ASTM E1963-22

Standard Guide for Conducting Terrestrial Plant Toxicity Tests

SIGNIFICANCE AND USE
5.1 Terrestrial phytotoxicity tests are useful in assessing the effects of environmental samples or specific chemicals as a part of an ecological risk assessment (3-6, 12, 13).  
5.2 Though inferences regarding higher-order ecological effects (population, community, or landscape) may be made from the results, these tests evaluate responses of individuals of one or more plant species to the test substance.  
5.3 This guide is applicable for: (a) establishing phytotoxicity of organic and inorganic substances; (b) determining the phytotoxicity of environmental samples; (c) determining the phytotoxicity of sludges and hazardous wastes, (d) assessing the impact of discharge of toxicants to land, and (e) assessing the effectiveness of remediation efforts.
SCOPE
1.1 This guide covers practices for conducting plant toxicity tests using terrestrial plant species to determine effects of test substances on plant growth and development. Specific test procedures are presented in accompanying annexes.  
1.2 Terrestrial plants are vital components of ecological landscapes. The populations and communities of plants influence the distribution and abundance of wildlife. Obviously, plants are the central focus of agriculture, forestry, and rangelands. Toxicity tests conducted under the guidelines and annexes presented herein can provide critical information regarding the effects of chemicals on the establishment and maintenance of terrestrial plant communities.  
1.3 Toxic substances that prevent or reduce seed germination can have immediate and large impacts to crops. In natural systems, many desired species may be sensitive, while other species are tolerant. Such selective pressure can result in changes in species diversity, population dynamics, and community structure that may be considered undesirable. Similarly, toxic substances may impair the growth and development of seedlings resulting in decreased plant populations, decreased competitive abilities, reduced reproductive capacity, and lowered crop yield. For the purposes of this guide, test substances include pesticides, industrial chemicals, sludges, metals or metalloids, and hazardous wastes that could be added to soil. It also includes environmental samples that may have had any of these test substances incorporated into soil.  
1.4 Terrestrial plants range from annuals, capable of completing a life-cycle in as little as a few weeks, to long-lived perennials that grow and reproduce for several hundreds of years. Procedures to evaluate chemical effects on plants range from short-term measures of physiological responses (for example, chlorophyll fluorescence) to field studies of trees over several years. Research and development of standardized plant tests have emphasized three categories of tests: (1) short-term, physiological endpoints (that is, biomarkers); (2) short-term tests conducted during the early stages of plant growth with several endpoints related to survival, growth, and development; and (3) life-cycle toxicity tests that emphasize reproductive success.  
1.5 This guide is arranged by sections as follows:  
Section  
Title  
1  
Scope  
2  
Referenced Documents  
3  
Terminology  
4  
Summary of Phytotoxicity Tests  
5  
Significance and Use  
6  
Apparatus  
7  
Test Material  
8  
Hazards  
9  
Test Organisms  
10  
Sample Handling and Storage  
11  
Calibration and Standardization  
12  
Test Conditions  
13  
Interference and Limitations  
14  
Quality Assurance and Quality Control  
15  
Calculations and Interpretation of Results    
16  
Precision and Bias  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate saf...

General Information

• Status: Published
• Publication Date: 31-Jul-2022
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM D4547-15 - Standard Guide for Sampling Waste and Soils for Volatile Organic Compounds
• Effective Date: 01-Sep-2015
• Refers: ASTM D5633-04(2012) - Standard Practice for Sampling with a Scoop
• Effective Date: 01-Sep-2012
• Refers: ASTM D4547-09 - Standard Guide for Sampling Waste and Soils for Volatile Organic Compounds
• Effective Date: 01-Jul-2009
• Refers: ASTM D5633-04(2008) - Standard Practice for Sampling with a Scoop
• Effective Date: 01-Sep-2008
• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E1733-95(2008) - Standard Guide for Use of Lighting in Laboratory Testing
• Effective Date: 01-Feb-2008
• Refers: ASTM D4447-06 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D4447-10 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D4547-06 - Standard Guide for Sampling Waste and Soils for Volatile Organic Compounds
• Effective Date: 01-Feb-2006
• Refers: ASTM D5633-04 - Standard Practice for Sampling with a Scoop
• Effective Date: 01-Apr-2004
• Refers: ASTM D4547-03 - Standard Guide for Sampling Waste and Soils for Volatile Organic Compounds
• Effective Date: 10-Jun-2003
• Refers: ASTM D4447-84(2003) - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 10-Mar-2003
• Refers: ASTM D1193-99e1 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999
• Refers: ASTM D1193-99 - Standard Specification for Reagent Water
• Effective Date: 10-Feb-1999

Overview

ASTM E1963-22: Standard Guide for Conducting Terrestrial Plant Toxicity Tests is a comprehensive guide developed by ASTM International to establish consistent procedures for assessing the effects of chemicals and environmental samples on terrestrial plant species. These plant toxicity tests are essential tools for ecological risk assessment, helping to determine the phytotoxic effects of a wide range of substances, including pesticides, industrial chemicals, sludges, metals, hazardous wastes, and contaminated soils. Adhering to this standard is critical for environmental professionals, researchers, and regulatory bodies involved in ecological protection, environmental monitoring, and soil remediation.

Key Topics

Purpose and Scope

• Provides standardized practices for conducting toxicity tests using terrestrial plants.
• Evaluates plant responses - including growth, survival, and development - to test substances under controlled conditions.
• Tests cover annual and perennial plants, with endpoints ranging from short-term physiological effects to long-term reproductive success.

Test Substances and Environmental Samples

• Applicable to a broad spectrum of substances:
  • Organic and inorganic chemicals
  • Environmental samples and contaminated soils
  • Sludges and hazardous wastes

Test Methods and Endpoints

• Procedures include:
  • Seed germination and root elongation assays
  • Short-term and life-cycle tests for growth and reproductive endpoints
• Endpoints measured:
  • Seed germination rate
  • Seedling growth and development
  • Survival and plant yield
  • Physiological responses such as chlorophyll fluorescence

Laboratory and Field Application

• Tests can be adapted for laboratory, greenhouse, or field conditions.
• Guidance on apparatus, test material preparation, sample handling, biological materials, quality control, and data interpretation.

Safety and Compliance

• Emphasizes the importance of safety, including appropriate use of personal protective equipment and proper disposal of test substances according to regulations.
• References applicable laws and related standards for sample collection, handling, and hazardous materials disposal.

Applications

ASTM E1963-22 supports a variety of practical applications across multiple industries:

• Ecological Risk Assessment: Evaluates the impact of pollutants and waste materials on plant health, informing site assessments and regulatory decisions.
• Soil and Remediation Projects: Assesses soil quality, detects phytotoxic contamination, and determines the effectiveness of soil remediation efforts.
• Agriculture and Land Management: Examines how industrial chemicals, pesticides, and waste materials affect crops and rangelands, supporting sustainable land use practices.
• Environmental Monitoring: Enables ongoing surveillance of terrestrial ecosystems near industrial sites, landfills, and remediation sites for early detection of adverse effects.

Organizations involved in environmental consulting, agriculture, soil science, and government regulation utilize these guidelines to ensure reliable, repeatable results in plant toxicity testing.

Related Standards

To meet regulatory requirements and ensure best practices, users of ASTM E1963-22 should be familiar with related standards and references, such as:

• ASTM D1193 - Specification for Reagent Water
• ASTM D4447 - Guide for Disposal of Laboratory Chemicals and Samples
• ASTM D4547 - Guide for Sampling Waste and Soils for Volatile Organic Compounds
• ASTM D5633 - Practice for Sampling with a Scoop
• ASTM E943 - Terminology Relating to Biological Effects and Environmental Fate
• ASTM E1733 - Guide for Use of Lighting in Laboratory Testing

For international projects, reference to OECD Guidelines and local environmental protection regulations may also be necessary.

---

By following ASTM E1963-22, professionals can generate high-quality, scientifically valid data to evaluate the risks and impacts of chemical exposure on terrestrial plant communities, supporting both environmental safety and regulatory compliance.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E1963 − 22
Standard Guide for
Conducting Terrestrial Plant Toxicity Tests
This standard is issued under the fixed designation E1963; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope tests conducted during the early stages of plant growth with
severalendpointsrelatedtosurvival,growth,anddevelopment;
1.1 Thisguidecoverspracticesforconductingplanttoxicity
and ( 3) life-cycle toxicity tests that emphasize reproductive
tests using terrestrial plant species to determine effects of test
success.
substances on plant growth and development. Specific test
procedures are presented in accompanying annexes. 1.5 This guide is arranged by sections as follows:
Section Title
1.2 Terrestrial plants are vital components of ecological
1 Scope
landscapes. The populations and communities of plants influ-
2 Referenced Documents
ence the distribution and abundance of wildlife. Obviously,
3 Terminology
4 Summary of Phytotoxicity Tests
plants are the central focus of agriculture, forestry, and range-
5 Significance and Use
lands. Toxicity tests conducted under the guidelines and
6 Apparatus
annexes presented herein can provide critical information 7 Test Material
8 Hazards
regarding the effects of chemicals on the establishment and
9 Test Organisms
maintenance of terrestrial plant communities.
10 Sample Handling and Storage
11 Calibration and Standardization
1.3 Toxic substances that prevent or reduce seed germina-
12 Test Conditions
tion can have immediate and large impacts to crops. In natural
13 Interference and Limitations
14 Quality Assurance and Quality Control
systems, many desired species may be sensitive, while other
15 Calculations and Interpretation of Results
species are tolerant. Such selective pressure can result in
16 Precision and Bias
changes in species diversity, population dynamics, and com-
1.6 The values stated in SI units are to be regarded as
munitystructurethatmaybeconsideredundesirable.Similarly,
standard. No other units of measurement are included in this
toxic substances may impair the growth and development of
standard.
seedlings resulting in decreased plant populations, decreased
1.7 This standard does not purport to address all of the
competitive abilities, reduced reproductive capacity, and low-
safety concerns, if any, associated with its use. It is the
ered crop yield. For the purposes of this guide, test substances
responsibility of the user of this standard to establish appro-
include pesticides, industrial chemicals, sludges, metals or
priate safety, health, and environmental practices and deter-
metalloids,andhazardouswastesthatcouldbeaddedtosoil.It
mine the applicability of regulatory limitations prior to use.
also includes environmental samples that may have had any of
Specific precautionary statements are given in Section 8.
these test substances incorporated into soil.
1.8 This international standard was developed in accor-
1.4 Terrestrial plants range from annuals, capable of com-
dance with internationally recognized principles on standard-
pleting a life-cycle in as little as a few weeks, to long-lived
ization established in the Decision on Principles for the
perennials that grow and reproduce for several hundreds of
Development of International Standards, Guides and Recom-
years. Procedures to evaluate chemical effects on plants range
mendations issued by the World Trade Organization Technical
from short-term measures of physiological responses (for
Barriers to Trade (TBT) Committee.
example,chlorophyllfluorescence)tofieldstudiesoftreesover
several years. Research and development of standardized plant
2. Referenced Documents
tests have emphasized three categories of tests: (1) short-term,
2.1 ASTM Standards:
physiological endpoints (that is, biomarkers); (2) short-term
D1193Specification for Reagent Water
D4447Guide for Disposal of Laboratory Chemicals and
Samples
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Aug. 1, 2022. Published September 2022. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
published in 1998. Last previous edition published 2014 as E1963–09(2014). DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E1963-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1963 − 22
D4547Guide for Sampling Waste and Soils for Volatile 3.2.5 leachate, n—water plus solutes that has percolated
Organic Compounds through a column of soil or waste.
D5633Practice for Sampling with a Scoop
3.2.6 test material, n—any formulation, dilution, etc. of a
E943Terminology Relating to Biological Effects and Envi-
test substance.
ronmental Fate
3.2.7 test substance, n—a chemical, formulation, eluate,
E1598Practice for Conducting Early Seedling GrowthTests
sludge, or other agent or substance that is the target of the
(Withdrawn 2003)
investigation in a toxicity test.
E1733Guide for Use of Lighting in Laboratory Testing
3.2.8 toxicant, n—anagentormaterialcapableofproducing
2.2 Code of Federal Regulations Standard:
an adverse response (effect) in a biological system, adversely
CFR 49
impacting structure or function or producing death.
2.3 Other useful references have described phytotoxicity test
3.2.9 toxicity endpoints, n—measurements of organism re-
procedures(1-11) .
sponse such as death, growth, developmental, or physiological
parameters resulting from exposure to toxic substances.
3. Terminology
3.3 Definitions of Terms Specific to This Standard:
3.3.1 chlorotic, adj—the discoloration of shoots that occurs
3.1 General Terminology—The words “must,” “should,”
as chlorophyll is degraded as a result of disease, toxic
“may,”“ can,” and “might” have very specific meanings in this
guide. “Must” is used to express an absolute requirement, that substances, nutrient deficiencies, or senescence.
is, to state that the test ought to be designed to satisfy the
3.3.2 coleoptile, n—the protective tissues surrounding the
specified condition, unless the purpose of the test requires a
growing shoot in a monocotyledonous plant.
differentdesign.“Must”isonlyusedinconnectionwithfactors
3.3.3 cotyledon, n—a primary leaf of the embryo in seeds,
that directly relate to the acceptability of the test (see Section
only one in the monocotyledons, two in dicotyledons. In many
14). “Should” is used to state that the specified condition is
of the latter, such as the bean, they emerge above ground and
recommended and ought to be met if possible. Although
appear as the first leaves.
violationofone“should”israrelyaseriousmatter,violationof
3.3.4 cutting, n—a vegetative segment of a plant, usually a
several will often render the results questionable. Terms such
stem that contains several nodes and associated buds, that can
as“isdesirable,”“isoftendesirable,”and“mightbedesirable”
be used to regenerate an entire plant.
are used in connection with less important factors. “May” is
3.3.5 dead test plants, n—those individuals that expired
used to mean “is (are) allowed to,” “can” is used to mean “is
during the test observation period as indicated by severe
(are) able to,” and “might” is used to mean “could possibly.”
desiccation, withering, chlorosis, necrosis, or other symptoms
Thus the classic distinction between “may” and “can” is
that indicate non-viability.
preserved, and “might” is never used as a synonym for either
“may” or “can.”
3.3.6 desiccated, adj—the plant, or portion of the plant, that
3.1.1 For definitions of terms used in this guide, refer to
is dried in comparison to the control plant.
Terminology E943.
3.3.7 development, n—the series of steps involving cell
3.2 Definitions: divisionandcelldifferentiationintovarioustissuesandorgans.
3.2.1 control, n—the treatment group in a toxicity test
3.3.8 dicotyledon, n—in the classification of plants, those
consisting of reference soil or artificial soil that duplicates all
having two seed leaves.
the conditions of the exposure treatments, but contains no test
3.3.9 dormancy, n—aspecialconditionofarrestedgrowthin
substance. The control is used to determine if there are any
which buds, embryos, or entire plants survive at lowered
statistical differences in endpoints related to the test substance.
metabolic activity levels. Special environmental cues such as
3.2.2 eluate, n—solutionobtainedfromwashingasolidwith
particular temperature regimes or photoperiods are required to
a solvent to remove adsorbed material.
activate metabolic processes and resume growth. Seeds that
require additional treatment besides adequate moisture and
3.2.3 hazardous substance, n—a material that can cause
deleterious effects to plants, microbes, or animals. (A hazard- moderatetemperaturetogerminatearesaidtobedormant.(See
quiescence.)
ous substance does not, in itself, present a risk unless an
exposure potential exists.)
3.3.10 emergence, n—following germination of a plant, the
early growth of a seedling that pushes the epicotyl through the
3.2.4 inhibition, n—a statistically lower value of any end-
soil surface.
point compared to the control values that is related to environ-
mental concentration or application rate.
3.3.11 enhanced growth and yield, n—when a treated plant
exhibits shoot growth, root elongation, lateral root growth, or
yield significantly greater than the control values, the plant is
The last approved version of this historical standard is referenced on
“enhanced” or “stimulated.”
www.astm.org.
Available from Standardization Documents Order Desk, DODSSP, Bldg. 4,
3.3.12 epicotyl, n—that portion of an embryo or seedling
Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098, http://
containing the shoot. It is delineated anatomically by the
www.dodssp.daps.mil.
transition zone which separates the epicotyl from the hypoco-
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this guide. tyl.
E1963 − 22
3.3.13 fruits, n—the reproductive tissues derived from the can be adapted to meet project-specific objectives. Test organ-
ovary in the case of epigenous flowers or the ovary and isms are maintained either as seeds or as cuttings until a
accessory tissues in the case of hypigenous flowers. particular test is to be conducted. A prescribed number of
individual plants are introduced into test treatments that
3.3.14 germination, n—the physiological events associated
includeanegativecontrol,aseriesofpositivecontrols,andone
withre-initiationofembryogrowthandmobilizationofreserve
ormoretest-substancetreatmentconcentrations.Thetreatment
nutrients in seeds. The emergence of the seedling radicle from
concentrations may be known or unknown; nominal or
the seed coat defines the end of germination and the beginning
measured, depending on the nature of the investigation. In the
of early seedling growth.
casewherethetestsubstanceisevaluatedasanadditivetosoil,
3.3.15 growth, n—a change in size or mass measured by
a range of concentrations is recommended. In tests of environ-
length, height, volume, or mass.
mental samples that already contain a putative phytotoxic
3.3.16 hypocotyl, n—that portion of an embryo or seedling
substance, the tests may be conducted with either the test soil
containing the root or radicle. It is delineated anatomically by
as collected from the field, or as diluted with a suitable
the transition zone which separates the epicotyl from the
reference soil. Another variant of the tests allows for
hypocotyl.
amendments, or spikes, of selected toxic substances to be
addedtoenvironmentalsamples.Finally,inthecaseoftheroot
3.3.17 inhibited plant growth and yield, n— plant growth,
root length and lateral root growth, or yield are “inhibited” elongation assay, eluates, effluents, or other aqueous deriva-
tives of a soil sample are tested.
whentheirmeasurementsaresignificantlylessthanthecontrol
values.
4.2 Plants are exposed to the test substances in the form
3.3.18 lateral roots, n—roots growing off the primary roots, described in the specific variations of the tests for a discrete
also referred to as secondary roots.
period of time that ranges from 96 h to several months. For
short tests, no nutrient additions or amendments are needed or
3.3.19 monocotyledon, n— in the classification of plants,
recommended as the amendments may interact with the toxi-
those having a single seed leaf.
cant and alter the toxicity response. For tests lasting more than
3.3.20 mottled, adj—marked with lesions, spots or streaks
two weeks, nutrient additives may be warranted, depending on
of different colors. This includes the discoloration of leaf
the test objectives, in order to maximize the potential for plant
margins.
growth and development. Thinning, culling, or replacing indi-
3.3.21 phytotoxicity, n—a lethal or sub-lethal response of
vidualplantsmustnotbedoneonceexposureofplantstoatest
plants to a toxicant.
substance has begun as such actions invalidate the test through
3.3.22 quiescence, n—a condition in buds, embryos, or the introduction of bias or variable test duration among test
entire plants characterized by lowered metabolic rates and organisms.At intermediate times, and at the conclusion of the
limitedornogrowth.Seedsthatgerminatewhensuppliedwith exposure period, tallies of survival and measures of shoot
adequate moisture and moderate temperature are said to be growth and development are made.
quiescent. (See dormancy.) –2 –1
4.3 Forphytotoxicitytests,100µmolto200µmolm s of
3.3.23 radicle, n—the emerging root of an embryo during
visible light (or photosynthetically active radiation, 400 ηmto
germination.
700ηm)hasbeenfoundtobeabroadlyapplicablefluencerate.
In some cases, different light levels or spectral ranges (for
3.3.24 seed, n—the propagule of a plant derived from an
example, solar ultraviolet) may be required. Guide E1733.
ovule. It consists of an embryo, a protective covering (seed
coat), and may have storage tissue (endosperm).
4.4 Measured endpoints and other observational data are
used to calculate response levels in terms of ECxx or ICxx
3.3.25 shoot, n—the above-ground portion of a plant con-
(where xx refers to a specified percentage response), or
sisting of stems, leaves, as well as any reproductive parts that
categorical descriptions of phytotoxic effects (for example,
may be attached.
proportion of plants exhibiting abnormal development or other
3.3.26 surviving plants, n—test plants that are alive at the
symptomatic indices that might be scored in qualitative terms)
time observations are recorded.
relative to controls. These are interpreted to characterize
3.3.27 viable, adj—plants capable of resuming metabolic
phytotoxic effects attributed to test substances.
functions and growth are considered “viable.” Buds, embryos,
or entire plants may be dormant or quiescent and therefore
5. Significance and Use
exhibit no growth during the period of observation. Distin-
5.1 Terrestrial phytotoxicity tests are useful in assessing the
guishing dead plants from viable plants with certainty is
effectsofenvironmentalsamplesorspecificchemicalsasapart
difficult without special training and sophisticated measures of
of an ecological risk assessment (3-6, 12, 13).
metabolic function.
5.2 Though inferences regarding higher-order ecological
3.3.28 withering, v—becoming limp or desiccated, deprived
effects (population, community, or landscape) may be made
of moisture; often the result of root damage.
fromtheresults,thesetestsevaluateresponsesofindividualsof
4. Summary of Phytotoxicity Tests one or more plant species to the test substance.
4.1 The terrestrial phytotoxicity tests covered under this 5.3 This guide is applicable for: ( a) establishing phytotox-
guide apply to a range of test conditions and test species that icity of organic and inorganic substances; (b) determining the
E1963 − 22
phytotoxicity of environmental samples; (c) determining the constituents of the test substance. Paper and plastic plant pots
phytotoxicity of sludges and hazardous wastes, (d) assessing should be disposed after one use.
the impact of discharge of toxicants to land, and (e) assessing
the effectiveness of remediation efforts. 7. Test Material
7.1 Chemical Substance:
6. Apparatus
7.1.1 General—The test substance should be reagent-grade
6.1 Facilities—Thepreparationofthetest,testsoilmedium,
orbetter,unlessatestonaformulation,commercialproduct,or
storageofsoilandseeds,andallstagesofatestproceduremust
technical-grade or use-grade substance is specifically needed.
take place in an atmosphere free from toxic contamination and
Before a test is initiated, the following information should be
vapors. The facility, whether a greenhouse or a growth
obtained about the test substance: identities and concentration
chamber, should have reasonable temperature control and
of major ingredients and major impurities, for example, impu-
monitoring, as well as supplemental lighting. In general, the
rities constituting more than about 1% of substance; solubility
facilityshouldbecapableofmaintaininguniformtemperatures
andstabilityindilutionwater;anestimateoftoxicitytothetest
in the 20°C to 30°C range. Lighting should provide at least
species (a range-finding study may be required); precision and
–2 –1
250 µmol m s Photosynthetically Active Radiation (PAR)
biasoftheanalyticalmethodattheplannedconcentration(s)of
controlled on a clock timer to maintain a specified diurnal
the test substance; and an estimate of toxicity to humans and
cycle.Seeappropriateannexforanyspecificrequirementsofa
other potentially exposed organisms.
given test.
7.1.2 Test Concentrations—Chemical concentrations in
soils are expressed as dry weight to dry weight. It is preferable
6.2 Equipment and Supplies:
6.2.1 Plant Pots—Containersshouldbechosentobeinertto to add the test substance directly to the test medium, however,
a stock solution may be prepared and aliquots added to each
test and control substances. The test or control substances
should not adhere to or react in any way with the container. test solution or test chamber. Special considerations regarding
chemicaldegradation,complexing,andvolatilizationandother
Glass, stainless steel, or paper containers with drainage holes
canbeusedasplantpots.Paperorothernaturalfibermaterials factorsthatmightinfluencebioavailabilityshouldbeevaluated
may absorb test substances. If pots with drainage holes are to determine the appropriate mixing, handling, and storage
used, then a secondary container or shallow dish should be procedures to be used. The number of selected test concentra-
usedtopreventcross-contaminationamongtestunits.Polyeth- tions should be based on the goal of the study. Multiple
concentrations can be used to calculate ICxx values, whereas,
ylene pots or other containers may be used, provided they are
free of toxic materials.The volume of the pot container should testing at a single concentration can be used to obtain rapid,
simple answers. When the interest is (a) in the effect of a
be large enough so as not to restrict seedling growth for the
duration of the test. It is suggested that the selection of growth specific concentration of test substance on the growth of the
test species or (b) whether or not the ICxx value is above or
containersnotbearbitrary,andthattheappropriatesize,shape,
color, and composition of the container be considered for each below a specific concentration, only one concentration and the
controls need to be tested.
plant species and toxicity test undertaken.
6.2.2 Balance—Sensitivity to 0.001 g.
7.1.3 Stock Solution— For compounds with low water
6.2.3 pH Meter—Sensitivity to 0.1 units.
solubility, a solvent can be used to make a stock solution. If a
6.2.4 Photometer (Radiometer)—Capable of measuring the
stocksolutionisused,theconcentrationandstabilityofthetest
photosynthetically active range. Fluence rate of incident light
substance in the stock should be determined before the
–2 –1
should be expressed as µmol m s .
beginning of the test. If the test substance is subject to
6.2.5 Thermometer—A continuous recording thermometer
photolysisorotherphoto-reactiveprocesses,thestocksolution
or a maximum-minimum thermometer that is checked daily.
should be shielded from light. If a solvent is necessary, its
Many continuous recording units also record humidity.
concentration in test solutions should be kept to a minimum
6.2.6 Industrial Mixer or Cement Mixer—A revolving or (not greater than 1% [volume to volume or weight to
rotating mixer is recommended for combining test substances volume]), and should be low enough that it does not affect
or test soils with large volumes of control or reference soil
either survival or growth of the test organisms. (These limita-
medium. tionsdonotapplytoanyingredientsofamixture,formulation,
6.2.7 Reagent Water—Unless otherwise indicated, refer- or commercial product unless an extra amount of solvent is
ences to water shall be understood to mean reagent water used in the preparation of the stock solution.) If the concen-
conforming to Specification D1193, Type III. Type III water tration of solvent is not the same in all test solutions that
maybepreparedbydistillation,ionexchange,reverseosmosis, contain test substance, either (a) a solvent test must be
or a combination of methods. conducted to determine whether either survival, or growth of
6.2.8 Equipment Care—Clean the test equipment after each the test species is related to the concentration of solvent over
use. Wash all new containers with a detergent and rinse therangeusedinthephytotoxicitytestor(b)suchasolventtest
thoroughlywithwater,pesticide-freeacetone,diluteacid(such must have already been conducted using the same dilution
as 5% hydrochloric acid), and at least twice with tap or clean water and test species. If either survival or growth is found to
water. Final rinses with Specification D1193 Type III water or be related to the concentration of solvent, a test would be
equivalent is recommended. Clean equipment, such as the unacceptable if any treatment contained a concentration of
mixer and mixer blades by a procedure known to remove solvent in the response range. If neither survival, or growth is
E1963 − 22
found to be related to the concentration of solvent, a toxicity the test material should be studied before a test is begun.
test with that same species in the same water may contain Special procedures might be necessary with radio-labeled test
solvent concentrations within the tested range, but the solvent materials (23, 24) and with test materials that are, or are
control must contain the highest concentration of solvent suspected of being, carcinogenic (25).
present in any of the other treatments.
8.2 Although disposal of stock solutions, test solutions, test
7.1.4 Soil Medium— Natural soil (free of chemical
soil, and test organisms pose no special problems in most
contamination), commercial potting soil, synthetic soil mixes,
cases, health and safety precautions and applicable regulations
orwashedquartzsandmaybeusedasthe“soilmedium.”Each
should be considered before beginning a test. Removal or
choice has substantive limitations for various phytotoxicity
degradation of the test substance in the test medium might be
investigations. Natural soils are not easily demonstrated to be
desirablebeforedisposalofstockandtestsolutions(seeGuide
free of toxic substances. Some commercial potting soils may
D4447). Hazardous materials must be disposed of in accor-
adversely affect growth and survival of some plants. Synthetic
dance with local, state, and federal regulations.
mixes may not be representative of real world conditions.
8.3 Because water is a good conductor of electricity, use of
Quartz sand or glass beads offer only a physical matrix; and
ground fault systems and leak detectors should be considered
thereforedonotprovidearealisticsoilconditionwithregardto
to help avoid electrical shocks.
binding and exchange sites. It may be especially important to
consider soil texture, pH, organic matter or other physical-
9. Test Organisms
chemical properties before embarking on a test. Preliminary
trials are often valuable to ascertain the suitability of a
9.1 Test Species—The majority of species routinely used in
particular soil medium for the test species and conditions to be
phytotoxicitytestshasbeenlimitedtoagronomicplants.Under
investigated.
FIFRAguidelines (4, 5),tenspeciesbelongingtoeightfamilies
7.2 Environmental Sample: are listed for toxicity testing (see Table 1). The United States
7.2.1 Liquid, Sludge, or Slurry—These environmental Food and Drug Administration (11, 26), has relied on plant
samples may be handled as chemical additives described tests similar to those for FIFRA (see Table 1). International
above. As complex mixtures, however, the test concentrations guidance (10) uses agronomic species, but has a broader
will most likely be handled as percentage dilutions of the selection of plants compared to United States guidance. CER-
100% sample concentration. In some cases, selected chemical CLA offers limited guidance with respect to plant testing.
analyses may be warranted as a means of expressing concen- GeneralmethodsrecommendedfortheRemedialInvestigation
trations of selected constituents in ppm or molar values.All of Baseline RiskAssessment portion of work listed by name only
the provisions described for single chemicals apply. the seed germination and root elongation assays (3, 6). Only
7.2.2 Soil—Site soils may be collected as cores or as bulk lettuce (Lactuca sativa) is listed as the standard species of the
samples from specified soil depths (for example, 0 to 15 cm test, although “other (taxa) can be used.” The Department of
depth). Sampling and handling procedures may be found in Interior in developing rules for Natural Resource Damage
Practices D4547 and D5633. The soil samples may be tested Assessment (27) referred to “economically important plant
directly (that is, 100% site soil) or diluted with an appropriate species.” Thirty-one plant taxa are explicitly identified in
federal and international test guidelines and standard test
reference soil or a synthetic soil mixture to achieve specified
relative concentrations. In some cases, selected chemical procedures (see Table 1). Many additional plant taxa including
aquatictaxawerereportedinphytotoxicityliterature(seeTable
analyses may be warranted as a means of expressing concen-
trations of selected constituents in ppm (dry weight basis) or 2). Nearly a hundred plant taxa (see Table 2) have been used
routinely to study phytotoxicity. In an early version of PHY-
molar values.
7.2.3 Eluates—Aqueous extracts of soils are sometimes TOTOX (28), 1569 plant species from 682 genera in 147
families were reported in the records. However, 42% of the
desired to evaluate the phytotoxicity of water-soluble soil
constituents.Theeluatesareusedinthesamemannerasliquid records referred to only 20 species.
environmental samples described above.
9.2 Purchase—Seeds of the most commonly used taxa
identified in FIFRA guidelines may be purchased from com-
8. Hazards
mercial seed companies. Many of the less common taxa are
8.1 Many materials can adversely affect humans if safety available from specialty seed companies, especially those that
precautionsareinadequate.Therefore,skincontactwithalltest service landscaping and restoration activities. When purchas-
materials and solutions of them should be minimized by such ing seeds, it is best to talk to technical staff of the supplier to
means as wearing appropriate protective gloves (especially gatherimportantinformationregardingtheseedlot,collection,
when washing equipment, putting hands in test solutions or handling and storage practices of the seed company, germina-
treated soil, or handling treated plant material), laboratory tion percentage expected, and any special conditions affecting
coats, aprons, and glasses. Special precautions, such as venti- germination. Generally, it is preferable to use untreated seeds
lating the area surrounding the flats should be taken when (thatis,nottreatedwithfungicide,repellents,orotherchemical
conducting tests on volatile materials or dust containing agents) in phytotoxicity tests, however, specific test objectives
hazardous substances. Respirators may be warranted. Informa- may permit use of treated seeds. The principal investigator
tion on toxicity to humans (14-18), recommended handling shoulddetailtherationaleforusingtreatedseeds.Seedsshould
procedures (19-22), and chemical and physical properties of be acquired at least annually. At a minimum, a sufficient
E1963 − 22
A
TABLE 1 List of Plant Species Identified in Regulatory Documents and in Standard Test Procedures
APHA ASTM
Family Species Common Name FIFRA TSCA FDA OECD
AWWA ESG
Chenopodiaceae Atriplex patula Seaside Greens =
Compositae Lactuca sativa Lettuce === = =
Cruciferae Brassica alba Mustard ==
Cruciferae Brassica campestris var. chinensis Chinese Cabbage ==
Cruciferae Brassica napus Rape ==
Cruciferae Brassica oleracea Cabbage === =
Cruciferae Brassica rapa Turnip ==
Cruciferae Lepidium sativum Cress ==
Cruciferae Raphanus sativus Radish ==
Cruciferae Rorippa nasturtium-aquaticum Watercress =
Cucurbitaceae Cucumis sativus Cucumber === =
Leguminosae Glycine max Soybean === =
Leguminosae Phaseolus aureus Mungbean ==
Leguminosae Phaseolus vulgaris Bean ==
Leguminosae Trifolium ornithopodioides Fenugreek ==
Leguminosae Trifolium pratense Red Clover ==
Leguminosae Vicia sativa Vetch ==
Liliaceae Allium cepa Onion == =
Nymphaeceae Nelumbo lutea American Lotus =
Poaceae Avena sativa Oat === = =
Poaceae Echinochloa cursgalli Japanese Millet =
Poaceae Leersia oryzoides Rice Cutgrass =
Poaceae Lolium perenne Perennial Ryegrass === = =
Poaceae Oryza sativa Rice == =
Poaceae Sorghum bicolor Sorghum ==
Poaceae Spartina alterniflora Smooth Cordgrass =
Poaceae Triticum aestivum Wheat == =
Poaceae Zea mays Corn === =
Poaceae Zizania aquatica Wild Rice =
Solonaceae Lycopersicon esculentum Tomato === =
Umbelliferae Daucus carota Carrot === =
A
FIFRA = Federal Insecticide, Fungicide, and Rodenticide Act (4)(5) ; TSCA = Toxic Substance Control Act (2) ; FDA = Federal Drug Administration (11)(26); OECD =
Organization for Economic Cooperation and Development (10);APHA=American Public HealthAssociation;AWWA=American Water WorksAssociation (1); andASTM
= American Society for Testing and Materials (Practice E1598).
quantity of seeds should be acquired to allow tests of all example can be separated mechanically using wire mesh
1 1 1 1 1 1 1 1
treatments (including controls) to be conducted with seeds screens: ⁄6 × ⁄28 in.; ⁄6 × ⁄30 in.; ⁄6 × ⁄32 in.; ⁄6 × ⁄34 in. Red
from the same batch. clover may be sized using perforated metal sheets with round
1 1 1 1
holes of the following diameters: ⁄19 in., ⁄18 in., ⁄17 in., ⁄16 in.
9.3 Collection—If seeds are collected from the field, care
9.4.2 Native Species— If this test uses native plant seeds
must be taken to ensure that seeds from only a single species
rather than commercially selected plants, considerable care
are obtained. The following minimum set of information
should be taken in sizing and sorting seeds collected. Numer-
should be recorded for each batch of seeds collected: the
ousstudieshaveshownthatthevariabilityinseedgermination
location of the collection site as precisely as practicable (for
is not entirely random within a population of a particular
example, section, township and range, county, state); the
species. The point during the growing season at which a lot of
persons collecting the seeds; date of collection; description of
seeds are produced and collected will affect germination in
noteworthy circumstances such as drought, flood, condition of
many species. Also, the location within a particular inflores-
surrounding landscape, and any indication of pesticide use in
cence (for example, with composites) will also affect germi-
the vicinity; and quantity of seeds collected.
nation. There can also be considerable intra-species variation
9.4 Grading and Sizing Seeds:
between remote populations. The test design becomes consid-
9.4.1 Domestic Species— Seeds of a given species vary in
erably more complicated to account for these and other
size, shape, and in some cases, color. These differences in
potential sources of variation.
external features of the seed are often associated with different
rates of germination or even different germination require- 9.5 Seed Storage and Maintenance—Seedsshouldbestored
ments.To minimize the variance in test results the investigator inadesiccatorandrefrigerateduntilneeded(preferablyat4°C
should determine whether such variants in seed size, shape, or 6 2°C). It is recommended no disinfecting agent such as
color are critical to the investigation. (For example, alfalfa hypochlorite be used. Exceptions may be warranted for some
seeds often come as a mixture of light-colored and dark- investigations if gnotobiotic conditions are desired, however,
colored seeds. The dark-colored seeds have low percentage such special cases must be described fully as exceptions to the
germination (;10%), while the light-colored seeds have high guide described here. Examples of exceptions would include,
percentage germination (;90%).) Separation of broken or but not be limited to, amendments with microbial inocula such
damaged seeds from the batch is important. Various sieves or asrhizobiaforlegumes,actinomycetesforactinorhizalspecies,
screens may be useful in separating the seeds. Lettuce for or mycorrhizal fungi.
E1963 − 22
TABLE 2 Continued
9.6 Seedlings or cuttings may be collected from the field,
Species Common Name Ref.
propagated by the investigator, or purchased from nurseries,
Raphanus sativus radish (29), (30)
horticulture supply houses, or research laboratories. As with
Rubus sp. raspberry (31)
seeds, it is important to document as much information as
Setaria italica foxtail millet (30)
Solanum tuberosum potato (30), (31)
reasonable for each batch of cuttings obtained. Care should be
Sorghum bicolor sundangrass; sorghum (29), (30), (31)
taken to limit the range of stem size, age, and developmental
Spartina alterniflora cordgrass (33)
stage of the plant.
Spinacia oleracea spinach (29), (31)
Spirea alba meadowsweet (31)
TABLE 2 Partial Listing of Plant Taxa studied for Toxicity
Spirea alba meadow sweet (31)
Effects
Tagetes sp. marigold (29)
Species Common Name Ref.
Thalassia testidinum seagrass (30)
Tradescantia paludosa spiderwort (30)
Agrostis alba red top (29)
Agrostis sp. bentgrass (29) Trifolium pratense clover (30)
Triticum aestivum wheat (30), (31)
Apocynum sp. milkweed (30)
Arabidopsis thaliana mouse-ear-cress (30) Vicia faba broad bean (30)
Vicia sp. vetch (29)
Arachis hypogaea peanut (29)
Avena sativa oats (29), (30), (31) Zea mays corn (30), (31)
Beta vulgaris beets (29), (30), (31)
Beta vulgaris chard (29) 10. Sample Handling and Storage
Beta vulgaris sugarbeet (30), (31)
10.1 The proper collection, packaging, and shipping of
Brassica campestris kale (29), (31)
Brassica nigra mustard (29), (30), (31)
waste site samples is critical. Proper sampling and shipping
Brassica oleracea broccoli (29)
ensures sample integrity, handling safety, and an adequate data
Brassica oleracea cauliflower (29)
base for sample processing and future sampling requirements.
Brassica rapa turnip (29)
Bromus smooth bromegrass (29)
Local, state, and federal shipping regulations should be con-
Bromus japonicus Japanese bromegrass (29)
sulted regarding size and quantity restrictions, labeling, and
Cenchrus ciliaris buffelgrass (29)
documentation requirements. Sample packaging depends upon
Chrysanthemum sp. chrysanthemum (31)
Citrus sinesnsis orange (31)
thetypeofsample.Doublebaggingisrecommended.Soilsand
Cucumis sativa cucumber (29), (30)
sedimentsmaybestoredinaplasticbagwhichisinturnplaced
Cyperus esculentus yellow nutsedge (32)
in a second protective plastic bag before placing in a pail. The
Dactylis glomerata orchardgrass (29)
Daucas carota carrot (29), (31)
plastic bags as well as the pail should be sealed with tape.
Echinochloa crusgalli barnyard grass (33)
Elodea densa elodea (30) 10.2 Proper labeling should be placed inside and outside of
Eragrostis curvula weeping lovegrass (29)
allcontainersduringthepackagingprocess.Allcontainerswill
Eragrostis lehmanniana Lehman lovegrass (29)
be identified in accordance with specific requirements and
Erysimum capitatum wall flower (31)
Fagopyrum esculentum buckwheat (31)
sampling and shipping information recorded on a sample data
Festuca arundinacea tall fescue (29), (30)
sheet. The U.S. Department of Transportation regulations
Festuca pratensis meadow fescue (31)
provide information governing shipping. Labeling must com-
Feasted rubber red fescue (29)
Forgery sp. strawberry (31) ply with Department of Transportation (DOT) CFR-49 speci-
Gladiolus sp. gladioli (31)
fications. These specifications are found in Section 172 of the
Glycine max soybean (29), (30)
DOT Hazardous Materials Shipping and Handling Regula-
Gossypium cotton (31)
Helianthus annuus sunflower (31)
tions.Theseregulationscanbefoundattheofficeofanycarrier
Hordeum vulgare barley (30), (31)
authorized to haul hazardous materials. If soils contain poten-
Lactuca sativa lettuce (29), (30), (31)
tial biohazards, special permits may be required to cross state
Lemna gibba duckweed (30)
Lemna minor duckweed (30)
lines or to be imported.
Lespedeza sp. lespedeza (29)
Lolium perenne perennial rye (29), (30)
11. Calibration and Standardization
Lotus corniculatus birdsfoot trefoil (29)
Ludwigia natans floating loosestrife (30)
11.1 Calibration and standardization of routine laboratory
Lupinus sp. lupine (29)
equipment and growth chambers used in this toxicity test will
Lycopersicon esculentum tomato (29), (31)
follow manufacturers’ recommended practices. In addition,
Medicago sativa alfalfa (29), (30), (31)
Melilotus albal white sweet clover (29), (30)
anyrelevantASTMmethodstoaparticularprocedurewillalso
Melilotus officinale yellow sweet clover (29)
be followed.
Musa paradislaca banana (31)
Nicotiana tabaccum tobacco (31)
12. Test Conditions
Oryza sativa rice (31)
Panicum miliaceum millet (30)
12.1 The annex for each specific test method should be
Panicum virgatum switchgrass (29)
consulted for detailed procedures. The investigator is urged to
Phaseolus sp. beans (30), (31)
Phaseolus vulgaris pinto beans (30)
developoptimaltesttreatmentstosatisfystatisticaldemandsof
Phleum pratense Timothy grass (29), (31)
each study. In some cases it may be advisable to adjust the
Pinus talda loblolly pine (30)
number of treatments and the number of replicates in order to
Pistia statiotes water lettuce (30)
Pisum sativum pea (31)
increase the power of the test. (Refer to Section 15 for
Poa pratense Kentucky bluegrass (29)
additionaldiscussionofstatisticalissuesrelatedtotestdesign.)
E1963 − 22
12.2 Negative Control— The negative control should con- 12.6 Test Condition Monitoring:
sistoftheidenticalsolution(water,organicsolvent,ornutrient
12.6.1 The light irradiance level (fluence rate) should be
solution) used to introduce the test substance into the soil
determined at the start and conclusion of a test with the
medium.
radiometer or quantum sensor that detects PAR. Light mea-
surements should be repeated anytime during the test if events
12.3 Positive Control— Boron as boric acid may be used as
that potentially affect the light sources occur (for example,
the positive control (34, 35, 36). A watering solution of boric
light bulb replacement). Adjustments to supplement lighting
acidatthedesiredconcentrationsisaddedtothetestsoil.A0.5
may be necessary. In some cases full spectrum (PAR plus
–1 –1 –1
dilution series (that is, 10 mg kg ,20mgkg ,40mgkg
Ultraviolet) light may be required (see Practice E1733).
–1 –1 –1 –1
,80mgkg ,160mgkg ,320mgkg ,and640mgkg soil
12.6.2 Airtemperatureshouldbemonitoredatleastdaily.It
dry weight) brackets sensitivity of most plant species tested to
is recommended that the air temperature and relative humidity
date. Once the range of sensitivity is established for a species,
be monitored continuously and recorded with the use of a
fewer test concentrations are needed. However, different soils
seven-day recorder. A thermal probe can be used to measure
alter the bioavailable fraction and therefore, preliminary tests
soil temperature of representative plant pots.
arerecommendedforeachnewsoilmediumtested.Alternative
12.6.3 The relative humidity may be monitored continu-
positive controls may be selected to meet the objectives of a
ouslyandrecordedusingaseven-dayrecorderoraninstrument
specific investigation. In selecting alternative substances for
equipped with an electronic datalogger. Relative humidity
use as positive controls, the investigator should consider
generally should be maintained above 30% (recommended
potential health effects to workers, interference of test sub-
approximately 50%). It may be necessary to increase the
stance with soil constituents, known mode of action of the
relative humidity in the growth chamber or the greenhouse if
substance and therefore appropriateness for use with different
the soil dries rapidly.
plant species, and disposal restrictions.
12.6.4 Soil pH (or pH in water) should be checked the day
12.4 Seed Planting— A template made of stainless steel or
the test soil medium is prepared, and again at the end of the
wood may be used to make holes approximately 2.5cm to 4.0
study. The soil pH is determined by placing 100 g of soil in a
cm deep in the soil for large seeds, (for example, corn and
250mL flask containing 100 mL of distilled water. The
beans),and1.0cmto1.5cmdeepforsmallerseeds.Templates
resulting slurry is mixed for 30 s to 1 min, left to stand for 1 h,
only help standardize planting in large scale testing; for most
then measured with the appropriate pH electrodes and meter
purposes manual planting will suffice. Seeds should be planted
(37). The pH of a soil may require adjusting if outside the
at a soil depth 1.5 to 2 times the seed diameter. It is suggested
optimumgrowingrangefrom6.0to7.5.ThepHofanacidsoil
that a minimum of 25 seeds be planted per concentration (for
can be raised by the addition of calcium carbonate. By adding
example,fivereplicatesoffiveormoreseedseach).Increasing
anacid,suchassulfuricacid,gypsum,orammoniumsulfateto
the number of seeds or plants per treatment improves the
a soil, the pH can be lowered (see Note 1). The addition of
ability to distinguish treatment effects.There may be instances
calcium carbonate, gypsum, ammonium sulfate, sulfuric acid,
that a single seed would be placed in a test container.After the
or other additives to change soil pH should be selected so that
seeds have been placed in the holes in the soil, tap the pots
they do not interfere with the test/control substances.
lightly to cover the seeds. Additional soil may be required to
NOTE1—Caution:Cautionshouldbeusedwhenworkingwithanacid.
fill the pots once they have settled. The plant pots that contain
thetestsubstancemixedthroughoutthesoilmediumshouldbe
13. Interference and Limitations
wateredtobringthemtofieldmoisturecapacity.Sub-irrigation
ispreferred,asthisminimizesdisturbancetotheplantedseeds. 13.1 Toxic substances can be introduced as contaminants in
Those pots that will be exposed via sub-irrigation can be
dilution water, glassware, sample hardware, and testing equip-
hydrated at this time. Excess water should be allowed to drain ment. In addition, high concentrations of suspended dissolved
from the pots that are sub-irrigated before placing them in an
solids, or both, can mask the presence of toxic substances.
environmental chamber or greenhouse.
Improper hazardous waste sampling and eluate preparation
alsocanaffecttestresultsadversely.Pathogenicorherbivorous
12.5 Soil Water Holding
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1963 − 09 (Reapproved 2014) E1963 − 22
Standard Guide for
Conducting Terrestrial Plant Toxicity Tests
This standard is issued under the fixed designation E1963; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This guide covers practices for conducting plant toxicity tests using terrestrial plant species to determine effects of test
substances on plant growth and development. Specific test procedures are presented in accompanying annexes.
1.2 Terrestrial plants are vital components of ecological landscapes. The populations and communities of plants influence the
distribution and abundance of wildlife. Obviously, plants are the central focus of agriculture, forestry, and rangelands. Toxicity tests
conducted under the guidelines and annexes presented herein can provide critical information regarding the effects of chemicals
on the establishment and maintenance of terrestrial plant communities.
1.3 Toxic substances that prevent or reduce seed germination can have immediate and large impacts to crops. In natural systems,
many desired species may be sensitive, while other species are tolerant. Such selective pressure can result in changes in species
diversity, population dynamics, and community structure that may be considered undesirable. Similarly, toxic substances may
impair the growth and development of seedlings resulting in decreased plant populations, decreased competitive abilities, reduced
reproductive capacity, and lowered crop yield. For the purposes of this guide, test substances include pesticides, industrial
chemicals, sludges, metals or metalloids, and hazardous wastes that could be added to soil. It also includes environmental samples
that may have had any of these test substances incorporated into soil.
1.4 Terrestrial plants range from annuals, capable of completing a life-cycle in as little as a few weeks, to long-lived perennials
that grow and reproduce for several hundreds of years. Procedures to evaluate chemical effects on plants range from short-term
measures of physiological responses (for example, chlorophyll fluorescence) to field studies of trees over several years. Research
and development of standardized plant tests have emphasized three categories of tests: (1) short-term, physiological endpoints (that
is, biomarkers); (2) short-term tests conducted during the early stages of plant growth with several endpoints related to survival,
growth, and development; and ( 3) life-cycle toxicity tests that emphasize reproductive success.
1.5 This guide is arranged by sections as follows:
Section Title
1 Scope
2 Referenced Documents
3 Terminology
4 Summary of Phytotoxicity Tests
5 Significance and Use
6 Apparatus
7 Test Material
This guide is under the jurisdiction of ASTM Committee E50 on Environmental Assessment, Risk Management and Corrective Action and is the direct responsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Oct. 1, 2014Aug. 1, 2022. Published December 2014September 2022. Originally published in 1998. Last previous edition published 20092014
as E1963E1963–09(2014).–09. DOI: 10.1520/E1963-09R14.10.1520/E1963-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1963 − 22
Section Title
8 Hazards
9 Test Organisms
10 Sample Handling and Storage
11 Calibration and Standardization
12 Test Conditions
13 Interference and Limitations
14 Quality Assurance and Quality Control
15 Calculations and Interpretation of Results
16 Precision and Bias
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 8.
1.8 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D4447 Guide for Disposal of Laboratory Chemicals and Samples
D4547 Guide for Sampling Waste and Soils for Volatile Organic Compounds
D5633 Practice for Sampling with a Scoop
E943 Terminology Relating to Biological Effects and Environmental Fate
E1598 Practice for Conducting Early Seedling Growth Tests (Withdrawn 2003)
E1733 Guide for Use of Lighting in Laboratory Testing
2.2 Code of Federal Regulations Standard:
CFR 49
2.3 Other useful references have described phytotoxicity test procedures(1-11) .
3. Terminology
3.1 General Terminology—The words “must,” “should,” “may,”“ can,” and “might” have very specific meanings in this guide.
“Must” is used to express an absolute requirement, that is, to state that the test ought to be designed to satisfy the specified
condition, unless the purpose of the test requires a different design. “Must” is only used in connection with factors that directly
relate to the acceptability of the test (see Section 14). “Should” is used to state that the specified condition is recommended and
ought to be met if possible. Although violation of one “should” is rarely a serious matter, violation of several will often render the
results questionable. Terms such as “is desirable,” “is often desirable,” and “might be desirable” are used in connection with 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 classic distinction between “may” and “can” is preserved, and “might” is never used as a synonym
for either “may” or “can.”
3.1.1 For definitions of terms used in this guide, refer to Terminology E943.
3.2 Definitions:
3.2.1 control, n—the treatment group in a toxicity test consisting of reference soil or artificial soil that duplicates all the conditions
of the exposure treatments, but contains no test substance. The control is used to determine if there are any statistical differences
in endpoints related to the test substance.
3.2.2 eluate, n—solution obtained from washing a solid with a solvent to remove adsorbed material.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
The last approved version of this historical standard is referenced on www.astm.org.
Available from Standardization Documents Order Desk, DODSSP, Bldg. 4, Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098, http://www.dodssp.daps.mil.
The boldface numbers in parentheses refer to the list of references at the end of this guide.
E1963 − 22
3.2.3 hazardous substance, n—a material that can cause deleterious effects to plants, microbes, or animals. (A hazardous substance
does not, in itself, present a risk unless an exposure potential exists.)
3.2.4 inhibition, n—a statistically lower value of any endpoint compared to the control values that is related to environmental
concentration or application rate.
3.2.5 leachate, n—water plus solutes that has percolated through a column of soil or waste.
3.2.6 test material, n—any formulation, dilution, etc. of a test substance.
3.2.7 test substance, n—a chemical, formulation, eluate, sludge, or other agent or substance that is the target of the investigation
in a toxicity test.
3.2.8 toxicant, n—an agent or material capable of producing an adverse response (effect) in a biological system, adversely
impacting structure or function or producing death.
3.2.9 toxicity endpoints, n—measurements of organism response such as death, growth, developmental, or physiological
parameters resulting from exposure to toxic substances.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 chlorotic, adj—the discoloration of shoots that occurs as chlorophyll is degraded as a result of disease, toxic substances,
nutrient deficiencies, or senescence.
3.3.2 coleoptile, n—the protective tissues surrounding the growing shoot in a monocotyledonous plant.
3.3.3 cotyledon, n—a primary leaf of the embryo in seeds, only one in the monocotyledons, two in dicotyledons. In many of the
latter, such as the bean, they emerge above ground and appear as the first leaves.
3.3.4 cutting, n—a vegetative segment of a plant, usually a stem that contains several nodes and associated buds, that can be used
to regenerate an entire plant.
3.3.5 dead test plants, n—those individuals that expired during the test observation period as indicated by severe desiccation,
withering, chlorosis, necrosis, or other symptoms that indicate non-viability.
3.3.6 desiccated, adj—the plant, or portion of the plant, that is dried in comparison to the control plant.
3.3.7 development, n—the series of steps involving cell division and cell differentiation into various tissues and organs.
3.3.8 dicotyledon, n—in the classification of plants, those having two seed leaves.
3.3.9 dormancy, n—a special condition of arrested growth in which buds, embryos, or entire plants survive at lowered metabolic
activity levels. Special environmental cues such as particular temperature regimes or photoperiods are required to activate
metabolic processes and resume growth. Seeds that require additional treatment besides adequate moisture and moderate
temperature to germinate are said to be dormant. (See quiescence.)
3.3.10 emergence, n—following germination of a plant, the early growth of a seedling that pushes the epicotyl through the soil
surface.
3.3.11 enhanced growth and yield, n—when a treated plant exhibits shoot growth, root elongation, lateral root growth, or yield
significantly greater than the control values, the plant is “enhanced” or “stimulated.”
E1963 − 22
3.3.12 epicotyl, n—that portion of an embryo or seedling containing the shoot. It is delineated anatomically by the transition zone
which separates the epicotyl from the hypocotyl.
3.3.13 fruits, n—the reproductive tissues derived from the ovary in the case of epigenous flowers or the ovary and accessory tissues
in the case of hypigenous flowers.
3.3.14 germination, n—the physiological events associated with re-initiation of embryo growth and mobilization of reserve
nutrients in seeds. The emergence of the seedling radicle from the seed coat defines the end of germination and the beginning of
early seedling growth.
3.3.15 growth, n—a change in size or mass measured by length, height, volume, or mass.
3.3.16 hypocotyl, n—that portion of an embryo or seedling containing the root or radicle. It is delineated anatomically by the
transition zone which separates the epicotyl from the hypocotyl.
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 control values.
3.3.18 lateral roots, n—roots growing off the primary roots, also referred to as secondary roots.
3.3.19 monocotyledon, n— in the classification of plants, those having a single seed leaf.
3.3.20 mottled, adj—marked with lesions, spots or streaks of different colors. This includes the discoloration of leaf margins.
3.3.21 phytotoxicity, n—a lethal or sub-lethal response of plants to a toxicant.
3.3.22 quiescence, n—a condition in buds, embryos, or entire plants characterized by lowered metabolic rates and limited or no
growth. Seeds that germinate when supplied with adequate moisture and moderate temperature are said to be quiescient.quiescent.
(See dormancy.)
3.3.23 radicle, n—the emerging root of an embryo during germination.
3.3.24 seed, n—the propagule of a plant derived from an ovule. It consists of an embryo, a protective covering (seed coat), and
may have storage tissue (endosperm).
3.3.25 shoot, n—the above-ground portion of a plant consisting of stems, leaves, as well as any reproductive parts that may be
attached.
3.3.26 surviving plants, n—test plants that are alive at the time observations are recorded.
3.3.27 viable, adj—plants capable of resuming metabolic functions and growth are considered “viable.” Buds, embryos, or entire
plants may be dormant or quiescientquiescent and therefore exhibit no growth during the period of observation. Distinguishing
dead plants from viable plants with certainty is difficult without special training and sophisticated measures of metabolic function.
3.3.28 withering, v—becoming limp or desiccated, deprived of moisture; often the result of root damage.
4. Summary of Phytotoxicity Tests
4.1 The terrestrial phytotoxicity tests covered under this guide apply to a range of test conditions and test species that can be
adapted to meet project-specific objectives. Test organisms are maintained either as seeds or as cuttings until a particular test is
to be conducted. A prescribed number of individual plants are introduced into test treatments that include a negative control, a
series of positive controls, and one or more test-substance treatment concentrations. The treatment concentrations may be known
or unknown; nominal or measured, depending on the nature of the investigation. In the case where the test substance is evaluated
E1963 − 22
as an additive to soil, a range of concentrations is recommended. In tests of environmental samples that already contain a putative
phytotoxic substance, the tests may be conducted with either the test soil as collected from the field, or as diluted with a suitable
reference soil. Another variant of the tests allows for amendments, or spikes, of selected toxic substances to be added to
environmental samples. Finally, in the case of the root elongation assay, eluates, effluents, or other aqueous derivatives of a soil
sample are tested.
4.2 Plants are exposed to the test substances in the form described in the specific variations of the tests for a discrete period of
time that ranges from 96 h to several months. For short tests, no nutrient additions or amendments are needed or recommended
as the amendments may interact with the toxicant and alter the toxicity response. For tests lasting more than two weeks, nutrient
additives may be warranted, depending on the test objectives, in order to maximize the potential for plant growth and development.
Thinning, culling, or replacing individual plants must not be done once exposure of plants to a test substance has begun as such
actions invalidate the test through the introduction of bias or variable test duration among test organisms. At intermediate times,
and at the conclusion of the exposure period, tallies of survival and measures of shoot growth and development are made.
–2 –1
4.3 For phytotoxicity tests, 100 μmol to 200 μmol m s of visible light (or photosynthetically active radiation, 400 ηm to 700
ηm) has been found to be a broadly applicable fluence rate. In some cases, different light levels or spectral ranges (for example,
solar ultraviolet) may be required. Guide E1733.
4.4 Measured endpoints and other observational data are used to calculate response levels in terms of ECxx or ICxx (where xx
refers to a specified percentage response), or categorical descriptions of phytotoxic effects (for example, proportion of plants
exhibiting abnormal development or other symptomatic indices that might be scored in qualitative terms) relative to controls. These
are interpreted to characterize phytotoxic effects attributed to test substances.
5. Significance and Use
5.1 Terrestrial phytotoxicity tests are useful in assessing the effects of environmental samples or specific chemicals as a part of
an ecological risk assessment (3-6, 12, 13).
5.2 Though inferences regarding higher-order ecological effects (population, community, or landscape) may be made from the
results, these tests evaluate responses of individuals of one or more plant species to the test substance.
5.3 This guide is applicable for: ( a) establishing phytotoxicity of organic and inorganic substances; (b) determining the
phytotoxicity of environmental samples; (c) determining the phytotoxicity of sludges and hazardous wastes, (d) assessing the
impact of discharge of toxicants to land, and (e) assessing the effectiveness of remediation efforts.
6. Apparatus
6.1 Facilities—The preparation of the test, test soil medium, storage of soil and seeds, and all stages of a test procedure must take
place in an atmosphere free from toxic contamination and vapors. The facility, whether a glasshousegreenhouse or a growth
chamber, should have reasonable temperature control and monitoring, as well as supplemental lighting. In general, the facility
should be capable of maintaining uniform temperatures in the 2020 °C to 30°C30 °C range. Lighting should provide at least
–2 –1
100250 μmol m s Photosynthetically Active Radiation (PAR) controlled on a clock timer to maintain a specified diurnal cycle.
See appropriate annex for any specific requirements of a given test.
6.2 Equipment and Supplies:
6.2.1 Plant Pots—Containers should be chosen to be inert to test and control substances. The test or control substances should not
adhere to or react in any way with the container. Glass, stainless steel, or paper containers with drainage holes can be used as plant
pots. Paper or other natural fiber materials may absorb test substances. If pots with drainage holes are used, then a secondary
container or shallow dish should be used to prevent cross-contamination among test units. Polyethylene pots or other containers
may be used, provided they are free of toxic materials. The volume of the pot container should be large enough so as not to restrict
seedling growth for the duration of the test. It is suggested that the selection of growth containers not be arbitrary, and that the
appropriate size, shape, color, and composition of the container be considered for each plant species and toxicity test undertaken.
6.2.2 Balance—Sensitivity to 0.001 g.
6.2.3 pH Meter—Sensitivity to 0.1 units.
E1963 − 22
6.2.4 Photometer (Radiometer)—Capable of measuring the photosynthetically active range. Fluence rate of incident light should
–2 – 1
be expressed as μmol m s .
6.2.5 Thermometer—A continuous recording thermometer or a maximum-minimum thermometer that is checked daily. Many
continuous recording units also record humidity.
6.2.6 Industrial Mixer or Cement Mixer—A revolving or rotating mixer is recommended for combining test substances or test soils
with large volumes of control or reference soil medium.
6.2.7 Reagent Water—Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to
Specification D1193, Type III. Type III water may be prepared by distillation, ion exchange, reverse osmosis, or a combination of
methods.
6.2.8 Equipment Care—Clean the test equipment after each use. Wash all new containers with a detergent and rinse thoroughly
with water, pesticide-free acetone, dilute acid (such as 5 % hydrochloric acid), and at least twice with tap or clean water. Final
rinses with Specification D1193 Type III water or equivalent is recommended. Clean equipment, such as the mixer and mixer
blades by a procedure known to remove constituents of the test substance. Paper and plastic plant pots should be disposed after
one use.
7. Test Material
7.1 Chemical Substance:
7.1.1 General—The test substance should be reagent-grade or better, unless a test on a formulation, commercial product, or
technical-grade or use-grade substance is specifically needed. Before a test is initiated, the following information should be
obtained about the test substance: identities and concentration of major ingredients and major impurities, for example, impurities
constituting more than about 1 % of substance; solubility and stability in dilution water; an estimate of toxicity to the test species
(a range-finding study may be required); precision and bias of the analytical method at the planned concentration(s) of the test
substance; and an estimate of toxicity to humans and other potentially exposed organisms.
7.1.2 Test Concentrations—Chemical concentrations in soils are expressed as dry weight to dry weight. It is preferable to add the
test substance directly to the test medium, however, a stock solution may be prepared and aliquots added to each test solution or
test chamber. Special considerations regarding chemical degradation, complexing, and volatilization and other factors that might
influence bioavailability should be evaluated to determine the appropriate mixing, handling, and storage procedures to be used. The
number of selected test concentrations should be based on the goal of the study. Multiple concentrations can be used to calculate
ICxx values, whereas, testing at a single concentration can be used to obtain rapid, simple answers. When the interest is (a) in the
effect of a specific concentration of test substance on the growth of the test species or (b) whether or not the ICxx value is above
or below a specific concentration, only one concentration and the controls need to be tested.
7.1.3 Stock Solution— For compounds with low water solubility, a solvent can be used to make a stock solution. If a stock solution
is used, the concentration and stability of the test substance in the stock should be determined before the beginning of the test. If
the test substance is subject to photolysis or other photo-reactive processes, the stock solution should be shielded from light. If a
solvent is necessary, its concentration in test solutions should be kept to a minimum (not greater than 1 % [volume to volume or
weight to volume]), and should be low enough that it does not affect either survival or growth of the test organisms. (These
limitations do not apply to any ingredients of a mixture, formulation, or commercial product unless an extra amount of solvent is
used in the preparation of the stock solution.) If the concentration of solvent is not the same in all test solutions that contain test
substance, either (a) a solvent test must be conducted to determine whether either survival, or growth of the test species is related
to the concentration of solvent over the range used in the phytotoxicity test or (b) such a solvent test must have already been
conducted using the same dilution water and test species. If either survival or growth is found to be related to the concentration
of solvent, a test would be unacceptable if any treatment contained a concentration of solvent in the response range. If neither
survival, or growth is found to be related to the concentration of solvent, a toxicity test with that same species in the same water
may contain solvent concentrations within the tested range, but the solvent control must contain the highest concentration of
solvent present in any of the other treatments.
7.1.4 Soil Medium— Natural soil (free of chemical contamination), commercial potting soil, synthetic soil mixes, or washed quartz
sand may be used as the “soil medium.” Each choice has substantive limitations for various phytotoxicity investigations. Natural
soils are not easily demonstrated to be free of toxic substances. Some commercial potting soils may adversely affect growth and
E1963 − 22
survival of some plants. Synthetic mixes may not be representative of real world conditions. Quartz sand or glass beads offer only
a physical matrix; and therefore do not provide a realistic soil condition with regard to binding and exchange sites. It may be
especially important to consider soil texture, pH, organic matter or other physical-chemical properties before embarking on a test.
Preliminary trials are often valuable to ascertain the suitability of a particular soil medium for the test species and conditions to
be investigated.
7.2 Environmental Sample:
7.2.1 Liquid, Sludge, or Slurry—These environmental samples may be handled as chemical additives described above. As complex
mixtures, however, the test concentrations will most likely be handled as percentage dilutions of the 100 % sample concentration.
In some cases, selected chemical analyses may be warranted as a means of expressing concentrations of selected constituents in
ppm or molar values. All of the provisions described for single chemicals apply.
7.2.2 Soil—Site soils may be collected as cores or as bulk samples from specified soil depths (for example, 0 to 15 cm depth).
Sampling and handling procedures may be found in Practices D4547 and D5633. The soil samples may be tested directly (that is,
100 % site soil) or diluted with an appropriate reference soil or a synthetic soil mixture to achieve specified relative concentrations.
In some cases, selected chemical analyses may be warranted as a means of expressing concentrations of selected constituents in
ppm (dry weight basis) or molar values.
7.2.3 Eluates—Aqueous extracts of soils are sometimes desired to evaluate the phytotoxicity of water-soluble soil constituents.
The eluates are used in the same manner as liquid environmental samples described above.
8. Hazards
8.1 Many materials can adversely affect humans if safety precautions are inadequate. Therefore, skin contact with all test materials
and solutions of them should be minimized by such means as wearing appropriate protective gloves (especially when washing
equipment, putting hands in test solutions or treated soil, or handling treated plant material), laboratory coats, aprons, and glasses.
Special precautions, such as ventilating the area surrounding the flats should be taken when conducting tests on volatile materials
or dust containing hazardous substances. Respirators may be warranted. Information on toxicity to humans (14-18), recommended
handling procedures (19-22), and chemical and physical properties of the test material should be studied before a test is begun.
Special procedures might be necessary with radio-labeled test materials (23, 24) and with test materials that are, or are suspected
of being, carcinogenic (25).
8.2 Although disposal of stock solutions, test solutions, test soil, and test organisms pose no special problems in most cases, health
and safety precautions and applicable regulations should be considered before beginning a test. Removal or degradation of the test
substance in the test medium might be desirable before disposal of stock and test solutions. solutions (see Guide D4447).
Hazardous materials must be disposed of in accordance with local, state, and federal regulations.
8.3 Because water is a good conductor of electricity, use of ground fault systems and leak detectors should be considered to help
avoid electrical shocks.
9. Test Organisms
9.1 Test Species— The majority of species routinely used in phytotoxicity tests has been limited to agronomic plants. Under
FIFRA guidelines (4, 5), ten species belonging to eight families are listed for toxicity testing (see Table 1). The United States Food
and Drug Administration (11, 26), has relied on plant tests similar to those for FIFRA (see Table 1). International guidance (10)
uses agronomic species, but has a broader selection of plants compared to United States guidance. CERCLA offers limited
guidance with respect to plant testing. General methods recommended for the Remedial Investigation Baseline Risk Assessment
portion of work listed by name only the seed germination and root elongation assays (3, 6). Only lettuce (Lactuca sativa) is listed
as the standard species of the test, although “other (taxa) can be used.” The Department of Interior in developing rules for Natural
Resource Damage Assessment (27) referred to “economically important plant species.” Thirty-one plant taxa are explicitly
identified in federal and international test guidelines and standard test procedures (see Table 1). Many additional plant taxa
including aquatic taxa were reported in phytotoxicity literature (see Table 2). Nearly a hundred plant taxa (see Table 2) have been
used routinely to study phytotoxicity. In an early version of PHYTOTOX (28), 1569 plant species from 682 genera in 147 families
were reported in the records. However, 42 % of the records referred to only 20 species.
9.2 Purchase—Seeds of the most commonly used taxa identified in FIFRA guidelines may be purchased from commercial seed
E1963 − 22
A
TABLE 1 List of Plant Species Identified in Regulatory Documents and in Standard Test Procedures
APHA ASTM
Family Species Common Name FIFRA TSCA FDA OECD
AWWA ESG
Chenopodiaceae Atriplex patula Seaside Greens =
Compositae Lactuca sativa Lettuce = = = = =
Cruciferae Brassica alba Mustard = =
Cruciferae Brassica campestris var. chinensis Chinese Cabbage = =
Cruciferae Brassica napus Rape = =
Cruciferae Brassica oleracea Cabbage = = = =
Cruciferae Brassica rapa Turnip = =
Cruciferae Lepidium sativum Cress = =
Cruciferae Raphanus sativus Radish = =
Cruciferae Rorippa nasturtium-aquaticum Watercress =
Cucurbitaceae Cucumis sativus Cucumber = = = =
Leguminosae Glycine max Soybean = = = =
Leguminosae Phaseolus aureus Mungbean = =
Leguminosae Phaseolus vulgaris Bean = =
Leguminosae Trifolium ornithopodioides Fenugreek = =
Leguminosae Trifolium pratense Red Clover = =
Leguminosae Vicia sativa Vetch = =
Liliaceae Allium cepa Onion = = =
Nymphaeceae Nelumbo lutea American Lotus =
Poaceae Avena sativa Oat = = = = =
Poaceae Echinochloa cursgalli Japanese Millet =
Poaceae Leersia oryzoides Rice Cutgrass =
Poaceae Lolium perenne Perennial Ryegrass = = = = =
Poaceae Oryza sativa Rice = = =
Poaceae Sorghum bicolor Sorghum = =
Poaceae Spartina alterniflora Smooth Cordgrass =
Poaceae Triticum aestivum Wheat = = =
Poaceae Zea mays Corn = = = =
Poaceae Zizania aquatica Wild Rice =
Solonaceae Lycopersicon esculentum Tomato = = = =
Umbelliferae Daucus carota Carrot = = = =
A
FIFRA = Federal Insecticide, Fungicide, and Rodenticide Act (4) (5) ; TSCA = Toxic Substance Control Act (2) ; FDA = Federal Drug Administration (11) (26); OECD =
Organization for Economic Cooperation and Development (10); APHA = American Public Health Association; AWWA = American Water Works Association (1); and ASTM
= American Society for Testing and Materials (Practice E1598).
companies. Many of the less common taxa are available from specialty seed companies, especially those that service landscaping
and restoration activities. When purchasing seeds, it is best to talk to technical staff of the supplier to gather important information
regarding the seed lot, collection, handling and storage practices of the seed company, germination percentage expected, and any
special conditions affecting germination. Generally, it is preferable to use untreated seeds (that is, not treated with fungicide,
repellents, or other chemical agents) in phytotoxicity tests, however, specific test objectives may permit use of treated seeds. The
principal investigator should detail the rationale for using treated seeds. Seeds should be acquired at least annually. At a minimum,
a sufficient quantity of seeds should be acquired to allow tests of all treatments (including controls) to be conducted with seeds
from the same batch.
9.3 Collection—If seeds are collected from the field, care must be taken to ensure that seeds from only a single species are
obtained. The following minimum set of information should be recorded for each batch of seeds collected: the location of the
collection site as precisely as practicable (for example, section, township and range, county, state); the persons collecting the seeds;
date of collection; description of noteworthy circumstances such as drought, flood, condition of surrounding landscape, and any
indication of pesticide use in the vicinity; and quantity of seeds collected.
9.4 Grading and Sizing Seeds:
9.4.1 Domestic Species— Seeds of a given species vary in size, shape, and in some cases, color. These differences in external
features of the seed are often associated with different rates of germination or even different germination requirements. To
minimize the variance in test results the investigator should determine whether such variants in seed size, shape, or color are critical
to the investigation. (For example, alfalfa seeds often come as a mixture of light-colored and dark-colored seeds. The dark-colored
seeds have low percentage germination (;10 %), while the light-colored seeds have high percentage germination (;90 %).)
Separation of broken or damaged seeds from the batch is important. Various sieves or screens may be useful in separating the seeds.
1 1 1 1 1 1 1 1
Lettuce for example can be separated mechanically using wire mesh screens: ⁄6 × ⁄28 in.; ⁄6 × ⁄30 in.; ⁄6 × ⁄32 in.; ⁄6 × ⁄34 in.
1 1 1 1
Red clover may be sized using perforated metal sheets with round holes of the following diameters: ⁄19 in., ⁄18 in., ⁄17 in., ⁄16 in.
9.4.2 Native Species— If this test uses native plant seeds rather than commercially selected plants, considerable care should be
E1963 − 22
taken in sizing and sorting seeds collected. Numerous studies have shown that the variability in seed germination is not entirely
random within a population of a particular species. The point during the growing season at which a lot of seeds are produced and
collected will affect germination in many species. Also, the location within a particular inflorescence (for example, with
composites) will also affect germination. There can also be considerable intra-species variation between remote populations. The
test design becomes considerably more complicated to account for these and other potential sources of variation.
9.5 Seed Storage and Maintenance—Seeds should be stored in a desiccator and refrigerated until needed (preferably at 44 °C 6
2°C).2 °C). It is recommended no disinfecting agent such as hypochlorite be used. Exceptions may be warranted for some
investigations if gnotobiotic conditions are desired, however, such special cases must be described fully as exceptions to the guide
described here. Examples of exceptions would include, but not be limited to, amendments with microbial inocula such as rhizobia
for legumes, actinomycetes for actinorhizal species, or mycorrhizal fungi.
9.6 Seedlings or cuttings may be collected from the field, propagated by the investigator, or purchased from nurseries, horticulture
supply houses, or research laboratories. As with seeds, it is important to document as much information as reasonable for each
batch of cuttings obtained. Care should be taken to limit the range of stem size, age, and developmental stage of the plant.
TABLE 2 Partial Listing of Plant Taxa studied for Toxicity Effects
Species Common Name Ref.
Agrostis alba red top (29)
Agrostis sp. bentgrass (29)
Apocynum sp. milkweed (30)
Arabidopsis thaliana mouse-ear-cress (30)
Arachis hypogaea peanut (29)
Avena sativa oats (29), (30), (31)
Beta vulgaris beets (29), (30), (31)
Beta vulgaris chard (29)
Beta vulgaris sugarbeet (30), (31)
Brassica campestris kale (29), (31)
Brassica nigra mustard (29), (30), (31)
Brassica oleracea broccoli (29)
Brassica oleracea cauliflower (29)
Brassica rapa turnip (29)
Bromus smooth bromegrass (29)
Bromus japonicus Japanese bromegrass (29)
Cenchrus ciliaris buffelgrass (29)
Chrysanthemum sp. chrysanthemum (31)
Citrus sinesnsis orange (31)
Cucumis sativa cucumber (29), (30)
Cyperus esculentus yellow nutsedge (32)
Dactylis glomerata orchardgrass (29)
Daucas carota carrot (29), (31)
Echinochloa crusgalli barnyard grass (33)
Elodea densa elodea (30)
Eragrostis curvula weeping lovegrass (29)
Eragrostis lehmanniana Lehman lovegrass (29)
Erysimum capitatum wall flower (31)
Fagopyrum esculentum buckwheat (31)
Festuca arundinacea tall fescue (29), (30)
Festuca pratensis meadow fescue (31)
Feasted rubber red fescue (29)
Forgery sp. strawberry (31)
Gladiolus sp. gladioli (31)
Glycine max soybean (29), (30)
Gossypium cotton (31)
Helianthus annuus sunflower (31)
Hordeum vulgare barley (30), (31)
Lactuca sativa lettuce (29), (30), (31)
Lemna gibba duckweed (30)
Lemna minor duckweed (30)
Lespedeza sp. lespedeza (29)
Lolium perenne perennial rye (29), (30)
Lotus corniculatus birdsfoot trefoil (29)
Ludwigia natans floating loosestrife (30)
Lupinus sp. lupine (29)
Lycopersicon esculentum tomato (29), (31)
Medicago sativa alfalfa (29), (30), (31)
Melilotus albal white sweet clover (29), (30)
Melilotus officinale yellow sweet clover (29)
Musa paradislaca banana (31)
Nicotiana tabaccum tobacco (31)
Oryza sativa rice (31)
E1963 − 22
TABLE 2 Continued
Species Common Name Ref.
Panicum miliaceum millet (30)
Panicum virgatum switchgrass (29)
Phaseolus sp. beans (30), (31)
Phaseolus vulgaris pinto beans (30)
Phleum pratense Timothy grass (29), (31)
Pinus talda loblolly pine (30)
Pistia statiotes water lettuce (30)
Pisum sativum pea (31)
Poa pratense Kentucky bluegrass (29)
Raphanus sativus radish (29), (30)
Rubus sp. raspberry (31)
Setaria italica foxtail millet (30)
Solanum tuberosum potato (30), (31)
Sorghum bicolor sundangrass; sorghum (29), (30), (31)
Spartina alterniflora cordgrass (33)
Spinacia oleracea spinach (29), (31)
Spirea alba meadowsweet (31)
Spirea alba meadow sweet (31)
Tagetes sp. marigold (29)
Thalassia testidinum seagrass (30)
Tradescantia paludosa spiderwort (30)
Trifolium pratense clover (30)
Triticum aestivum wheat (30), (31)
Vicia faba broad bean (30)
Vicia sp. vetch (29)
Zea mays corn (30), (31)
10. Sample Handling and Storage
10.1 The proper collection, packaging, and shipping of waste site samples is critical. Proper sampling and shipping ensures sample
integrity, handling safety, and an adequate data base for sample processing and future sampling requirements. Local, state, and
federal shipping regulations should be consulted regarding size and quantity restrictions, labeling, and documentation
requirements. Sample packaging depends upon the type of sample. Double bagging is recommended. Soils and sediments may be
stored in a plastic bag which is in turn placed in a second protective plastic bag before placing in a pail. The plastic bags as well
as the pail should be sealed with tape.
10.2 Proper labeling should be placed inside and outside of all containers during the packaging process. All containers will be
identified in accordance with specific requirements and sampling and shipping information recorded on a sample data sheet. The
U.S. Department of Transportation regulations provide information governing shipping. Labeling must comply with Department
of Transportation (DOT) CFR-49 specifications. These specifications are found in Section 172 of the DOT Hazardous Materials
Shipping and Handling Regulations. These regulations can be found at the office of any carrier authorized to haul hazardous
materials. If soils contain potential biohazards, special permits may be required to cross state lines or to be imported.
11. Calibration and Standardization
11.1 Calibration and standardization of routine laboratory equipment and growth chambers used in this toxicity test will follow
manufacturers’ recommended practices. In addition, any relevant ASTM methods to a particular procedure will also be followed.
12. Test Conditions
12.1 The annex for each specific test method should be consulted for detailed procedures. The investigator is urged to develop
optimal test treatments to satisfy statistical demands of each study. In some cases it may be advisable to adjust the number of
treatments and the number of replicates in order to increase the power of the test. (Refer to Section 15 for additional discussion
of statistical issues related to test design.)
12.2 Negative Control— The negative control should consist of the identical solution (water, organic solvent, or nutrient solution)
used to introduce the test substance into the soil medium.
12.3 Positive Control— Boron as boric acid may be used as the positive control (34, 35, 36). A watering solution of boric acid
–1 –1
at the desired concentrations is added to the test soil. A 0.5 dilution series (that is, 10, 20, 40,10 mg kg 80, 160, 320,, 20 mg kg
–1 –1 –1 –1 –1
, 40 mg kg , 80 mg kg , 160 mg kg , 320 mg kg , and 640 mg kg soil dry weight) brackets sensitivity of most plant species
tested to date. Once the range of sensitivity is established for a species, fewer test concentrations are needed. However, different
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soils alter the bioavailable fraction and therefore, preliminary tests are recommended for each new soil medium tested. Alternative
positive controls may be selected to meet the objectives of a specific investigation. In selecting alternative substances for use as
positive controls, the investigator should consider potential health effects to workers, interference of test substance with soil
constituents, known mode of action of the substance and therefore appropriateness for use with different plant species, and disposal
restrictions.
12.4 Seed Planting— A template made of stainless steel or wood may be used to make holes approximately 2.52.5 cm to 4.0 cm
deep in the soil for large seeds, (for example, corn and beans), and 1.01.0 cm to 1.5 cm deep for smaller seeds. Templates only
help standardize planting in large scale testing; for most purposes manual planting will suffice. Seeds should be planted at a soil
depth 1.5 to 2 times the seed diameter. It is suggested that a minimum of 25 seeds be planted per concentration (for example, five
replicates of five or more seeds each). Increasing the number of seeds or plants per treatment improves the ability to distinguish
treatment effects. There may be instances that a single seed would be placed in a test container. After the seeds have been placed
in the holes in the soil, tap the pots lightly to cover the seeds. Additional soil may be required to fill the pots once they have settled.
The plant pots that contain the test substance mixed throughout the soil medium should be watered to bring them to field moisture
capacity. Sub-irrigation is preferred, as this minimizes disturbance to the planted seeds. Those pots that will be exposed via
sub-irrigation can be hydrated at this time. Excess water should be allowed to drain from the pots that are sub-irrigated before
placing them in an environmental chamber or greenhouse.
12.5 Soil Water Holding Capacity—In some testing situations, it is desirable to know the quantity of water that can be stored in
a soil. For some species, germination is improved if the soil is maintained at approximately 85 % water holding capacity. Whether
test soils are saturated or maintained at less than saturation (for example, 85 %), all treatments and replicates should be handled
similarly. Water holding capacity is expressed as a percentage of soil dry weight. To determine the water holding capacity of a soil,
saturate a volume of soil with water and allow to drain for one hour. After the excess water has drained from the soil, measure
the weight of the saturated soil. The soil is then dried in an oven (105°C)(105 °C) until constant weight is achieved. The water
held by the soil is determined as the difference in saturated weight and the dry weight.
12.6 Test Condition Monitoring:
12.6.1 The light irradiance level (fluence rate) should be determined at the start and conclusion of a test with the radiometer or
quantum sensor that detects PAR. Light measurements should be repeated anytime during the test if events that potentially affect
the light sources occur (for example, light bulb replacement). Adjustments to supple
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