1、Designation: E1963 09 (Reapproved 2014)Standard Guide forConducting Terrestrial Plant Toxicity Tests1This standard is issued under the fixed designation E1963; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisi
2、on. 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 guide covers practices for conducting plant toxicitytests using terrestrial plant species to determine effects of testsubsta
3、nces on plant growth and development. Specific testprocedures are presented in accompanying annexes.1.2 Terrestrial plants are vital components of ecologicallandscapes. The populations and communities of plants influ-ence the distribution and abundance of wildlife. Obviously,plants are the central f
4、ocus of agriculture, forestry, and range-lands. Toxicity tests conducted under the guidelines andannexes presented herein can provide critical informationregarding the effects of chemicals on the establishment andmaintenance of terrestrial plant communities.1.3 Toxic substances that prevent or reduc
5、e seed germina-tion can have immediate and large impacts to crops. In naturalsystems, many desired species may be sensitive, while otherspecies are tolerant. Such selective pressure can result inchanges in species diversity, population dynamics, and com-munity structure that may be considered undesi
6、rable. Similarly,toxic substances may impair the growth and development ofseedlings resulting in decreased plant populations, decreasedcompetitive abilities, reduced reproductive capacity, and low-ered crop yield. For the purposes of this guide, test substancesinclude pesticides, industrial chemical
7、s, sludges, metals ormetalloids, and hazardous wastes that could be added to soil. Italso includes environmental samples that may have had any ofthese test substances incorporated into soil.1.4 Terrestrial plants range from annuals, capable of com-pleting a life-cycle in as little as a few weeks, to
8、 long-livedperennials that grow and reproduce for several hundreds ofyears. Procedures to evaluate chemical effects on plants rangefrom short-term measures of physiological responses (forexample, chlorophyll fluorescence) to field studies of trees overseveral years. Research and development of stand
9、ardized planttests have emphasized three categories of tests: (1) short-term,physiological endpoints (that is, biomarkers); (2) short-termtests conducted during the early stages of plant growth withseveral endpoints related to survival, growth, and development;and ( 3) life-cycle toxicity tests that
10、 emphasize reproductivesuccess.1.5 This guide is arranged by sections as follows:Section Title1 Scope2 Referenced Documents3 Terminology4 Summary of Phytotoxicity Tests5 Significance and Use6 Apparatus7 Test Material8 Hazards9 Test Organisms10 Sample Handling and Storage11 Calibration and Standardiz
11、ation12 Test Conditions13 Interference and Limitations14 Quality Assurance and Quality Control15 Calculations and Interpretation of Results16 Precision and Bias1.6 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standar
12、d 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 prior to use. Specific precau-tionary statem
13、ents are given in Section 8.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD4547 Guide for Sampling Waste and Soils for VolatileOrganic CompoundsD5633 Practice for Sampling with a ScoopE1598 Practice for Conducting Early Seedling Growth Tests(Withdrawn 2003)3E1733 Gu
14、ide for Use of Lighting in Laboratory Testing1This guide is under the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment, Risk Management and Corrective Action and is the direct responsibil-ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.Current edition approved Oct
15、. 1, 2014. Published December 2014. Originallypublished in 1998. Last previous edition published 2009 as E196309. DOI:10.1520/E1963-09R14.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume
16、information, refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States12.2 Code of Federal Regul
17、ations Standard:CFR 4942.3 Other useful references have described phytotoxicity testprocedures(1-11) .53. Terminology3.1 General TerminologyThe words “must,” “should,”“may,”“ can,” and “might” have very specific meanings in thisguide. “Must” is used to express an absolute requirement, thatis, to sta
18、te that the test ought to be designed to satisfy thespecified condition, unless the purpose of the test requires adifferent design. “Must” is only used in connection with factorsthat directly relate to the acceptability of the test (see Section14). “Should” is used to state that the specified condit
19、ion isrecommended and ought to be met if possible. Althoughviolation of one “should” is rarely a serious matter, 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 facto
20、rs. “May” isused 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 classic distinction between “may” and “can” ispreserved, and “might” is never used as a synonym for either“may” or “can.”3.2 Definitions:3.2.1 control, nthe
21、treatment group in a toxicity testconsisting of reference soil or artificial soil that duplicates allthe conditions of the exposure treatments, but contains no testsubstance. The control is used to determine if there are anystatistical differences in endpoints related to the test substance.3.2.2 elu
22、ate, nsolution obtained from washing a solid witha solvent to remove adsorbed material.3.2.3 hazardous substance, na material that can causedeleterious effects to plants, microbes, or animals. (A hazard-ous substance does not, in itself, present a risk unless anexposure potential exists.)3.2.4 inhib
23、ition, na statistically lower value of any end-point compared to the control values that is related to environ-mental concentration or application rate.3.2.5 leachate, nwater plus solutes that has percolatedthrough a column of soil or waste.3.2.6 test material, nany formulation, dilution, etc. of at
24、est substance.3.2.7 test substance, na chemical, formulation, eluate,sludge, or other agent or substance that is the target of theinvestigation in a toxicity test.3.2.8 toxicant, nan agent or material capable of producingan adverse response (effect) in a biological system, adverselyimpacting structu
25、re or function or producing death.3.2.9 toxicity endpoints, nmeasurements of organism re-sponse such as death, growth, developmental, or physiologicalparameters resulting from exposure to toxic substances.3.3 Definitions of Terms Specific to This Standard:3.3.1 chlorotic, adjthe discoloration of sho
26、ots that occursas chlorophyll is degraded as a result of disease, toxicsubstances, nutrient deficiencies, or senescence.3.3.2 coleoptile, nthe protective tissues surrounding thegrowing shoot in a monocotyledonous plant.3.3.3 cotyledon, na primary leaf of the embryo in seeds,only one in the monocotyl
27、edons, two in dicotyledons. In manyof the latter, such as the bean, they emerge above ground andappear as the first leaves.3.3.4 cutting, na vegetative segment of a plant, usually astem that contains several nodes and associated buds, that canbe used to regenerate an entire plant.3.3.5 dead test pla
28、nts, nthose individuals that expiredduring the test observation period as indicated by severedesiccation, withering, chlorosis, necrosis, or other symptomsthat indicate non-viability.3.3.6 desiccated, adjthe plant, or portion of the plant, thatis dried in comparison to the control plant.3.3.7 develo
29、pment, nthe series of steps involving celldivision and cell differentiation into various tissues and organs.3.3.8 dicotyledon, nin the classification of plants, thosehaving two seed leaves.3.3.9 dormancy, na special condition of arrested growth inwhich buds, embryos, or entire plants survive at lowe
30、redmetabolic activity levels. Special environmental cues such asparticular temperature regimes or photoperiods are required toactivate metabolic processes and resume growth. Seeds thatrequire additional treatment besides adequate moisture andmoderate temperature to germinate are said to be dormant.
31、(Seequiescence.)3.3.10 emergence, nfollowing germination of a plant, theearly growth of a seedling that pushes the epicotyl through thesoil surface.3.3.11 enhanced growth and yield, nwhen a treated plantexhibits shoot growth, root elongation, lateral root growth, oryield significantly greater than t
32、he control values, the plant is“enhanced” or “stimulated.”3.3.12 epicotyl, nthat portion of an embryo or seedlingcontaining the shoot. It is delineated anatomically by thetransition zone which separates the epicotyl from the hypoco-tyl.3.3.13 fruits, nthe reproductive tissues derived from theovary i
33、n the case of epigenous flowers or the ovary andaccessory tissues in the case of hypigenous flowers.3.3.14 germination, nthe physiological events associatedwith re-initiation of embryo growth and mobilization of reservenutrients in seeds. The emergence of the seedling radicle fromthe seed coat defin
34、es the end of germination and the beginningof early seedling growth.4Available from Standardization Documents Order Desk, DODSSP, Bldg. 4,Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098, http:/www.dodssp.daps.mil.5The boldface numbers in parentheses refer to the list of references at the en
35、d ofthis guide.E1963 09 (2014)23.3.15 growth, na change in size or mass measured bylength, height, volume, or mass.3.3.16 hypocotyl, nthat portion of an embryo or seedlingcontaining the root or radicle. It is delineated anatomically bythe transition zone which separates the epicotyl from thehypocoty
36、l.3.3.17 inhibited plant growth and yield, n plant growth,root length and lateral root growth, or yield are “inhibited”when their measurements are significantly less than the controlvalues.3.3.18 lateral roots, nroots growing off the primary roots,also referred to as secondary roots.3.3.19 monocotyl
37、edon, n in the classification of plants,those having a single seed leaf.3.3.20 mottled, adjmarked with lesions, spots or streaksof different colors. This includes the discoloration of leafmargins.3.3.21 phytotoxicity, na lethal or sub-lethal response ofplants to a toxicant.3.3.22 quiescence, na cond
38、ition in buds, embryos, orentire plants characterized by lowered metabolic rates andlimited or no growth. Seeds that germinate when supplied withadequate moisture and moderate temperature are said to bequiescient. (See dormancy.)3.3.23 radicle, nthe emerging root of an embryo duringgermination.3.3.2
39、4 seed, nthe propagule of a plant derived from anovule. It consists of an embryo, a protective covering (seedcoat), and may have storage tissue (endosperm).3.3.25 shoot, nthe above-ground portion of a plant con-sisting of stems, leaves, as well as any reproductive parts thatmay be attached.3.3.26 su
40、rviving plants, ntest plants that are alive at thetime observations are recorded.3.3.27 viable, adjplants capable of resuming metabolicfunctions and growth are considered “viable.” Buds, embryos,or entire plants may be dormant or quiescient and thereforeexhibit no growth during the period of observa
41、tion. Distin-guishing dead plants from viable plants with certainty isdifficult without special training and sophisticated measures ofmetabolic function.3.3.28 withering, vbecoming limp or desiccated, deprivedof moisture; often the result of root damage.4. Summary of Phytotoxicity Tests4.1 The terre
42、strial phytotoxicity tests covered under thisguide apply to a range of test conditions and test species thatcan be adapted to meet project-specific objectives. Test organ-isms are maintained either as seeds or as cuttings until aparticular test is to be conducted. A prescribed number ofindividual pl
43、ants are introduced into test treatments thatinclude a negative control, a series of positive controls, and oneor more test-substance treatment concentrations. The treatmentconcentrations may be known or unknown; nominal ormeasured, depending on the nature of the investigation. In thecase where the
44、test substance is evaluated as an additive to soil,a range of concentrations is recommended. In tests of environ-mental samples that already contain a putative phytotoxicsubstance, the tests may be conducted with either the test soilas collected from the field, or as diluted with a suitablereference
45、 soil. Another variant of the tests allows foramendments, or spikes, of selected toxic substances to beadded to environmental samples. Finally, in the case of the rootelongation assay, eluates, effluents, or other aqueous deriva-tives of a soil sample are tested.4.2 Plants are exposed to the test su
46、bstances in the formdescribed in the specific variations of the tests for a discreteperiod of time that ranges from 96 h to several months. Forshort tests, no nutrient additions or amendments are needed orrecommended as the amendments may interact with the toxi-cant and alter the toxicity response.
47、For tests lasting more thantwo weeks, nutrient additives may be warranted, depending onthe test objectives, in order to maximize the potential for plantgrowth and development. Thinning, culling, or replacing indi-vidual plants must not be done once exposure of plants to a testsubstance has begun as
48、such actions invalidate the test throughthe introduction of bias or variable test duration among testorganisms. At intermediate times, and at the conclusion of theexposure period, tallies of survival and measures of shootgrowth and development are made.4.3 For phytotoxicity tests, 100 to 200 mol m2s
49、1ofvisible light (or photosynthetically active radiation, 400 to 700m) has been found to be a broadly applicable fluence rate. Insome cases, different light levels or spectral ranges (forexample, solar ultraviolet) may be required. Guide E1733.4.4 Measured endpoints and other observational data areused to calculate response levels in terms of ECxx or ICxx(where xx refers to a specified percentage response), orcategorical descriptions of phytotoxic effects (for example,proportion of plants exhibiting abnormal development or othersymptomatic i
