ASTM E1415-91(2012)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E1415 − 91 (Reapproved 2012)
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 tion unacceptably affects the growth of the test species or
whether the IC50 is above or below a specific concentration.
1.1 Thisguidedescribesproceduresforobtaininglaboratory
Another end point that may be calculated is the no observed
data concerning the adverse effects of a text material added to
effect concentration (NOEC).
growth medium on a certain species of duckweed (Lemna
gibba G3) during a 7-day exposure using the static technique. 1.5 The sections of this guide appear as follows:
These procedures will probably be useful for conducting
Title Section
toxicity tests with other species of duckweed and other floating
Referenced Documents 2
vascular plants, although modifications might be necessary.
Terminology 3
Summary of Guide 4
1.2 Special needs or circumstances might also justify modi-
Significance and Use 5
fication of this standard. Although using appropriate proce-
Hazards 6
dures is more important than following prescribed procedures, Apparatus 7
Facilities 7.1
results of tests conducted using unusual procedures are not
Test Chambers 7.2
likely to be comparable to results of many other tests. Com-
Cleaning 7.3
parison of results obtained using modified and unmodified
Acceptability 7.4
Growth Medium 8
versions of these procedures might provide useful information
Test Material 9
concerning new concepts and procedures for conducting tests
General 9.1
with duckweed. Stock Solution 9.2
Test Concentration(s) 9.3
1.3 The procedures in this guide are applicable to most
Test Organisms 10
Species 10.1
chemicals, either individually or in formulations, commercial
Source 10.2
products, or known mixtures. With appropriate modifications
Stock Culture 10.3
these procedures can be used to conduct tests on temperature
Procedure 11
Experimental Design 11.1
and pH and on such other materials as aqueous effluents (see
Temperature 11.2
also Guide E1192), leachates, oils, particulate matter, sedi-
Illumination 11.3
ments and surface waters.These procedures do not specifically
Beginning the Test 11.4
Duration of Test 11.5
address effluents because to date there is little experience using
Biological Data 11.6
duckweedsineffluenttestingandsuchtestsmayposeproblems
Other Measurements 11.7
with acclimation of the test organisms to the receiving water.
Analytical Methodology 12
Acceptability of Test 13
Statictestsmightnotbeapplicabletomaterialsthathaveahigh
Calculation of Results 14
oxygen demand, are highly volatile, are rapidly biologically or
Report 15
chemically transformed in aqueous solution, or are removed
1.6 This standard does not purport to address all of the
from test solutions in substantial quantities by the test cham-
safety concerns, if any, associated with its use. It is the
bers or organisms during the test.
responsibility of the user of this standard to establish appro-
1.4 Results of toxicity tests performed using the procedures
priate safety and health practices and to determine the
in this guide should usually be reported in terms of the 7-day
applicability of regulatory limitations prior to use. Specific
IC50 based on inhibition of growth. In some situations it might
hazard statements are given in Section 6.
only be necessary to determine whether a specific concentra-
2. Referenced Documents
2.1 ASTM Standards:
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 Dec. 1, 2012. Published December 2012. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1991. Last previous edition approved in 2004 as E1415 – 91 (2004). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/E1415-91R12. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1415 − 91 (2012)
E729 Guide for Conducting Acute Toxicity Tests on Test selected concentration of test material has been added. Speci-
Materials with Fishes, Macroinvertebrates, and Amphib- fied data concerning growth of duckweed in each test chamber
ians are obtained during the test and are usually analyzed to
E943 Terminology Relating to Biological Effects and Envi- determine the IC50 or NOEC based on inhibition of growth.
ronmental Fate
E1023 Guide for Assessing the Hazard of a Material to 5. Significance and Use
Aquatic Organisms and Their Uses
5.1 The term duckweed commonly refers to members of the
E1192 Guide for ConductingAcute Toxicity Tests onAque-
family Lemnaceae. This family has many species world-wide
ous Ambient Samples and Effluents with Fishes,
in 4 genera.This guide is designed for toxicity testing with one
Macroinvertebrates, and Amphibians
particular clone of one species of duckweed that has been
E1218 Guide for Conducting Static Toxicity Tests with
extensively studied, Lemna gibba G3, although other species
Microalgae
such as Lemna minor or Spirodela spp. can probably also be
IEEE/ASTM SI 10 American National Standard for Use of
tested using the procedures described herein.
theInternationalSystemofUnits(SI):TheModernMetric
5.2 Duckweeds are widespread, free-floating aquatic plants,
System
ranging in the world from tropical to temperate zones. Duck-
3. Terminology
weeds are a source of food for waterfowl and small animals
and provide food, shelter, and shade for fish. The plants also
3.1 The words must, should, may, can, and might have very
serve as physical support for a variety of small invertebrates.
specific meanings in this guide. Must is used to express an
Duckweed is fast growing and reproduces rapidly compared
absolute requirement, that is, to state that the test ought to be
with other vascular plants (1). Under conditions favorable for
designed to satisfy the specified condition, unless the purpose
its growth, it can multiply quickly and form a dense mat in
of the test requires a different design. Must is only used in
lakes, ponds, and canals, primarily in fresh water, but also in
connection with factors that directly relate to the acceptability
estuaries. It also grows well in effluents of wastewater treat-
of the test (see Section 13). Should is used to state that the
ment plants and has been suggested as a means of treating
specified condition is recommended and ought to be met if
wastewaters (2). A dense mat of duckweed can block sunlight
possible. Although violation of one should is rarely a serious
and aeration and cause fish kills (3).
matter, violation of several will often render the results
questionable. Terms such as is desirable, is often desirable,
5.3 Duckweed is small enough that large laboratory facili-
might be desirable are used in connection with less important
ties are not necessary, but large enough that effects can be
factors. May is used to mean is (are) allowed to, can is used to
observed visually.
meanis(are)ableto,and mightisusedtomeancouldpossibly.
5.4 Because duckweed is a floating macrophyte, it might be
Thus the classic distinction between may and can is preserved,
particularly susceptible to surface active and hydrophobic
and might is never used as a synonym for either may or can.
chemicals that concentrate at the air-water interface. Results of
3.2 Definitions of Terms Specific to This Standard:
duckweed tests on such chemicals, therefore, might be sub-
3.2.1 frond—individual leaf-like structure on a duckweed
stantially different from those obtained with other aquatic
plant.
species.
3.2.2 IC50—a statistically or graphically estimated concen-
5.5 Results of toxicity tests with duckweed might be used to
tration of test material that is expected to cause a 50 %
predicteffectslikelytooccuronduckweedinfieldsituationsas
inhibition of one or more specified biological processes (such
a result of exposure under comparable conditions.
as growth or reproduction), for which the data are not
5.6 Results of tests with duckweed might be used to
dichotomous, under specified conditions.
compare the toxicities of different materials and to study the
3.3 For definitions of other terms used in this guide, refer to
effectsofvariousenvironmentalfactorsonresultsofsuchtests.
Terminology E943, and Guides E729 and E1023. For an
explanation of units and symbols, refer to Practice IEEE/ 5.7 Results of tests with duckweed might be an important
consideration when assessing the hazards of materials to
ASTM SI 10 .
aquatic organism (see Guide E1023) or when deriving water
4. Summary of Guide
quality criteria for aquatic organisms (4).
4.1 In each of two or more treatments, plants of Lemna
5.8 Results of tests with duckweed might be useful for
gibba G3 are maintained for 7 days in two or more test
studying biological availability of, and structure-activity rela-
chambers using the static technique. In each of the one or more
tionships between test materials.
controltreatments,theplantsaremaintainedingrowthmedium
5.9 Results of tests with duckweed will depend on
to which no test material has been added in order to provide a
temperature, composition of the growth medium, condition of
measure of the acceptability of the test by giving an indication
thetestorganisms,andotherfactors.Thegrowthmediathatare
of the quality of the duckweed and the suitability of the growth
medium,testconditions,handlingprocedures,andsoforth,and
the basis for interpreting data obtained from the other treat-
ments. In each of the one or more other treatments, the
The boldface numbers in parentheses refer to the list of references at the end of
duckweed plants are maintained in growth medium to which a this guide.
E1415 − 91 (2012)
usually used for tests with duckweed contain concentrations of and 250 or 500-mLErlenmeyer flasks have been used success-
salts, minerals, and nutrients that greatly exceed those in most fully (9-11). The ratio of the size of the test chamber to the
surface waters. volume of test solution should be 5 to 2 (that is, 100 mL in a
250-mL Erlenmeyer flask, 200 mL in a 500-mL Erlenmeyer
flask). Plastic chambers may be used only if duckweed does
6. Hazards
not adhere to the walls and the test material does not sorb onto
6.1 Many materials can affect humans adversely if precau-
the plastic more than it does to glass. Chambers should be
tions are inadequate. Therefore, skin contact with all test
covered to keep out extraneous contaminants and to reduce
materials and solutions of them should be minimized by such
evaporation of test solution and test material. Beakers should
means as wearing appropriate protective gloves (especially
be covered with a clear watch glass and flasks should be
when washing equipment or putting hands in test solutions),
covered with loose-fitting caps such as foam plugs, stainless
laboratory coats, aprons, and glasses. Special precautions, such
steel caps, Shimadzu enclosures, glass caps, or screw caps.
as covering test chambers and ventilating the area surrounding
(Theacceptabilityoffoamplugsshouldbeinvestigatedpriorto
the chambers, should be taken when conducting tests on
use because some brands have been found to be toxic.) All
volatile materials. Information on toxicity to humans (5),
chambers and covers in a test must be identical.
recommended handling procedures (6), and chemical and
physicalpropertiesofthetestmaterialshouldbestudiedbefore 7.3 Cleaning—Test chambers and equipment used to pre-
a test is begun. Special procedures might be necessary with pare and store growth medium, stock solutions, and test
solutions should be cleaned before use. New items should be
radio-labeled test materials (7) and with materials that are, or
are suspected of being, carcinogenic (8). washed with detergent and rinsed with water, a water-miscible
organic solvent, water, acid (such as 10 % concentrated hydro-
6.2 Although disposal of stock solutions, test solutions, and
chloric acid), and at least twice with deionized or distilled
test organisms poses no special problems in most cases, health
water. (Some lots of some organic solvents might leave a film
and safety precautions and applicable regulations should be
that is insoluble in water.)Adichromate-sulfuric acid cleaning
considered before beginning a test. Removal or degradation of
solution may be used in place of both the organic solvent and
test material might be desirable before disposal of stock and
the acid. At the end of the test, all items that are to be used
test solutions.
again should be immediately (a) emptied, (b) rinsed with
6.3 Cleaning of equipment with a volatile solvent such as
water, (c) cleaned by a procedure appropriate for removing the
acetone should be performed only in a well-ventilated area in
test material (for example, acid to remove metals and bases;
whichnosmokingisallowedandnoopenflame,suchasapilot
detergent, organic solvent, or activated carbon to remove
light, is present.
organic chemicals), (d) cleaned with a nonphosphate detergent
6.4 Acidic solutions and hypochlorite solutions should not using a stiff bristle brush to loosen any attached materials, and
(e) rinsed at least twice with deionized or distilled water.Acid
be mixed because hazardous fumes might be produced.
is often used to remove mineral deposits. Chambers should be
6.5 To prepare dilute acid solutions, concentrated acid
dried in an oven at 50 to 100°C, capped with appropriate
should be added to water, not vice versa. Opening a bottle of
closures, autoclaved for 20 min at 121°C and 1.1 kg/cm . Test
concentratedacidandaddingconcentratedacidtowatershould
chambers should be rinsed with growth medium just before
be performed only in a fume hood.
use.
6.6 Because growth medium and test solutions are usually
7.4 Acceptability—Before a toxicity test is conducted with
good conductors of electricity, use of ground fault systems and
duckweed in new test facilities, it is desirable to conduct a
leak detectors should be considered to help prevent electrical
nontoxicant test, in which all test chambers contain growth
shocks.
medium with no added test material, to determine before the
firsttoxicitytestwhetherduckweedwillgrowacceptablyinthe
7. Apparatus
new
...