1、Designation: E 2317 04Standard Guide forConducting Renewal Microplate-Based Life-Cycle ToxicityTests with a Marine Meiobenthic Copepod1This standard is issued under the fixed designation E 2317; the number immediately following the designation indicates the year oforiginal adoption or, in the case o
2、f revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide describes procedures for obtaining laboratorydata concerning the adverse effects o
3、f 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 are checked and
4、 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) brood extrusi
5、on(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-cycle toxicity test
6、 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 chemicals,eith
7、er 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 materialsas aqueous ef
8、fluents (see also Guide E 1192), 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 test solutionsin
9、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 solutions moreoft
10、en.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 use.2. Referenc
11、ed Documents2.1 ASTM Standards:2E 380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)E 729 Guide for Conducting Acute Toxicity Tests withFishes, Macroinvertebrates, and AmphibiansE 943 Terminology Relating to Biological Effects and En-vironmental FateE 1023 G
12、uide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1191 Guide for Conducting Life-Cycle Toxicity Testswith Saltwater MysidsE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Effluents with Fishes, Macroinvertebrates, andAmphibiansE 1218 Guide for Conducting Stati
13、c 96-h Toxicity Testswith MicroalgaeE 1847 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 to express an absolute requirement,that i
14、s, to state that the test ought to be designed to satisfy the1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Pub
15、lished July 2004.2For 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.1Copyright ASTM International, 100 Barr Harb
16、or Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.specified condition, unless the purpose of the test requires adifferent design. “Must” 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 t
17、hat the specified condition isrecommended and ought to be met if possible. Althoughviolation of one “should” is rarely a serious matter, violation ofseveral will often render the results questionable. Terms suchas “is desirable,” “is often desirable,” and “might be desirable”are used in connection w
18、ith 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 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 d
19、efinitions of other terms in this guide, refer toGuide E 729, Terminology E 943, and Guide E 1023. For anexplanation of units and symbols, refer to Practice E 380.4. Summary of Guide4.1 In each of one or more treatments and a control(s),individually isolated A. tenuiremis are maintained and reared i
20、n144 or more individual microwell (300 L total volume) testchambers from immediately after birth (less than 24-h old)until sexual maturity and production of progeny. Microwells(test chambers) are dispersed among at least three replicate96-well microplates per treatment. Number of treatments, testcha
21、mbers 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. Normally, five sublethaltreatment concentrations that cause # 10 % acute naupliarmortality may be selected based on an acute 96-h range finding
22、test with 20 nauplii in duplicate microplates over a log-orderseries of test chemical concentrations spanning zero mortalityto 100 % mortality. Beginning with the maximum 10 % lethal-ity concentration, test concentrations normally should decreaseby 40 % with each successively lower treatment. In eac
23、h of oneor more control treatments, at least 144 individually isolatedcopepods are maintained in seawater containing the carriersolvent where appropriate but to 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 qua
24、lity of thecopepods and the suitability of the seawater, food, test condi-tions, handling procedures, etc.; and (b) the basis for interpret-ing data obtained from the other treatments. In each of one ormore other treatments, at least 144 individual copepods aremaintained in seawater to which a selec
25、ted concentration oftest material has been added. Copepod survival and the numberof days it takes for development from a first stage nauplius toa reproductively mature copepod are checked and recordeddaily. Once copepods have matured to adults, the gender ofeach copepod is determined, and individual
26、 male:female mat-ing pairs are allowed to mate for twelve days in new, isolatedmicrowell test chambers with seawater containing solvent(where appropriate) (control) or test material. The solutions inthe microwell test chambers during reproduction are renewedevery third day as appropriate under 1.2.
27、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 provide a sufficient six day supply of food eventhough microwell test solutions are renewed every third day.After single pairing of adult male
28、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 brood, number of days between first (and subsequent)brood extrusion(s) and hatching of first- (and subsequent)brood nauplii, number of hatche
29、d and surviving nauplii,number of unhatched or necrotic eggs and aborted eggsacs, andthe total number of females able to produce viable offspringover the entire mating period. The test is terminated 12 dayspast the median time of first brood release in the controltreatment(s) to allow for delays in
30、first brood release bycopepods exposed to the test material. This microplate-basedfull life-cycle toxicity test has a duration of approximately 24days at 25C. Specified data on the concentration of the testmaterial in test solutions and the survival, growth, genderratios, and reproduction of each co
31、pepod pair are obtained andanalyzed to determine the effect(s) of the test material onstage-specific survival, development rates, gender ratios, fer-tility, and reproduction of the test organisms.5. Significance and Use5.1 Protection of a species requires prevention of unaccept-able effects on the n
32、umber, health, and uses of individuals ofthat species. A life-cycle toxicity test is conducted to determinechanges in the numbers of individuals and offspring of a testspecies resulting from effects of the test material on survival,growth, gender ratios, endocrine function, genetic expression,fertil
33、ity 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 sensitivities of severalmeiobenthic copepods to several common metals and organictoxicants have been reviewed (5). For most compounds
34、tested/published to date, A. tenuiremis is acutely less sensitive thanmysid and penaeid shrimp, similarly sensitive as amphipods,and often more sensitive than cladocerans (daphniids, specifi-cally). Reference 96-h aqueous toxicity tests with cadmium at30 g/kg salinity showed an LC50 for A. tenuiremi
35、s 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. tenuiremis is a comparativelynew toxicity test organism, and an extensive database ofspecies sensitivity to multiple aqueous test compounds
36、is notyet available. Relative to other harpacticoid copepod studies inthe literature, A. tenuiremis is more chronically sensitive thanall other species published to date where there is comparativedata (5).5.3 Results of life-cycle tests with A. tenuiremis can be usedto predict long-term effects at t
37、he 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 tests with A. tenuiremis might beused to compare the chronic sensitivities of different species3The boldface numbers in parentheses re
38、fer to the list of references at the end ofthis standard.E2317042and the chronic toxicities of different materials, and also studythe effects of various environmental factors such as tempera-ture, pH, and ultraviolet light on results of such tests.5.5 Results of life-cycle tests with A. tenuiremis m
39、ight be animportant consideration when assessing the hazards of materi-als to aquatic organisms (see Guide E 1023) or when derivingwater quality criteria for aquatic organisms (6).5.6 Results of a life-cycle test with A. tenuiremis might beuseful for predicting the results of chronic tests on the sa
40、me 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 acute toxicity tests, and so theusefulness of the results from a life-cycle toxicity test with A.tenuiremis is greatly increased by also r
41、eporting the results ofan acute toxicity test (see Guide E 729) conducted under thesame environmental conditions.5.7 Results of life-cycle tests with A. tenuiremis might beuseful for studying the biological availability of, and structure-activity relationships between, test materials.5.8 Results of
42、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-cycle tests with A. tenuiremis are conducted oncopepods reared individually in microwells of 96-well micro-plates. Thus they can be useful f
43、or studying endocrine, pre-zygotic and gender-specific toxicities of test materials (1-3).6. Apparatus6.1 FacilitiesFlow-through seawater:sediment brood-stock cultures and static-renewal, but not flow-through, mi-crowell test chambers should be maintained in constant-temperature areas or in incubato
44、rs. 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 recirculating seawater tanks for brood-stock culturesand containers used to prepare test solutions. The headboxshould be equipped for tem
45、perature 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 0.22-m bacterial filter might be desirable. The facilityshould be well ventilated and free of fumes. To further reducethe possibility
46、of contamination by test materials and othersubstances, especially volatile ones, the recirculating seawatertanks for brood-stock cultures should not be in a room wherestock solutions or test solutions are prepared, or equipment iscleaned. During testing, organisms should be shielded fromdisturbance
47、s (that is, maintained within a temperature-regulated incubator) to prevent unnecessary stress. White-lightfluorescent bulbs (40 W) with light output equal to 3150lumens per bulb should be used for culturing and testing. Atiming device within the incubator should be used to provide a12-h light and 1
48、2-h dark photoperiod. A 15- to 30-min 50 %full-intensity light transition period (11) should be providedwhenever the lights go on or off to reduce the possibility ofcopepods being stressed by instantaneous changes in lightintensity.6.2 Construction MaterialsEquipment and facilities thatcontact stock
49、 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 be used whenever possibleto minimize dissolution, leaching, and sorption. Stainless steelshould not be used for tests on metals. High-densit
