1、Designation: E2317 04 (Reapproved 2012)Standard Guide forConducting Renewal Microplate-Based Life-Cycle ToxicityTests with a Marine Meiobenthic Copepod1This standard is issued under the fixed designation E2317; the number immediately following the designation indicates the year oforiginal adoption o
2、r, in the case of revision, the year 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 describes procedures for obtaining laboratorydata concerning the ad
3、verse effects of a test material added toseawater, but not to food, on the marine copepod Amphiascustenuiremis, during continuous exposures of individuals, fromimmediately after birth, until after the beginning of reproduc-tion using a 200 L renewal microplate-culturing technique.The following data
4、are checked and recorded during the testperiod: stage-specific survival, number of days it takes fordevelopment from a first stage nauplius to a reproductivelymature copepod, gender ratios, number of days for a female toextrude first and subsequent broods, number of days betweenfirst (and subsequent
5、) brood extrusion(s) and hatching offirst-generation nauplii, number of hatched and survivingnauplii, number of unhatched or necrotic eggs and abortedunhatching eggsacs, and the total number of females able toproduce viable offspring over the entire mating period. Thismicroplate-based full life-cycl
6、e toxicity test has a duration ofapproximately 17 days for toxicants that do not delay devel-opment. These procedures probably will be useful for conduct-ing life-cycle toxicity tests with other species of copepods,although modifications might be necessary.1.2 These procedures are applicable to most
7、 chemicals,either individually, or in formulations, commercial products, orknown mixtures, that can be measured accurately at thenecessary concentration in water. With appropriate modifica-tions these procedures can be used to conduct tests ontemperature, dissolved oxygen, and pH and on such materia
8、lsas aqueous effluents (see also Guide E1192), sediment porewaters, and surface waters. Renewal microplate tests might notbe applicable to materials that have a high oxygen demand, arehighly volatile, are rapidly transformed (biologically or chemi-cally) in aqueous solutions, or are removed from tes
9、t solutionsin substantial quantities by the test chambers or organismsduring the test. If the concentration of dissolved oxygen fallsbelow 50 % of saturation, or the concentration of test materialin the test solution decreases by more than 20 % betweenrenewals, it might be desirable to renew the sol
10、utions moreoften.1.3 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 safety and health practices and determine the applica-bility of regulatory requirements prior to u
11、se.2. Referenced Documents2.1 ASTM Standards:2E380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)E729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects a
12、nd Envi-ronmental FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE1191 Guide for Conducting Life-Cycle Toxicity Tests withSaltwater MysidsE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates
13、, and AmphibiansE1218 Guide for Conducting Static Toxicity Tests withMicroalgaeE1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM Guidelines3. Terminology3.1 The words “must,” “should,” “may,” “can,” and “might”have very specific meanings in this guide.3.1.1 “Must” is used
14、 to express an absolute requirement,that is, to state that the test ought to be designed to satisfy thespecified condition, unless the purpose of the test requires a1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fateand is the direct responsibility
15、 of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published January 2013. Originallyapproved in 2004. Last previous edition approved in 2004 as E2317 04. DOI:10.1520/E2317-04R12.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orc
16、ontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1different design. “M
17、ust” is used only in connection with factorsthat directly relate to the acceptability of the test (see Section13).3.1.2 “Should” is used to state that the specified condition isrecommended and ought to be met if possible. Althoughviolation of one “should” is rarely a serious matter, violation ofseve
18、ral will often render the results questionable. Terms suchas “is desirable,” “is often desirable,” and “might be desirable”are used in connection with less important factors.3.1.3 “May” is used to mean “is (are) allowed to,” “can,” isused to mean “is (are) able to,” and “might” is used to mean“could
19、 possibly.” Therefore, the classic distinction betweenmay and can is preserved, and might is never used as asynonym for either may or can.3.2 For definitions of other terms in this guide, refer toGuide E729, Terminology E943, and Guide E1023. For anexplanation of units and symbols, refer to Practice
20、 E380.4. Summary of Guide4.1 In each of one or more treatments and a control(s),individually isolated A. tenuiremis are maintained and reared in144 or more individual microwell (300 L total volume) testchambers from immediately after birth (less than 24-h old)until sexual maturity and production of
21、progeny. Microwells(test chambers) are dispersed among at least three replicate96-well microplates per treatment. Number of treatments, testchambers and organisms per treatment should be based on thepurpose of the life-cycle test and the type of data analysis thatis to be used to calculate results.
22、Normally, five sublethaltreatment concentrations that cause 10 % acute naupliarmortality may be selected based on an acute 96-h range findingtest with 20 nauplii in duplicate microplates over a log-orderseries of test chemical concentrations spanning zero mortalityto 100 % mortality. Beginning with
23、the maximum 10 % lethal-ity concentration, test concentrations normally should decreaseby 40 % with each successively lower treatment. In each of oneor more control treatments, at least 144 individually isolatedcopepods are maintained in seawater containing the carriersolvent where appropriate but t
24、o which no test material hasbeen added in order to provide (a) a measure of the accept-ability of the test by giving an indication of the quality of thecopepods and the suitability of the seawater, food, testconditions, handling procedures, etc.; and (b) the basis forinterpreting data obtained from
25、the other treatments. In each ofone or more other treatments, at least 144 individual copepodsare maintained in seawater to which a selected concentration oftest material has been added. Copepod survival and the numberof days it takes for development from a first stage nauplius toa reproductively ma
26、ture copepod are checked and recordeddaily. Once copepods have matured to adults, the gender ofeach copepod is determined, and individual male:female mat-ing pairs are allowed to mate for twelve days in new, isolatedmicrowell test chambers with seawater containing solvent(where appropriate) (control
27、) or test material. The solutions inthe microwell test chambers during reproduction are renewedevery third day as appropriate under 1.2. Copepods are fed a1:1:1 mixed algal cell suspension (107cells/mL) every sixthday. Sufficient numbers of algal cells will settle to the microw-ell bottoms to provid
28、e a sufficient six day supply of food eventhough microwell test solutions are renewed every third day.After single pairing of adult male and female copepods in eachcontrol and treatment solution, each mating pair is checkeddaily for the following: number of days for a female to extrudethe first broo
29、d, number of days between first (and subsequent)brood extrusion(s) and hatching of first- (and subsequent)brood nauplii, number of hatched and surviving nauplii,number of unhatched or necrotic eggs and aborted eggsacs, andthe total number of females able to produce viable offspringover the entire ma
30、ting period. The test is terminated 12 dayspast the median time of first brood release in the controltreatment(s) to allow for delays in first brood release bycopepods exposed to the test material. This microplate-basedfull life-cycle toxicity test has a duration of approximately 24days at 25C. Spec
31、ified data on the concentration of the testmaterial in test solutions and the survival, growth, genderratios, and reproduction of each copepod pair are obtained andanalyzed to determine the effect(s) of the test material onstage-specific survival, development rates, gender ratios,fertility, and repr
32、oduction of the test organisms.5. Significance and Use5.1 Protection of a species requires prevention of unaccept-able effects on the number, health, and uses of individuals ofthat species.Alife-cycle toxicity test is conducted to determinechanges in the numbers of individuals and offspring of a tes
33、tspecies resulting from effects of the test material on survival,growth, gender ratios, endocrine function, genetic expression,fertility and reproduction (1-3).3Information might also beobtained on effects of the material on the health (4) and uses ofthe species.5.2 Published information about the s
34、ensitivities of severalmeiobenthic copepods to several common metals and organictoxicants have been reviewed (5). For most compounds tested/published to date, A. tenuiremis is acutely less sensitive thanmysid and penaeid shrimp, similarly sensitive as amphipods,and often more sensitive than cladocer
35、ans (daphniids, specifi-cally). Reference 96-h aqueous toxicity tests with cadmium at30 g/kg salinity showed an LC50 for A. tenuiremis adults of213 to 234 g/L (Chandler, unpub.). Reference toxicant testswith sodium dodecyl sulfate showed a 96-h LC50 of 13.3 to15.5 mg/L (Chandler,unpubl.). A. tenuire
36、mis is a comparativelynew toxicity test organism, and an extensive database ofspecies sensitivity to multiple aqueous test compounds is notyet available. Relative to other harpacticoid copepod studies inthe literature, A. tenuiremis is more chronically sensitive thanall other species published to da
37、te where there is comparativedata (5).5.3 Results of life-cycle tests with A. tenuiremis can be usedto predict long-term effects at the individual and populationlevels likely to occur on copepods in field situations as a resultof exposure under comparable conditions (1,2).5.4 Results of life-cycle t
38、ests with A. tenuiremis might beused to compare the chronic sensitivities of different species3The boldface numbers in parentheses refer to the list of references at the end ofthis standard.E2317 04 (2012)2and the chronic toxicities of different materials, and also studythe effects of various enviro
39、nmental factors such astemperature, pH, and ultraviolet light on results of such tests.5.5 Results of life-cycle tests with A. tenuiremis might be animportant consideration when assessing the hazards of materi-als to aquatic organisms (see Guide E1023) or when derivingwater quality criteria for aqua
40、tic organisms (6).5.6 Results of a life-cycle test with A. tenuiremis might beuseful for predicting the results of chronic tests on the same testmaterial with the same species in another water or with anotherspecies in the same or a different water. Most such predictionstake into account results of
41、acute toxicity tests, and so theusefulness of the results from a life-cycle toxicity test with A.tenuiremis is greatly increased by also reporting the results ofan acute toxicity test (see Guide E729) conducted under thesame environmental conditions.5.7 Results of life-cycle tests with A. tenuiremis
42、 might beuseful for studying the biological availability of, and structure-activity relationships between, test materials.5.8 Results of life-cycle tests with A. tenuiremis will dependon temperature, quality of food, composition of seawater,condition of test organisms, and other factors.5.9 Life-cyc
43、le tests with A. tenuiremis are conducted oncopepods reared individually in microwells of 96-well mi-croplates. Thus they can be useful for studying endocrine,pre-zygotic and gender-specific toxicities of test materials(1-3).6. Apparatus6.1 FacilitiesFlow-through seawater:sediment brood-stock cultur
44、es and static-renewal, but not flow-through, mi-crowell test chambers should be maintained in constant-temperature areas or in incubators. If seawater is not preparedin a batch, it is usually piped directly from the source to anelevated headbox so it can be filtered at 0.45 m and gravity-fed into re
45、circulating seawater tanks for brood-stock culturesand containers used to prepare test solutions. The headboxshould be equipped for temperature control and aeration. Airused for aeration should be free of fumes, oil, and water; filtersto remove oil and water are desirable. Filtration of air througha
46、 0.22-m bacterial filter might be desirable. The facilityshould be well ventilated and free of fumes. To further reducethe possibility of contamination by test materials and othersubstances, especially volatile ones, the recirculating seawatertanks for brood-stock cultures should not be in a room wh
47、erestock solutions or test solutions are prepared, or equipment iscleaned. During testing, organisms should be shielded fromdisturbances (that is, maintained within a temperature-regulated incubator) to prevent unnecessary stress. White-lightfluorescent bulbs (40 W) with light output equal to 3150lu
48、mens per bulb should be used for culturing and testing. Atiming device within the incubator should be used to provide a12-h light and 12-h dark photoperiod. A15- to 30-min 50 %full-intensity light transition period (11) should be providedwhenever the lights go on or off to reduce the possibility ofc
49、opepods being stressed by instantaneous changes in lightintensity.6.2 Construction MaterialsEquipment and facilities thatcontact stock solutions, test solutions, or any seawater intowhich copepods will be placed should not contain substancesthat can be leached or dissolved by aqueous solutions inamounts that adversely affect copepods. In addition, equipmentand facilities that contact stock solutions or test solutionsshould be chosen to minimize sorption of test materials fromwater. Glass, Type 316 stainless steel, nylon, and polytetrafluo-roethylene (PTFE) plastics should b
