.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–113 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1000 Special Considerations for Conducting Aquatic Laboratory Studies

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1000 Special considerations for conducting aquatic laboratory studies. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) [Reserved] (b) Introduction. (1) This guideline provides additional information on how to design and conduct aquatic laboratory studies with emphasis on the importance of adequate characterization of the test material and proper understanding of how the material behaves under test conditions. This guideline also attempts to interpret those areas that need to be defined and set limits for designing and conducting laboratory studies.(2) Agency guidance for performing aquatic testing sets forth a reasonable position and approach for testing criteria, limits, and standards. However, standards are set with the recognition that certain problems will arise and provisions must be made to accommodate unavoidable problems. This document provides for exceptions, while at the same time maintaining a high level of scientific integrity so that testing will provide information that is scientifically defensible and protective of the environment, while taking into consideration the chemistry of the test material. (c) General considerations. (1) Note that for aquatic toxicity testing,the solubility and stability of the test material must be known for the conditions under which it will be tested and chemical analysis of the batch test material must be performed. Determining the solubility and stability of the test material in the mixture or test solution is an important part of these studies. (2) The behavior of a test material should be based on experiments which are conducted under the same conditions as those occurring during the test. These include but are not limited to: (i) Test solution characteristics (salt or freshwater). (ii) Temperature, pH, conductivity, lighting. (iii) With test organisms in place. (iv) Use of the same test containers. (v) Use of the same flow-through systems where appropriate. (3) All chemistry methods used in preliminary trials, in range-findingtests, in establishing percent purity of batches of test material, or in measuring concentrations in test containers must be submitted with the study. The documentation must include a complete description of the method so

### 2 that a bench chemist can determine the necessary equipment and performthe analysis. It must also include the raw data, standards, and chromatograms from a representative analysis using the method. This representative analysis must be conducted with the specific media for which it will be used during the test—i.e., analysis should be performed undertest conditions. The actual limit of detection (LOD) and limit of quantification (LOQ) must be identified. (d) Definitions.

EEC is the effective environmental concentration.

LOD is the limit of detection below which the qualitative presence of the material is uncertain.

LOEC is the lowest-observable-effect-concentration. LOQ is the limit of quantification below which the quantitative amount of the material is uncertain relative the amount.
Measured concentration is an analytically derived measure above the LOQ.
NOEC is the no-observed-effect-concentration.
Nominal concentration is, for aquatic tests, the nominal test level, which is the concentration that would exist if all test material added to the test solution was completely dissolved and did not dissipate in any way. Recommended means that the procedure or test is preferred in order to avoid problems, but it is not required. If the recommended procedure or test is not performed, the study will not necessarily be rejected. Solubility is defined as the amount of chemical retained in the supernatant of a conventionally centrifuged sample of test medium. (e) Stability. (1) A test material is considered to be stable under testconditions if, under those conditions, it does not degrade, volatilize, dissipate, precipitate, sorb to test container walls, or otherwise decline to concentrations less than 70 percent of the day–0 measured concentration during the study period. If it is expected to decline to less than 70 percent of the day–0 measured concentration during the study period, either static renewal or flow-through design is needed to try to ensure that the test concentration is maintained at levels greater than or equal to 70 percent. The only exception is testing with algae and diatoms, which cannot betested in static renewal or flow-through systems (see discussion in paragraph (m) of this guideline on testing with algae and diatoms).(2) Static renewal is one method to ensure relatively continuous concentrations when the test material is not stable under test conditions. At

### 3 a minimum, the renewal cycle should be based on the stability of the test material under test conditions. The time to renewal (renewal cycle) should be shorter than the time it takes for the concentration of the test material to decline to < 70 percent. (The renewal cycle may be shorter than required by stability characteristics of the test material because of other factors, such as dissolved oxygen, feeding, etc.) (f) Sample storage. If samples of growth medium, stock solutions,or test solutions collected for chemical analysis cannot be analyzed immediately, they should be handled and stored appropriately to minimize loss of the test material. Loss could be caused by such processes as microbialdegradation, hydrolysis, oxidation, photolysis, reduction, sorption, or volatilization. Stability determination under storage conditions, whether it refers to storing the test material before testing or storing samples awaiting analysis, is required by GLP regulation. (g) Preliminary trials. (1) The Agency recommends preliminary testing for problem chemicals. The information about stability and solubilityof problem chemicals should be developed under test conditions. This information can be gained while doing the currently required range-finding studies. A list of recommended preliminary tests is as follows: (i) Stability trials should be conducted under test conditions. These trials must be documented and submitted to the Agency for review with the study to which they apply. (ii) Solubility trials should be conducted under test conditions. These trials must be documented and submitted with the study to the Agency for review. Surfactants and charged polymers will be self-dispersing in water and should be tested at or below their dispersability limits. (iii) If solubility is a problem (<100 ppm), trials should be conductedunder test conditions using various solvents that are most likely to be effective and that are widely recognized as being nontoxic and other means to ensure that the appropriate methods are used during the laboratory tests to enhance solubility. Once a solvent is chosen based upon more simplistic,comparative evaluations, the decision should be confirmed in the preliminary trials with only that solvent. (iv) Chemical analysis methods as detailed in paragraph (l) of this guideline. (v) Stability of the test material in the samples to be collected for chemical analyses should be determined during the laboratory studies. This includes determining whether and how samples can be stored for future analysis.

### 4(2) Laboratory studies must be designed taking into account this preliminary information. This means the trials described are to be conducted before the definitive laboratory studies are initiated. (h) Toxicity tests with poorly soluble materials. (1) Existing OPP guidelines for aquatic toxicity tests require that chemicals be tested up to a maximum dissolved concentration of 100 ppm (milligrams per liter) for pesticides or 1,000 ppm for industrial chemicals in an effort to obtain an LC50 or EC50. This amount of test material is considered to representa conservative measure of the most bioavailable fraction, which may include some colloidal material not removed by centrifugation in addition to the truly dissolved fraction. (2) Applicants must demonstrate the technique used to maximizechemical dissolution in the test media under standard conditions. Consideration of the optimum technique should include use of nontoxic solvents,saturation (solubility) columns, sonication, minor adjustments to environmental conditions (i.e., temperature, pH, etc.), as appropriate. Minor adjustments should not extend outside the recommended range of conditions for the specific test organism. (3) Current policy allows chemicals that are poorly soluble (solubility <100 ppm) or dispersible in water to be tested up to the maximum water solubility or dispersibility limit obtainable for the given test conditions employed, provided that certain prerequisites apply:(i) Concentrations of test chemical in test media are measured at appropriate intervals and from appropriate test chambers of all test levelsare determined from centrifuged supernatant or other appropriate separation (e.g., filtrate). Self-dispersing industrial chemicals (e.g., surfactants, detergents, or charged [polymers) should be sampled directly. (ii) Testing is also performed with a more soluble formulation e.g.,emulsifiable concentrate, if one exists (in addition to testing with the technical-grade material). Testing with a more soluble formulation will not, however, be required if it does not provide a twofold increase in solubility.(4) Studies that involve radical changes in environmental test conditions outside the recommended range of values for temperature, salinity, pH, etc., will be considered on a case-by-case basis. (i) Methods for solubility enhancement—(1) Saturator columns. The use of saturation columns as an aid in the dissolution of test material and in confirming maximum solubility is recommended but not required for nonvolatile test chemicals with test media solubilities of 10 ppm or less. Methods for using these columns in aquatic toxicity tests can be adapted from the methods established for their use in determining water solubility under OECD’s Column Elution Method (see OPPTS 830.7840). Saturator columns may be considered to generate test solutions for static

### 5 studies or for flow-through studies. Furthermore, saturator columns for these studies need only be considered if conventional techniques for dosing the water do not result in water concentrations within twice the stated solubility of the compound. (2) Emulsifiers and formulation testing. Testing with a more soluble formulation, if one exists and which may contain emulsifiers, dispersants, solubilizing agents, etc., is required for all active ingredients subject to aquatic organism testing and having a water solubility less than100 ppm and less than an EC/LC50. A defined EC/LC50 provides a greatly improved basis for risk assessment. (3) Effect of temperature. Solubility is a function of temperatureand is especially sensitive at the limits of solubility. Generally, below saturation, increases of as much as 10 ° C may affect the solubility up to a factor of 2. However if test solutions are close to saturation, small changes in temperature may result in supersaturated solutions. In addition, control of temperature is important because of its well-known effects on the actual toxicity of the compound. (4) Centrifugation. Conventional centrifugation is required for alltest media where undissolved test material, precipitate, flocculant, or colloidal suspension except for surfactants or charged polymers) are observed in the test chambers or where the solubility and (hence bioavailability)are in question. Filtration may be used instead of centrifugation if the analytical method is validated over a range of acceptable concentrations. (j) Measurement at initiation and termination of testing—(1) Initial analysis. (i) Analysis at the 0–hour: A 30–min interval is generally required between the addition of the test substance and the introductionof the test organisms. 0-hour measurement should be made when test organisms are added. Industry will have to justify an exception from the 30– min requirement for adding test organisms if the characteristics of the test material and test system require a longer equilibration time. If preliminary trials have been performed, this delay should be predictable.(ii) In flow-through tests, the study should be conducted with knowledge of the time it will take for the test material to reach equilibrium or steady-state in the test container. Initiation of the test and scheduling of the sampling times must be based on this information. In some cases, a flow-through system may have to be run for an extended-time pretestin an attempt to achieve equilibrium or steady-state conditions. If equilibrium or steady-state cannot be achieved, and/or it appears that the measured concentrations will be substantially below (< 70 percent) nominal, the study report should reflect that the laboratory was aware of this problem. The study report should clearly identify the problem, indicate the stepstaken to mitigate it and justify the study design and dosing levels. However, if sufficient analytical methods are available and acceptable toxicity

### 6 data are produced, additional testing and evaluation with the sole objective of obtaining initial measured concentrations greater than 70 percent of nominal will not be required. (2) Analysis at test termination. Where indicated, measurement at test termination is considered necessary to determine if the test organisms were exposed to the test material throughout the entire study and at what levels. A significant change in test concentration during the last part ofthe study may substantially alter the results. For example, if the test concentration dropped dramatically during the last few days of a study, the effects that may have been caused by such exposure may not occur. The EC50 or LC50 developed from that study would be misleading if it is called a ‘‘96–h LC50’’. (k) Replicates and concentration measurement. (1) Average concentrations of replicates are used in regression analysis. When replicate test containers and measurement of test concentration are required, each replicate in each test concentration must be analyzed separately becausethe responses in each replicate are viewed as independent and it is necessary to know what the concentrations were so variation can be determined. Exceptions to this occur when: (i) Replicate treatment containers under static tests or static renewal conditions are filled from a bulk preparation. In this case, only samples from the bulk supply for each test level must be analyzed. (ii) A ‘‘splitter’’ is used in a flow-through test to feed more than one replicate. In this case, only samples from one replicate per treatment level require analysis. It is recommended that samples be collected from all replicates and be stored in case anomalous concentrations are measured in the one that is analyzed. Analyzing the other replicates may shed light on the cause and extent of the anomalous measurements. (2) Replicates receiving flow from a splitter should be sampled and analyzed alternately. In other words, if there are two replicates (A and B), replicate A should be analyzed in the first week and replicate B in the second week, etc. (3) To the extent possible, variability in measured concentrations should be minimized. The goal for limiting variability of measurements between replicates of the same concentration, and over time in the same concentration, is maintaining the ratio of the highest concentration to the lowest concentration at 1.5:1 or less. Generally, variability above this amount is not acceptable. (4) An important factor in considering the limits of variability is the avoidance of overlapping mean test concentrations between test levels. High variability puts into question the reliability of the environmental chemistry method and/or the concentrations on which to base statistical

### 7 analysis and toxicological conclusions. If variability beyond the 1.5:1 ratio occurs, an exception to it should be justified. (5) This justification should clearly state the problem, explain why it occurred, provide scientific justification, and identify all measures takento mitigate the problem. The justification also should include the fully developed chemistry method, including the documentation necessary for a bench chemist to review and evaluate it. (6) For cases in which variability problems are suspected, preliminary trials are strongly recommended. If it becomes clear that high variability cannot be avoided, an exception should be justified. Any justificationshould be provided in advance. Agency scientists will decide on the validity of the rationale for the exception, and may recommend other methods to reduce potential variability. (l) Use of chemical analysis to confirm exposure in aquatic testing—(1) Acute static tests. Except for acute aquatic algae and diatom studies (which can only be conducted as static tests), acute static tests may be conducted only if, among other things, the test material has been shown to be stable under the test conditions, as defined in paragraph (e)of this guideline. (Other factors not addressed in this guideline may preclude conducting a static test even if the test material is stable under test conditions. These include, but are not limited to, problems in maintaining dissolved oxygen levels, feeding requirements, and concern for bacterial/ microbial contaminants.) In an acute static test with a test material that is stable and readily soluble at the treatment levels, measurements of each test concentration are not absolutely required. However: (i) For static tests, the concentration of toxicant should be measuredat the beginning and end of the test in all test chambers. Further, measurement of the toxicant’s degradation products is desirable, but not required. (ii) The study may be rejected if the following occurs: (A) The test material was not stable under test conditions. (B) Precipitates formed. (C) Solubility was likely to have been a problem at the levels tested. (iii) If the recommended chemical measurements were made to verify exposure levels, the study may not be rejected. Whether the study design was modified in a scientifically defensible attempt to accommodate these chemical characteristics will also be considered. (iv) If variability is expected to be a problem, it is recommended that measurements of test concentrations be made at each test level at 0–hour, 48–h and, for tests longer than 48 h, at test termination. Replicate

### 8 test containers should be measured separately, except as explained under paragraph (k) of this guideline. (2) Acute static renewal. Refer to the general discussion of replicates under paragraph (k) of this guideline. If a static renewal test is conducted, each test chamber must be sampled for chemical analysis at the 0–hour,at the end of the first (or longest) cycle, and at test termination. It is recommended that measurements be made at the end of each renewal cycle acute flow-through. (3) Acute flow-through. If a flow-through test is conducted, each test concentration must be measured at the 0–hour and at test termination. It is recommended that for 96–h tests, an intermediate measurement be made at 48–h to verify midtest exposure if variability is expected to be a problem. (All acute aquatic algae and diatom tests must be conducted as static. Flow-through and static renewal systems are not recommended for these tests, since they are conducted with microscopic organisms that cannot be protected from loss when renewing or draining water from the test containers. Static tests for Lemna gibba can be conducted, regardless of stability.) (4) Chronic static renewal. Refer to the general discussion of replicates under paragraph (k) of this guideline. Concentrations must be measured at each test level at 0–hour, at the end of the last renewal cycle (at test termination), and at the beginning and end of an intervening cycle at least once per week. The longest cycle in a sequence should be used if variable-cycle periods are employed. (5) Chronic flow-through. Refer to the general discussion of replicates under paragraph (k) of this guideline. In each concentration, measure at 0–hour, every 7 days, and at test termination. At the beginning of a study, the exact flow of the system and water output at each splittermust be documented. In addition, system flow must be metered and monitored visually or mechanically on a daily basis (every 24 h), and it is recommended that the system flow be metered and monitored twice a day (approximately every 12 h). Measurement of test concentration is required each time metering fluctuation or malfunction is detected or observed. A record of the regular inspections must be maintained and provided with the study report. (m) Measured concentrations versus nominal concentrations. This section describes acceptable limits of deviation of measured from nominal concentrations. (1) Test endpoints are used as if the organisms were exposed to the test material at the statistically developed value (LC50 or EC50) for theentire test duration. One aspect of the risk assessment is to compare concern levels based on the LC50 is to initial immediate concentrations. However, field conditions may exist in which concentrations that may be of

### 9 acute concern may last longer or occur frequently enough to be comparable to the 48–h, 96–h or 120–h test duration. Even though a pesticide may degrade rapidly under one environmental condition (in water, for example),the possibility of repeated exposure needs to be considered. Repeated exposure from reservoirs of the active ingredient, occurring in environmental compartments where persistence is greater, may occur. The Agency takes these eventualities into account in order to generate risk assessments that adequately address hazard to the aquatic ecosystem. (2) Presumably, a safer chemical one that may degrade rapidly, has low solubility, and is used at low rates. While these characteristics may result in lower exposure levels in the field, the risk they represent can only be determined if the actual toxicity of the pesticide is known or thelevel below which the pesticide is not likely to result in 50 percent mortality (i.e., an LC50 > X-concentration situation.) When potentially low, realistic exposure levels are calculated and used for risk assessments, it isimperative that the actual toxicity of the pesticide at those levels be determined. If the test is conducted using nominal concentrations, the results could reflect a higher apparent effect concentration (e.g. LC50, EC50, or NOEL). As a result, potential risk may be missed because the comparison would be between a low ‘‘realistic’’ exposure and a high nominal test level that was not the true toxicity level. A risk assessment based on such a comparison and data would be faulty and could not be scientifically defended. (3) Pesticide chemicals that are used at very low levels tend to have high biological activity. For this reason, it is imperative that the toxicitydata developed for these pesticides be accurate and scientifically defensible. (4) Measured concentrations are used when they are available because they indicate what the exposure was in the test chambers. When measured concentrations are indicated, they are considered necessary because: (i) There are concerns that the actual concentrations to which the test organisms are exposed may differ from ‘‘nominal.’’ This variation maybe due to chemical characteristics, test conditions, or mechanical apparatus. (ii) Measured concentrations confirm that the test system was designed appropriately and is operating acceptably. Characteristics that make testing difficult (low solubility, short half-life, high binding potential, etc.) must be accounted for in the exposure estimates. They are not a reason for developing misleading toxicity values from laboratory tests.(5) Measurement of test concentrations is not performed just to determine if the technician knows how to mix the test solution once. Among other things, it also ensures that the test solution was mixed correctly each

### 10 time. It corroborates the precision of the technician or mechanics of the test system. (6) If test levels are not measured, the nominal values are used tocalculate the LC50, EC50, NOEC and LOEC. If the test material has degraded or has become unavailable because of insolubility or sorption, the pesticide may be characterized as less toxic than it really is. For example, if based on nominal test levels, the LC50 is 5 ppm, the pesticide would be considered moderately toxic. No higher tier testing would be required and that value (5 ppm) would be the basis for developing concern levels with which to compare EECs. But if, in reality, the concentrations to which the organism was actually exposed were only between 0.1 and 1 ppm, the LC50 may well be closer 0.5 ppm. For pesticides this would result in labeling, and could trigger higher tier tests. More importantly, it would yield substantially lower concern levels with which to compare exposure levels. (7) When a laboratory test design has been specifically modified to accommodate the instability of test material or other factors likely to cause variability in test concentrations, and the design is judged adequate based on sufficient preliminary information, the study will not be rejected solelyon the grounds that measured concentrations varied by more than 30 percent of the nominal concentration. (This assumes that the preliminary stability tests were conducted under test conditions essentially identical to the actual test conditions.) An increase in measured test concentration of more than 30 percent from the nominal concentration during the test will generally not result in rejection, provided that the following conditions are met: (i) A reasonable and scientific explanation is given, and the variabilityof results produced by the chemical analysis method is adequately characterized. (ii) All test containers exhibit a similar (but not necessarily identical)shift. (If concentrations in some containers go up substantially (>30 percent) and test concentrations in other containers go down substantially (>30 percent), they will not be considered to have exhibited a similar shift. The most important criterion is that test levels must not experience a shift in ‘‘order.’’ That is, the highest test level should remain highest, the next should remain second, etc. If orders are shifted, the test may be rejected, since regression analysis would not yield statistically sound median lethal concentrations and confidence limits.) (iii) The variability of the measured concentrations is acceptable. (iv) A statistically valid endpoint can be derived from the measured concentrations (either an LC50, EC50, or that the LC50 or EC50 is greater than 100 ppm).

### 11 (v) The preliminary stability information is provided with complete documentation and description of methods used to derive such information. (8) In some cases, high variability cannot be avoided because the testconcentrations are approaching the limit of detection or because of unavoidable binding of the test material to the chemical analysis apparatus.When the ratio of the highest concentration to the lowest measured concentration is expected to vary by more than 1.5, the registrant is stronglyadvised to justify an exception to this requirement in advance of conducting the aquatic laboratory studies. This exception justification should consist of: (i) Documentation of the preliminary trials indicating this problem. (ii) The specific steps that will be taken to reduce the variation. (iii) The fully developed chemical analysis method. (iv) The raw data, standards, and chromatogram from a representativeanalysis using the method. For each chemistry method, the actual minimum detection level and level of quantification must be identified.(9) The Agency will decide on each exception justification on a caseby-case basis. However, if a series of aquatic tests are to be conducted with one chemical and it is anticipated that these limits will be exceeded, one exception justification may cover more than one study. The Agency will then exercise judgment in evaluating studies with test materials that are difficult to measure. (10) Conducting flow-through or static renewal tests with aquatic algae is not feasible with the current state of the practice. Therefore, the following is recommended for a test material that, based on preliminarystability testing, is expected to degrade to less than 70 percent of the nominal concentration. The study should be conducted normally, with concentrations measured at 0-hour and at test termination. Although it is undesirable to allow the concentrations to decline throughout the study, the problem may be unavoidable. In this case, the LC50 regression analysisis based on the mean measured concentration. If the concentration is expected to decline to less than the minimum detection level before the end of the study, then it is recommended that interim chemical measurements be made to determine the decline rate. (11) For purposes of consistency, the aquatic test with a vascular plant (Lemna gibba ) need not be done using a flow-through or static renewal system with the sole purpose of maintaining test concentrations. There may be other reasons for conducting a static renewal study..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–114 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1010 Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1010 Aquatic invertebrate acute toxicity test, freshwater daphnids. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline are 40 CFR 797.1300 Daphnid Acute Toxicity Test; OPP 72–2 Acute Toxicity Test for Freshwater Aquatic Invertebrates (Pesticide Assessment Guidelines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024, 1982; and OECD 202 Daphnia sp. Acute Immobilisation Test and Reproduction Test. (b) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures (‘‘chemicals’’) subject to environmental effects test regulations. This guideline prescribes an acute toxicity test in which daphnids (Daphnia magna or D. pulex)are exposed to a chemical in static and flow-through systems. The Environmental Protection Agency will use data from this test in assessing the hazard a chemical may present in the aquatic environment. (c) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA) and 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. In addition, the following definitions apply to this guideline:

Brood stock means the animals which are cultured to produce test organisms through reproduction.

EC50 means that experimentally derived concentration of test substance in dilution water that is calculated to affect 50 percent of a test population during continuous exposure over a specified period of time. In this guideline, the effect measured is immobilization. Ephippium means a resting egg which develops in daphnids under the carapace in response to stress conditions. Flow-through means a continuous or an intermittent passage of test solution or dilution water through a test chamber or culture tank with no recycling. Immobilization means the lack of movement by the test organisms. Loading means the ratio of daphnid biomass (grams, wet weight) or number of daphnids to the volume (liters) of test solution in a test chamber at a point in time, or passing through the test chamber during a specific interval.

### 2 Static system means a test system in which the test solution and test organisms are placed in the test chamber and kept there for the duration of the test without renewal of the test solution. Static-renewal system means a static test system in which the test solution is renewed every 24 h. (d) Test procedures—(1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. In the flow-through test, the flow of dilution water through each chamber is adjusted to the rate desired. The test chemical is introduced into each treatment chamber. The addition of test chemical in the flow-through system is conducted ata rate which is sufficient to establish and maintain the desired concentration in the test chamber. The test is started within 30 min after the test chemical has been added and uniformly distributed in static test chambersor after the concentration of test chemical in each flow-through test chamber reaches the prescribed level and remains stable. In static-renewal testing the dilution water and test chamber are renewed periodically. At the initiation of the test, daphnids which have been cultured and acclimatedin accordance with the test design are randomly placed into the test chambers. Daphnids in the test chambers are observed periodically during the test, the immobile daphnids removed, and the findings recorded.(ii) Dissolved oxygen concentration (DOC), pH, temperature, the concentration of test chemical and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to develop concentration-response curves and determine EC50 values for the test chemical at the end of 24 and 48 h. (2) Range-finding test. (i) A range-finding test should be conducted to establish test solution concentrations for the definitive test. (ii) The daphnids should be exposed to a series of widely spaced concentrations of the test chemical (e.g. 1, 10, 100 mg/L, etc.).(iii) A minimum of five daphnids should be exposed to each concentration of test chemical for a period of 48 h. The exposure period may be shortened if data suitable for the purpose of the range-finding test canbe obtained in less time. No replicates are required and nominal concentrations of the chemical are acceptable. (3) Definitive test. (i) The purpose of the definitive test is to determine the concentration-response curves and the 24– and 48–h EC50 values. (ii) A minimum of 20 daphnids per concentration should be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, and 64 mg/L). An equal number of daphnids should be placed in two or more

### 3 replicates. If solvents, solubilizing agents, or emulsifiers have to be used,they should be commonly used carriers and should not possess a synergistic or antagonistic effect on the toxicity of the test chemical. If carriersare absolutely necessary, the amount used should be the minimum necessary to achieve solution of the test substance. Triethylene glycol and dimethyl formamide are preferred, but ethanol and acetone can be used if necessary. Carrier concentrations should be kept constant at all treatmentlevels. The concentration of solvent should not exceed 100 mg/L. The concentration ranges should be selected to determine the concentration-response curves and EC50 values at 24 and 48 h. Concentration of test chemical in test solutions should be analyzed prior to use. (iii) Every test should include controls consisting of the same dilution water, conditions, and procedures, and daphnids from the same population (culture container), except that none of the test chemical is added.(iv) The DOC, temperature, and pH should be measured at the beginning and end of the test in each chamber. (v) The test duration is 48 h. The test is unacceptable if more than 10 percent of the control organisms are immobilized during the 48–h test period. Each test chamber should be checked for immobilized daphnids at 24 and 48 h after the beginning of the test. Concentration-responsecurves and 24–h and 48–h EC50 values for immobilization should be determined along with their 95 percent confidence limits. (vi) In addition to immobility, any abnormal behavior or appearance should also be reported. (vii) Test organisms should be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shouldbe positioned in a random manner or in a way in which appropriate statistical analyses can be used to determine the variation due to placement. (viii) The concentration of the test chemical in the chambers should be measured as often as is feasible during the test. In the static test the concentration of test chemical should be measured in each test chamberat a minimum at the beginning and at the end of the test. In the staticrenewal test, the test concentration of test chemicals should be measured in each test chamber at a minimum at the beginning and at the end ofthe renewal period. In the flow-through test the concentration of test chemical should be measured in each chamber at a minimum at the beginningof the test and at 48 h after the start of the test, and in at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Among replicate test chambers of a treatment concentration, the measured concentration of the test chemical should not vary more than ± 20 percent.

### 4 (4) Analytical measurements—(i) Test chemical. Deionized water should be used in making stock solutions of the test chemical. Standard analytical methods should be used whenever available in performing theanalyses. The analytical method used to measure the amount of test chemical in a sample should be validated before beginning the test by appropriate laboratory practices. Any analytical method is not acceptable if likely degradation products of the test chemical, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and mathematically corrected. (ii) Numerical. The number of immobilized daphnids should be counted during each definitive test. Appropriate statistical analyses should provide a goodness-of-fit determination for the concentration-response curves. A 24– and 48–h EC50 and corresponding 95 percent interval should be calculated. (e) Test conditions—(1) Test species—(i) Selection. (A) The cladocerans, D. magna or D. pulex, are the test species to be used in this test. Either species may be used for testing of a particular chemical. The species identity of the test organisms should be verified using appropriate systematic keys. First instar daphnids, ≤ 24 h old, are to be used to start the test. (B) Daphnids to be used in acute toxicity tests should be cultured at the test facility. Records should be kept regarding the source of the initial stock and culturing techniques. All organisms used for a particular test should have originated from the same culture population.(C) Stock daphnids may be tested periodically to determine any genetic changes in the populations which may alter the sensitivity to test chemicals. (D) Daphnids should not be used for a test: ( 1 ) If cultures contain ephippia. (2 ) If adults in the cultures do not produce young before day 12. (3 ) If more than 20 percent of the culture stock die during the 2 days preceding the test. (4 ) If adults in the culture do not produce an average of at least three young per adult per day over the 7–day period prior to the test. (5) If daphnids have been used in any portion of a previous test, either in a treatment or in a control. (ii) Acclimation. (A) Brood daphnids should be maintained in 100-percent dilution water at the test temperature for at least 48 h prior to the start of the test. This is easily accomplished by culturing them in

### 5 the dilution water at the test temperature. During production of neonates, daphnids should not be fed. (B) During culturing and acclimation to the dilution water, daphnidsshould be maintained in facilities with background colors and light intensities similar to those of the testing area. (iii) Care and handling. (A) Daphnids should be cultured in dilution water under environmental conditions similar to those used in the test.Organisms should be handled as little as possible. When handling is necessary it should be done as gently, carefully, and quickly as possible. During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, physical damage, and mortality. Dead and abnormal individuals should be discarded. Organisms that touch dry surfaces or are dropped or injured in handling should be discarded.(B) Smooth glass tubes (I.D. greater than 5 mm), equipped with rubber bulbs, should be used for transferring daphnids with minimal culture media carry-over. Care should be exercised to introduce the daphnids below the surface of any solution to avoid trapping air under the carapace. (iv) Feeding. A variety of foods (e.g. unicellular green algae) have been demonstrated to be adequate for daphnid culture. Daphnids should not be fed during testing. (2) Facilities—(i) Apparatus. (A) Facilities needed to perform this test include: ( 1 ) Containers for culturing and acclimating daphnids. (2 ) A mechanism for controlling and maintaining the water temperature during the culturing, acclimation, and test periods. (3 ) Apparatus for straining particulate matter, removing gas bubbles, or aerating the water as necessary. (4 ) An apparatus for providing a 16–h light and 8–h dark photoperiod with a 15– to 30–min transition period. (5) In addition, the flow-through system should contain appropriate test chambers in which to expose daphnids to the test chemical and an appropriate test substance delivery system.(B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organisms. (C) Test chambers should be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions.

### 6 (ii) Construction materials. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from the dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect the test results. (B) For static tests, daphnids can be conveniently exposed to the test chemical in 250–mL beakers or other suitable containers.(C) For flow-through tests, daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. The containers should be suspended in the test chamber in such a manner to ensure that the test solution flows regularly into and out of the container and that the daphnids are always submerged in at least 5 cm of test solution. Test chambers can be constructed using 250–mL beakers or other suitable containers equipped with screened overflow holes, standpipes, or V-shaped notches. (iii) Dilution water. (A) Surface or ground water, reconstituted water or dechlorinated tap water are acceptable as dilution water if daphnids will survive in it for the duration of the culturing, acclimation, and testing periods without showing signs of stress. The quality of the dilution water should be constant and should meet the specifications in the following Table 1.: Table 1.—Water Quality Parameters

Substance Maximum concentration

Hardness as CaCO 3 ......................................................................................... 180 mg/L Particulate matter

............................................................................................. 20 mg/L Total organic carbon or

.................................................................................... 2 mg/L Chemical oxygen demand

........................................................................ 5 mg/L Un-ionized ammonia

........................................................................................ 20 μg/L Residual chlorine

.............................................................................................. < 3 μg/L Total organophosphorus pesticides

................................................................. 50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or

..... 50 ng/L Organic chlorine

........................................................................................ 25 ng/L

(B) The water quality parameters should be measured at least twice a year or whenever it is suspected that these characteristics may have changed significantly. If dechlorinated tap water is used, daily chlorine analysis should be performed. (C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilledwater. Glass distilled or carbon-filtered deionized water with a conductivity less than 0.1 mS/m is acceptable as the diluent for making reconstituted water.

### 7 (iv) Cleaning. All test equipment and test chambers should be cleaned before each use using standard laboratory procedures. (v) Test substance delivery system. In flow-through tests, proportional diluters, metering pump systems, or other suitable devices should be used to deliver test chemical to the test chambers. The system should be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test chemical in each chamber. The general operation of the test substance delivery systemshould be checked twice during a test. The 24–h flow through a test chamber should be equal to at least 5 × the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent from any one test chamber to another. (3) Test parameters. Environmental parameters of the water contained in test chambers should be maintained as specified below: (i) The test temperature should be 20 ° C. Excursions from the test temperature should be no greater than ± 2 ° C. (ii) DOC between 60 and 105 percent saturation. Do not aerate daphnid toxicity tests. A single air bubble can get under the carapace of the daphnid and kill it, or float the daphnid to the surface where it will get trapped. (iii) The number of daphnids placed in a test chamber should not affect test results. Loading should not exceed 40 daphnids per liter of test solution in the static system. In the flow-through test, loading limits will vary depending on the flow rate of dilution water. Loading should not cause the DOC to fall below the recommended levels. (iv) Photoperiod of 16 h light and 8 h darkness. (f) Reporting. The sponsor must submit to the EPA all data developed by the test that are suggestive or predictive of acute toxicity andall concomitant gross toxicological manifestations. In addition to the reporting requirements prescribed in 40 CFR Part 792—Good Laboratory Practice Standards, the reporting of test data should include the following: (1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing. (2) A detailed description of the test chemical including its source, lot number, composition (identity and concentration of major ingredients(percent active ingredient of chemical) and major impurities), known physical and chemical properties and any carriers or other additives used and their concentrations.

### 8 (3) The source of the dilution water, its chemical characteristics (e.g. conductivity, hardness, pH, etc.), and a description of any pretreatment, carriers and/or additives used, and their concentrations. (4) Carriers and/or additives used and their concentrations.(5) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture method. The age of the daphnids used in the test should be reported. (6) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g. conditioning, test chemical additions), number of test organisms per test chamber, number of replicatesper treatment, lighting, method of test chemical introduction or test substance delivery system, renewal schedule (in static-renewal tests), and flow rate (in flow-through test) expressed as volume additions per 24 h. (7) The concentration of the test chemical in each test chamber at times designated for static and flow-through tests. (8) The number and percentage of organisms that were immobilized or showed any adverse effects in each test chamber at each observation period.(9) Utilizing the average measured test chemical concentration, concentration-response curves should be fitted to immobilization data at 24 and 48 h. A statistical test of goodness-of-fit should be performed and the results reported. (10) The 24– and 48–h EC50 values and their respective 95 percent confidence limits using the mean measured test chemical concentration,and the methods used to calculate both the EC50 values and their confidence limits.(11) All chemical analyses of water quality and test chemical concentrations, including methods, method validations, and reagent blanks.(12) The data records of the culture, acclimation, and test temperatures. (13) Any deviation from this test guideline and anything unusual about the test, e.g. diluter failure, temperature fluctuations, etc. (14) If it is observed that the stability or homogeneity of the test substance cannot be maintained, care should be taken in the interpretation of the results, and note made that the results may not be reproducible..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–118 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1075 Fish Acute Toxicity Test, Freshwater and Marine

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1075 Fish acute toxicity test, freshwater and marine. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline are 40 CFR 797.1400 Fish Acute Toxicity Test; OPP 72–1 Acute Toxicity Test for Freshwater Fish and 72–3 Acute Toxicity Test for Estuarine and Marine Organisms (Pesticide AssessmentGuidelines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09-82-024, 1982; and OECD 203 Fish Acute Toxicity Test. (b) Purpose. The purpose of the acute toxicity test with fish species is to help in the assessment of possible risk to similar species in natural environments, as an aid in determination of possible water quality criteria for regulatory purposes, and for use in correlation with acute testing ofother species for comparative purposes. Data on a cold and warm freshwater species are generally required. The rainbow trout, Oncorhynchus mykiss , and bluegill sunfish, Lepomis macrochirus , are preferred species to meet this requirement since they are sensitive indicator species and alarge data base which characterizes the response to environmental contaminants is available. Other species as identified in paragraph (e)(4)(i)(A) of this guideline may be used. However, under certain circumstances, when potential environmental exposures may lead to significant risks, data on the preferred species may be required for risk assessment purposes so that the Agency can conduct comparative analyses with alternative chemical substances. Historically, it appears that many chemical classes are subjectto comparative analyses. Development of a good data base could ultimately result in the use of other species in comparative analyses. In anycase, the results of such a study should not be construed to represent behavior of the test material in the natural environment where other factors may come into play, but rather as a indicator of effects which might occur under comparable conditions as those utilized in the study. (c) Principle of the test—(1) Definitive test. The goal of the definitive test is to determine concentration-response curves for fish mortality, the LC50’s, and the 95 percent confidence intervals for each species tested at 24, 48, 72, and 96 h in a static, static-renewal, or flow-through system. (2) Range-finding or limit testing. Definitive testing may be waived if limit testing with at least 30 organisms shows LC50 levels to be greater than 1,000 mg/L based on 100 percent active ingredients (AI), or the limits of water solubility or dispersibility. For pesticides, a lower level of 100 mg AI/L may be tested when estimated environmental concentrations are not expected to exceed 100 mg/L (ppm) as might occur with pesticide use. Prior to selection of definitive test concentrations it may be advisable

### 2 to conduct a range-finding test. Results of any range-finding and limit tests should be reported with results of the definitive test. (3) Information on the test substance. The material to be testedshould be technical grade unless the test is designed to test a specific formulation, mixture, or effluent. The degree of purity must be recorded for technical ingredients and mixtures. The percentage of each impurity shouldbe reported and percentages should total 100 percent. A complete description of physicochemical characteristics (i.e. solubility, vapor pressure, hydrolysis in pH 5, 7, and 9) should be included with description of theAI used in specific chemical testing. A reliable analytical method for quantification of test substance concentrations must be available. (d) Validity of the test. (1) Maximum-allowable control or solvent control mortality is 10 percent (or 1 mortality if 7 to 10 control fish are used) for a 96–h period of testing. If the test is continued past 96 h, the maximum-allowable additional mortality is 10 percent.(2) Constant conditions must be maintained throughout the test period. Flow-through procedures are preferred over static-renewal or semistatic procedures and static-renewal procedures are preferred over a static test procedure. (3) In static tests, the dissolved oxygen (DO) in each replicate should at all times be greater than 60 percent saturation. In flow-through tests, the DO should be maintained above 75 percent saturation.(4) Measured concentrations are required if the test chemical is unstable or a flow-through diluter system is employed. Exception may be made in cases where hydrolysis studies indicate chemical to be stable (< 5 percent degradation) in 96 h at a pH comparable to test dilution water. In any case there must be evidence that test concentrations remained at least 80 percent of the nominal concentrations throughout the test or that mean measured concentrations are an accurate representation of exposure levels maintained throughout the test period. (e) Description of the method—(1) Apparatus. Normal laboratory equipment and especially the following is necessary: (i) Equipment for determination of water hardness, etc. (ii) Adequate apparatus for temperature control. (iii) Tanks constructed of chemically inert material and of suitable capacity to allow recommended loading levels. (2) Water. (i) Clean surface or ground water, seawater (for estuarine or marine species), and reconstituted water are acceptable as dilution water. Dechlorinated water should not be used because some forms of chlorination are difficult to remove adequately. If dechlorinated tap water

### 3 is used, then daily chlorine analysis should be performed. Reconstituted or natural water is preferred.(ii) Chemical analysis of water used in testing should include the following elements and limitations on maximum concentrations based on at least biannual testing:

Substance Maximum concentration

Particulate matter

................................................................................... 20.0 mg/L Chemical oxygen demand (COD)

.......................................................... 5.0 mg/L Total organic carbon (TOC)

................................................................... 2.0 mg/L Boron and fluoride

.................................................................................. <100.0 mg/L Residual chlorine

.................................................................................... 0.003 mg/L Un-ionized ammonia

.............................................................................. 0.020 mg/L Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, and zinc

. 0.001 mg/L Cadmium, mercury, and silver

............................................................... <0.100 μg/L Total organophosphorus pesticides

....................................................... 0.050 μg/L Total organochlorine pesticides + PCBs or organic chlorine

................ 0.050 μg/L Specific conductivity

............................................................................... <1.0 μohms (iii) Salinity should be 20 ± 5 ppt for estuarine species. (iv) Hardness should range between 40 and 180 mg/L as CaCO 3 for freshwater species. (v) Water hardness or salinity, as appropriate, should be measured at the beginning of each test. (vi) In marine flow-through tests, salinity should be recorded at the beginning of the test, on day 4, and if extended, on days 7 and 14. (3) Solutions of test water. (i) Distilled water should be used in making stock solutions of the test substance. If the stock volume is more than 10 percent of the test solution volume, dilution water should be used. If a carrier, i.e. a solvent and/or dispersant, is absolutely necessary to dissolvethe test substance, the amount used should not exceed the minimum volume necessary to dissolve or suspend the test substance in the dilutionwater. If the test substance is a mixture, formulation, or commercial product, none of the ingredients is considered a carrier unless an extra amount is used to prepare the stock solution. (ii) Solvent concentration may not exceed 0.5 mL/L in static-renewal or static testing, and 0.1 mL/L in flow-through testing. (iii) Preferred solvents are dimethyl formamide, triethylene glycol, methanol, acetone, or ethanol. Solvent use should be avoided if possible. (iv) Solvent concentrations selected should be kept constant in thesolvent control and all test solutions. The concentration of solvent in highest treatment level should be used in the solvent control.

### 4 (v) The use of a solubility (saturation) column is permitted in thepreparation of stock solutions. This may help to ensure the aqueous solubility limit is attained for poorly soluble test materials.(vi) The pH should not be adjusted after the addition of the test chemical or stock solution into dilution water. (vii) The pH should be measured in each replicate at the beginning of the test and every 24 h thereafter.(viii) The pH must be monitored in low, medium, and high test concentrations and must remain > 6.0 and < 8.0 for freshwater testing and > 7.5 and < 8.5 for marine testing. (ix) The pH may be adjusted in stock solutions to match the pH of dilution water if pH change does not affect stability of compound in water. HCl and NaOH may be used for this adjustment if warranted. (4) Selection of test species—(i) Test species. One or more of the following species may be used: (A) Freshwater species—Atlantic salmon, Salmo salar ; bluegill sunfish, Lepomis macrochirus ; brook trout, Salvelinus fontinalis ; channel catfish, Ictalurus punctatus ; coho salmon, Oncorhynchus kisutch ; common carp, Cyprinus carpio ; fathead minnnow, Pimephales promelas ; guppy, Poecilia reticulata ; rainbow trout, Oncorhynchus mykiss ; red killifish, Oryzias latipes ; threespine stickleback, Gasterosteus aculeatus ; and zebrafish, Brachydanio rerio

. (B) Saltwater species—Atlantic silverside, Menidia menidia ; sheepshead minnow, Cyprinodon variegatus ; and tidewater silverside, Menidia penisulae

. (C) Data on both a warm and a cold freshwater species are generally required. The preferred warm water species is the bluegill sunfish. The rainbow trout is the preferred cold water species. When data on a marine or estuarine species is desired, the Atlantic silversides is preferred. (ii) Acclimation. (A) A minimum 12–day acclimation period is required with 14 days recommended. A minimum of 7 days of the acclimation period must be performed in test dilution water.(B) Holding water should come from the same source as the test dilution water, if not, acclimation to the dilution water should be done gradually over a 48–h period. (C) No disease treatments may be administered within 48 h of test initiation or during testing. (D) No feeding is permitted within 48 h of test initiation.

### 5 (E) Pretest mortality must be < 5 percent during acclimation. If pretest mortality is > 10 percent, then the entire batch must be rejected and a new batch begun in acclimation. (F) Any changes in water temperature should not exceed 3 ° C per day. Fish should be held for a minimum of 7 days at the test temperature prior to testing. (G) During the final 48 h of acclimation fish should be maintained in facilities with background colors and light intensities similar to those of testing area. (iii) Age and size of test fish. (A) Juvenile fish must be tested. Juvenile fish < 3.0 g should be used and the longest should not be more than twice the length of the shortest. The fish should be of normal size and appearance for their age. All fish must be of the same age. (B) Wild caught fish may be used to satisfy testing guidelines if size, age, and source requirements are satisfied. Wild caught fish should be quarantined 7 days before acclimation procedures begin. (C) Fish must originate from the same source and population. Records should be kept regarding the source of the initial stock and/or culturing techniques. (D) Fish should not be used for a test if they appear stressed, or if more than 5 percent die during the 48 h immediately prior to the test, or if they were used in previous tests for treatments or controls.

(iv) Temperature. The recommended test temperatures are:

Species Temperature, ° C

Atlantic salmon

....................................................................... 12 ± 2.0 Atlantic silverside

.................................................................... 22 ± 2.0 Bluegill sunfish

........................................................................ 22 ± 2.0 Brook trout

.............................................................................. 12 ± 2.0 Channel catfish

....................................................................... 22 ± 2.0 Coho salmon

.......................................................................... 12 ± 2.0 Common carp

......................................................................... 22 ± 2.0 Fathead minnnow

................................................................... 23 ± 2.0 Guppy

..................................................................................... 23 ± 2.0 Rainbow trout

......................................................................... 12 ± 2.0 Red killifish

............................................................................. 23 ± 2.0 Sheepshead minnow

.............................................................. 22 ± 2.0 Threespine stickleback

........................................................... 10 ± 2.0 Tidewater silverside

................................................................ 22 ± 2.0 Zebra-fish

................................................................................ 23 ± 2.0
(v) Feeding. Feeding of test fish daily until 48 h prior to test initiation is suggested.

### 6 (f) Performance of the test—(1) Test design—(i) Test duration. Acute testing must be performed for a minimum of 96 h. (ii) Controls. Every test should include controls consisting of the same dilution water, conditions, procedures, and test population, except that no test substance is added. Solvent (carrier) controls are also required if a solvent was used. (iii) Introduction of fish. Fish should be added to test chambers within 30 min of addition of the test material to dilution water. Fish may beadded prior to addition of test material. Fish should be introduced randomly to individual replicates. (iv) Number of test organisms. A minimum of seven fish per replicate is required. The use of 10 fish per replicate is preferred to obtain a more statistically accurate representation of the dose-response curve, to allow for mortality which may occur, yet be unrelated to chemical effect, and to avoid unnecessary repetitions of the test due to excessive control mortality. (v) Replicates. (A) Two replicates per test concentration are preferred to avoid test repetition due to system failures, and to provide a stronger statistical baseline. (B) Each test chamber should contain an equal volume of test solutionand equal numbers of test fish. Replicate test chambers should be physically separated. (vi) Loading. (A) The number of fish placed in each replicate should not be so great as to affect the test results. (B) In static or static-renewal tests, loading should not exceed 0.8 g (fresh weight) of fish per liter of test solution in a replicate at any one time. (C) In flow-through tests, loading should not exceed 0.5 g fresh weight of fish (FWF) per liter of test solution passing through a replicate within 24 h. (vii) Test chambers and support equipment. (A) Construction materials and equipment that contact the stock solution, test solution, or dilution water should not contain substances that can be leached or dissolvedinto aqueous solutions in quantities that can affect the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test chemicals. Glass, no. 316 stainless steel, nylon screen, and perfluorocarbon plastic (e.g. Teflon) are acceptable materials and should be used whenever possible. Concrete, fiberglass, or plastic (e.g. PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be thoroughly conditioned before use. Rubber, copper, brass, galvanized metal, epoxy glues, lead, and flexible tubing

### 7 should not come in contact with the dilution water, stock solution, or test solution. (B) Test chambers should be loosely covered to reduce evaporation and to minimize the entry of dust or other particulates into solutions and to prevent loss of test fish.(C) Size. Many different sizes of test chambers have been used successfully. The size, shape, and depth of the test chamber is acceptable if the specified flow rate and loading requirements can be achieved. Test vessels must be of adequate size to maintain a load rate of FWF > 0.8 g FWF/L for static or static-renewal tests, or FWF > 0.5 g FWF/L for flow-through tests. (D) Test substance delivery system. ( 1 ) In flow-through tests, proportional diluters, metering pump systems, or other suitable systems should be used to deliver the test chemical to the test chambers. The choice ofa specific delivery system depends on the specific properties and requirements of the test substance. (2 ) The system should be calibrated before and after each test. Calibration includes determining the flow rate and test concentration in each replicate. The apparatus used should accurately and precisely deliver the appropriate amount of stock solution and dilution water to each replicate. (3 ) A closed flow-through system may be used to test volatile compounds when more than 20 percent of the test substance would be lost through volatility or the test substance would cause oxygen levels may fall below 60 percent of the saturation level. A design description of this type of system should be included in the study report. (E) Aeration. Gentle aeration of test vessels used in static systems during the exposure period is permitted only in cases where oxygen levels are in danger of dropping below 60 percent saturation due to chemical characteristics of the test material. Test concentrations must be measured during the test if aeration is used. No aeration of actual test vessels may be utilized in flow-through tests. (viii) Light. (A) The photoperiod with 15 to 30 min transition periods is suggested. Photoperiods may range from 12D/12N to 16D/8N, where D = day, and N = night.(B) Light intensity should range from 30 to 100 lm at the water surface; the intensity selected should be duplicated as closely as possible in all replicates. (ix) Temperature. (A) Temperatures must be recorded in all replicates at the beginning of the test and every 24 h thereafter. The temperature should be recorded at least hourly in one replicate throughout the

### 8 test. Temperature should vary no more then 1.0 ° C in any given 24–h period.(B) The test system should be equipped with an automatic alarm system to alert staff of temperature changes in excess of 2.0 ° C. (C) If the water is heated, precautions should be taken to ensure that supersaturation of dissolved gases is avoided. (x) Dissolved oxygen. DO concentrations should be measured in each replicate at the beginning of the test and every 24 h thereafter. (xi) Feeding. Fish may not be fed during the treatment period. (xii) Disturbances. Any disturbance which might change the behavior of the test fish should be avoided. (2) Test concentrations. (i) A minimum of five test concentrations must be employed. (ii) Five or more concentrations in a geometric series should be tested. Test concentrations must be at least 50 percent greater than the next lowest test concentration (not to exceed 120 percent). Range-finding studies prior to testing may allow more accurate selection of test concentrations.(iii) No more than 25 percent variation is allowed between test concentrations within the same treatment during the test.(iv) Concentration selection. (A) Test concentrations should be selected to produce a no-observable-effect concentration (NOEC) and, preferably, at least two partial mortalities, i.e. one greater than and the other less than 50 percent, after 96 h. The highest test concentration should not exceed the chemical’s aqueous solubility limit if the chemical is not a surfactant or the chemical’s self-dispersibility limit if the chemical is a surfactant or a charged polymer. (B) Exceptions may be required in testing certain pesticide AIs asproducts. Product formulations may increase the solubility of the AI beyond its aqueous solubility limit.(v) Concentration analysis. (A) Concentration analysis must be performed at initiation and every 48 h of the study thereafter. (B) In static tests, the test substance concentration should be measured in each replicate minimally at the beginning (0–hour, before test organisms are added), at 48 h, and at the end of the test. (C) In static-renewal tests, the test substance should be measured in each replicate at the beginning and end of test and just before and after each renewal.

### 9 (D) In flow-through tests, the test substance should be measured as follows: ( 1 ) In each replicate at 0, 48, and 96 h, and every 96 h thereafter, as long as the test is continued. (2 ) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. (3) Collection of samples for measurement. (i) Water samples must be removed from a central point within the test vessel, not from inflow or outflow points.(ii) These samples should not contain any surface particulates or material dislodged from the bottom or sides. Samples should be analyzed immediately, or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photodegradation, chemical reaction, volatilization, or sorption. (iii) The test solution volume should not be reduced during the test by more than 10 percent as a result of sampling. (iv) Samples from each test concentration replicate should not be pooled for analyses. (v) Diluter systems must be monitored for proper adjustment, and operation every 24 h, and should be monitored during the first hour of operation. (vi) Surface films and precipitates must be reported should they occur.(vii) The flow rate to each replicate should be measured at the beginning and end of each test. (viii) During a test, the flow rates should not vary more than a factor of 10 from any one replicate to another. (ix) Minimum number of test vessel replacements should be 6 to 10 per 24–h period for flow-through testing. (4) Observations. (i) Mortality observations should be recorded at 6, 24, 48, 72, and 96 h. (ii) If the test is continued past 96 h, additional observations should be made every 24 h until termination.(iii) In addition to mortality, any abnormal behavior should be recorded, such as, but not limited to, erratic swimming, loss of reflex, increased excitability, lethargy, and changes in appearance or physiology such as discoloration, excessive mucous production, hyperventilation, opaque eyes, curved spine, or hemorrhaging.

### 10 (g) Data and reporting—(1) Treatment of results. The cumulativepercentage mortality for each exposure period is plotted against concentration on logarithmic paper. Normal statistical procedures are then employedto calculate the LC50 for the appropriate exposure period. Confidence limits (CI) with p = 0.95 for the calculated LC50 values are to be included. (2) Test report. (i) The test report must include the following: (ii) Test facilities, test dates, and personnel must be reported. (iii) Identification of the test substance and purity. (iv) Water quality characteristics as reported in the laboratory recordsfor the study. These must include 24-h records of DO, pH, and temperature. (v) Methods of stock solution preparation and the concentrations used in definitive testing.(vi) All test concentrations measured during the test and at termination.(vii) The number of test organisms in each replicate and/or test concentration.(viii) The LC50 concentration-response curves, LC50 values, and associated 95 percent CI should be determined for 24, 48, 72 and 96 h, whenever sufficient data exist. (ix) A graph of the concentration-mortality curve at test termination. Any control mortality observed during the acclimation or study period. (x) An NOEL for the 96–h test should also be reported. (xi) If no LC50 value is determined, but it can be demonstrated thatthe concentrations tested were the highest possible due to the test chemical’s aqueous solubility limit, self-dispersibility limit, or other physicochemical limitations, then the data will be considered for acceptance. Explanation should include details of the solvents which were tried prior to initiation of the final study. (xii) Any abnormal behavior displayed by the test fish.(xiii) Any protocol deviations or occurrences which may have influenced the final results of the test. (xiv) A quality control methods and quality assurance statement should accompany all final study reports. (xv) Raw data must be available to support study author’s conclusions and should be presented with the study report.

### 11 (xvi) Methods of statistical analysis should be reported. (xvii) Methods used in analysis of test concentrations of the test chemical should be described. The accuracy of the method (i.e. detection limit and quantification limit) should be given. (h) References. The following references should be consulted for additional background material on this test guideline. (1) Standard Guide for Conducting Acute Toxicity Tests with Fishes,Macroinvertebrates, and Amphibians, E 729–88a. American Society Testing Materials, Philadelphia, PA. Approved Nov. 21, 1988.(2) Organization of Economic Cooperation and Development, Guidelines for Testing of Chemicals, Guideline 203 ‘‘Fish Acute Toxicity Test.’’ Adopted July 17, 1992.(3) Test Guideline EG–9, Fish Acute Toxicity Test, Office of Pollution Prevention and Toxics, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington DC. (4) Standard Evaluation Procedure Acute Toxicity Test for FreshwaterFish, EPA–540/9–85–006, Office of Pesticide Programs, Office of Prevention Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington DC. Revised June 1985.(5) Acute Toxicity Test for Estuarine and Marine Organisms (Estuarine Fish 96–Hour Acute Toxicity Test), EPA 540/9–85–009, Office ofPesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington DC. Revised June 1985. (6) Federal Insecticide, Fungicide, Rodenticide Act, Subdivision E, Hazard Evaluation, Wildlife and Aquatic Organisms, U.S. Environmental Protection Agency. October 1982. (7) Finney, D.J., Probit Analysis. 3rd Edition. Cambridge University Press: London and New York (1971).(8) Stephen, C.E., ‘‘Methods for Calculating an LC50’’ Aquatic Toxicology and Hazard Evaluation, ASTM STP 634, American Society of Testing and Materials, Philadelphia, PA (1977). (9) Canada, Environment Canada. Biological test method: acute lethality test using threespine stickleback ( Gasterosteus aculeatus ). Environmental Protection, Conservation and Protection, Environment Canada, Report EPS 1/RM/10 (1990)..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–117 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1085 Fish Acute Toxicity Mitigated by Humic Acid

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1085 Fish acute toxicity mitigated by humic acid. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline is 40 CFR 797.1460 Fish Acute Toxicity Mitigated by Humic Acid. (b) Purpose. This guideline may be used to develop data on the acute toxicity of chemical substances and mixtures under static or static renewalconditions, subject to environmental effects testing. This guideline prescribes procedures to be used to develop data on the acute toxicity ofchemicals to fish with and without the presence of naturally occurring dissolved organic substances (.e.g., humic acids and their salts). EPA willuse data from these tests in assessing the hazard of a chemical to the environment. For additional background information on this test guideline see OPPTS 850.1075. (c) Definitions. In addition to the definitions in section 3 of the Toxic Substances Control Act (TSCA), and the definitions in 40 CFR Part 792— Good Laboratory Practice Standards, the following definitions also apply to this test guideline:
Acclimation means the physiological compensation by test organisms to new environmental conditions (e.g. temperature, hardness, pH). Acute toxicity test means a method used to determine the concentration of a substance that produces a toxic effect on a specified percentage of test organisms in a short period of time (e.g. 96 h). In this guideline,death is used as the measure of toxicity under static or static renewal conditions only.

Carrier means a solvent used to dissolve a test substance prior to delivery to the test chamber.
Death means the lack of opercular movement by a test fish. Dissolved organic carbon (DOC) means various organic molecules occurring in lotic and lentic ecosystems, which in this test are restricted to a heterogeneous group of humic substances. Total organic carbon (TOC) means the sum of all organic carbon molecules, which are dissolved, particulate, and suspended, occurring in test dilution waters.
Humic substances means humic acids (HAs), fulvic acids, and humin fractions, and their various salts, resulting from chemical fractionation of this heterogeneous naturally-occurring organic substance. For purposes of

### 2 this test, HA, sodium salt (e.g. Aldrich Catalog No. H1,675–2; mentionof a commercial company or product does not constitute approval or endorsement by the Agency) may be used as the source of DOC.
LC50 means that the test substance concentration calculated from experimentally-derived mortality data is lethal to 50 percent of a test population during continuous exposure over a specified period of time. Loading means the ratio of fish biomass (in grams, wet weight) to the volume (in liters) of test solution in a test chamber or passing through it in a 24–h period.
Static means the test solution is not renewed during the period of the test.
Test solution means the dilution water containing the dissolved test substance to which test organisms are exposed. (d) Test procedures—(1) Summary of the test. (i) This test is designed to determine the acute effects of the test substance on one of three species of fish with HA. Test chambers are filled with appropriate volumes of dilution water. (ii) The test substance is introduced into each test chamber. Some test chambers contain only dilution water; other contain a concentration of spiked HA. (iii) Test fish which have been acclimated in accordance with the test design are introduced into the test and control chambers by stratified random assignment. (iv) Fish in the test and control chambers are observed periodically during the test; dead fish are removed at least twice each day and the findings are recorded. (v) The dissolved oxygen (DO) concentration, pH, and temperature are measured at intervals in selected test chambers.(vi) A concentration-response curve and LC50 value for the test substance in dilution water spiked with a known amount of HA are developed from the mortality data collected during the test. (2) Range-finding test. (i) If the toxicity of the test substance in HAis not already known, a range-finding test should be performed to determine the range of concentrations to be used in the definitive test. The highest concentration of test substance for use in the range-finding testshould not exceed its solubility in water or the permissible amount of carrier used. (ii) Initially, two fish test is performed at 20 mg/L of HA. In some cases, the 20 mg HA/L concentration may be so high that no toxicity will

### 3 be present due to the formation of viscous, colloidal complexes. If this occurs, the 20 mg HA/L concentration should be decreased to 15 mg/L, or an appropriately lower concentration. (3) Definitive test. (i) A minimum of 20 fish should be exposed to each of five or more test substance concentrations in dilution water spiked with a known amount of HA. The range of test substance concentrations to which the fish are exposed should be such that in 96 h there are at least two partial mortality exposures bracketing 50 percent survival. (ii) For exposure to each concentration of a test substance in dilution water spiked with a known amount of HA, an equal number of test fish should be placed in two or more replicate test chambers. Test fish should be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. (iii) Every test should include a control consisting of the same dilution water, conditions, procedures, and fish from the same group used in the test, except that none of the test substance is added. Every test should also include negative controls consisting of dilution water with HA alone. (iv) Mortality data collected during the test are used to calculate a 96–h LC50 value. The 24–, 48–, and 72–h values should be calculated whenever there is sufficient mortality data to determine such values. (v) Test fish should not be fed while they are being exposed to the test substance under static conditions. (4) Test results . (i) Death is the primary criterion used in this test guideline to evaluate the toxicity of the test substances on the presence of a known amount of HA.(ii) In addition to death, any abnormal behavior such as, but not limited to, erratic swimming, loss of reflex, increased excitability, lethargy,or any changes in appearance of physiology, such as discoloration, excessive mucous production, hyperventilation, opaque eyes, curved spine, or hemorrhaging should be recorded. (iii) Observations on compound solubility and/or dispersibility should be recorded. The investigator should report the appearance of surface slicks, precipitates, or material adhering to the sides of the test chamber. (iv) Each test and control chamber should be checked for dead fish and observations recorded at 24, 48, 72, and 96 h after the beginning of the test or within 1 h of the designated times. If the test is continuedpast 96 h, additional observations should be made every 24 h until termination. (v) The mortality data are used to calculate LC50 values and their 95 percent confidence limits, and to plot concentration-response curves

### 4for each time interval whenever sufficient data exists. The methods recommended for use in calculating LC50 values include probit, logit, binomial, and moving average angle. (vi) A test is be unacceptable if more than 10 percent of the control fish die or exhibit abnormal behavior during a 96–h test. (5) Analytical measurements—(i) Water quality analysis. (A) The hardness, acidity, alkalinity, pH, conductivity, TOC, or chemical oxygen demand (COD), and total suspended solids (TSS) of the dilution watershould be measured at the beginning of each static test. The month-tomonth variation of the above values should be less than 10 percent and the pH should vary less than 0.4 units. (B) During static tests, the DO concentration, temperature, and pH should be measured in each test chamber at the beginning and end of the test. The test solution volume should not be reduced by more than 10 percent as a result of these measurements. (ii) Dissolved organic carbon. The naturally-occurring DOC selected for this test should be HA, which is available from the Aldrich catalog, (No. H1,675–2). (iii) Collection of samples for measurement of TOC. Samples to be analyzed for TOC should be taken from the control chambers midway between the top, bottom, and sides of the test chamber. These samples should not include any surface scum or material dislodged from the bottom or sides. (iv) Measurement of TOC. (A) For static tests, DOC should be measured (as TOC) at a minimum in each test chamber at the beginning (time 0, before fish are added) of the test. Three TOC measurements should be made and the average reported. (B) The analytical methods used to measure the TOC in a sample should be validated before beginning the test. The accuracy of a methodshould be verified by a method such as using known additions. This involves adding a known amount of the dissolved organic carbon source to three water samples taken from a chamber containing dilution waterto be used in the test. The normal concentration of dissolved organic carbon in those samples should span the TOC concentration range to be used in the test. (C) The nominal concentration of test substance based on 100 percent active ingredient (AI) should be used to calculate all LC50 values and to plot all concentration-response curves. (e) Test conditions—(1) Test species— (i) Selection . The test species for this test are the rainbow trout ( Oncorhynchus mykiss = Salmo

### 5 gairdneri), bluegill (Lepomis macrochirus ), and fathead minnow (Pimephales promelas ). (ii) Age and condition of fish. Juvenile fish should be used. Fish used in a particular test should be the same age and be of normal size and appearance for their age. The longest fish should not be more thantwice the length of the shortest. All newly acquired fish should be quarantine and observed for at least 14 days prior to use in a test. Fish should not be used for a test if they appear stressed or if more than 5 percent die during the 48 h immediately prior to the test. (iii) Acclimation of test fish . If the holding water is not from the same source as the test dilution water, acclimation to the dilution water should be done gradually over a 48–h period. The fish should be held an additional 14 days in the dilution water prior to testing. Any changes in water temperature should not exceed 3 ° C per day. Fish should be held for a minimum of 7 days at the test temperature prior to testing. During the final 48–h of acclimation, fish should be maintained in facilities with background colors and light intensities similar to those of the testing area and should not be fed. (2) Facilities—(i) General . Facilities needed to perform this test include: (A) Flow-through tanks for holding and acclimating fish.(B) A mechanism for controlling and maintaining the water temperature during the holding, acclimation, and test periods. (C) Apparatus for straining particulate matter, removing gas bubbles, or insufficient dissolved oxygen, respectively. (D) Apparatus for providing a 16–h light and 8–h dark photoperiod with a 15– to 30–min transition period. (E) Chambers for exposing test fish to the test substance. (ii) Construction materials . Construction materials and commercially purchased equipment that may contact the stock solution, test solution, or dilution water should not contain substances that can be leachedor dissolved into aqueous solutions in quantities that can alter the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test chemicals. Glass, stainless steel, and perfluorocarbon plastic should be used whenever possible. Concrete, fiberglass, or plastic (e.g. PVC) may be used for holding tanks, acclimationtanks, and water supply systems, but they should be thoroughly conditioned before use. If cast iron pipe is used in freshwater supply systems, colloidal iron may leach into the dilution water and strainers or filters should be used to remove rust particles. Rubber, copper, brass, galvanized

### 6 metal, epoxy glues, and lead should not come in contact with the dilution water, stock solution, or test solution. (iii) Test chambers . Test chambers made of stainless steel should be welded, not soldered. Test chambers made of glass should be fused or bonded using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the chamber. (iv) Cleaning of test system . Test chambers should be cleaned before each test. They should be washed with detergent and rinsed in sequence with clean water, pesticide-free acetone, clean water, and 5 percent nitric acid, followed by two or more changes of dilution water. (v) Dilution water. (A) Clean surface or ground water, reconstituted water, or dechlorinated tap water is acceptable as dilution water if the the test fish will survive in it for the duration of the holding, acclimating, and testing periods without showing signs of stress, such as discoloration, hemorrhaging, disorientation, or other unusual behavior. The quality of the clean dilution water (without spiked HA) should be constant and should meet the specifications in the following Table 1., measured at least twice a year: Table 1.—Specifications for Dilution Water

Substance Maximum Concentration Total suspended solids

................................................................ 20 mg/L Total organic carbon (TOC), or chemical oxygen demand (COD)

. 2 mg/L, or 5 mg/L, respectively

Un-ionized ammonia

.................................................................... 20 μg/L Residual chlorine

......................................................................... 1 μg/L Total organophosphorus pesticides

............................................. 50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls (PCBs),or organic chlorine

. 50 ng/L, or 25 ng/L, respectively Hardness (as CaCO 3 during testing)

........................................... 180 mg/L

The quality of the dilution water after spiking with HA should meet all the previous specifications except for TOC or COD. (B) The DO concentration in the dilution water should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/L for tests with trout, and 8.0 to 8.9 mg/L for tests with bluegill or fathead minnow at sea level. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use. Buffered soft water should be aerated before but not after the addition of buffers. (C) Diseased organisms present in the dilution water in sufficientnumber to cause infection of the fish should be killed or removed by suitable equipment.

### 7(D) Glass-distilled or carbon-filtered deionized water with a conductivity less than 1 μ S/cm is acceptable for use in making reconstituted water. If the reconstituted water is prepared from a ground or surface water source, conductivity and TOC should be measured on each batch. (vi) Carriers. Only distilled water should be used in making stock solutions of the test substance. However, if the stock volume is more than10 percent of the test solution volume, dilution water should be used. Carbon-based carriers cannot be used in this test. If necessary, stock solution pH should be adjusted to pH 7. (3) Test parameters—(i) Loading. The number of fish placed in a test chamber should not be so great as to affect the results of the test. The loading should not be so great that the test substance concentrations are decreased by more than 20 percent due to uptake by the fish. Loading should not exceed 0.5 g of fish/L of solution in the test chamber at any one time. These loading rates should be sufficient to maintain the DOconcentration above the recommended levels and the ammonia concentration below 20 μg/L. (ii) Dissolved oxygen concentration. During static tests with rainbow trout, the DO should be maintained above 5.5 mg/L in each test chamber. In tests with bluegill and fathead minnow, the DO should be greater than 4.5 mg/L in each test chamber. (iii) Temperature. The test temperature should be 22 ° C for bluegill and fathead minnow, and 12 ° C for rainbow trout. Deviations from the test temperature should be no greater than ±2 ° C. The temperature should be measured at least hourly in one test chamber. (iv) Light. A 16–h light and 8–h dark photoperiod should be maintained. (f) Reporting . The sponsor should submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In additionto the reporting requirements prescribed in 40 CFR Part 792—Good Laboratory Practice Standards, the reported test data should include the following: (1) The source of the dilution water, a description of any pretreatment, and the measured hardness, acidity, alkalinity, pH, conductivity, TOC, COD, and total suspended solids. (2) The source of the HA (e.g., batch number), as well as a complete description and chemical characterization.(3) A description of the test chambers, the depth and volume of solution in the chamber, and the specific way the test was begun (e.g., conditioning and test substance additions).

### 8 (4) Detailed information about the test fish, including the scientific name and method of verification, average weight (grams, wet weight), standard length, age, source, history, observed diseases, treatments and mortalities, acclimation procedures, and food use.(5) The number of replicates used, the number of organisms per replicate, and the loading rate. (6) The measured DO, pH, and temperature and the lighting regime. (7) A description of preparation of the stock solution. If the pH of the stock solution was adjusted, describe the adjustment. (8) The concentrations of the dissolved organic carbon as TOC from the HA control just before the start of the test, all triplicate measurements, and average TOC values. (9) Results from any range-finding tests performed at 20 mg/L of HA. (10) The number of dead and live tests organisms, the percentage of organisms that died, and the number that showed any abnormal effects in the control and in each test chamber at each observation period. (11) The 96–h LC50, and when sufficient data have been generated, the 24–, 48–, 72–h LC50 values, their 95 percent confidence limits, and the methods used to calculate the LC50 values and their confidence limits.(12) When observed, the no-observed-effect-concentration (the highest concentration tested at which there were no mortalities, abnormal behavioral, or physiological effects) in treatments. (13) The concentration-response curve at each observation period for which LC50 values are calculated.(14) Methods and data records of all chemical analyses of water quality parameters, TOC, including method validations and reagent blanks..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–120 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1300 Daphnid Chronic Toxicity Test

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1300 Daphnid chronic toxicity test. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline are 40 CFR 797.1330 Daphnid Chronic Toxicity Test; OPP 72–4 Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies (Pesticide Assessment Guidelines, Subdivision E— Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09- 82-024, 1982; and OECD 202, Daphnia sp. Acute Immobilisation Test and Reproduction Test. (a) Purpose. This guideline prescribes a chronic toxicity test in whichdaphnids are exposed to a chemical either in a static-renewal or a flowthrough system. The Environmental Protection Agency will use data from this test in assessing the hazard a chemical may present to the aquatic environment. No preference is given in this guideline on the type of test system to be used, either static-renewal or flow-through. However, the former works well if individual daphnids need to be monitored during the test. The latter works well with chemicals that are volatile, have low water solubilities, and an oxygen demand, and for those that degrade, hydrolyze,or photolyze easily. Flow-through systems allow maintenance of near constant chemical concentrations throughout the test. (b) Definitions. The definitions in section 3 of the Toxic SubstancesControl Act (TSCA) and the definitions in 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this test guideline.

Brood stock means the animals which are cultured to produce test organisms through reproduction. Chronic toxicity test means a method used to determine the concentration of a substance in water that produces an adverse effect on a test organism over an extended period of time. In this test guideline, mortality and reproduction (and optionally, growth) are the criteria of toxicity.

EC50 means that experimentally derived concentration of test substance in dilution water that is calculated to affect 50 percent of a test population during continuous exposure over a specified period of time. In this guideline, the effect measured is immobilization. Ephippium means a resting egg which develops under the carapace in response to stress conditions in daphnids.

### 2 Flow-through means a continuous or intermittent passage of test solution or dilution water through a test chamber or culture tank with no recycling. Immobilization means the lack of movement by daphnids except for minor activity of the appendages. Loading means the ratio of daphnid biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber at a point in time or passing through the test chamber during a specific interval.

LOEC (lowest observed effect concentration) means the lowest concentration of a material used in this test that has an adverse effect on the test organisms and is the test concentration immediately above the NOEC.

MATC (maximum acceptable toxicant concentration) means the maximum concentration at which a chemical can be present and not be toxic to the test organism.

NOEC (no observed effect concentration) means the highest concentration of a material used in this test that does not have an adverse effect on the test organisms and is the test concentration immediately below the LOEC. Static-renewal system means the technique in which test organisms are periodically transferred to fresh test solution of the same composition. (c) Test procedures—(1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. In the flow-through test the flow of dilution water through each chamber is then adjusted to the rate desired. The test substance is introduced into each test chamber. The addition of test substance in the flow-through system is done at a rate which is sufficient to establish and maintain the desired concentration of test substance in the test chamber. (ii) The test is started within 30 min after the test substance has been added and uniformly distributed in the test chambers in the static-renewal test or after the concentration of test substance in each test chamber ofthe flow-through test system reaches the prescribed level and remains stable. At the initiation of the test, daphnids which have been cultured or acclimated in accordance with the test design, are randomly placed into the test chambers. Daphnids in the test chambers are observed periodically during the test, immobile adults and offspring produced are counted and removed, and the findings are recorded. Dissolved oxygen concentration,pH, temperature, the concentration of test substance, and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to determine any significant differences (p ≤ 0.05) in immobilization and reproduction as compared to the

### 3 control. At the end of the test, the growth of surviving adults is measured as the total body length or dry weight or both. (2) Range-finding test. (i) A range-finding test should be conducted to establish test solution concentrations for the definitive test. (ii) The daphnids should be exposed to a series of widely spaced concentrations of the test substance (e.g. 1, 10, 100 mg/L), usually under static conditions.(iii) A minimum of five daphnids should be exposed to each concentration of test substance for a period of time which allows estimation of appropriate chronic test concentrations. No replicates are required and nominal concentrations of the chemical are acceptable. (3) Definitive test. (i) The purpose of the definitive test is to determine concentration-response curves, EC50 values, and effects of a chemical on immobilization and reproduction during chronic exposure. (ii) A minimum of 10 daphnids per concentration should be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, 64 mg/L). In flow-through testing, an equal number of daphnids (minimum of 20 per concentration) should be placed in two or more replicates ortest chambers, e.g. four replicates each with five daphnids, for each concentration. In static-renewal tests, 10 or more replicates of one daphnid each, for each concentration, should be used. The concentration ranges should be selected to determine the concentration-response curves, EC50values, and MATC. Solutions should be analyzed for chemical concentration at designated times during the test. (iii) Every test should include controls consisting of the same dilutionwater, conditions, procedures and daphnids from the same population (culture container), except that none of the chemical is added. (iv) The test duration is 21 days. The test is invalid and unacceptable if any of the following occur:(A) More than 20 percent of the control organisms appear to be immobilized, stressed, or diseased during the test. (B) Each control daphnid living the full 21 days produces an average of less than 60 young. (C) Any ephippia are produced by control animals. (v) The number of immobilized daphnids in each chamber should be recorded on day 21 of the test. After offspring are produced, they shouldbe counted and removed from the test chambers every 2 or 3 days. Concentration-response curves, EC50 values, and associated 95 percent con-

### 4 fidence limits for adult immobilization should be determined for day 21.An MATC should be determined for the most sensitive test criteria measured (number of adult animals immobilized, number of young per adult, and number of immobilized young per adult). (vi) Growth of daphnids is determined by measuring total body length or dry weight, or both, of each surviving adult. It is preferred that both measures be taken. (vii) In addition, any abnormal behavior or appearance should also be reported. (viii) Test organisms should be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shouldbe positioned in a random manner as in a way in which appropriate statistical analyses can be used to determine the variation due to placement. (4) Analytical measurements—(i) Test chemical. Deionized water should be used in making stock solutions of the test substance. Standard analytical methods should be used whenever available in performing theanalyses. The analytical method used to measure the amount of test substance in a sample should be validated before beginning the test by appropriate laboratory practices. An analytical method is not acceptable if likelydegradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences which cannot be systematically identified and corrected mathematically. (ii) Numerical. The number of immobilized adults, total offspring per adult, and immobilized offspring per adult should be counted duringeach test. Appropriate statistical analyses should provide a goodness-offit determination for the adult immobilization concentration-responsecurves calculated on day 21. A 21–day EC50 based on adult immobilization and corresponding 95 percent confidence intervals should also be calculated. Appropriate statistical tests (e.g. analysis of variance, mean separation test) should be used to test for significant chemical effects on chronic test criteria (cumulative number of immobilized adults, cumulative number of offspring per adult and cumulative number of immobilized offspring per adult) on day 21. An MATC should be calculated using these chronic test criteria. (d) Test conditions—(1) Test species—(i) Selection. (A) The cladocerans, Daphnia magna or D. pulex , are the species to be used in this test. Either species can be utilized for testing of a particular chemical.The species identity of the test organisms should be verified using appropriate systematic keys. (B) First instar daphnids, ≤ 24 h old, are to be used to start the test.

### 5 (ii) Acquisition. (A) Daphnids to be used in chronic toxicity tests should be cultured at the test facility. Records should be kept regarding the source of the initial stock and culturing techniques. All organisms usedfor a particular test should have originated from the same culture population. (B) Daphnids should not be used for a test if: ( 1 ) Cultures contain ephippia. (2 ) Adults in the cultures do not produce young before day 12. (3 ) More than 20 percent of the culture stock die in the 2 days preceding the test. (4 ) Adults in the culture do not produce an average of at least three young per adult per day over the 7–day period prior to the test. (5) Daphnids have been used in any portion of a previous test either in a treatment or in a control. (iii) Feeding. (A) During the test the daphnids should be fed the samediet and with the same frequency as that used for culturing and acclimation. All treatments and controls should receive, as near as reasonably possible, the same ration of food on a per-animal basis.(B) The food concentration depends on the type used. Food concentrations should be sufficient to support normal growth and developmentand to allow for asexual (parthenogenic) reproduction. For automatic feeding devices, a suggested rate is 5 to 7 mg food (either solids or algal cells, dry weight) per liter of dilution water or test solution. For manual once-a-day feeding, a suggested rate is 15 mg food (dry weight) per liter of dilution water or test solution. (iv) Loading. The number of test organisms placed in a test chamber should not affect test results. Loading should not exceed 40 daphnids per liter in the static-renewal system. In the flow-through test, loading limits will vary depending on the flow rate of the dilution water. Loading shouldnot cause the dissolved oxygen concentration to fall below the recommended level. (v) Care and handling of test organisms. (A) Daphnids should be cultured in dilution water under similar environmental conditions to those used in the test. A variety of foods has been demonstrated to be adequate for daphnid culture. They include algae, yeasts, and a variety of mixtures. (B) Organisms should be handled as little as possible. When handling is necessary it should be done as gently, carefully, and quickly as possible. During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, physical damage, and mortality.

### 6 Dead and abnormal individuals should be discarded. Organisms that touch dry surfaces or are dropped or injured during handling should be discarded. (C) Smooth glass tubes (I.D. > 5 mm) equipped with a rubber bulbcan be used for transferring daphnids with minimal culture media carryover. (D) Care should be exercised to introduce the daphnids below the surface of any solution in order not to trap air under the carapace. (vi) Acclimation. (A) Brood daphnids should be maintained in 100 percent dilution water at the test temperature for at least 48 h prior tothe start of the test. This is easily accomplished by culturing them in dilution water at the test temperature. During acclimation, daphnids should be fed the same food as will be used for the definitive test. (B) During culturing and acclimation to the dilution water, daphnidsshould be maintained in facilities with background colors and light intensities similar to those of the testing area. (2) Facilities—(i) General. (A) Facilities needed to perform this test include: ( 1 ) Containers for culturing and acclimating daphnids. (2 ) A mechanism for controlling and maintaining the water temperature during the culturing, acclimation, and test periods. (3 ) Apparatus for straining particulate matter, removing gas bubbles, or aerating the water when water supplies contain particulate matter, gas bubbles, or insufficient dissolved oxygen, respectively. (4 ) An apparatus for providing a 16–h light and 8–h dark photoperiod. (5) An apparatus to introduce food if continuous or intermittent feeding is used. ( 6) In addition, the flow-through test should contain appropriate testchambers in which to expose daphnids to the test substance and an appropriate test substance delivery system. (B) Facilities should be well ventilated and free of fumes and other disturbances that may affect the test organisms. (ii) Test chambers. (A) Materials and equipment that contact test solutions should be chosen to minimize sorption of test chemicals from the dilution water and should not contain substances that can be leached into aqueous solution in quantities that can affect test results. (B) For static-renewal tests, daphnids can be conveniently exposed to the test solution in 250–mL beakers or other suitable containers.

### 7(C) For flow-through tests daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. Such containers should be suspended in the test chamber in such a manner to ensure that the test solution flows regularly into and out of the containerand that the daphnids are always submerged in at least 5 cm of test solution. Test chambers can be constructed using 250–mL beakers or other suitable containers equipped with screened overflow holes, standpipes, or V-shaped notches. (D) Test chambers should be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (iii) Test substance delivery system. (A) In the flow-through test, proportional diluters, metering pump systems, or other suitable systems should be used to deliver the test substance to the test chambers. (B) The test substance delivery system should be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance in each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24–h flow rate through a test chamber should be equal to at least 5 × the volume of the test chamber. During a test, the flow rates should not vary more than 10 percent from any one test chamber to another. For the static-renewal test, test substance dilution water should be completely replaced at least once every 3 days. (iv) Dilution water . (A) Surface or ground water, reconstituted water, or dechlorinated tap water are acceptable as dilution water if daphnids will survive in it for the duration of the culturing, acclimation, and testing periods without showing signs of stress. The quality of the dilution water should be constant and should meet the specifications in the following Table 1.: Table 1.—Specifications for Dilution Water

Substance

Particulate matter

.................................................................................... 20 mg/L Total organic carbon or:

.......................................................................... 2 mg/L Chemical oxygen demand

..................................................... 5 mg/L Un-ionized ammonia

................................................................................ 20 μg/L Residual chlorine

..................................................................................... < 3 μg/L Total organophosphorus pesticides

........................................................ 50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or:

......................................................................................................... 50 ng/L Organic chlorine

.................................................................... 25 ng/L

(B) The water quality characteristics listed above should be measured at least twice a year or when it is suspected that these characteristics may

### 8have changed significantly. If dechlorinated tap water is used, daily chlorine analysis should be performed. (C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilledwater. Glass-distilled or carbon-filtered deionized water with a conductivity of less than 1 μohm/cm is acceptable as the diluent for making reconstituted water. (D) If the test substance is not soluble in water, an appropriate carrier should be used at a concentration ≤ 0.1mL/L. Triethylene glycol and dimethyl formamide are preferred solvents, but ethanol or acetone can be used if necessary. (v) Cleaning of test system. All test equipment and test chambersshould be cleaned before each use following standard laboratory procedures. Cleaning of test chambers may be necessary during the testing period. (3) Test parameters. (i) Environmental conditions of the water contained in test chambers should be maintained as specified in this paragraph: (A) The test temperature should be 20 ° C. Excursions from the test temperature should be no greater than ± 1 ° C.(B) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, should be done before the addition of the test substance. All treatment and control chambers should be given the same aeration treatment. (C) Photoperiod of 16–h light and 8–h darkness. (ii) Additional measurements include: (A) The concentration of the test substance in the chambers should be measured during the test. (B) At a minimum, the concentration of test substance should be measured as follows: ( 1 ) In each chamber before the test. (2 ) In each chamber on days 7, 14, and 21 of the test. (3 ) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis. Among replicate test chambers of a treatment concentration,

### 9 the measured concentration of the test substance should not vary more than 20 percent. (C) The dissolved oxygen concentration, temperature, and pH should be measured at the beginning of the test and on days 7, 14, and 21 inat least two chambers of the high, middle, low, and control test concentrations. (e) Reporting. The sponsor should submit to the EPA all data developed by the test that are suggestive or predictive of chronic toxicity andall associated toxicologic manifestations. In addition to the reporting requirements prescribed under Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the reporting of test data should include the following: (1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing. (2) A detailed description of the test substance including its source, lot number, composition (identity and concentration of major ingredients, percent active ingredient, and major impurities), known physical andchemical properties, and any carriers or other additives used and their concentrations. (3) The source of the dilution water, its chemical characteristics (e.g. conductivity, hardness, pH), and a description of any pretreatment.(4) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture methods. The age of the daphnids used in the test should be reported. (5) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g. conditioning, test substance additions), the number of test organisms per test chamber, the number ofreplicates per treatment, the lighting, the static-renewal process and schedule for the static-renewal chronic test, the test substance delivery system and flow rate expressed as volume additions per 24 h for the flow-through chronic test, and the method of feeding (manual or continuous), and type of food. (6) The concentration of the test substance in test chambers at times designated for static-renewal and flow-through tests. (7) The number and percentage of organisms that show any adverse effect in each test chamber at each observation period. (8) The cumulative adult and offspring immobilization values and the progeny produced at designated observation times, the time (days) to first brood, the number of offspring per adult in the control replicates and in

### 10 each treatment replicate, and the growth of surviving adults measured as total length or dry weight or both.(9) All chemical analyses of water quality and test substance concentrations, including methods, method validations, and reagent blanks.(10) The data records of the culture, acclimation, and test temperatures. (11) Any deviation from this test guideline, and anything unusual about the test, e.g. dilution failure, temperature fluctuations. (12) The MATC to be reported is calculated as the geometric meanbetween the lowest measured test substance concentration that had a significant (p ≤ 0.05) effect (LOEC) and the highest measured test substance concentration that had no significant (p ≤ 0.05) effect (NOEC) on day 21 of the test. The most sensitive of the test criteria (number of adult animals immobilized, the number of young per surviving female, the number of immobilized young per female, and the growth of surviving females) isused to calculate the MATC. The criterion selected for MATC computation is the one which exhibits an effect (a statistically significant difference between treatment and control groups; p ≤ 0.05) at the lowest test substance concentration for the shortest period of exposure. Appropriate statistical tests (analysis of variance, mean separation test) should be used to test for significant test substance effects. The statistical tests employed and the results of these tests should be reported. (13) Concentration-response curves utilizing the average measuredtest substance concentration should be fitted to cumulative adult immobilization data at 21 days. A statistical test of goodness-of-fit should be performed and the results reported.(14) An EC50 value based on adult immobilization with corresponding 95 percent confidence limits when sufficient data are present for day21. These calculations should be made using the average measured concentration of the test substance..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–121 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1400 Fish Early-Life Stage Toxicity Test

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1400 Fish early-life stage toxicity test. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline are 40 CFR 797.1600 Fish Early Life Stage Toxicity Test; OPP 72–4 Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies (Pesticide Assessment Guidelines, Subdivision E— Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09- 82-024, 1982; and OECD 210 Fish Early-Life Stage Toxicity Test. (b) Introduction. (1) Tests with the early-life stages of fish are intended to define the lethal and sublethal effects of chemicals on the stages and species tested. They yield information of value for the estimation ofthe chronic lethal and sublethal effects of the substance on other fish species. (2) This guideline is based on a proposal from the United Kingdom which was discussed at a meeting of OECD experts convened at Medmenham (United Kingdom) in November 1988. (c) Definitions. The definitions in section 3 of the Toxic SubstancesControl Act (TSCA) and the definitions in 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this test guideline.

LOEC (Lowest-observed-effect-concentration) is the lowest tested concentration of a test substance at which the substance is observed to have a significant effect (at p < 0.05) when compared with the control. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC.

NOEC (No-observed-effect-concentration) is the test concentration immediately below the LOEC. (d) Principle of the test. The early-life stages of fish are exposedto a range of concentrations of the test substance dissolved in water, preferably under flow-through conditions, or where appropriate, semistatic conditions. The test is begun by placing fertilized eggs in the test chambers and is continued at least until all the control fish are free-feeding. Lethal and sublethal effects are assessed and compared with control values to determine the LOEC and the NOEC. (e) Information on the test substance. (1) Results of an acute toxicity test (see OPPTS 850.1075), preferably performed with the specieschosen for this test, should be available. This implies that the water solubility and the vapor pressure of the test substance are known and a reliable

### 2analytical method for the quantification of the substance in the test solutions with known and reported accuracy and limit of detection is available. (2) Useful information includes the structural formula, purity of the substance, stability in water and light, pKa , P ow , and results of a test for ready biodegradability. (See OPPTS 835.3110 through 835.3160.) (f) Validity of the test. For a test to be valid the following conditions apply: (1) The dissolved oxygen concentration must be between 60 and 100 percent of the air saturation value throughout the test. (2) The water temperature must not differ by more than ± 1.5 ° C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Tables 4. and 5. under paragraphs (h)(1)(ii) and (h)(1)(iii) of this guideline). (3) Evidence must be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ± 20 percent of the mean measured values.(4) Overall survival of fertilized eggs in the controls and, where relevant, in the solvent-only controls must be greater than or equal to the limits defined in Tables 4. and 5. under paragraphs (h)(1)(ii) and (h)(1)(iii) of this guideline.(5) When a solubilizing agent is used it must have no significant effect on survival nor produce any other adverse effects on the early-life stages as revealed by a solvent-only control. (g) Description of the method—(1) Test chambers. Any glass,stainless steel, or other chemically inert vessels can be used. The dimensions of the vessels should be large enough to allow compliance with loading rate criteria given below. It is desirable that test chambers be randomly positioned in the test area. A randomized block design with each treatmentbeing present in each block is preferable to a completely randomized design. The test chambers should be shielded from unwanted disturbance. (2) Selection of species. (i) Recommended fish species are: (A) Freshwater—rainbow trout, Oncorhynchus mykiss ; fathead minnow, Pimephales promelas ; zebra fish, Danio rerio ; ricefish, Oryzias latipes

. (B) Salt water—sheepshead minnow, Cyprinodon variegatus

. (ii) The Office of Prevention, Pesticides and Toxic Substances prefers rainbow trout ( O. mykiss ) or fathead minnow (P. promelas ) as test species for freshwater fish and sheepshead minnow ( C. variegatus ) or silverside

### 3 (Menidia menidia , M. beryllina , or M. peninsulae ) as test spcies for estuarine or marine fish. This does not preclude the use of other species. Examples of other well-documented species which have also been used are: (A) Freshwater—coho salmon, Oncorhynchus kisutch ; chinook salmon, Oncorhynchus tschawytscha ; brown trout, Salmo trutta ; Atlantic salmon, Salmo salar ; brook trout, Salvelinus fontinalis , lake trout, Salvelinus namaycush ; northern pike, Esox lucius ; white sucker, Catostomus commersoni ; bluegill, Lepomis macrochirus ; channel catfish, Ictalurus punctatus ; flagfish, Jordanella floridae ; three-spined stickleback, Gasterosteus aculeatus ; common carp, Cyprinus carpio

. (B) Salt water—Atlantic silverside, M. menidia ; Tidewater silverside, M. peninsulae

. (iii) Feeding and handling requirements of brood and test animals, test conditions, duration, and survival criteria for these species can be found in Tables 1. and 2. under paragraph (g)(3) of this guideline, and Tables 4. and 5. under paragraphs (h)(1)(i) and (h)(1)(ii) of this guideline. (iv) The test procedure might have to be adapted to provide suitabletest conditions. The rationale for the selection of the species and the experimental method should be reported in this case. (3) Holding of the brood fish. (i) Details on holding the brood stock under satisfactory conditions may be found in the references cited under paragraphs (j)(1), (j)(2), and (j)(3) of this guideline. (ii) Conditions for recommended species are provided in the following Table 1. Abbreviations in the table have the following meanings: BW = body weight; FBS = frozen brine shrimp—adults Artemia sp.; BSN = brine shrimp nauplii—newly hatched; BSN48 = brine shrimp nauplii—48 h old.

### 4 Table 1.—Feeding and Handling Requirements of Brood and Test Animals of Recommended Species

Species Food Post-Hatch Transfer

Time 1 Time to First Feeding Brood Fish NewlyHatched Larvae Juveniles Type Amount Frequency

Freshwater Oncorhynchus

mykiss , rainbow trout trout food none 2 trout starter 4% BW per day 2–4 feeds/ day 14–16 days post-hatchor at swimup 3 19 days posthatch or at swim-up Pimephales

promelas , fathead minnow FBS BSN BSN48 ad libitum once hatching is 90% within 2 days of hatching

Danio rerio , Zebra fish BSN48, flake food protozoa 4 protein 5 BSN48 not necessary 6–7 days after spawning Oryzias latipes , ricefish flake food BSN, flake food (or protozoa or rotifers) BSN48, flake food (or rotifers) BSN once daily; flake food twice daily or flake food and rotifers once daily from hatch to swim-up within 24 h ofhatch/swimup

Saltwater Cyprinodon

variegatus ,Sheepshead minnow FBS or flake food BSN BSN48 2–3 feeds per day not applicable within 1 day first hatch

1 if applicable 2 yolk-sac larvae require no food 3 not essential 4 filtered from mixed culture 5 granules from fermentation process (iii) Conditions for other species are provided in the following Table 2.:Abbreviations in the table have the following meanings: BSN = brine shrimp nauplii, newly hatched; BSN48 = brine shrimp nauplii, 48 hours old; FBS = frozen brine shrimp; adult Artemia sp.

Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other WellDocumented Species

Species Food Post-Hatch Transfer Time (if applicable) Time To First Feeding Brood fish Newlyhatched larvae Juveniles Type Amount Frequency

Freshwater Oncorhynchus

kisutch , Coho salmon trout food none 1 trout starter 4% BW per day 2–4 feeds/ day 26–36 days post-hatchor at swimup after swim-up at transfer

Oncorhynchus

tschawytscha, Chinook salmon trout food none trout starter 4% BW per day 2–4 feeds/ day 26–36 days post-hatchor at swimup 26 days posthatch at swim-up

Salmo trutta , brown trout trout food none trout starter 4% BW per day 5 feeds/day 21 days posthatch or at swim-up at swim-up

Salmo salar Atlantic

salmon trout food none trout starter 4% BW per day 5 feeds/day 21 days posthatch or at swim-up at swim-up

Salvelinus fontinalis , brook trout trout food none trout starter 4% BW per day 5 feeds/day 21 days posthatch or at swim-up at swim-up

### 5Table 2.—Feeding and Handling Requirements of Brood and Test Animals of Other WellDocumented Species—Continued

Species Food Post-Hatch Transfer Time (if applicable) Time To First Feeding Brood fish Newlyhatched larvae Juveniles Type Amount Frequency

Salvelinus

namaycush , lake trout trout food none trout starter 4% BW per day 5 feeds/day 21 days posthatch or at swim-up at swim-up

Esox lucius, Northern pike live minnows BSN48 larval fish transfer hatched fish daily 1 week posthatch or swimming yolk-sac stage Catostomus

commersoni, white sucker FBS none BSN48 3 feeds/day once all embryos have hatched 7–8 days post-hatchor at swimup Lepomis

macrochirus , bluegill FBS, trout food BSN BSN48 3 feeds per day at swim-up

Ictalurus punctatus , channel catfish catfish food modified Oregon modified Oregon at least 3 feeds per day 6–7 days at 26 ° C 2 within 48 h of swim-up

Jordanella floridae , flagfish FBS, flake food, BSN BSN48, flake food, or protozoa/ rotifers 3 BSN48, flake food Artemia nauplii once daily; flake food twice daily or flake food and protozoa & rotifers once daily from hatch to swim-up within 24 h of hatch

Gasterosteus

aculeatus, threespined stickleback Tetramin FBS Brachionus rubens (rotifer) BSN48, Tetramin BSN48, 2–3 feeds per day; Tetramin once daily several hours after hatch 2 within 24 hours of hatch

Cyprinus carpio , common carp Proprietary carp food;freezedried tubifex or trout food BSN BSN48, ground;trout starter, or flake food 3–4 feeds per day once hatching complete 36–48 h posthatch

Saltwater Menidia menidia, Atlantic silverside BSN48, flake food days 1–8 rotifers 3 3 feeds per day not applicable within 24 h of first hatch days 9–11 BSN48 and rotifers 3 2 feeds per day not applicable within 24 h of first hatch days 11–end BSN48 not applicable within 24 h of first hatch Menidia peninsulae , Tidewater silverside BSN48, flake food days 1–8 rotifers 3 3 feeds per day not applicable within 24 h of first hatch days 9–11 BSN48 and rotifers 3 2 feeds per day not applicable within 24 h of first hatch days 11–end BSN48 not applicable within 24 h of first hatch

1 yolk-sac larvae require no food 2 fish may be handled with a 6 mm internal diameter glass siphon tube 3 rotifers—Brachionus plicatilis (4) Handling of embryos and larvae. (i) Initially, embryos and larvae may be exposed within the main vessel in smaller glass or stainlesssteel vessels, fitted with mesh sides or ends to permit a flow of test solution through the vessel. Nonturbulent flow through these small vessels may

### 6 be induced by suspending them from an arm arranged to move the vessel up and down but always keeping the organisms submerged. Fertilized eggs of salmonid fishes can be supported on racks or meshes with apertures sufficiently large to allow larvae to drop through after hatching. (ii) Where egg containers, grids, or mesh have been used to hold eggs within the main test vessel, these restraints should be removed after the larvae hatch, according to the advice in Table 1. except that mesh should be retained to prevent the escape of the fish. If there is a need to transfer the larvae, they should not be exposed to the air, and nets should not be used to release fish from egg containers. The timing of this transfer varies with the species and transfer may not always be necessary. (5) Water. Any water in which the test species shows control survival, at least as good as that described in Table 4. under paragraph (h)(1)(ii) of this guideline, and Table 5. under paragraph (h)(1)(iii) of this guideline, is suitable as a test water. It should be of constant quality during the period of the test. In order to ensure that the dilution water will notunduly influence the test result (for example, by complexation of test substance) or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg, Na, K, Cl, sulfate), pesticides, total organic carbon, and suspended solids should be made, for example, every 3 months where a dilution water is known to be relatively constant in quality. Some chemical characteristics of an acceptable dilution water are listed in the following Table 3: Table 3.—Some Chemical Characteristics of an Acceptable Dilution Water

Substance Maximum Concentration

Particulate matter

.................................................................................... < 20 mg/L Total organic carbon

................................................................................ < 2 mg/L Un-ionized ammonia

................................................................................ < 1 μg/L Residual chlorine

..................................................................................... < 10 μg/L Total organophosphorus pesticides

........................................................ < 50 ng/L Total organochlorine pesticides plus polychlorinated biphenyls

............. < 50 ng/L Total organic chlorine

.............................................................................. < 25 ng/L

(6) Test solutions. (i) For flow-through tests, a system which continually dispenses and dilutes a stock solution of the test substance (e.g. metering pump, proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals during the test and should not vary by more than 10 percent throughout the test. A flow rate equivalent to at least five test chamber volumes per 24 h has been found suitable (see paragraph (j)(1) of this guideline).

### 7 (ii) The use of solvents or dispersants (solubilizing agents) may be required in some cases in order to produce a suitably concentrated stock solution. (iii) For the semistatic technique, two different renewal procedures may be followed. Either new test solutions are prepared in clean vessels and surviving eggs and larvae gently transferred into the new vessels, or the test organisms are retained in the test vessels while a proportion (at least two-thirds) of the test water is changed. (h) Procedure. Useful information on the performance of fish earlylife stage tests is available in the literature, some examples of which are included under paragraphs (j)(1), and (j)(4) through (j)(8) of this guideline. (1) Conditions of exposure—(i) Duration. The test should start as soon as possible after the eggs have been fertilized, the embryos preferablybeing immersed in the test solutions before cleavage of the blastodisc commences, or as close as possible after this stage. The test should continue at least until all the control fish have been free-feeding. Test duration will depend upon the species used. (A) Data for recommended species are provided in the following Table 4.:

Table 4.—Test Conditions, Duration, and Survival Criteria for Recommended Species

Species Test Conditions Recommended Duration of Test Survival of Controls (minimum percent) Temperature ( ° C) Photoperiod (hours) Hatching Success Post-Hatch Success

Freshwater Oncorhynchus mykiss , Rainbow trout

. 10 ± 2 1 12 ± 2 2 3 14 4 2 weeks after controls are free-feeding (or 60 days post-hatch) > 66 70

Pimephales promelas , Fathead minnow

. 25 ± 2 16 32 days from start of test (or 28 days post-hatch) > 66 70

Danio rerio , Zebra fish

................. 25 ± 2 12–16 6 30 days post-hatch 70 Oryzias latipes , ricefish

............... 24 ± 1 (a) 23 ± 2 2 5 12–16 6 30 days post-hatch 80
Saltwater Cyprinodon variegatus, Sheepshead minnow 7

. 25 ± 2 12–16 6 32 days from start of test (or 28 days post-hatch) > 75 80

1 for embryos 2 for larvae and juvenile fish 3the particular strain of rainbow trout tested may necessitate the use of other temperatures; brood stock must be held at the same temperature as that to be used for the eggs

4 darkness for larvae until one week after hatching except when they are being inspected, then subdued lighting throughout test (12–16 h photoperiod (6))

5this supersedes the requirement for temperature control given earlier on in the test 6for any given test conditions, light regime should be constant 7 salinity shall be at 15–30; for any given test this shall be performed to ± 2 percent.

### 8 (B) Data for other species are provided in the following Table 5: Table 5.—Test Conditions, Duration and Survival Criteria for Other Well-Documented Species

Species Test Conditions Recommended Duration of Test Survival of Controls (minimum percent) Temperature ( ° C) Photoperiod (hours) Hatching Success Post-Hatch Success

Freshwater Oncorhynchus kisutch , Coho salmon

. 10 1 , 12 2 12–16 3 60 days post-hatch > 66 70 Oncorhynchus tschawytscha , Chinook salmon

. 10 1 , 12 2 12–16 3 60 days post-hatch > 66 70

Salmon trutta , brown trout

........... 10 12–16 3 60 days post-hatch > 66 70 Salmo salar , Atlantic salmon

....... 10 12–16 3 60 days post-hatch > 66 70 Salvelinus fontinalis , brook trout

. 10 14 3 60 days post-hatch > 66 70 Salvelinus namaycush , Lake trout 12–18 16 60 days post-hatch > 66 70 Esox lucius , Northern pike .......... 7 12–16 3 32 days from start of test > 66 70 Catostomus commersoni , White sucker

. 15 16 32 days from start of test > 66 80 Lepomis macrochirus , Bluegill

..... 28 16 32 days from start of test 75 Ictalurus punctatus, Channel catfish

. 26 16 32 days from start of test 65 (overall)

Jordanella floridae , Flagfish

........ 24–26 16 Gasterosteus aculeatus, Threespined stickleback

. 18–20 12–16 28 days 80 80 Cyprinus carpio , common carp

... 21–25 12–16 28 days post-hatch > 80 75 Saltwater Menidia menidia , Atlantic silverside 4

. 22–25 13 28 days > 80 60 Menidia peninsulae , Tidewater silverside 4

. 22–25 13 28 days > 80 60

1 for embryos 2 for larvae and juvenile fish 3 darkness for larvae until 1 week after hatching except when they are being inspected, then subdued lighting throughout test (12–16 h photoperiod unless otherwise specified, but constant regime for a given test)

4 salinity 20 (ii) Loading. The number of fertilized eggs at the start of the testshould be sufficient to meet statistical requirements. They should be randomly distributed among treatments, and at least 60 eggs, divided equallybetween at least two replicate test chambers, should be used per concentration. The loading rate (biomass per volume of test solution) should be low enough in order that a dissolved oxygen concentration of at least 60percent of the air saturation value (ASV) can be maintained without aeration. For flow-through tests, a loading rate not exceeding 0.5 g/L/24 h and not exceeding 5 g/L of solution at any time has been recommended (see paragraph (j)(1) of this guideline). (iii) Light and temperature. The photoperiod and water temperatureshould be appropriate for the test species as given in Table 4. under paragraph (h)(1)(ii) of this guideline. (iv) Feeding. Food and feeding are critical, and it is essential that the correct food for each stage should be supplied at an appropriate time and at a level sufficient to support normal growth. Feeding should be ad libitum while minimizing the surplus. Surplus food and feces should be removed as necessary to avoid accumulation of waste. Detailed feeding regimes are given in Table 1. under paragraph (g)(3)(ii) of this guideline,

### 9 but, as experience is gained, food and feeding regimes are continually being refined to improve survival and optimize growth. Effort shouldtherefore be made to confirm the proposed regime with acknowledged experts. (v) Test concentrations. (A) Normally five concentrations of the test substance spaced by a constant factor not exceeding 3.2 are required. The curve relating LC50 to period of exposure in the acute study should be considered when selecting the range of test concentrations. The use of fewer than five concentrations, for example in limit tests, and a narrowerconcentration interval may be appropriate in some circumstances. Justification should be provided if fewer than five concentrations are used. Concentrations of the substance higher than the 96–h LC50 or 10 mg/L, whichever is the lower, need not be tested. (B) Where a solubilizing agent is used, its concentration should notbe greater than 0.1 mL/L and should be the same in all test vessels. However, every effort should be made to avoid the use of such materials. (vi) Controls. One dilution-water control and also, if relevant, one control containing the solubilizing agent should be run in addition to the test series. (2) Frequency of analytical determinations and measurements. (i) During the test, the concentrations of the test substance are determinedat regular intervals to check compliance with the validity criteria. A minimum of five determinations is necessary. In studies lasting more than 1 month, determinations should be made at least once a week. Samples may need to be filtered (e.g. using a 0.45 μm pore size) or centrifugedto ensure that the determinations are made on the substance in true solution. (ii) During the test, dissolved oxygen, pH, total hardness and salinity (if relevant), and temperature should be measured in all test vessels. As a minimum, dissolved oxygen, salinity (if relevant), and temperature should be measured weekly, and pH and hardness should be measured at the beginning and end of the test. Temperature should preferably be monitored continuously in at least one test vessel. (3) Observations—(i) Stage of embryonic development . The embryonic stage at the beginning of exposure to the test substance should be verified as precisely as possible. This can be done using a representative sample of eggs suitably preserved and cleared. (ii) Hatching and survival. Observations on hatching and survival should be made at least once daily and numbers recorded. Dead embryos, larvae, and juvenile fish should be removed as soon as observed since they can decompose rapidly and may be broken up by the actions of the other fish. Extreme care should be taken when removing dead individuals

### 10not to knock or physically damage adjacent eggs/larvae, these being extremely delicate and sensitive. Criteria for death vary according to life stage:(A) For eggs: Particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance. (B) For embryos: Absence of body movement and/or absence of heart-beat.(C) For larvae and juvenile fish: Immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of central nervous system and/or lack of reaction to mechanical stimulus. (iii) Abnormal appearance. The number of larvae or fish showingabnormality of body form should be recorded at adequate intervals depending on the duration of the test and the nature of the abnormality described. It should be noted that abnormal embryos and larvae occur naturally and can be of the order of several percent in the controls in some species. Abnormal animals should only be removed from the test vessels on death. (iv) Abnormal behavior. Abnormalities, e.g. hyperventilation, uncoordinated swimming, atypical quiescence, and atypical feeding behavior should be recorded at adequate intervals depending on the duration of the test. These effects, although difficult to quantify, can, when observed, aid in the interpretation of mortality data and influence a decision to extend the exposure period beyond the recommended duration. (v) Weight. At the end of the test all surviving fish must be weighed. Individual weights are preferred but, if the fish are especially small, they may be weighed in groups by test vessel. Dry weights (24 h at 60 ° C) are preferable to wet weights (blotted dry). (vi) Length . At the end of the test, measurement of individual lengths is recommended: Standard, fork, or total length may be used. If however, caudal fin rot or fin erosion occurs, standard lengths should be used. (vii) Data for statistical analysis. These observations will result in some or all of the following data being available for statistical analysis: (A) Cumulative mortality. (B) Numbers of healthy fish at end of test. (C) Time to start of hatching and end of hatching. (D) Numbers of larvae hatching each day. (E) Length and weight of surviving animals.

### 11 (F) Numbers of deformed larvae. (G) Numbers of fish exhibiting abnormal behavior. (i) Data and reporting—(1) Treatment of results. (i) It is recommended that a statistician be involved in both the design and analysis of the test results since this test guideline allows for considerable variation in experimental design as, for example, in the number of test chambers,number of test concentrations, starting number of fertilized eggs, and number of parameters measured. (ii) In view of the options available in test design, specific guidance on statistical procedures is not given here. However, it will be necessary for variations to be analyzed within each set of replicates using analysisof variance or contingency table procedures. To make a multiple comparison between the results at the individual concentrations and those for the controls, Dunnett’s method might be found useful (see paragraphs (j)(9) and (j)(10) of this guideline). However, care must be taken where applying such a method to ensure that chamber-to-chamber variability is estimatedand is acceptably low. Other useful methods are also available (see paragraphs (j)(1), (j)(6), and (j)(11) of this guideline). (2) Interpretation of results. The results should be interpreted with caution where measured toxicant concentrations in test solutions occur at levels near the detection limit of the analytical method. (3) Test report. The test report must include the following information: (i) Test substance. (A) Physical nature and, where relevant, physicochemical properties. (B) Chemical identification data. (ii) Test species. Scientific name, strain, source and method of collection of the fertilized eggs, and subsequent handling. (iii) Test conditions. (A) Test procedure used (e.g. semistatic or flowthrough design). (B) Photoperiods. (C) Test design (e.g. number of test chambers and replicates, number of embryos per replicate).(D) Method of preparation of stock solutions and frequency of renewal (the solubilizing agent and its concentration must be given, when used). (E) Nominal test concentrations, means of the measured values, their standard deviations in the test vessels, and the method by which these

### 12 were attained, and evidence that measurements refer to concentrations of the test substance in true solution.(F) Dilution water characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), totalorganic carbon, suspended solids, salinity of the test medium (if measured), and any other measurements made. (G) Water quality within test vessels: pH, hardness, temperature, and dissolved oxygen concentration. (H) Detailed information on feeding (e.g. type of feed, source, amount given, and frequency). (iv) Results. (A) Evidence that controls met the overall survival acceptability standard of the test species (Tables 4. and 5.). (B) Data on mortality/survival at embryo, larval, and juvenile stages and overall mortality/survival. (C) Days to hatch and numbers hatched. (D) Data for length and weight. (E) Incidence and description of morphological abnormalities, if any. (F) Incidence and description of behavioral effects, if any. (G) Statistical analysis and treatment of data. (H) NOEC for each response assessed. (I) LOEC (at p = 0.05) for each response assessed. (J) Any concentration-response data and curves available. (v) Discussion of the results. [Reserved] (j) References. The following references should be consulted for additional background material on this test guideline. (1) American Society for Testing and Materials (ASTM). Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM E 1241–92, p. 180–207, Philadelphia, PA (1992).(2) Brauhn, J.L. and Schoettger, R.A., Acquisition and Culture of Research Fish: Rainbow trout, Fathead minnows, Channel catfish and Bluegills. p. 54, Ecological Research Series, EPA–660/3–75–011, Duluth, MN (1975).(3) Brungs, W.A. and Jones, B.R., Temperature Criteria for Freshwater Fish: Protocol and Procedures. p. 128, Ecological Research Series EPA–600/3–77–061, Duluth, MN (1977).

### 13 (4) Hansen, D.J. and Parrish, P.R., Suitability of sheepshead minnows ( Cyprinodon variegatus ) for life-cycle toxicity tests. In Aquatic Toxicology and Hazard Evaluation (edited by F.L. Mayer and J.L. Hamelink), pp. 117–126, ASTM STP 634 (1977). (5) McKim, J.M. et al., Metal toxicity to embryos and larvae of eight species of freshwater fish-II: Copper. Bulletin of Environmental and Contamination Toxicology 19:608–616 (1978).(6) Rand, G.M. and Petrocelli, S.R., Fundamentals of Aquatic Toxicology. Hemisphere Publication Corporation, NY (1985).(7) USEPA, Recommended Bioassay Procedure for Fathead Minnows, Pimephales promelas (Rafinesque), Chronic Tests. p. 13, National Water Quality Laboratory, Duluth, MN (1972). (8) USEPA, Recommended Bioassay Procedure for Bluegill. Lepomis macrochirus (Rafinesque), Partial Chronic Tests. p. 11, National Water Quality Laboratory, Duluth, MN (1972). (9) Dunnett, C.W., A multiple comparisons procedure for comparing several treatments with a control. Journal of the American Statistical Association 50: 1096–1121 (1955).(10) Dunnett, C.W., New tables for multiple comparisons with a control. Biometrics 20:482–491 (1964). (11) McClave, J.T. et al., Statistical Analysis of Fish Chronic Toxicity Test Data. Proceedings of 4th Aquatic Toxicology Symposium, ASTM, Philadelphia, PA (1980).United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–129 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.1730 Fish BCF

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information. This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies. These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail. guidelines@epamail.epa.gov.
To Submit Comments. Interested persons are invited to submit comments. By mail. Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person.bring to. Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to. guidelines@epamail.epa.gov.
Final Guideline Release. This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp. fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.1730 Fish BCF (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ). (2) Background. The source material used in developing this harmonized OPPTS test guideline is 40 CFR 797.1520 Fish Bioconcentration Test; OPP guideline 72–6 Aquatic Organism Bioavailability/Biomagnification/Toxicity Tests (Pesticide Assessment Guidelines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 540/09–82–024, 1982 and OPP 165–4 Laboratory Studies of Pesticide Accumulation in Fish (Pesticide Assessment Guidelines, Subdivision N— Environmental Fate) EPA report 540/09–82–031, 1982; and OECD 305E Bioaccumulation: Flow-Through Fish Test. (b) Introduction—(1) Purpose. The purpose of the study is to determine uptake and depuration rate constants and bioconcentration factors (BCFs) for fish exposed to a test chemical in aqueous solution. Another purpose is to identify and quantify major degradates at steady state. BCF values for the test chemical should always be based on concentrations of the chemical in fish tissue and exposure water, and not on total radiolabeled residues. BCFs may be used to help assess risks to the fish and to nontarget organisms (including humans) above them in the food chain. (2) Criteria for performing test. The test is most commonly required for chemicals that are relatively persistent (stable) in water and have a relatively high potential for bioaccumulation as indicated by log P ow (log of the octanol/water partition coefficient) values less than or equal to 1.0. (3) Criteria for degradate characterization. BCFs based on total radiolabeled residues in fish tissue and exposure water can be used to help determine whether major degradates should be identified and quantified. If the BCF in terms of total radiolabeled residues is greater than or equal to 1,000, OPP requires that an attempt be made to identify and quantify pesticide degradates representing greater than or equal to 10 percent of total residues in fish tissues at steady state. If degradates representing greater than or equal to 10 percent of total radiolabeled residues in the fish tissue are identified and quantified, then degradates in the test water should also be identified and quantified. (4) Desired information on the test chemical. To determine whethera BCF test is warranted (see paragraph (b)(2) of this guideline), it is necessary to know aqueous fate characteristics of the test chemical that determine its persistence in water and its octanol/water partition coefficient. Aqueous fate characteristics include rates of abiotic hydrolysis, biodegradation, direct photolysis in natural sunlight, and volatilization from water. Henry’s law constant (approximated by the ratio of the chemi-

### 2cal’s vapor pressure to its solubility in water) is a good indicator of volatilization potential. It is necessary to know the test chemical’s solubility in water to ensure that exposure concentrations do not exceed it. It is alsonecessary to know the toxicity of the chemical to test fish to ensure exposure concentrations do not adversely effect them (see paragraph (g)(2) of this guideline). The purity of the test chemical should be known as well as its radiopurity if radiolabeled. The structure and radiolabeled positions should be known. An appropriate analytical method, of known accuracy, precision, and sensitivity, for the quantification of the substance in the test solutions and in biological material must be available, together with details of sample preparation and storage. Analytical detection limit of test substance in both water and fish tissues should also be known. (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards (GLP) apply to this test guideline. The following definitions also apply to this test guideline.

Bioconcentration/bioaccumulation is the increase in concentration of the test substance in or on an organism (specified tissues thereof) relative to the concentration of test substance in the surrounding medium. The bioconcentration factor (BCF or KB) at any time during the uptake phase of this accumulation test is the concentration of test substance (expressed in milligrams per gram or parts per million) in/on the fish or specified tissues thereof, divided by the concentration of the chemical in the surrounding medium (BCF = C f /C w ).

The depuration (loss) rate constant (k2) is the numerical value defining the rate of reduction in the concentration of the test substance in the test fish (or specified tissues thereof) following the transfer of the test fish from a medium containing the test substance to a medium free of that substance (k2 is expressed in day -1 ).

The exposure or uptake phase is the time during which fish are exposed to the test chemical. Kinetic concentration factors (BCFK) are bioconcentration factors calculated directly from kinetic rate constants (k 1 /k2).

The octanol-water partition coefficient (P ow ) is the ratio of the solubility of a chemical in n -octanol and water at equilibrium and can also be expressed as Kow . Log P ow is used as an indication of a chemical’s potential for bioconcentration by aquatic organisms.

A plateau or steady-state is reached when the the plot of yhe concentration of test substance in fish (C f ) against time becomes parallel to the time axis and three successive analyses of C f made on samples taken at intervals of at least 2 days are within ± 20 percent of each other, and there are no significant differences among the three sampling periods. At

### 3least four successive analyses are required when pooled samples are analyzed. For test substances which are taken up slowly, the intervals would more appropriately be 7 days.

The postexposure or depuration (loss) phase is the time, following the transfer of the test fish from a medium containing test substance to a medium free of that substance, during which the depuration (or the net loss) of the substance from the test fish (or specified tissue thereof) is studied.

The steady state bioconcentration factor is found when the BCF does not change significantly over a prolonged period of time, the concentration of the test substance in the surrounding medium being constant during this period of time.

The uptake rate constant (k 1 ) is the numerical value defining the rateof increase in the concentration of test substance in/on test fish (or specified tissues thereof) when the fish are exposed to that chemical (k 1 is expressed in day–1 ). (d) Principle of test—(1) Uptake and depuration phase. The test consists of two phases—the exposure (uptake) and postexposure (depuration) phases. During the uptake phase, separate groups of fish of one species are exposed to at least two concentrations of the test substanceuntil steady state is achieved or to a maximum of 28–60 days (see paragraph (g)(3) of this guideline). They are then transferred to a medium freeof the test substance for a depuration phase of adequate duration (see paragraph (g)(4) of this guideline). The concentration of the test substance in/ on the fish (or specified tissue thereof) and in water is followed through both phases of the test. (2) Determination of rate constants and BCFs. (i) Concentrationsof the test chemical in fish tissue and water as a function of time throughout the uptake and depuration phases are used to determine the uptake (k 1 ) and depuration (k2) rate constants (see paragraph (i)(1) of this guideline. (ii) Both the steady state and kinetic bioconcentration factors should be calculated (see paragraph (i)(2) of this guideline). The steady statebioconcentration factor (BCFs) is calculated as the ratio of the concentration in the fish (C f ) and to that in the water (C w ) at apparent steady-state. The kinetic bioconcentration factor (BCFK) is calculated as the ratio of the uptake rate constant (k 1 ) to the depuration rate constant (k2) assuming first-order kinetics (iii) At a minimum, BCFs should be computed for the whole fish. Whenever possible, they should also be calculated for edible and nonedible tissue. BCFs should be related to both the weight and lipid content of

### 4 the fish. If first-order kinetics are obviously not obeyed, more complex models should be employed under paragraph (d)(2)(iv) of this guideline. (iv) Model discrimination. Most bioconcentration data have been assumed to be reasonably well described by a simple two-compartment/twoparameter model, as indicated by the rectilinear curve which approximates to the points for concentrations in fish, during the depuration phase, whenthese are plotted on semilog paper. (Where these points cannot be described by a rectilinear curve then more complex models should be employed, see paragraph (k)(20) of this guideline.)(A) Graphical method for determination of depuration (loss) rate constant k2 . Plot the concentration of the test substance found in each sample of fish against sampling time on semilog paper. The slope of that line is k2 . k2 = ln (C f1 /C f2)/(t2 – t 1 ) Note that deviations from a straight line may indicate a more complex depuration pattern than first order kinetics. A graphical method may beapplied for resolving types of depuration deviating from first order kinetics. (B) Graphical method for determination of uptake rate constant k 1 . Given k2 , calculate k 1 as follows: Equation 1

k 1 = C f k2/C w Τ1× (1 – e -k2t)

The value of C f is read from the midpoint of the smooth uptake curve produced by the data when log concentration is plotted versus time (on an arithmetical scale). (C) Computer method for calculation of uptake and depuration (loss) rate constants. The preferred means for obtaining the bioconcentration factor and k 1 and k2 rate constants is to use nonlinear parameter estimation methods on a computer. These programs find values for k 1 and k2 given a set of sequential time concentration data and the model: Equation 2

C f = C w × k 1 /k2 × (1 – e -k2t) 0 < t < tc

### 5 Equation 3
C f = C w × k 1 /k2 × (e–k2 ( t - tc ) – ek-2t t > tc where tc = time at the end of the uptake phase. This approach provides standard deviation estimates of k 1 and k2 . (D) As k2 in most cases can be estimated from the depuration curvewith relatively high precision, and because a strong correlation exists between the two parameters k 1 and k2 if estimated simultaneously, it may be advisable first to calculate k2 from the depuration data only, and subsequently calculate k 1 from the uptake data using nonlinear regression. (e) Materials—(1) Exposure tanks and tubes. Care should be takento avoid the use of materials, for all parts of the equipment, that can dissolve, sorb or leach and have an adverse effect on the fish. Standard rectangular or cylindrical tanks, made of chemically inert material and of a suitable capacity in compliance with loading rate (see paragraph (e)(7) ofthis guideline), can be used. The use of soft plastic tubing should be minimized. Use Teflon, stainless steel and/or glass tubing. Experience has shown that for substances with high adsorption coefficients, such as the synthetic pyrethroids, silanized glass may be required. In these situations the equipment will have to be discarded after use. (2) Diluter. For flow-through tests, a system which continuously dispenses and dilutes a stock solution of the test substance (e.g. metering pump, proportional diluter, saturator system) is required to deliver the test concentrations to the test chambers. Preferably allow at least five volume replacements through each test chamber per day. The flow rates of stock solutions and dilution water should be checked both 48 hours before and then at least daily during the test. Include in this check the determination of the flow-rate through each test chamber and ensure that it does not vary by more than 20 percent either within or between chambers. The flow-through mode is to be preferred, but where this is not possible (e.g. when the test organisms are adversely affected) a semi-static technique may be used provided that the validity criteria are satisfied (see paragraph (g)(11) of this guideline). (3) Dilution water. (i) Natural water is generally used in the test and should be obtained from uncontaminated and uniform quality source. The dilution water must be of a quality that will allow the survival of the chosen fish species for the duration of the acclimation and test periods without them showing any abnormal appearance or behavior. Ideally, itshould be demonstrated that the test species can survive, grow and reproduce in the dilution water (e.g. in laboratory culture or a life-cycle toxicity test). The water should be characterized at least by pH, hardness, total solids, total organic carbon and, preferably also ammonium, nitrite and alkalinity and, for marine species, salinity. Although the parameters which

### 6are important for optimal fish well-being are not fully known, the following Table 1. gives recommended maximum concentrations of a number of parameters for fresh and marine test waters. Table 1.—Some chemical characteristics of an acceptable dilution water

Substance Limit concentration Particulate matter ................................................................................. 5 mg/L Total organic carbon ............................................................................ 2 mg/L Un-ionized ammonia ............................................................................ 1 mg/L Residual chlorine ................................................................................. 10 mg/L Total organophosphorus pesticides ..................................................... 50 ng/L Total organochlorine pesticides ........................................................... 50 ng/L plus polychlorinated biphenyls .............................................. 25 ng/L Total organic chlorine .......................................................................... 1 μ g/L Aluminium ............................................................................................ 1 μ g/L Arsenic ................................................................................................. 1 μ g/L Chromium ............................................................................................. 1 μ g/L Cobalt ................................................................................................... 1 μ g/L Copper ................................................................................................. 1 μ g/L Iron ....................................................................................................... 1 μ g/L Lead ..................................................................................................... 1 μ g/L Nickel ................................................................................................... 1 μ g/L Zinc ...................................................................................................... 1 μ g/L Cadmium .............................................................................................. 100 ng/L Mercury ................................................................................................ 100 ng/L Silver .................................................................................................... 100 ng/L (ii) The water should be of constant quality during the period of a test. The pH value should be within the range 6.0 to 8.5, but during a given test it should be within a range of ± 0.5 pH units. In order to ensurethat the dilution water will not unduly influence the test result (for example, by complexation of the test substance) or adversely affect the performance of the stock of fish, samples should be taken at intervals for analysis. Determination of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca, Mg, Na, K, C1, SO 4 ), pesticides (e.g. totalorganophosphorus and total organochlorine pesticides), total organic carbon and suspended solids should be made, for example, every 3 months where a dilution water is known to be relatively constant in quality. If water quality has been demonstrated to be constant over at least 1 year, determinations can be less frequent and intervals extended (e.g. every 6 months). (iii) The natural particle content as well as the total organic carbon(TOC) of the dilution water should be as low as possible to avoid adsorption of the test substance to organic matter which may reduce its bioavailability. The maximum acceptable value is 5 mg/L for particulate matter (dry matter, not passing a 0.45 μm filter) and 2 mg/L for total

### 7 organic carbon. If necessary, the water should be filtered before use. Thecontribution 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 notexceed the concentration of organic carbon originating from the test substance and, if used, the solubilizing agent by more than 10 mg/L (± 20 percent). (4) Test chemical. Whether radiolabeled or not, the chemical purity of the test chemical should be as high as practical (preferably greater than or equal to 98 percent). If radiolabeled, the radiopurity should be greater than or equal to 95 percent. (5) Test chemical stock solutions. Prepare a stock solution of thetest substance at a suitable concentration. The stock solution should preferably be prepared by simply mixing or agitating the test substance in the dilution water. The use of solvents or dispersant (solubilizing agents) is not recommended; however this may occur in some cases in order to produce a suitably concentrated stock solution. Solvents which may be used are, ethanol, methanol, ethylene glycol monomethyl ether, ethyleneglycol dimethyl ether, dimethylformamide and triethylene glycol. Dispersant which may be used are Cremophor RH40, Tween 80, methylcellulose0.01 percent and HCO-40. Care should be taken when using readily biodegradable agents as these can cause problems with bacterial growth in flow-through tests. (6) Test species. (i) Important criteria in the selection of species are that they are readily available, can be obtained in convenient sizes andcan 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 the following Table 2. Other species may be used but the test procedure may have to be adapted toprovide suitable test conditions. The rationale for the selection of the species and the experimental method should be reported in this case.

### 8 Table 2.—Fish species recommended for testing

Species Test temperature Total lengthof test animal ( ° C) (cm)

Danio rerio 1 (Teleostei, Cyprinidae) (Hamilton-Buchanan) Zebra-fish ......................................................... 20–25 3.0 ± 0.5 Pimephales promelas (Teleostei, Cyprinidae) (Rafinesque) Fathead minnow ....................................... 20–25 5.0 ± 2.0 Cyprinus carpio (Teleostei, Cyprinidae) (Linnaeus) Common carp ........................................................................ 20–25 5.0 ± 3.0 Oryzias latipes (Teleostei, Poecilliidae) (Temminck and Schlegel) Ricefish .......................................................... 20–25 4.0 ± 1.0 Poecilia reticulata (Teleostei, Poeciliidae) (Peters) Guppy 20–25 3.0 ± 1.0 Lepomis macrochirus (Teleostei Centrarchidae) (Rafinesque) Bluegill ...................................................... 20–25 5.0 ± 2.0 Oncorhynchus mykiss (Teleostei Salmonidae (Walbaum) Rainbow trout ................................................................. 13–17 8.0 ± 4.0 Gasterosteus aculeatus (Teleostei, (Gasterosteidae) (Linnaeus) Three-spined stickleback ................................... 18–20 3.0 ± 1.0

1 Meyer A. and G. Orti. Proceedings of the Royal Society of London 252 (Series B):231 (1993).

(ii) Various estuarine and marine species have been used in different countries, for example: Spot (Leiostomus xanthurus ); Sheepshead minnow ( Cyprinodon variegatus ); Silverside (Menidia beryllina ); Shiner perch (Cymatogaster aggregata ); English sole (Parophrys vetulus ); Staghorn sculpin (Leptocottus armatus ); Three-spined stickleback ( Gasterosteus aculeatus ); Sea bass (Dicentracus labrax); Bleak ( Alburnus alburnus ) (iii) The fresh water fish listed are easy to rear and/or are widelyavailable throughout the year, whereas the availability of marine and estuarine species is partially confined to the respective countries. 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. (7) Reference chemicals. The use of reference compounds of known bioconcentration potential would be useful in checking the experimentalprocedure, when required. However, specific substances cannot yet be recommended. (f) Fish care and health—(1) Acclimation. Acclimate the stock population of fish for at least 2 weeks in water at the test temperature and

### 9 feed throughout on a sufficient diet (see paragraph (f)(3) of this guideline) and of the same type to be used during the test. (2) Pretest mortality and health. (i) Following a 48–h settling-inperiod (during acclimation), mortalities are recorded and the following criteria applied: (A) Mortalities of greater than 10 percent of population in 7 days, reject the entire batch. (B) Mortalities of between 5 and 10 percent of population in 7 days, acclimate for 7 additional days. (C) Mortalities of less than 5 percent of population in 7 days, accept the batch. If more than 5 percent mortality during the second 7 days, reject the entire batch. (ii) Ensure that fish used in tests are free from observable diseasesand abnormalities. Discard any diseased fish. Fish should not receive treatment for disease in the two weeks preceding the test, or during the test. (3) Feeding. (i) 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 percent of body weight per day; this keeps the lipid concentration in most species of fish at a relatively constant level during the test. The amount of feed should be recalculated, for example,once per week, in order to maintain consistent body weight and lipid content. For this calculation, the weight of the fish in each test chamber can be estimated from the weight of the fish sampled most recently in that chamber. Do not weigh the fish remaining in the chamber. (ii) Siphon uneaten food and faeces daily from the test chambersshortly after feeding (30 min to 1 h). Keep the chambers as clean as possible throughout the test so that the concentration of organic matter is kept as low as possible (see paragraph (e)(3), since the presence of organic carbon may limit the bioavailability of the test substance under paragraph (k)(6) of this guideline. (iii) Since many feeds are derived from fishmeal, the feed should be analyzed for the test substance. It is also desirable to analyze the feed for pesticides and heavy metals. (g) Exposure conditions during test—(1) Optional preliminary test to determine optimal conditions. It may be useful to conduct a 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.

### 10 (2) Exposure concentrations of test chemical. (i) During the uptakephase, expose fish under flow-through conditions to at least two concentrations of the test substance in water. Normally, select the higher (or highest)concentration of the test substance to be about 1 percent of its acute asymptotic LC50, and to be at least tenfold higher than its detection limit in water by the analytical method used. The highest test concentration can also be determined by dividing the acute 96–h LC50 by an appropriate acute/chronic ratio (e.g. appropriate ratios for some chemicals are about 3, but a few are above 100). If possible, choose the other concentrations such that it differs from the one above by a factor of 10. If this is not possible because of the 1 percent of LC50 criterion and the analytical limit, a lower factor than 10 can be used or the use of 14 C labeled test substance should be considered. (ii) No exposure concentration used should be above the solubility in water of the test substance.(iii) Where a solubilizing agent is used in the stock solution, its diluted concentration in the exposure water should not be greater than0.1 mL/L and should 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. However, every effort should be made to avoid the use of such materials.(iv) Minimize results reported as ‘‘not detected at the limit of detection’’ by pretest method development and experimental design, since such results cannot be used for rate constant calculations. Pretest results can be used to determine the exposure concentrations necessary to ensure that concentrations in fish tissue are generally above method detection limits. (3) Duration of uptake phase. (i) A prediction of the duration of the uptake phase and time required to reach steady state can be obtained from practical experience (e.g. from a previous study or an accumulationstudy on a structurally related chemical) or from certain empirical relationships utilizing knowledge of either the solubility in water or the octanol/ water partition coefficient of the test substance (see paragraph (g)(5) of this guideline). (ii) The uptake phase should be run for 28 days unless it can be demonstrated that equilibrium has been reached earlier. If the steady-state has not been reached by 28 days, the uptake phase should be extended, taking further measurements, until steady-state is reached or 60 days, whichever is shorter. The depuration phase is then begun. (4) Duration of depuration phase. (i) The depuration period is begun by transferring the fish to the same medium but without the test substance in another clean vessel. A depuration phase is always necessaryunless uptake of the substance during the uptake phase has been insignificant (e.g. the BCF is less than 10).

### 11(ii) A period of half the duration of the uptake phase is usually sufficient for an appropriate (e.g. 95 percent) reduction in the body burden of the substance to occur (see paragraph (g)(5) of this guideline for an explanation of the estimation). If the time required to reach 95 percent loss is impractically long, exceeding for example twice the normal duration of the uptake phase (i.e. more than 56 days) a shorter period may be used (e.g. until the concentration of test substance is less than 10 percent of steady-state concentration). However, for substances having more complexpatterns 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. The period may, however, be governed by the period over which the concentration of test substance in the fish remains above the analytical detection limit. (5) Prediction of the duration of the uptake and depuration phases—(i) Prediction of the duration of the uptake phase. (A) Before performing the test, an estimate of k2 and hence some percentage of thetime needed to reach steady-state may be obtained from empirical relationships between k2 and the n -octanol/water partition coefficient (P ow ) or k2 and the aqueous solubilities. (B) ( 1 ) An estimate of k2 (day–1) may be obtained from the following empirical relationship (see paragraph (k)(20) of this guideline): Equation 1 log k2 = –0.414 log P ow + 1.47(r2 = 0.95) For other relationships see see paragraph (k)(14) of this guideline. (2 ) If the partition coefficient (P ow ) is not known, an estimate can be made (see paragraph (k)(4) of this guideline) from a knowledge of the aqueous solubility (s) of the substance using: Equation 2 log P ow = 0.862 log(s) + 0.710(r2 = 0.994) where s = solubility expressed as moles per liter: (n=36) (3 ) These relationships apply only to chemicals with log P ow values between 2 and 6.5 (see paragraph (k)(12) of this guideline). The time to reach some percentage of steady-state may be obtained by applying the k2 -estimate, from the general kinetic equation describing uptake and depuration (first-order kinetics):

dC f /dt = k 1 C w – k2C f or, if C w is constant:

### 12 Equation 3

C f = k 1 /k2 . C w (1 – (exp) –k2t When steady-state is approached (as t approaches infinity), equation 3 may be reduced (see paragraphs (k)(3) and (k)(9) of this guideline) to:

C f = k 1 /k2C w

or C f /C w = k 1 /k2 = BCF

Then k 1 /k2 . Cw is an approach to the concentration in the fish at steadystate (C f,s ). Equation 3 may be transcribed to:

C f = C f,s (1 – (exp) –k2t

or Equation 4

C f /C f,s = 1 – e–k2t Applying equation 4, the time to reach some percentage of steady-state may be predicted when k2 is preestimated using equation 1 or 2. As a guideline, the statistically optimal duration of the uptake phase for the production of statistically acceptable data (BCFK) is that period which is required for the curve of the logarithm of the concentration of the test substance in fish plotted against linear time to reach its midpoint, or 1.6/k2 , or 80 percent of steady-state but not more than 3.0/k2 or 95 percent of steady-state (see paragraph (k)(19) of this guideline). The time to reach 80 percent of steady-state is (equation 4):

0.8 = 1 – e–k2t

or Equation 5

t 80 = 1.6/k2 Similarly 95 percent of steady-state is: Equation 6

t95 - 3.0/k2 For example, the duration of the uptake phase (up) for a test substance with log P ow = 4 would be (using equations 1, 5, and 6):

### 13 log k2 = –0.414.(4) + 1.47

k2 = 0.652 days –1

up (80 pct)= 1.6/0.652 i.e. 2.45 days (59 h)

or

up (95 pct)= 3.0/0.652 i.e. 4.60 days (110 h) Similarly, for a test substance with s = 10–5 mol/L, (log(s) = – 5.0), the duration of up would be (using equations 1, 2 and 5, 6): log (P ow ) = –0.862 (–5.0) + 0.710 = 5.02 log k2 = –0.414 (5.02) + 1.47 k2 = 0.246 days –1

up (80 pct) = 1.6/0.246, i.e. 6.5 days (156 hours)

or

up (95 pct) = 3.0/0.246, i.e. 12.2 days (293 hours) Alternatively, the expression:

teq = 6.54 × 10–3 P ow + 55.31 (hours) may be used to calculate the time for effective steady-state to be reached (see paragraph (k)(12) of this guideline). (ii) Prediction of the duration of the depuration phase. (A) A prediction of the time needed to reduce the body burden to some percentage of the initial concentration may also be obtained from the general equation describing uptake and depuration (first order kinetics) (see paragraphs (k)(13) and (k)(20) of this guideline. For the depuration phase, C w is assumed to be zero. The equation may then be reduced to:
dC f /dt = –k2C f

or C f = C f,0 (exp) –k2t
where C f,0 is the concentration at the start of the depuration period. 50 percent depuration will then be reached at the time (t50):

or Similarly 95 percent depuration will be reached at:

### 14 t95 = 3.0/k2 If 80 percent uptake is used for the first period (1.6/k2) and 95 percent loss in the depuration phase (3.0/k2), then depuration phase is approximately twice the duration of the uptake phase. It is important to note, however, that the estimations are based on the assumption that uptake and depuration patterns will follow first order kinetics. If first order kinetics are obviously not obeyed, more complex models should be employed (e.g. paragraph (k)(16) of this guideline). (6) Numbers and characteristics of test fish. (i) Select the numbers of fish per test concentration such that a minimum of four fish per sample are available at each sampling. If greater statistical power is required, more fish per sample will be necessary. (ii) If adult fish are used, report whether male or female, or both are used in the experiment. If both sexes are used, differences in lipid content between sexes should be documented to be nonsignificant before the start of the exposure; pooling all male and all female fish may be necessary. (iii) In any one test, select fish of similar weight such that the smallest are no smaller than two-thirds of the weight of the largest. All should be of the same year-class and come from the same source. Since weight and age of a fish appear sometimes to have a significant effect on BCFvalues (see paragraph (k)(6) of this guideline) record these details accurately. It is recommended that a sub-sample of the stock of fish is weighed before the test in order to estimate the mean weight (see paragraph (h)(2) of this guideline). (7) Loading of fish. (i) Use high water-to-fish ratios in order to minimize the reduction in C w caused by the addition of the fish at the start of the test and also to avoid decreases in dissolved oxygen concentration. It is important that the loading rate is appropriate for the test species used. In any case, a loading rate of 0.1–1.0 g of fish (wet weight) per liter of water per day is normally recommended. High loading rates can be used if it is shown that the required concentration of test substance can be maintained within ± 20 percent limits, and that the concentration of dissolved oxygen does not fall below 60 percent saturation.(ii) In choosing appropriate loading regimes, take account of the normal habitat of the fish species. For example, bottom-living fish may demand a larger bottom area of the aquarium for the same volume of water than pelagic fish species. (8) Light and temperature. The photoperiod is usually 12 to 16 h and the temperature (± 2 ° C) should be appropriate for the test species (see Table 3. under paragraph (e)(6)(i) of this guideline). The type and

### 15 characteristics of illumination should be known. Caution should be givento the possible phototransformation of the test substance under the irradiation conditions of the study. Appropriate illumination should be used avoiding exposure of fish to unnatural photoproducts. In some cases it may be appropriate to use a filter to screen out UV irradiation below 290 nm. (9) Water quality measurements. During the test, dissolved oxygen,TOC, pH and temperature should be measured in all vessels. Total hardness and salinity (if relevant) should be measured in the controls and one vessel at the higher (or highest) concentration. As a minimum, dissolvedoxygen and salinity (if relevant) should be measured 3 times—at the beginning, around the middle, and end of the uptake period—and once a week in the depuration period. TOC should be measured at the beginning of the test (24 h and 48 h prior to test initiation of uptake phase) before addition of the fish and, at least once a week, during both uptake anddepuration phases. Temperature should be measured daily, pH at the beginning and end of each period and hardness once each test. Temperature should preferably be monitored continuously in at least one vessel. (10) Controls. In addition to the two test concentrations, a control group of fish is held under identical conditions except for the absence of the test substance, to relate possible adverse effects observed in the bioconcentration test to a matching control group and to obtain background concentrations of test substance. One dilution water control and if relevant, one control containing the solubilizing agent should be run. (11) Validity of test. For a test to be valid the following conditions apply: (i) The temperature variation is less than ± 2 ° C. (ii) The concentration of dissolved oxygen does not fall below 60 percent saturation.(iii) The concentration of the test substance in the chambers is maintained within ± 20 percent of the mean of the measured values during the uptake phase. (iv) The mortality or other adverse effects/disease in both control and treated fish is less than 10 percent at the end of the test; where the test is extended over several weeks or months, death or other adverse effects in both sets of fish should be less than 5 percent per month and not exceed 30 percent in all. (h) Sampling and analysis of fish and water—(1) Fish and water sampling schedule. (i) Sample water from the test chambers for the determination of test substance concentration before addition of the fish and during both uptake and depuration phases. As a minimum, sample the

### 16 water at the same time as the fish and before feeding. During the uptake phase, the concentrations of test substance are determined in order to checkcompliance with the validity criteria (see paragraph (g)(11) of this guideline).(ii) Sample fish on at least five occasions during the uptake phase and at least on four occasions during the depuration phase. Since on some occasions it will be difficult to calculate a reasonably precise estimate of the BCF value based on this number of samples (especially when otherthan simple first-order depuration kinetics are indicated), it may be advisable to take samples at a higher frequency in both periods (see the following Table 3.) Store the extra samples as described in paragraph (h)(3) andanalyze them only if the results of the first round of analyses prove inadequate for the calculation of the BCF with the desired precision. (iii) An example of an acceptable sampling schedule is given in the following Table 3. Table 3.—Theoretical example of sampling schedule for bioconcentration tests of substances with log P ow = 4

Fish Sampling Sample time schedule

No. of water samples** No. of fish per sample**Minimal required frequency (days) Additional sampling

(days)

Uptake phase ............................ –1 2* ................................................ 0 2 Add 45-80 fish 1st ............................................... 0.3 2 4 ................................................ 0.3 (2) (4) 2nd ............................................. 0.6 2 4 ................................................ 0.9 (2) (4) 3rd .............................................. 1.2 2 4 ................................................ 1.7 (2) (4) 4th .............................................. 2.4 2 4 ................................................ 3.3 (2) (4) 5th .............................................. 4.7 2 6 Depuration phase

..................... Transfer fish to water free of test chemical 6th .............................................. 5.0 4 ................................................ 5.3 (4) 7th .............................................. 5.9 4 ................................................ 7.0 (4) 8th .............................................. 9.3 4 ................................................ 11.2 (4) 9th .............................................. 14.0 6 ................................................ 17.5 (4)*Sample water after minimum of 3 ‘‘chamber-volumes’’ have been delivered. **Values in parentheses are numbers of samples (water, fish) to be taken if additional sampling is carried out.

### 17 Note: Pretest estimate of k2 for log P ow of 4.0 is 0.652 days –1 . The total duration of the experiment is set to 3 × up = 3 × 4.6 days = 14 days. For the estimation of up see paragraph (g)(5) of this guideline. Other schedules can readily be calculated using other assumed values of P ow to calculate the exposure time for 95 percent uptake. (iv) Continue sampling during the uptake phase until a steady-statehas been established or for 28 days, whichever is the shorter. If the steadystate has not been reached within 28 days continue until a steady-state has been attained or 60 days, whichever is shorter. Before beginning the depuration phase transfer the fish to clean tanks. (2) Sampling methodology. (i) Obtain water samples for analysis by siphoning through inert tubing from a central point in the test chamber. Since neither filtration nor centrifuging appears always to separate the nonbioavailable fraction of the test substance from that which is bioavailable (especially for superlipophilic chemicals, those chemicals with a log P ow greater than or equal to 5) (see paragraphs (k)(6) and (k)(8)of this guideline), samples may not be subjected to those treatments. Instead, measures should be taken to keep the tanks as clean as possible and the content of total organic carbon should be monitored during both the uptake and depuration phases (see paragraph (g)(9) of this guideline). (ii) Remove an appropriate number of fish (normally a minimum of four) from the test chambers at each sampling time. Rinse the sampled fish quickly with water, blot dry, kill instantly, using the most appropriate and humane method, and then weigh. (3) Sample storage. (i) It is preferable to analyze fish and water immediately after sampling in order to prevent degradation or other lossesand to calculate approximate uptake and depuration rates as the test proceeds. Immediate analysis also avoids delay in determining when a plateau has been reached. (ii) Failing immediate analysis, store the samples by an appropriatemethod. Obtain information on the proper method of storage for the particular test substance before the beginning of the study—for example, deep-freezing, holding at 4 ° C, duration of storage, extraction, etc. (4) Analysis of fish samples. (i) Radiolabeled test substances canfacilitate the analysis of water and fish samples, and may be used to determine whether degradate identification and quantification should be made. BFCs based on total radiolabeled residues (e.g. by combustion or tissue solubilization) can serve as one of the criteria for determining if degradesidentification and quantification is necessary. However, BCF determinations for the parent compound should be based upon the concentrationof the parent compound in fish and water, not upon total radiolabeled residues.

### 18 (ii) If the BCF in terms of total radiolabeled residues is greater thanor equal to 1,000, it may be advisable, and for certain categories of chemicals such as pesticides strongly recommended, to identify and quantify degradates representing greater than or equal to 10 percent of total residues in fish tissues at steady state. If degradates representing greater than or equal to 10 percent of total radiolabeled residues in the fish tissue areidentified and quantified, then it is also recommended to identify and quantify degradates in the test water. The major metabolites may be characterized at steady-state or at the end of the uptake phase, whichever is the sooner. It is possible to combine a fish metabolism study with a bioconcentration study to identify and quantify residues in tissues.(iii) The concentration of the test substance should usually be determined for each weighed individual fish. If this is not possible, pooling of the samples on each sampling occasion may be done but pooling does restrict the statistical procedures which can be applied to the data. If a specific statistical procedure and power are important considerations, then an adequate number of fish to accommodate the desired pooling, procedure and power, should be included in the test. See paragraphs (k)(7) and (k)(10) of this guideline for an introduction to relevant pooling procedures. (5) Determination of lipid content. BCF should be expressed both as a function of total wet weight and, for high lipophilic substances, as a function of the lipid content. Determine the lipid content of the fish on each sampling occasion if possible. Suitable methods should be used for determination of lipid content (see paragraphs (k)(5) and (k)(15) ofthis guideline). Chloroform/methanol extraction technique may be recommended as standard method (see paragraph (k)(11) of this guideline). The various methods do not give identical values (see paragraph (k)(18) of this guideline), so it is important to give details of the method used. When possible, the analysis for lipid should be made on the same extract as that produced for analysis for the test substance, since the lipids often have to be removed from the extract before it can be analyzed chromatographically. The lipid content of the fish (as mg/kg wet weight) at the end of the experiment should not differ from that at the start by

more ± 25 percent. The tissue percent solids should also be reported to allow conversion of lipid concentration from a wet to a dry basis. (6) Quality of analytical method. Since the whole procedure is governed essentially by the accuracy, precision, and sensitivity of the analytical method used for the test substance, check the precision and reproducibility of the chemical analysis experimentally, as well as recovery of the test substance from both water and fish to ensure that they are satisfactoryfor the particular method. Also, check that the test substance is not detectable in the dilution water used. If necessary, correct the values of C w and C f obtained from the test for the recoveries and background values of controls. Handle the fish and water samples throughout in such a manner as

### 19 to minimize contamination and loss (e.g. resulting from adsorption by the sampling device). (i) Data analysis—(1) Determination of uptake and depuration rate constants. (i) Obtain the uptake and depuration curves of the test substance by plotting its concentration in/on fish (or specified tissues) in the uptake and in the depuration phase against time on arithmetic scales. The depuration rate constant (k2) is usually determined from the depuration curve (i.e. a plot of the decrease in test substance concentration in the fish with time). The uptake rate constant (k 1 ) is then calculated given k2 and a value of C f which is derived from the uptake curve. See paragraph(d)(2)(iv) of this guideline for a description of these methods. The preferred method for obtaining BCFK and the rate constants, k 1 and k2 , isto use nonlinear parameter estimation methods on a computer (see paragraph (k)(15) of this guideline). Otherwise, graphical methods may be used to calculate k 1 and k2 . If the depuration curve is obviously not first-order, then more complex models should be employed (see paragraphs (k)(3),(k)(4), (k)(9), (k)(12), (k)(13), (k)(14), (k)(19, and (k)(20) of this guideline) and advice sought from a biostatistician. (ii) The uptake rate constant, the depuration (loss) rate constant (or constants, where more complex models are involved), the bioconcentrationfactor, 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. (iii) The results should be interpreted with caution where measured concentrations of test solutions occur at levels near the detection limit ofthe analytical method. Clearly defined uptake and loss curves are an indication of good quality bioconcentration data. The variation in uptake/ depuration constants between the two test concentrations should be less than 20 percent. Observed significant differences in uptake/depuration rates between the two applied test concentrations should be recorded and possible explanations given. Generally the confidence limit of BCFs from well-designed studies approach ± 20 percent. (2) Determination of the steady state and kinetic BCFs. (i) Obtain the uptake curve of the test substance by plotting its concentration in/on fish (or specified tissues) in the uptake phase against time on arithmetic scales. If the curve has reached a plateau, that is, become approximatelyasymptotic to the time axis, calculate the steady state BCFs from the following relationship:

C f at steady state (mean)/C w at steady state (mean) (ii) When no steady state is reached, it may be possible to calculate a BCFs of sufficient precision for hazard assessment from a steady-state at 80 percent (1.6/k2) or 95 percent (3.0/k2) of equilibrium.

### 20 (iii) Determine the concentration factor (BCFK) as the ratio k 1 /k2 , the two first-order kinetic constants. (iv) The BCF is expressed as a function of the total wet weight of the fish. However, for special purposes, specified tissues or organs (e.g. muscle, liver), may be used if the fish are sufficiently large or the fish may be divided into edible (fillet) and nonedible (viscera) fractions. Since,for many organic substances, there is a clear relationship between the potential for bioconcentration and lipophilicity, there is also a corresponding relationship between the lipid content of the test fish and the observedbioconcentration of such substances. Thus, to reduce this source of variability in test results for those substances with high lipophilicity (i.e. with log P ow greater than or equal to 3), bioconcentration should be expressed in relation to lipid content in addition to whole body weight. The lipid content should be determined on the same biological material as is used to determine the concentration of the test substance, when feasible. (j) Test report. The test report must include the following information. (1) Summary. Test chemical and test species, uptake and depuration rate constants, and steady state and kinetic BCFs (2) Materials. (i) Exposure tanks and tubes--material and size of tanks. (ii) Diluter-type and description. (iii) Dilution water. Source, description of any pretreatment, andwater characteristics including pH, hardness, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon, suspended solids, salinity of the test medium (if appropriate) and any other measurements made.(iv) Test substance. Physical nature and, where relevant, physicochemical properties; chemical identification data (including the organic carbon content, if appropriate); if radio-labeled, the precise position of thelabeled atoms and the percentage of radioactivity associated with impurities. (v) Stock solutions. Method of preparation of stock solutions and frequency of renewal (the solubilizing agent, its concentration and its contribution to the organic carbon content of test water must be given, when used). (vi) Test species. Scientific name, strain, source, any pretreatment, age, size-range, etc.

### 21 (vii) Care of fish. Acclimation, pretest mortality and health, feeding (e.g. type of foods, source, composition—at least lipid and protein content if possible, amount given and frequency). (3) Test conditions— (i) Test design. Number and size of test chambers, water volume replacement rate, number of replicates, number of fish per replicate tank, number of test concentrations, controls. (ii) Exposure concentrations. The nominal concentrations, the means of the measured values and their standard deviations in the test vessels. (iii) Length of uptake and depuration phases. Give the lengths of the uptake and depuration phases and the rationale behind them (iv) Light. Type and characteristics of illumination used and photoperiods. (v) Water quality within test vessels. pH, hardness, TOC, temperature and dissolved oxygen concentration. (4) Sampling and analysis. (i) Sampling frequency for fish and water samples. (ii) Sample storage. (iii) Sample extraction and analysis. (iv) Detection and quantification limits. (v) Accuracy and precision—results of spike and replicate analyses (5) Results. (i) Data obtained in any preliminary test. (ii) Validity of the test. Fish mortality and/or abnormal behavior for exposed and control, variations in exposure concentrations, variations in temperature, and minimum dissolved oxygen with respect to test validity criteria. (iii) Lipid content of the test fish.(iv) Uptake and depuration curves of the test chemical in fish; graphical representation of data.(v) Concentrations of parent in fish tissue and exposure water. Tabular representation of data; C f and C w (with standard deviation and range, if appropriate) for all sampling times (C f expressed in milligrams per gam of wet weight (parts per million) of whole body or specified tissues thereof e.g. lipid, and C w expressed in milligrams per gam of wet weight (parts per million). C w values for the control series (background should also be reported).

### 22 (vi) Uptake and depuration rate constants. Give values and 95 percentconfidence limits for the uptake and depuration (loss) rate constants, describe the computation. (vii) Steady state and kinetic BCFs. The BCFs and the BCFK (bothexpressed 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). (viii) Degradate concentrations. Where radiolabeled substances are used, and when required, the accumulation of any major metabolites at steady state or at the end of the uptake phase. (ix) Deviations and/or unusual observations. Report anything unusual about the test, any deviation from these procedures, and any other relevant information. (k) References. The following references should be consulted for additional background material on this test guideline. (1) American Society for Testing and Materials. ASTM E-1022-84. Standard Practice for conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (1988). (2) Bintein, S. et al. Nonlinear dependance of fish bioconcentration

on n -octanol/water partition coefficient. Environmental Research 1:29–390 (1993). (3) Branson, D.R. et al. Transactions of the American Fisheries Society 104:785–792 (1975).(4) Chiou, C.T. and Schmedding D.W. Partitioning of organic compounds in octanol-water systems. Environmental Science and Technology 16:4–10 (1982). (5) Compaan, H. Chapter 2.3, Part II in The determination of thepossible effects of chemicals and wastes on the aquatic environment: degradation, toxicity, bioaccumulation. Government Publishing Office, The Hague, The Netherlands (1980). (6) Connell, D.W. Bioaccumulation behavior of persistent chemicals with aquatic organisms. Reviews of Environmental Contaminant Toxicology 102:117–156 (1988). (7) 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).

### 23 (8) Environmental Protection Agency. 822–R–94–002. Great Lake Water Quality Initiative Technical Support Document for the Procedure to Determine Bioaccumulation Factors (1994). (9) Ernst W. Accumulation in Aquatic Organisms. In: Appraisal of tests to predict the environmental behavior of chemicals. Ed. by Sheehman P., Korte F., Klein W. and Bourdeau P.H. Part 4.4 pp 243-255. 1985 SCOPE, John Wiley & Sons Ltd., New York (1985). (10) Food and Drug Administration. Pesticide analytical manual. Vol. 1. 5600 Fisher’s Lane, Rockville, MD 20852, (1975). (11) Gardner et al. Limnology and Oceanography 30:1099–1105 (1995). (12) Hawker, D.W. and D.W. Connell D.W. Influence of partition coefficient of lipophilic compounds on bioconcentration kinetics with fish. Water Research 22: 701–707. (13) Ko  ̈ nemann, H. and K. Van Leeuwen Toxicokinetics in Fish: Accumulation and Elimination of Six Chlorobenzenes by Guppies. Chemosphere 9:3–19 (1980). (14) Kristensen P. (1991) Bioconcentration in fish: comparison of bioconcentration factors derived from OECD and ASTM testing methods; influence of particulate organic matter to the bioavailability of chemicals. Water Quality Institute, Denmark.(15) Kristensen, P. and N. Nyholm. CEC. Bioaccumulation of chemical substances in fish: the flow-through method—Ring Test Programme, 1984–1985 Final report, March 1987. (16) Organization for Economic Cooperation and Development. Guidelines for testing of chemicals. Paris (1993). (17) OECD, Paris (1995). Direct Phototransformation of chemicals in water. Guidance Document. February 1996. (18) Randall R.C., Lee H., Ozretich R.J., Lake J.L. and Pruell R.J.(1991). Evaluation of selected lipid methods for normalizing pollutant bioaccumulation. Environ. Toxicol. Chem. Vol.10, pp. 1431–1436.(19) Reilly P.M. et al. Guidelines for the optimal design of experiments to estimate parameters in first order kinetic models. Canadian Journal of Chemical Engineering 55:614–622 (1977). (20) Spacie, A. and J.L. Hamelink Alternative models for describing the bioconcentration of organics in fish. Environmental Toxicology and Chemistry 1:309–320 (1982)..... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–164 April 1996 

# Ecological Effects Test Guidelines OPPTS 850.5400 Algal Toxicity, Tiers I and II

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).
Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0135 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 850.5400 Algal toxicity, Tiers I and II. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source material used in developing this harmonized OPPTS test guideline are 40 CFR 797.1050 Algal Acute Toxicity Test; OPP 123–2 Growth and Reproduction of Aquatic Plants (Tier 2) (Pesticide Assessment Guidelines, Subdivision J—Hazard Evaluation; Nontarget Plants) EPA report 540/09-82-020, 1982; and OECD 201, Algal Growth Inhibition Test. (b) Purpose. This guideline is intended for use in developing dataon the acute toxicity of chemical substances and mixtures (chemicals) subject to environmental effects test regulations, and was written specifically for Selenastrum capricornutum and Skeletonema costatum (see paragraph (d)(2)(iii) of this guideline). Use of Anabaena flos-aquae or Navicula pelliculosa may require some specific modifications in test procedures. This guideline prescribes test procedures and conditions using freshwater and marine algae to develop data on the phytotoxicity of chemicals. EPA will use data from these tests in assessing the hazard of a chemical to the environment. (c) Definitions. The definitions in section 3 of the Toxic SubstancesControl Act (TSCA) and the definitions in 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this guideline: Algicidal means having the property of killing algae. Algistatic means having the property of inhibiting algal growth.

ECX means the experimentally derived chemical concentration that is calculated to effect X percent of the test criterion.

Growth means an increase in an algal population based on the number and/or weight of algal cells per volume of nutrient medium or test solution in a specified period of time. Static system means for this test a system in which old nutrient medium (test solution) is not renewed or replaced during the period of the test. (d) Test procedures—(1) Summary of the test. (i) In preparationfor the test, fill test containers with appropriate volumes of nutrient medium and/or test solution. Start the test by introducing algae into the test and control containers in the growth chambers. Environmental conditions within the growth chambers are established at predetermined limits.

### 2 (ii) At the end of 96 h, and at the end of 24, 48, and 72 h if possible,enumerate the algal cells in all containers to determine inhibition or stimulation of growth and the pattern of growth in test containers comparedto controls. Use data to define the concentration-response curve, and calculate the EC50 value at these times. (2) Range-finding test. (i) A range-finding test should be conductedto determine if definitive testing is necessary, and if so, test chemical concentrations for the definitive test. Water solubility of the test chemical (as well as other physical chemical characteristics, e.g. volatility) should be determined before definitive testing. A validated analytical method should also be developed prior to any toxicity testing. (ii) Algae are exposed to a widely spaced (e.g. log interval) chemical concentration series. The lowest value in the series, exclusive of controls, should be at the chemical’s detection limit. The upper value, for water soluble compounds, should be the saturation concentration. A minimum of three replicates is required: Nominal concentrations of the chemical are acceptable if it is determined that definitive testing is not required. (iii) The test is performed once for each of the recommended algal species or selected alternates. Test chambers should contain equal volumes of test solution and approximately 1 × 10 4 S. capricornutum (misidentified previously as as Monoraphidium capricornutum , correctly also known as Raphidocelis subcapitata Korsikov), N. pelliculosa , or A. flos-aquae cells per milliliter or 7.7 × 10 4 S. costatum cells per milliliter of test solution. The algae should be exposed to each concentration of test chemical for up to 96 h. (iv) Definitive testing may not be necessary if the highest chemical concentration tested (water saturation concentration or 1,000 mg/L) resultsin less than a 50 percent reduction in growth. However, if the lowest concentration tested (analytical detection limit) results in greater than a 50 percent reduction in growth definitive testing is necessary. If testing a pesticide under FIFRA at the maximal labeled dosage, a minimum of three replicates for each test chemical concentration is required. (3) Definitive test. (i) The purpose of the definitive test is to determine the concentration response curves, the EC50’s for algal growth foreach species tested, with a minimum amount of testing beyond the rangefinding test. (ii) Algae should be exposed to five or more concentrations of the test chemical in a geometric series in which the ratio is between 1.5 and 2.0 (e.g. 2, 4, 8, 16, 32, and 64 mg/L). Often it is possible to choosetest chemical concentrations based on the anticipated slope of the concentration-response curve, and these concentrations should bracket the expected test end-points. Algae are to be placed in a minimum of three replicate test containers for each concentration of test chemical and control.

### 3 With the exception of the use of four replicates for N. pelliculosa , more than three replicates may be required to provide sufficient data. Each test chamber should contain equal volumes of test solution and approximately 1 × 10 4 Selenastrum , Navicula , or Anabaena cells per milliliter or 7.7 × 10 4 Skeletonema cells per milliliter of test solution. The chemical concentrations should result in greater than 90 percent of algal growth being inhibited or stimulated at the highest concentrations of test substance compared to controls or that the test concentrations should bracket the expected EC50 value. (iii) Every test is to include a control (negative control) consisting of the same nutrient medium, conditions, procedures, and algae from the same culture, except that none of the test substance is added. If a carrier is present in any of the test chambers, a separate carrier control is required. (iv) Positive controls using zinc chloride as a reference chemical should also be run periodically. The purpose of a positive control with a reference chemical is to determine that the test algae are responding to a known chemical in the expected manner. If the algae are responding to subsequent reference chemical tests consistently, it is assumed that thealgae will respond to other chemicals consistently. Changes in algal response caused by such factors as poor nutrition, genetic drift, and contaminants may not be detected by negative controls, yet may still influence test results. At least three concentrations of the reference chemical are run at or near the expected median effect level. (v) The test begins when algae (inocula) from 3– to 7–day–old stock cultures are placed in the test chambers containing test solutions having the appropriate concentrations of the test substance. The mean cell volume of inocula should be approximately 35–45 μg/m 3 at the onset of testing. Algal growth in controls should reach the logarithmic growth phase by 96 h (at which time the number of algal cells should be approximately 1.5 × 10 6/mL for Skeletonema or 3.5 × 10 6/mL for Selenastrum . If logarithmic growth cannot be demonstrated, the test is to be repeated. At the end of 96 h, and, if possible, at the end of 24, 48, and 72 h, the algal growth response (number or weight of algal cells per milliliter) in all test containers and controls is to be determined by an indirect (spectrophotometry, electronic cell counters, dry weight, etc.) or a direct(actual microscopic cell count of at least 400 cells per flask) method. Indirect methods are to be calibrated by a direct microscopic count or data should be presented that relate electronic counts with microscopic counts. The percentage inhibition or stimulation of growth for each concentration, EC50, and the concentration-response curves are determined from these counts. (vi) A particle counter or microscopic counting cannot be used for Anabaena unless the filaments are broken up and dispersed using a syringe, ultrasonic bath, or blender. Limited use of sonification is allowed

### 4 for Anabaena . The procedure used to break up the filaments should result in consistent filament lengths across treatments and replicates. Sonification, ultrasonic bath, blender, syringe, or any other methods of cell separation, other than manual or rotary shaking are not allowed for Selenastrum , Skeletonema , or Navicula

. (vii) At the end of the definitive test, the following additional analyses of algal growth response are to be performed: (A) Determine whether the altered growth response between controls and test algae (in highest test chemical concentrations) was due to a change in relative cell numbers, cell sizes, or both. Also note any unusual cell shapes, color differences, differences in chloroplast morphology, flocculations, adherence of algae to test containers, or aggregation of algal cells. These observations are qualitative and descriptive, and are not used in end-point calculations. They can be useful in determining additional effects of tested chemicals. (B) In test concentrations where growth is maximally inhibited,algistatic effects may be differentiated from algicidal effects by the following two methods. ( 1 ) Add 0.5 mL of a 0.1 percent solution (weight/volume) of Evans blue stain to a 1–mL aliquot of algal suspension from a control container and to a 1–mL aliquot of algae from the test container having the lowest concentration of test chemical which completely inhibited algal growth (if algal growth was not completely inhibited, select an aliquot of algae for staining from the test container having the highest concentration of testchemical which inhibited algal growth). Wait 10 to 30 min, examine microscopically, and determine the percent of the cells which stain blue (indicating cell mortality). A staining control is to be performed concurrently using heat-killed or formaldehyde-preserved algal cells; 100 percent of these cells should stain blue.This method will work for Skeletonema and possibly Navicula , but probably will not work with Selenastrum or Anabaena

. (2 ) Remove 0.5 mL aliquots of test solution containing growth-inhibited algae from each replicate test container having the concentration of test substance evaluated in paragraph (d)(3)(vii)(B)( 1 ) of this guideline.Combine these aliquots into a new test container and add a sufficient volume of fresh nutrient medium to dilute the test chemical to a concentrationwhich does not affect growth. Incubate this subculture under the environmental conditions used in the definitive test for a period of up to 9 days, and observe periodically (e.g. every other day) for algal growth (direct or indirect methods) to determine if the algistatic effect noted after the 96–h test is reversible. This subculture test may be discontinued as soon as growth occurs.

### 5 (4) Analytical measurements—(i) Chemical. (A) Water of sufficient quality (e.g. ASTM Type I water) is to be used in the preparation of thenutrient medium. The pH of the test solution and controls is to be measured at the beginning and at the end of the definitive test. The concentration of test chemical in the test containers is to be determined at the beginning and end of the definitive test by standard analytical methods which have been validated prior to the test. An analytical method is unacceptableif likely degradation products of the chemical, such as hydrolysis and oxidation products, give positive or negative interference to the method. Tobe acceptable, the analytical method must be corrected for these interferences. (B) At the end of the test and after aliquots have been removed for algal growth-response determinations, microscopic examination, mortal staining, or subculturing, the replicate test containers for each chemical concentration may be pooled into one sample. An aliquot of the pooledsample may then be taken and the concentration of test chemical determined after all algal cells have been removed. In addition, the concentration of test chemical associated with the algae alone may be determined. Separate and concentrate the algal cells from the test solution by centrifuging or filtering the remaining pooled sample and measure the test substance concentration in the algal-cell concentrate. (ii) Numerical. Algal growth response (as percent of inhibition or stimulation in the test solutions compared to the controls) is calculated at the end of the test. Mean and standard deviation should be calculated and plotted for each treatment and control. Appropriate statistical analyses(see paragraphs (g)(1) and (g)(2) of this guideline) should provide a goodness-of-fit determination for the concentration response curves. The concentration response curves are plotted using the mean measured test solution concentrations obtained in the test chambers at the end of the test.Results from the recovery phase are used to determine the algistatic concentration (refer to paragraph (g)(3) of this guideline). Various statistical procedures for modeling continuous toxicity data are available and can be used (see paragraph (g)(4) of this guideline). (e) Test conditions—(1) Test species. Species of algae recommended as test organisms for this test are the freshwater green alga, S. capricornutum (or R. subcapitata ), the marine diatom, S. costatum , the freshwater diatom, N. pelliculosa , and the blue-green alga or cyanobacterium, A. flos-aquae . Algae to be used in acute toxicity testsmay be initially obtained from commercial sources and subsequently cultured using sterile technique. Toxicity testing should not be performeduntil algal cultures are shown to be actively growing (i.e. capable of logarithmic growth within the test period) in at least two subcultures lasting 7 days each prior to the start of the definitive test. All algae used for a particular test should be from the same source and the same stock cul-

### 6 ture. Also, the clone of all species should be specified. Test algae should not have been used in a previous test, either in a treatment or a control. (2) Facilities—(i) General. (A) Facilities needed to perform this test include: A growth chamber or a controlled environment room that canhold the test containers and will maintain the air temperature, lighting intensity, and photoperiod specified in this test guideline, apparatus for culturing and enumerating algae, a source of water of acceptable quality, and apparatus for carrying out analyses of the test chemical. (B) Disposal facilities should be adequate to accommodate spent glassware, algae, and test solutions at the end of the test, and any bench covering, lab clothing, or other contaminated materials. (ii) Test containers. Erlenmeyer flasks should be used for test containers. The flasks may be of any volume between 125 and 500 mL as long as the same size is used throughout a test and the test solution volume does not exceed 50 percent of the flask volume. (iii) Cleaning and sterilization. New test containers may contain substances which inhibit growth of algae. They are therefore to be cleaned thoroughly and used several times to culture algae before being used in toxicity testing. All glassware used in algal culturing or testing is to be cleaned and sterilized prior to use according to standard good laboratory practices. These practices include rinsing in a solvent followed by rinsing with acid. (iv) Conditioning. Test containers should be conditioned, if needed, by a rinse with the appropriate test solutions prior to the start of the test. Decant and add fresh test solutions after an appropriate conditioning period for the test chemical. (v) Nutrient medium. (A) Formulation and sterilization of nutrientmedium used for algal culture and preparation of test solutions should conform to those currently recommended by the EPA for freshwater and marine algal bioassays under paragraph (g)(1) of this guideline. Chelating agents are included in the nutrient medium for optimum cell growth. No chelating agents are to be included in the nutrient medium used for test solution preparation if it is suspected that the chelater will interact with the test chemical. For some chemicals, the stock solution must be prepared with dilution water (e.g. ASTM Type I water). Volumetric addition should be limited to prevent dilution of the nutrients. Nutrient medium should be freshly prepared for algal testing and may be dispensed in appropriate volumes in test containers and sterilized by autoclaving or filtering by using a clean water source. The pH of the nutrient medium is to be 7.5 (± 0.1) for Selenastrum , 8.1 (± 0.1) for Skeletonema , 7.5 (± 0.1) for Navicula , and 7.5 (± 0.1) for Anabaena at the start of the test and may be adjusted prior to test chemical addition with 0.1N or 1N NaOH or HC1.

### 7 (B) Dilution water used for preparation of nutrient medium and testsolutions should be of sufficient quality (e.g. ASTM Type I water). Saltwater for marine algal nutrient medium and test solutions should be prepared by adding a commercial synthetic sea salt formulation or a modifiedsynthetic seawater formulation to distilled/deionized water to a concentration of 30 ppt (24 to 35 g/kg). (vi) Carriers. Nutrient medium is to be used in making stock solutions of the test chemical. If a carrier (or solvent) other than nutrient medium is absolutely necessary to dissolve the chemical, the volume used should not exceed the minimum volume necessary to dissolve or suspend the chemical in the test solution. The upper limit of carrier volume is0.5 mL/L and the same amount of carrier should be added to each concentration (refer to paragraph (g)(1) of this guideline). (3) Test parameters. (i) The test temperature is to be 24 ° C for Selenastrum , Navicula , and Anabaena , and 20 ° C for Skeletonema. Excursions from the test temperature should be no greater than ± 2 ° C. Temperature should be recorded hourly during the test. A continuous recording device is suitable for this purpose. (ii) Test chambers containing Selenastrum , Navicula , and Anabaena , must be illuminated continuously; those containing Skeletonema are to be provided a 14–h light/10–h dark photoperiod. Fluorescent lights providing 4.3 K lx (4,306 lm/m2 or 400 ± 10% fc) for Selenastrum , Skeletonema , and Navicula , and 2.2 K lx for Anabaena are to be used. These lamps should have a photosynthetically active radiation of approximately 66.5 ± 10% μEin/m2/sec. Light intensities (or light fluence rate) should be measured at each test chamber position at the approximate level of the test solution.(iii) Stock algal cultures should be shaken on a rotary shaking apparatus. Test containers also should be placed on a rotary shaking apparatus and oscillated at approximately 100 cycles/min for Selenastrum and at approximately 60 cycles/min for Skeletonema during the test. If clumping of Skeletonema is experienced or anticipated, hand shaking once or twice a day is acceptable. The rate of oscillation should be determined at the beginning of the test or at least once daily during testing if the shaking rate is changed or changes. (iv) The pH of nutrient medium in which algae are subcultured is to be 7.5 (± 0.1) for Selenastrum , 8.1 (± 0.1) for Skeletonema , 7.5 (± 0.1) for Navicula , and 7.5 (± 0.1) for Anabaena , and is not adjusted after the addition of the algae. The pH of all test solutions is to be measured at the beginning and end of the test. If the test chemical is highly acidic and reduces the pH of the test solution below 5.0 at the first measurement, appropriate adjustments to pH should be considered, and the test solution measured for pH on each day of the test.

### 8 (v) Light intensity should be monitored at the beginning of the test at the level of the test solution or at each test chamber position. If it is suspected that light intensity has changed, monitoring more often during the test will be necessary. (f) Reporting. The sponsor must submit to the EPA all data developed by the test including those that are suggestive or predictive of acute phytotoxicity. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information is to be reported:(1) Detailed information about the test organisms, including the scientific name, method of verification, strain, and source. (2) Control charts of growth in the nontreatment and solvent controls for each toxicity test. (3) A description of the test chambers and containers, the volumes of solution in the containers, the way the test was begun (e.g. conditioning, test substance additions, etc.), the number of replicates, the temperature,the lighting, and method of incubation, oscillation rates, and type of apparatus. Specific modifications in test procedures due to using Anabaena or Navicula must be noted. (4) The concentration of the test chemical in the control and in each treatment at the end of the test and the pH of the solutions.(5) The number of algal cells per milliliter in each treatment and control and the method used to derive these values at the beginning, at 24, 48, and 72 h, and at the end of the test; the percentage of inhibition or stimulation of growth relative to controls; and other adverse effect in the control and in each treatment.(6) The 96–h EC50 values, and when sufficient data have been generated, the 24–, 48–, and 72–h EC50s and 95 percent confidence limits, the methods used to derive these values, the data used to define the shape of the concentration-response curve and the goodness-of-fit determination. Electronic data submission (raw data) is encouraged to reduce data entry time required to conduct statistical analyses.(7) Methods and data records of all chemical analyses and test substance concentrations, including method validations and reagent blanks. (8) The results of analyses such as: Microscopic appearance of algae, size or color changes, percent mortality of cells and the fate of subcultured cells, the concentration of test substance associated with algae and test solution supernate or filtrate. (9) If the range-finding test showed that the highest concentration of the chemical tested (not less than 1,000 mg/L or saturation concentration)

### 9had no effect on the algae, report the results and concentration and a statement that the chemical is of minimum phytotoxic concern.(10) If the range-finding test showed greater than a 50 percent inhibition of algal growth at a test concentration at or below the analytical detection limit, report the results, concentration, and a statement that the chemical is phytotoxic at or below the analytical detection limit. (g) References. The following references should be consulted for additional background material on this test guideline. (1) American Society for Testing and Materials. ASTM E1218–20. Standard guide for conducting 96–h toxicity tests with microalgae. In: 1991 Annual Book of ASTM Standards, Vol. 11.04: Pesticides; resourcerecovery; hazardous substances and oil spill response; waste disposal; biological effects. pp 845–856 (1991). (2) American Society for Testing and Materials. ASTM D 3978–80. Standard practice for algal growth potential testing with Selenastrum capricornutum . In: 1991 Annual Book of ASTM Standards, Vol. 11.04: Pesticides; resource recovery; hazardous substances and oil spill response; waste disposal; biological effects. pp 32–36 (1991). (3) Payne, A.G. and R.H. Hall. A method for measuring algal toxicity and its application to the safety assessment of new chemicals. pp 171- 180 in L.L. Marking and R.A. Kimerle (eds.). Aquatic Toxicology, ASTM STM 667, American Society for Testing and Materials, Philadelphia, PA (1979).(4) Bruce, R.D. and D.J. Versteeg. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494 (1992).