1、Designation: E 1841 04Standard Guide forConducting Renewal Phytotoxicity Tests With FreshwaterEmergent Macrophytes1This standard is issued under the fixed designation E 1841; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、 of 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.1. Scope1.1 This test guide is designed to give general guidance forassessing the potential phytotoxicity of water soluble te
3、stmaterial to freshwater emergent macrophytes.1.2 This renewal test continuously exposes selected plantspecies, growing in sediment, to various concentrations of testmaterial, dissolved in a nutrient solution.1.3 This test guide is based on the Toxic Substances ControlAct (TSCA) guidelines for condu
4、cting toxicity tests withterrestrial plants (1)2and is applicable to most water solublechemicals, either individually or in formulations, commercialproducts, or known mixtures (see Guides E 1193 and E 1598).With slight modifications the procedure also might be used foreffluents (see Guide E 1192).1.
5、4 Results from this toxicity test can be used to report anIC50 or NOEC (see Section 3) based on the concentration ofchlorophyll extracted from the plants (see Guides D 3731 andE 1218). In some situations, it might be necessary to only testat one concentration to determine whether or not that specifi
6、cconcentration is toxic to the plants.1.5 This test method is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Hazards 7Apparatus and Reagents 6Facilities 6.1Test Chambers 6.2Cleaning 6.3HPLC 6.4Reagents 6.5Nutrient Solution 8Test Material 9Genera
7、l 9.1Test Concentrations 9.2Stock Solution 9.3Controls 9.4Sediments 10Test Organisms 11Recommended Species 11.1Alternate Species 11.2Culturing 11.3Procedure 12Experimental Design 12.1Beginning of Test 12.2Evaluation of Test 12.3Calculation 13Acceptability of Test 14Report 15Precision and Bias 16Keyw
8、ords 17Appendix X1References1.6 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 and determine the applica-bility of regulatory limitations
9、prior to use. Specific hazardstatements are given in Section 7.2. Referenced Documents2.1 ASTM Standards:3D 3731 Practices for Measurement of Chlorophyll Contentof Algae in Surface WatersE 729 Guide for Conducting Acute Toxicity Tests withFishes, Macroinvertebrates, and AmphibiansE 943 Terminology R
10、elating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Test on Aque-ous Effluents with Fishes, Macroinvertebrates, and Am-phibiansE 1193 Guide for Conducting Renewal Life-Cy
11、cle ToxicityTests with Daphnia magnaE 1218 Guide for Conducting Static 96-h Toxicity Testswith MicroalgaeE 1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and
12、Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published May 2004. Originallyapproved in 1996. Last previous edition approved in 1996 as E184196.2The boldface numbers in parentheses refer to the l
13、ist of references at the end ofthis test method.3For 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 onthe ASTM website.1Copyright AS
14、TM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.E 1598 Practice for Conducting Early Seedling GrowthTestsE 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants with Fresh Water In-vertebratesE 1733 Guide for Use of Ligh
15、ting in Laboratory Testing3. Terminology3.1 The words “must,” “should,”“ may,” “can,” and “might”have very specific meanings in this guide. “Must” is used toexpress an absolute requirement, that is, to state that the testought to be designed to satisfy the specified condition, unlessthe purpose of t
16、he test requires a different design. “Must” isonly used in connection with factors that directly relate to theacceptability of the test (see Section 14). “Should” is used tostate that the specified condition is recommended and ought tobe met if possible. Although violation of one “should” is rarelya
17、 serious matter, violation of several will often render theresults questionable. Terms such as “is desirable,” is often“desirable,” and “might be desirable” are used in connectionwith less important factors. “May” is used to mean “is (are)allowed to,”“ can” is used to mean “is (are) able to,” and“mi
18、ght” 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.”3.2 DefinitionsFor definitions of other terms used in thisstandard, refer to Terminology E 943 and Practice E 1598.3.3 Definitions
19、 of Terms Specific to This Standard:3.3.1 IC50, na statistically or graphically estimated con-centration of test material that, under specified conditions, isexpected to cause one or more specified effects in 50 % of agroup of organisms, for which the data are not dichotomous.3.3.2 emergent macrophy
20、tevascular plant that typicallyhas a well defined root system that anchors the plant insediments and long linear erect leaves that emerge above thewater surface.3.3.3 rhizome, nunderground horizontal stems fromwhich leaves and roots can develop.3.3.4 surrogate species, nplant species that may be use
21、dto gage or measure a response that might be demonstrated byanother plant species exposed to similar conditions.3.3.5 tuber, nshort, thickened, fleshy part of an under-ground stem, used for photosynthate storage.4. Summary of Guide4.1 Tubers, rhizomes or seeds of selected freshwater emer-gent macrop
22、hytes are planted in pots containing sediment.4.2 The sediment is kept saturated constantly by placing thepots in trays that are kept filled with water so that the waterlevel is below the rim of the pots. The plants are allowed togrow, and once firmly established, the phytotoxicity test maybegin. De
23、pending on the species and culture conditions thistime period may be two to six weeks.4.3 Pots containing the actively growing plants are placed inindividual trays. This constitutes the test chamber. Each traywill contain a selected concentration of the test materialdissolved in a nutrient solution.
24、 The amount of solution is notcritical as long as there is a continuous supply. The testsolutions including the control are renewed three times a week(see Guide E 1193).4.4 Following a two-week exposure to the test solution, theplants are harvested by cutting the stems at the soil level.4.5 To deter
25、mine treatment differences, it is recommendedthat chlorophyll be extracted from the leaf material (2) andanalyzed using High-Performance Liquid Chromatograph(HPLC). A spectrophotometer or fluorometer also may be usedto determine treatment differences (3-5).4.6 A variety of procedures can be used to
26、calculate theresults of a growth test. Means comparison procedure can beused to determine if treatments are different from the controlwhile regression may be used to determine IC50s.5. Significance and Use5.1 Increased emphasis is being placed on protecting wet-lands (6) and several agencies includi
27、ng U.S. EnvironmentalProtection Agency and Environment Canada are beginning torequire, for the registration of pesticides, data regardingtoxicity of test materials to rooted aquatic vascular plants(7,8,9).5.2 Much research is being conducted with vascular plants,both terrestrial and aquatic (10), ho
28、wever, protocols for phy-totoxicity testing with freshwater emergent macrophytes stillare not well defined.5.3 This guide is designed to assess potential detrimentaleffects of water soluble chemical substances on selectedsurrogate species of freshwater emergent macrophytes.5.4 This guide focuses on
29、diminishment of chlorophyllcontent in leaves as the measurable endpoint, however, not allchemicals affect chlorophyll production. Dry weight can beused as the endpoint for O. sativa, however, exposure timesmay need to be extended to detect treatment differences. Dryweight is not a recommended endpoi
30、nt for any of the testspecies started as rhizomes or tubers. Other endpoints, such asperoxidase activity (11) or chlorophyll fluorescence (12) couldpossibly be used.5.5 This guide could be used to provide early indication ofpotential problems, identify hazardous substances before con-tamination of w
31、etlands occurs, and establish “margins ofsafety” for specific chemicals within wetlands (see GuideE 1023).5.6 This guide is not designed to replace field assessmentsor other aquatic testing procedures. It is designed to compli-ment such testing, so that a more complete assessment ispossible.6. Appar
32、atus and Reagents6.1 FacilitiesPlants are cultured and tests are conductedin areas where light and temperature can be controlled. Agreenhouse or culture room is preferable. Light can be pro-vided either by natural sunlight, fluorescent/incandescent lightsor a mixture of both (see Guide E 1733). With
33、 the design of thetest chambers having open water, humidity around the plantsshould be adequate for plant growth. To minimize interference,such as drafts, the plants can be shielded with curtains orpartitions. Testing facilities should be kept separate fromculturing facilities to prevent cross conta
34、mination.E18410426.2 Test ChambersPlastic pots with drainage holes in thebottom are used for culturing and exposing the plants in thephytotoxicity test. Pots should be large enough to prevent theplants from becoming root bound. Each pot is placed in anindividual test tray that is larger in diameter
35、than the pot andcan hold the test solution.6.3 CleaningThe pots and test trays containing the plantsshould be disposable. All other equipment, except plastic, thatwill come in contact with the test solutions should be washedwith a mild detergent and rinsed with water, a water-miscibleorganic solvent
36、, water, acid, such as 10 % concentrated hydro-chloric acid, and at least twice with ASTM Type I water.6.4 HPLCA system capable of performing binary orternary linear gradients at a constant flow rate and capable ofinjecting 50 to 200 L aliquots is recommended. The systemshould have a stainless steel
37、 HPLC column, packed with 5-mC-18 reverse-phase packing and a column flow rate of 150L/min for a 250-mm long by 4.6-mm inside diameter column.For columns with different dimensions, the flow rate should beadjusted appropriately. The absorbance detector should becapable of detecting light in the visib
38、le region (400700 nm).A data system or integrator for measuring peak areas isrecommended as well.6.5 Reagents:6.5.1 Dimethylsulfoxide (DMSO), solvent grade.6.5.2 Chlorophyll StandardChlorophyll A from spinachprepared in DMSO (see 12.3.10).6.5.3 Water for HPLC AnalysisHPLC grade or obtainedfrom a wat
39、er purification system capable of producing waterwith a resistivity 12 mV/cm. Filter and degas (by vacuum orhelium purging) before use.6.5.4 Ethyl Acetate, HPLC grade. Filter and degas (byvacuum or helium purging) before use.6.5.5 Methanol, HPLC grade. Filter and degas (by vacuumor helium purging) b
40、efore use.7. Hazards7.1 It is recommended that the material safety data sheet(MSDS) be reviewed for safety, storage, and disposal precau-tions for each test substance.7.2 Many materials can affect humans adversely if precau-tions are inadequate. Contact with all test materials andsolutions, therefor
41、e, should be minimized by wearing protec-tive gloves, especially when washing equipment or puttinghands in test solutions, laboratory coats, aprons, glasses, andrespirators if necessary. Information on toxicity to humans(13-17), recommended handling procedures (18-21), andchemicals and physical prop
42、erties of the test material should bestudied before a test is started.7.3 Although 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 d
43、egradation oftest material might be desirable before disposal of stock andtest solutions.7.4 Cleaning of equipment with a volatile solvent, such asacetone, should be performed only in a well-ventilated areawhere no smoking, open flame, such as a pilot light, orsparking electrical equipment are prese
44、nt.8. Nutrient Solution8.1 The nutrient solution is one-half strength Hoaglandssolution (Appendix X1) and is prepared by adding specifiedstock solutions to ASTM Type I water or other dilution water.8.2 It is preferable to prepare the nutrient solution fromASTM Type I water. Alternatively, a constant
45、 source ofdilution water, acceptable to the test organisms and available inadequate supply, should be used to make the Hoaglandssolution. The minimal requirement for an acceptable dilutionwater is that healthy test species survive through germination,growth, and testing without showing signs of stre
46、ss.8.3 The quality of water from a well or spring usually ismore uniform than surface water. Distilled or deionized wateralso is acceptable. Chlorinated water should not be used as thedilution water because it may be toxic to the plants. Dechlo-rinated, municipal drinking water should be used only a
47、s a lastresort because the dechlorination process often is incomplete,and because the water may contain unacceptably high concen-trations of copper, lead, zinc, and fluoride.8.4 The water source should be analyzed several times ayear (see Guide E 729) for physical and chemical factorsincluding metal
48、s and other inorganic chemicals, and organicchemicals including pesticides. The concentrations in thedilution water should be below detection limit or the lowestconcentration that has been shown to adversely affect the testspecies (22).9. Test Material9.1 GeneralThe test material should be reagent-g
49、rade orbetter, unless a test on a formulation, commercial product, ortechnical-grade material specifically is needed. Before a test isinitiated, the following information should be obtained aboutthe test material:9.1.1 Identities and concentrations of major ingredients andmajor impurities, that is, impurities constituting more than 1 %of the material.9.1.2 Solubility and stability in dilution water.9.1.3 Potential for microbial degradation, transformation,sorption etc., of the test substance within the sediment matrix(see Test Method E 1706).9.1.4 An estimate of toxi
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