5.1 Mysids are an important component of both the pelagic and epibenthic community. They are preyed upon by many species of fish, birds, and larger invertebrate species, and they are predators of smaller crustaceans and larval stages of invertebrates. In some cases, they feed upon algae. Mysids are sensitive to both organic and inorganic toxicants (1).3 The ecological importance of mysids, their wide geographical distribution, ability to be cultured in the laboratory, and sensitivity to contaminants make them appropriate acute toxicity test organisms.5.2 An acute toxicity test is conducted to obtain information concerning the immediate effects of a short-term exposure to a test material on a test organism under specified experimental conditions. An acute toxicity test provides data on the short-term effects that are useful for comparisons to other species but does not provide information on delayed effects.5.3 Results of acute toxicity tests can be used to predict acute effects likely to occur on aquatic organisms in field conditions except that mysids might avoid exposure when possible.5.4 Results of acute toxicity tests might be used to compare the acute sensitivities of different species and the acute toxicities of different test materials, and to study the effects of various environmental factors on results of such tests.5.5 Results of acute toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023) or when deriving water quality criteria for aquatic organisms (2).5.6 Results of acute toxicity tests might be useful for studying biological availability of, and structure activity relationships between test materials.5.7 Results of acute toxicity tests will depend, in part, on the temperature, quality of the food, condition of test organisms, test procedures, and other factors.1.1 This guide describes procedures for obtaining data concerning the adverse effects of a test material (not food) added to marine and estuarine waters on certain species of marine and estuarine mysids during 96 h of continuous exposure. Juvenile mysids used in these tests are taken from cultures shortly after release from the brood and exposed to varying concentrations of a toxicant in static or flow-through conditions. These procedures will be useful for conducting toxicity tests with other species of mysids, although modifications might be necessary.1.2 Modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests with other species of mysids.1.3 The procedures given in this guide are applicable to most chemicals, either individually or in formulations, commercial products, and known or unknown mixtures. With appropriate modifications these procedures can be used to conduct acute tests on factors such as temperature, salinity, and dissolved oxygen. These procedures can also be used to assess the toxicity of potentially toxic discharges such as municipal wastes, oil drilling fluids, produced water from oil well production, and other types of industrial wastes.1.4 Results of acute toxicity tests with toxicants experimentally added to salt and estuarine waters should usually be reported in terms of a LC50 (median lethal concentration).1.5 This guide is arranged as follows:  Section   Referenced Documents  2Terminology  3Summary of Guide  4  5Apparatus  6 Facilities  6.1 Construction Materials  6.2 Metering Systems  6.3 Test Chambers  6.4 Cleaning  6.5 Acceptability  6.6Safety Precautions  7Dilution Water  8 Requirements  8.1 Source  8.2 Treatment  8.3 Characterization  8.4Test Material  9 General  9.1 Stock Solution  9.2 Test Concentrations  9.3Test Organisms 10 Species 10.1 Age 10.2 Source 10.3 Brood Stock 10.4 Food 10.5 Handling 10.6 Harvesting Young 10.7 Quality 10.8Procedure 11 Experimental Design 11.1 Dissolved Oxygen 11.2 Temperature 11.3 Loading 11.4 Salinity 11.5 Light 11.6 Beginning of Test 11.7 Feeding 11.8 Duration of Test 11.9 Biological Data 11.10 Other Measurements 11.11Analytical Methodology 12Acceptability of Test 13Interpretation of Results 14Report 15Appendixes   Holmesimysis costata X1 Neomysis mercedis X21.6 The values stated in SI units are to be regarded as the standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 7.

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5.1 The USEPA's policy for whole-effluent monitoring stresses, an integrated approach to toxicity testing (1, 5) tests and other measures of toxicity, should be systematically employed and should be related to certain aquatic-system factors, such as the type of habitats available (benthic and water column), flow regime, and physicochemical quality of the site water and sediment. The determination of toxicity is generally accomplished with a few surrogate species for four major reasons: a regulatory agency can compare test results between sites and over time in order to help prioritize enforcement efforts, tests using these species are relatively inexpensive since the organisms can be cultured year-round under laboratory conditions, the reliability of test methods utilizing surrogate species is better established than for other species, and surrogate species are better integrated into toxicity identification evaluations than other species. For regulatory purposes, under the National Pollution Discharge Elimination System (NPDES), USEPA considers it unnecessary to conduct whole effluent toxicity tests with resident or indigenous species (6). An alternate testing procedure protocol is provided by USEPA for validating toxicity methods using species not already approved (6,7). In systems where surrogate species are not found, erroneous predictions might be obtained of environmental impact or water and sediment quality impairment based on toxicity tests using surrogate species (8).5.2 This guide is intended to assist researchers and managers in selecting appropriate resident species for site-specific toxicity assessments. This guide could be used to select a resident species for use in predicting the potential toxic effects of a substance in certain types of aquatic environments. Another use might be for selecting a number of indigenous species from the aquatic community, that when tested, might indicate potential toxic effects of the test substance or material on the ecological integrity of that community. Selection of a suitable test species is very important because species might respond quite differently to toxic compounds (9). Species suggested as test organisms by regulatory agencies might not occur in the receiving waters of interest and their sensitivity to a toxic substance might not be representative of the sensitivity exhibited by resident species. Since aquatic ecosystem structure and function is often determined by a few key species (10, 11, 12, 13), toxicological tests with these resident species might be very important.5.3 This guide can be used in the selection of representative test species for certain site-specific assessments, such as the Resident-Species Criteria Modification Procedure (1), the Recalculation Procedure (14), and ecological risk assessment studies.5.4 This guide can be used as a general framework for researchers who desire to develop or modify existing toxicity test methods for previously untested species.5.5 Researchers in countries other than the United States and Canada might obtain useful information from this guide regarding potential test species or test methods for sites of local interest.1.1 This guide along with Guide E1192 and guidance from the U.S. Environmental Protection Agency (1,2)2 covers the use of resident species in toxicity testing, particularly if site-specific information is desired. For example, in those systems where particular species are considered to be economically or aesthetically important, it might be more appropriate to utilize resident species for testing (3). For this reason, the USEPA allows development of site-specific chemical standards, using resident species, in order to reflect local conditions (1). This guide is designed to guide the selection of resident species for use as test organisms in aquatic and sediment toxicity tests. It presupposes that the user is familiar with the taxonomy of aquatic and benthic species and has some field experience.1.2 Because toxicological information is often limited for many aquatic species, it is assumed that the majority of testing applications will be acute tests. Therefore, much of the guidance presented in this guide pertaining to the species selection process is applicable when acute toxicity testing is the desired goal. However, the principles discussed in this guide pertain to chronic toxicity test applications as well, although it should be clearly understood that such testing requires substantially greater effort, time, and resources than acute testing.1.3 The procedures for selecting resident species in toxicity testing are necessarily general at this time because information is often lacking for specific taxa or groups of taxa. This guide attempts to give specific information when appropriate.1.4 This guide is not intended to be inclusive. References listed provide a starting point from which to approach the literature. This guide deals solely with aquatic toxicity test situations. Terrestrial, arboreal, or atmospheric species are not considered in this guide.1.5 This guide is arranged as follows:  Section    1  Referenced Documents 2  Terminology 3  Summary of Guide 4   5  Species Selection Process 6  Collection of Information 6.1Obtaining Resident Species for Toxicity Testing 6.2Criteria for Selection 6.3Test Performance Characterization 6.4Interferences 7  Safety Precautions 8  Documentation 9  Keywords 10   AppendixesPotential Test Species  Appendix X1  Algae  X1.1Aquatic Floating Macrophytes  X1.2Protozoa  X1.3Rotifera  X1.4Attached and Benthic Fauna  X1.5Fish  X1.6Amphibia  X1.7Examples of Resident Species  Table X1.1Taxonomic Keys—Partial Listing  Appendix X2  Flow Chart of Factors to Consider For Selecting A Resident Species  Appendix X3  1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. All safety precautions and health-related practices are the responsibility of the user. Specific safety practices are suggested in Section 8.1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Polychaetes are an important component of the benthic community, in which they generally comprise 30 to 50 % of the macroinvertebrate population. They are preyed upon by many species of fish, birds, and larger invertebrate species. Larger polychaetes feed on small invertebrates, larval stages of invertebrates, and algae. Polychaetes are especially sensitive to inorganic toxicants and, to a lesser extent, to organic toxicants (1).4 The ecological importance of polychaetes and their wide geographical distribution, ability to be cultured in the laboratory, and sensitivity to contaminants make them appropriate acute and chronic toxicity test organisms. Their relatively short life cycle enables the investigator to measure the effect of contaminants on reproduction.5.2 An acute toxicity or chronic text is conducted to obtain information concerning the immediate effects of an exposure to a test material on a test organism under specified experimental conditions. An acute toxicity test provides data on the short-term effects, which are useful for comparisons to other species but do not provide information on delayed effects. Chronic toxicity tests provide data on long-term effects.5.3 A life-cycle toxicity test is conducted to determine the effects of the test material on survival, growth, and reproduction of the test species. Additional sublethal endpoints (for example, biochemical, physiological, and histopathological) may be used to determine the health of the species under field conditions.5.4 The results of acute, chronic, and life-cycle toxicity tests can be used to predict effects likely to occur on marine organisms under field conditions.5.5 The results of acute, chronic, or life-cycle toxicity tests might be used to compare the sensitivities of different species and the toxicities of different test materials, as well as to study the effects of various environmental factors on the results of such tests.5.6 The results of acute, chronic, or life-cycle toxicity tests might be an important consideration when assessing the hazards of materials to marine organisms (see Guide E1023) or when deriving water quality criteria for aquatic organisms (2).5.7 The results of acute, chronic, or life-cycle toxicity tests might be useful for studying the biological availability of, and structure activity relationships between, test materials.5.8 The results of acute, chronic, and life-cycle toxicity tests will depend partly on the temperature, quality of food, condition of test organisms, test procedures, and other factors.1.1 This guide covers procedures for obtaining data concerning the adverse effects of a test material added to marine and estuarine waters on certain species of polychaetes during short- or long-term continuous exposure. The polychaete species used in these tests are either field collected or from laboratory cultures and exposed to varying concentrations of a toxicant in static or static-renewal conditions. These procedures may be useful for conducting toxicity tests with other species of polychaetes, although modifications might be necessary.1.2 Modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using unusual procedures are not likely to be comparable to those of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute, chronic, or life-cycle tests with other species of polychaetes.1.3 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, and known or unknown mixtures. With appropriate modifications, these procedures can be used to conduct these tests on factors such as temperature, salinity, and dissolved oxygen. These procedures can also be used to assess the toxicity of potentially toxic discharges such as municipal wastes, sediments/soils, oil drilling fluids, produced water from oil well production, and other types of industrial wastes. An LC50 (medial lethal concentration) may be calculated from the data generated in each acute and chronic toxicity test when multiple concentrations are tested. Growth, determined by a change in measured weight, and reproduction, as the change in total number of organisms, are used to measure the effect of a toxicant on life-cycle tests; data are analyzed statistically to indicate that concentration at which a significant difference occurs between the test solutions and control(s).1.4 The results of dose-response acute or chronic toxicity tests with toxicants added experimentally to salt water should usually be reported in terms of an LC50 (mortality), or EC50 (medial effect concentration). The results of life-cycle toxicity tests with toxicants added experimentally to salt water should be reported as that concentration at which a statistically significant difference in the number of offspring or growth (determined by weight) is produced with reference to the control(s).1.5 Where appropriate, this standard has been designed to be consistent with or complementary to other methods for assessing toxicity to invertebrates described in Test Methods E1367 and E1706, and Guides E1391, E1525, E1611, and E1688.1.6 This guide is arranged as follows:  SectionReferenced Documents  2Terminology  3Summary of Guide  4  5Apparatus  6 Facilities  6.1 Construction Materials  6.2 Test Chambers  6.3 Cleaning  6.4 Acceptability  6.5Safety Precautions  7Dilution Water  8 Requirements  8.1 Source  8.2 Treatment  8.3 Characterization  8.4Test Material  9 General  9.1 Stock Solution  9.2 Test Concentrations  9.3Test Organisms 10 Species 10.1 Age 10.2 Source 10.3 Feeding 10.4 Holding 10.5 Quality 10.6Procedure 11 Experimental Design 11.1  Acute Test 11.1.1  Chronic Test 11.1.2  Life-Cycle Test 11.1.3 Test Condition Specifications 11.2  Dissolved Oxygen 11.2.1  Temperature 11.2.2  Loading 11.2.3  Salinity 11.2.4  Light 11.2.5 Beginning the Test 11.3 Feeding 11.4 Duration of Test 11.5 Biological Data 11.6 Other Measurements 11.7Hazards  Analytical Methodology 13Acceptability of Test 14Calculation of Results 15Report 16Keywords 17Appendixes:   Neanthes arenaceodentata Appendix X1 Capitella capitata Appendix X2 Ophryotrocha diadema Appendix X3 Dinophilus gyrociliatus Appendix X41.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 7.1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 An acute toxicity test is conducted to assess effects of a short-term exposure of organisms to a test material under specific experimental conditions. An acute toxicity test does not provide information concerning whether delayed effects will occur and typically evaluates effects on survival. A chronic test is typically longer in duration and includes a sublethal endpoint to assess effects on a population that might occur beyond the exposure period. Because the echinoderm embryo development test includes a sublethal endpoint, but is also short in duration, these tests are considered to be short-term chronic tests, consistent with EPA guidance.5.2 Because embryos and larvae are usually assumed to be the most sensitive life stages of these echinoid species, and because some of these species are commercially and recreationally important, the results of these tests are often considered to be a good indication of the acceptability of pollutant concentrations to saltwater species in general. The results of these toxicity tests are often assumed to be an important consideration when assessing the hazard of materials to other saltwater organisms (see Guides E724 and E1023) or when deriving water quality criteria for saltwater organisms (7).5.3 The results of short-term chronic toxicity tests might be used to predict effects likely to occur to aquatic organisms in field situations as a result of exposure under comparable conditions, except that toxicity to benthic species might depend on sorption or settling of the test material onto the substrate.5.4 The results of short-term chronic tests might be used to compare the sensitivities of different species and the acute toxicities of different test materials, and to determine the effects of various environmental factors on the results of such tests.5.5 The results of short-term chronic toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.5.6 The results of any toxicity tests will depend on temperature, composition of the dilution water, condition of the test organisms, and other factors.5.7 Results of short-term chronic toxicity tests might be used to predict effects likely to occur to aquatic organisms exposed to suspended particulates of dredged sediments disposed through the water column.5.8 Results of short-term chronic toxicity tests might be used to predict effects likely to occur to aquatic organisms exposed to bedded whole sediments.1.1 This guide covers procedures for obtaining laboratory data concerning the short-term chronic effects of a test material on echinoderm embryos and the resulting larvae (sea urchins and sand dollars) during static 48- to 96-h exposures. These procedures have generally been used with U.S. East Coast (Arbacia punctulata and Strongylocentrotus droebachiensis ) (1)3 and West Coast species (Strongylocentrotus purpuratus, S. droebachiensis, and Dendraster excentricus) (2). The basic procedures described in this guide first originated in Japan and Scandanavia (3), and parallel procedures have been used with foreign species, especially in Japan and the Mediterranean (4). These procedures will probably be useful for conducting static toxicity tests with embryos of other echinoid species, although modifications might be necessary.1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using procedures appropriate to a particular species or special needs and circumstances is more important than following prescribed procedures, the results of tests conducted by using unusual procedures are not likely to be comparable with those of many other tests. The comparison of results obtained by using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting tests starting with embryos of echinoids.1.3 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications, these procedures can be used to conduct tests on temperature, dissolved oxygen, and pH and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, surface waters, effluents, and sediments (Annex A1). Renewal tests might be preferable to static tests for materials that have a high oxygen demand, are highly volatile, are rapidly transformed biologically or chemically in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test.1.4 Results of short-term chronic toxicity tests with echinoid embryos should usually be reported as the 50 % effect concentration (EC50) based on the total abnormally developed embryos and larvae. In some situations, it might only be necessary to determine whether a specific concentration is toxic to embryos or whether the EC50 is above or below a specific concentration.1.5 This guide is arranged as follows:  Section 1 Referenced Documents 2 Terminology 3 Summary of Guide 4  5 Apparatus 6  Facilities 6.1  Construction Materials 6.2  Test Chambers 6.3  Cleaning 6.4  Acceptability 6.5 Safety Precautions 7 Dilution Water 8  Requirements 8.1  Source 8.2  Treatment 8.3  Characterization 8.4 Test Material 9  General 9.1  Stock Solution 9.2  Test Concentration(s) 9.3 Test Organisms 10  Species 10.1  Age 10.2  Source of Embryos 10.3  Handling 10.4  Test Animal Source and Condition 10.5  Spawning and Fertilization 10.6  Quality 10.7 Procedure 11  Experimental Design 11.1  Dissolved Oxygen 11.2  Temperature 11.3  Beginning the Test 11.4  Feeding 11.5  Duration of Test 11.6  Biological Data 11.7  Control Performance 11.8  Other Measurements 11.9 Analytical Methods 12 Acceptability of Test 13 Calculation of Results 14 Report 15 Keywords 16 Annex   Sediment Tests Annex A1 1.6 The values stated in SI units are to be regarded as the standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 7.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Ceriodaphnia  was first used as a toxicity test organism by Mount and Norberg (2). Introduced for use in effluent and ambient water evaluations, Ceriodaphnia have also been a valuable addition to single chemical test procedures.5.2 Protection of a population requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species, or species for which the test species serves as a surrogate. A three-brood toxicity test is conducted to help determine changes in survival and the number of neonates produced that result from exposure to the test material.5.3 Results of three-brood toxicity tests with C. dubia might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions.5.4 Results of three-brood toxicity tests with C. dubia might be compared with the chronic sensitivities of different species and the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.5.5 Results of three-brood toxicity tests with C. dubia might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most such predictions are based on the results of acute toxicity tests, and so the usefulness of the results of a three-brood toxicity test with C. dubia might be greatly increased by also reporting the results of an acute toxicity test (see Guides E729 and E1192) conducted under the same conditions. In addition to conducting an acute test with unfed C. dubia, it might also be desirable to conduct an acute test in which the organisms are fed the same as in the three-brood test, to see if the presence of that concentration of that food affects the results of the acute test and the acute chronic ratio (see 10.4.1).5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be obtained from a three-brood toxicity test with a known test material, but often all the concentrations in the test will be below the EC50 or LC50. In addition, it is usually desirable to know the EC50 or LC50 before beginning the three-brood test, as a means to determine the concentrations for use in the chronic test (see 10.4.1). It should be noted that results from an acute test may not necessarily correspond to those of a chronic test, due to the addition of food to the chronic test.5.6 Three-brood toxicity tests with C. dubia might be useful for studying biological availability of, and structure activity relationships between, test materials.5.7 Results of three-brood toxicity tests with C. dubia can vary with temperature, quality and quantity of food, dissolved ion concentrations, quality of the dilution water, condition of the test organisms, and other factors.5.8 Results of three-brood toxicity tests with C. dubia might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023), or when deriving water quality criteria for aquatic organisms.1.1 This guide describes procedures for obtaining data concerning the adverse effects of an effluent or a test material (added to dilution water, but not to food) on Ceriodaphnia dubia Richard 1894, during continuous exposure throughout a portion of the organism's life. These procedures should also be useful for conducting life cycle toxicity tests with other Cladocera (Guide E1193), although modifications will be necessary.1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, dissolved ions, and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments (see also Guide E1706), and surface waters. Renewal tests might not be applicable to materials that have high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed, or sorb to test chambers. If the concentration of dissolved oxygen falls below 4 mg/L or the concentration of test material decreases by more than 20 % in test solution(s) at any concentration between renewals, more frequent renewals might be necessary.1.3 Other modifications of these procedures might be justified by special needs or circumstances. Results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting three-brood toxicity tests with C. dubia.1.4 This guide is arranged as follows:  Section Referenced Documents 2Terminology 3Summary of Guide 4 5Apparatus 6 Facilities 6.1 Construction Materials 6.2 Test Chambers 6.3 Cleaning 6.4Reagents and Materials 7Hazards 8Dilution Water 9 Requirements 9.1 Source 9.2 Treatment 9.3 Characterization 9.4Test Material 10 General 10.1 Stock Solution 10.2 Effluent 10.3 Test Concentration(s) 10.4 Collection 10.5 Sample Containers 10.6 Preservation 10.7 Treatment 10.8Test Organisms 11 Species 11.1 Age 11.2 Source 11.3 Brood Stock 11.4 Food 11.5 Handling 11.6 Quality 11.7Procedure 12 Demonstration of Feasibility 12.1 Experimental Design 12.2 Dissolved Oxygen 12.3 Temperature 12.4 Preparing Test Solutions 12.5 Conditioning Test Chambers 12.6 Beginning a Test 12.7 Renewing Test Solutions 12.8 Duration of Test 12.9 Biological Data 12.10 Other Measurements 12.11 Test Material 12.12Analytical Methodology 13Acceptability of Test 14Calculation 15Report 16Appendixes   Food Appendix X1 Culture Techniques Appendix X2 Test Chambers Appendix X3 Statistical Guidance Appendix X41.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 8.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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4.1 Application of this guide will provide information on the acute toxicity of water-miscible metalworking fluids and will assist the user in evaluating the potential health hazards of the fluid and developing appropriate work practices. A water-miscible metalworking fluid is a concentrate designed to be diluted in water for use.4.2 Water-miscible metalworking fluids are complex chemical mixtures. The United States Occupational Safety and Health Administration (OSHA) Hazard Communication Standard (see A1.8) outlines procedures for the hazard determination of mixtures and states that if a mixture has not been tested as a whole, then the mixture shall be assumed to present the same hazards as do the components that comprise 1 % (by weight or volume) or greater of the mixture, except that the mixture shall be assumed to present a carcinogenic hazard if it contains a component in concentrations of 0.1 % or greater, which is considered to be a carcinogen (as defined in OSHA Standard 29 CFR 1910.1200). The determination of when to test a mixture as a whole and which toxicity tests are appropriate for the product must be made by a health professional qualified in evaluating toxicological data.4.3 Acute toxicology testing of water-miscible metalworking fluids consists of several individual tests including acute oral, dermal, or inhalation toxicity, eye irritation, skin irritation or corrosion, or both, skin sensitization, and sensory irritation. Certain protocols for acute oral, dermal, and inhalation toxicity tests are limit tests; further multi-dose testing (for example, Test Method E1103) should take place if mortality is noted on any of these tests. The referenced protocols specify the species and number of animals required. Selection of tests conducted should be designed to minimize the number of animals used.4.3.1 Acute Oral Toxicity—Acute oral toxicity tests (see A1.1) provide information on health hazards likely to arise from short-term exposure by the oral route. Results of this type of test are used to develop warning statements on labels as may be required by OSHA Hazard Communication Standard 29 CFR 1910.1200 (see A1.8) or Federal Hazardous Substances Act (see A1.10). These are also used to establish a dosage regimen for subchronic and other testing. Endpoint: mortality.4.3.2 Acute Dermal Toxicity—Acute dermal toxicity tests (see A1.2) provide information on health hazards likely to arise from short-term exposure by the dermal route and may provide initial information on dermal absorption and the mode of toxic action of a substance. In addition, some measure of irritation caused by the fluid may be obtained by observing local tissue damage at the sight of application. Endpoint: mortality.4.3.3 Acute Inhalation Toxicity—Acute inhalation toxicity tests give an indication of relative toxicity (see A1.3). The results provide an indication of the potential of the fluid to cause death and other adverse health effects when inhaled for a specified time period. Endpoint: mortality.4.3.4 Eye Irritation—Eye irritation tests provide an indication of the potential of the fluid to cause eye irritation or damage upon direct contact (see A1.4). An irritant is defined as a chemical that is not corrosive, but causes a reversible inflammatory effect on living tissue by chemical action at the site of contact. Endpoint: degree of irritation.4.3.5 Skin Irritation or Corrosion—Skin irritation or corrosion tests indicate the potential of the fluid to produce irritation or damage to skin (see A1.5). A corrosive chemical is one that causes visible destruction of, or irreversible alterations in, living tissue by chemical action at the site of contact. Endpoint: irritation or corrosion.4.3.6 Skin Sensitization—A chemical sensitizer is a material that causes a substantial proportion of exposed people or animals to develop an allergic reaction in normal tissue after repeated exposure to the chemical. A number of methods are available for measuring skin sensitization, however, there are differences in opinion on the most appropriate method. These are due to variations in compound administration and degree of reaction to a sensitizing substance. Refer to the Code of Federal Regulations (CFR) for the various protocols (see A1.6). Additionally, toxicology testing contract labs may have standard procedures for conducting these assays. Endpoint: sensitization.4.3.7 Sensory Irritation—Upon exposure to a sensory irritant, humans experience discomfort or a burning sensation of the eyes, nose, and throat, and may also cough. Test Method E981 (see A1.2.5) provides a means to evaluate the sensory irritant potential of airborne chemicals and mixtures, as well as a means to assess the comparative irritancy of compounds and formulations. However, this test method cannot be used to evaluate the relative obnoxiousness of odors. End point: upper respiratory tract irritation.4.4 A number of federal guidelines can be used to establish general procedures for testing acute toxicity of metalworking fluids. Several references are cited in Annex A1. Regardless of the method used, Good Laboratory Practices, as outlined by the United States Environmental Protection Agency (EPA 40 CFR 792) (see A1.9) must be followed. The OSHA Hazard Communication Standard (see A1.8) outlines the responsibilities of chemical manufacturers, importers, and employers in the determination of chemical hazards and communication of information on those hazards.4.5 The methods referenced in this guide, or appropriate alternate methods such as those suggested by the Organization for Economic Cooperation and Development (OECD), are acceptable for testing the acute toxicity of water-miscible metalworking fluids. For each test outlined in A1.1 – A1.5, a table is included that highlights the similarities and differences between the test protocols.1.1 This guide defines acute animal toxicity tests and sets forth the references for procedures to assess the acute toxicity of water-miscible metalworking fluids as manufactured.1.2 Although water-miscible metalworking fluids are typically used at high dilution, dilution rates vary widely. Additionally, there is potential for exposure to the metalworking fluid as manufactured.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This practice provides a means of measuring the susceptibility of an avian species to a test substance in its diet under controlled conditions. The LC50 obtained in this test is a conditional measure of subacute toxicity because consumption is voluntary, and because the dietary route may introduce metabolic transformations of the test substance that might be absent in other exposure techniques.5.2 Use of this practice contributes to the evaluation of the hazards of chemicals to birds because exposure is analogous to most field exposures, that is, through dietary intake.5.3 The use of this practice allows for observation of signs of toxicity in addition to mortality.5.4 The dose-response curve provides additional information about the response of birds to a test substance.5.5 This practice can be used to study the effects of test substances in combination in order to simulate situations where birds may be exposed to more than one substance simultaneously (1).35.6 This practice provides one basis for deciding whether additional toxicity testing should be conducted with birds.1.1 This practice describes a procedure for determining the subacute dietary toxicity of a test substance administered to birds in their daily diet. The LC50 value time to mortality and slope of the dose response curve may also be derived.1.2 This practice is applicable to substances that can be mixed uniformly into the diet.1.3 This practice is intended primarily to be used with the young of the following species: northern bobwhite (Colinus virginianus), Japanese quail (Coturnix japonica), mallard (Anas platyrhynchos), and ring-necked pheasant (Phasianus colchicus). Other species or age groups, for example, with wild-trapped birds, may be used with appropriate husbandry modifications to the practice.1.4 This standard is used routinely to address avian regulatory testing requirements. Modifications to the procedures described in this standard have been proposed and are being evaluated to better address the needs of the latest risk assessment procedures. Specifically, the latest procedures call for individual bird feed consumption measurements so that a more precise dose can be determined. While such procedures may replace procedures described in the current standard, there is no certainty that the newest procedures will work as anticipated, and validation is not complete. Therefore, the current guideline has utility prior to validation and acceptance of a modified standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For specific precautionary statements see Section 6.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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4.1 The use of statistical analysis will enable the investigator to make better, more informed decisions when using the information derived from the analyses.4.1.1 The goals when performing statistical analyses, are to summarize, display, quantify, and provide objective measures for assessing the relationships and anomalies in data. Statistical analyses also involve fitting a model to the data and making inferences from the model. The type of data dictates the type of model to be used. Statistical analysis provides the means to test differences between control and treatment groups (one form of hypothesis testing), as well as the means to describe the relationship between the level of treatment and the measured responses (concentration effect curves), or to quantify the degree of uncertainty in the end-point estimates derived from the data.4.1.2 The goals of this practice are to identify and describe commonly used statistical procedures for toxicity tests. Fig. 1, Section 6, following statistical methods (Section 5), presents a flow chart and some recommended analysis paths, with references. From this guideline, it is recommended that each investigator develop a statistical analysis protocol specific to his test results. The flow chart, along with the rest of this guideline, may provide both useful direction, and service as a quality assurance tool, to help ensure that important steps in the analysis are not overlooked.FIG. 1  Flow Chart for Practice for Statistical AnalysisFIG. 1  Flow Chart for Practice for Statistical Analysis (continued)FIG. 1  Flow Chart for Practice for Statistical Analysis (continued)FIG. 1  Flow Chart for Practice for Statistical Analysis (continued)1.1 This practice covers guidance for the statistical analysis of laboratory data on the toxicity of chemicals or mixtures of chemicals to aquatic or terrestrial plants and animals. This practice applies only to the analysis of the data, after the test has been completed. All design concerns, such as the statement of the null hypothesis and its alternative, the choice of alpha and beta risks, the identification of experimental units, possible pseudo replication, randomization techniques, and the execution of the test are beyond the scope of this practice. This practice is not a textbook, nor does it replace consultation with a statistician. It assumes that the investigator recognizes the structure of his experimental design, has identified the experimental units that were used, and understands how the test was conducted. Given this information, the proper statistical analyses can be determined for the data.1.1.1 Recognizing that statistics is a profession in which research continues in order to improve methods for performing the analysis of scientific data, the use of statistical methods other than those described in this practice is acceptable as long as they are properly documented and scientifically defensible. Additional annexes may be developed in the future to reflect comments and needs identified by users, such as more detailed discussion of probit and logistic regression models, or statistical methods for dose response and risk assessment.1.2 The sections of this guide appear as follows:Title      Section Referenced Documents 2Terminology 3 4Statistical Methods 5Flow Chart 6Flow Chart Comments 7Keywords 8References  1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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4.1 This practice is intended to help assess the biocompatibility of materials used in medical devices. It is an acute toxicological test designed to detect the presence of injurious leachable substances.4.2 This practice may not be appropriate for all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.4.3 The only limitation applicable is the extract preparation. Refer to Sections 4.3 and 4.4 of Practice F619 for a description of this limitation.1.1 This practice covers a nonspecific, acute toxicity test used for detecting leachables from materials used in medical devices.1.2 The liquids injected into the mouse are those obtained by Practice F619 where the extraction vehicles are saline, vegetable oil, or other liquids simulating human body fluids.1.3 Two procedures are outlined: Method A for intravenous injection and Method B for intraperitoneal injection.1.4 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 The test procedure covered in this guide is not intended to simulate exactly the exposure of benthic polychaetes to chemicals under natural conditions, but rather to provide a conveniently rapid, standard toxicity test procedure yielding a reasonably sensitive indication of the toxicity of materials in marine and estuarine sediments.5.2 The protection of a community of organisms requires averting detrimental contaminant-related effects on the number and health of individuals and species within that population. Sediment toxicity tests provide information on the toxicity of test materials in sediments. Theoretically, projection of the most sensitive species within a community will protect the community as a whole.5.3 Polychaetes are an important component of the benthic community. They are preyed upon by many species of fish, birds, and larger invertebrate species, and they are predators of smaller invertebrates, larval stages of invertebrates, and, in some cases, algae, as well as organic material associated with sediment. Polychaetes are sensitive to both organic and inorganic chemicals (1, 2).5 The ecological importance of polychaetes, their wide geographical distribution and ability to be cultured in the laboratory, and sensitivity to chemicals, make them appropriate toxicity test organisms.5.4 An acute or 10-day toxicity test is conducted to obtain information concerning the immediate effects to a test material on a test organism under specified experimental conditions for a short period of time. An acute toxicity test does not necessarily provide information concerning whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, could provide such information.5.5 The results of acute sediment toxicity tests can be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible and (2) toxicity to benthic organisms can be dependent on sediment characteristics, the dynamics of equilibrium partitioning, and the route of exposure to the benthic organisms.5.6 The polychaete sediment toxicity test might be used to determine the temporal or spatial distribution of sediment toxicity. Test methods can be used to detect horizontal and vertical gradients to toxicity. Mortality data can be used to indicate the relative toxicity of field-collected sediments.5.7 The results of acute tests with toxicants added experimentally to sediments can be used to compare the acute sensitivities of different species and acute toxicities of different test materials, and to define the effects of various environmental factors on the results of such tests.5.8 The results of acute sediment toxicity tests are useful for studying the biological availability of, and structure-activity relationships between, test materials in sediment.5.9 The results of acute sediment toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023) or when deriving the sediment quality for aquatic organisms (3). Sediment toxicity tests might be useful for making decisions regarding the extent of remedial action necessary for contaminated sites.5.10 A 10-day test provides data on the short-term effects that are useful for comparisons to other species but does not provide information on delayed effects. Results of the 20-day to 28-day sediment toxicity test, which measures growth in addition to survival, can be useful indicators of the effects of contaminated sediments over a longer time period.1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of potentially contaminated sediment, or of a test material added experimentally to contaminated or uncontaminated sediment, on marine or estuarine infaunal polychaetes during 10-day or 20 to 28-day exposures. These procedures are useful for testing the effects of various geochemical characteristics of sediments on marine and estuarine polychaetes and could be used to assess sediment toxicity to other infaunal taxa, although modifications of the procedures appropriate to the test species might be necessary. Procedures for the 10-day static test are described for Neanthes arenaceodentata and Alitta virens 2 (formerly Nereis virens and Neanthes virens) and for the 20 to 28-day static-renewal sediment toxicity for N. arenaceodentata.1.2 Modifications of these procedures might be appropriate for other sediment toxicity test procedures, such as flow-through or partial life-cycle tests. The methods outlined in this guide should also be useful for conducting sediment toxicity tests with other aquatic taxa, although modifications might be necessary. Other test organisms might include other species of polychaetes, crustaceans, and bivalves.1.3 Other modifications of these procedures might be appropriate for special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using unusual procedures are not likely to be comparable to those of many other tests. Comparisons of the results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting sediment tests with infaunal organisms.1.4 These procedures are applicable to sediments contaminated with most chemicals, either individually or in formulations, commercial products, and known or unknown mixtures. These procedures can be used with appropriate modifications to conduct sediment toxicity tests on factors such as temperature, salinity, dissolved oxygen (DO), and natural sediment characteristics (for example, particle size distribution, organic carbon content, and total solids). These procedures can also be used to conduct bioconcentration tests and in situ tests, and to assess the toxicity of potentially contaminated field sediments, or of materials such as sewage sludge, oils, particulate matter, and solutions of toxicants added to sediments. A median lethal concentration (LC50) or median sublethal effect concentration (EC50) of toxicants or of highly contaminated sediment mixed into uncontaminated sediment can be determined. Materials adhering to sediment particles or dissolved in interstitial water can be tested.1.5 The results of 10-day toxicity tests with contaminated sediments can be reported as a LC50 if a series of concentrations is tested or as a percent mortality relative to a control or reference sediment. The results of 20 to 28-day toxicity tests with contaminated sediments can be reported as a LC50 if a series of concentrations is tested or as a percent mortality or growth relative to a control or reference sediment.1.6 This guide is arranged as follows:  SectionReferenced Documents  2Terminology  3Summary of Guide  4  5Interferences  6Apparatus  7 Facilities  7.1 Construction Materials  7.2 Test Chambers  7.3 Cleaning  7.4 Acceptability  7.5Hazards  8Test Water  9 General Requirements  9.1 Source  9.2 Preparation  9.3 Characterization  9.4Test and Control Sediments 10 General 10.1 Characterization 10.2 Control Sediment 10.3 Field-Collected Test Sediment 10.4 Reference Sediment 10.5 Laboratory-Spiked Test Sediment 10.6 Test Concentration(s) 10.7 Addition of Toxicant to Sediment 10.8Test Organisms 11  Species 11.1  Age 11.2  Feeding 11.3  Source 11.4  Collection and Handling 11.5  Quality 11.6 Experimental Design 12  Controls 12.2  Field Survey Design 12.3  Laboratory Experiments 12.4 Procedure 13  Dissolved Oxygen 13.1  Temperature 13.2  Salinity 13.3  Light 13.4  Feeding 13.5  Beginning of Test 13.6  Duration of Test 13.7  Biological Data 13.8  Other Measurements 13.9 Analytical Methodology 14 Acceptability of Test 15 Interpretation of Results 16 Report 17 Keywords 18 Annexes    Neanthes arenaceodentata Annex A1  Alitta virens Annex A21.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazards statements are given in Section 8.1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This test method should generate data to identify the majority of chronic effects and shall serve to define long-term dose response relationships. In addition the test should allow for the detection of general toxic effects including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.5.2 This test method should provide information on target organs, the possibilities of accumulation, and may be used for establishing safety criteria for human exposure. It provides information on potential health hazards likely to arise from repeated exposure over a long period of time.1.1 This test method covers a long-term study to determine the effects of a substance in a mammalian species such as the rat following prolonged and repeated oral exposure. Under the conditions of the chronic toxicity test, effects that require a long latency period or that are cumulative should become manifest.1.2 This test method assumes that the user is knowledgeable in mammalian toxicology and related pertinent areas, and relies heavily on the judgment of the evaluator.1.3 The values stated in SI units are to be regarded as the standard. The inch-pound units given in parentheses are for information only.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific hazard statements, see Section 6.

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This specification covers performance requirements for biodegradable hydraulic fluids with low aquatic toxicity used in industrial/mobile hydraulic applications. There are some cases where biodegradable fluids have been found to perform differently than traditional mineral oils, which makes separate performance requirements desirable.1.1 This specification covers performance requirements for biodegradable hydraulic fluids with low aquatic toxicity used in industrial/mobile hydraulic applications.1.2 In some cases, biodegradable fluids have been found to perform differently than traditional mineral oils, thus separate performance requirements are desirable.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. Toxicity tests can be used provide information about the toxicity of a test material to a specific life stage of a particular species of mussel. The primary adverse effects studied are reduced survival or growth. 5.2 Results of toxicity tests might be used to predict effects likely to occur on mussels in field situations as a result of an exposure under comparable conditions. 5.3 Results of toxicity tests might be used to compare the sensitivities of different mussel species and the toxicity of different test materials, and to study the effects of various environmental factors on results of such tests. 5.4 Results of toxicity tests conducted with mussels might be an important consideration when assessing the risks of test materials to aquatic organisms or when deriving environmental guideline values for toxicants. 5.5 An acute toxicity test is conducted to obtain information concerning the immediate effects on mussels of a short exposure to a test material under specific experimental conditions. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, might provide such information (Guide E729). 5.6 Results of chronic (at least 28 d) toxicity tests with mussels might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions. 5.7 Short-term chronic toxicity tests are conducted for 7 d, a complementary test duration in the USEPA shot-term methods for estimating the chronic toxicity of effluents and receiving waters to fathead minnow (Pimephales promelas; USEPA 2002) (31) and provides a more direct estimate of the safe concentrations of effluents and receiving waters than acute toxicity tests, at a slightly lower level of effort compared to chronic 28 d toxicity test. 5.8 Results of toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials. 5.9 Results of toxicity tests will depend on temperature, composition of the dilution water, condition of the test organisms, and other factors. 5.10 Interferences—A number of factors can impede or prevent selection and use of freshwater mussels for toxicity testing (Guide E1850). The following should be considered when selecting a test species and measuring the sensitivity of the test species during toxicity tests. 5.10.1 Handling of field-collected adult mussels resulting from collection or transport to the laboratory might cause excessive mortality or sublethal effects. 5.10.2 The age, health, and physical condition of adult mussels (for example, the presence of parasites, bacteria, and disease) collected from a resident population might not be adequately known. 5.10.3 The physical characteristics of the testing environment (such as water quality, temperature, water flow, light) and food requirements might affect the ability of the test organisms to acclimate, recover from handling, or adapt to the laboratory environment conditions. 5.10.4 The degree of contamination and the history of contamination at the collection of the adult mussels might not be adequately known. 5.10.5 In the field, mussels may be exposed to contaminants in water, sediment, or food. This standard only addresses effects associated with exposure of mussels to contaminants in water. Methods for conducting sediment toxicity tests with juvenile mussels are included in Guide E1706. 5.10.6 There are insufficient data available to determine if juvenile mussels are able to avoid exposure to chemicals by valve closure. If it is suspected that juvenile mussels are avoiding exposure to a chemical in a toxicity test, it may be desirable to place the suspected live test organisms into dilution water that does not contain any added test material for 1 d to 2 d after the end of the toxicity test to determine whether these test organisms are alive or dead (section A1.4.7; Guide E729). 1.1 This standard guide describes methods for conducting laboratory toxicity tests with early life stages of freshwater mussels including glochidia and juvenile mussels in water-only and effluent exposures (Annex A1). Future revisions to this standard may describe methods for conducting toxicity tests with endpoints of reproduction, behaviors, and biomarkers. 1.2 Freshwater mussels (order Unionida) are one of the most imperiled groups of animals in the world, and environmental contamination has been linked as a contributing factor to the decline of mussel populations (Lydeard et al. 2004 (1); Strayer et al. 2004 (2); Haag 2012 (3); Lopes-Lima et al. 2017 (4)).2 Three critical life stages (glochidia, juvenile mussels, and adults) have been used in toxicity assessments and the toxicity studies are separated according to the medium of exposure (water, sediment, and host fish (Ingersoll et al. 2007 (5)). Recent studies on early life stages of mussels have demonstrated that the mussels are among the most sensitive freshwater species to a variety of contaminants, including ammonia, some metals (for example, aluminum, copper, nickel, and zinc), and major ions (for example, chloride, nitrate, potassium, and sulfate) (Bringolf et al. 2007 (6); Newton et al. 2007 (7); Wang et al. 2007ab, 2010, 2011ab, 2016, 2017ab, 2018abc, 2020ab (8-20); Cope et al. 2008 (21); Gillis et al. 2008, 2010, 2011, 2021 (22-25); Miao et al. 2010 (26); Salerno et al. 2020 (27)). These studies indicate that environmental guideline values for individual chemicals established for the protection of aquatic organisms may not be adequately protective of sensitive stages of freshwater mussels. For example, when freshwater mussel toxicity data were included in an update to the United States Environmental Protection Agency (USEPA) ambient water quality criteria (WQC) for ammonia, the acute criterion decreased by about a 1.4 fold and the chronic criterion decreased by 2.4 fold (USEPA 2013) (28). 1.3 Summary of Life History of Freshwater Mussels:  1.3.1 Freshwater mussels are bivalve mollusks belonging to the taxonomic Order Unionida (section 10.1). Like most bivalves, mussels are totally aquatic, relatively sedentary, filter-feeding animals, and spend most of their lives partially or completely burrowed in the substrate of streams, rivers, or lakes. Freshwater mussels have an unusual and complex life cycle that includes a larval stage, the glochidium, that is briefly parasitic on fish (Fig. 1). 1.6 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only. 1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7. 1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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6.1 The primary goal of this practice is to extract representative samples from PV modules for TCLP toxicity testing purposes in order to receive unbiased, comparable and repeatable toxicity test results from independent TCLP testing laboratories.6.2 Solar photovoltaic (PV) modules in the United States and the world reaching end-of-life due to failure, underperformance or breakage due to extreme weather have to be recycled or otherwise safely disposed of following the Resource Conservation and Recovery Act (RCRA) regulation [United States, Resource Conservation and Recovery Act. Pub.L. 94–580, October 1976]. For end-of-life PV modules, the U.S. Environmental Protection Agency (EPA) Method 1311 (TCLP) is used for waste characterization based on leaching potential under simulated landfill conditions.6.3 Commercial PV modules contain compounds and alloys of various metals (for example, Ag, Al, Cd, Cu, Ga, In, Ni, Pb, Se, Sn, Te, Zn) which are used in semiconductor compounds and electrical contacts.5 Modules that pass the EPA Method 1311 TCLP test, and state protocols (if applicable), can be disposed of in a regular landfill. Otherwise, they are classified as hazardous waste and must go through a more onerous and expensive disposal process. Currently, there is no national or international standard, nor a standardized protocol available for removal of test samples from PV modules for toxicity testing per the EPA Method 1311 standard.6.4 The validity of the toxicity test results heavily depends on the location of extracted samples in the module, specifically within the laminate area, and the particle size of the extracted samples. Therefore, it is critical that the sample extraction procedure be properly designed to avoid biased or otherwise inaccurate toxicity test results.6.5 The development and application of a homogeneous and representative sampling standard will help utilities and manufacturers to limit the number of variables and to obtain repeatable test results.1.1 The purpose of this practice is to describe a representative and repeatable sample preparation methodology to conduct toxicity testing on solar photovoltaic (PV) modules for use with EPA Test Method 1311: Toxicity Characteristic Leaching Procedure (TCLP).1.2 This practice refers to the extraction and preparation of PV module samples by EPA Method 1311, the testing for eight (8) distinct metals – mercury (by Method 7470A), arsenic, barium, cadmium, chromium, lead, selenium and silver (by Method 6010C) as well as the analysis and interpretation of the test results on a module level.1.3 This practice applies to only (1) standard crystalline silicon (c-Si) modules, multi and mono-crystalline silicon with aluminum back surface field (Al-BSF) cell technology and (2) cadmium telluride (CdTe) PV modules.1.4 Other and newer PV technologies and module architectures, for example, passivated emitter and rear cell (PERC), interdigitated back contact (IBC), hetero-junction technology (HJT), multiwire, half cut, shingled etc., have not been evaluated with this practice, although the concept and practice can be easily extended and applied to other technologies following the conceptual approach presented in this document.1.5 The sample extraction/removal methodology applied in this practice is the waterjet cutting sampling method. Sample extraction with mechanical cutting has been extensively evaluated but the variability of TCLP test results based on the mechanical cut samples tend to be much higher (30 %) than that of the waterjet cut samples (8 %).2 Therefore, the mechanical cut method is not presented in this practice.1.6 Only the laminate area of the PV module is considered for TCLP testing, as other possible module parts, such as aluminum frame, junction box and cables contain recyclable materials that are already well-documented and are not specific to the PV modules.1.7 The material gravimetric density (g/cm3) throughout the laminate area is considered constant.1.8 This practice was developed to be consistent with three fundamental requirements:1.8.1 Sample pieces with particle size not to exceed the allowed size limit of EPA 1311 standard which is 9.5 mm,1.8.2 The particle size used in this practice as sample piece is consistent with the median particle size expected in landfill disposal2, and1.8.3 An assumption that each laminate sample piece will result in 100 % glass coverage area, due to the presence of bonding encapsulant layers once it is broken in the landfill.1.9 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.10 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.11 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Waste samples collected using this practice provide representative samples for analysis in a laboratory using the TCLP.5.2 The TCLP is used to simulate the transfer of lead from buried lead-containing waste into the ground water system upon codisposal of the lead-containing waste and municipal solid waste in unlined solid-waste landfills. The TCLP attempts to simulate rain or ground water leaching, or both. For the procedure to yield a predictor of the subsurface (in-ground) leaching process, a representative sample of the volume of the waste must be selected and submitted for leaching and analysis. The result of the sampling, leaching, and analysis process is used to determine the waste handling and disposal protocols to be followed and to document compliance with applicable laws, regulations, and requirements. This practice addresses the sampling process by defining a component-volume-based method to collect and assemble a representative sample of a solid waste stream that may contain heterogeneous components.5.3 The collection of a volume-based sample of the waste stream is based on the fact that the TCLP leachate lead concentration limit, like other such TCLP limits, was developed based on the spatial dimensions of landfills.5.4 Individuals who use this practice are expected to be trained in the proper and safe conduct of sampling of lead-containing wastes, qualified/certified/licensed as required by those authorities having jurisdiction over such activities, and properly utilize tools and safety equipment when conducting these procedures.5.5 This practice may involve use of various hand and power tools for sampling the components of the waste. It is intended that such tools should be properly and safely used by persons trained and familiar with their performance and use.5.6 In general terms, building components are drilled, sawed, snipped, etc., to collect samples of the various components in proportion to the volume of those components in the entire building. The component samples are assembled, and the resulting assembled sample is analyzed according to the TCLP protocol.1.1 This practice describes a method for selecting samples of building components coated with paints suspected of containing lead. The samples are collected from the debris waste stream created during demolition, renovation, lead hazard control, or abatement projects. The samples are subsequently analyzed in the laboratory for lead.1.1.1 The debris waste stream is assumed to have more than one painted component, for example, metal doors, wood doors, and wood window trim.1.2 This practice is intended for use when sampling to test for lead only and does not include sampling considerations for other metals or for organic compounds. This practice also does not include consideration of sampling for determination of other possible hazardous characteristics of the waste.1.3 This practice assumes that the individual component types comprising the debris waste stream are at least partially segregated and that the volume of each type of component in the debris waste stream may be estimated.1.4 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are provided for information only and are not considered standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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