
  Office of Chemical Safety and Pollution Prevention (7101)
                                                               EPA 712-C-16-013
                                                                  October 2016 

Ecological Effects
Test Guidelines



OCSPP 850.1010:

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




OCSPP 850.1010: Aquatic invertebrate acute toxicity test, freshwater daphnids
(a) Scope.
      (1) Applicability. This guideline is intended for use in meeting testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.). It describes procedures that, if followed, would result in data that would generally be of scientific merit for the purposes described in paragraph (b) of this guideline.
      (2) Background. The source materials used in developing this harmonized OCSPP test guideline are 40 CFR 797.1300 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, see paragraph (j)(6) of this guideline); Standard Evaluation Procedure: Acute Toxicity Test for Freshwater Invertebrates (see paragraph (j)(7) of this guideline); EPA Pesticide Reregistration Rejection Rate Analysis: Ecological Effects (see paragraph (j)(8) of this guideline); OECD 202 Daphnia sp. Acute Immobilisation Test, 2004 (see paragraph (j)(4) of this guideline); and ASTM E729-96 (07), Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians (see paragraph (j)(1) of this guideline).
(b) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations. This guideline describes an acute toxicity test in which daphnids (Daphnia magna or Daphnia pulex) are exposed to a test substance in static, static-renewal, or flow-through systems. The Environmental Protection Agency will use data from this test to assess the hazards and risks a test substance may present in the aquatic environment.
(c) Definitions. The definitions in OCSPP 850.1000 apply to this test guideline. In addition, the following more specific definitions also apply to this guideline:
      Effect concentration, median (EC50) is the experimentally derived concentration of test substance in dilution water that would be expected to cause a defined adverse effect in 50 percent (%) of a group of test organisms under specified exposure conditions. In this guideline, the effect measured is immobilization as a surrogate for death.
      Ephippium is a resting egg that develops in daphnids under the carapace in response to stress conditions.
      Immobilization refers to a lack of movement except for minor spontaneous, random activity of appendages; those animals that are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobilized even if they can still move their antennae. Immobilization, as defined for this guideline, is used as a surrogate for death. Dead animals are counted as immobilized.
(d) General considerations.
      (1) Summary of the test. Young daphnids less than (<) 24 hours old at test initiation are exposed to the test substance and to appropriate controls (i.e., dilution water control and vehicle (solvent) control, if a vehicle is used) for 48 hours, during which observations are made on immobilization (death) and other toxic effects. The test is designed to determine the relationship between aqueous concentrations of the test substance and immobilization of daphnids over the full concentration-response curve. The results of the test are expressed as the 48-hour median effect concentration (48-h EC50) for immobilization and the slope of the concentration-response relationship. Although the 48-h EC50 for immobility is the primary toxicity endpoint, information on other signs of toxicity such as abnormal appearance and behavior and concentration-response curves is useful in understanding the toxic response.
      (2) General test guidance. The general guidance in OCSPP 850.1000 applies to this guideline except as specifically noted herein.
      (3) Range-finding test. A range-finding test is usually conducted to establish the appropriate test solution concentrations to be used in the definitive test. In the range-finding test, the test organisms are generally exposed to a series of widely-spaced concentrations of the test substance (e.g., 1, 10, 100 milligrams per liter (mg/L)). The details of the range-finding test do not have to be the same as for definitive testing in that the number of replicates, the number of test organisms, and duration of exposure may be less than that used in definitive testing. In addition, the types of observations made on test organisms may not be as detailed or as frequently observed as that of a definitive test. 
      (4) Definitive test. The primary goal of the definitive test is to determine the 48-hour concentration-response curve for immobilization; the 48-h EC50, its standard error and 95% confidence interval; and the slope of the concentration-response curve, its standard error and 95% confidence interval. Where sufficient data are available, these values are also calculated for the 24-hour concentration-response curve. A minimum of 5 concentrations of the test substance, plus appropriate controls, should be tested. The selected test concentrations should bracket the 48-h EC50. Clinical signs of toxicity such as abnormal appearance and behavior, if any, should be reported. Analytical confirmation of dissolved test concentrations should be performed as described in OCSPP 850.1000. Summaries of the test conditions are presented in Table 1 of this guideline. Test validity elements are listed in Table 2.
      (5) Limit test. In some situations, it is only necessary to ascertain that the 48-h EC50 is above a certain limit concentration (i.e., 48-h EC50 greater than (>) limit concentration). In a limit test, at least 20 daphnids, divided equally into a minimum of 2 replicates, are exposed to a single "limit concentration," with the same number of organisms in appropriate controls. For most industrial chemicals, the lower of 100 mg/L or the limit of water solubility or dispersion is considered appropriate as the limit concentration. For pesticides, the lower of 100 milligrams active ingredient per liter (mg a.i./L), when estimated environmental concentrations are not expected to exceed 100 mg/L, or the limit of water solubility may be used as the limit concentration. Except for the number of test concentrations, limit tests should follow the same test procedures, have the same duration as the multiple-concentration definitive test (see Table 1 of this guideline), and have both a dilution water control and a vehicle (solvent) control, if a vehicle is used. Limit tests, like definitive tests, should include analytical confirmation of the dissolved concentration of the test substance. Clinical signs of toxicity such as abnormal appearance and behavior, if any, should be reported. For pesticides, if any daphnid is immobilized in the limit test concentration, a multiple-concentration 48-hour test should be conducted.
(e) Test standards.
      (1) Test substance. The substance to be tested should be technical or reagent grade unless the test is designed to evaluate a specific formulation, mixture, or end-use product. For pesticides, if more than one active ingredient constitutes a technical product, the technical grade of each active ingredient should be tested separately, in addition to the combination, if applicable. OCSPP 850.1000 lists the type of information that should be known about the test substance before testing and discusses methods for preparation of test solutions.
	(2) Test duration. The test duration is a minimum of 48 hours.
	(3) Test organism.
            (i) Species. The test species are Daphnia magna or Daphnia pulex. First instar daphnids, <24 hours old, should be used to start the test. 
            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 cultures established from the same stock of daphnids.
            (ii) Holding and Acclimation. During culturing and acclimation to the dilution water, stock animals should be maintained in culture conditions (e.g., temperature, light intensity) similar to those to be used in the test. Any changes in the temperature and chemistry of the dilution water used for culturing the test organisms to those of the test should be gradual, and a group of brood daphnids should be maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test. Within a 24-hour period, changes in water temperature should not exceed 1 degree Celsius (°C). 
            (iii) Health status and condition. To reduce variability, it is strongly recommended that daphnids used in the test not be first brood progeny. 
            Daphnids should not be used for a test:
                  (A) If the culture contains ephippia;
                  (B) If more than 5% of the culture stock dies or shows signs of stress (e.g., exhibits abnormal behavior) during the 48 hours preceding the test;		
                  (C) If adults in the cultures do not produce young before day 12;
                  (D) If adults in the culture do not produce an average of at least 3 young per adult per day over the 7-day period prior to the test;
                  (E) If they have been used in a previous test, either in a treatment or in a control group.
            (iv) Care and handling. Organisms should be handled as little as possible, but when necessary, it should be done as carefully and quickly as possible. During culturing, holding, 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 during handling should also be discarded. Smooth glass tubes (inner diameter >5 millimeters (mm)) equipped with rubber bulbs have been used in 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.
            (v) Diet and feeding. Daphnids should not be fed during testing. Various combinations of trout chow, yeast, alfalfa, and algae have been successfully used for culturing daphnids; however, research has demonstrated that it is best to incorporate algae into the diet to maintain consistently healthy daphnids (see paragraphs (j)(2) and (j)(9) of this guideline). 
	(4) Administration of test substance.
            (i) Preparation of test solutions. Preparation of test solutions depends on the solubility and stability of the test substance. Guidance for preparation of test solutions, especially for difficult or low solubility test substances, is provided in OCSPP 850.1000. Dilution water source and quality used in the test are described in OCSPP 850.1000 and paragraph (e)(7)(vi) of this guideline. 
            The concentration of vehicle solvent should not exceed 0.1 milliliters per liter (mL/L). A previous review recommends that solvent concentrations as low as 0.02 mL/L of dilution water be used (see paragraph (j)(3) of this guideline). 
            The pH of stock solutions may be adjusted to match the pH of dilution water or to a neutral pH if pH change does not affect the stability of the test substance in water. The pH of test solutions may be adjusted after the addition of the test substance or stock solution into the dilution water. However, all pH adjustments need to be made prior to the addition of test organisms. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) may be used for this adjustment if warranted. 
            See additional information about pH during testing in (e)(8)(ii).
            (ii) Exposure technique. The test may be conducted using one of three basic exposure techniques: static, static-renewal, or flow-through. Guidance on the selection of the appropriate exposure technique is provided in OCSPP 850.1000.
            (iii) Treatment concentrations. At least 5 test solution concentrations should be used for definitive testing, plus the appropriate control(s). A range-finding test can be used to establish the appropriate test solution concentrations for the definitive test (see paragraph (d)(3) of this guideline). For scientifically sound estimates of a given point estimate (e.g., EC50), test substance concentrations should immediately bracket the point estimate(s) of concern. OCSPP 850.1000 provides guidance on selection of test concentrations. For a limit test, there is a single treatment concentration, plus the appropriate control(s). Guidance on the limit concentration is provided in paragraph (d)(5) of this guideline.
      (5) Controls. Every test includes a dilution water control and a vehicle (solvent) control, if a vehicle is used. Controls consist of the same dilution water, conditions, procedures, and test population as the test solutions, except that no test substance is added. 
      A test is not acceptable if more than 10% of the organisms in any control shows signs of disease, stress (e.g., discoloration, unusual behavior, immobilization), and/or death.
      (6) Number of test organisms and replicates. For definitive and limit tests, the minimum number of daphnids per test concentration is 20 divided into a minimum of 2 replicates, each with 10 daphnia. Each test vessel should contain an equal volume of test solution and an equal number of daphnids. Replicate test vessels should be physically separated, since the test vessel is the experimental unit.
            (i) Loading. The number of daphnids placed in a test vessel should not be so large as to cause the dissolved oxygen concentration to fall below the recommended levels or affect the results of the test. In static or static-renewal tests, loading should not exceed 1 daphnid per 20 mL of test solution (50 daphnids per liter). In flow-through tests, loading requirements will vary depending upon the flow rate of dilution water, but should not exceed 0.5 grams wet weight of organism per liter (g/L) of solution passing through the test vessel in 24 hours or 5 g/L at any time. 
            (ii) Introduction of test organisms. The test should be started by introducing <24-hour old daphnids, from acclimated parents, into the test vessels after the test substance has been added. Test vessels for treatment levels should be randomly or indiscriminately located within the test area, and test organisms should be randomly or indiscriminately distributed among test vessels. Further guidance is provided in OCSPP 850.1000.
       (7) Facilities, apparatuses, and supplies. Normal laboratory equipment should be used, especially the following: 
            (i) Facilities. Facilities for culturing, holding, acclimating, and testing daphnids that are well ventilated and free of fumes and disturbances which may affect the test organisms. Equipment for culturing and/or handling food sources for daphnids.
            (ii) Environmental control equipment. Mechanisms for controlling and maintaining the water temperature and lighting during the culturing, holding, acclimation, and test periods. Apparatus for aerating dilution water and removing gas bubbles as necessary. For flow-through tests, apparatus for aerating the dilution water in the head box before mixing with the test substance or delivery to test vessels. An apparatus providing a 30-minute lighting transition period may be needed.
            (iii) Water quality testing instruments. Equipment for determination of water quality characteristics (pH, hardness, temperature, etc.).
            (iv) Cleaning of test system. Test substance delivery systems and test vessels should be cleaned before each test. See OCSPP 850.1000 for further information.
            (v) Test containers and delivery system. Construction materials and equipment that may contact the stock solution, test solution, or dilution water should not contain substances that can be leached or dissolved into aqueous solutions in quantities that can affect the test results. Construction materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test substances. Refer to OCSPP 850.1000 for additional information on appropriate construction materials. Test vessels, which should be constructed of chemically inert material, should be of a capacity to maintain the loading rate and environmental conditions. Test vessels should be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. A flow-through system, if used, should contain an appropriate test substance delivery system.
            For flow-through tests, daphnids can be retained in glass or stainless steel containers (i.e., test compartments) with stainless steel or nylon screen bottoms within the test vessel. The containers should be suspended in the test vessel in such a manner as to ensure that the test solution flows regularly into and out of the container and that the daphnids are submerged in generally at least 5 centimeters (cm) of test solution. 
            (vi) Dilution water. Clean surface water, ground water, or reconstituted water are acceptable as dilution water if daphnids will survive in it for the duration of the culturing, holding, acclimation, and testing periods without showing signs of stress. Dechlorinated tap water is not recommended because some forms of chlorination are difficult to remove adequately. If dechlorinated tap water is used, recommended maximum chlorine levels as well as other ways to demonstrate suitability as a dilution water source can be found in OCSPP 850.1000. The addition of selenium and vitamin B12 may also be desirable in reconstituted water (see paragraph (j)(2) of this guideline). Daphnids may also be cultured and tested in a defined medium (see paragraph (j)(5) of this guideline). 
            Dissolved oxygen in the dilution water (prior to use in a test) should be between 90 and 100% saturation. If necessary, the dilution water can be aerated before the addition of the test substance.
            Hardness, alkalinity, and conductivity should be measured in the dilution water at the beginning of the test. When using soft water (40-50 mg/L hardness), the test species should be D. pulex, as D. magna is intolerant of soft water. 
            Measurement of total organic carbon (TOC) or chemical oxygen demand (COD) in the dilution water at the beginning of the test is recommended, but at a minimum, TOC and COD should be analyzed periodically in the dilution water source to document and characterize their magnitude and variability. For tests with cationic substances, TOC or COD should be measured at the beginning of the test.  
            Specifications for dilution water quality and constancy are described in OCSPP 850.1000.
      (8) Environmental conditions. Environmental parameters during the test should be maintained as specified below. The number and frequency of measurements recommended for documenting and confirming the magnitude and variability of water quality parameters (e.g., temperature, dissolved oxygen, and pH) in test solutions during the test are described in detail in OCSPP 850.1000.
            (i) Temperature. The water temperature selected should be within the range of 18 to 22 °C, preferably 20 °C. During a given test, the selected temperature should be constant within plus or minus (+-) 1 °C.
            (ii) pH. The pH should be between 6.0 and 8.5 and vary less than 1 pH unit during the test within a test vessel and between test concentrations (including control(s)).
            (iii) Lighting and photoperiod. A photoperiod should be selected from regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark. For any given test, the light regime should be constant. Light intensity should range from 540 to 1080 lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition period between light and dark is suggested. 
            (iv) Dissolved oxygen. The dissolved oxygen concentration should be between 60 and 100% saturation during the test. If aeration is needed to achieve an appropriate dissolved oxygen level, it should be done before the addition of the test substance. For flow-through exposures, the dilution water may be aerated vigorously prior to delivery to the test vessels (e.g., in the diluter head box) such that the dissolved oxygen concentration is at or near 90 to 100% saturation. If the water is heated, precautions should be taken to ensure that supersaturation of dissolved gases is avoided. Aeration of test solutions during the test is strongly discouraged because a single air bubble can become caught under the carapace of a daphnid and kill it or float the daphnid to the surface where it will become trapped. Therefore, gentle aeration of test vessels during the exposure period may only be used in cases where the dissolved oxygen levels are in danger of dropping below 60% saturation. In such cases, assurances should be made that the use of aeration does not stress the test organisms; test substance concentrations should be measured during the test to ensure that they are not affected by the use of aeration; and all treatment and control vessels should be given the same aeration treatment.
            (v) Flow in a flow-through system. During a test, the flow rates should not vary more than 10% between any one replicate and another. The minimum number of test vessel volume replacements should be five per 24-hour period. It is recommended that diluter systems be monitored for proper adjustment and operation at least twice daily throughout the test period to better ensure that the target test concentrations are achieved and maintained. The flow rate to each test vessel should be measured at the beginning and end of the test.
	(9) Observations.
            (i) Measurement of test substance. OCSPP 850.1000 describes the recommended sampling methods, frequency of sampling, and sample processing (especially of low solubility test substances) for analytical confirmation of dissolved test concentrations and characterization of test substance stability throughout the test. The analytical methods used to measure the amount of dissolved test substance in a sample should be validated before beginning the test, as described in OCSPP 850.1000, and the relevant method detection limit(s) and limit(s) of quantification should be reported.
            (ii) Test solution appearance. Observations on test solution appearance and test substance solubility should be made daily and at the beginning and end of the test. The appearance of surface slicks, precipitates, or material adhering to the sides of the test vessels or in any part of the mixing and delivery system should be recorded at a minimum at the beginning and end of the test and during the test when the test solution appearance changes.
		(iii) Measures of effect.
                  (A) Immobilized daphnids. The number of immobilized daphnids in each test vessel should be counted and recorded at 24 and 48 hours. An observation period at <12 hours is desirable. Immobilized daphnids should be removed from test vessels at the time of observation.
                  (B) Appearance and behavior. In addition to immobilization, any abnormal behavior or appearance, and the number of individuals exhibiting these characteristics, should be recorded at the same time as observations of immobilization. 
(f) Treatment of results.
	(1) Summary statistics.
            (i) Immobilization. The number of daphnids exposed at test initiation in each treatment and replicate and the cumulative number of immobilized daphnids should be summarized in tabular form by time of observation, treatment, and replicate.
            (ii) Appearance and behavior. The number of daphnids exhibiting abnormal appearance or behavioral symptoms should be summarized in tabular form by time of observation, treatment, and replicate.
      (2) Percent immobilization. The percent of daphnids immobilized at each treatment level and in the controls at 24 hours and at test termination (48 hours) should be calculated.
      (3) Evaluation of limit test results. For pesticides, if any daphnid is immobilized in the limit test concentration, a multiple-concentration 48-hour test should be conducted.
	(4) Evaluation of multiple-concentration definitive test. 
            (i) Concentration-response curve, slope, and EC50. Statistical procedures should be employed to calculate the 48-h EC50 (standard error and 95% confidence interval) based upon immobilization. If a concentration-response curve model (e.g., probit) was fit to the data to determine the EC50, the model parameters (e.g., slope) and uncertainty estimates (e.g., standard error) should be recorded. The 24-h EC50 values should also be calculated if the magnitude of immobilization response allows.
            (ii) No observed effect concentration (NOEC). While calculation of the NOEC and lowest observed effect concentration (LOEC) is usually not part of the experimental design for the regression-based definitive test, reporting these values when possible is useful for understanding the toxic response. 
            (iii) Statistical methods. Statistical procedures for modeling quantal data should be used. Additional discussion about endpoints and statistical procedures can be found in OCSPP 850.1000.
(g) Tabular summary of test conditions. Table 1 lists the important conditions that should prevail during the multiple-concentration definitive test. The same conditions are recommended for a limit test, except for differences in the number of test concentrations. Meeting these test conditions will help ensure the satisfactory performance of the test.
Table 1.--Summary of Test Conditions for Freshwater Daphnid Acute Toxicity Test
Test type
Static, static-renewal, or flow-through
Test species
Daphnia magna or Daphnia pulex
Test duration
48 hours
Temperature
Selected from a range of 18 to 22 °C (preferably 20 °C) (constant during test within +-1 °C of selected test temperature)
Light quality
Ambient laboratory illumination
Light intensity
540-1080 lux (approximately 50-100 ft-c)
Photoperiod
Selected from among 12 hours light:12 hours dark to 16 hours light:8 hours dark schemes
pH
Between 6.0 and 8.5 (constant during test within +1 pH unit)
Water hardness (as CaCO3) 
<250 mg/L (preferably <180 mg/L); for testing with metals, 40-50 mg/L 
TOC
<=2 mg/L
Age of test organisms
<24 hours old
Number of test organisms per concentration
20 (minimum)
Number of replicate test vessels per concentration
2 (minimum) 
Loading
Static or static-renewal tests: >=20 mL per daphnid; 
Flow-through test: <=0.5 g/L per 24 hours and <5 g/L at any time
Feeding regime
No feeding during test
Test vessel aeration
Strongly discouraged because a single air bubble can become caught under the carapace of a daphnid and kill it or float the daphnid to the surface where it will become trapped. Gentle aeration of test vessels may only be used in cases where the dissolved oxygen levels are in danger of dropping below 60% saturation. In such cases, assurances should be made that the use of aeration does not stress the test organisms; test substance concentrations should be measured during the test; and all treatment and control vessels should be given the same aeration treatment.
Test concentrations
Definitive test: minimum of 5 test concentrations chosen in a geometric series plus a dilution water control and a vehicle (solvent) control, if a vehicle is used
Vehicle concentration, if used
<=0.1 mL/L for recommended solvents (see OCSPP 850.1000)
Measure of effect or measurement endpoint
48-h EC50 based on immobilization


(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or more of the conditions in Table 2 occurred. These parameters are not the only elements considered when evaluating the acceptability of a test, and it is possible that a test could be found unacceptable or invalid based on other considerations. However, except for the conditions listed in Table 2 and in OCSPP 850.1000, it is unlikely that a test will be rejected when there are only slight variations from guideline environmental conditions and test design unless the control organisms are significantly affected and/or significant biases are introduced in defining the magnitude of effect on measurement endpoints as compared to guideline conditions. Before departing significantly from this guideline (such as deviating from the organism age), the investigator should contact the Agency to discuss the reason for the departure and the effect the change(s) may have on test acceptability. In the test report, all departures from the guideline should be identified, reasons for the changes given, and any resulting effects on test endpoints noted and discussed.
Table 2.--Test Validity Elements for the Acute Toxicity Test, Freshwater Daphnids
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1. All test vessels were not identical.
2. Treatments were not randomly or indiscriminately assigned to individual test vessel locations, or individual test organisms were not randomly or indiscriminately assigned to test vessels.
3. A dilution water control (and vehicle (solvent) control, if a vehicle was used) was not included in the test.
4. More than 10% of the organisms in either the dilution water or vehicle (solvent) controls showed signs of disease, stress (e.g., discoloration, unusual behavior, immobilization), and/or death.
5. Daphnids were fed during the test.
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6. A surfactant or dispersant was used in the preparation of a stock or test solution. (However, adjuvants may be used when testing pesticide typical end-use products.)
(i) Reporting.
      (1) Background information. Paragraph (k)(1) of OCSPP 850.1000 describes the minimum background information to be supplied in the report.
      (2) Guideline deviations. Provide a statement of the guideline or protocol followed. Include a description of any deviations from the test guideline or any occurrences which may have influenced the results of the test, the reasons for these changes, and any resulting effects on test endpoints noted and discussed.
      (3) Test substance. 
            (i) Identification of the test substance: common name, IUPAC and CAS names, CAS number, structural formula, source, lot or batch number, chemical state or form of the test substance, purity (i.e., for pesticides, the identity and concentration of active ingredient(s)), and radiolabeling, if any, including the location of label(s) and radiopurity.
            (ii) Storage conditions of the test chemical or test substance and stability of the test chemical or test substance under storage conditions if stored prior to use.
            (iii) Methods of preparation of the test substance and the treatment concentrations used in the range-finding and definitive tests, or limit test. Identify whether the nominal concentrations are corrected or uncorrected for purity of the test substance.
            (iv) Physicochemical properties of the test substance such as water solubility, vapor pressure, UV absorption, pKa, and Kow.
            (v) If a vehicle (solvent) is used to prepare stock or test substance, provide the name and source of the vehicle, the nominal concentration(s) of the test substance in the vehicle in stock solutions or mixtures, and the vehicle concentration(s) used in the treatments and vehicle control. If different vehicle concentrations are used at different treatment levels, the report should, at a minimum, identify the maximum vehicle concentration used. It is helpful to support the vehicle choice by including a description of any measures that were taken to identify an appropriate vehicle for use in the test, such as the types and concentrations of vehicles used and their corresponding effects on solubility during any preliminary work.
            (vi) If a positive control is used, provide the name and source of positive control and the nominal concentration(s) of the positive control material in stock solutions or mixtures. 
	(4) Test organisms. 
		(i) Scientific name and common name.
		(ii) Method for verifying the species.
            (iii) Information about the daphnids used as brood stock for the test: source, culture practices, and holding and acclimation procedures and conditions, including acclimation period, water used, feeding history, and health status (mortality of brood stock before test initiation and any preventative or disease treatments). Feed should be analyzed periodically to identify background contaminants such as heavy metals (e.g., arsenic, cadmium, lead, mercury, and selenium) and persistent pesticides, especially chlorinated insecticides.	
            (iv) Age of test organisms at test initiation.
      (5) Test system and conditions. Provide a description of the test system and conditions used in the definitive or limit test and any preliminary range-finding tests.
		(i) Description of the test vessels: size, type, material, and fill volume.
            (ii) Description of the exposure technique: static, static-renewal, flow-through, open or closed system. If static-renewal, the frequency of test solution renewal, and if flow-through, a description of the flow-through system, including flow rates and test vessel turnover rate. For closed systems, a description of the closed system design. For all systems, a description of the calibration and validation methods. 
            (iii) Description of the dilution water and any water pretreatment: source/type; temperature; pH; hardness and alkalinity; dissolved oxygen; total organic carbon or chemical oxygen demand; particulate matter; conductivity; metals, pesticides, and residual chlorine concentrations (mean, standard deviation, range). Describe the frequency and sample date(s) for documenting dilution water quality and consistency. 
            (iv) Use of aeration, if any, and location of aeration within exposure system (e.g., test solution or dilution water prior to test substance addition).
		(v) Number of test organisms added to each test vessel at test initiation.
		(vi) Number of test vessels (replicates) per treatment level and control(s).
            (vii) Methods used for treatment randomization and assignment of test organisms to test vessels.
		(viii) Date of introduction of test organisms to test solutions and test duration.
		(ix) Loading rate.
		(x) Photoperiod and light source.
            (xi) Methods and frequency of environmental monitoring performed during the definitive or limit test for test solution temperature, dissolved oxygen, pH, and light intensity.
            (xii) Methods and frequency of measuring dissolved test substance to confirm exposure concentrations.
            (xiii) Methods and frequency of counting number of immobilized daphnids and measuring any other toxic symptoms.
            (xiv) For definitive and limit tests, description of all analytical procedures, accuracy of the method, method detection limit, and limit of quantification. 
	(6) Results. 
            (i) Nominal exposure concentrations and a tabulation of test substance analytical results by treatment group and test vessel (provide raw data) and descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation).
            (ii) Environmental monitoring data results (test solution temperature, dissolved oxygen, pH, and light intensity) in tabular form (provide raw data for measurements not made on a continuous basis) and descriptive statistics (mean, standard deviation, minimum, maximum).
            (iii) For preliminary range-finding test, if conducted, a tabulation of the number and percentage of immobilized daphnids in each test vessel, for all treatment levels and control(s), at each observation period. A description and count of any other appearance or behavioral effects, if recorded, at each treatment level and in the control(s).
            (iv) For limit test, a tabulation of the number and percentage of immobilized daphnids in each test vessel, for the limit concentration and control(s), at each observation time (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum).
            (v) For definitive test, a tabulation of the number and percentage of immobilized daphnids in each test vessel, for all treatment levels and control(s), at each observation period (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum).
            (vi) For limit and definitive tests, a description and tabulation of abnormal appearance and behavioral signs of toxicity by test vessel, treatment, and observation time (provide raw data).
		(vii) Graphs of the concentration-response data for percent immobilization.
            (viii) For limit test, conclusion about the 48-h EC50 for immobilization being above the limit concentration.
            (ix) For definitive test, where sufficient data exist to fit a model (e.g., probit), a tabulation of the 48-hour slope of the concentration-response curve, its standard error and 95% confidence interval, and any goodness-of-fit results.
            (x) For definitive test, the 48-h EC50 value for immobilization, its standard error and 95% confidence interval.
            (xi) For definitive test, results for the 24-h EC50 value if the magnitude of immobilization response allows.
		(xii) For definitive test, the 48-hour NOEC for immobilization, if determined.
            (xiii) Description of statistical method(s) used for point estimates, including the software package for determining EC50 values and fitting the concentration-response model, and the basis for the choice of method. Provide results of any goodness-of-fit tests.
            (xiv) Description of statistical method(s) used for NOEC and LOEC determination, including the software package, and the basis for the choice of method.
(j) References. The following references should be consulted for additional background material on this test guideline.
      (1) American Society for Testing and Materials. ASTM E729-96, Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved October 1, 2014.
      (2) American Society for Testing and Materials. ASTM E1193-97, Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved April 1, 2004.
      (3) Hutchinson, T.H., N. Shillabeer, M.J. Winter and D.B. Pickford, 2006. Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Aquatic Toxicology, 76, 69-92.
      (4) Organization for Economic Cooperation and Development, 2004. Guidelines for Testing of Chemicals, Guideline 202, Daphnia sp., Acute Immobilisation Test.
      (5) Organization for Economic Cooperation and Development, 2012. OECD Guidelines for Testing of Chemicals, Guideline 211, Daphnia magna Reproduction Test.
      (6) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Office of Pesticide and Toxic Substances, Washington, DC, EPA 540/9-82-024.
      (7) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard Evaluation Procedure: Acute Toxicity Test for Freshwater Invertebrates, Office of Pesticides Programs, Washington, DC, EPA-540/9-85-005.
      (8) U.S. Environmental Protection Agency, 1994. Pesticides Reregistration Rejection Rate Analysis: Ecological Effects, Prevention, Pesticides and Toxic Substances, Washington, DC, EPA 738-R-94-035.
      (9) U.S. Environmental Protection Agency, 2002. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, Fifth edition, October 2002. EPA-821-R-02-012.
