ASTM E1415-22

Standard Guide for Conducting Static Toxicity Tests With <emph type="ital">Lemna gibba</emph> G3

SIGNIFICANCE AND USE
5.1 The term duckweed commonly refers to members of the family Lemnaceae. This family has many species world-wide in 4 genera. This guide is designed for toxicity testing with one particular clone of one species of duckweed that has been extensively studied, Lemna gibba G3, although other species such as Lemna minor or Spirodela spp. can probably also be tested using the procedures described herein.  
5.2 Duckweeds are widespread, free-floating aquatic plants, ranging in the world from tropical to temperate zones. Duckweeds are a source of food for waterfowl and small animals and provide food, shelter, and shade for fish. The plants also serve as physical support for a variety of small invertebrates. Duckweed is fast growing and reproduces rapidly compared with other vascular plants (1).3 Under conditions favorable for its growth, it can multiply quickly and form a dense mat in lakes, ponds, and canals, primarily in fresh water, but also in estuaries. It also grows well in effluents of wastewater treatment plants and has been suggested as a means of treating wastewaters (2). A dense mat of duckweed can block sunlight and aeration and cause fish kills (3).  
5.3 Duckweed is small enough that large laboratory facilities are not necessary, but large enough that effects can be observed visually.  
5.4 Because duckweed is a floating macrophyte, it might be particularly susceptible to surface active and hydrophobic chemicals that concentrate at the air-water interface. Results of duckweed tests on such chemicals, therefore, might be substantially different from those obtained with other aquatic species.  
5.5 Results of toxicity tests with duckweed might be used to predict effects likely to occur on duckweed in field situations as a result of exposure under comparable conditions.  
5.6 Results of tests with duckweed might be used to compare the toxicities of different materials and to study the effects of various environmental factors on results of such tests.  
5.7...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the adverse effects of a text material added to growth medium on a certain species of duckweed (Lemna gibba G3) during a 7-day exposure using the static technique. These procedures will probably be useful for conducting toxicity tests with other species of duckweed and other floating vascular plants, although modifications might be necessary.  
1.2 Special needs or circumstances might also justify modification of this standard. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting tests with duckweed.  
1.3 The procedures in this guide are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications these procedures can be used to conduct tests on temperature and pH and on such other materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments and surface waters. These procedures do not specifically address effluents because to date there is little experience using duckweeds in effluent testing and such tests may pose problems with acclimation of the test organisms to the receiving water. Static tests might not be applicable to materials that have a high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test.  
1.4 Results of toxicity tests performed using the procedures in this guide should usually be reported in terms of the 7-day IC5...

General Information

• Status: Published
• Publication Date: 31-Oct-2022
• ICS: 11.100.01 - Laboratory medicine in general
• Technical Committee: E50 - Environmental Assessment, Risk Management and Corrective Action
• Drafting Committee: E50.47 - Biological Effects and Environmental Fate

Relations

• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E1192-97(2008) - Standard Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Feb-2008
• Refers: ASTM E729-96(2007) - Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Oct-2007
• Refers: ASTM E1023-84(2007) - Standard Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses
• Effective Date: 01-Oct-2007

Overview

ASTM E1415-22: Standard Guide for Conducting Static Toxicity Tests With Lemna gibba G3 provides guidelines for laboratory procedures to assess the adverse effects of chemicals and materials on the aquatic plant Lemna gibba G3, commonly known as duckweed. This guide outlines methods for performing static toxicity tests over a 7-day exposure period, supporting environmental risk assessment and ecological toxicity studies. While the primary focus is on Lemna gibba G3, the principles can also apply to other duckweed species and floating aquatic plants, with potential modification to suit specific research or regulatory needs.

Key Topics

• Test Organism: The standard centers on Lemna gibba G3, a widely distributed, rapidly growing floating aquatic plant commonly used in toxicity evaluation due to its sensitivity and ease of laboratory handling.
• Test Design: Static toxicity testing procedures are detailed, including experimental setup, selection and maintenance of test organisms, test chamber requirements, preparation of growth mediums, and control treatments.
• Exposure and Assessment: Describes how to expose duckweed to chemicals or environmental samples and monitor biological endpoints, typically evaluating inhibition of growth as measured by increases in frond number or biomass.
• Applicability: The guide explains its suitability for a broad range of test substances including chemicals (individually or in mixtures), commercial products, and environmental samples such as effluents, leachates, and surface waters.
• Data Reporting: Results are recommended to be expressed as the 7-day IC50 (inhibition concentration for 50% effect) or NOEC (no observed effect concentration), providing benchmarks for environmental hazard evaluation.

Applications

ASTM E1415-22 is essential for organizations and laboratories involved in:

• Ecotoxicological Risk Assessment: Evaluates how contaminants, pollutants, or commercial products impact aquatic plant life, aiding in environmental impact studies and regulatory compliance.
• Wastewater and Effluent Testing: The rapid growth and wide distribution of duckweed make it valuable for assessing the toxicity of freshwater discharges, including those from municipal or industrial wastewater treatment plants.
• Comparative Toxicity Studies: Enables comparison between the toxicities of different chemicals or substances under controlled conditions, supporting research into structure-activity relationships.
• Field Predictability: Laboratory results using Lemna gibba G3 often reflect possible responses in real-world aquatic environments, supporting environmental monitoring programs and water quality management.
• Environmental Regulation and Water Quality Criteria: Results can be used to help develop or meet regulatory guidelines and water quality standards for the protection of aquatic ecosystems.

Related Standards

For enhanced reliability and broader application, ASTM E1415-22 references and complements several other ASTM and international standards in aquatic toxicity testing and environmental assessment, including:

• ASTM E729: Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians.
• ASTM E943: Terminology Relating to Biological Effects and Environmental Fate.
• ASTM E1023: Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses.
• ASTM E1192: Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians.
• ASTM E1218: Guide for Conducting Static Toxicity Tests with Microalgae.

Practical Value

• Accessibility: Testing with duckweed requires minimal laboratory facilities and resources, making it a practical approach for routine and large-scale screening.
• Versatility: The method can be adapted for a wide range of chemical substances and sample types, including waterborne pollutants, industrial chemicals, agricultural runoff, and more.
• Relevance to Aquatic Health: Results contribute directly to understanding and managing risks to primary producers in freshwater habitats, supporting sustainable environmental stewardship.

By following ASTM E1415-22, laboratories and regulatory authorities can conduct reproducible, standardized static toxicity tests with Lemna gibba G3, generating data crucial for environmental protection, regulatory submissions, and scientific research in aquatic ecotoxicology.

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: E1415 − 22
Standard Guide for
Conducting Static Toxicity Tests With Lemna gibba G3
This standard is issued under the fixed designation E1415; 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 only be necessary to determine whether a specific concentra-
tion unacceptably affects the growth of the test species or
1.1 Thisguidedescribesproceduresforobtaininglaboratory
whether the IC50 is above or below a specific concentration.
data concerning the adverse effects of a text material added to
Another end point that may be calculated is the no observed
growth medium on a certain species of duckweed (Lemna
effect concentration (NOEC).
gibba G3) during a 7-day exposure using the static technique.
These procedures will probably be useful for conducting
1.5 The sections of this guide appear as follows:
toxicity tests with other species of duckweed and other floating
Title Section
vascular plants, although modifications might be necessary.
Referenced Documents 2
1.2 Special needs or circumstances might also justify modi-
Terminology 3
fication of this standard. Although using appropriate proce-
Summary of Guide 4
Significance and Use 5
dures is more important than following prescribed procedures,
Hazards 6
results of tests conducted using unusual procedures are not
Apparatus 7
likely to be comparable to results of many other tests. Com- Facilities 7.1
Test Chambers 7.2
parison of results obtained using modified and unmodified
Cleaning 7.3
versions of these procedures might provide useful information
Acceptability 7.4
concerning new concepts and procedures for conducting tests
Growth Medium 8
Test Material 9
with duckweed.
General 9.1
Stock Solution 9.2
1.3 The procedures in this guide are applicable to most
Test Concentration(s) 9.3
chemicals, either individually or in formulations, commercial
Test Organisms 10
products, or known mixtures. With appropriate modifications
Species 10.1
Source 10.2
these procedures can be used to conduct tests on temperature
Stock Culture 10.3
and pH and on such other materials as aqueous effluents (see
Procedure 11
also Guide E1192), leachates, oils, particulate matter, sedi-
Experimental Design 11.1
Temperature 11.2
ments and surface waters.These procedures do not specifically
Illumination 11.3
address effluents because to date there is little experience using
Beginning the Test 11.4
duckweedsineffluenttestingandsuchtestsmayposeproblems
Duration of Test 11.5
Biological Data 11.6
with acclimation of the test organisms to the receiving water.
Other Measurements 11.7
Statictestsmightnotbeapplicabletomaterialsthathaveahigh
Analytical Methodology 12
oxygen demand, are highly volatile, are rapidly biologically or
Acceptability of Test 13
Calculation of Results 14
chemically transformed in aqueous solution, or are removed
Report 15
from test solutions in substantial quantities by the test cham-
1.6 This standard does not purport to address all of the
bers or organisms during the test.
safety concerns, if any, associated with its use. It is the
1.4 Results of toxicity tests performed using the procedures
responsibility of the user of this standard to establish appro-
in this guide should usually be reported in terms of the 7-day
priate safety, health, and environmental practices and deter-
IC50 based on inhibition of growth. In some situations it might
mine the applicability of regulatory limitations prior to use.
Specific hazard statements are given in Section 6.
1.7 This international standard was developed in accor-
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
dance with internationally recognized principles on standard-
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
ization established in the Decision on Principles for the
Current edition approved Nov. 1, 2022. Published November 2022. Originally
Development of International Standards, Guides and Recom-
approved in 1991. Last previous edition approved in 2012 as E1415 – 91 (2012),
mendations issued by the World Trade Organization Technical
which was withdrawn January 2021 and reinstated in November 2022. DOI:
10.1520/E1415-22. Barriers to Trade (TBT) Committee.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1415 − 22
2. Referenced Documents controltreatments,theplantsaremaintainedingrowthmedium
2 to which no test material has been added in order to provide a
2.1 ASTM Standards:
measure of the acceptability of the test by giving an indication
E729 Guide for Conducting Acute Toxicity Tests on Test
of the quality of the duckweed and the suitability of the growth
Materials with Fishes, Macroinvertebrates, and Amphib-
medium,testconditions,handlingprocedures,andsoforth,and
ians
the basis for interpreting data obtained from the other treat-
E943 Terminology Relating to Biological Effects and Envi-
ments. In each of the one or more other treatments, the
ronmental Fate
duckweed plants are maintained in growth medium to which a
E1023 Guide for Assessing the Hazard of a Material to
selected concentration of test material has been added. Speci-
Aquatic Organisms and Their Uses
fied data concerning growth of duckweed in each test chamber
E1192 Guide for ConductingAcute Toxicity Tests onAque-
are obtained during the test and are usually analyzed to
ous Ambient Samples and Effluents with Fishes,
determine the IC50 or NOEC based on inhibition of growth.
Macroinvertebrates, and Amphibians
E1218 Guide for Conducting Static Toxicity Tests with
5. Significance and Use
Microalgae
5.1 The term duckweed commonly refers to members of the
IEEE/ASTM SI 10 American National Standard for Use of
family Lemnaceae. This family has many species world-wide
theInternationalSystemofUnits(SI):TheModernMetric
in 4 genera.This guide is designed for toxicity testing with one
System
particular clone of one species of duckweed that has been
3. Terminology extensively studied, Lemna gibba G3, although other species
such as Lemna minor or Spirodela spp. can probably also be
3.1 The words must, should, may, can, and might have very
tested using the procedures described herein.
specific meanings in this guide. Must is used to express an
absolute requirement, that is, to state that the test ought to be 5.2 Duckweeds are widespread, free-floating aquatic plants,
designed to satisfy the specified condition, unless the purpose ranging in the world from tropical to temperate zones. Duck-
of the test requires a different design. Must is only used in weeds are a source of food for waterfowl and small animals
connection with factors that directly relate to the acceptability and provide food, shelter, and shade for fish. The plants also
of the test (see Section 13). Should is used to state that the serve as physical support for a variety of small invertebrates.
specified condition is recommended and ought to be met if Duckweed is fast growing and reproduces rapidly compared
possible. Although violation of one should is rarely a serious with other vascular plants (1). Under conditions favorable for
matter, violation of several will often render the results its growth, it can multiply quickly and form a dense mat in
questionable. Terms such as is desirable, is often desirable, lakes, ponds, and canals, primarily in fresh water, but also in
might be desirable are used in connection with less important estuaries. It also grows well in effluents of wastewater treat-
factors. May is used to mean is (are) allowed to, can is used to ment plants and has been suggested as a means of treating
meanis(are)ableto,and mightisusedtomeancouldpossibly. wastewaters (2). A dense mat of duckweed can block sunlight
Thus the classic distinction between may and can is preserved, and aeration and cause fish kills (3).
and might is never used as a synonym for either may or can.
5.3 Duckweed is small enough that large laboratory facili-
3.2 Definitions of Terms Specific to This Standard: ties are not necessary, but large enough that effects can be
observed visually.
3.2.1 frond—individual leaf-like structure on a duckweed
plant.
5.4 Because duckweed is a floating macrophyte, it might be
3.2.2 IC50—a statistically or graphically estimated concen- particularly susceptible to surface active and hydrophobic
tration of test material that is expected to cause a 50 %
chemicals that concentrate at the air-water interface. Results of
inhibition of one or more specified biological processes (such
duckweed tests on such chemicals, therefore, might be sub-
as growth or reproduction), for which the data are not
stantially different from those obtained with other aquatic
dichotomous, under specified conditions.
species.
3.3 For definitions of other terms used in this guide, refer to
5.5 Results of toxicity tests with duckweed might be used to
Terminology E943, and Guides E729 and E1023. For an
predicteffectslikelytooccuronduckweedinfieldsituationsas
explanation of units and symbols, refer to Practice IEEE/
a result of exposure under comparable conditions.
ASTM SI 10 .
5.6 Results of tests with duckweed might be used to
compare the toxicities of different materials and to study the
4. Summary of Guide
effectsofvariousenvironmentalfactorsonresultsofsuchtests.
4.1 In each of two or more treatments, plants of Lemna
5.7 Results of tests with duckweed might be an important
gibba G3 are maintained for 7 days in two or more test
consideration when assessing the hazards of materials to
chambers using the static technique. In each of the one or more
aquatic organism (see Guide E1023) or when deriving water
quality criteria for aquatic organisms (4).
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 boldface numbers in parentheses refer to the list of references at the end of
the ASTM website. this guide.
E1415 − 22
5.8 Results of tests with duckweed might be useful for the possibility of contamination by test materials and other
studying biological availability of, and structure-activity rela- substances, especially volatile ones, the culture chambers
tionships between test materials.
should not be in a room in which toxicity tests are conducted,
stock solutions or test solutions are prepared, or equipment is
5.9 Results of tests with duckweed will depend on
cleaned.
temperature, composition of the growth medium, condition of
thetestorganisms,andotherfactors.Thegrowthmediathatare
7.2 Test Chambers—In a toxicity test with aquatic
usually used for tests with duckweed contain concentrations of
organisms, test chambers are defined as the smallest physical
salts, minerals, and nutrients that greatly exceed those in most
unitsbetweenwhichnowaterconnectionsexist.Glass250-mL
surface waters.
beakers, 200-mL flat-bottomed test tubes, 250-mL fruit jars,
and 250 or 500-mLErlenmeyer flasks have been used success-
6. Hazards
fully (9-11). The ratio of the size of the test chamber to the
6.1 Many materials can affect humans adversely if precau-
volume of test solution should be 5 to 2 (that is, 100 mL in a
tions are inadequate. Therefore, skin contact with all test
250-mL Erlenmeyer flask, 200 mL in a 500-mL Erlenmeyer
materials and solutions of them should be minimized by such
flask). Plastic chambers may be used only if duckweed does
means as wearing appropriate protective gloves (especially
not adhere to the walls and the test material does not sorb onto
when washing equipment or putting hands in test solutions),
the plastic more than it does to glass. Chambers should be
laboratory coats, aprons, and glasses. Special precautions, such
covered to keep out extraneous contaminants and to reduce
as covering test chambers and ventilating the area surrounding
evaporation of test solution and test material. Beakers should
the chambers, should be taken when conducting tests on
be covered with a clear watch glass and flasks should be
volatile materials. Information on toxicity to humans (5),
covered with loose-fitting caps such as foam plugs, stainless
recommended handling procedures (6), and chemical and
steel caps, Shimadzu enclosures, glass caps, or screw caps.
physicalpropertiesofthetestmaterialshouldbestudiedbefore
(Theacceptabilityoffoamplugsshouldbeinvestigatedpriorto
a test is begun. Special procedures might be necessary with
use because some brands have been found to be toxic.) All
radio-labeled test materials (7) and with materials that are, or
chambers and covers in a test must be identical.
are suspected of being, carcinogenic (8).
7.3 Cleaning—Test chambers and equipment used to pre-
6.2 Although disposal of stock solutions, test solutions, and
pare and store growth medium, stock solutions, and test
test organisms poses no special problems in most cases, health
solutions should be cleaned before use. New items should be
and safety precautions and applicable regulations should be
washed with detergent and rinsed with water, a water-miscible
considered before beginning a test. Removal or degradation of
organic solvent, water, acid (such as 10 % concentrated hydro-
test material might be desirable before disposal of stock and
chloric acid), and at least twice with deionized or distilled
test solutions.
water. (Some lots of some organic solvents might leave a film
6.3 Cleaning of equipment with a volatile solvent such as
that is insoluble in water.)Adichromate-sulfuric acid cleaning
acetone should be performed only in a well-ventilated area in
solution may be used in place of both the organic solvent and
whichnosmokingisallowedandnoopenflame,suchasapilot
the acid. At the end of the test, all items that are to be used
light, is present.
again should be immediately (a) emptied, (b) rinsed with
6.4 Acidic solutions and hypochlorite solutions should not
water, (c) cleaned by a procedure appropriate for removing the
be mixed because hazardous fumes might be produced.
test material (for example, acid to remove metals and bases;
detergent, organic solvent, or activated carbon to remove
6.5 To prepare dilute acid solutions, concentrated acid
organic chemicals), (d) cleaned with a nonphosphate detergent
should be added to water, not vice versa. Opening a bottle of
using a stiff bristle brush to loosen any attached materials, and
concentratedacidandaddingconcentratedacidtowatershould
(e) rinsed at least twice with deionized or distilled water.Acid
be performed only in a fume hood.
is often used to remove mineral deposits. Chambers should be
6.6 Because growth medium and test solutions are usually
dried in an oven at 50 to 100°C, capped with appropriate
good conductors of electricity, use of ground fault systems and
closures, autoclaved for 20 min at 121°C and 1.1 kg/cm . Test
leak detectors should be considered to help prevent electrical
chambers should be rinsed with growth medium just before
shocks.
use.
7. Apparatus
7.4 Acceptability—Before a toxicity test is conducted with
duckweed in new test facilities, it is desirable to conduct a
7.1 Facilities—Culture and test chambers should be main-
tained in an environmental chamber, incubator, or room with nontoxicant test, in which all test chambers contain growth
medium with no added test material, to determine before the
constant temperature (see 11.2) and appropriate illumination
(see 11.3). A water bath is generally not acceptable because it firsttoxicitytestwhetherduckweedwillgrowacceptablyinthe
prevents proper illumination of the test chambers. The facility new facilities, whether the growth medium, handling
should be well-ventilated and free of fumes. To further reduce procedures, and so forth, are acceptable, whether there are any
E1415 − 22
location effects on growth, and the magnitudes of the within- and should be low enough that it does not affect growth of
chamber and between-chamber variances. duckweed. Because of its low toxicity to aquatic organisms,
low volatility, and high ability to dissolve many organic
8. Growth Medium
chemicals, triethylene glycol is often a good organic solvent
for preparing stock solutions. Other water-miscible organic
8.1 Growth medium is prepared by adding appropriate
solvents such as methanol, ethanol, and acetone may also be
amounts of specified reagent-grade chemicals to deionized or
used, but they might stimulate undesirable growths of micro-
distilled water. Recommended growth media are given in
organisms; acetone is also quite volatile. If an organic solvent
Appendix X1.
is used, it should be reagent-grade or better and its concen-
9. Test Material tration in any test solution should not exceed 0.5 mL/L. A
4 surfactant should not be used in the preparation of a stock
9.1 General—The test material should be reagent-grade or
solutionbecauseitmightaffecttheformandtoxicityofthetest
better, unless a test on a formulation, commercial product, or
materialinthetestsolutions.(Theselimitationsdonotapplyto
technical-grade or use-grade material is specifically needed.
any ingredient of a mixture, formulation, or commercial
Before a test is begun, the following should be known about
product unless an extra amount of solvent is used in the
the test material:
preparation of the stock solution.)
9.1.1 Identities and concentrations of major ingredients and
9.2.3 If a solvent other than growth medium or water is
major impurities, for example, impurities constituting more
used, at least one solvent control, using solvent from the same
than about 1 % of the material,
batch used to make the stock solution, must be included in the
9.1.2 Solubility, stability, photodegradability, and volatility
test, and a growth medium control should be included in the
in the growth medium,
test. If no solvent other than growth medium or water is used,
9.1.3 Measured or estimated toxicity to duckweed (if noth-
a growth medium control must be included in the test.
ing is known about the toxicity to duckweed, a range-finding
9.2.3.1 If a solvent control is required and the concentration
test is suggested),
of solvent is the same in all test solutions that contain test
9.1.4 Precision and bias of the analytical method at the
material, the solvent control must contain the same concentra-
planned concentration(s) of test material, if the test concentra-
tion of solvent.
tion(s) are to be measured,
9.1.5 Estimate of toxicity to humans, and 9.2.3.2 If a solvent control is required and the concentration
9.1.6 Recommended handling procedures (see 6.1).
of solvent is not the same in all test solutions that contain test
material, either (a) a solvent test must be conducted to
9.2 Stock Solution—In some cases the test material can be
determine whether growth of duckweed is related to the
addeddirectlytothegrowthmedium,butusuallyitisdissolved
concentration of the solvent over the range used in the toxicity
in a solvent to form a stock solution that is then added to
testor(b)suchasolventtestmusthavealreadybeenconducted
growth medium. If a stock solution is prepared, the concentra-
usingthesamegrowthmedium.Ifgrowthisfoundtoberelated
tion and stability of the test material in it should be determined
to the concentration of solvent, a toxicity test in that medium is
before the beginning of the test. If the test material is subject to
unacceptable if any treatment contained a concentration of
photolysis, the stock solution should be shielded from light.
solvent in that range. If growth is not found to be related to the
9.2.1 Except possibly for tests on hydrolyzable, oxidizable,
concentration of solvent, a toxicity test in that same medium
and reducible materials, the preferred solvent is growth me-
may contain solvent concentrations within the tested range, but
dium. Distilled or deionized water may also be used as a
the solvent control must contain the highest concentration of
solvent, but the amount of water added to growth medium to
solvent present in all of the other treatments.
prepare the test solutions should be kept to less than 10 % of
9.2.3.3 If the test contains both a growth medium control
the total volume to avoid dilution of the growth medium.
and a solvent control, the growth of the duckweed in the two
Several techniques have been specifically developed for pre-
controls should be compared using a t-test. Adjustments for
paring aqueous stock solutions of slightly soluble materials
chamber-to-chamber heterogeneity might be necessary. The
(12). The minimum necessary amount of a strong acid or base
use of a large alpha level (for example, 0.25) will make it more
may be used in the preparation of an aqueous stock solution,
difficult to accept the null hypothesis when it should not be
but such reagents might affect the pH of test solutions
accepted. The test statistic, its significance level, the minimum
appreciably. Use of a more soluble form of the test material,
detectable difference, and the power of the test should be
such as chloride or sulfate salts of organic amines, sodium, or
reported.
potassium salts of phenols and organic acids, and chloride or
nitrate salts of metals, might affect the pH even more than the 9.2.3.4 If a statistically significant difference in growth is
use of the minimum necessary amount of a strong acid or base. detected between the two controls, only the solvent control can
9.2.2 If a solvent other than growth medium is used, its be used for meeting the requirements of 13.1.3 and as the basis
concentration in test solutions should be kept to a minimum for calculation of results. If no statistically significant differ-
ence is detected, the data from both controls should be used for
meeting the requirements of 13.1.3 and as the basis for
“Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-
calculation of results.
cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
9.2.4 Ifasolventotherthangrowthmediumorwaterisused
the American Chemical Society, see “Analar Standards for Laboratory U.K.
Chemicals,” BDH Ltd., Poole, Dorset, and the “United States Pharmacopeia.” to prepare a stock solution, it might be desirable to conduct
E1415 − 22
simultaneous tests on the test material using two chemically 11. Procedure
unrelated solvents or two different concentrations of the same
11.1 Experimental Design:
solvent to obtain information concerning possible effects of the
11.1.1 Decisions concerning such aspects of experimental
solvent on the results of the test.
design as the dilution factor, number of treatments, and
9.3 Test Concentration(s):
numbers of test chambers and fronds per treatment should be
9.3.1 If the test is intended to allow calculation of the 7-day
based on the purpose of the test and the type of procedure that
IC50, the test concentrations (see 11.1.1.1) should bracket the
is to be used to calculate results (see Section 14). One of the
predicted IC50.Aprediction might be based on the results of a
following two types of experimental design will probably be
testonthesameorasimilarmaterialwiththesameorasimilar
appropriate in most cases.
species. If a useful prediction is not available, it is usually
11.1.1.1 A test intended to allow calculation of an IC50
desirable to conduct a range-finding test in which the test
usually consists of one or more control treatments and a
species is exposed to a control and three to five concentrations
geometric series of at least five concentrations of test material.
of the test material that differ by a factor of 10. The greater the
In the medium or solvent controls, or both, (see 9.2.3),
similaritybetweentherange-findingtestandtheactualtest,the
duckweed is exposed to growth medium to which no test
more useful the range-finding test will be.
material has been added. Except for the control(s) and the
9.3.1.1 If necessary, concentrations above solubility should
highest concentration, each concentration should be at least
be used because organisms in the real world are sometimes
60 % of the next higher one, unless information concerning the
exposed to concentrations above solubility and because solu-
concentration-effect curve indicates that a different dilution
bility is often not well known. The use of concentrations that
factor is more appropriate. At a dilution factor of 0.6, five
are more than ten times greater than solubility is probably not
properly chosen concentrations are a reasonable compromise
worthwhile. With some test materials it might be found that
between cost and the risk of all concentrations being either too
concentrations above solubility do not affect growth any more
high or too low. If the estimate of toxicity is particularly
than does the concentration that is the solubility limit; such
nebulous (see 9.3.1), six or seven concentrations might be
information is certainly worth knowing.
desirable.
9.3.2 In some (usually regulatory) situations, it is only
11.1.1.2 If it is only necessary to determine whether a
necessary to determine whether a specific concentration of test
specific concentration unacceptably affects growth or whether
material unacceptably affects growth of the test species or
the IC50 is above or below a specific concentration (see 9.3.2),
whether the IC50 is above or below a specific concentration.
only that concentration and the control(s) are necessary. Two
For example, the specific concentration might be the concen-
additional concentrations at about one-half and two times the
tration occurring in a surface water, the concentration resulting
specific concentration of concern are desirable to increase
from the direct application of the material to a body of water,
confidence in the results.
or the solubility limit of the material in water. When there is
11.1.1.3 If an IC near the extremes of toxicity, such as an
only interest in a specific concentration, it is often only
IC5 or IC95, is to be calculated, at least one concentration of
necessary to test that specific concentration (see 11.1.1.2), and
test material should have inhibited growth by a percentage,
it is not necessary to actually determine the IC50.
other than 0 or 100 %, near the percentage for which the IC is
to be calculated. This requirement might be met in a test
10. Test Organisms
designed to determine an IC50, but a special test with appro-
10.1 Species—The test species is Lemna gibba G3. It is
priate concentrations of test material will usually be necessary.
widely distributed, easily handled in the laboratory, and has a
11.1.2 The primary focus of the physical and experimental
history of successful use. The identity of the organism should
design of the test and the statistical analysis of the data is the
be verified using an appropriate taxonomic key (13).Itis
experimental unit, which is defined as the smallest physical
important to identify the clone (1), because it has been shown
entitytowhichtreatmentscanbeindependentlyassigned.Thus
that different clones of the same species can have different
the test chamber, as defined in 7.2, is the experimental unit.As
sensitivities (14).
the number of test chambers (that is, experimental units) per
10.2 Stock Culture—Plants used in testing must be obtained treatment increases, the number of degrees of freedom
from laboratory stock cultures that have been actively growing increases, and, therefore, the width of the confidence interval
in growth medium under constant warm-white fluorescent onapointestimatedecreasesandthepowerofasignificanttest
illumination of approximately 580 to 620 fc (6200 to 6700 lx) increases. With respect to factors that might affect results
and temperature of 25 6 2°C for at least the eight weeks within test chambers and, therefore, results of the test, all test
immediately preceding the start of the test. Maintenance of chambers in the test should be treated as similarly as possible.
axenic stock cultures is recommended. Plants should be asep- For example, the temperature in all test chambers should be as
tically transferred on a regular schedule (weekly is suggested) similar as possible unless the purpose of the test is to study the
into fresh growth medium. effect of temperature. Test chambers are usually arranged in
one or more rows. Treatments must be randomly assigned to
individual test chamber locations and may be randomly reas-
There is currently no commercial source of Lemna gibba G3. It may be
signed during the test. A randomized block design (with each
available from: Dr. Elaine Tobin, UCLA, Biology De
...