1、Designation: E 1913 04Standard Guide forConducting Static, Axenic, 14-Day Phytotoxicity Tests inTest Tubes with the Submersed Aquatic Macrophyte,Myriophyllum sibiricum Komarov1This standard is issued under the fixed designation E 1913; the number immediately following the designation indicates the y
2、ear oforiginal adoption or, in the case of revision, the year 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 Submersed rooted aquatic macrophytes are importantco
3、mponents of aquatic systems. They contribute to primaryproductivity, improve water quality, cycle nutrients, generateoxygen, affect flow patterns, provide habitat and food for otherorganisms, and stabilize the sediment. These plants can beadversely affected when pesticides are sprayed to controlaqua
4、tic weeds and algal blooms or when phytotoxic chemicalsenter the waterway through atmospheric fallout, soil erosion,industrial effluent, sewage discharge, spills or drift from aerialor ground applications.1.2 This guide is designed to give guidance for assessing thepotential phytotoxicity of a test
5、material added to a sterileliquid growth medium on a species of freshwater submersedmacrophyte (Myriophyllum sibiricum Komarov) during a 14-day static exposure. A sterile system is recommended todetermine the direct effect of the test chemical upon individualparameters of the submersed macrophyte be
6、cause there is nodegradation of the test item by micro-organisms. For similarreasons, other aquatic plant testing, such as those of, Lemnaand algae, is commonly conducted in an axenic fashion.Overall environmental impact can not be directly determined.The main other disadvantage of the axenic test s
7、ystem is thedifficulty in preventing accidental contamination. These proce-dures could possibly be useful for conducting toxicity testswith other species of submersed macrophytes, although modi-fications might be necessary (1-8)2.1.3 The procedures in this guide are applicable to mostchemicals, eith
8、er individually or in formulations, commercialproducts, or known mixtures. These procedures might be usedto conduct tests for dependency on temperature, light, nutrientsand pH. With appropriate modification, these procedures mightbe used to conduct tests for contaminated surface waters andaqueous ef
9、fluents (see Guide E 1192). This static, axenictoxicity test might not be applicable to materials that containmicroorganisms unless the sample can be filter sterilizedwithout removing the toxicant. If the test materials are highlyvolatile, care should be taken to ensure that the test chambersare iso
10、lated. It might be necessary to replace the test materialon a regular basis if the test material is rapidly biologically orchemically transformed in aqueous solution, or is removedfrom the test solutions in substantial quantities by the testchambers or organisms during the test. This toxicity test i
11、s notsuitable for testing interactions between aquatic plants andother organisms, such as plant pathogens.1.4 Results from the toxicity test outlined in this guide canbe reported in terms of a 14-day IC25, IC50, or NOEC. Thisparameter may be based on several endpoints including inhi-bition of plant
12、growth during the 14-day period, inhibition ofshoot length, inhibition of root number and length, inhibition offresh or dry weight (see Guide E 1415), inhibition of oxygenproduction, change in membrane permeability, and change inchlorophyll a, chlorophyll b and carotenoid content extractedfrom secti
13、ons of the plants (see Practice D 3731 and GuideE 1218) (9-18). All or some of these endpoint parameters maybe examined depending upon the mode of phytotoxic action orresearcher preference. It might be necessary to conduct thetoxicity test at only one concentration to determine whether ornot that sp
14、ecific concentration is inhibitory to plant growth anddevelopment.1.5 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Definitions 3.1Definitions of Terms Specific to this Standard 3.2Summary of Guide 4Significance and Use 5Interferences 6Apparatus 7Facilities 7.1Test Cham
15、bers 7.2Equipment 7.3Cleaning 7.4Acceptability 7.5Reagents 8Hazards 9Nutrient Solution 10Test Material 111This 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
16、.Current edition approved April 1, 2004. Published May 2004. Originallyapproved in 1997. Last previous edition approved in 1997 as E 1913 97.2The boldface numbers given in parentheses refer to a list of references at theend of the text.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C70
17、0, West Conshohocken, PA 19428-2959, United States.SectionGeneral 11.1Test Concentrations 11.2Stock Solutions 11.3Controls 11.4Test Organism 12Recommended Species 12.1Alternate Species 12.2Culturing 12.3Procedure 13Experimental Design 13.1Temperature 13.2Illumination 13.3Beginning the Test 13.4Durat
18、ion of Test 13.5Evaluation of Test 13.6Analytical Methodology 14Calculations 15Acceptability of Test 16Report 17Precision and Bias 18Keywords 19Appendixes X1, X2References1.6 The values stated in SI units are to be regarded as thestandard.1.7 This standard does not purport to address all of thesafet
19、y 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 prior to use. This standard mayinvolve hazardous materials, operations, and equipment. Se
20、eSection 9 for specific hazard statements.2. Referenced Documents2.1 ASTM Standards:3D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 3731 Practices for Measurement of Chlorophyll Contentof Algae in Surface WatersD 3978 Practice for Algal Growth Potential Testing withSelen
21、astrum capricornutumE 729 Guide for Conducting Acute Toxicity Tests withFishes, Macroinvertebrates, and AmphibiansE 943 Terminology Relating 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
22、Acute Toxicity Tests onAqueous Effluents with Fishes, Macroinvertebrates, andAmphibiansE 1218 Guide for Conducting Static 96-h Toxicity Testswith MicroalgaeE 1415 Guide for Conducting Static Toxicity Tests WithLemna gibba G3E 1598 Practice for Conducting Early Seedling GrowthTestsE 1733 Guide for th
23、e Use of Lighting in Laboratory TestingE 1841 Guide for Conducting Renewal Phytotoxicity TestsWith Freshwater Emergent MacrophytesE 1847 Practice for Statistical Analysis of Toxicity TestsConducted under ASTM Guidelines3. Terminology3.1 Definitions:3.1.1 The words must, should, may, can, and might h
24、avevery specific 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 conditions, unless the purposeof the test requires a different design. Must is only used inconnection with factors that directly relat
25、e to acceptability ofthe test (see Section 15). 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 resultsquestionable. Terms such as is desirable, i
26、s often desirable, andmight 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 and can is preserved,and might is never used as
27、a synonym for either may or can.3.1.2 For definitions of other terms used in this guide, referto Terminology D 1129, Guide E 729, Terminology E 943, andPractices E 1598 and E 1847.3.2 Definitions of Terms Specific to This Standard:3.2.1 apexthe uppermost portion of a plant containing theactively gro
28、wing tissue or apical meristem.3.2.2 axenic or sterilefree from other organisms, bothactive and dormant.3.2.3 culturethe stock of organisms that is raised undercontrolled conditions to produce test organisms throughasexual reproduction.3.2.4 submersed macrophytea rooted freshwater vascularplant that
29、 remains covered with water during the growingseason.3.2.5 toxicity testa standardized procedure that measuresthe concentration at which a test material has a defined effectupon the test organism.3.2.6 turionan asexual reproductive structure formed onlateral branches in response to lower autumn temp
30、eratures.Turions develop into new plants when environmental condi-tions become favorable (19-23).4. Summary of Guide4.1 Axenically cultured aspices of Myriophyllum sibiricumare exposed in a static system to a single concentration or adilution series of a test substance. At the end of a 14-day testpe
31、riod under standardized conditions, growth and developmentof plants exposed to the test material is compared with thegrowth and development of plants in an appropriate control. Atest substance is considered biologically active when a statis-tically significant concentration-dependent inhibition of p
32、lantgrowth occurs.4.2 The axenic toxicity testing technique involves exposingthe test organism to selected concentrations of the test chemicalin individual tubes. Each test tube contains a rooting substrateand 40 mL of nutrient medium previously spiked with the test3For referenced ASTM standards, vi
33、sit 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.E1913042chemical. In this axenic testing system,3gofTurfacet4hasproven successful as an artific
34、ial rooting medium (9). Withouta rooting substrate, the plants roots may push the plant segmentupwards and out of the nutrient medium. The use of anotherrooting medium would need to be validated. A 3cm apicalsegment of M. sibiricum is added to the tube. The tubes areincubated (16 h light (fluence ra
35、te = 100 150 mol m2s1)and 8 h dark at 25C during the light and 20C during the darkphase) for 14 days, during which time the increase in plantheight over time may be measured and growth curves estab-lished. On Day 14, other possible test endpoints that may bemeasured include final shoot length, root
36、number and total rootlength, total fresh weight, chlorophyll a, chlorophyll b andcarotenoid content, membrane integrity, oxygen evolution, andchlorophyll fluorescence.5. Significance and Use5.1 Protection of aquatic areas is currently being empha-sized by several agencies including the U.S. Environm
37、entalProtection Agency and Environment Canada. For pesticideregistration, these agencies are beginning to require dataregarding the toxicity of test chemicals to aquatic rootedvascular plants (25-28).5.2 Toxicological research with terrestrial and aquatic vas-cular plants has been initiated (29) inc
38、luding the developmentof a protocol for testing with emergent macrophytes (GuideE 1841) (30). However, protocols for phytotoxicity testing withfreshwater submersed plants still require development. Toxi-cological research has been conducted using submersed mac-rophytes (1-8, 31-35) but standardizati
39、on of the methods isrequired.5.3 This guide is designed to assess the phytotoxic effects ofchemicals upon a selected freshwater species of submersedaquatic macrophyte, Myriophyllum sibiricum Komarov. Thisspecies is an ecologically important submersed aquatic dicoty-ledon with a north temperate distr
40、ibution. It is readily culturedin test tubes in the laboratory (36). Lower temperatures inautumn initiate the formation of turions on lateral branches thatdevelop into new plants when environmental conditions be-come favorable (19-23). Toxicological testing with this specieshas demonstrated that it
41、is an ideal species for laboratorytesting since it grows readily under laboratory conditions, thetoxic response is reproducible and there is very little variationbetween experimental replicates (9-15).5.4 It is a common practice to use sterile plant culture whentesting the direct effects of test mat
42、erials upon a plant species.Sterile plant culture and toxicity testing have been conductedwith algae (Practice D 3978, 37-39), floating aquatic macro-phytes (Guide E 1415, 37, 40) and submersed aquatic plantspecies (2-5). An axenic testing system is designed to deter-mine the direct effect of the te
43、st material upon the test species.There is nothing except the plant within the test system thatcould degrade or otherwise change the test chemical. Hydroly-sis or phytolysis may occur but degradation studies candetermine the rate of degradation by these means. Axenic testsare especially valuable dur
44、ing the initial stages of examining anew compound (for example, pesticide evaluation and regis-tration (Tier 1 and Tier 2) (25-27). In studies with otherspecies of aquatic macrophytes, it has been shown that thepresence of filamentous algae can cause a reduction in newshoot growth, fresh weight and
45、chlorophyll a content of themacrophytes when compared to macrophytes grown in theabsence of algae (41). The test tubes are recommended fortesting because they require a small incubation area, smallamount of plant tissue, small volume of test material and allowfor the maintenance of a sterile culture
46、 (2, 3, 36). Furthermore,test tubes permit height measurements in situ (36).5.5 There are numerous possible physiological and morpho-logical endpoints that can be utilized to assess the toxicity ofchemicals to this aquatic plant species. The test material effectmay be assessed as a change in total p
47、lant height, growth rate,fresh or dry weight, number and total length of roots, chloro-phyll a, chlorophyll b, carotenoids, membrane integrity oroxygen evolution, or any combination of these parameters.Peroxidase activity (31-35) and chlorophyll fluorescence (42)might be other endpoints that could b
48、e explored. Someendpoints have been compared (43, 44), but selected appropri-ate endpoint(s) based upon mode of action or route ofexposure.5.6 This toxicity test may be utilized during the pesticideregistration process, to provide an early warning of potentialecosystem problems, identify hazardous c
49、hemicals before con-tamination of aquatic systems occurs, and help establish“margins of safety” for specific chemicals within wetlands (seeGuide E 1023). Plants cultured using this method have alsobeen used to determine the effects of toxicants on plants inmicrocosms (45).5.7 This test is not designed to replace field assessments oftest material damage or other aquatic testing procedures, butshould be used as a screening tool. It should compliment othertesting so that a more complete environmental assessment ispossible. It is difficult to interpret effects observed in t
