1、Designation: E1440 91 (Reapproved 2012)Standard Guide forAcute Toxicity Test with the Rotifer Brachionus1This standard is issued under the fixed designation E1440; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last re
2、vision. 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 acute toxicity of chemicals and aqueouseffluents
3、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 describesmodifi
4、cations 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 accordance wi
5、thGuide E729 and Guide E1192.1.2 Modifications of these procedures might be justified byspecial needs or circumstances. Although using appropriateprocedures is more important than following prescribedprocedures, the results of tests conducted using modifiedprocedures might not be comparable to rotif
6、er acute tests thatfollow the protocol described here. Comparison of the resultsusing modified procedures might provide useful informationconcerning new concepts and procedures for conducting acutetoxicity tests on chemicals and aqueous effluents.1.3 This guide is organized as follows:SectionScope 1
7、Referenced 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,either indivi
8、dually 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. This guidemi
9、ght 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 theresponsib
10、ility 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:2E380 Practice for Use of the International System
11、 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 and Envi-ronmental FateE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Sampl
12、es and Effluents with Fishes,Macroinvertebrates, and Amphibians3. 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. Rotifer cysts are
13、 often1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fateand is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published December 2012. Originallyapproved in 1991. Last pr
14、evious edition approved in 2004 as E1440 91 (2004).DOI: 10.1520/E1440-91R12.2For 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 onth
15、e ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1incorrectly referred to as resting eggs. Upon hydration, embry-onic development resumes until a neonate female emergesfrom the cyst.3.1.2 rotifer neonatea newly hatched, f
16、reely 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 dynamic
17、s 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 test
18、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 a seri
19、ous 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 “might”
20、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. Theseneona
21、tes 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 Use5.1
22、 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-ton an
23、d bacteria, rotifers exert substantial grazing pressure thatat times exceeds that of the larger crustacean zooplankton (1,2).3Rotifer grazing on phytoplankton is highly selective (2-4)and can influence phytoplankton composition, the coexistenceof competitors, and overall water quality (5). The contr
24、ibutionof 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 larval fish
25、, 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 ecological ro
26、le 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 factors
27、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 of the
28、 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 are e
29、cologically 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 described here
30、 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 cysts ar
31、e 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 toxi-can
32、ts. 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 speciesspecific, but the sensitivity of B. calyciflorus is generallycomparable to that of Daphnia
33、(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 best since
34、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 for this te
35、st 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, a3The boldface numbers in parentheses refer to the list of refer
36、ences at the end ofthis standard.E1440 91 (2012)2stereomicroscope capable of 10 to 15 magnification, 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 NaHCO3,60mgCaSO42H2O, 60 mg MgSO47H2O, and
37、 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 for up to s
38、even 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 Other reconstitu
39、ted dilution waters may be used asdescribed in Guide E729. In addition, natural dilution watersometimes might be desirable (Guide E729). Cyst hatching andLC50s in these dilution waters might differ from those previ-ously reported (37).8. Hazards8.1 Many materials can affect humans adversely if preca
40、u-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, andpipets sh
41、ould 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(46-49), and
42、 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 solutions, te
43、st 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 Cleaning of e
44、quipment 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 produced.8.5
45、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 systems and
46、 leak detectors should be used to helpavoid electrical shocks.9. Test Material9.1 Single ChemicalGuide E729, sections on stocksolutions, solvents, solvent controls, and test concentrationsapply to this test.9.2 EffluentsGuide E1192, sections on collection,preservation, treatment, and test concentrat
47、ions of effluents,apply to this 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 (see 7.1) and incubat
48、ingat 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 alkalinity can all delay
49、hatching. 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, number of trea
