1、Designation: E1218 04 (Reapproved 2012)Standard Guide forConducting Static Toxicity Tests with Microalgae1,2This standard is issued under the fixed designation E1218; 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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers procedures for obtaining laboratorydata concerning the adverse effects of a test material added togrowth
3、 medium on growth of certain species of freshwater andsaltwater microalgae during a static exposure. These proce-dures will probably be useful for conducting short-term toxic-ity tests with other species of algae, although modificationsmight be necessary.Although the test duration is comparable toan
4、 acute toxicity test with aquatic animals, an algal toxicity testof short duration (72, 96 or 120 h) allows for examination ofeffects upon multiple generations of an algal population andthus should not be viewed as an acute toxicity test.1.2 Other modifications of these procedures might be justi-fie
5、d by special needs or circumstances. Although using appro-priate procedures is more important than following prescribedprocedures, results of tests conducted using unusual proceduresare not likely to be comparable to results of many other tests.Comparison of results obtained using modified and unmod
6、ifiedversions of these procedures might provide useful informationconcerning new concepts and procedures for conducting tox-icity tests with microalgae.1.3 These procedures are applicable to many chemicals,either individually or in formulations, commercial products, orknown mixtures. With appropriat
7、e modifications, these proce-dures can be used to conduct tests on temperature, and pH andon such materials as aqueous effluents (see also Guide E1192),leachates, oils, particulate matter, sediments, and surface wa-ters. Static tests might not be applicable to materials that arehighly volatile, are
8、rapidly biologically or chemically trans-formed in aqueous solutions, or are removed from test solu-tions in substantial quantities by the test vessels or organismsduring the test. However, practical flow-through test proce-dures with microalgae have not been developed.1.4 Results of tests using mic
9、roalgae should usually bereported in terms of the 96-h (or other time period) IC50 (see3.2.5) based on reduction in growth. In some situations, itmight only be necessary to determine whether a specificconcentration unacceptably affects the growth of the testspecies or whether the IC50 is above or be
10、low a specificconcentration.1.5 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Hazards 7Apparatus 6Facilities 6.1Equipment 6.2Test Vessels 6.3Cleaning 6.4Acceptability 6.5Growth Medium 8Test Material 9General 9.1Stock Solution 9.2T
11、est Concentration(s) 9.3Test Organisms 10Species 10.1Source 10.2Culture 10.3Quality 10.4Procedure 11Experimental Design 11.1Temperature 11.2Illumination 11.3Beginning the Test 11.4Gas Exchange 11.5Duration of Test 11.6Biological Data 11.7Other Measurements 11.8Determination of Algistatic and Algicid
12、al Effects 11.8.5Analytical Methodology 12Acceptability of Test 13Calculation 14Report 15Keywords 161.6 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 heal
13、th practices and determine the applica-bility of regulatory limitations prior to use. Specific hazardstatements are given in Section 7.1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fateand is the direct responsibility of SubcommitteeE47.01 on Aqua
14、tic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published December 2012. Originallyapproved in 1990. Last previous edition approved in 2004 as E1218-04. DOI:10.1520/E1218-04R12.2This standard guide is a document, developed using the consensus mechanismsof ASTM, that provides gui
15、dance for the selection of procedures to accomplish aspecific test, but which does not stipulate specific procedures.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12. Referenced Documents32.1 ASTM Standards:D1129 Terminology Relating
16、 to WaterD3731 Practices for Measurement of Chlorophyll Content ofAlgae in Surface WatersD3978 Practice for Algal Growth Potential Testing withPseudokirchneriella subcapitataD4447 Guide for Disposal of Laboratory Chemicals andSamplesE380 Practice for Use of the International System of Units(SI) (the
17、 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 FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE1192
18、Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE1733 Guide for Use of Lighting in Laboratory TestingE1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM Guidelines3. Terminology3.1 Definitions
19、:3.1.1 The words “must,” “should,” “may,”“ can,” and“might” have very specific meanings in this guide.3.1.1.1 mayis 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 between“may” and “can” is pr
20、eserved, and “might” is never used as asynonym for either “may” or “can”.3.1.1.2 mustis used 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 adifferent design. “Must” is only used in co
21、nnection with factorsthat directly relate to the acceptability of the test (see 13.1).3.1.1.3 shouldis used to state that the specified conditionis recommended and ought to be met if possible. Althoughviolation of one “should” is rarely a serious matter, violation ofseveral will often render the res
22、ults questionable. Terms suchas “is desirable,” “is often desirable,” and“ might be desirable”are used in connection with less important factors.3.2 Definitions of Terms Specific to This Standard:3.2.1 algicidalhaving the propeerty of killing algae.3.2.2 algistatichaving the property of inhibiting a
23、lgalgrowth.3.2.3 biomassthe dry weight of living matter present in apopulation and expressed in terms of a given area or volume,for example, mg algae per liter. Because biomass is difficult tomeasure accurately, surrogate measures of biomass, such ascell counts, are typically used in this test.3.2.4
24、 growth ratethe increase in biomass per unit of time.3.2.5 IC50a statistically or graphically estimated concen-tration that is expected to cause a 50 % inhibition of one ormore specified biological processes (such as growth or repro-duction) for which the data are not dichotomous, underspecified con
25、ditions. Alternative values for inhibition, such as10 % or 20 %, are referred to as IC10 or IC20.3.2.6 standing cropthe algal biomass at the end of the test.3.2.7 yieldthe algal biomass at the end of the test minusthe algal biomass at the beginning of the test.3.3 For definitions of other terms used
26、 in this standard, referto Guides E729 and E1023 and Terminology E943. Forexplanation of units and symbols, refer to Practice E380.4. Summary of Guide4.1 In each of two or more treatments, organisms of onespecies of microalgae are maintained in replicate test vesselsusing the static technique. The t
27、est duration is typically 96 h,but shorter periods (for example, 72 h) have been used forfast-growing algae and longer periods (for example, 120 h)may be necessary for slower-growing algae. In each of the oneor more control treatments, the algae are maintained in growthmedium to which no test materi
28、al has been added in order toprovide the following: a measure of the acceptability of the testby giving an indication of the quality of the algae and thesuitability of the growth medium, test conditions, handlingprocedures, and so forth, and the basis for interpreting dataobtained from the other tre
29、atments. In each of the one or moreother treatments, the algae are maintained in growth medium towhich test material has been added to achieve a selectedconcentration. Specified data on population growth are ob-tained during the test and are usually analyzed to determine theIC50 based on reduction i
30、n growth.5. Significance and Use5.1 Tests with algae provide information on the toxicity oftest materials to an important component of the aquatic biotaand might indicate whether additional testing (1)4is desirable.5.2 Algae are ubiquitous in aquatic ecosystems, where theyincorporate solar energy in
31、to biomass, produce oxygen, func-tion in nutrient cycling and serve as food for animals. Becauseof their ecological importance, sensitivity to many toxicants,ready availability, ease of culture, and fast growth rates(rendering it possible to conduct a multi-generation test in ashort period of time),
32、 algae are often used in toxicity testing.5.3 Results of algal toxicity tests might be used to comparethe sensitivities of different species of algae and the toxicitiesof different materials to algae and to study the effects of variousenvironmental factors on results of such tests.5.4 Results of alg
33、al toxicity tests might be an importantconsideration when assessing the hazards of materials to3For 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 Documen
34、t Summary page onthe ASTM website.4The boldface numbers given in parentheses refer to a list of references at theend of the text.E1218 04 (2012)2aquatic organisms (See Guide E1023) or deriving water qualitycriteria for aquatic organisms (2).5.5 Results of algal toxicity tests might be useful forstud
35、ying biological availability of, and structure-activity rela-tionships between, test materials.5.6 Results of algal toxicity tests will depend on thetemperature, composition of the growth medium, and otherfactors. These tests are conducted in solutions that containconcentrations of salts, minerals,
36、and nutrients that greatlyexceed those in most surface waters. These conditions mayover- or under-estimate the effects of the test material ifdischarged to surface waters.6. Apparatus6.1 FacilitiesCultures and test vessels should be main-tained in rooms, incubators, or environmental chambers withcon
37、stant temperatures (see 11.2) and appropriate illumination(see 11.3). A water bath is generally not acceptable because itprevents proper illumination of the test vessels. The facilityshould be well ventilated and free of fumes. To further reducethe possibility of contamination by test materials and
38、othersubstances, especially volatile ones, algae should not becultured in a room in which toxicity tests are conducted, stocksolutions or test solutions are prepared, or equipment iscleaned.6.2 EquipmentSome or all of the following will beneeded:6.2.1 Centrifuge,6.2.2 Centrifuge Tubes, glass or poly
39、carbonate with screw-cap lids,6.2.3 Rotary or Oscillation Shaker, with variable speedcontrol capable of 100 r/min (or oscillations per minute),6.2.4 Erlenmeyer Flasks, borosilicate glass, or polycarbon-ate6.2.5 Stainless Steel Caps, Shimatsu Enclosures, FoamPlugs, Glass Caps, or Standard Screw Caps,
40、 (plastic/bakelite)(all closures should be loose-fitting),6.2.6 Pipets, Eppendorf or equivalent,6.2.7 Filtration Apparatus,6.2.8 Membrane Filters, with 0.45 and 0.22-m pore size,6.2.9 Analytical Balance,6.2.10 Autoclave or Microwave Oven,6.2.11 pH Meter,6.2.12 Calibrated Light Meter, reading in mol
41、m2s1orlumens,6.2.13 Microscope, capable of 100 to 400 magnification,6.2.14 Hemacytometer Counting Chamber or PlanktonCounting Chamber and Ocular Micrometer,6.2.15 Particle Counter, with 70- or 100-m aperture tube,and (optional) mean cell volume computer, or6.2.16 Fluorometer, equipped to measure chl
42、orophyll a,or6.2.17 Spectrophotometer, to measure cell densities in logphase cultures.6.3 Test Vessels:6.3.1 In a toxicity test with aquatic organisms, test chambers(also referred to as test vessels) are defined as the smallestphysical units between which there are no water connections.Vessels shoul
43、d be covered to keep out extraneouscontaminants, especially bacteria and undesirable algae. Be-cause algae consume carbon dioxide, the covers used for algaltests must not prevent the passage of air.All vessels and coversin a test must be identical.6.3.2 Sterile Erlenmeyer flasks of borosilicate glas
44、s orpolycarbonate are usually used as test and culture vessels. Anysize flask can be used as long as the test solution volume doesnot exceed 50 % of the flask volume for tests conducted on ashaker, and not more than 20 % of the flask volume for testsnot conducted on a shaker. The proper solution/vol
45、ume ratioshould be determined for each test species in each laboratorybecause the ratio is dependent on the species and conditions.6.4 Cleaning:6.4.1 Test vessels and equipment used to prepare and storegrowth medium, stock solutions, and test solutions should becleaned before use. New items should b
46、e washed with deter-gent and rinsed with water, a water-miscible organic solvent,water, acid (such as 10 % concentrated hydrochloric acid), andat least twice with deionized or distilled water. (Some lots ofsome organic solvents might leave a film that is insoluble inwater.)At the end of the test, al
47、l items that are to be used againshould be immediately emptied, rinsed with water, cleaned bya procedure appropriate for removing the test material (forexample, acid to remove metals and bases; detergent, organicsolvent, or activated carbon to remove organic chemicals),cleaned with a non-phosphate d
48、etergent using a stiff bristlebrush to loosen any attached materials and rinsed at least twicewith deionized or distilled water. Acid is often used to removemineral deposits.6.4.2 If an electronic particle counter is to be used to countalgal cells, the final rinse should be with water that has beenf
49、iltered through a 0.22-m membrane filter.6.4.3 Test vessels may be dried in an oven at 50 to 100Cand capped with either stainless steel, foam or glass caps, orShimatsu closures. Glassware should be sterilized by autoclav-ing for 20 min at 121C and 1.1 kg/cm2or by microwaving (7).Hand-made cotton plugs should not be used. The acceptabilityof foam plugs should be investigated prior to use because somebrands have been reported to be toxic.6.5 AcceptabilityBefore a toxicity test is conducted withalgae in new test facilities, it is desirable to conduct a“non-toxicant”
