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 variables on the toxicity of a chemical substance.5.8 Results of behavioral toxicity tests can be useful in guiding decisions regarding the extent of remedial action needed for contaminated aquatic and terrestrial sites.5.9 The behavioral characteristics of a particular organism need to be understood and defined before a response can be used as a measure of toxicity (11). Swimming, feeding, and social behavior varies among species as well as among life stages within a species; the most effective test methods are therefore those tailored to a particular life stage of a single species. The range of variability of any behavioral response of unexposed organisms is influenced by genetic, experiential, physiological, and environmental factors. It is thus important to avoid selecting test organisms from populations that may vary in these factors.5.10 Results of behavioral toxicity tests will depend on the behavioral response measured, testing conditions, water quality, species, genetic strain, life stage, health, and condition of test organisms. The behavioral response may therefore be affected by the test environment.5.11 No numerical value or range of values has been defined as the norm for swimming, feeding, or social behavior for any fish; the detection of abnormal activity is therefore based on comparisons of the responses of exposed fish, either with activity measured during a baseline or pre-exposure period or observations of fish under a control treatment (10).5.12 These measures are incorporated readily into standard toxicity test protocols, with minimal stress to the test organism.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  1Referenced Documents  2Terminology  3Summary of Guide  4  5Interferences  6Safety Precautions  7Responses Measured  8Test Organisms  9Facility 10Qualitative Behavioral Assessment Method 11Quantitative Behavioral Measurements 12Experimental Design 13Calculation of Test Results 14Report 151.6 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 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. While some safety considerations are included in this guide, it is beyond the scope of this guide to encompass all safety requirements necessary to conduct behavioral toxicity tests. Specific hazards statements are given in Section 7.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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This practice covers determination of the quantitative and qualitative species composition of fish in a specified area. The successful use of this technique is dependent on: (1) preventing fish from escaping the sample area and (2) retrieving all affected fish, which may take up to three days. This practice is useful in both short- and long-term studies for management and impact assessment purposes. The sample area is blocked off with a small mesh net(s) and the volume of water to be treated is calculated. The required quantity of rotenone is diluted and distributed throughout the water column in the sample area. All fish should be affected and they should be collected for processing.1.1 This practice covers determination of the quantitative and qualitative species composition of fish in a specified area. The successful use of this technique is dependent on: (1) preventing fish from escaping the sample area and (2) retrieving all affected fish, which may take up to three days.1.2 Advantages: 1.2.1 Easily detoxified.1.2.2 All native freshwater fish are susceptible, but it has low toxicity to mammals and birds.1.2.3 At low concentrations fish toxicity depends on species, age, and size.1.2.4 The suffocating action is reversible.1.3 Limitations: 1.3.1 It is less effective in cold (below 20°C) and highly alkaline water.1.3.2 Smaller fish and those without air bladders usually do not float.1.3.3 Completely random selection of sample areas is not possible.1.3.4 Overkill beyond sample area can sometimes occur.1.3.5 Food web organisms may be eliminated.1.4 Applications—This practice is useful in both short- and long-term studies for management and impact assessment purposes. It is adaptable to both lotic and lentic situations in littoral and limnetic areas.1.5 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.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For specific hazards, see Section 7.1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Responses that reflect oxygen consumption or utilization have often been targeted as useful indicators of incipient toxic conditions (26, 27, 28, 29, 30). In addition, sustained acute fish ventilatory behavioral responses reflect a physiological change in the organism and therefore might have ecological relevance.5.2 For some time, the technological means have been available to log and display ventilatory signals over time. As a result, there are a considerable number of studies which examined ventilatory behavior of fish and other aquatic organisms. A large number of substances at lethal levels have been shown to elicit ventilatory responses relatively quickly (13, 19, 20, 31, 32, 33, 34). For many pollutants, a significant response was often generated in less than 1 h of exposure to concentrations approaching the 96 h LC50. Studies performed using subacutely toxic samples of effluents or individual pollutants (concentrations well below the reported LC50 concentration), often documented responses within 1 to 10 h of exposure (11, 18, 21, 30, 35, 36) .5.3 Given the data obtained thus far, it appears that fish ventilatory behavior may be a very sensitive and rapid indicator of acute toxicity if various aspects of this behavior (that is, rate and amplitude) are assessed and analyzed simultaneously. It appears that the more aspects of ventilatory behavior that are assessed, the more sensitive and rapid the system is (11, 12, 21, 22).5.4 Although a variety of organisms have been examined including crayfish (37), aquatic insect larvae (31), and bivalves (13), most research in aquatic ventilatory behavior has used freshwater fish species. This is largely because fish are generally more ecologically “visible” in their importance in aquatic systems and many species (particularly the salmonids and centrarchids) have large opercular flaps that yield relatively clear ventilatory signals for measurement and evaluation. Species eliciting relatively small bioelectric ventilatory signals are more difficult to use given the electrode and amplification systems referenced in this guide.5.5 Changes in ventilatory behavior have been shown to be a reliable indicator of accidental toxic spills or “slugs” of pollutants in wastewater and drinking water systems (15, 20, 23, 24, 33).1.1 This guide covers information on methods to measure and interpret ventilatory behavioral responses of freshwater fish to contaminants.1.2 Ventilatory responses are often some of the first prelethal symptoms exhibited by animals to environmental stressors (1, 2, 3, 4, 5, 6, 7, 8, 9, 10).2 Continued, abnormal ventilatory behavior (that is, rapid or shallow breathing, erratic breathing) can indicate physiological damage that may be irreversible. Such damage could eventually result in decreased survival, growth, or reproduction of the organism, or all of these.1.3 Ventilatory responses of some fish species can be measured relatively easily and quickly, providing a useful tool for biomonitoring studies of wastewaters, pure chemicals, surface water, and ground water.1.4 Appropriate studies of ventilatory responses can yield definitive endpoints such as no observable effect concentration (NOEC) or an EC50, often more rapidly than standard toxicity test methods (11, 12).1.5 The mode of action of test substances and the type of chemical toxicant can be determined by examining ventilatory behavioral responses in conjunction with other physiological responses (8, 9, 10, 11, 12).1.6 Fish ventilatory behavior can be assessed in real-time using appropriate computer hardware and software (12, 13, 14, 15, 16, 17, 18, 19) . Such systems have proved useful for long-term, on-line monitoring of wastewater effluents, pure chemicals, and surface waters (12, 15, 20, 21, 22, 23, 24, 25) . These systems are usually technically complex and will not be discussed in this guide.1.7 Given the technological constraints of electrical components, it is currently not feasible to monitor bioelectric signals, such as those elicited in ventilatory behavior, in saline (>2 ppt) or high conductivity (>3000 μmhos/cm) water using the procedures discussed in this guide. Therefore, this guide is restricted to the testing of freshwater matrices.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific safety precautions, see Section 6.1.9 This guide is arranged as follows:  Section Number  1Referenced Documents  2Terminology  3Summary of Guide  4  5Safety Precautions  6Responses Measured  7Test System  8Test Procedure  9Data Collection and Analysis 10Interferences 11Documentation 12References 13

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4.1 Fish sampling includes a number of lethal and non-lethal practices.4.2 This guide provides an overview of commonly used fish sampling practices.4.3 This summary serves as a brief accounting of options available to personnel responsible for determining the fish sampling practice or practices that best serve the sampling objectives.1.1 This guide covers the use of lethal and non-lethal collection practices for fish.1.1.1 Lethal practices include the use of rotenone and antimycin which are used to collect or eradicate fish; numerous chemicals have been used but presently only rotenone and antimycin are U.S. Environmental Protection Agency (EPA)-approved for this use.1.2 Non-lethal collection practices typically do not cause mortality to fish.1.2.1 Non-lethal practices include surface or bank observation, underwater observation, gill netting, beach seines, hoop nets, fyke nets, trap nets, electroshocking, minnow traps, enclosure (pop drop and throw) traps, angler surveys, commercial surveys.1.3 The focus of this guide is to provide sampling practices for fish collection. This standard does not cover the identification of species or any statistical methods for the sampling data.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this 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. Refer to the MSDSs for all chemicals used in this procedure.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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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.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.21.2 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.

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5.1 This procedure is used to determine the effects of water-related contaminants on the odor and taste of exposed fish. This procedure may be used as evidence in showing compliance with regulatory procedures.5.2 This guide is designed for use by fish processors or research laboratories for evaluations by a trained and monitored sensory panel under the supervision of a sensory professional.1.1 This guide covers procedures for determination of the effects of water-related contaminants on the odor and taste of live fish or fishery products after alleged exposure where flavor impairment is a suspected issue.1.2 This guide addresses safety, harvested quality, sample preparation, assessor selection and training, testing procedures with assessor instructions, as well as test environment and parameters.1.3 This guide is applicable to product categories from aquaculture and wild-caught sectors. The range of contaminated products could be from a small-scale water source, such as an estuary, or a limited river system, to a large-scale source, such as a bay, gulf or portion of an ocean. For details on how these methods compare to field- or laboratory-exposed fishery samples, see Ref (1).21.4 Also covered in this guide are fish species, harvest method (wild-caught versus aquaculture/farmed fish), post-harvest handling, processing methods, and storage.1.5 This guide provides suggested procedures and is not meant to exclude alternate procedures that may be effective. It also does not address all of the nuances of testing throughout the world. It is the responsibility of the user to be aware of their local guidelines and apply them as needed. Some useful resources are also cited in this guide.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 safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazards statements are given in Section 7.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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