1、Designation: E 1440 91 (Reapproved 2004)Standard Guide forAcute Toxicity Test with the Rotifer Brachionus1This standard is issued under the fixed designation E 1440 ; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last
2、 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 acute toxicity of chemicals and aqueousefflue
3、nts released into fresh, estuarine, or marine waters. Acutetoxicity is measured by exposing Brachionus newly hatchedfrom cysts to a series of toxicant concentrations under con-trolled conditions. This guide describes a test for using B.calyciflorus, a fresh water rotifer, and the Appendix describesm
4、odifications of this test for estuarine and marine waters usingB. plicatilis. These procedures lead to an estimation of acutetoxicity, including the concentration expected to kill 50 % ofthe test rotifers (LC50) in 24 h. Procedures not specificallystated in this guide should be conducted in accordan
5、ce withGuide E 729 and Guide E 1192.1.2 Modifications of these procedures might be justified byspecial needs or circumstances. Although using appropriateprocedures is more important than following prescribed pro-cedures, the results of tests conducted using modified proce-dures might not be comparab
6、le to rotifer acute tests that followthe protocol described here. Comparison of the results usingmodified procedures might provide useful information con-cerning new concepts and procedures for conducting acutetoxicity tests on chemicals and aqueous effluents.1.3 This guide is organized as follows:S
7、ectionScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Dilution Water 7Hazards 8Test Material 9Test Organisms 10Test Procedure 11Calculation of Results 12Acceptability of the Test 13Report 14Keywords 151.4 These procedures are applicable to most chemicals,
8、either individually or in formulations, commercial products, ormixtures. This guide can also be used to conduct investigationsof the effects on rotifer survival of pH, hardness, and salinityand on materials such as aqueous effluents, leachates, oils,particulate matter, sediments, and surface waters.
9、 This guidemight not be appropriate for materials with high oxygendemand, with high volatility, subject to rapid biological orchemical transformation, or that readily sorb to test chambers.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is
10、 theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. For specific hazardsstatements, see Section 8.2. Referenced Documents2.1 ASTM Standards:2E 380 Practice for Use of the Intern
11、ational System of Units(SI) (the Modernized Metric System)E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-ians3E 943 Terminology Relating to Biological Effects and En-vironmental Fate3E 1192 Guide for Conducting Acute Toxicity Tests onAque
12、ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and Amphibians33. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 rotifer cysta rotifer embryo arrested at an early stagein development, enclosed in an envelope and resistant todesiccation and temperature extremes.
13、 Rotifer cysts are oftenincorrectly referred to as resting eggs. Upon hydration, embry-onic development resumes until a neonate female emergesfrom the cyst.1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of Subc
14、ommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004.Originally ap-proved in 1991. Last previous edition approved in 1998 as E 1440 91 (1998).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Serv
15、ice at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.2 rotifer neonatea newly hatc
16、hed, freely swimmingrotifer. All neonates hatched from cysts are females.3.1.3 straina geographically identified population of asingle species. Strains are usually separated by considerabledistances and can be characterized genetically through isozymeanalysis or physiologically by their population d
17、ynamics andsensitivity to toxicants.3.1.4 The words “must,” “should,” “may,” “can,” and“might” have very specific meanings in this guide. “Must” isused to express an absolute requirement, that is, to state that thetest ought to be designed to satisfy the specified condition,unless the purpose of the
18、 test requires a different design.“Must” is used only in connection with factors directly relatingto the acceptability of the test (see 13.1). “Should” is used tostate that the specified condition is recommended and ought tobe met if possible. Although violation of one “should” state-ment is rarely
19、a serious matter, violation of several will oftenrender the results questionable. Terms such as “is desirable,”“is often desirable,” and “might be desirable” are used inconnection with less important factors. “May” is used to mean“is (are) allowed to,” “can” is used to mean “is (are) able to,”and “m
20、ight” is used to mean “could possibly.” Thus, the classicdistinction between “may” and “can” is preserved, and “might”is never used as a synonym for either “may” or “can.”4. Summary of Guide4.1 Rotifer cysts are induced to hatch in 16 to 22 h byincubating them at 25C in standard dilution water. Thes
21、eneonates are then exposed immediately to two or moreconcentrations of test material plus a control in covered dishes.After 24 h, the percent of dead animals in each dish is recorded.An appropriate statistical method is used to calculate an LC50or some other appropriate endpoint.5. Significance and
22、Use5.1 An important goal of aquatic toxicology is to determinethe effects of toxic compounds on species that play a centralrole in aquatic communities. Rotifers have a major impact onseveral important ecological processes in freshwater andcoastal marine environments. As filter-feeders on phytoplank-
23、ton and bacteria, rotifers exert substantial grazing pressure thatat times exceeds that of the larger crustacean zooplankton (1,2).4Rotifer grazing on phytoplankton is highly selective (2-4)and can influence phytoplankton composition, the coexistenceof competitors, and overall water quality (5). The
24、 contributionof rotifers to the secondary production of many aquaticcommunities is substantial (6-9). In fresh water, rotifers oftenaccount for the major fraction of zooplankton biomass atcertain times of the year (10, 11). Rotifers and other zooplank-ton are a significant food source for many larva
25、l fish, plank-tivorous adult fish (12, 13), and several invertebrate predators(14-16). The high metabolic rates of rotifers contribute to theirrole in nutrient cycling, which might make rotifers moreimportant than crustaceans in certain communities (17, 18).5.2 In addition to their important ecologi
26、cal role in aquaticcommunities, rotifers are attractive organisms for toxicologicalstudies because an extensive database exists on the basicbiology of this group. Techniques have been published for theculture of many rotifer species (3, 19). The rotifer life cycle iswell defined (20, 21), and the fa
27、ctors regulating it are reason-ably well understood (22-25). Several aspects of rotifer behav-ior have been examined closely (26-29). The biogeography ofmany rotifer species has been characterized (30, 31), and thesystematics of the group are well described (32, 33).5.3 Toxicity tests with rotifers
28、of the genus Brachionus aremore easily performed than with many other aquatic animalsbecause of their rapid reproduction, short generation times,sensitivity (34), and the commercial availability of rotifercysts. Brachionus spp. have a cosmopolitan distribution thatspans six continents (31), and they
29、 are ecologically importantmembers of many aquatic communities impacted by pollution.The use of B. plicatilis in an acute toxicity test for estuarineand marine environments and B. rubens in fresh water has beendescribed, as well as their sensitivity to several toxicants (35,36).5.4 The test describe
30、d here is fast, easy to execute, sensitive,and cost-effective. Obtaining test animals from cysts greatlyreduces some of the major problems in routine aquatic toxico-logical testing such as the limited availability of test animalsand the inconsistency of sensitivity over time. Rotifers hatchedfrom cy
31、sts are of similar age and are physiologically uniform,thus eliminating pre-test conditions as a source of variability inthe toxicity test. Cysts can be shipped inexpensively world-wide, allowing all laboratories to use standard, geneticallydefined strains that have been calibrated with reference to
32、xi-cants. The convenience of an off-the-shelf source of testanimals that require no pre-conditioning is likely to permit newapplications of aquatic toxicity tests.5.5 Sensitivity to toxicants is compound and species spe-cific, but the sensitivity of B. calyciflorus is generally compa-rable to that o
33、f Daphnia (37).5.6 Rotifer cysts are commercially available, but they canalso be obtained from natural populations and from laboratorycultures. Techniques for rotifer cyst production in laboratorypopulations have been described (24, 25, 38, 39). However,using a well-characterized rotifer strain is b
34、est since strains areknown to have differing toxicant sensitivities.6. Apparatus6.1 Laboratory FacilitiesPreparation of the test, storageof the dilution water, and all stages of the test procedure shouldtake place in an atmosphere free from dust and toxic vapors.6.2 EquipmentThe equipment required f
35、or this test in-cludes: a constant temperature bath or environmental chambercapable of maintaining 25C, petri dishes with covers ormultiwell tissue culture plates, micropipets with smoothedopenings, test tubes or petri dishes for hatching cysts, astereomicroscope capable of 10 to 153 magnification,
36、and a 20to 40 W fluorescent light.7. Dilution Water7.1 Reconstituted fresh water is prepared with high-qualitydeionized or distilled water to which 96 mg of NaHCO3,60mg4The boldface numbers in parentheses refer to the list of references at the end ofthis standard.E 1440 91 (2004)2CaSO42H2O, 60 mg Mg
37、SO47H2O, and 4 mg KCl are addedper litre (40). This moderately hard dilution water (with ahardness of 80 to 100 mg CaCO3per litre and alkalinity of 60to 70 mg per litre) is stirred for 24 h and adjusted to pH 7.5using concentrated hydrochloric acid or sodium hydroxide.This dilution water may be used
38、 for up to seven days, but thenit should be discarded. The dissolved oxygen content should beat least 90 % of saturation at the beginning of the test.Unexpected and inconsistent results can often be traced toproblems with the dilution water, so it should be prepared andstored very carefully.7.2 Othe
39、r reconstituted dilution waters may be used asdescribed in Guide E 729. In addition, natural dilution watersometimes might be desirable (Guide E 729). Cyst hatchingand LC50s in these dilution waters might differ from thosepreviously reported (37).8. Hazards8.1 Many materials can affect humans advers
40、ely if precau-tions are inadequate. Therefore, skin contact with all testmaterials and solutions should be minimized by wearingappropriate protective gloves, especially when washing equip-ment or putting hands in test solutions. Laboratory coats,aprons, and protective glasses should always be worn,
41、andpipets should be used to remove organisms from test solutions.Special precautions, such as covering test chambers andventilating the area surrounding the chambers, should be takenwhen conducting tests on volatile materials. Information ontoxicity to humans (41-45), recommended handling procedures
42、(46-49), and chemical and physical properties of the testmaterial should be studied before a test is begun. Specialprocedures might be necessary with radiolabeled test materials(50, 51) and with test materials that are, or are suspected ofbeing, carcinogenic (52).8.2 Although the disposal of stock s
43、olutions, test solutions,and test organisms poses no special problems in most cases,health and safety precautions and applicable regulations shouldbe considered before beginning a test. Removal or degradationof the test material might be desirable before disposal of thestock and test solutions.8.3 C
44、leaning of equipment with a volatile solvent such asacetone should be performed only in a well-ventilated area inwhich no smoking is allowed and no open flame, such as a pilotlight, is present.8.4 An acidic solution should not be mixed with a hypochlo-rite solution because hazardous fumes might be p
45、roduced.8.5 To prepare dilute acid solutions, concentrated acidshould be added to water, not vice versa. Opening a bottle ofconcentrated acid and adding concentrated acid to water shouldbe performed only in a well-ventilated area.8.6 Becuase water is such a good conductor of electricity,ground fault
46、 systems and leak detectors should be used to helpavoid electrical shocks.9. Test Material9.1 Single ChemicalGuide E 729, sections on stock solu-tions, solvents, solvent controls, and test concentrations applyto this test.9.2 EffluentsGuide E 1192, sections on collection, preser-vation, treatment, a
47、nd test concentrations of effluents, apply tothis test.10. Test Organisms10.1 Test animals are obtained by hatching cysts. Rotifercyst hatching should be initiated approximately 16 h before thestart of the toxicity test. Hatching is initiated by placing B.calyciflorus cysts in the dilution water (se
48、e 7.1) and incubatingat 25C at an illumination level of 1000 to 3000 lux. Hatchingshould begin after approximately 15 h, and by 20 h approxi-mately 50 % of the cysts should have hatched. A hatchingpercent of 50 % is common. Cooler temperatures, low or highpH, low light, elevated hardness, and alkali
49、nity can all delayhatching. If hatching is delayed, the cysts should be checkedhourly to ensure collection of the test animals within 0 to 2 hof hatching. It is important to obtain 0 to 2-h-old animals forthe test because there is no feeding during the toxicity test.Consequently, food deprivation begins to cause mortality afterabout 32 h at 25C. If rotifers are older than 32 h at the end ofthe test, excessive control mortality might result.11. Test Procedure11.1 Experimental Design:11.1.1 Decisions concerning aspects of the experimentaldesign, such as the dilution factor
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