1、Designation: E 1415 91 (Reapproved 2004)e1Standard Guide forConducting Static Toxicity Tests With Lemna gibba G31This standard is issued under the fixed designation E 1415; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year o
2、f 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.e1NOTETable X1.3 was editorially corrected in August 2006.1. Scope1.1 This guide describes procedures for obtaining laboratoryd
3、ata concerning the adverse effects of a text material added togrowth medium on a certain species of duckweed (Lemnagibba G3) during a 7-day exposure using the static technique.These procedures will probably be useful for conductingtoxicity tests with other species of duckweed and other floatingvascu
4、lar plants, although modifications might be necessary.1.2 Special needs or circumstances might also justify modi-fication of this standard. Although using appropriate proce-dures is more important than following prescribed procedures,results of tests conducted using unusual procedures are notlikely
5、to be comparable to results of many other tests. Com-parison of results obtained using modified and unmodifiedversions of these procedures might provide useful informationconcerning new concepts and procedures for conducting testswith duckweed.1.3 The procedures in this guide are applicable to mostc
6、hemicals, either individually or in formulations, commercialproducts, or known mixtures. With appropriate modificationsthese procedures can be used to conduct tests on temperatureand pH and on such other materials as aqueous effluents (seealso Guide E 1192), leachates, oils, particulate matter, sedi
7、-ments and surface waters. These procedures do not specificallyaddress effluents because to date there is little experience usingduckweeds in effluent testing and such tests may pose problemswith acclimation of the test organisms to the receiving water.Static tests might not be applicable to materia
8、ls that have a highoxygen demand, are highly volatile, are rapidly biologically orchemically transformed in aqueous solution, or are removedfrom test solutions in substantial quantities by the test cham-bers or organisms during the test.1.4 Results of toxicity tests performed using the proceduresin
9、this guide should usually be reported in terms of the 7-dayIC50 based on inhibition of growth. In some situations it mightonly be necessary to determine whether a specific concentra-tion unacceptably affects the growth of the test species orwhether the IC50 is above or below a specific concentration
10、.Another end point that may be calculated is the no observedeffect concentration (NOEC).1.5 The sections of this guide appear as follows:Title SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Hazards 6Apparatus 7Facilities 7.1Test Chambers 7.2Cleaning 7.3Acceptabilit
11、y 7.4Growth Medium 8Test Material 9General 9.1Stock Solution 9.2Test Concentration(s) 9.3Test Organisms 10Species 10.1Source 10.2Stock Culture 10.3Procedure 11Experimental Design 11.1Temperature 11.2Illumination 11.3Beginning the Test 11.4Duration of Test 11.5Biological Data 11.6Other Measurements 1
12、1.7Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 151.6 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 health practices a
13、nd to determine 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. Published April 2004. Originallyapproved in 1
14、991. Last previous edition approved in 1998 as E 1415 91 (1998).1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.applicability of regulatory limitations prior to use. Specifichazard statements are given in Section 6.2. Referenced Docu
15、ments2.1 ASTM Standards:2E 380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)3E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates andAmphibiansE 943 Terminology Relating to Biological Effects and En-vironmental Fa
16、teE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Texts onAqueous Ambient Smaples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1218 Guide for Conducting 96-h Toxicity Tests withMicroalgae3. Terminology3
17、.1 The words must, should, may, can, and might have veryspecific meanings in this guide. Must is used to express anabsolute requirement, that is, to state that the test ought to bedesigned to satisfy the specified condition, unless the purposeof the test requires a different design. Must is only use
18、d inconnection with factors that directly relate to the acceptabilityof the test (see Section 13). Should is used to state that thespecified condition is recommended and ought to be met ifpossible. Although violation of one should is rarely a seriousmatter, violation of several will often render the
19、 resultsquestionable. Terms such as is desirable, is often desirable,might be desirable are used in connection with less importantfactors. May is used to mean is (are) allowed to, can is used tomean is (are) able to, and might is used to mean could possibly.Thus the classic distinction between may a
20、nd can is preserved,and might is never used as a synonym for either may or can.3.2 Definitions of Terms Specific to This Standard:3.2.1 frondindividual leaf-like structure on a duckweedplant.3.2.2 IC50a statistically or graphically estimated concen-tration of test material that is expected to cause
21、a 50 %inhibition of one or more specified biological processes (suchas growth or reproduction), for which the data are not dichoto-mous, under specified conditions.3.3 For definitions of other terms used in this guide, refer toTerminology E 943, and Guides E 729 and E 1023. For anexplanation of unit
22、s and symbols, refer to Practice E 380.4. Summary of Guide4.1 In each of two or more treatments, plants of Lemnagibba G3 are maintained for 7 days in two or more testchambers using the static technique. In each of the one or morecontrol treatments, the plants are maintained in growth mediumto which
23、no test material has been added in order to provide ameasure of the acceptability of the test by giving an indicationof the quality of the duckweed and the suitability of the growthmedium, test conditions, handling procedures, and so forth, andthe basis for interpreting data obtained from the other
24、treat-ments. In each of the one or more other treatments, theduckweed plants are maintained in growth medium to which aselected concentration of test material has been added. Speci-fied data concerning growth of duckweed in each test chamberare obtained during the test and are usually analyzed todet
25、ermine the IC50 or NOEC based on inhibition of growth.5. Significance and Use5.1 The term duckweed commonly refers to members of thefamily Lemnaceae. This family has many species world-widein 4 genera. This guide is designed for toxicity testing with oneparticular clone of one species of duckweed th
26、at has beenextensively studied, Lemna gibba G3, although other speciessuch as Lemna minor or Spirodela spp. can probably also betested using the procedures described herein.5.2 Duckweeds are widespread, free-floating aquatic plants,ranging in the world from tropical to temperate zones. Duck-weeds ar
27、e a source of food for waterfowl and small animalsand provide food, shelter, and shade for fish. The plants alsoserve as physical support for a variety of small invertebrates.Duckweed is fast growing and reproduces rapidly comparedwith other vascular plants (1).4Under conditions favorable forits gro
28、wth, it can multiply quickly and form a dense mat inlakes, ponds, and canals, primarily in fresh water, but also inestuaries. It also grows well in effluents of wastewater treat-ment plants and has been suggested as a means of treatingwastewaters (2). A dense mat of duckweed can block sunlightand ae
29、ration and cause fish kills (3).5.3 Duckweed is small enough that large laboratory facili-ties are not necessary, but large enough that effects can beobserved visually.5.4 Because duckweed is a floating macrophyte, it might beparticularly susceptible to surface active and hydrophobicchemicals that c
30、oncentrate at the air-water interface. Results ofduckweed tests on such chemicals, therefore, might be sub-stantially different from those obtained with other aquaticspecies.5.5 Results of toxicity tests with duckweed might be used topredict effects likely to occur on duckweed in field situations as
31、a result of exposure under comparable conditions.5.6 Results of tests with duckweed might be used tocompare the toxicities of different materials and to study theeffects of various environmental factors on results of such tests.5.7 Results of tests with duckweed might be an importantconsideration wh
32、en assessing the hazards of materials toaquatic organism (see Guide E 1023) or when deriving waterquality criteria for aquatic organisms (4).5.8 Results of tests with duckweed might be useful forstudying biological availability of, and structure-activity rela-tionships between test materials.2For re
33、ferenced 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.3Withdrawn.4The boldface numbers in parentheses refer to the list of
34、 references at the end ofthis guide.E 1415 91 (2004)e125.9 Results of tests with duckweed will depend on tempera-ture, composition of the growth medium, condition of the testorganisms, and other factors. The growth media that areusually used for tests with duckweed contain concentrations ofsalts, mi
35、nerals, and nutrients that greatly exceed those in mostsurface waters.6. Hazards6.1 Many materials can affect humans adversely if precau-tions are inadequate. Therefore, skin contact with all testmaterials and solutions of them should be minimized by suchmeans as wearing appropriate protective glove
36、s (especiallywhen washing equipment or putting hands in test solutions),laboratory coats, aprons, and glasses. Special precautions, suchas covering test chambers and ventilating the area surroundingthe chambers, should be taken when conducting tests onvolatile materials. Information on toxicity to h
37、umans (5),recommended handling procedures (6), and chemical andphysical properties of the test material should be studied beforea test is begun. Special procedures might be necessary withradio-labeled test materials (7) and with materials that are, orare suspected of being, carcinogenic (8).6.2 Alth
38、ough disposal of stock solutions, test solutions, andtest organisms poses no special problems in most cases, healthand safety precautions and applicable regulations should beconsidered before beginning a test. Removal or degradation oftest material might be desirable before disposal of stock andtest
39、 solutions.6.3 Cleaning 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.6.4 Acidic solutions and hypochlorite solutions should notbe mixed because hazardous fumes
40、might be produced.6.5 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 fume hood.6.6 Because growth medium and test solutions are usuallygood conductors
41、 of electricity, use of ground fault systems andleak detectors should be considered to help prevent electricalshocks.7. Apparatus7.1 FacilitiesCulture and test chambers should be main-tained in an environmental chamber, incubator, or room withconstant temperature (see 11.2) and appropriate illuminat
42、ion(see 11.3). A water bath is generally not acceptable because itprevents proper illumination of the test chambers. The facilityshould be well-ventilated and free of fumes. To further reducethe possibility of contamination by test materials and othersubstances, especially volatile ones, the culture
43、 chambersshould not be in a room in which toxicity tests are conducted,stock solutions or test solutions are prepared, or equipment iscleaned.7.2 Test ChambersIn a toxicity test with aquatic organ-isms, test chambers are defined as the smallest physical unitsbetween which no water connections exist.
44、 Glass 250-mLbeakers, 200-mL flat-bottomed test tubes, 250-mL fruit jars,and 250 or 500-mL Erlenmeyer flasks have been used success-fully (9-11). The ratio of the size of the test chamber to thevolume of test solution should be 5 to 2 (that is, 100 mL in a250-mL Erlenmeyer flask, 200 mL in a 500-mL
45、Erlenmeyerflask). Plastic chambers may be used only if duckweed doesnot adhere to the walls and the test material does not sorb ontothe plastic more than it does to glass. Chambers should becovered to keep out extraneous contaminants and to reduceevaporation of test solution and test material. Beake
46、rs shouldbe covered with a clear watch glass and flasks should becovered with loose-fitting caps such as foam plugs, stainlesssteel caps, Shimadzu enclosures, glass caps, or screw caps.(The acceptability of foam plugs should be investigated prior touse because some brands have been found to be toxic
47、.) Allchambers and covers in a test must be identical.7.3 CleaningTest chambers and equipment used to pre-pare and store growth medium, stock solutions, and testsolutions should be cleaned before use. New items should bewashed with detergent and rinsed with water, a water-miscibleorganic solvent, wa
48、ter, acid (such as 10 % concentrated hydro-chloric acid), and at least twice with deionized or distilledwater. (Some lots of some organic solvents might leave a filmthat is insoluble in water.) A dichromate-sulfuric acid cleaningsolution may be used in place of both the organic solvent andthe acid.
49、At the end of the test, all items that are to be usedagain should be immediately (a) emptied, (b) rinsed withwater, (c) cleaned by a procedure appropriate for removing thetest material (for example, acid to remove metals and bases;detergent, organic solvent, or activated carbon to removeorganic chemicals), (d) cleaned with a nonphosphate detergentusing a stiff bristle brush to loosen any attached materials, and(e) rinsed at least twice with deionized or distilled water. Acidis often used to remove mineral deposits. Chambers should bedried in an oven at 50 to 100C, capped with
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