
Office of Chemical Safety and Pollution Prevention
(7101)
                                                               EPA 712-C-16-001
                                                                   October 2016

Ecological Effects Test Guidelines



OCSPP 850.1740:

Spiked Whole Sediment 10-Day Toxicity Test, Saltwater Invertebrates

                                       



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  NOTICE
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  This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline's number. Any test guidelines developed after April 22, 2010 will use the new acronym (OCSPP) in their title.
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  	The OCSPP harmonized test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In this guidance, the use of "should" with regard to an action means that the action is recommended rather than mandatory. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis. 
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  	For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.1740: Spiked whole sediment 10-day toxicity test, saltwater invertebrates
(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 ASTM E 1367-03 (2008), Standard Guide for Conducting 10-day Static Sediment Toxicity Tests with Marine and Estuarine Amphipods (see paragraph (j)(1) of this guideline); and U.S. EPA Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods (EPA/600/R-94-025) (see paragraph (j)(7) of this guideline). Other source materials are listed in paragraph (j) of this guideline. Mention of trade names or commercial products in this guideline does not constitute endorsement or recommendation for use.
(b) Purpose. This guideline is intended for use in determining the toxicity to benthic invertebrates from sub-chronic exposure to sediment concentrations of chemical substances and mixtures subject to environmental effects test regulations. This guideline prescribes a test in which benthic saltwater macroinvertebrates are exposed to test substances incorporated in sediment. Naturally-derived clean sediment or formulated sediment is spiked with different concentrations of test substance. The test results are used to determine relationships between the test substance in sediment and a biological response such as mortality in benthic invertebrates. The Environmental Protection Agency will use data from this test in assessing the hazards and risks a test substance may present in the aquatic benthic environment.
(c) Definitions. The definitions in OCSPP 850.1000 apply to this test guideline. In addition, the following more specific definitions apply to this guideline:
      Clean refers to a sediment or water that does not contain organisms or concentrations of constituents such as metals, synthetic organic compounds, oil and grease, petroleum hydrocarbons, ammonia levels, etc. which cause apparent stress to the test organisms or reduce their survival or growth (optional endpoint).
      Interstitial water or pore water is water occupying the space between sediment or soil particles.
	Overlying water is the water placed over sediment in a test vessel during a test.
      Sediment refers to deposits of organic matter, sand, soil, and other particulate matter which form a substrate in an aquatic system. In this guideline, the definition also includes formulated or artificially-prepared particulate material that is intended to form a substrate below overlying water in a test vessel. 
      Sediment, bulk (or whole) is the solid-phase sediment plus its associated pore water which has undergone minimal manipulation. The term bulk sediment is used synonymously with whole sediment.
      Sediment concentration is the ratio of the weight of test substance(s) to the dry weight of bulk (or whole) sediment.
      Sediment concentration, acid-volatile sulfide normalized refers to the normalization of the sediment concentration to the acid-volatile sulfide content of the sediment and is the ratio of the weight of test substance to the weight of acid-volatile sulfide in the whole sediment sample.
      Sediment concentration, organic-carbon normalized refers to the normalization of the sediment concentration to the organic-carbon content of the sediment and is the ratio of the weight of test substance to the weight of organic-carbon in the whole sediment sample.
      Spiked sediment refers to natural or artificial sediment to which a test substance has been added and incorporated for experimental or study purposes.
(d) General considerations
      (1) Summary of the test. Whole sediment toxicity test procedures are outlined for saltwater amphipods (Ampelisca abdita, Eohaustorius estuarius, Rhepoxynius abronius, or Leptocheirus plumulosus). The duration of sub-chronic whole sediment tests is 10 days using spiked sediment as well as an appropriate control(s). Test organisms are monitored during the test for sediment avoidance and other toxic effects observable without disturbing the sediment. At test termination, survival, reburial (optional for E. estuarius, L. plumulosus and R. abronius), and growth (optional for L. plumulosus) are determined. The results of the test can be expressed as the 10-day median lethal concentration (10-d LC50), the 10-day median growth effect concentration (10-d EC50), or the 10-day no observed effect concentration (10-d NOEC) and 10-day lowest observed effect concentration (10-d LOEC) for survival and growth. For pesticides, the NOEC and LOEC are the preferred 10-d endpoints. For industrial chemicals, the LC50 and/or EC50 are the preferred 10-d endpoints.
      (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 substance sediment concentrations to be used for 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 kilogram (mg/kg) dry weight). 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, and nominal sediment concentrations of the test substance are acceptable. 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. It may be possible that "water-only" range-finding tests are sufficient to establish the concentrations for definitive testing, based upon predicted pore-water concentrations. 
      (4) Definitive test. The goal of the definitive test for pesticides is to determine the NOEC and LOEC values for the most sensitive measure of effect, either mortality or growth (optional). The selected test concentrations should bracket the NOEC and LOEC values. If growth is to be measured, then additional procedures described below need to be implemented in order to ensure adequate growth during the test (e.g., feeding, renewal of overlying water).
      The test may also be designed to determine the concentration-response curves for mortality and growth effects, the 10-d LC50 for mortality and the 10-d EC50 for growth along with their respective 95% confidence intervals, the slopes of the concentration-response curves, their associated standard errors, and 95% confidence intervals. The selected test concentrations should bracket the expected 10-d LC50 for mortality and 10-d EC50 for growth (if measured), or whichever is more sensitive (if measuring both mortality and growth). 
       An additional optional measure is the ability of surviving amphipods to rebury to determine effective mortality (i.e., the sum of dead animals plus those survivors that fail to rebury). The optional measures should be considered for those test substances where there is information that indicates such measures will be more sensitive than mortality such as results from similar chemicals. 
      Either test design should use at least five sediment concentrations of the test substance, plus appropriate control(s). Test concentrations are typically expressed as mg/kg dry weight for sediment and mg/L for interstitial (pore) water. Depending on the test substance, endpoints may be normalized to organic carbon content or acid-volatile sulfide content of the sediment and to dissolved pore water concentrations. Analytical confirmation of test concentrations in bulk sediment and interstitial (pore) water should be performed. Summaries of the test conditions are presented in Table 1 of this guideline. The test validity elements are listed in Table 2.
      (5) Limit test. In some situations, it is only necessary to ascertain that the 10-d NOEC or 10-d LC50/EC50 for survival and growth is above a certain limit concentration (i.e., 10-d NOEC or 10-d LC50/EC50>limit concentration). In a limit test, at least eight replicate test vessels with 20 test organisms per replicate are exposed to a single "limit concentration," with the same number of replicates and test organisms in appropriate control(s). For pesticides, 100 milligrams of active ingredient per kilogram of sediment (mg a.i./kg) (dry weight) , when estimated environmental concentrations are not expected to exceed 100 mg a.i./kg, may be used as the limit concentration. For most industrial chemicals, 100 milligrams per kilogram of sediment (mg/kg) (dry weight) is considered appropriate as the limit concentration. These sediment limit concentrations should be appropriately adjusted upwards if use, production, disposal, or other releases result in sediment concentrations above these limits. Except for the number of test concentrations, an acceptable limit test follows the same test procedures, is the same duration, and has the same number of control(s) as the multiple-concentration definitive test (see Table 1). Limit tests, like definitive tests, include analytical confirmation of the limit concentration in bulk sediment and the interstitial water concentration. For pesticides, if there is a statistically significant reduction in mortality or growth (if measured) at the limit concentration as compared to the control(s) (i.e., no observed effect concentration (NOEC) less than (<) limit concentration), a definitive test should be conducted. For industrial chemicals, if the effect level for mortality or inhibition of growth at the limit concentration compared to the control(s) is 50% or greater, then a definitive test should be conducted.
(e) Test standards
     (1) Test substance. The test substance should be reagent grade (or technical grade for pesticides) unless the test is designed for a specific formulation, mixture or degradate. 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. The test substance is spiked into the sediment as described in OCSPP 850.1000 (f)(2)(i). 
     (2) Test duration. The duration of the sub-chronic test with saltwater amphipods is 10 days.
     (3) Test organisms
            (i) Species. The recommended test species are the amphipods Ampelisca abdita, Eohaustorius estuarius, Rhepoxynius abronius, or Leptocheirus plumulosus. The preferred species for pesticide testing is Leptocheirus plumulosus. These test species have contact with the sediment, tolerate varying sediment physical and chemical characteristics, have a database demonstrating relative sensitivity to various chemicals, and have been used in interlaboratory studies (paragraph (j)(1)). The test organism used should be identified using an appropriate taxonomic key. The species of amphipod to be used in testing should be selected based upon availability, sensitivity to test substances, tolerance to ecological conditions (e.g., temperature, salinity, and grain size), ecological importance, and ease of handling in the laboratory. The submitter should consult with the Agency prior to using other benthic invertebrate species in testing. Information about the life history of these species can be found in the references in paragraph (j)(1), (j)(7) and (j)(8).
            R. abronius is a free-burrowing amphipod. It is found in clean, fine, sandy sediments in areas of salinity 25 parts per thousand (ppt) or greater. E. estuarius is a free-burrowing detritivore. It lives in intertidal sands and is a desirable test species for sediments which have interstitial salinities ranging from 2 to 28 ppt. Since both of these species normally inhabit sandy sediments, a fine-sediment control should be included in the experimental design if a naturally derived sediment that is predominantly silt or clay is tested. 
            
            A. abdita is a tube-dwelling amphipod, constructing its tube from fine sand grains. The tubes are approximately 3.5 centimeters (cm) in length and 2 to 3 millimeters (mm) wide with most of the tube constructed below the substrate and only about 1 cm above the surface sediment. This species feeds both on suspended particles (including algae, sediment grains, and organic detritus) and on those from the sediment surface (sand grains and detritial material) surrounding its tube. It is euryhaline and has been found in waters ranging from fully marine to 10 ppt. This species generally inhabits sediments from fine sand to mud and silt, and is often abundant in sediments with high organic content. 
            
            L. plumulosus is an infaunal amphipod. It can tolerate a broad salinity range (0-33 ppt) and constructs U-shaped burrows in sediments ranging from fine sand to silty clays. 
            All organisms should be as uniform as possible in age and size. For L. plumulosus, it is recommended that the test be initiated with 7- to 8-day old (immature) organisms using laboratory cultures. If organisms are field collected the size range should be comparable to what is expected for 7- to 8-day old organisms. The size range of amphipods used for testing should be 3-5 mm for the other three species and mature female amphipods, which are distinguishable by the presence of embryos in the brood pouch or oviduct, should not be selected for testing. Additionally, mature male A. abdita should not be used for testing. The size range of test organisms should be kept to a minimum. 
            All organisms in a test should be from the same source. Organisms may be obtained from laboratory cultures, from culture facilities, or field collected. Use of field-collected organisms should be reserved for situations where the sensitivity of field populations of a species is the objective of the sediment toxicity study or when culture methods have not been established. Culture methods have been established for L. plumulosus and are described in paragraph (j)(1) and (j)(9). Culture methods for A. abdita are described in paragraph (j)(5). Culture methods have not been established for E. estuarius and R. abronius. For species that can be cultured in the laboratory, obtaining organisms from wild populations should be avoided unless organisms are cultured through several generations in the laboratory and the sensitivity of the wild population can be documented. If organisms are shipped, temperature and dissolved oxygen should be measured upon arrival to determine if the organisms might have been unacceptably stressed. The methods for field collection and shipping of test organisms are species-specific and details may be found in paragraph (j)(7). 
            Subtidal species, such as A. abdita and L. plumulosus, may be collected with a small dredge or grab apparatus (e.g., PONAR, Smith-McIntyre, or Van Veen) and may also be collected by skimming the sediment surface with a long-handled, fine-mesh net. E. estuarius and R. abronius are found both subtidally and intertidally. The aforementioned methods are suitable for subtidal populations. Intertidal populations may be collected using a shovel.
            Approximately one-third more organisms should be collected than are required for testing. Collected organisms should be handled as carefully and as quickly as possible. Collected amphipods should be sieved in the field by slowly immersing the sieve into collection site water. A. abdita and L. plumulosus should be isolated from the collection site sediment by using a 0.5 mm mesh sieve. A. abdita which remain in tubes should be left undisturbed for 20 to 30 minutes to allow for natural exit of the organisms. A 1.0 mm sieve should be used to isolate E. estuarius and R. abronius. Sieved amphipods should be separated from detritus and predators and transferred gently to transport containers containing 2 centimeters (cm) of collection site sediment. Sieved test organisms should be kept submerged in ambient collection water at all times. Direct exposure of amphipods out of sediment to sunlight should be avoided. Salinity and temperature at the surface and bottom of the water column at the collection site should be measured and recorded. Amphipods should be transported in coolers with a layer of material sufficiently thick to prevent movement over a layer of ice packs. Aeration is recommended for transport times exceeding 1 hour. Sediment from the field collection site should be used as holding sediment in the laboratory and potentially as an additional reference control test sediment.
            
            (ii) Holding and acclimation. Depending on temperature and salinity at the collection site, amphipods may require acclimation to standard testing conditions. If shipped animals do no exhibit active swimming behavior upon placement in water, full digestive tracts, and normal coloration, they should not be used in a test. Changes from site to testing conditions should be gradual (<3 degrees Celsius (°C) and 5 ppt in salinity per hour). Amphipods should be held under test conditions for at least 48 hours prior to test initiation. Amphipods should be held and acclimated (if necessary) in containers (4 to 8 L volume) that contain a 2 to 4 cm layer of collection site sediment that has been sieved through a 0.5 mm mesh screen. Amphipod density should not exceed 1 amphipod per square centimeter. Lighting should be constant and continuous throughout the holding and acclimation period for E. estuarius and R. abronius. A. abdita require a 16 hours light:8 hours dark photoperiod to promote feeding. For cultures of L. plumulosus,a photoperiod should be selected from regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark. Similar to the definitive test, light quality during holding and acclimation should range from approximately 540 to 1080 lux (approximately 50 to 100 foot-candles (ft-c)). 
            During the acclimation period, mortalities should be recorded, and the following recommendations should be applied:
                  (A) Mortalities of greater than 10% of the population during acclimation: rejection of entire batch;
                  (B) Mortalities of between 5 and 10% of the population during acclimation: acclimation continued for additional 7 days;
                  (C) Mortalities of less than 5% of the population during acclimation: acceptance of batch.
            
             (iii) Health status and condition. A group of organisms should not be used for a test:
                  (A) If more than 5% of the culture dies or shows signs of stress during the 2 days preceding the test; 
                  (B) If they have been used in a previous test, either in a treatment or in a control group.
            (iv) Care and handling. Test organisms should be handled as little as possible but when necessary it should be done as carefully and quickly as possible. Test organisms should be introduced into the overlying water below the air-water interface. A wide-bore pipette or a smooth glass tube has been used in transferring organisms. 
            (v) Diet and feeding. The four species of amphipods used in this method are typically not fed during testing. During the holding/acclimation period, A. abdita and L. plumulosus need supplemental feeding. A. abdita should be fed daily, while L. plumulosus may be fed two to three times a week. Algae have been used for both species, and L. plumulosus may also be fed finely ground fish flake food.
            For assessing the optional growth endpoint, feeding of L. plumulosus during the test should generally follow recommendations in (j)(9). Specifically, a commercial fish flake food (e.g, TetraMin(R) (Tetra Holding (US), Inc., Blacksburg, VA) or functional equivalent) only diet is recommended. With this diet, water renewal is strongly recommended to ensure adequate dissolved oxygen levels. Three times per week, following renewal, a flake food slurry is delivered to each chamber in 1-mL aliquots. The flake food is fed at a rate of 20 mg per test chamber over the duration of the test. To prepare the slurry, the flake food is finely ground with a food mill (blender, mortar and pestle, or a similar device) and sieved through a 0.25-mm screen. Test water is added to the appropriate amount of flake food, and the slurry is mixed on a stir plate for 15 min. The slurry is prepared fresh for each use and should be mixed continuously during feeding to prevent the flake food from settling.
      (4) Administration of test substance. Depending upon the objectives of the test, the test substance is either spiked into naturally-derived sediment or formulated sediment. If naturally-derived sediment is used, it should be fully-characterized and free of organisms that might compete with or consume the test organisms.
            (i) Preparation of spiked sediment. Spiked sediment of a given concentration is prepared by: 1) adding the test substance directly to the sediment as an aqueous solution for soluble test substances; 2) adding the test substance directly to the sediment in a dry form for a water insoluble solid; or 3) by sorbing the test substance to sand and then mixing the treated sand into the sediment for water insoluble test substances. Stock solutions of test substance in organic solvents should not be added to the sediment mixture because they can affect the concentration of dissolved organic carbon in pore water. See OCSPP 850.1000 paragraph (f)(2)(i) for guidance on preparation of spiked sediment. 
            If a vehicle is used, a solvent sediment control should be included in the test in addition to the negative sediment control. The selected vehicle should not affect the test organisms at the concentration used or interfere with test results. The solvent sediment control should be prepared from the same batch of solvent and should contain the same concentration of vehicle used in the test treatments (preferably) or the highest concentration of vehicle used in any test treatment. See (e)(5) of this guideline for additional details on negative and solvent controls.
             (ii) Placement of sediment in test vessels. Test sediment (unspiked control sediment and spiked sediment at various test concentrations and aged as described in OCSPP 850.1000 paragraph (f)(2)(i)(C)) should be thoroughly mixed and added to test vessels the day before (day -1) the start of the test. The degree of homogeneity should be inspected visually. Homogeneity may be quantified by taking replicate subsamples and analyzing for total organic carbon (TOC), test substance sediment concentration, and particle size.
            Equal amounts of sediment should be added to each test vessel on the basis of volume or weight. To minimize disturbance of sediment, overlying water should be poured gently along the sides of the test vessels or poured over a turbulence reducer (e.g., a disk cut from polyethylene, nylon, or Teflon, or a glass Petri dish attached to a glass pipette) positioned above the sediment. Turbulence reducers should be rinsed with overlying water between replicates, and individual turbulence reducers used between treatments. For this test, 1 L test vessels with 175 mL of sediment and 800 mL of overlying water been used successfully. The renewal of overlying water should commence on day -1. The test begins once organisms are added to the test vessels (day 0).
            (iii) Sediment test concentrations. At least five sediment test concentrations are used for definitive testing, plus the appropriate control(s). A range-finding test can be used to establish the appropriate sediment test concentrations for the definitive test (see paragraph (d)(3) of this guideline). For scientifically sound estimates of a given point estimate on the dose-response curve (e.g., LC50, IC50) test substance concentrations should immediately bracket the point estimates(s) of concern. For a hypothesis-based endpoint study, the lowest test treatment level should be at or below the NOEC for the most sensitive effect measure (survival). OCSPP 850.1000 provides guidance on selection of test concentrations. For a limit test, there is a single sediment treatment concentration, plus the appropriate control(s). Guidance on the limit sediment concentration is provided in paragraph (d)(5) of this guideline.
       (5) Controls
            (i) Every test includes a negative sediment control, and a solvent sediment control is also included if a vehicle is used. Sediment controls are created using the same sediment, overlying water, and animal test populations and undergo the same manipulations and procedures as the test substance treatment groups, except that no test substance is added. Sediment controls are kept under the same environmental conditions as test substance treatment groups. Control sediment is essentially free of contaminants and is used routinely to assess the acceptability of a test. Comparing test sediments to control sediments is a measure of the toxicity of a test sediment beyond inevitable background contamination.
            (ii) A test is unacceptable if mean survival in either the negative sediment control or solvent sediment control is less than 80% of exposed amphipods at the end of the test.
            (iii) For pesticides, if a solvent (vehicle) is used, both a negative and solvent control are recommended. If a non-volatile solvent is used, both a negative and solvent control are needed. However, when using a volatile solvent (e.g., acetone), and the solvent is evaporated to completeness as recommended in paragraph (f)(2)(i) of the OCSPP 850.1000 guideline, use of only one control (solvent) may be allowed provided  the testing laboratory demonstrate to the Agency that the solvent control is functionally equivalent to a negative control.  Factors to be considered when demonstrating functional equivalency are:
            
  Concentration of solvent in the test sediment after evaporation (e.g., analytically determined);
                  
  Levels of the solvent that are known to affect organism health;
               
  The potential for impurities in the solvent and their potential impact on organism health and;
                  
                 Historical organism performance of solvent vs. negative controls. 
      (6) Number of test organisms and replicates. The minimum recommended number of replicates varies with the objectives of the test and the statistical method used for the analysis of the data. See OCSPP 850.1000 for guidance. For tests designed to solely establish regression-based endpoints (ECx and/or LCx), at least three replicates per test concentration is recommended. For hypothesis or analysis of variance-based endpoints (NOEC), if a power analysis is not performed, at least eight replicates per test concentration should be used (see reference in paragraph (j)(8) of this guideline). For pesticides, as the objective is to establish analysis of variance-based (NOEC) values, the minimum number of replicates per treatment is eight. For industrial chemicals, it is recommended that the Agency be consulted prior to test initiation to ascertain the appropriate study design to address the concerns for the test substance. 
      The recommended number of organisms in each test replicate is 20.
      Each test vessel should contain an equal amount of sediment and overlying water and an equal number of test organisms. In addition to the recommended minimum number of test vessels for conducting the effects analysis, separate replicate test vessels should be set up which can be destructively sampled for test substance concentrations during the test as described in paragraph (e)(9) of this guideline. Replicate test vessels should be physically separated, since the test vessel is the experimental unit.
            (i) Loading. The number of test organisms placed in a test vessel should not be so great as to affect the results of the test. Experience has indicated that the use of 20 organisms in a test vessel containing 175 mL of sediment and 800 mL of overlying water is acceptable. The loading should not cause the dissolved oxygen to fall below the recommended level of 4.4 mg/L or un-ionized ammonia to rise to levels that negatively impact organism health.
            (ii) Introduction of test organisms. The test is started by adding the test organisms to the test vessels which contain test sediment and overlying water. Twenty amphipods are impartially or randomly added to each test vessel in batches of 5 to 10 on day 0, following the addition of sediment and overlying water on day -1. One-third more amphipods than necessary are sieved from culture or control sediment and transferred to sorting trays. Recommended sieve sizes are 0.5 mm for A. abdita and L. plumulosus and 1.0 mm for E. estuarius and R. abronius. For pesticide testing, starting with known-age 7- to 8-day old L. plumulosus is preferred to sieving. Isolated amphipods are transferred from the sorting tray into test vessels by pipetting the organisms directly into the overlying water just below the air-water interface. The test organisms are observed for injury or stress after addition. If any E. estuarius, L. plumulosus, and R. abronius have not burrowed within 5 to 10 minutes, they should be replaced. A. abdita that have not burrowed within 1 hour should also be replaced. Organisms expressing sediment avoidance should be removed and recorded, but not replaced. Test vessels for treatment levels are randomly or indiscriminately located within the test area (see OCSPP 850.1000).
      (7) Facilities, apparatus, and supplies. Normal laboratory equipment should be used, especially the following:
            (i) Facilities. Facilities for culturing or holding and acclimating and testing amphipods that are well ventilated and free of fumes and disturbances which may affect the test organisms. Equipment for culturing and/or handling of food sources for amphipods. If measuring growth, drying ovens, aluminum weighing pans, and an analytical balance capable of accurately weighing to 0.01 mg.
            (ii) Environmental control equipment. A mechanism for controlling and maintaining the water temperature and lighting during the culturing, holding, acclimation, and test periods. 
            (iii) Water quality testing instruments. Equipment and instruments for determination of water quality and sediment quality characteristics (pH, salinity, temperature, etc.). For monitoring water and sediment quality, the use of instruments that do not require removal of water or sediment samples is recommended. Both dissolved oxygen and pH may be measured in overlying water using a probe. Probes should be thoroughly inspected between samples to make sure that organisms are not attached.
            (iv) Cleaning of test system. 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, sediment, or overlying 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 matrices containing test substances 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 of a capacity to maintain the recommended loading level and environmental conditions. Test vessels consisting of 1 L glass beakers or jars with an inner diameter of 10 cm have been used successfully. Test vessels should be loosely covered to reduce the loss of test solution or overlying water due to evaporation and to minimize entry of dust or other particles into the solutions. 
            (vi) Overlying water. Clean natural seawater or artificial salt water are acceptable as overlying water if it is of uniform quality, and allows satisfactory survival of the test organisms. Organisms cultured and tested in the selected saltwater should not show signs of disease or stress. Guidance in OCSPP 850.1000 on dilution water is applicable to overlying water used in this test.
            
             Natural seawater should be filtered through a filter with a pore size of <20 microns (um) prior to use in a test.Although natural seawater is preferable, artificial saltwater is acceptable. Artificial saltwater can be prepared by adding commercially available formulations or by adding specific amounts of reagent-grade chemicals to reagent water (deionized, distilled, or reverse osmosis water), surface water, or ground water. Dechlorinated tap water is not recommended for preparation of artificial saltwater or dilution of natural seawater because some forms of chlorination are difficult to remove adequately. If dechlorinated tap water is used, recommended maximum chlorine levels as well as other ways to demonstrate suitability as a dilution water source are in OCSPP 850.1000. 
            
            During testing, the recommended overlying water salinity is 28 ppt for A. abdita and R. abronius and 20 ppt for E. estuarius and L. plumulosus. 
            Dissolved oxygen in the dilution (overlying) water (prior to use in a test) should be between 90 and 100% saturation. If necessary, the overlying water can be aerated before it is introduced into the test vessels.
            Measurement of TOC or chemical oxygen demand (COD) in the overlying water source is recommended, but at a minimum TOC and COD should be measured periodically in the 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. 
            Additional dilution water specifications (e.g., metals, organic compounds) listed in OCSPP 850.1000 is applicable to overlying water used in this test.
            (vii) Sediment. For pesticide testing, the use of natural or formulated sediment as described in OCSPP 850.1000 is acceptable. If natural sediment is used for testing of pesticides, it should be free of contaminants and organisms that may interfere with the test and should be typical of sediments naturally encountered by the test organism. For testing of industrial chemicals, the use of natural sediment or formulated sediment depends on the objective of the study; it is recommended that the submitter consult the Agency prior to study initiation to ascertain the appropriate sediment to use to address the concerns about the test substance. The characteristics of all sediment should be determined; specific characteristics are described in OCSPP 850.1000.
            Formulated sediments consist of mixtures of materials designed to mimic natural sediments. The use of formulated sediment eliminates or controls the variation in sediment physico-chemical characteristics and provides a consistent method for evaluating the fate and effects of chemicals in sediments. Detailed information on formulated sediments may be found in OCSPP 850.1000.
            
            Natural sediments may also be used; procedures for handling, storage, and manipulation of natural sediments can be found in OCSPP 850.1000. Ammonia concentrations in field-collected sediments may be toxic to amphipods. When ammonia concentrations exceed the water column no-effect levels given in paragraph (j)(7), any mortality observed during the 10-d sediment test may be due to the ammonia. Ammonia levels should be measured in the overlying water at approximately 1 cm above the sediment surface prior to initiation of a test, and if necessary, reduced prior to the addition of test organisms (Day 0) by sufficiently flushing overlying water and/or increasing renewal during the study up to 4x/day.
            The range of salinity tolerance is variable for the four amphipod species. For sediment testing, two scenarios for test salinity are acceptable if appropriate conditions are met. In laboratory sediment testing, the overlying water salinity can be based on the standard salinity for each test species or adjusted to match the salinity of the pore water. It is critical that pore water salinity be measured prior to test initiation and that the appropriate species be used. Overlying water salinity tolerance ranges for A. abdita, E. estuarius, L. plumulosus, and R. abronius are 20-32, 2-34, 1.5-32, and 25-32 ppt, respectively. Conversely, a range of pore water salinities in which a given species can survive for 10 days when using the species-specific overlying water salinity can also be used. Pore water salinity ranges for A. abdita with overlying water salinity of 28 to 32 ppt, E. estuarius with overlying water salinity of 20 ppt, L. plumulosus with overlying salinity of 20 ppt, and R. abronius with overlying water salinity of 28-32 ppt are 0-34, 2-34, 1.5-32, and 25-34 ppt, respectively. See recommended overlying water salinity concentrations during testing in paragraph (e)(7)(vi) of this guideline.
            While the four estuarine/marine test species generally tolerate a wide range of sediment types, grain size may adversely affect some species of amphipods. When this possibility exists, a clean control/reference sediment should be incorporated into the test design to facilitate distinction of test chemical effects versus particle size effects. Species-specific responses to various ranges of grain sizes are described below:
                  (A) L. plumulosus: This species can tolerate a wide range of grain sizes in clean sediments. 
                  (B) A. abdita: Survival may be impacted in sediments containing 95% or more sand. Test sediment should contain less than 95% sand.
                  (C) E. estuarius: Survival is unaffected by clean sediments containing 0.6 to 100% sand. Increased mortality may be associated with increased proportions of fine-grained sediment. In these cases, an appropriate control/reference should be included. 
                  (D) R. abronius: Very fine grains, particularly silts and clays, may reduce survival of this species. When test sediments contain silts and clays, the use of control/reference groups with particle sizes characteristic of the test sediment is recommended. 

      (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, salinity, and pH) in the overlying water and sediment during the test are described in detail in OCSPP 850.1000. 
      If removing overlying water samples, they should be removed with a pipette from 1 to 2 cm above the sediment surface without disturbance. Caution should be exercised to avoid removing test organisms when sampling. Checking the pipette to make sure no organisms are removed during sampling of overlying water is recommended. Both dissolved oxygen and pH may be measured in overlying water using a probe. All test vessels should be checked daily for air flow to overlying water. Small amounts of saltwater should be added daily to maintain the initial volume of overlying water if a static system is used.
      To allow for measurements in sediment, these measurements may be made from the separate replicates resembling the biological replicates used to provide the required sample for chemical analysis.
            (i) Temperature. The respective selected test temperatures are representative of the summertime thermal maximum for each species. E. estuarius and R. abronius should be tested at 15 °C, A. abdita should be tested at 20 °C, and L. plumulosus should be tested at 25 °C. During a given test, the selected temperature should be constant within +-1 °C. 
            (ii) pH. pH should be measured in both overlying and pore water, and should vary less than one pH unit in overlying water during the test within a test vessel and between test concentrations (including control(s)). 
            (iii) Light. For L. plumulosus, a photoperiod should be selected from regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark. For  E. estuaries, R. abronius, and A. abdita, the photoperiod should be 16 hours light: 8 hours dark.  For any given test, the light regime should be constant. Light intensity should range from 540 to1080 lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition period between light and dark is suggested. .
            (iv) Dissolved oxygen in overlying water. Dissolved oxygen in overlying water should be maintained above 4.4 mg/L (60% saturation) during the course of the test and above 3.6 mg/L (50% saturation) on any given day. If a static system is used, the test vessels should be continuously aerated from day -1 to day 10 except when test organisms are being added. Compressed air, previously filtered and free of oil, should be bubbled through a glass or plastic pipette and attached plastic tubing. The tip of the pipette should be suspended 2 to 3 cm above the surface of the sediment layer so as not to disturb the sediment surface. Aeration should be gentle enough that it does not disturb or resuspend sediment.
            
            (v) Salinity. During testing, salinity should be 20 plus or minus (+-) 3 ppt for E. estuarius and L. plumulosus. Salinity should be 28 +- 3 ppt for A. abdita and R. abronius.  Pore water salinity should be measured prior to the start of the test (See paragraph (e)(7)(vii) of this guideline for more information on salinity). Alternatively, the salinity of the overlying water can be adjusted to the salinity of the pore water at the site of interest in tests. If tests are conducted at different salinities, additional tests should be conducted to determine comparability of results (see reference in paragraph (j)(1)).
            
            (vi) Ammonia and sediment Eh. Ammonia concentrations in overlying water should be measured on day 0, and again on day 10. Ammonia should also be measured in the pore water and the beginning, mid-test, and end of the test. The sediment redox potential (Eh) should be measured at test initiation, mid-test (day 5), and at test termination.
            (vii) Renewal of Overlying Water
            
                  (A) Historically, 10-day tests of marine amphipods have been conducted in static systems where overlying water is not renewed. This use of static systems reflected a focus on assessing effects on survival in which organisms were not fed. If organisms are fed (e.g., for assessing effects on growth), overlying water should be renewed in order to avoid undesirable impacts on water quality. Even if organisms are not fed, it may be preferable to renew overlying water in order to avoid potential problems with overlying water quality.
                  (B) If water is renewed during the test, the renewal system should be designed such that volumes delivered to each exposure chamber do not differ by more than about 10%.
                  (C) Minimum number of test vessel volume replacements should be 2 per 24-hour period. If low dissolved oxygen requires action, the frequency/volume of overlying water addition can be increased from 2 up to a maximum of 4 volume additions/day.
                  It is highly recommended that water removal and replacement be completed using procedures that minimize disturbance to sediment in the test chambers. Water can be removed by siphoning through a tube with fine-meshed screening over the intake to prevent uptake of amphipods. A pump can also be used to remove water. Water should not be poured from test chambers because this practice can re-suspend and disturb the sediment. A separate turbulence-reducer should be used for each treatment when water is replaced to avoid cross contamination).	
      
            
      
      (9) Observations
            (i) Measurement of test substance. As described in OCSPP 850.1000, analytical confirmation of test substance concentrations should be performed on a subsample of each batch of spiked sediment for each test group to ensure that spiking is uniform. In addition, for pesticides with an aerobic soil or aerobic aquatic metabolism half-life of <=10 days, analytical confirmation of test substance concentrations, and concentrations of major degradation products, if applicable, is performed in bulk sediment, interstitial water, and overlying water at a minimum at the beginning and end of the test in each test group. Analytical confirmation at the middle of the test (i.e., day 5) is also recommended. For longer-lived pesticides and industrial chemicals, sampling at the beginning and end of the test in each test group may be appropriate. Sampling frequency should be adequate to characterize exposure concentrations in the bulk sediment and interstitial water throughout the duration of the test. 
            
            Measurements in interstitial water and overlying water should be of the dissolved or bioavailable form (see OCSPP 850.1000) whereas measurements in bulk sediment should be of the total form. Separate test vessels should be set up at the beginning of the test which can be destructively sampled during the test. The test vessels for test substance analytical measurements are set up and treated in the same way as those used for biological observations, including the presence of test organisms, except no biological observations are made. The analytical methods used to measure the amount of test substance in a sample should be validated before beginning the test, as described in OCSPP 850.1000.
            The concentration of test substance in overlying water is measured by pipetting water samples from 1 to 2 cm above the sediment surface. Caution should be used to eliminate the presence of any surface debris, material from the sides of the vessel, or sediment in the overlying water sample. Measurement of test substance concentration in sediment can be taken by siphoning most of the overlying water without disturbing the surface of the sediment, then removing appropriate aliquots of the bulk sediment for chemical analysis. The suggested method for isolation of interstitial water from a bulk sediment sample is by centrifugation without filtration. Centrifugation at about 10,000 g and 4 °C for 30 minutes is the recommended procedure to isolate interstitial water. In some cases, pooling replicates may be necessary to analyze concentrations in the pore water. When pooling, the volume of interstitial water from each replicate should be equal.
            (ii) Overlying water and sediment appearance. Observations are made daily on appearance of overlying water and test sediment. The appearance of surface slicks, precipitates, mold or fungus on sediment, or material adhering to the sides of the test vessels or in any part of the overlying water delivery or outflow system should be recorded.
            
            
            (vi) Measures of effect
                  (A) Monitoring of test organisms. All test vessels should be checked daily. Any test organisms trapped in the air-water interface should be gently pushed back down using a glass rod or pipette. Test organisms should be observed for abnormal behavior, such as sediment avoidance. However, the test organisms are often not visible during the exposure. The number of animals that appear to be dead should be noted. For L. plumulosus, the number of animals that appear to be dead should be noted, but organisms should not be removed or otherwise disturbed during the test. Exuviae may be mistaken for dead amphipods; therefore, care should be taken in identifying animals as dead.

                  (B) Survival. At test termination, all test organisms are collected by sieving the sediment with the overlying water, and the number of living, dead, and missing organisms is determined for each replicate in each test group. The amount of time taken to recover test organisms should be consistent across replicates.
                  Sieves should not exceed 0.5 mm. Small portions of test sediment should be washed into sorting trays using saltwater and should be examined carefully. Caution should be taken to ensure that no tube-dwelling organisms remain trapped on the sieve. Slapping the sieve forcefully against the surface of the water should successfully dislodge all A. abdita. The tubes of A. abdita should be teased apart under a dissecting microscope to ensure that all organisms are accounted for. The remaining species should be easily separated by the sieving process. Animals failing to respond to a gentle prodding are recorded as dead. 

                  (C) Reburial (optional). Data on the ability of E. estuarius, L. plumulosus, and R. abronius to rebury in clean sediment in a set amount of time may be determined as an optional additional measure of sublethal effects on behavior. At test termination, surviving amphipods should be transferred to containers holding a 2-cm layer of 0.5 mm sieved control sediment and overlying test saltwater (2 cm). Salinity of the test saltwater for reburial should be the same as that measured in the test vessel. The number of surviving amphipods unable to rebury in control sediment after one hour is recorded for each test vessel and is used to calculate effective mortality. 
                  
                  (D) Growth (optional). For L. plumulosus, growth may be measured either by weight or length. If growth is measured, the organisms are fed during the test. 
                  
                           (1) Dry weight. Dry weight of amphipods can be determined as follows: (1) transferring the archived amphipods from a replicate out of the preservative into a crystallizing dish; (2) rinsing amphipods with deionized water; (3) transferring these rinsed amphipods to a preweighed aluminum pan; (4) drying these samples to constant weight at 60°C; and (5) weighing the pan and dried amphipods on a balance to the nearest 0.01 mg. Average dry weight of individual amphipods in each replicate is calculated from these data. Due to the small size of the amphipods, caution should be taken during weighing. The average per-capita dry weight of adult amphipods for each replicate is the difference between the tared weight of the boat and the total weight of the boat plus dried amphipods, divided by the number of amphipods in the weigh boat. Weigh pans need to be carefully handled using powderless gloves and the balance should be calibrated with standard weights with each use. Use of small aluminum pans will help reduce variability in measurements of dry weight. Weigh boats can also be constructed from sheets of aluminum foil. 
                        
                          (2) Length. Amphipod body length (+-0.1 mm) can be measured from the base of the first antennae to the tip of the third uropod along the curve of the dorsal surface.

(f) Treatment of results
      (1) Response variable calculation. Survival is the primary endpoint recorded. For measures of survival, surviving organisms are counted at test termination. For E. estuarius, L. plumulosus, and R. abronius, a calculation of effective mortality, which is the sum of dead animals plus those survivors that fail to rebury in clean control sediment, has also been used. For measures of growth, the response variables used in the analyses are the average weight or length of surviving test organisms.	
      Average test organism weight. The response measure for test organism weight for a test vessel is the average test organism weight () which is calculated using Equation 1.
		Equation 1
            where:
            j = index number of the replicates in a test group from 1 to the total number of test vessels in a test group;
            TWj = total dry weight of surviving test organisms (dry weight for amphipods measured as described in paragraph (e)(9)(vi)(D)) in replicate j; and
            NSj = number of surviving test organisms in replicate j (that were weighed).
      Average test organism length. The response measure for test organism length is the average test organism length for a test vessel () which is calculated using Equation 2. 
		Equation 2
            where:
            j = index number of the replicates in a test group from 1 to the total number of test vessels in a test group;
            k = index number of length measurement in test vessel j from 1 to NSj; and
            NSj = number of surviving test organisms in replicate j that were measured.
      (2) Summary statistics
            (i) Environmental conditions. For overlying water, interstitial (pore) water, and sediment homogeneity: 
                  (A) Overlying water. Calculate descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) of environmental conditions (temperature, dissolved oxygen, pH, un-ionized ammonia) and salinity monitored during the test.
                  (B) Interstitial (pore) water. Calculate descriptive statistics (mean, standard deviation, coefficient of variation, minimum, maximum) of interstitial (pore) water environmental conditions (Eh, pH, salinity, and un-ionized ammonia) monitored during the test.
                  (C) Sediment homogeneity. Calculate descriptive statistics (mean standard deviation, coefficient of variation, minimum, maximum) of bulk sediment characteristics, such as TOC, particle size distribution (percent of sand, silt, clay), percent water content, and acid volatile solids, if applicable.
            (ii) Test substance concentration
                  (A) Overlying and Interstitial water. Calculate descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation) by test vessel and treatment level of the test substance soluble concentration in overlying water and interstitial water.
                  (B) Bulk sediment. Calculate descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation) by test vessel and treatment level of the test substance concentration in bulk sediment. Where applicable calculate descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation) by test vessel and treatment level of the major toxic degradate(s) concentration in bulk sediment. Test concentrations are typically expressed as mg/kg dry weight for sediment. In some cases, it is also desirable to normalize sediment concentrations of test substance to factors other than sediment dry weight, such as organic carbon content for nonionic organic compounds or acid volatile sulfides for certain metals.
            (iii) Mortality. Number of organisms exposed at test initiation in each treatment and replicate and the cumulative number of dead test organisms should be summarized in tabular form by time of observation, treatment, and replicate.
            (iv) Reburial (optional). Following the 10-d test termination for survival, an optional endpoint is the 1-h reburial for E. estuarius, R. abronius and L. plumulosus. The number of surviving test organisms in each test vessel placed in clean sediment at test termination for reburial observations and the number not able to rebury should be summarized by treatment level and replicate.
            (v) Growth (optional). Average weight and/or length of surviving test organisms at test termination by treatment and replicate.
            (vi) Sediment Avoidance. Number of organisms appearing to avoid the sediment (e.g., observed in overlying water or on top of the sediment when should be under cover) should be summarized in tabular form by time of observation, treatment, and replicate.
            (vii) Appearance and behavior. Number of organisms exhibiting abnormal appearance or behavioral symptoms should be summarized in tabular form time of observation, treatment, and replicate.
	(3) Percent mortality
            (i) Mortality. Calculate the percent mortality at each treatment level and in the control(s) at test termination.
            (ii) Effective mortality. Infaunal test organisms unable to rebury are not considered likely to survive in nature. Effective mortality is calculated as the percent of test organisms dead at test termination in each test group plus those survivors in the test group unable to rebury. If all survivors in a test group are tested for the ability to rebury, the percent effective mortality is calculated using Equation 3; if only a subset of survivors in a test group are tested for reburial, the percent effective mortality is calculated using Equation 4.
            			Equation 3
            where:
            N0j = total number of test organisms exposed in treatment j at test initiation;
            ndeadj = total number of dead test organisms in treatment j at test termination; and
            nfailj = total number of survivors at test termination in treatment j that fail to rebury in a set time span.
            
            		Equation 4
            where:
            N0j = total number of test organisms exposed in treatment j at test initiation;
            ndeadj = total number of dead test organisms in treatment j at test termination;
            nfailj = total number of survivors placed in dishes at test termination in treatment j that fail to rebury in a set time span; and
            NSj = total number of survivors in test group j placed in clean dishes for reburial testing.
      (4) Percent inhibition. For survival and growth (weight and/or length), calculate and plot the mean percent inhibition for survival and growth at test termination in each treatment level as compared to the controls 
      (5) Evaluation of limit test results. To ascertain that there is no observable effect on survival at the limit concentration (i.e., NOEC>limit concentration), the limit treatment response is compared to the control treatment response. For pesticides, if a significant effect is detected in survival at the limit concentration, it is preferred that a multiple-concentration sub-chronic 10-d test be conducted. For industrial chemicals, if the effect level for mortality or inhibition of growth at the limit concentration compared to the control(s) is 50% or greater, then a definitive test should be conducted.
      (6) Evaluation of multiple-concentration definitive test
            (i) Concentration-response curve, slope, and LC50 and EC50. Statistical procedures are employed to calculate the 10-d LC50 (standard error and 95% confidence interval) based upon mortality and 10-d EC50 (standard error and 95% confidence intervals) for growth (weight and/or length) when appropriate to the test design. The slope of the concentration-response curve, its standard error, and 95% confidence interval should also be reported. If a concentration-response curve model was fit to the data to determine the LC50 or EC50, the model parameters (e.g., slope) and their uncertainty estimates (e.g., standard error) should be recorded.
            (ii) NOEC. Calculate the NOEC and LOEC values for survival and growth (weight, length) when appropriate to the test design. Hypothesis testing procedures described in OCSPP 850.1000 can be used to determine NOEC and LOEC values. 
            (iii) Statistical methods. Statistical procedures for modeling quantal data should be used for calculating the LC50. Statistical procedures for modeling continuous toxicity data should be used for calculating the EC50 (see references in paragraphs (j)(3) and (j)(4) of this guideline). Analysis-of-variance testing procedures are used to determine NOEC and LOEC values. Additional discussion about endpoints and statistical procedures is found in OCSPP 850.1000 and in paragraphs (j)(1) and (j)(9).
(g) Tabular summary of test conditions. Table 1 lists the important conditions that should prevail during this test. 
Table 1.--Summary of Test Conditions for 10-Day Sediment Toxicity with Saltwater Amphipods
Test type
Spiked-sediment toxicity test
Test species
Recommended: Ampelisca abdita, Eohaustorius estuarius, Rhepoxynius abronius, or Leptocheirus plumulosus 
Test duration
10 days
Test matrix
Formulated or natural clean sediment with overlying water
Overlying water 
Saltwater (natural or artificial)
Temperature
E. estuarius and R. abronius: 15 ºC 
A. abdita: 20 ºC 
L. plumulosus: 25 ºC
(constant during test within +-2 ºC)
Light quality 
Ambient laboratory illumination
Light intensity
5-1080 lux (50-100 foot-candles)
Photoperiod
For L. plumulosus, selected from among 12 hours light:12 hours dark to 16 hours light:8 hours dark schemes. For other species, 16 hours light:8 hours dark
Overlying water pH
pH not specified, however should be constant within +-1 pH unit within a test vessel and between test concentrations (including control(s) during test
Overlying water salinity
A. abdita and R. abronius: 28 ppt 
E. estuarius and L. plumulosus: 20 ppt 
(constant during test within +-3 ppt)
Sediment pore water salinity
Should be within the salinity range of the chosen amphipod species (see paragraph (e)(7)(vii))
Overlying water TOC
<=2 mg/L
Sediment/overlying volume
175 mL/800mL in 1-L glass beaker or jar with 10-cm inner diameter has been used successfully 
Renewal of overlying water
None if a static system is used. 2 volume replacements/day if water is renewed. Renewal is highly recommended if organisms are fed.
Size or age and life stage of test organisms

A. abdita: 3-5 mm (no mature males or females)
E. estuarius: 3-5 mm
L. plumulosus: 7- to 8-days old (no mature males or females) 
R. abronius: 3-5 mm
Number of test organisms per test vessel
20
Number of replicate test vessels per concentration
For pesticides, minimum of 8 for NOEC/LOEC determinations 
For industrial chemicals, minimum of 3 for LC50 determinations 
Additional replicates may be needed for use in analytical determinations of test substance concentrations in bulk sediment, pore water, and overlying water and sediment chemistry during the test.
Feeding regime
None during test if a static system is used. If growth is being measured and water is renewed, 20 mg flake food per chamber 3x/week.
Test vessel aeration
If a static system is used, overlying water in each test vessel should be aerated overnight before start of test and throughout the test.
Test concentrations
Unless performing a limit test, a minimum of 5 test concentrations chosen in a geometric series plus appropriate control(s)
Test concentration preparation
Addition of the test substance directly to the sediment as an aqueous solution for a soluble test substance, directly to the sediment in a dry form for a water insoluble solid, or by sorbing the test substance to sand and then mixing the treated sand into the sediment for a water insoluble test substance
Measures of Effect or Measurement Endpoints
For pesticides, 10-d NOEC and LOEC for survival and growth; if desired, 10-d LC50 (mortality) and 10-d EC50 (growth based on weight or length, or for effective mortality)

For industrial chemicals, 10-d LC50, 10-d EC50 (growth based on weight or length, or for effective mortality)

(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 study will be rejected when there are only slight variations from guideline environmental conditions and study 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 size/age), the investigator should contact the Agency to discuss the reason for the departure and the effect the change(s) will have on test acceptability. In the test report, all departures from the guideline should be identified, reasons for these changes given, and any resulting effects on test endpoints noted and discussed.
 Table 2. -- Test Validity Elements for the 10-Day Sediment Toxicity Test with
--------------------------------------------------------------------------------
Saltwater Amphipods

1. All test vessels were not identical or did not contain the same amount of sediment and overlying water.

2. Test organisms were not randomly or impartially assigned to test vessels.

3. A negative sediment control and a solvent sediment control, if applicable, were not included in a test.

4. Average survival of amphipods in the either the negative sediment control or solvent sediment control was not greater than or equal to 80% at test termination.
_____________________________________________________________________

(i) Data 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, and its purity (i.e. for pesticides, the identity and concentration of active ingredient(s)), radiolabeling if any, 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 test, 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, Kow.
            
            (v) If a vehicle (solvent) is used to prepare stock or test substance, provide: the name and source of the solvent, the nominal concentration(s) of the test substance in the solvent in stock solutions or mixtures, and the solvent concentration(s) used in the treatments and solvent sediment control. If different solvent concentrations are used at different treatment levels, the report should, at a minimum, identify the maximum solvent concentration used. It is helpful to support the solvent choice by including a description of any measures that were taken to identify an appropriate solvent for use in the study, such as the types and concentrations of solvents 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 organism 
		(i) Scientific name and common name.
		(ii) Method for verifying the test species.
            (iii) Information about the organisms used in the test: source, date of collection, duration of quarantine for organisms collected from a natural population, culture practices, and holding and acclimation procedures and conditions, including environmental conditions, acclimation period, water used, substrate, feeding history, and health status of culture used to obtain larvae or instars (mortality of stock before test initiation and any preventative or disease treatments). 
            (iv) Age (days, developmental stage) and size (weight, length) of test organisms in the test population 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 container used: size, type, material, fill volume.
            (ii) For naturally derived sediment, description of the sediment source and procedures for collection and handling: location, time, core depth, water depth, collection equipment, shipping method, pooling, pretreatment (sieving, UV treatment), and storage methods and duration.
            (iii) For an artificial sediment, description of the source, type and amounts of ingredients used to prepare formulated sediment, preparation date, mixing and homogenization procedures, and storage methods and duration.
            (iv) Type and magnitude of biota present in naturally-derived sediment at test initiation.
            (v) Physical and chemical characteristics of the bulk sediment: pore water salinity, color, pH, Eh, particle size distribution (percent sand, silt, clay), TOC, cation exchange capacity, moisture content (percent water content of the sediment) and if from a naturally-derived source, the background concentration of metals, synthetic organic compounds, oil and grease, petroleum hydrocarbons, and ammonia, the biological oxygen demand (BOD), and COD of the sediment. If metals are tested, the concentration of acid volatile sulfides should also be reported. Describe the frequency and sampling methods and sampling date(s) for documenting physical and chemical characteristics and the homogeneity of the sediment physical and chemical characteristics.
            (vi) Description of the overlying water and if test was conducted as a static, static renewal, or intermittent flow-through. If static renewal, the frequency of test solution renewal, and if intermittent 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 used.
            (vii) Description of the source of clean water used for overlying water and any water pretreatment: source/type; temperature; salinity; pH; hardness and alkalinity; 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 source water quality and consistency.
            (viii) Methods of adding the test substance to sediment and concentrations used in definitive or limit testing.
            (ix) Equilibration phase of the spiked sediment-water system: duration and conditions.
      (x) Depth and volume of sediment and of overlying water in test vessels.
      (xi) Use of aeration and method. 
            (xii) Number of test organisms added to each test vessel at test initiation and the method of introduction.
      (xiii) Number of test vessels (replicates) per treatment level and control(s).
            (xiv) Methods used for treatment randomization and assignment of test organisms to test vessels.
		(xv) Date of introduction of test organisms to test vessels and test duration.
            (xvi) The photoperiod and light source and quality.
            (xvii) Description of feeding protocols: source and composition of food; preparation of food; frequency of feeding and food ration; frequency and sample date(s) for documenting the contaminant status (heavy metals, persistent or chlorinated pesticides) of the feed and tabulation of the results of the analysis.
            (xviii) Methods and frequency of environmental monitoring performed during the definitive or limit study for overlying water temperature, dissolved oxygen, pH, ammonia, conductivity, and alkalinity. 
            (xix) Methods and frequency of monitoring performed during the definitive or limit study for pore water pH and ammonia.
            (xx) Methods and frequency of monitoring performed during the definitive or limit study for sediment Eh.
            (xxi) Methods and frequency of measuring test substance (and major degradates where appropriate) in bulk sediment, pore water, and overlying water to verify exposure concentrations.
            (xxii) Methods and frequency of counting number of surviving and dead test organisms and measuring dry weight, length, sediment avoidance and any other toxic symptoms.
            (xxiii) For definitive and limit tests, a description of all analytical procedures used, accuracy of the method, method detection limit, and limit of quantification.
	(6) Results 
            (i) Nominal sediment exposure concentrations and a tabulation of test substance analytical results for bulk sediment, pore water, and overlying water by treatment group and test vessel (provide raw data) and descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation). For testing with organic compounds, also provide a tabulation of test substance sediment analytical results normalized to organic carbon content of sediment. For testing with certain types of metals, also provide a tabulation of sediment analytical results normalized to acid volatile sulfide content of sediment.
            (ii) Environmental monitoring data results 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 tests, if conducted, a tabulation of the number and percentage of organisms that died in each test vessel, including all treatment levels and control(s), at each observation period. A description and count of any test organisms exhibiting avoidance or other appearance or behavioral effects at each treatment level and in the control(s).
		(iv) For the limit and definitive tests, if conducted, a tabulation of: 
                  (A) The number of dead test organisms and the percent mortality in each test vessel, including all treatment levels and control(s), at test termination (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum).
                  (B) The number of organisms exhibiting avoidance behavior or other abnormal behavior or appearance by test vessel, treatment level, and observation period (provide the raw data).
                  (C) The dry weight or length for L. plumulosus, if measured, in each vessel, including all treatment levels and control(s), at test termination (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum).
                  (D) The percent reduction in survival and percent inhibition in growth (weight, length), if measured, for the limit concentration as compared to the control(s).
            (v) Graphs of the concentration-response data for percent mortality and percent inhibition of in survival.
            (vi) For limit tests, conclusion about the 10-d NOEC for survival and growth (weight, length), if measured.
            (vii) For definitive tests with industrial chemicals, the 10-d LC50 and EC50 value, its standard error and 95% confidence interval, and where sufficient data exist to fit a regression model (e.g. probit), the slope of the concentration-response curve, its standard error and 95% confidence intervals, and any goodness of fit results for mortality.
            (viii) For definitive tests with pesticides, the 10-d NOEC and LOEC for survival and growth (weight, length), if measured.
             (ix) Description of statistical method(s) used for point estimates, including software package, for determining the LC50 and EC50 values,, fitting the concentration-response model, and the basis for the choice of method. Provide results of any goodness-of-fit tests.
            (x) Description of statistical method(s) used for NOEC and LOEC determination, including software package, and the basis for the choice of the 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 E 1367-03e1, Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Invertebrates, American Society for Testing and Materials, West Conshohocken, PA. Current edition approved 2008. http://www.astm.org/Standards/E1367.htm
      (2) American Society for Testing and Materials. ASTM E-1391-03, Standard Guide for Collection, Storage, Characterization, and Manipulation of Sediments for Toxicological Testing and for Selection of Samplers Used to Collect Benthic Invertebrates, American Society for Testing and Materials, West Conshohocken, PA. Current edition approved 20088. http://www.astm.org/Standards/E1391.htm
      (3) Bruce, R.D. and D.J. Versteeg, 1992. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
      (4) European Commission, 1994. Sediment toxicity tests for poorly water-soluble substances, final report to the European Commission, Report No. EC 3738, programme co-ordinator R. Fleming.
      (5) Redmond, M., K. Scott, R Swartz, and J Jones, 1994. Preliminary culture and life-cycle experiments with benthic amphipod Ampelisca abdita. Environmental Toxicology and Chemistry 13(8): 1355-1365.
      (6) Society of Environmental Toxicology and Chemistry - Europe, 1994. Guidance document on sediment toxicity tests and bioassays for freshwater and marine environments, from the Workshop on sediment toxicity assessment, held 8-10 November, 1993, Hill, I.R., Matthiessen, P. and Heimbach, F., eds. 
      (7) U.S. Environmental Protection Agency, 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine Amphipods, EPA/600/R-94/025, June 1994.
      (8) U.S. Environmental Protection Agency. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates, Second Edition, EPA 600/R-99/064, March 2000. http://www.epa.gov/waterscience/cs/freshfact.html
      (9) U.S. Environmental Protection Agency, 2001. Methods for Assessing the Chronic Toxicity of Marine and Estuarine Sediment-associated Contaminants with the Amphipod Leptocheirus plumulosus, EPA/600/R-01/020, March 2001. http://water.epa.gov/polwaste/sediments/cs/leptofact.cfm 
      
