E50.47 - Biological Effects and Environmental Fate

SIGNIFICANCE AND USE
5.1 HACCP is a proactive management tool that serves to reduce hazards potentially expressed as adverse biological or environmental effects, for example, associated with chemical releases, changes in natural resource or engineering practices and their related impacts, and accidental or intentional releases of biological stressors such as invasive species.  
5.2 Sequential implementation of HACCP and feedback in the iterative HACCP process allows for technically-based judgments concerning, for example, natural resources or the use of natural resources. Implementing the HACCP process serves to reduce adverse effects potentially associated with a particular material or process, and provides guidance for testing and evaluation of products or processes, through a pre-emptive procedure focused on information most pertinent to a system’s characterization. For example, identification of CCPs assure that processes and practices can be managed to achieve hazard reduction. For different processes and situations, HA may be based on substantially different amounts and kinds of, for example, biological, chemical, physical, and toxicological data, but the identification of CCPs serving to reduce hazard is key to successful implementation of HACCP.  
5.3 HACCP should never be considered complete for all time, and continuing reassessment is a characteristic of HACCP evaluations, especially if there should be changes in, for example, production volumes of a material, or its use or disposal increases, new uses are discovered, or new information on biological, chemical, physical, or toxicological properties becomes available. Similarly, HACCP should be considered an ongoing process serving as a key component in engineering practices, for example, related to construction activities and land-use changes, and natural resource management practices, for example, related to habitat use, enhancement, and species introductions such as fish-stocking programs. Periodic review of a system’s per...
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1.1 This guide describes a stepwise procedure for using existing information, and if available, supporting field and laboratory data concerning a process, materials, or products potentially linked to adverse effects likely to occur in the environment as a result of an event associated with a process such as the dispersal of a potentially invasive species or the release of material (for example, a chemical or a physical substance) or its derivative products to the environment. Hazard Analysis-Critical Control Point (HACCP) evaluations were historically linked to food safety (Hulebak and Schlosser W. 2002 (1);2 Mortimer and Wallace 2013 (2)), but the process has increasingly found application in planning processes such as those occurring in health sciences ; Quattrin et al. 2008 (3); Hjarno et al. 2007 (4); Griffith 2006 (5) or; Noordhuizen and Welpelo 1996 (6)), in natural resource management (US Forest Service 2014 a,b,c (7, 8, 9), (US EPA, 2006 (10); see also    
http://www.waterboards.ca.gov/water_issues/programs/swamp/ais/prevention_planning.shtml; (last accessed October 16, 2023)
or in supporting field operations wherein worker health and natural resource management issues intersect.  
1.2 HACCP evaluation is a simple linear process or a network of linear processes that represents the structure of any event; the hazard analysis (HA) depends on the data quality and data quantity available for the evaluation process, especially as that relates to critical control points (CCPs) characterized in completing HACCP. Control measures target CCPs and serve as limiting factors or control steps in a process that reduce or eliminate the hazards that initiated the HACCP evaluation. The main reason for implementing HACCP is to prevent problems associated with a specific process, practice, material, or product.  
1.3 This guide assumes that the reader is knowledgeable in specific resource management or engineering practices us...

SIGNIFICANCE AND USE
5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks may be introduced in many ways, directly by genotoxic compounds, through the induction of apoptosis or necrosis, secondarily through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes (20-22). In addition to a linkage with cancer, studies have demonstrated that increases in cellular DNA damage precede or correspond with reduced growth, abnormal development, and reduced survival of adults, embryos, and larvae (16, 23, 24).  
5.1.1 The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells  (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay ((2, 27) .  
5.1.2 These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained (28). Only fluorescent staining methods will be described in this guide. The microscopic determination of DNA migration can be made either by eye using an ocular micrometer or with the use of image analysis software. Scoring by eye can be performed using a calibrated ocular...
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1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.  
1.1.1 The Comet assay has been used to screen the genotoxicity of a variety of compounds on cells in vitro and in vivo (5-7), as well as to evaluate the dose-dependent anti-oxidant (protective) properties of various compounds (3, 8-11). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).  
1.2 This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair...

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide a logical, tiered approach in the development of environmental health criteria coincident with level and effort in the research, development, testing, and evaluation of new materials for military use. Various levels of uncertainty are associated with data collected from previous stages. Following the recommendation in the guide should reduce the relative uncertainty of the data collected at each developmental stage. At each stage, a general weight of evidence qualifier shall accompany each exposure/effect relationship. They may be simple (for example, low, medium, or high confidence) or sophisticated using a numerical value for each predictor as a multiplier to ascertain relative confidence in each step of risk characterization. The specific method used will depend on the stage of development, quantity and availability of data, variation in the measurement, and general knowledge of the dataset. Since specific formulations, conditions, and use scenarios may not be known until the later stages, exposure estimates can be determined when practical (for example, Engineering and Manufacturing Development; see 6.6). Exposure data can then be used with other toxicological data collected from previous stages in a quantitative risk assessment to determine the relative degree of hazard.  
5.2 Data developed from the use of this guide are designed to be consistent with criteria required in weapons and weapons system development (for example, programmatic environment, safety and occupational health evaluations, environmental assessments/environmental impact statements, toxicity clearances, and technical data sheets).  
5.3 Information shall be evaluated in a flexible manner consistent with the needs of the authorizing program. This requires proper characterization of the current problem. For example, compounds may be ranked relative to the environmental criteria of the prospective alternatives, the replacement compound, and within bo...
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1.1 This guide is intended to determine the relative environmental influence of new substances, consistent with the research and development (R&D) level of effort and is intended to be applied in a logical, tiered manner that parallels both the available funding and the stage of research, development, testing, and evaluation. Specifically, conservative assumptions, relationships, and models are recommended early in the research stage, and as the technology is matured, empirical data will be developed and used. Munition constituents are included and may include propellants, oxidizers, explosives, binders, stabilizers, metals, dyes, and other compounds used in the formulation to produce a desired effect. Munition systems range from projectiles, grenades, rockets/missiles, training simulators, to smokes and obscurants. Given the complexity of issues involved in the assessment of environmental fate and effects and the diversity of the systems used, this guide is broad in scope and not intended to address every factor that may be important in an environmental context. Rather, it is intended to reduce uncertainty at minimal cost by considering the most important factors related to human health and environmental impacts of energetic materials. This guide provides an outline for collecting data useful in a relative ranking procedure to provide the systems scientist with a sound basis for prospectively determining a selection of candidates based on environmental and human health criteria. The general principles in this guide are applicable to substances other than energetics if intended to be used in a similar manner with similar exposure profiles.  
1.2 The scope of this guide includes:  
1.2.1 Energetic and other new/novel materials and compositions in all stages of research, development, test and evaluation.  
1.2.2 Environmental assessment, including:
1.2.2.1 Human and ecological effects of the unexploded energetics and ...

SIGNIFICANCE AND USE
5.1 An important goal of aquatic toxicology is to determine the effects of toxic compounds on species that play a central role in aquatic communities. Rotifers have a major impact on several important ecological processes in freshwater and coastal marine environments. As filter-feeders on phytoplankton and bacteria, rotifers exert substantial grazing pressure that at times exceeds that of the larger crustacean zooplankton  (1, 2).4 Rotifer grazing on phytoplankton is highly selective (2-4) and can influence phytoplankton composition, the coexistence of competitors, and overall water quality (5). The contribution of rotifers to the secondary production of many aquatic communities is substantial (6-9). In fresh water, rotifers often account for the major fraction of zooplankton biomass at certain times of the year (10, 11) . Rotifers and other zooplankton are a significant food source for many larval fish, planktivorous adult fish (12, 13), and several invertebrate predators (14-16). The high metabolic rates of rotifers contribute to their role in nutrient cycling, which might make rotifers more important than crustaceans in certain communities (17, 18).  
5.2 In addition to their important ecological role in aquatic communities, rotifers are attractive organisms for toxicological studies because an extensive database exists on the basic biology of this group. Techniques have been published for the culture of many rotifer species (3, 19). The rotifer life cycle is well defined (20, 21), and the factors regulating it are reasonably well understood (22-25). Several aspects of rotifer behavior have been examined closely (26-29). The biogeography of many rotifer species has been characterized (30, 31), and the systematics of the group are well described (32, 33).  
5.3 Toxicity tests with rotifers of the genus Brachionus are more easily performed than with many other aquatic animals because of their rapid reproduction, short generation times, sensitivity (34), and the c...
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1.1 This guide describes procedures for obtaining laboratory data concerning the acute toxicity of chemicals and aqueous effluents released into fresh, estuarine or marine waters. Acute toxicity is measured by exposing  Brachionus  newly hatched from cysts to a series of toxicant concentrations under controlled conditions. This guide describes a test for using  B. calyciflorus, a freshwater rotifer, and the Appendix describes modifications of this test for estuarine and marine waters using  B. plicatilis. These procedures lead to an estimation of acute toxicity, including the concentration expected to kill 50 % of the test rotifers (LC50) in 24 h. Procedures not specifically stated in this guide should be conducted in accordance with Guide E729 and Guide E1192.  
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 modified procedures might not be comparable to rotifer acute tests that follow the protocol described here. Comparison of the results using modified procedures might provide useful information concerning new concepts and procedures for conducting acute toxicity tests on chemicals and aqueous effluents.  
1.3 This guide is organized as follows:    
Section    
Scope  
1    
Referenced Documents  
2    
Terminology  
3    
Summary of Guide  
4    
Significance and Use  
5    
Apparatus  
6    
Dilution Water  
7    
Hazards  
8    
Test Material  
9    
Test Organisms  
10    
Test Procedure  
11    
Calculation of Results  
12    
Acceptability of the Test  
13    
Report  
14    
Keywords  
15  
1.4 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or mixtures. This guide can also be used to investigate the effects on rotifer su...

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1.1 This terminology document defines terms commonly used in standards developed by ASTM Subcommittee E50.47 on Biological Effects and Environmental Fate. This terminology document is intended to be consistent with the use of terms in ASTM standards related to this field and, to the extent possible, with use by other organizations.  
1.1.1 If a specific Subcommittee E50.47 standard uses one of these terms in a different context, then the term should be defined in that standard. A term used only in a specific ASTM standard need not be included in this terminology document.  
1.2 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.

SIGNIFICANCE AND USE
5.1 A microcosm test is conducted to obtain information concerning toxicity or other effects of a test material on the interactions among three trophic levels (primary, secondary, and detrital) and the competitive interactions within each trophic level. As with most natural aquatic ecosystems, the microcosms depend upon algal production (primary production) to support the grazer trophic level (secondary production), which along with the microbial community are primarily responsible for the nutrient recycling necessary to sustain primary production. Microcosm initial condition includes some detritus (chitin and cellulose) and additional detritus is produced by the system. The microcosms include ecologically important processes and organisms representative of ponds and lakes, but are non-site specific. To the extent possible, all solutions are mixtures of distilled water and reagent grade chemicals (see Section 8) and all organisms are available in commercial culture collections.  
5.2 The species used are easy to culture in the laboratory and some are routinely used for single species toxicity tests (Guide E729; Practice D3978, Guides E1192 and E1193). Presumably acute toxicity test results with some of these species would be available prior to the decision to undertake the microcosm test. If available, single species toxicity results would aid in distinguishing between indirect and direct effects.  
5.3 These procedures are based mostly on published methods (4-6), interlaboratory testing (7-10, 11), intermediate studies (12-23, 24), statistical studies  (25-27) and mathematical simulation results  (28). Newer studies on jet fuels have been reported (29)(See 15.1 for multivariate statistical analyses) and on the implications of multispecies testing for pesticide registration (30). Environmental Protection Agency, (EPA) and Food and Drug Administration, (FDA) published similar microcosm tests (31). The methods described here were used to determine the criteria for A...
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1.1 This practice covers procedures for obtaining data concerning toxicity and other effects of a test material to a multi-trophic level freshwater community, independent of the location of the test.  
1.2 These procedures also might be useful for studying the fate of test materials and transformation products, although modifications and additional analytical procedures might be necessary.  
1.3 Modification 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting multi-trophic level tests.  
1.4 This practice is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Practice  
4  
Significance and Use  
5  
Apparatus  
6  
Facilities  
6.1  
Container  
6.2  
Equipment  
6.3  
Hazards  
7  
Microcosm Components  
8  
Medium  
8.1  
Medium Preparation  
8.2  
Sediment  
8.3  
Microcosm Assembly  
8.4  
Test Material  
9  
General  
9.1  
Stock Solution  
9.2  
Nutrient Control  
9.3  
Test Organisms  
10  
Algae  
10.1  
Animals  
10.2  
Specificity of Organisms  
10.3  
Sources  
10.4  
Algal Culture Maintenance  
10.5  
Animal Culture Maintenance  
10.6  
Procedure  
11  
Experimental Design  
11.1  
Inoculation  
11.2  
Culling  
11.3  
Addition of Test Material  
11.4  
Measurements  
11.5  
Reinoculations  
11.6  
Analytical Methodology  
12  
Data Processing  
13  
Calculations of Variables from Measurements  
14  
Statistical Analyses ...

SIGNIFICANCE AND USE
5.1 An acute toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term 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. Bioavailability of the test substance may also differ between real-world exposures and laboratory exposures due to site-specific water quality conditions (see Guides E1192, E1563, and E2455).  
5.2 Results of acute toxicity tests might 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 might be dependent on sorption or settling of the test material onto the substrate.  
5.3 Results of acute 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.  
5.4 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 (3).  
5.5 Results of acute toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.  
5.6 Results of acute toxicity tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.
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1.1 This guide (1)2 describes procedures for obtaining laboratory data concerning the adverse effects (for example, lethality and immobility) of a test material added to dilution water, but not to food, on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, usually during 2 to 4-day exposures, depending on the species. These procedures will probably be useful for conducting acute toxicity tests with many other aquatic species, although modifications might be necessary.  
1.2 Other modifications of these procedures might be justified by special needs or circumstances such as meeting specific study goals, regulatory needs, or to accommodate specific test organism life stages. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual or novel procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests.  
1.3 This guide describes tests using three basic exposure techniques: static, renewal, and flow-through. Selection of the technique to use in a specific situation will depend on the needs of the investigator and on available resources. Tests using the static technique provide the most easily obtained measure of acute toxicity, but conditions often change substantially during static tests; therefore, static tests should not last longer than 96 h, and test organisms should not be fed during such tests unless the test organisms are severely stressed without feeding over 48 h. Static tests should probably not be conducted on 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. Because the pH and concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed, tests using ren...

ABSTRACT
This guide covers procedures for obtaining laboratory data concerning the adverse effects of aqueous ambient samples and effluents on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, during a short-term exposure, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests on aqueous effluents with many other aquatic species, although modifications might be necessary. Static tests might not be applicable to effluents that have a high oxygen demand, or contain materials that (1) are highly volatile, (2) are rapidly biologically or chemically transformed in aqueous solutions, or (3) are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Results of acute toxicity tests should usually be reported in terms of a median lethal concentration (LC50) or median effective concentration (EC50). An acute toxicity test does not provide information about whether delayed effects will occur. Specified requirements involving the following are detailed: (1) hazards; (2) apparatus: facilities, special requirements, construction materials, metering system, test chambers, cleaning, and acceptability; (3) dilution water requirements, source, treatment, and characterization; (4) effluent sampling point, collection, preservation, treatment, and test concentrations; (5) test organism species, age, source, care and handling, feeding, disease treatment, holding, acclimation, and quality; (6) procedure: experimental design, dissolved oxygen, temperature, loading, beginning the test, feeding, duration of test, biological data, and other measurements; (7) analytical methodology; (8) acceptability of test; (9) calculation of results; and (1) report of results.
SIGNIFICANCE AND USE
5.1 An acute effluent toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term exposure to an effluent under specific experimental conditions. One can directly examine acute effects of complex mixtures of chemicals as occurs in effluents and some ambient waters. Acute effluent toxicity tests can be used to evaluate the potential for designated-use or aquatic life impairment in the receiving stream, lake, or estuary. 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.  
5.2 Results of acute effluent tests might 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, (2) toxicity to benthic species might be dependent on sorption or settling of components of the effluent onto the substrate, and (3) the effluent might physically or chemically interact with the receiving water.  
5.3 Results of acute effluent tests might be used to compare the acute sensitivities of different species and the acute toxicities of different effluents, and to study the effects of various environmental factors on results of such tests.  
5.4 Acute tests are usually the first step in evaluating the effects of an effluent on aquatic organisms.  
5.5 Results of acute effluent tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.
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1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of an aqueous effluent on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, usually during 2 day to 4 day exposures, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests on ...

SIGNIFICANCE AND USE
5.1 General:  
5.1.1 Sediment provides habitat for many aquatic organisms and is a major repository for many of the more persistent chemicals that are introduced into surface waters. In the aquatic environment, most anthropogenic chemicals and waste materials including toxic organic and inorganic chemicals eventually accumulate in sediment. Mounting evidences exists of environmental degradation in areas where USEPA Water Quality Criteria (WQC; Stephan et al.(66)) are not exceeded, yet organisms in or near sediments are adversely affected Chapman, 1989  (67). The WQC were developed to protect organisms in the water column and were not directed toward protecting organisms in sediment. Concentrations of contaminants in sediment may be several orders of magnitude higher than in the overlying water; however, whole sediment concentrations have not been strongly correlated to bioavailability Burton, 1991 (68). Partitioning or sorption of a compound between water and sediment may depend on many factors including: aqueous solubility, pH, redox, affinity for sediment organic carbon and dissolved organic carbon, grain size of the sediment, sediment mineral constituents (oxides of iron, manganese, and aluminum), and the quantity of acid volatile sulfides in sediment Di Toro et al. 1991(69) Giesy et al. 1988 (70). Although certain chemicals are highly sorbed to sediment, these compounds may still be available to the biota. Chemicals in sediments may be directly toxic to aquatic life or can be a source of chemicals for bioaccumulation in the food chain.  
5.1.2 The objective of a sediment test is to determine whether chemicals in sediment are harmful to or are bioaccumulated by benthic organisms. The tests can be used to measure interactive toxic effects of complex chemical mixtures in sediment. Furthermore, knowledge of specific pathways of interactions among sediments and test organisms is not necessary to conduct the tests Kemp et al. 1988, (71). Sediment tests can be used ...
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1.1 This test method covers procedures for testing estuarine or marine organisms in the laboratory to evaluate the toxicity of contaminants associated with whole sediments. Sediments may be collected from the field or spiked with compounds in the laboratory. General guidance is presented in Sections 1 – 15 for conducting sediment toxicity tests with estuarine or marine amphipods. Specific guidance for conducting 10-d sediment toxicity tests with estuarine or marine amphipods is outlined in Annex A1 and specific guidance for conducting 28-d sediment toxicity tests with  Leptocheirus plumulosus is outlined in Annex A2.  
1.2 Procedures are described for testing estuarine or marine amphipod crustaceans in 10-d laboratory exposures to evaluate the toxicity of contaminants associated with whole sediments (Annex A1; USEPA 1994a (1)). Sediments may be collected from the field or spiked with compounds in the laboratory. A toxicity method is outlined for four species of estuarine or marine sediment-burrowing amphipods found within United States coastal waters. The species are Ampelisca abdita, a marine species that inhabits marine and mesohaline portions of the Atlantic coast, the Gulf of Mexico, and San Francisco Bay; Eohaustorius estuarius, a Pacific coast estuarine species; Leptocheirus plumulosus, an Atlantic coast estuarine species; and Rhepoxynius abronius , a Pacific coast marine species. Generally, the method described may be applied to all four species, although acclimation procedures and some test conditions (that is, temperature and salinity) will be species-specific (Sections 12 and Annex A1). The toxicity test is conducted in 1-L glass chambers containing 175 mL of sediment and 775 mL of overlying seawater. Exposure is static (that is, water is not renewed), and the animals are not fed over the 10-d exposure period. The endpoint in the toxicity test is survival with reburial of surviving amphipods as an additional m...

SIGNIFICANCE AND USE
5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. A life-cycle toxicity test is conducted to determine what changes in the numbers and weights of individuals of the test species result from effects of the test material on survival, growth, and reproduction. Information might also be obtained on effects of the material on the health and uses of the species.  
5.2 Results of life-cycle tests with mysids might be used to predict long-term effects likely to occur on mysids in field situations as a result of exposure under comparable conditions.  
5.3 Results of life-cycle tests with mysids might be used to compare the chronic sensitivities of different species and the chronic toxicities of different materials, and also to study the effects of various environmental factors on results of such tests.  
5.4 Results of life-cycle tests with mysids 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).4  
5.5 Results of a life-cycle test with mysids 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 (2). Most such predictions take into account results of acute toxicity tests, and so the usefulness of the results from a life-cycle test with mysids is greatly increased by also reporting the results of an acute toxicity test (see Guide E729) conducted under the same conditions.  
5.6 Results of life-cycle tests with mysids might be useful for studying the biological availability of, and structure-activity relationships between, test materials.  
5.7 Results of life-cycle tests with mysids might be useful for predicting population effects on the same species in another water or with another species in the same or a different...
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1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a test material added to dilution water, but not to food, on certain species of saltwater mysids during continuous exposure from immediately after birth until after the beginning of reproduction using the flow-through technique. These procedures will probably be useful for conducting life-cycle toxicity tests with other species of mysids, although modifications might be necessary.  
1.2 Other 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting life-cycle toxicity tests with saltwater mysids.  
1.3 These procedures are applicable to all 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, and pH and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments, and surface waters.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Hazards  
7  
Apparatus  
6  
Facilities  
6.1  
Construction Materials  
6.2  
Metering System  
6.3  
Test Chambers  
6.4  
Cleaning  
6.5  
Acceptability  
6.6  
Dilution Water  
8  
Requirements  
8.1  
Source  
8.2  
Treatment  
8.3  
Characterizat...

SIGNIFICANCE AND USE
5.1 Sediment toxicity evaluations are a critical component of environmental quality and ecosystem impact assessments, and are used to meet a variety of research and regulatory objectives. The manner in which the sediments are collected, stored, characterized, and manipulated can influence the results of any sediment quality or process evaluation greatly. Addressing these variables in a systematic and uniform manner will aid the interpretations of sediment toxicity or bioaccumulation results and may allow comparisons between studies.  
5.2 Sediment quality assessment is an important component of water quality protection. Sediment assessments commonly include physicochemical characterization, toxicity tests or bioaccumulation tests, as well as benthic community analyses. The use of consistent sediment collection, manipulation, and storage methods will help provide high quality samples with which accurate data can be obtained for the national inventory and for other programs to prevent, remediate, and manage contaminated sediment.  
5.3 It is now widely known that the methods used in sample collection, transport, handling, storage, and manipulation of sediments and interstitial waters can influence the physicochemical properties and the results of chemical, toxicity, and bioaccumulation analyses. Addressing these variables in an appropriate and systematic manner will provide more accurate sediment quality data and facilitate comparisons among sediment studies.  
5.4 This standard provides current information and recommendations for collecting and handling sediments for physicochemical characterization and biological testing, using procedures that are most likely to maintain in situ conditions, most accurately represent the sediment in question, or satisfy particular needs, to help generate consistent, high quality data collection.  
5.5 This standard is intended to provide technical support to those who design or perform sediment quality studies under a variety of ...
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1.1 This guide covers procedures for obtaining, storing, characterizing, and manipulating marine, estuarine, and freshwater sediments, for use in laboratory sediment toxicity evaluations and describes samplers that can be used to collect sediment and benthic invertebrates (Annex A1). This standard is not meant to provide detailed guidance for all aspects of sediment assessments, such as chemical analyses or monitoring, geophysical characterization, or extractable phase and fractionation analyses. However, some of this information might have applications for some of these activities. A variety of methods are reviewed in this guide. A statement on the consensus approach then follows this review of the methods. This consensus approach has been included in order to foster consistency among studies. It is anticipated that recommended methods and this guide will be updated routinely to reflect progress in our understanding of sediments and how to best study them. This version of the standard is based primarily on a document developed by USEPA (2001 (1))2 and by Environment Canada (1994 (2)) as well as an earlier version of this standard.  
1.2 Protecting sediment quality is an important part of restoring and maintaining the biological integrity of our natural resources as well as protecting aquatic life, wildlife, and human health. Sediment is an integral component of aquatic ecosystems, providing habitat, feeding, spawning, and rearing areas for many aquatic organisms (MacDonald and Ingersoll 2002 a, b (3)(4)). Sediment also serves as a reservoir for contaminants in sediment and therefore a potential source of contaminants to the water column, organisms, and ultimately human consumers of those organisms. These contaminants can arise from a number of sources, including municipal and industrial discharges, urban and agricultural runoff, atmospheric deposition, and port operations.  
1.3 Contaminated sediment can cause lethal ...

SIGNIFICANCE AND USE
6.1 Contaminated sediments may affect natural populations of aquatic organisms adversely. Sediment-dwelling organisms may be exposed directly to contaminants by the ingestion of sediments and by the uptake of sediment-associated contaminants from interstitial and overlying water. Contaminated sediments may affect water column species directly by serving as a source of contaminants to overlying waters or a sink for contaminants from overlying waters. Organisms may also be affected when contaminated sediments are suspended in the water column by natural or human activities. Water column species and nonaquatic species may also be affected indirectly by contaminated sediments by the transfer of contaminants through ecosystems (7, 8).  
6.2 The procedures described in this guide may be used and adapted for incorporation in basic and applied research to determine the ecological effects of contaminated sediments. These same methods may also be used in the development and implementation of monitoring and regulatory programs designed to prevent and manage sediment contamination.  
6.3 Sediment tests with aquatic organisms can be used to quantify the acute and chronic toxicity and the bioavailability of new and presently used materials. Sediment toxicity may also result from environmental processes such as ammonia generation, pH shifts, or dissolved oxygen fluctuation. In many cases, consideration of the adverse effects of sediment-associated contaminants is only one part of a complete hazard assessment of manufactured compounds that are applied directly to the environment (for example, pesticides) and those released (for example, through wastewater effluents) as by-products from the manufacturing process or from municipalities (7).  
6.4 Sediment tests can be used to develop exposure-response relationships for individual toxicants by spiking clean sediments with varying concentrations of a test chemical and determining the concentration that elicits the target response in...
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1.1 As the contamination of freshwater and saltwater ecosystems continues to be reduced through the implementation of regulations governing both point and non-point source discharges, there is a growing emphasis and concern regarding historical inputs and their influence on water and sediment quality. Many locations in urban areas exhibit significant sediment contamination, which poses a continual and long-term threat to the functional condition of benthic communities and other species inhabiting these areas (1).2 Benthic communities are an important component of many ecosystems and alterations of these communities may affect water-column and nonaquatic species.  
1.2 Biological tests with sediments are an efficient means for evaluating sediment contamination because they provide information complementary to chemical characterizations and ecological surveys (2). Acute sediment toxicity tests can be used as screening tools in the early phase of an assessment hierarchy that ultimately could include chemical measurements or bioaccumulation and chronic toxicity tests. Sediment tests have been applied in both saltwater and freshwater environments (2-6). Sediment tests have been used for dredge material permitting, site ranking for remediation, recovery studies following management actions, and trend monitoring. A particularly important application is for establishing contaminant-specific effects and the processes controlling contaminant bioavailability(7).  
1.3 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Application  
4  
Summary of Guide  
5  
Significance and Use  
6  
Hazards  
7  
Sediment Test Types  
8  
Biological Responses  
9  
Test Organisms  
10  
Experimental Design Considerations  
11  
Data Interpretation  
12  
Keywords  
13  
1.4 The values stated in SI units are to be regarded as the standard. The values g...

SIGNIFICANCE AND USE
5.1 This procedure can be used to limit the need for screening tests prior to performing a test for estimating the LC50 of a non-reactive and non-electrolytic chemical to the fathead minnow. By eliminating the screening test, fewer fish need be tested. The time used for preparing and performing the screening test can also be saved. The value obtained in this procedure can be used as the preliminary estimate of the LC50 in a full-scale test.  
5.2 Estimates can be used to set testing priority of groups of non-reactive and non-electrolytic chemicals.  
5.3 If the estimated value is more than 0.3 times the experimental value, the mechanism of action is probably narcosis. If less, the effect concentration is considered to reflect a different mechanism of action.  
5.4 This practice estimates a maximum LC50, that is, non-reactive and non-electrolytic chemicals are at least as toxic as the practice predicts, but may have a lower LC50 if acting by a more specific mechanism. Data on a chemical indicating a lower toxicity than predicted should be considered suspect or an artifact because of limited solubility of the test material.
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1.1 This practice covers a procedure for estimating the fathead minnow (Pimephales promelas) 96-h LC50 of nonreactive (that is, covalently bonded without unsaturated residues) and nonelectrolytic (that is, require vigorous reagents to facilitate substitution, addition, replacement reactions and are non-ionic, non-dissociating in aqueous solutions) organic chemicals acting solely by narcosis, also referred to as Meyer-Overton toxicity relationship.2  
1.2 This procedure is accurate for organic chemicals that are toxic due to narcosis and are non-reactive and non-electrolytic. Examples of appropriate chemicals are: alcohols, ketones, ethers, simple halogenated aliphatics, aromatics, and aliphatic substituted aromatics. It is not appropriate for chemicals whose structures include a potential toxiphore (that structural component of a chemical molecule that has been identified to show mammalian toxicity, for example CN is known to be reponsible for inactivation of enzymes, NO2 for decoupling of oxidative phosphorylation, both leading to mammalian toxicity). Examples of chemicals inappropriate for this practice are: carbamates, organophosphates, phenols, beta-gamma unsaturated alcohols, electrophiles, and quaternary ammonium salts.  
1.3 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.

SIGNIFICANCE AND USE
5.1 Adverse effects on natural populations of aquatic organisms and their uses have demonstrated the need to assess the hazards of many new, and some presently used, materials. The process described herein will help producers, users, regulatory agencies, and others to efficiently and adequately compare alternative materials, completely assess a final candidate material, or reassess the hazard of a material already in use.  
5.2 Sequential assessment and feedback allow appropriate judgments concerning efficient use of resources, thereby minimizing unnecessary testing and focusing effort on the information most pertinent to each material. For different materials and situations, assessment of hazard will appropriately be based on substantially different amounts and kinds of biological, chemical, physical, and toxicological data.  
5.3 Assessment of the hazard of a material to aquatic organisms and their uses should never be considered complete for all time. Reassessment should be considered if the amount of production, use, or disposal increases, new uses are discovered, or new information on biological, chemical, physical, or toxicological properties becomes available. Periodic review will help assure that new circumstances and information receive prompt appropriate attention.  
5.4 If there is substantial transformation to another material, the hazard of both materials may need to be assessed.  
5.5 In many cases, consideration of adverse effects should not end with completion of the hazard assessment. Additional steps should often include risk assessment, decisions concerning acceptability of identified hazards and risks, and mitigative actions.  
5.6 Because this practice deals mostly with adverse effects on aquatic organisms and their uses, it is important that mitigative actions, such as improved treatment of aqueous effluents, not result in unacceptable effects on non-aquatic organisms. Thus, this standard should be used with other information in order to a...
SCOPE
1.1 This guide describes a stepwise process for using information concerning the biological, chemical, physical, and toxicological properties of a material to identify adverse effects likely to occur to aquatic organisms and their uses as a result of release of the material to the environment. The material will usually be a specific chemical, although it might be a group of chemicals that have very similar biological, chemical, physical, and toxicological properties and are usually produced, used, and discarded together.  
1.2 The hazard assessment process is complex and requires decisions at a number of points; thus, the validity of a hazard assessment depends on the soundness of those decisions, as well as the accuracy of the information used. All decisions should be based on reasonable worst-case analyses so that an appropriate assessment can be completed for the least cost that is consistent with scientific validity.  
1.3 This guide assumes that the reader is knowledgeable in aquatic toxicology and related pertinent areas. A list of general references is provided (1).2  
1.4 This guide does not describe or reference detailed procedures for estimating or measuring environmental concentrations, or procedures for determining the maximum concentration of test material that is acceptable in the food of predators of aquatic life. However, this guide does describe how such information should be used when assessing the hazard of a material to aquatic organisms and their uses.  
1.5 Because assessment of hazard to aquatic organisms and their uses is a relatively new activity within aquatic toxicology, most of the guidance provided herein is qualitative rather than quantitative. When possible, confidence limits should be calculated and taken into account.  
1.6 This guide provides guidance for assessing hazard but does not provide guidance on how to take into account social considerations in order to judge the acceptabil...

SIGNIFICANCE AND USE
5.1 The term duckweed commonly refers to members of the family Lemnaceae. This family has many species world-wide in 4 genera. This guide is designed for toxicity testing with one particular clone of one species of duckweed that has been extensively studied, Lemna gibba G3, although other species such as Lemna minor or Spirodela spp. can probably also be tested using the procedures described herein.  
5.2 Duckweeds are widespread, free-floating aquatic plants, ranging in the world from tropical to temperate zones. Duckweeds are a source of food for waterfowl and small animals and provide food, shelter, and shade for fish. The plants also serve as physical support for a variety of small invertebrates. Duckweed is fast growing and reproduces rapidly compared with other vascular plants (1).3 Under conditions favorable for its growth, it can multiply quickly and form a dense mat in lakes, ponds, and canals, primarily in fresh water, but also in estuaries. It also grows well in effluents of wastewater treatment plants and has been suggested as a means of treating wastewaters (2). A dense mat of duckweed can block sunlight and aeration and cause fish kills (3).  
5.3 Duckweed is small enough that large laboratory facilities are not necessary, but large enough that effects can be observed visually.  
5.4 Because duckweed is a floating macrophyte, it might be particularly susceptible to surface active and hydrophobic chemicals that concentrate at the air-water interface. Results of duckweed tests on such chemicals, therefore, might be substantially different from those obtained with other aquatic species.  
5.5 Results of toxicity tests with duckweed might be used to predict effects likely to occur on duckweed in field situations as a result of exposure under comparable conditions.  
5.6 Results of tests with duckweed might be used to compare the toxicities of different materials and to study the effects of various environmental factors on results of such tests.  
5.7...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a text material added to growth medium on a certain species of duckweed (Lemna gibba G3) during a 7-day exposure using the static technique. These procedures will probably be useful for conducting toxicity tests with other species of duckweed and other floating vascular plants, although modifications might be necessary.  
1.2 Special needs or circumstances might also justify modification of this standard. 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting tests with duckweed.  
1.3 The procedures in this guide 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 and pH and on such other materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments and surface waters. These procedures do not specifically address effluents because to date there is little experience using duckweeds in effluent testing and such tests may pose problems with acclimation of the test organisms to the receiving water. Static tests might not be applicable to materials that have a high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed 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 toxicity tests performed using the procedures in this guide should usually be reported in terms of the 7-day IC5...

SIGNIFICANCE AND USE
3.1 This practice pertains to all forms of toxicological testing (acute, subchronic, or chronic) performed by any route of administration (inhalation, oral, dermal, ocular, or other).  
3.2 The U.S. Environmental Protection Agency, Good Laboratory Practices for Nonclinical Laboratory Studies, as listed in 40 CFR, requires that a testing facility maintain specific standard operating procedures (SOPs) including an SOP covering clinical observations in test animals.  
3.3 This practice serves as a basis for consistency in clinical observations and is not meant to serve as a comprehensive list of observations that may be observed. Actual procedures and forms to be used in recording observations must be described in individual study protocols.
SCOPE
1.1 This practice describes the terms used in observing and recording cutaneous, gastrointestinal, respiratory, reproductive, neuromuscular, ocular, and general clinical signs of animals undergoing toxicological testing. This practice also assists in properly observing and assessing laboratory animals for signs of disease or adverse effects of compound administration.  
1.2 This practice includes codes and descriptions for a wide variety of clinical signs, anatomical locations, and other descriptive qualifiers, and a technique for scoring the extent or severity of clinical signs.  
1.3 This practice assumes that the reader is knowledgeable in animal toxicology and related pertinent areas and is trained in making clinical observations.  
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.

SIGNIFICANCE AND USE
5.1 Terrestrial phytotoxicity tests are useful in assessing the effects of environmental samples or specific chemicals as a part of an ecological risk assessment (3-6, 12, 13).  
5.2 Though inferences regarding higher-order ecological effects (population, community, or landscape) may be made from the results, these tests evaluate responses of individuals of one or more plant species to the test substance.  
5.3 This guide is applicable for: (a) establishing phytotoxicity of organic and inorganic substances; (b) determining the phytotoxicity of environmental samples; (c) determining the phytotoxicity of sludges and hazardous wastes, (d) assessing the impact of discharge of toxicants to land, and (e) assessing the effectiveness of remediation efforts.
SCOPE
1.1 This guide covers practices for conducting plant toxicity tests using terrestrial plant species to determine effects of test substances on plant growth and development. Specific test procedures are presented in accompanying annexes.  
1.2 Terrestrial plants are vital components of ecological landscapes. The populations and communities of plants influence the distribution and abundance of wildlife. Obviously, plants are the central focus of agriculture, forestry, and rangelands. Toxicity tests conducted under the guidelines and annexes presented herein can provide critical information regarding the effects of chemicals on the establishment and maintenance of terrestrial plant communities.  
1.3 Toxic substances that prevent or reduce seed germination can have immediate and large impacts to crops. In natural systems, many desired species may be sensitive, while other species are tolerant. Such selective pressure can result in changes in species diversity, population dynamics, and community structure that may be considered undesirable. Similarly, toxic substances may impair the growth and development of seedlings resulting in decreased plant populations, decreased competitive abilities, reduced reproductive capacity, and lowered crop yield. For the purposes of this guide, test substances include pesticides, industrial chemicals, sludges, metals or metalloids, and hazardous wastes that could be added to soil. It also includes environmental samples that may have had any of these test substances incorporated into soil.  
1.4 Terrestrial plants range from annuals, capable of completing a life-cycle in as little as a few weeks, to long-lived perennials that grow and reproduce for several hundreds of years. Procedures to evaluate chemical effects on plants range from short-term measures of physiological responses (for example, chlorophyll fluorescence) to field studies of trees over several years. Research and development of standardized plant tests have emphasized three categories of tests: (1) short-term, physiological endpoints (that is, biomarkers); (2) short-term tests conducted during the early stages of plant growth with several endpoints related to survival, growth, and development; and (3) life-cycle toxicity tests that emphasize reproductive success.  
1.5 This guide is arranged by sections as follows:  
Section  
Title  
1  
Scope  
2  
Referenced Documents  
3  
Terminology  
4  
Summary of Phytotoxicity Tests  
5  
Significance and Use  
6  
Apparatus  
7  
Test Material  
8  
Hazards  
9  
Test Organisms  
10  
Sample Handling and Storage  
11  
Calibration and Standardization  
12  
Test Conditions  
13  
Interference and Limitations  
14  
Quality Assurance and Quality Control  
15  
Calculations and Interpretation of Results    
16  
Precision and Bias  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this 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 saf...

SIGNIFICANCE AND USE
5.1 The information in this guide is designed to allow investigators conducting research or tests of environmental relevance to select appropriate light sources.  
5.2 Investigators will be able to make reasonable selections of light sources based on cost, the requirements of the test organisms, and the properties of the test chemicals.  
5.3 These methods have major significance for the comparison of results between laboratories. Investigators at different sites will be able to select similar light sources. This will provide standardization of a factor that can have major impact on the effects of hazardous chemicals.
SCOPE
1.1 The use of artificial lighting is often required to study the responses of living organisms to contaminants in a controlled manner. Even if the test organism does not require light, the investigator will generally need light to manipulate the samples, and the test might be conducted under the ambient light of the laboratory. One will need to consider not only whether the particular test organism requires light for growth, but also whether the environmental compartment relevant to the test is exposed to light and, if so, what the attributes of light are in that compartment. The light could affect growth of the organism or toxicity of a contaminant, or both. For instance, it has been shown that the toxicity of some organic pollutants is enhanced dramatically by the ultraviolet (UV) radiation present in sunlight (1, 2) .2 Furthermore, the level of ambient lighting in the laboratory (which might affect the test) is not standardized, nor is it comparable to natural environments. It is thus important to consider lighting in all forms of environmental testing. When light is used in the test, one should determine whether the spectral distribution of the radiation source mimics sunlight adequately to be considered environmentally relevant. Also, the container or vessel for the experiment must be transparent, at the point of light entry, to all of the spectral regions in the light source needed for the test.  
1.2 It is possible to simulate sunlight with respect to the visible:UV ratio with relatively inexpensive equipment. This guide contains information on the types of artificial light sources that are commonly used in the laboratory, compositions of light sources that mimic the biologically relevant spectral range of sunlight, quantification of irradiance levels of the light sources, determination of spectral outputs of the light sources, transmittance properties of materials used for laboratory containers, calculation of biologically effective radiation, and considerations that should go into designing a relevant light source for a given test.  
1.3 Special needs or circumstances will dictate how a given light source is constructed. This is based on the requirements of the test and the environmental compartment to which it is targeted. Using appropriate conditions is most important for any experiment, and it is desirable to standardize these conditions among laboratories. In extreme cases, tests using unusual lighting conditions might render a data set incomparable to other tests.  
1.4 The lighting conditions described herein are applicable to tests with most organisms and using most chemicals. With appropriate modifications, these light sources can be used under most laboratory conditions with many types of laboratory vessels.  
1.5 The attributes of the light source used in a given study should list the types of lamps used, any screening materials, the light level as an energy fluence rate (in W m−2 ) or photon fluence rate (in μmol m−2 s−1 ), and the transmission properties of the vessels used to hold the test organism(s). If it is relevant to the outcome of a test, the spectral quality of the light source should be measured with a spectroradiometer and the emission spectrum provided graphically for reference.  
1.6 The sections of this guide are arranged as foll...

SIGNIFICANCE AND USE
5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, nematodes have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Bacterial-feeding nematodes such as C. elegans feed on soil microbes and contribute to the breakdown of organic matter. They are also of extreme importance in the cycling and degradation of key nutrients in soil ecosystems (9). Soil nematodes also serve as a source of prey and nutrients for fauna and microflora such as soil nematophagous fungi (10). A major change in the abundance of soil invertebrates such as nematodes, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.  
5.2 Results from soil tests might be an important consideration when assessing the hazards of materials to terrestrial organisms.  
5.3 The soil test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity.  
5.4 Results of soil tests could be used to compare the sensitivities of different species.  
5.5 An understanding of the effect of these parameters on toxicity may be gained by varying soil characteristics such as pH, clay content, and organic material.  
5.6 Results of soil tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations.  
5.6.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites.  
5.6.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation c...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of chemicals associated with soil to nematodes from soil toxicity tests. This standard is based on a modification to Guide E1676. The methods are designed to assess lethal or sublethal toxic effects on nematodes in short-term tests in terrestrial systems. Soils to be tested may be (1) references soils or potentially toxic soil sites; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Caenorhabditis elegans (see Annex A1). Methods described in this guide may also be useful for conducting soil toxicity tests with other terrestrial species, although modifications may be necessary.  
1.2 Summary of Previous Studies—Initial soil toxicity testing using the free-living, bacterivorous soil nematode Caenorhabditis elegans was developed by Donkin and Dusenbery (1).2 Following the development of an effective method of recovery of C. elegans  from test soils, the organism was used to identify factors that affect the toxicity of zinc, cadmium, copper, and lead (2) . Freeman et al. further refined the nematode bioassay by decreasing the quantity of soil and spiking solution volumes, determining test acceptability criteria, and developing control charts to assess worm health using copper as a reference toxicant (3). More recently, the toxicological effects of nitrate and chloride metallic salts in two natural soils were compared (4) . LC50 values for C. elegans exposed for 24-h to nitrate salts of cadmium, copper, zinc, lead and nickel in an artificial soil (see Annex A2) were found to be similar to LC50 values for the earthworm, Eisenia fetida  (5). Increasing the exposure time to 48-h resulted in much lower LC50 values (6). However, longer exposure times necessitate the additio...

SIGNIFICANCE AND USE
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 ...
SCOPE
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 crite...

SIGNIFICANCE AND USE
5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. An early life-stage toxicity test provides information about the chronic toxicity of a test material to a species of fish. The primary adverse effects studied are reduced survival and growth.  
5.2 Results of early life-stage toxicity tests are generally useful estimates of the results of comparable life-cycle tests with the same species (1).4 However, results of early life-stage tests are sometimes under estimative of those obtained with the same species in the longer life-cycle tests (2).  
5.3 Results of early life-stage toxicity tests might be used to predict long-term effects likely to occur on fish in field situations as a result of an exposure under comparable conditions, except that motile organisms might avoid exposure when possible.  
5.4 Results of early life-stage toxicity tests might be used to compare the chronic sensitivities of different fish 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 early life-stage 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 (3).  
5.6 Results of an early life-stage test 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 take into account the results of acute toxicity tests, and so the usefulness of the results of an early life-stage test is greatly increased by reporting also the results of an acute toxicity test (see Guide E729) conducted with juveniles of the same species under the same conditions.  
5.7 Results of early life-stage toxicity tests might be useful for...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a test material added to dilution water—but not to food—on certain species of freshwater and saltwater fishes during 28 day to 120 day (depending on species) continuous exposure, beginning before hatch and ending after hatch, using flow-through exposures. This guide will probably be useful for conducting early life-stage toxicity tests with some other species of fish, although modifications might be necessary.  
1.2 Other 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting early life-stage toxicity tests with fishes.  
1.3 These procedures are applicable to all 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, and pH and on such materials as aqueous effluents (see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Standard  
4  
Significance and Use  
5.1  
Hazards  
6  
Apparatus  
7  
Facilities  
7.1  
Construction Materials  
7.2  
Metering System  
7.3  
Test Chambers and Incubation Cups  
7.4  
Cleaning  
7.5  
Acceptability  
7.6  
Dilution Water  
8  
Requirements  
8.1  
S...

SIGNIFICANCE AND USE
5.1 The ecological importance of bivalves, their wide geographic distribution, ease of handling in the laboratory and the field, and their ability to filter and ingest large volumes of water and sediment particles make them appropriate species for conducting field bioassays to assess bioaccumulation potential and associated biological effects. The test procedures in this guide are intended to provide guidance for conducting controlled experiments with caged bivalves under “natural,” site-specific conditions. It is important to acknowledge that a number of “natural” factors can affect bivalve growth and the accumulation of chemicals in their tissues (Section 6, Interferences). This field bioassay can also be conducted in conjunction with laboratory bioassays to help answer questions raised in the field exposures. The field exposures can also be used to validate the results of laboratory bioassays.  
5.2 The ultimate resources of concern are communities. However, it is often difficult or impossible to adequately assess the ecological fitness or condition of the community or identify and test the most sensitive species. Bivalves are recommended as a surrogate test species for other species and communities for the following reasons: (1) They readily accumulate many chemicals and show sublethal effects associated with exposure to those chemicals (2); (2) they accumulate many chemicals through multiple pathways of exposure, including water, sediment, and food  (24, 25, 26, 27, 28, 29), and (3) caged bivalves have been shown to represent effects on the benthos more accurately than traditional laboratory tests (30, 31). Although bivalve species might be considered insensitive because of their wide use as indicators of chemical bioavailability, it has been suggested that sensitivity is related to the type of test, end points being measured, and duration of exposure  (2). In relatively short-term toxicity assessments in which survival is typically determined as the measureme...
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1.1 This guide describes procedures for conducting controlled experiments with caged bivalves under field conditions. The purpose of this approach is to facilitate the simultaneous collection of field data to help characterize chemical exposure and associated biological effects in the same organism under environmentally realistic conditions. This approach of characterizing exposure and effects is consistent with the US EPA ecological risk assessment paradigm. Bivalves are useful test organisms for in-situ field bioassays because they (1) concentrate and integrate chemicals in their tissues and have a more limited ability to metabolize most chemicals than other species, (2) exhibit measurable sublethal effects associated with exposure to those chemicals, (3) provide paired tissue chemistry and response data which can be extrapolated to other species and trophic levels, (4) provide tissue chemistry data which can be used to estimate chemical exposure from water or sediment, and (5) facilitate controlled experimentation in the field with large sample sizes because they are easy to collect, cage, and measure (1, 2)2. The experimental control afforded by this approach can be used to place a large number of animals of a known size distribution in specific areas of concern to quantify exposure and effects over space and time within a clearly defined exposure period. Chemical exposure can be estimated by measuring the concentration of chemicals in water, sediment, or bivalve tissues, and effects can be estimated with survival, growth, and other sublethal end points (3). Although a number of assessments have been conducted using bivalves to characterize exposure by measuring tissue chemistry or associated biological effects, relatively few assessments have been conducted to characterize both exposure and biological effects simultaneously (2, 4, 5). This guide is specifically designed to help minimize the variability in tissue che...

SIGNIFICANCE AND USE
5.1 A bioconcentration test is conducted to obtain information concerning the ability of an aquatic species to accumulate a test material directly from water. This guide provides guidance for designing bioconcentration tests on the properties of the test material so that each material is tested in a cost-effective manner.  
5.2 Because steady-state is usually approached from the low side and the definition of apparent steady-state is based on a statistical hypothesis test, the apparent steady-state BCF will usually be lower than the steady-state BCF. With the variation and sample sizes commonly used in bioconcentration tests, the actual steady-state BCF will usually be no more than twice the apparent BCF.  
5.3 When both are determined in the same test, the projected steady-state BCF will usually be higher than the apparent steady-state BCF because the models used to calculate the projected BCF assume that the BCF steadily increases until infinite time.  
5.4 The BCFs and rates and extents of uptake and depuration will depend on temperature, water quality, the species and its size, physiological condition, age, and other factors (1).4 Although organisms are fed during tests, uptake by means of sorption onto food is probably negligible during tests.  
5.5 Results of bioconcentration tests are used to predict concentrations likely to occur in aquatic organisms in field situations as a result of exposure under comparable conditions, except that mobile organisms might avoid exposure when possible. Under the experimental conditions, particulate matter is deliberately minimized compared to natural water systems. Exposure conditions for the tests may therefore not be comparable for an organic chemical that has a high octanol-water partition coefficient or for an inorganic chemical that sorbs substantially onto particulate matter. The amount of the test substance in solution is thereby reduced in both cases, and therefore the material is less available to many organisms...
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1.1 This guide describes procedures for obtaining laboratory data concerning bioconcentration of a test material added to dilution water—but not to food—by freshwater and saltwater fishes and saltwater bivalve mollusks using the flow-through technique. These procedures also should be useful for conducting bioconcentration tests with other aquatic species, although modifications might be necessary.  
1.2 Other 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. The comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting bioconcentration tests.  
1.3 These procedures are applicable to all chemicals that can be measured accurately at the necessary concentrations in water and in appropriate tissues. Bioconcentration tests are usually conducted on individual chemicals but can be conducted on mixtures if appropriate measurements can be made. Some techniques described in this guide were developed for tests on non-ionizable organic chemicals (see 11.1.2.1) and might not apply to ionizable or inorganic chemicals.  
1.4 Results of bioconcentration tests should usually be reported in terms of apparent steady-state and projected steady-state bioconcentration factors (BCFs) and uptake and depuration rate constants. Results should be reported in terms of whole body for fishes and in terms of total soft tissue for bivalve mollusks. For fishes and scallops consumed by humans, some results should also be reported in terms of the edible portion, especially if ingestion of the test material by humans is a major concern. For tests on organic and organometallic chemicals, the ...

SIGNIFICANCE AND USE
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 li...
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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...

SIGNIFICANCE AND USE
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 obtaine...
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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  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Apparatus ...

SIGNIFICANCE AND USE
5.1 While federal criteria and state standards exist that define acute and chronic “safe” levels in the water column, effects levels in the sediment are poorly defined and may be dependent upon numerous modifying factors. Even where USEPA recommended Water Quality Criteria (WQC, (49)) are not exceeded by water-borne concentrations, organisms that live in or near the sediment may still be adversely affected (50). Therefore, simply measuring the concentration of a chemical in the sediment or in the water is often insufficient to evaluate its actual environmental toxicity. Concentrations of contaminants in sediment may be much higher than concentrations in overlying water; this is especially true of hydrophobic organic compounds as well as inorganic ions that have a strong affinity for organic ligands and negatively-charged surfaces. Higher chemical concentrations in sediment do not, however, always translate to greater toxicity or bioaccumulation (51), although research also suggests that amending sediment with organic matter actually increases the bioaccumulation of contaminant particles (52, 53). Other factors that can potentially influence sediment bioaccumulation and toxicity include pH mineralogical composition, acid-volatile sulfide (AVS) grain size, and temperature (54-56). Laboratory toxicity tests provide a direct and effective way to evaluate the impacts of sediment contamination on environmental receptors while providing empirical consideration of all of the physical, chemical and biological parameters that may influence toxicity.  
5.2 Amphibians are often a major ecosystem component of wetlands around the world, however limited data are available regarding the effects of sediment-bound contaminants to amphibians (39, 41, 43, 55, 57, 58). Laboratory studies such as the procedure described in this standard are one means of directly assessing sediment toxicity to amphibians in order to evaluate potential ecological risks in wetlands.  
5.3 Results from sed...
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1.1 This standard covers procedures for obtaining laboratory data concerning the toxicity of test material (for example, sediment or hydric soil (that is, a soil that is saturated, flooded, or ponded long enough during the growing season to develop anaerobic (oxygen-lacking) conditions that favor the growth and regeneration of hydrophytic vegetation)) to amphibians. This test procedure uses larvae of the northern leopard frog (Lithobates pipiens). Other anuran species (for example, the green frog (Lithobates clamitans), the wood frog (Lithobates sylvatica), the American toad (Bufo americanus)) may be used if sufficient data on handling, feeding, and sensitivity are available. Test material may be sediments or hydric soil collected from the field or spiked with compounds in the laboratory.  
1.2 The test procedure describes a 10-d whole sediment toxicity test with an assessment of mortality and selected sublethal endpoints (that is, body width, body length). The toxicity tests are conducted in 300 to 500-mL chambers containing 100 mL of sediment and 175 mL of overlying water. Overlying water is renewed daily and larval amphibians are fed during the toxicity test once they reach Gosner stage 25 (operculum closure over gills). The test procedure is designed to assess freshwater sediments, however, R. pipiens can tolerate mildly saline water (not exceeding about 2500 mg Cl-/L, equivalent to a salinity of about 4.1 when Na+ is the cation) in 10-d tests, although such tests should always include a concurrent freshwater control. Alternative test durations and sublethal endpoints may be considered based on site-specific needs. Statistical evaluations are conducted to determine whether test materials are significantly more toxic than the laboratory control sediment or a field-collected reference sample(s).  
1.3 Where appropriate, this standard has been designed to be consistent with previously developed methods for assessing s...

SIGNIFICANCE AND USE
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...
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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...

SIGNIFICANCE AND USE
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, t...
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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...

SIGNIFICANCE AND USE
5.1 The significance of measuring algal growth potential in water samples is that differentiation can be made between the nutrients of a sample determined by chemical analysis and the nutrients that are actually available for algal growth. The addition of nutrients (usually nitrogen and phosphorus singly or in combination) to the sample can give an indication of which nutrient(s) is (are) limiting for algal growth  (1,10,11,12,13,14).
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1.1 This practice measures, by Pseudokirchnereilla subcapitata growth response, the biological availability of nutrients, as contrasted with chemical analysis of the components of the sample. This practice is useful for assessing the impact of nutrients, and changes in their loading, upon freshwater algal productivity. Other laboratory or indigenous algae can be used with this practice. However, Pseudokirchnereilla subcapitata must be cultured as a reference alga along with the alternative algal species.  
1.2 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 a specific precautionary statement, see Section 16.  
1.3 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.

SIGNIFICANCE AND USE
5.1 Tests with algae provide information on the toxicity of test materials to an important component of the aquatic biota and might indicate whether additional testing (2) is desirable. Specific testing procedures under various regulatory jurisdictions follow procedures similar to those described in this Guide (3, 4). Users should consult with any specific regulatory requirements to determine the applicability and consistency of this standard with such requirements.  
5.2 Algae are ubiquitous in aquatic ecosystems, where they incorporate solar energy into biomass, produce oxygen, function in nutrient cycling and serve as food for animals. Because of their ecological importance, sensitivity to many toxicants, ready availability, ease of culture, and fast growth rates (rendering it possible to conduct a multi-generation test in a short period of time), algae are often used in toxicity testing.  
5.3 Results of algal toxicity tests might be used to compare the sensitivities of different species of algae and the toxicities of different materials to algae and to study the effects of various environmental factors on results of such tests.  
5.4 Results of algal toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (See Guide E1023) or deriving water quality criteria for aquatic organisms (5).  
5.5 Results of algal toxicity tests might be useful for studying biological availability of, and structure-activity relationships between, test materials.  
5.6 Results of algal toxicity tests will depend on the temperature, composition of the growth medium, and other factors. These tests are conducted in solutions that contain concentrations of salts, minerals, and nutrients that greatly exceed those in most surface waters. These conditions may over- or under-estimate the effects of the test material if discharged to surface waters.
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1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of a test material added to growth medium on growth of certain species of freshwater and saltwater microalgae during a static exposure. These procedures will probably be useful for conducting short-term toxicity tests with other species of algae, although modifications might be necessary. Although the test duration is comparable to an acute toxicity test with aquatic animals, an algal toxicity test of short duration (72, 96 or 120 h) allows for examination of effects upon multiple generations of an algal population and thus should not be viewed as an acute toxicity test.  
1.2 Other 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting toxicity tests with microalgae.  
1.3 These procedures are applicable to many chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications, these procedures can be used to conduct tests on temperature, and pH and on such materials as aqueous effluents (see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters. Static tests might not be applicable to materials that are highly volatile, are rapidly biologically or chemically transformed in aqueous solutions, or are removed from test solutions in substantial quantities by the test vessels or organisms during the test. (1)3 However, practical flow-through test procedures with microalgae have not been developed.  
1.4 Results of tests using microalgae should usually be reported in terms of the 96-h (or other time period) IC50 (see 3.2.5) b...

SIGNIFICANCE AND USE
5.1 An acute toxicity test is conducted to assess the 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 bivalve embryo development test includes a sublethal endpoint, but is also short in duration, these tests are considered to be short-term chronic tests.  
5.2 Because embryos and larvae are usually assumed to be the most sensitive life stages of these bivalve mollusc species and because these species are commercially and recreationally important, results of these acute tests are often considered to be a good indication of the acceptability of pollutant concentrations to saltwater molluscan species in general. Results of these acute toxicity tests are often assumed to be an important consideration when assessing the hazard of materials to other saltwater organisms (see Guide E1023) or when deriving water quality criteria for saltwater organisms (3) .  
5.3 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 Results of short-term chronic tests might be used to compare the sensitivities of different species to different test materials, and to determine the effects of various environmental factors on results of such tests.  
5.5 Results of short-term chronic toxicity tests might be useful for studying biological availability of, and structure activity relationships between, test materials.  
5.6 Results of any toxicity test will depend on temper...
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1.1 This guide describes procedures for obtaining laboratory data concerning the acute effects of a test material on embryos and the resulting larvae of four species of saltwater bivalve molluscs (Pacific oyster, Crassostrea gigas Thunberg; eastern oyster,  Crassostrea virginica Gmelin; quahog or hard clam,  Mercenaria mercenaria Linnaeus; and the mussel species complex (Mytilus spp.) including the blue mussel,  Mytilus edulis Linnaeus; the Mediterranean mussel, Mytilus galloprovincialis Lamark; and the Northern Bay Mussel, Mytilus trossulus Gould) during static 48-h exposures. These procedures will probably be useful for conducting static short-term chronic toxicity tests starting with embryos of other bivalve species (1)2 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, results of tests conducted by using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained by using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting 48-h acute tests starting with embryos of bivalve molluscs.  
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 acute tests on temperature, dissolved oxygen, and pH and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments, and surface waters. Renewal tests might be preferable to static tests for materials that have a high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed...

SIGNIFICANCE AND USE
4.1 This guide provides a test procedure for evaluating the potential ecological impacts and environmental transport of a chemical in an agricultural (tilled, low-till, or no-till) or natural field soil ecosystem that may be released or spilled into the environment. The suggested test procedures are designed to supply site-specific information for a chemical without having to perform field testing. (See EPA 560/6-82-002 and EPA 560/6-82-003.)  
4.2 This guide is not specifically designed to address fate of chemicals in soils of forested ecosystems. However, with some modifications, it may be adapted for that purpose by the individual investigator.  
4.3 Specifically, this guide is used to determine the effect of a chemical on (1) growth and reproduction of either natural grassland vegetation or crops, and (2) nutrient uptake and cycling within the soil/plant system. Additionally, the soil-core microcosm will provide information on (1) potential for bioaccumulation (enrichment) of the chemical into plant tissues, and (2) the potential for and rate of transport of the chemical through soil to groundwater.  
4.4 The results of this test should be used in conjunction with information on the chemical and biological activity of the test substance to assess the relative environmental hazard and the potential for environmental movement once released.  
4.5 The test methods described in this guide are designed specifically for liquid or solid materials. Significant modifications of the exposure system would be necessary to accommodate chemicals that are volatile or that may be released in a gaseous or aerosolized form. For methods that could be adapted for use with volatile or gaseous test substances see Refs (3, 4, 5, 6).  
4.6 Results of a multi-year soil-core microcosm test have been correlated with data derived from a series of multi-year field plot tests for a limited number of materials. Information on the correlation between microcosm and field results can be fo...
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1.1 This guide defines the requirements and procedures for using soil-core microcosms to test the environmental fate, ecological effects, and environmental transport of chemicals that may enter terrestrial ecosystems. The approach and the materials suggested for use in the microcosm test are also described.  
1.2 This guide details a procedure designed to supply site-specific or possibly regional information on the probable chemical fate and ecological effects in a soil system resulting from the release or spillage of chemicals into the environment in either liquid or solid form.  
1.3 Experience has shown that microcosms are most helpful in the assessment process after preliminary knowledge about the chemical properties and biological activity have been obtained. Data generated from the test can then be used to compare the potential terrestrial environmental hazards of a chemical.  
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.

SIGNIFICANCE AND USE
5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, lumbricid earthworms and enchytraeid potworms have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Earthworms may ingest large quantities of soil, have a close relationship with other soil biomasses (for example, invertebrates, roots, humus, litter, and microorganisms), constitute up to 92 % of the invertebrate biomass of soil, and are important in recycling nutrients (1, 2).4 Enchytraeids contribute up to 5.2 % of soil respiration, constitute the second-highest biomass in many soils (the highest in acid soils in which earthworms are lacking) and effect considerably nutrient cycling and community metabolism (3-5). Earthworms and potworms accumulate and are affected by a variety of organic and inorganic compounds (2-10, 11-14). In addition, earthworms and potworms are important in terrestrial food webs, constituting a food source for a very wide variety of organisms, including birds, mammals, reptiles, amphibians, fish, insects, nematodes, and centipedes  (15, 16, 3). A major change in the abundance of soil invertebrates such as lumbricids or enchytraeids, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.  
5.2 A number of species of lumbricids and enchytraeid worms have been used in field and laboratory investigations in the United States and Europe. Although the sensitivity of various lumbricid species to specific chemicals may vary, from their study of four species of earthworms (including E. fetida) exposed to ten organic compounds representing six classes of chemicals, Neuhauser, et al  (7)  suggest that the selection of earthworm test species does not affect the a...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of contaminants (for example, chemicals or biomolecules) associated with soil to earthworms (Family Lumbricidae) and potworms (Family Enchytraeidae) from soil toxicity or bioaccumulation tests. The methods are designed to assess lethal or sublethal toxic effects on earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-term tests (14 to 42 days) in terrestrial systems. Soils to be tested may be (1) reference soils or potentially toxic site soils; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Eisenia fetida (see Annex A1) and for the species Enchytraeus albidus (see Annex A4). Methods described in this guide may also be useful for conducting soil toxicity tests with other lumbricid and enchytraeid terrestrial species, although modifications may be necessary.  
1.2 Modification of these procedures might be justified by special needs. The results of tests conducted using atypical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity and bioaccumulation tests with terrestrial worms.  
1.3 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints (see Section 14). These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter c...

SIGNIFICANCE AND USE
5.1 Protection of an aquatic species requires prevention of unacceptable effects on populations in natural habitats. Toxicity tests are conducted to provide data that may be used to predict what changes in numbers and weights of individuals might result from similar exposure to the test material in the natural aquatic environment. Information might also be obtained on the effects of the material on the health of the species.  
5.2 Results of life-cycle tests with D. magna are used to predict chronic effects likely to occur on daphnids in field situations as a result of exposure under comparable conditions.  
5.2.1 Life-cycle tests with D. magna are used to compare the chronic sensitivities of different species, the chronic toxicities of different materials, and study the effects of various environmental factors on the results of such tests.  
5.2.2 Life-cycle tests with D. magna are used to assess the risk of materials to aquatic organisms (see Guide E1023) or derive water quality criteria for aquatic organisms (1).3  
5.2.3 Life-cycle tests with D. magna are used to extrapolate the results of chronic toxicity 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 take into account the results of acute toxicity tests, and so the usefulness of the results of a life-cycle test with D. magna may be increased by reporting the results of an acute toxicity test (see Guide E729) conducted under the same conditions. In addition to conducting an acute toxicity test with unfed D. magna, it may be relevant to conduct an acute test in which the daphnids are fed the same as in the life-cycle test to see if the presence of that concentration of that food affects the results of the acute test and the acute-chronic ratio (ACR) (see 10.3.1).  
5.2.4 Life-cycle tests are used to evaluate the biological availability of, and structure-activity relationships between, test materials and t...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of a test material (added to dilution water, but not to food) on Daphnia magna Straus, 1820, during continuous exposure throughout a life-cycle using the renewal or flow-through techniques. These procedures also should be useful for conducting life-cycle toxicity tests with other invertebrate species and cladocerans from the same genus (for example, Daphnia pulex), although modifications might be necessary.  
1.2 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, pH, and on such materials as aqueous effluents (also see Guide E1192), leachates, oils, particulate matter, sediments, and surface waters. The technique, (renewal or flow-through), will be selected based on the chemical characteristics of the test material such as high oxygen demand, volatility, susceptibility to transformation (biologically or chemically), or sorption to glass.  
1.3 Modification 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 standard test procedures. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting life-cycle toxicity tests with D. magna. Appendix X3 provides modifications for conducting the chronic toxicity test method with D. pulex Leydig, 1860.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Si...

SIGNIFICANCE AND USE
5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, and health of that species. Behavioral toxicity provides information concerning sublethal effects of chemicals and signals the presence of toxic test substances.  
5.1.1 The behavioral responses of all organisms are adaptive and essential to survival. Major changes in the behavioral responses of fish, amphibians, and macroinvertebrates may result in a diminished ability to survive, grow, or reproduce and cause significant changes in the natural population (8).  
5.2 The results from behavioral toxicity tests may be useful for measuring injury in the assessment of damages resulting from the release of hazardous materials (9) .  
5.3 Behavioral toxicity test methods may be useful for long-term monitoring of effluents (10) .  
5.4 The results from behavioral toxicity data can be used to predict the effects of exposure on fish, amphibians, and aquatic invertebrates likely to occur in field situations as a result of exposure under similar conditions, including the avoidance of exposure by motile organisms (11).  
5.5 The results from behavioral toxicity tests might be an important consideration for assessing the hazard of materials to aquatic organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebrate organisms.  
5.6 The results from behavioral toxicity tests can be used to compare the sensitivities of different species, relative toxicity of different chemical substances on the same organism, or effect of various environmental variables on the toxicity of a chemical substance.  
5.7 The results from behavioral toxicity tests can be used to predict the effects of long-term exposure.  
5.8 The results of behavioral toxicity tests can be useful for guiding decisions regarding the extent of remedial action needed for contaminated aquatic and terrestrial sites.  
5.9 The behavioral charac...
SCOPE
1.1 This guide covers some general information on the selection and application of behavioral methods useful for determining the sublethal effects of chemicals to fish, amphibians, and macroinvertebrates.  
1.2 Behavioral toxicity occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce a behavioral change that exceeds the normal range of variability  (1).2 Behavior includes all observable, recordable, or measurable activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants  (2).  
1.3 Behavioral methods can be used in biomonitoring, the determination of no-observed-effect and lowest-observed-effect concentrations, and the prediction of hazardous chemical impacts on natural populations (3).  
1.4 Behavioral methods can be applied to fish, amphibians, and macroinvertebrates in standard laboratory toxicity tests, tests of effluents, and sediment toxicity tests.  
1.5 The various behavioral methods included in this guide are categorized with respect to seven interdependent, functional responses that fish, amphibians, and macroinvertebrates must perform in order to survive. These functional responses include respiration, locomotion, habitat selection, feeding, predator avoidance, competition, and reproduction (4). These responses can be documented visually or through video or acoustic imagery. Electronically recorded information can be derived through manual techniques or through the use of digital image analysis software (5, 6, 7).  
1.5.1 The functional responses are not necessarily mutually exclusive categories. For instance, locomotion, of some form of movement, is important to all behavioral functions.  
1.6 Additional behavioral methods for any category may be added when new tests are developed as well as when methods are adapted to different species or different life stages of an organism.  
1.7 This gui...

SIGNIFICANCE AND USE
5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, health, and uses of individuals of that species. Behavioral toxicity tests provide information concerning the sublethal effects of chemicals and signal the presence of toxic test substances.  
5.1.1 The locomotory, feeding, and social responses of fish are adaptive and essential to survival. Major changes in these responses may result in a diminished ability to survive, grow, avoid predation, or reproduce and cause significant changes in the natural population (8). Fish behavioral responses are known to be highly sensitive to environmental variables as well as toxic substances.  
5.2 Results from behavioral toxicity tests may be useful for measuring injury resulting from the release of hazardous materials (9).  
5.3 Behavioral responses can also be qualitatively assessed in a systematic manner during toxicity tests to discern trends in sublethal contaminant effects (5).  
5.4 The assessment of locomotory, feeding, and social behaviors is useful for monitoring effluents and sediments from contaminated field sites as well as for defining no-effect concentrations in the laboratory or under controlled field conditions. Such behavioral modifications provide an index of sublethal toxicity and also indicate the potential for subsequent mortality.  
5.5 Behavioral toxicity data can be used to predict the effects of exposure likely to occur in the natural environment (10).  
5.6 Results from behavioral toxicity tests might be an important consideration when assessing the hazard of materials to aquatic organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebrate organisms.  
5.7 Results from behavioral toxicity tests can be used to compare the sensitivities of different species, the relative toxicity of different chemical substances on the same organism, or the effect of various environmental...
SCOPE
1.1 This guide covers some general information on methods for qualitative and quantitative assessment of the behavioral responses of fish during standard laboratory toxicity tests to measure the sublethal effects of exposure to chemical substances. This guide is meant to be an adjunct to toxicity tests and should not interfere with those test procedures.  
1.2 Behavioral toxicosis occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce a behavioral change that exceeds the normal range of variability (1). Behavior includes all of the observable, recordable, or measurable activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants (2).  
1.3 Behavioral methods can be used in biomonitoring, in the determination of no-observed-effect and lowest-observed-effect concentrations, and in the prediction of hazardous chemical impacts on natural populations (3).  
1.4 The behavioral methods described in this guide include locomotory activity, feeding, and social responses, which are critical to the survival of fish (4).  
1.5 This guide is arranged as follows:    
Section Number  
Scope  
1  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Interferences  
6  
Safety Precautions  
7  
Responses Measured  
8  
Test Organisms  
9  
Facility  
10  
Qualitative Behavioral Assessment Method  
11  
Quantitative Behavioral Measurements  
12  
Experimental Design  
13  
Calculation of Test Results  
14  
Report  
15  
1.6 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard. For an explanation of units and symbols, refer to IEEE/ASTM SI 10.  
1.7 This standard does ...

SIGNIFICANCE AND USE
5.1 Sediment provides habitat for many aquatic organisms and is a major repository for many of the more persistent chemicals that are introduced into surface waters. In the aquatic environment, most anthropogenic chemicals and waste materials including toxic organic and inorganic chemicals can accumulate in sediment, which can in turn serve as a source of exposure for organisms living on or in sediment. Contaminated sediments may be directly toxic to aquatic life or can be a source of contaminants for bioaccumulation in the food chain.  
5.2 The objective of a sediment test is to determine whether chemicals in sediment are harmful to or are bioaccumulated by benthic organisms. The tests can be used to measure interactive toxic effects of complex chemical mixtures in sediment. Furthermore, knowledge of specific pathways of interactions among sediments and test organisms is not necessary to conduct the tests. Sediment tests can be used to: (1) determine the relationship between toxic effects and bioavailability, (2) investigate interactions among chemicals, (3) compare the sensitivities of different organisms, (4) determine spatial and temporal distribution of contamination, (5) evaluate hazards of dredged material, (6) measure toxicity as part of product licensing or safety testing, (7) rank areas for clean up, and (8) estimate the effectiveness of remediation or management practices.  
5.3 Results of toxicity tests on sediments spiked at different concentrations of chemicals can be used to establish cause and effect relationships between chemicals and biological responses. Results of toxicity tests with test materials spiked into sediments at different concentrations may be reported in terms of a LC50 (median lethal concentration), an EC50 (median effect concentration), an IC50 (inhibition concentration), or as a NOEC (no observed effect concentration) or LOEC (lowest observed effect concentration). However, spiked sediment may not be representative of chemicals a...
SCOPE
1.1 Relevance of Sediment Contamination—Sediment provides habitat for many aquatic organisms and is a major repository for many of the more persistent chemicals that are introduced into surface waters. In the aquatic environment, both organic and inorganic chemicals may accumulate in sediment, which can in turn serve as a source of exposure for organisms living on or in sediment. Contaminated sediments may be directly toxic to aquatic life or can be a source of contaminants for bioaccumulation in the food chain.  
1.2 Sediment Assessment Tools—Several types of information may be useful in assessing the risk, or potential risk, posed by sediment contaminants, including: (1) chemical analysis of sediment contaminants; (2) sediment toxicity tests, (3) bioaccumulation tests; and (4) surveys of benthic community structure. Each of these provides a different type of information to the assessment, and integrating information from all four lines of evidence may often provide the most robust assessments.  
1.3 Strengths of Toxicity Testing of Contaminated Sediments—Directly assessing the toxicity of contaminated sediments provides some of the same advantages to sediment assessment that whole effluent toxicity testing provides to management of industrial and municipal effluents. As for effluent tests, direct testing of sediment toxicity allows the assessment of biological effects even if: (1) the identities of toxic chemicals present are not (or not completely) known; (2) the influence of site-specific characteristics of sediments on toxicity (bioavailability) is not understood; and (3) the interactive or aggregate effects of mixtures of chemicals present are not known or cannot be adequately predicted. In addition, testing the response of benthic or epibenthic organisms exposed via sediment provides an assessment that is based on the same routes of exposure that would exist in nature, rather than only through water column expos...

SIGNIFICANCE AND USE
5.1 Sediment exposure evaluations are a critical component for both ecological and human health risk assessments. Credible, cost-effective methods are required to determine the rate and extent of bioaccumulation given the potential importance of bioaccumulation by benthic organisms. Standardized test methods to assess the bioavailability of sediment-associated contaminants are required to assist in the development of sediment quality guidelines (1, 2, 3)5 and to assess the potential impacts of disposal of dredge materials (4).  
5.2 The extent to which sediment-associated contaminants are biologically available and bioaccumulated is important in order to assess their direct effects on sediment-dwelling organisms and assess their transport to higher trophic levels. Controlled studies are required to determine the potential for bioaccumulation that can be interpreted and modeled for predicting the impact of accumulated chemicals. The data collected by these methods should be correlated with the current understanding of toxicity or human health risks to augment the hazard interpretation for contaminated sediments.
SCOPE
1.1 This guide covers procedures for measuring the bioaccumulation of sediment-associated contaminants by infaunal invertebrates. Marine, estuarine, and freshwater sediments are a major sink for chemicals that sorb preferentially to particles, such as organic compounds with high octanol-water-partitioning coefficients (Kow) (for example, polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT)) and many metals. The accumulation of chemicals into whole or bedded sediments (that is, consolidated rather than suspended sediments) reduces their direct bioavailability to pelagic organisms but increases the exposure of benthic organisms. Feeding of pelagic organisms on benthic prey can reintroduce sediment-associated contaminants into pelagic food webs. The bioaccumulation of sediment-associated contaminants by sediment-dwelling organisms can therefore result in ecological impacts on benthic and pelagic communities and human health from the consumption of contaminated shellfish or pelagic fish.  
1.2 Methods of measuring bioaccumulation by infaunal organisms from marine, estuarine, and freshwater sediments containing organic or metal contaminates will be discussed. The procedures are designed to generate quantitative estimates of steady-state tissue residues because data from bioaccumulation tests are often used in ecological or human health risk assessments. Eighty percent of steady-state is used as the general criterion. Because the results from a single or few species are often extrapolated to other species, the procedures are designed to maximize exposure to sediment-associated contaminants so that residues in untested species are not underestimated systematically. A 28-day exposure with sediment-ingesting invertebrates and no supplemental food is recommended as the standard single sampling procedure. Procedures for long-term and kinetic tests are provided for use when 80 % of steady-state will not be obtained within 28 days or when more precise estimates of steady-state tissue residues are required. The procedures are adaptable to shorter exposures and different feeding types. Exposures shorter than 28 days may be used to identify which compounds are bioavailable (that is, bioaccumulation potential) or for testing species that do not live for 28 days in the sediment (for example, certain Chironomus). Non-sediment-ingestors or species requiring supplementary food may be used if the goal is to determine uptake in these particular species because of their importance in ecological or human health risk assessments. However, the results from such species should not be extrapolated to other species.  
1.3 Standard test methods are still under development, and much of this guide is based on techniques used in successful studies and expert opinion rather than experimen...

SIGNIFICANCE AND USE
3.1 This test method was developed to meet the following criteria:  
3.1.1 It provides positive recognition of sensory irritants of widely varying potencies.  
3.1.2 It is sufficiently simple to permit the testing of large numbers of materials.  
3.1.3 This test method is capable of generating concentration-response curves for purposes of compound comparison.  
3.1.4 This test method has good reproducibility.  
3.2 This test method can be used for a variety of divergent purposes, including the assessment of comparative irritancy of compounds or formulations and setting interim exposure levels for the workplace  (1, 2).2  
3.3 It has been shown that for a wide variety of chemicals and mixtures, a perfect rank order correlation exists between the decreases in respiratory rate in mice and subjective reports of sensory irritation in man (1, 3, 4, 5).  
3.4 A quantitative estimate of the sensory irritancy of a wide variety of materials can be obtained from concentration-response curves developed using this method (1, 3, 4, 6, 7, 8, 9).  
3.5 Although this test method is intended to measure sensory irritation of the nasal mucosa, the cornea is innervated by the same nerve. This animal model will, therefore, allow an estimate of the irritant potential of cosmetic ingredients or other household products to the eye, assuming that they can be aerosolized (10).  
3.6 This test method is recommended for setting interim guidelines for exposure of humans to chemicals in the workplace, to assess acute sensory irritation resulting from inadvertent spills of household products, and to assess the comparative irritancy of formulations or materials intended for a variety of uses (see Appendix X2).
FIG. 1 Typical Tracing of Normal Mouse Respiration (Top), and of a“ Moderate” Sensory Irritant Response (Bottom)  
Note 1: Taken from Ref. (3).
FIG. 2 Typical Tracing of Normal Mouse Respiration (Top), a Moderate Pulmonary Irritant Response (Center), and an Extreme Pulmonary Irr...
SCOPE
1.1 This laboratory test method provides a rapid means of determining sensory irritant potential of airborne chemicals or mixtures. It may also be used to estimate threshold limit values (TLV) for man. However, it cannot be used to evaluate the relative obnoxiousness of odors.  
1.2 This test method is intended as a supplement to, not a replacement for, chronic inhalation studies used to establish allowable human tolerance levels.  
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. Specific hazard information is given in Section 6.  
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.

SIGNIFICANCE AND USE
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...
SCOPE
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:    
Sec...

SIGNIFICANCE AND USE
5.1 FETAX is a rapid test for identifying potential developmental toxicity. Data may be extrapolated to other species including mammals. FETAX might be used to prioritize samples for further tests which use mammals. Validation studies using compounds with known mammalian or human developmental toxicity, or both, suggest that the predictive accuracy will exceed 85 % (2) . When evaluating a test material for mammalian developmental toxicity, FETAX must be used with and without a metabolic activation system (MAS). Use of this exogenous MAS should increase the predictive accuracy of the assay to approximately 95 %. The accuracy rate compares favorably with other currently available “ in vitro  teratogenesis screening assays” (3). Any assay employing cells, parts of embryos, or whole embryos other than in vivo  mammalian embryos is considered to be an  in vitro assay.  
5.2 It is important to measure developmental toxicity because embryo mortality, malformation, and growth inhibition can often occur at concentrations far less than those required to affect adult organisms.  
5.3 Because of the sensitivity of embryonic and early life stages, FETAX provides information that might be useful in estimating the chronic toxicity of a test material to aquatic organisms.  
5.4 Results from FETAX might be useful when deriving water quality criteria for aquatic organisms (4).  
5.5 FETAX results might be useful for studying structure-activity relationships between test materials and for studying bioavailability.
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the developmental toxicity of a test material. The test utilizes embryos of the African clawed frog, Xenopus laevis  and is called FETAX (Frog Embryo Teratogenesis Assay-Xenopus) (1).2 Some of these procedures will be useful for conducting developmental toxicity tests with other species of frogs although numerous modifications might be necessary. A list of alternative anurans is presented in Appendix X1.  
1.2 A renewal exposure regimen and the collection of the required mortality, malformation, and growth-inhibition data are described. Special needs or circumstances might require different types of exposure and data concerning other effects. Some of these modifications are listed in Appendix X2 although other modifications might also be necessary. Whenever these procedures are altered or other species used, the results of tests might not be comparable between modified and unmodified procedures. Any test that is conducted using modified procedures should be reported as having deviated from the guide.  
1.3 These procedures are applicable to all chemicals either individually or in formulations, commercial products or mixtures that can be measured accurately at the necessary concentrations in water. With appropriate modification these procedures can be used to conduct tests on the effects of temperature, dissolved oxygen, pH, physical agents, and on materials such as aqueous effluents (see Guide E1192), surface and ground waters, leachates, aqueous and solid phase extracts, and solid phase samples, such as soils and sediments, particulate matter, sediment, and whole bulk soils and sediment.  
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 guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Safety Precautions  
6  
Apparatus  
7  
Water for Culturing Xenopus  adults  
8  
Requirements  
8.1  
Source  
8.2  
Treatment  
8.3  
Characterization  
8.4  
FETAX Solution Water  
9  
Requirements  
9.1  
Formulation  
9.2   ...

SIGNIFICANCE AND USE
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).3  
5.6 This practice provides one basis for deciding whether additional toxicity testing should be conducted with birds.
SCOPE
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.

SIGNIFICANCE AND USE
5.1 This test method is of principal value in minimizing the number of animals required to estimate the acute oral toxicity (LD50). It also incorporates measures of variance (95 % CI) and a slope from which to make relative toxicity comparisons.  
5.2 This test method is inappropriate for materials typically producing death two or more days after administration of the test compound unless the observation time between dosages is increased. This test method can be successfully applied, however, for materials producing only an occasional death two or more days after administration.  
5.3 The LD50 is valuable as a measure of the relative acute toxicity of a material and can be used to make an estimate of potential hazard to humans when pesticides, other chemicals, or mixtures are ingested.  
5.4 This test method allows for observation of signs of toxicity in addition to mortality. This information can be useful in planning additional toxicity testing.
SCOPE
1.1 This test method determines the lethality (LD50 value, slope and 95 % confidence interval (CI)) and signs of acute toxicity from a material using a limited number of rats. The technique used in this test method is referred to as the “Stagewise, Adaptive Dose Method.”2 This test method is an alternative to the classical LD50 test and is applicable to both liquids and solids.  
1.2 This test method is not recommended for test materials which typically produce deaths beyond two days postdosing.  
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.

SIGNIFICANCE AND USE
5.1 The RSA method provides risk and resource managers with an enhanced understanding of the ecological health concerns at the sites they oversee because unlike conventional terrestrial ERAs, actual site mammals are the ones evaluated. Additionally, the HQs of desktop efforts report only on the contaminant exposure route of ingestion, and can only evaluate chemicals singly, whereas RSA findings reflect all three exposure routes as well as the combined effects of multiple chemicals on a highly valued endpoint. Critically, the RSA method incorporates site history considerations that necessarily influence the phenomenon of biological response. If reproductive impacts at contaminated sites were ever to be elicited, such would be apparent today because evaluated sites have, at a minimum, continuously exposed their ecological receptors to chemicals for multiple decades during which time tens and often more than one hundred generations have passed (5).  
5.2 Application of the subject guide familiarizes remedial decision-makers and risk managers with two concepts. First, rather than attempting to predict health effects arising in site receptors, there may be more value in documenting demonstrated health effects, should such exist in actual site-exposed mammalian receptors. Second, the possibility exists that site receptors never experienced stress or impact over the years since a site first became contaminated.  
5.3 Application of the subject guide can allow for substantial cost savings. Often, the outcomes of HQ-based assessments are summarily relied upon to conduct ongoing studies, monitor sites, or implement site cleanups, all of which may be unnecessary. Where RSA applications should demonstrate that maximally site-exposed mammalian receptors (as defined in section 4.1) are not experiencing compromise with regard to the sensitive endpoint of reproductive success, it can become apparent that soil remediation efforts on behalf of mammals are not needed.  
5.4 The des...
SCOPE
1.1 This guide describes the procedures for obtaining and interpreting data associated with a direct health status assessment for mammalian receptors at chemically contaminated terrestrial sites where ERA work is either scheduled or ongoing, and irrespective of the number and type of chemicals that may be present. Through reviewing sperm features, the RSA method reports on the reproductive health of male rodents in their natural environmental settings, with these animals serving as surrogates for other (and larger) site mammals (4).  
1.2 These procedures are applicable at any terrestrial property that supports small mammals (for example, mice, voles, rats, squirrels) and has contaminated soil. Importantly, chemicals of concern in site soils need not be spermatoxins. Additionally, the RSA method considers that any combination of chemicals or other site stressors might collectively act to compromise reproduction, held to be a sensitive toxicological endpoint for mammals. The anticipated primary application of the method will be at historically contaminated sites (such as Superfund sites). The procedures describe tasks conducted in the field and in a laboratory. For the latter, tasks may be conducted either in an on-site mobile laboratory, or in a more conventional laboratory setting. For certain tasks, a make-shift work space may be suitable as well (see 7.3).  
1.3 Initial determinations of compromised or non-compromised reproduction in resident male small rodents are made through a cautious comparative review of sperm parameters. Briefly, for the rodents of a given species collected at both a contaminated site and a habitat-matched (non-contaminated) reference location, arithmetic means are first computed for each of the three sperm parameters of count, motility, and morphology. If one or more of the parameter means of the contaminated site rodents reflect an unfavorable shift (that is, count or motility is less than ...

SIGNIFICANCE AND USE
5.1 Vapor pressure values can be used to predict volatilization rates (5). Vapor pressures, along with vapor-liquid partition coefficients (Henry's Law constant) are used to predict volatilization rates from liquids such as water. These values are thus particularly important for the prediction of the transport of a chemical in the environment (6).
SCOPE
1.1 This test method describes procedures for measuring the vapor pressure of pure liquid or solid compounds. No single technique is able to measure vapor pressures from 1 × 10−11 to 100 kPa (approximately 10−10 to 760 torr). The subject of this standard is gas saturation which is capable of measuring vapor pressures from 1 × 10–11 to 1 kPa (approximately 10–10 to 10 torr). Other methods, such as isoteniscope and differential scanning calorimetry (DSC) are suitable for measuring vapor pressures above 0.1 kPa An isoteniscope (standard) procedure for measuring vapor pressures of liquids from 1 × 10−1 to 100 kPa (approximately 1 to 760 torr) is available in Test Method D2879. A DSC (standard) procedure for measuring vapor pressures from 2 × 10−1 to 100 kPa (approximately 1 to 760 torr) is available in Test Method E1782. A gas-saturation procedure for measuring vapor pressures from 1 × 10−11 to 1 kPa (approximately 10−10 to 10 torr) is presented in this test method. All procedures are subjects of U.S. Environmental Protection Agency Test Guidelines.  
1.2 The gas saturation method is very useful for providing vapor pressure data at normal environmental temperatures (–40 to +60°C). At least three temperature values should be studied to allow definition of a vapor pressure-temperature correlation. Values determined should be based on temperature selections such that a measurement is made at 25°C (as recommended by IUPAC) (1),2 a value can be interpolated for 25°C, or a value can be reliably extrapolated for 25°C. Extrapolation to 25°C should be avoided if the temperature range tested includes a value at which a phase change occurs. Extrapolation to 25°C over a range larger than 10°C should also be avoided. If possible, the temperatures investigated should be above and below 25°C to avoid extrapolation altogether. The gas saturation method was selected because of its extended range, simplicity, and general applicability (2). Examples of results produced by the gas-saturation procedure during an interlaboratory evaluation are given in Table 1. These data have been taken from Reference (3).  (A) Sr is the estimated standard deviation within laboratories, that is, an average of the repeatability found in the separate laboratories.(B) SR is the square root of the component of variance between laboratories.(C) SR is the between-laboratory estimate of precision.  
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 problems, 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.

SIGNIFICANCE AND USE
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.
SCOPE
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.

SIGNIFICANCE AND USE
6.1 Explicit consideration of landscape features to characterize the quality of habitat for assessment species can enhance the ecological relevance of an EcoRA. This can help avoid assessing exposure in areas in which a wildlife species would be absent because of a lack of habitat or to bound exposure estimates in areas with low habitat quality. The measure of habitat quality is used in place of the commonly used Area Use Factor (AUF). Greater ecological realism and more informed management decisions can be realized through better use of landscape features to characterize sites.
SCOPE
1.1 Ecological Risk Assessments (EcoRAs) typically focus on valued wildlife populations. Regulatory authority for conducting EcoRAs derives from various federal laws [for example, Comprehensive Environmental Response, Compensation and Liability Act 1981, (CERCLA), Resource Conservation Recovery Act (RCRA), and Federal Insecticide, Fungicide, and Rodenticide Act, (FIFRA)]. Certain procedures for conducting EcoRAs (1-4)2 have been standardized [E1689-95(2003) Standard Guide for Developing Conceptual Site Models for Contaminated Sites; E1848-96(2003) Standard Guide for Selecting and Using Ecological Endpoints for Contaminated Sites; E2020-99a Standard Guide for Data and Information Options for Conducting an Ecological Risk Assessment at Contaminated Sites; E2205/E2205M-02 Standard Guide for Risk-Based Corrective Action for Protection of Ecological resources; E1739-95(2002) Standard Guide for Risk-Based Corrective Action Applied at Petroleum Release Sites]. Specialized cases for reporting data have also been standardized [E1849-96(2002) Standard Guide for Fish and Wildlife Incident Monitoring and Reporting] as have sampling procedures to characterize vegetation [E1923-97(2003) Standard Guide for Sampling Terrestrial and Wetlands Vegetation].  
1.2 Most states have enacted laws modeled after the federal acts and follow similar procedures. Typically, estimates of likely exposure levels to constituents of potential concern (CoPC) are compared to toxicity benchmark values or concentration-response profiles to establish the magnitude of risk posed by the CoPC and to inform risk managers considering potential mitigation/remediation options. The likelihood of exposure is influenced greatly by the foraging behavior and residence time of the animals of interest in the areas containing significant concentrations of the CoPC. Foraging behavior and residence time of the animals are related to landscape features (vegetation and physiognomy) that comprise suitable habitat for the species. This guide presents a framework for incorporating habitat quality into the calculation of exposure levels for use in EcoRAs.  
1.3 This guide is intended only as a framework for using measures of habitat quality in species specific habitat suitability models to assist with the calculation of exposure levels in EcoRA. Information from published Habitat Suitability Index (HSI) models (5) is used in this guide. The user should become familiar with the strengths and limitations of any particular HSI model used in order to characterize uncertainty in the exposure assessment (5-7). For species that do not have published habitat suitability models, the user may elect to develop broad categorical descriptions of habitat quality for use in estimating exposure.

SIGNIFICANCE AND USE
5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. A life-cycle toxicity test is conducted to determine what changes in the numbers and weights of individuals of the test species result from effects of the test material on survival, growth, and reproduction. Information might also be obtained on effects of the material on the health and uses of the species.  
5.2 Results of life-cycle tests with mysids might be used to predict long-term effects likely to occur on mysids in field situations as a result of exposure under comparable conditions.  
5.3 Results of life-cycle tests with mysids might be used to compare the chronic sensitivities of different species and the chronic toxicities of different materials, and also to study the effects of various environmental factors on results of such tests.  
5.4 Results of life-cycle tests with mysids 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).4  
5.5 Results of a life-cycle test with mysids 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 (2). Most such predictions take into account results of acute toxicity tests, and so the usefulness of the results from a life-cycle test with mysids is greatly increased by also reporting the results of an acute toxicity test (see Guide E729) conducted under the same conditions.  
5.6 Results of life-cycle tests with mysids might be useful for studying the biological availability of, and structure-activity relationships between, test materials.  
5.7 Results of life-cycle tests with mysids might be useful for predicting population effects on the same species in another water or with another species in the same or a different...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a test material added to dilution water, but not to food, on certain species of saltwater mysids during continuous exposure from immediately after birth until after the beginning of reproduction using the flow-through technique. These procedures will probably be useful for conducting life-cycle toxicity tests with other species of mysids, although modifications might be necessary.  
1.2 Other 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. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting life-cycle toxicity tests with saltwater mysids.  
1.3 These procedures are applicable to all 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, and pH and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments, and surface waters.  
1.4 This guide is arranged as follows:    
Section  
Referenced Documents  
2  
Terminology  
3  
Summary of Guide  
4  
Significance and Use  
5  
Hazards  
7  
Apparatus  
6  
Facilities  
6.1  
Construction Materials  
6.2  
Metering System  
6.3  
Test Chambers  
6.4  
Cleaning  
6.5  
Acceptability  
6.6  
Dilution Water  
8  
Requirements  
8.1  
Source  
8.2  
Treatment  
8.3  
Characterizat...

SIGNIFICANCE AND USE
5.1 An acute toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term 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. Bioavailability of the test substance may also differ between real-world exposures and laboratory exposures due to site-specific water quality conditions (see Guides E1192, E1563, and E2455).  
5.2 Results of acute toxicity tests might 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 might be dependent on sorption or settling of the test material onto the substrate.  
5.3 Results of acute 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.  
5.4 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 (3).  
5.5 Results of acute toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.  
5.6 Results of acute toxicity tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.
SCOPE
1.1 This guide (1)2 describes procedures for obtaining laboratory data concerning the adverse effects (for example, lethality and immobility) of a test material added to dilution water, but not to food, on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, usually during 2 to 4-day exposures, depending on the species. These procedures will probably be useful for conducting acute toxicity tests with many other aquatic species, although modifications might be necessary.  
1.2 Other modifications of these procedures might be justified by special needs or circumstances such as meeting specific study goals, regulatory needs, or to accommodate specific test organism life stages. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual or novel procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests.  
1.3 This guide describes tests using three basic exposure techniques: static, renewal, and flow-through. Selection of the technique to use in a specific situation will depend on the needs of the investigator and on available resources. Tests using the static technique provide the most easily obtained measure of acute toxicity, but conditions often change substantially during static tests; therefore, static tests should not last longer than 96 h, and test organisms should not be fed during such tests unless the test organisms are severely stressed without feeding over 48 h. Static tests should probably not be conducted on 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. Because the pH and concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed, tests using ren...

SIGNIFICANCE AND USE
5.1 An important goal of aquatic toxicology is to determine the effects of toxic compounds on species that play a central role in aquatic communities. Rotifers have a major impact on several important ecological processes in freshwater and coastal marine environments. As filter-feeders on phytoplankton and bacteria, rotifers exert substantial grazing pressure that at times exceeds that of the larger crustacean zooplankton  (1, 2).3 Rotifer grazing on phytoplankton is highly selective  (2-4)  and can influence phytoplankton composition, the coexistence of competitors, and overall water quality (5). The contribution of rotifers to the secondary production of many aquatic communities is substantial (6-9). In fresh water, rotifers often account for the major fraction of zooplankton biomass at certain times of the year (10, 11) . Rotifers and other zooplankton are a significant food source for many larval fish, planktivorous adult fish (12, 13), and several invertebrate predators  (14-16) . The high metabolic rates of rotifers contribute to their role in nutrient cycling, which might make rotifers more important than crustaceans in certain communities (17, 18) .  
5.2 In addition to their important ecological role in aquatic communities, rotifers are attractive organisms for toxicological studies because an extensive database exists on the basic biology of this group. Techniques have been published for the culture of many rotifer species (3, 19). The rotifer life cycle is well defined (20, 21), and the factors regulating it are reasonably well understood (22-25). Several aspects of rotifer behavior have been examined closely (26-29). The biogeography of many rotifer species has been characterized (30, 31), and the systematics of the group are well described (32, 33).  
5.3 Toxicity tests with rotifers of the genus Brachionus are more easily performed than with many other aquatic animals because of their rapid reproduction, short generation times, sensitivity (34), and ...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the acute toxicity of chemicals and aqueous effluents released into fresh, estuarine, or marine waters. Acute toxicity is measured by exposing  Brachionus  newly hatched from cysts to a series of toxicant concentrations under controlled conditions. This guide describes a test for using  B. calyciflorus, a fresh water rotifer, and the Appendix describes modifications of this test for estuarine and marine waters using  B. plicatilis. These procedures lead to an estimation of acute toxicity, including the concentration expected to kill 50 % of the test rotifers (LC50) in 24 h. Procedures not specifically stated in this guide should be conducted in accordance with Guide E729 and Guide E1192.  
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 modified procedures might not be comparable to rotifer acute tests that follow the protocol described here. Comparison of the results using modified procedures might provide useful information concerning new concepts and procedures for conducting acute toxicity tests on chemicals and aqueous effluents.  
1.3 This guide is organized as follows:    
Section    
Scope  
1    
Referenced Documents  
2    
Terminology  
3    
Summary of Guide  
4    
Significance and Use  
5    
Apparatus  
6    
Dilution Water  
7    
Hazards  
8    
Test Material  
9    
Test Organisms  
10    
Test Procedure  
11
Calculation of Results  
12    
Acceptability of the Test  
13    
Report  
14    
Keywords  
15  
1.4 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or mixtures. This guide can also be used to conduct investigations of the...