1、Designation: E1841 04 (Reapproved 2012)Standard Guide forConducting Renewal Phytotoxicity Tests With FreshwaterEmergent Macrophytes1This standard is issued under the fixed designation E1841; the number immediately following the designation indicates the year oforiginal adoption or, in the case of re
2、vision, the year of last 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 test guide is designed to give general guidance forassessing the potential phytotoxicity of w
3、ater soluble testmaterial 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) guide
4、lines for conducting toxicity tests withterrestrial plants (1)2and is applicable to most water solublechemicals, either individually or in formulations, commercialproducts, or known mixtures (see Guides E1193 and E1598).With slight modifications the procedure also might be used foreffluents (see Gui
5、de E1192).1.4 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 D3731 andE1218). In some situations, it might be necessary to only testat one concentration to determine whether or not th
6、at specificconcentration 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 Materi
7、al 9General 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 B
8、ias 16Keywords 17Appendix X1References1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the u
9、ser 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 Documents2.1 ASTM Standards:3D3731 Practices for Measurement of Chlorophyll Content ofAlg
10、ae in Surface WatersE729 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 Guide
11、for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,1This 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
12、 approved Dec. 1, 2012. Published January 2013. Originallyapproved in 1996. Last previous edition approved in 2004 as E184104. DOI:10.1520/E1841-04R12.2The boldface numbers in parentheses refer to the list of references at the end ofthis test method.3For referenced ASTM standards, visit the ASTM web
13、site, 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.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United Stat
14、es1Macroinvertebrates, and AmphibiansE1193 Guide for Conducting Daphnia magna Life-CycleToxicity TestsE1218 Guide for Conducting Static Toxicity Tests withMicroalgaeE1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing andfor Selection of Sampl
15、ers Used to Collect Benthic Inver-tebratesE1598 Practice for Conducting Early Seedling Growth Tests(Withdrawn 2003)4E1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater InvertebratesE1733 Guide for Use of Lighting in Laboratory Testing3. Terminology3.1 The
16、 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 the test requires a different design. “Must” iso
17、nly 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 serious matter, violation of several will often
18、 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“might” is used to mean “could possibly.” Thus, the
19、 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 E943 and Practice E1598.3.3 Definitions of Terms Specific to This Standard:3.3.1 IC50, na
20、 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 macrophytevascular plant that typicallyhas a well defined
21、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 usedto gage or measure a response that might be demon
22、strated 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 macrophytes are planted in pots containing sediment.4.2
23、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. Depending on the species and culture conditions this
24、time 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. The amount of solution is notcritical as long as
25、there is a continuous supply. The testsolutions including the control are renewed three times a week(see Guide E1193).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 determine treatment differences, it is recommendedthat c
26、hlorophyll be extracted from the leaf material (2) andanalyzed using High-Performance Liquid Chromatograph(HPLC).Aspectrophotometer or fluorometer also may be usedto determine treatment differences (3-5).4.6 A variety of procedures can be used to calculate theresults of a growth test. Means comparis
27、on 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 including U.S. EnvironmentalProtection Agency and Environmen
28、t 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), however, protocols for phy-totoxicity testing with fres
29、hwater 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 diminishment of chlorophyllcontent in leaves as the m
30、easurable 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 endpoint for any of the testspecies started as rhizomes or
31、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 wetlands occurs, and establish “margins ofsafety” for
32、specific chemicals within wetlands (see GuideE1023).4The last approved version of this historical standard is referenced onwww.astm.org.E1841 04 (2012)25.6 This guide is not designed to replace field assessmentsor other aquatic testing procedures. It is designed to compli-ment such testing, so that
33、a more complete assessment ispossible.6. Apparatus and Reagents6.1 FacilitiesPlants are cultured and tests are conducted inareas where light and temperature can be controlled. A green-house or culture room is preferable. Light can be providedeither by natural sunlight, fluorescent/incandescent light
34、s or amixture of both (see Guide E1733). With the design of the testchambers 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 fr
35、omculturing facilities to prevent cross contamination.6.2 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
36、test tray that is larger in diameter 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 w
37、ater, a water-miscibleorganic solvent, 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. Th
38、e systemshould have a stainless steel 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 beca
39、pable of detecting light in the visible 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 Ana
40、lysisHPLC grade or obtainedfrom a water purification system capable of producing waterwith a resistivity 12 m/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
41、degas (by vacuumor helium purging) before 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 t
42、est materials andsolutions, therefore, 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
43、-21), andchemicals and physical properties 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 b
44、efore beginning a test. Removal or degradation 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, orspa
45、rking electrical equipment are present.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 Typ
46、e I water. Alternatively, a constant 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 t
47、esting without showing signs of stress.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.Dechlorinated, municipal dr
48、inking water should be used onlyas a last resort because the dechlorination process often isincomplete, and because the water may contain unacceptablyhigh concentrations of copper, lead, zinc, and fluoride.8.4 The water source should be analyzed several times ayear (see Guide E729) for physical and
49、chemical factorsincluding metals 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-grade 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 conce
