1、Designation: E 1963 02Standard Guide forConducting Terrestrial Plant Toxicity Tests1This standard is issued under the fixed designation E 1963; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in
2、parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 testsubstances on plant g
3、rowth 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 focus of agricul
4、ture, 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 reduce seed germina-
5、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 undesirable. Similarl
6、y,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 chemicals, sludges, met
7、als 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 long-livedpere
8、nnials 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 standardized plantte
9、sts 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 emphasize repr
10、oductivesuccess.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 Standardization12 Test Co
11、nditions13 Interference and Limitations14 Quality Assurance and Quality Control15 Calculations and Interpretation of Results16 Precision and Bias1.6 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 stand
12、ard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Specific precau-tionary statements are given in Section 8.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 4547 Practice for Sampling W
13、aste and Soils for VolatileOrganicsD 5633 Practice for Sampling with a ScoopE 1598 Practice for Conducting Early Seedling GrowthTestsE 1733 Guide for Use of Lighting in Laboratory Testing2.2 Code of Federal Regulations Standard:1This guide is under the jurisdiction of ASTM Committee E47 on Biologica
14、lEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.02 on Terrestrial Assessment and Toxicology.Current edition approved Dec. 10, 2002. Published April 2003. Originallypublished 1998. Last previous edition published 1998, as E 196398.2For referenced ASTM standards, vi
15、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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-
16、2959, United States.CFR 4932.3 Other useful references have described phytotoxicitytest procedures (1-11) .43. 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, t
17、o state 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 c
18、ondition 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
19、factors. “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,
20、nthe 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.
21、2 eluate, nsolution obtained from washing a solidwith a 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
22、inhibition, 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.
23、of atest 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 st
24、ructure 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 o
25、f shoots 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 mono
26、cotyledons, 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 tes
27、t plants, 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 d
28、evelopment, 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 growthin which buds, embryos, or entire plants survive at
29、 loweredmetabolic 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 dorm
30、ant. (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 t
31、han the 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 hypo-cotyl.3.3.13 fruits, nthe reproductive tissues derived from theov
32、ary in 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
33、defines the end of germination and the beginningof early seedling growth.3.3.15 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 whi
34、ch separates the epicotyl from thehypocotyl.3Available from Standardization Documents Order Desk, Bldg. 4, Section D,700 Robbins Ave., Philadelphia, PA 191115094, Attn: NPODS.4The boldface numbers in parentheses refer to the list of references at the end ofthis guide.E19630223.3.17 inhibited plant g
35、rowth 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 monocotyledon, n in the classificat
36、ion 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 condition in buds, embryos, or
37、entire 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.3.3.24 seed, nthe propagule of
38、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 surviving plants, ntest plan
39、ts 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 observation. Distin-guishing dead
40、 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 terrestrial phytotoxicity tests
41、 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 plants are introduced into t
42、est 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 or mea-sured, depending on the nature of the investigation. In the casewhere the test substance is evalua
43、ted as an additive to soil, arange 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 soil. Another variant o
44、f the tests allows for amend-ments, or spikes, of selected toxic substances to be added toenvironmental samples. Finally, in the case of the root elonga-tion assay, eluates, effluents, or other aqueous derivatives of asoil sample are tested.4.2 Plants are exposed to the test substances in the formde
45、scribed 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. For tests lasting more
46、 thantwo weeks, nutrient additives may be warranted, depending onthe test objectives, in order to maximize the potential for plantgrowth and development. At intermediate times, and at theconclusion of the exposure period, tallies of survival andmeasures of shoot growth and development are made.4.3 F
47、or phytotoxicity tests, 100 to 200 mol m2s1ofvisible light (or photosynthetically active radiation, 400 to 700hm) 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 E 1733.4.4 Measured
48、 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 indices th
49、at might be scored in qualitative terms)relative to controls. These are interpreted to characterizephytotoxic effects attributed to test substances.5. Significance and Use5.1 Terrestrial phytotoxicity tests are useful in assessing theeffects of environmental samples or specific chemicals as a partof an ecological risk assessment (3-6, 12, 13).5.2 Though inferences regarding higher-order ecologicaleffects (population, community, or landscape) may be madefrom the results, these tests evaluate responses of individuals ofone or more plant species t
