1、Designation: E 1218 04e1Standard Guide forConducting Static Toxicity Tests with Microalgae1, 2This standard is issued under the fixed designation E 1218; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A
2、number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.e1NOTESection A1.3.9 was editorially corrected in February 2007.1. Scope1.1 This guide covers procedures for obtaining laboratorydata concerning t
3、he adverse effects of a test material added togrowth 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 nec
4、essary.Although the test duration is comparable toan 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
5、modifications of these procedures might be justi-fied 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.Com
6、parison of results obtained using modified and unmodifiedversions 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, co
7、mmercial products, orknown mixtures. With appropriate modifications, these proce-dures can be used to conduct tests on temperature, and pH andon such materials as aqueous effluents (see also Guide E 1192),leachates, oils, particulate matter, sediments, and surface wa-ters. Static tests might not be
8、applicable to materials that arehighly volatile, are 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 h
9、ave not been developed.1.4 Results of tests using microalgae 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
10、of the testspecies or whether the IC50 is above or below 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
11、Medium 8Test Material 9General 9.1Stock Solution 9.2Test 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 Mea
12、surements 11.8Determination of Algistatic and Algicidal Effects 11.8.5Analytical Methodology 121This 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 e
13、dition approved April 1, 2004. Published May 2004. Originallyapporved in 1990. Last previous edition approved in 1997 as E 1218-97a.2This standard guide is a document, developed using the consensus mechanismsof ASTM, that provides guidance for the selection of procedures to accomplish aspecific test
14、, but which does not stipulate specific procedures.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.SectionAcceptability of Test 13Calculation 14Report 15Keywords 161.6 This standard does not purport to address all of thesafety concer
15、ns, 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 limitations prior to use. Specific hazardstatements are given in Section 7.2. Referenced Documents32.1 ASTM S
16、tandards:D 1129 Terminology Relating to WaterD 3731 Practice for Measurement of Chlorophyll Contentof Algae in Surface WatersD 3978 Practice for Algal Growth Potential Testing withPseudokirchneriella subcapitataD 4447 Guide for the Disposal of Laboratory Chemicalsand SamplesE 380 Practice for Use of
17、 the International System of Units(SI) (the Modernized Metric System)E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Terminology Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Mat
18、erial toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1733 Guide for the Use of Lighting in Laboratory TestingE 1847 Practice for Statistical Analysis of Toxicity TestsConducted
19、 Under ASTM Guidelines3. Terminology3.1 Definitions: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 t
20、he classic distinction between“may” and “can” is preserved, 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 re
21、quires adifferent design. “Must” is only used in connection 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 ma
22、tter, violation ofseveral 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.3.2 Definitions of Terms Specific to This Standard:3.2.1 algicidalhaving the propeerty of killing algae.
23、3.2.2 algistatichaving the property of inhibiting algalgrowth.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 a
24、scell counts, are typically used in this test.3.2.4 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 whi
25、ch the data are not dichotomous, underspecified conditions. 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
26、of the test.3.3 For definitions of other terms used in this standard, referto Guides E 729 and E 1023 and Terminology E 943. Forexplanation of units and symbols, refer to Practice E 380.4. Summary of Guide4.1 In each of two or more treatments, organisms of onespecies of microalgae are maintained in
27、replicate test vesselsusing the static technique. The test 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
28、 are maintained in growthmedium to which no test material 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
29、 basis for interpreting dataobtained from the other treatments. 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 usu
30、ally analyzed to determine theIC50 based on reduction in 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 aq
31、uatic ecosystems, where theyincorporate solar energy into 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 rates3For referenced ASTM standard
32、s, 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.4The boldface numbers given in parentheses refer to a list of references at theend of the
33、text.E121804e12(rendering it possible to conduct a multi-generation test in ashort period of time), 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 a
34、nd to study the effects of variousenvironmental factors on results of such tests.5.4 Results of algal toxicity tests might be an importantconsideration when assessing the hazards of materials toaquatic organisms (See Guide E 1023) or deriving waterquality criteria for aquatic organisms (2).5.5 Resul
35、ts of algal toxicity tests might be useful forstudying 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 solutio
36、ns that containconcentrations of salts, minerals, 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 ro
37、oms, incubators, or environmental chambers withconstant 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 p
38、ossibility of contamination by test materials and 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.
39、1 Centrifuge,6.2.2 Centrifuge Tubes, glass or polycarbonate 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 Enclosur
40、es, FoamPlugs, Glass Caps,orStandard Screw Caps, (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 Mete
41、r,6.2.12 Calibrated Light Meter, reading in mol m2s1orlumens,6.2.13 Microscope, capable of 100 to 4003 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 comput
42、er, or6.2.16 Fluorometer, equipped to measure chlorophyll 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 w
43、hich there are no water connections.Vessels should be covered to keep out extraneous contami-nants, especially bacteria and undesirable algae. Because algaeconsume carbon dioxide, the covers used for algal tests mustnot prevent the passage of air. All vessels and covers in a testmust be identical.6.
44、3.2 Sterile Erlenmeyer flasks of borosilicate glass 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 tests
45、not conducted on a shaker. The proper solution/volume 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 solutio
46、ns should becleaned before use. New items should be 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 tha
47、t is insoluble inwater.) At the end of the test, all 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 remov
48、e organic chemicals),cleaned with a non-phosphate detergent 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,
49、the final rinse should be with water that has beenfiltered 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 facil
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