significantly 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 4. -- Test Validity Elements for the Oyster 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.  The uptake phase was terminated before either apparent steady-state or 28 days was reached.
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4. 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.)
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5. Evidence of spawning was observed.
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6. For tests of standard durations, more than 10% of the organisms in the control group [dilution or solvent control] showed mortality or adverse sublethal effects.  For tests that are extended for several weeks or months, 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.
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7.  Oysters in the test treatments showed evidence of adverse effects (e.g., excessive mucus production (stringy material floating suspended from oysters) lack of feeding, shell gaping, poor shell closing in response to prodding, 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.  A statement of the guideline or protocol followed should be provided.  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 should be included, 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.
	(4) Test organism.  
      (i) The scientific and common name.
		(ii) Method and person verifying the species.
            (iii) The mean and range of the size of the oysters (i.e. valve height) and of the weight of the oyster tissue (shucked blotted dry) at test initiation.
            (iv). Information about the oysters used: source, food and feeding history, prophylactic or disease treatments, and health status.
		(v) Method of confirmation of prespawn condition.
		(vi) Acclimation and holding procedures and conditions.
      (5) Test system and conditions.  Provide a description of the test system and conditions used in the bioconcentration study.
		(i) Description of the test container used: size, type, material, fill volume.
            (ii) Description of the exposure technique used: flow-through system design, flow rates, and test vessel turnover rate.  For closed systems include a description of the closed system design.
            (iii) Use of aeration, if any, and location within exposure system of aeration (e.g., test solution or dilution water prior to test substance addition).
            (iv) Detailed description of the diet: source, composition, amount provided and frequency.
            (v) The number of test substance and control treatments and the nominal solution concentrations.
		(vi) Number of oysters added to each test vessel at test initiation.
            (vii) Methods used for treatment randomization and assignment of oysters to test vessels.
		(ix) Date of introduction of test organisms to test solutions and test duration.
		(x) The photoperiod and light source and description.
            (xi) Detailed information on feeding (e.g., type of feed, source, 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.
            (xii) Methods and sampling frequency of environmental monitoring performed during the study.
            (xiii) Sampling schedule for tissue and water samples and a description of the tissue and water samples analyzed.
		(xiv) Methods used to obtain, prepare and store tissue and water samples.
            (xv) Methods used in the analysis of concentrations of the test substance in water and in tissue should be described.  The accuracy of the method, method detection limit, and limit of quantitation should be given.
            (xvi) Methods used for measurements of total lipids in tissue.  The accuracy of the method, method detection limit, and limit of quantitation should be given.
	(6) Results.  
            (i) The percentage of oysters that died or showed any abnormal effects in the control and in each test vessel and whether or not spawning was observed.
            (ii) Environmental monitoring data results (e.g., test solution temperature, dissolved oxygen, light intensity, pH) in tabular form (provide raw data for measurements not made on a continuous basis), and descriptive statistics (mean, standard deviation, minimum, maximum).
		(iii) Results of total lipid measurements.
            (iv) The mean and range of the size of the oysters (i.e. valve height) and weight of the oyster tissue (shucked blotted dry) at the beginning and end of the uptake phase and at the end of the depuration phase.
      (v) Length of uptake and depuration phases and the rationale behind them. 
            (vi) Tabular summary of concentrations of parent compound in oyster tissue and exposure water by sampling time (raw data).  Tabular representation of data; Coy and Cw (with mean, standard deviation and range, if appropriate) for all sampling times (Coy expressed in mg/kg of wet weight (ppm) of oyster, and Cw expressed in mg/L (ppm)).  Cw values for the control series (background should also be reported).
		(vii) Graphical representations of uptake and depuration curves.
		(viii) The time to steady state in oysters.
            (ix) The time to 95% elimination of accumulated residues of the test substance from oysters.
		(x) Uptake and depuration rate constants with 95% confidence limits. 
            (xi) The steady state and kinetic BCF values (both expressed in relation to the whole body and the total lipid content, if measured), confidence limits and standard deviation (as available).
            (xii) Where radiolabeled test substances are used, and when BCF is greater than e.g. 500, the accumulation of any major degradates or metabolites at steady state or at the end of the uptake phase. 
            (xiii) Description of statistical method(s) used for calculation of the k1 and k2, including software package, and the basis for the choice of method.  Provide results of any goodness-of-fit tests.
      (xiv) Provide the results of any preliminary or supplementary studies performed.
            
(j) References.
      (1) American Society for Testing and Materials.  ASTM E 1022-94 (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) 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
      (4) Chiou, C.T. and D.W. Schmedding (1982)  Partitioning of organic compounds in octanol-water systems.  Environmental Science and Technology 16:4-10.
      (5) Compaan, H. 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). 
      (6) Environmental Protection Agency. 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). 
      (7) Ernst W.  Accumulation in Aquatic Organisms.  In: Appraisal of tests to predict the environmental behavior of chemicals.  Ed. by Sheehman P., Korte F., Klein W. and Bourdeau P.H.  Part 4.4 pp 243-255.  1985 SCOPE, John Wiley & Sons Ltd., New York (1985).
      (8) Food and Drug Administration. Pesticide analytical manual. Vol. 1. 5600 Fisher's Lane, Rockville, MD 20852, (1975). 
      (9) Gardner, W.S., W.A. Frez and E.A. Cichocki (1985) Micromethod for lipids in aquatic invertebrates. Limnology and Oceanography 30:1099-1105.
      (10) Hawker, D.W. and D.W. Connell (1986) Bioconcentration of lipophilic compounds by some aquatic organisms, Ecotoxicology and Environmental Safety 11:184-197.
      (11) 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.
      (12) Kristensen, P. and N. Nyholm. CEC. Bioaccumulation of chemical substances in fish: the flow-through method--Ring Test Programme, 1984-1985 Final report, March 1987.
      (13) Organization for Economic Cooperation and Development. Guidelines for testing of chemicals. Paris (1993). 
      (14) Organization for Economic Cooperation and Development. OECD Guidelines for Testing of Chemicals 211.  1998.  Daphnia magna Reproduction Test.  Annex 6. Calculation of a Time-Weighted Mean.
      (15) OECD Guidelines for Testing of Chemicals 305.  2012. Bioaccumulation in Fish: Aqueous and Dietary Exposure. Adopted October 2, 2012. 72 pp.
      (16) Randall R.C., H. Lee, R.J. Ozretich, J.L. Lake and R.J. Pruell (1991). Evaluation of selected lipid methods for normalizing pollutant bioaccumulation. Environ. Toxicol. Chem. Vol.10, pp. 1431-1436. 
      (17) Reilly P.M., R. Bajramovic, G.E. Blau, D.R. Branson, and M.W. Sauerhoff (1977)  Guidelines for the optimal design of experiments to estimate parameters in first order kinetic models. The Canadian Journal of Chemical Engineering 55:614-622.
      (18) Schimmel, S.C. and R.L. Garnas, Interlaboratory comparison of the ASTM bioconcentration test method using the eastern oyster, pp. 277-287. In R.C. Bahner and R.T. Hansen (eds.), Aquatic Toxicology and Hazard Assessment: Eighth Symposium, ASTM STP 891, American Society for Testing and Materials, Philadelphia, PA (1985).
      (19) Spacie, A. and J.L. Hamelink  (1982) Alternative models for describing the bioconcentration of organics in fish.  Environmental Toxicology and Chemistry 1:309-320.
      (20) United States Environmental Protection Agency (EPA).  2000.  Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, 2[nd] edition.  Office of Research and Development, Mid-Continent Ecology Division, Duluth, Minnesota and Office of Science and Technology, Office of Water, Washington, DC.  EPA 600/R-99/064.
      (21) United States Environmental Protection Agency (EPA).  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.
