1、Designation: E1768 95 (Reapproved 2013)Standard Guide forVentilatory Behavioral Toxicology Testing of FreshwaterFish1This standard is issued under the fixed designation E1768; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the yea
2、r of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers information on methods to measureand interpret ventilatory behavioral responses of freshwaterfi
3、sh to contaminants.1.2 Ventilatory responses are often some of the first prele-thal symptoms exhibited by animals to environmental stressors(1, 2, 3, 4, 5, 6, 7, 8, 9, 10).2Continued, abnormal ventilatorybehavior (that is, rapid or shallow breathing, erratic breathing)can indicate physiological dama
4、ge 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 bemeasured relatively easily and quickly, providing a useful toolfor biomonitoring studies of wastewaters,
5、 pure chemicals,surface water, and ground water.1.4 Appropriate studies of ventilatory responses can yielddefinitive endpoints such as no observable effect concentration(NOEC) or an EC50, often more rapidly than standard toxicitytest methods (11, 12).1.5 The mode of action of test substances and the
6、 type ofchemical toxicant can be determined by examining ventilatorybehavioral responses in conjunction with other physiologicalresponses (8, 9, 10, 11, 12).1.6 Fish ventilatory behavior can be assessed in real-timeusing appropriate computer hardware and software (12, 13, 14,15, 16, 17, 18, 19). Suc
7、h systems have proved useful forlong-term, on-line monitoring of wastewater effluents, purechemicals, and surface waters (12, 15, 20, 21, 22, 23, 24, 25).These systems are usually technically complex and will not bediscussed in this guide.1.7 Given the technological constraints of electricalcomponen
8、ts, it is currently not feasible to monitor bioelectricsignals, such as those elicited in ventilatory behavior, in saline(2 ppt) or high conductivity (3000 mhos/cm) water usingthe procedures discussed in this guide. Therefore, this guide isrestricted to the testing of freshwater matrices.1.8 The val
9、ues stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.9 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate sa
10、fety and health practices and determine the applica-bility of regulatory limitations prior to use. For specific safetyprecautions, see Section 6.1.10 This guide is arranged as follows:SectionNumberScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Safety Precautions 6R
11、esponses Measured 7Test System 8Test Procedure 9Data Collection and Analysis 10Interferences 11Documentation 12References 132. Referenced Documents2.1 ASTM Standards:3E729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Rela
12、ting to Biological Effects and Envi-ronmental FateE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and Amphibians1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fateand is the
13、direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved March 1, 2013. Published March 2013. Originallyapproved in 1995. Last previous edition approved in 2008 as E1768 95(2013).DOI: 10.1520/E1768-95R13.2The boldface numbers given in parentheses refe
14、r to a list of references at theend of the text.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright AST
15、M International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1E1241 Guide for Conducting Early Life-Stage Toxicity Testswith FishesE1604 Guide for Behavioral Testing in Aquatic Toxicology3. Terminology3.1 The words “must,” “ should,” “ may,” “can,” and“might” h
16、ave very specific meanings. “Must” is used to expressan absolute requirement, that is, to state that the test ought tobe designed to satisfy the specified condition, unless thepurpose of the test requires a different design. “Must” is onlyused in connection with the factors that directly relate to t
17、heacceptability of the test. “ Should” is used to state that thespecified condition is recommended and ought to be met ifpossible. Although a violation of one “should” is rarely aserious matter, violation of several will often render the resultsquestionable. Terms such as “is desirable,” “is oftende
18、sirable,” and “might be desirable” are used in connectionwith less important factors. “ May” is used to mean “is (are)allowed to,” “can” is used to mean“ is (are) able to,” and “might” is used to mean “could possibly.” Thus the classicdistinction between “ may” and “can” is preserved, and“might” is
19、never used as a synonym for either “may”or“can.”3.2 Definitions of Terms Specific to This Standard:3.2.1 coughgill purge in fish; when a fish reverses orgreatly increases the flow of water over the gills and back outto the ambient water. Such activity is used to cleanse the gillsby removing particle
20、s or other material on the gill plate(s).3.2.2 electrodedevice (metallic or chemical based) thatreceives bioelectric signals from the organism.3.2.3 ventilationbreathing, respiratory process of organ-ism.3.2.4 waveformrepresentation of analog electrical signaldepicting breathing response of organism
21、 over time, usuallyrepresented on a strip chart recorder or computer monitor.4. Summary of Guide4.1 The potential toxicity of water or a pure chemical inwater is assessed by measuring changes in fish ventilatorybehavior during exposure using a flow-through system. Sig-nificant effects are determined
22、 by comparing specific ventila-tory responses of fish under control conditions with responsesof those same fish during exposure conditions. A set of controlfish may also be used in the test design in order to evaluatenon-toxic changes in ventilatory response over time, particu-larly when longer-term
23、 monitoring is desired.4.2 Ventilatory responses are observed by using non-invasive metallic or chemically-based electrodes, a signalamplification and filtration system, and strip chart recorder (orother recording device) to display the ventilatory waveform. Inshort-term tests (24h in length or in c
24、ontinuous real-time monitoring applications,ventilatory waveform data are aquisitioned, analyzed, andstored via a microcomputer equipped with an analog to digitalprocessor, disk or magnetic tape storage, and appropriatesoftware. With the aid of a computer and analog to digitalboard, responses can be
25、 monitored and analyzed on a real-timebasis. The computer-analyzed response reduces potential sub-jective biases due to manual analysis of strip-chart recordings.5. Significance and Use5.1 Responses that reflect oxygen consumption or utiliza-tion have often been targeted as useful indicators of inci
26、pienttoxic conditions (26, 27, 28, 29, 30). In addition, sustainedacute fish ventilatory behavioral responses reflect a physiologi-cal change in the organism and therefore might have ecologicalrelevance.5.2 For some time, the technological means have beenavailable to log and display ventilatory sign
27、als over time. As aresult, there are a considerable number of studies whichexamined ventilatory behavior of fish and other aquatic organ-isms. A large number of substances at lethal levels have beenshown to elicit ventilatory responses relatively quickly (13, 19,20, 31, 32, 33, 34). For many polluta
28、nts, a significant responsewas often generated in less than1hofexposure to concentra-tions approaching the 96 h LC50. Studies performed usingsubacutely toxic samples of effluents or individual pollutants(concentrations well below the reported LC50 concentration),often documented responses within 1 t
29、o 10 h of exposure (11,18, 21, 30, 35, 36).5.3 Given the data obtained thus far, it appears that fishventilatory behavior may be a very sensitive and rapid indica-tor of acute toxicity if various aspects of this behavior (that is,rate and amplitude) are assessed and analyzed simultaneously.It appear
30、s that the more aspects of ventilatory behavior that areassessed, the more sensitive and rapid the system is (11, 12, 21,22).5.4 Although a variety of organisms have been examinedincluding crayfish (37), aquatic insect larvae (31), and bivalves(13), most research in aquatic ventilatory behavior has
31、usedfreshwater fish species. This is largely because fish are gener-ally more ecologically “visible” in their importance in aquaticsystems and many species (particularly the salmonids andcentrarchids) have large opercular flaps that yield relativelyclear ventilatory signals for measurement and evalu
32、ation.Species eliciting relatively small bioelectric ventilatory signalsare more difficult to use given the electrode and amplificationsystems referenced in this guide.5.5 Changes in ventilatory behavior have been shown to bea reliable indicator of accidental toxic spills or “slugs” ofpollutants in
33、wastewater and drinking water systems (15, 20,23, 24, 33).6. Safety Precautions6.1 Many substances may pose health risks to humans ifadequate precautions are not taken. Information on toxicity tohumans, recommended handling procedures, and chemical andphysical properties of the test material should
34、be studied and allpersonnel informed before an exposure is initiated.(WarningSpecial procedures might be necessary with radio-labeled test materials and with test materials that are, or aresuspected of being carcinogenic.)E1768 95 (2013)26.2 Many materials can adversely affect humans if precau-tions
35、 are inadequate. Contact with test material, sediments, andwater should be minimized. Where appropriate, protectivegloves, laboratory coats, aprons, protective clothing, and safetyglasses should be worn and dip nets, sieves, or tubes should beused to remove test organisms. When handling potentiallyh
36、azardous materials, proper handling procedures may includemanipulating test materials under a ventilated hood or in anenclosed glovebox, enclosing and ventilating the exposurechambers, and use of respirators, aprons, safety glasses andgloves.7. Responses Measured7.1 Ventilatory parameters in fish th
37、at have been shown tobe affected by toxicity include ventilatory rate (opercularmovement over time), depth of ventilation (amplitude), cough-ing or gill purge rate, and erratic episode frequency due tosudden movement of the organism. Most commonly, changesin ventilatory rate (Fv) have been used as a
38、 bioindicator of toxicconditions (11, 12, 13, 19, 20, 21, 30, 31, 33, 34, 35, 36).However, depth of ventilation and cough rate have beenreported to be more sensitive indicators of toxicity for somecompounds (11, 19, 38, 39, 40).7.2 Manually, changes in ventilatory rate are often deter-mined by chang
39、es in the number of peaks per unit area on astrip-chart recording. Depth of ventilation (tidal volume) orsignal amplitude, is measured from top to the bottom of thewaveform (see Fig. 1).7.3 Cough rate has been more difficult to determine becauseseveral different types of coughs may be evident, each
40、with itsown characteristic wave form pattern (see Fig. 1 and (11, 39,40). Also, without the use of video techniques, (11, 41), theactual occurrence of a cough is not always clear. Researcherswho have investigated cough responses have interpreted mostabnormal peaks or pattern changes on a strip-chart
41、 recording asa cough. Work by Diamond et al. (11) however, indicated thatmany of these changes may in fact be due to general activity,and not coughing responses. Various aspects of monitoring fishcoughs have been reviewed by Drummond and Carlson (40).7.4 Erratic episode frequency or activity episode
42、 frequencyhas also proved to be a useful response in some studies (11, 12,21). These episodes are represented on a strip chart recordingas a multi-peak, high frequency (and often high amplitude)cluster of signals which can be easily distinguished from thenormal ventilatory signal (Fig. 1).8. Test Sy
43、stem8.1 Several techniques have been developed to monitor fishventilatory behavior. The simplest and most reliable methodmonitors the bioelectric potentials generated during ventilatorymovements by means of noninvasive electrodes (11, 12, 16, 20,21, 29) or silver/silver chloride (15). These electrod
44、es gener-ally consist of stainless steel wire or screen and are attached tothe monitoring chamber such that the fish is not restrained orstressed (see Fig. 2).8.2 The spatial orientation of the electrodes within themonitoring chamber affects the intensity with which theventilatory signal is received
45、 and recorded. Since reception ofthe bioelectric signal is dependent on there being a polarity orelectrical gradient between the electrodes, electrodes areplaced opposite each other in the monitoring chamber toachieve maximum sensitivity. Several different electrode ar-rangements have been utilized
46、including top and bottom of thechamber (see Fig. 2(a) and (12, 15, 21), front and back (29),and sides of the chamber (see Fig. 2(b) and (11, 14, 20). Eachof these arrangements may have advantages and disadvantagesin terms of signal reception and the ability to detect subtlechanges in amplitude, body
47、 movement, or cough rates. Infor-mation at this time suggests that a top and bottom electrodearrangement (see Fig. 2(a), will reduce ventilatory signalalteration due to changes in fish position relative to theelectrodes in comparison with a side electron orientation (12).Test chambers must be clean
48、prior to testing as described inPractice E729, and made of appropriate construction materialssuch as glass or plexiglass (see Guide E1241 and PracticeE729).8.3 Test organisms and chambers must be isolated so as toreduce external stimuli such as experimenter movement,vibration, and visual cues. This
49、is generally achieved byplacing a single fish in each chamber and by placing opaquedividers between test chambers. The entire system (all testchambers) should be isolated within a light-proof box orcontinuous light compartment.8.4 The electrical signal (microvolts), generated by ventila-tory movements, that is received by the electrodes, must beconditioned prior to use. First, the electrical components of thesystem must be properly grounded to avoid erratic signalreception. Second, electrical noise, particularly that arisingfrom normal 60 cycle electrical current (such as f
