ASTM E1218-21

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: E1218 − 21
Standard Guide for
1,2
Conducting Static Toxicity Tests with Microalgae
This standard is issued under the fixed designation E1218; 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 during the test. (1) However, practical flow-through test
procedures with microalgae have not been developed.
1.1 This guide covers procedures for obtaining laboratory
1.4 Results of tests using microalgae should usually be
data concerning the adverse effects of a test material added to
growth medium on growth of certain species of freshwater and reported in terms of the 96-h (or other time period) IC50 (see
3.2.5) based on reduction in growth. In some situations, it
saltwater microalgae during a static exposure. These proce-
dures will probably be useful for conducting short-term toxic- might only be necessary to determine whether a specific
concentration unacceptably affects the growth of the test
ity tests with other species of algae, although modifications
mightbenecessary.Althoughthetestdurationiscomparableto species or whether the IC50 is above or below a specific
concentration.
anacutetoxicitytestwithaquaticanimals,analgaltoxicitytest
of short duration (72, 96 or 120 h) allows for examination of
1.5 This guide is arranged as follows:
effects upon multiple generations of an algal population and
Section
thus should not be viewed as an acute toxicity test.
Referenced Documents 2
Terminology 3
1.2 Other modifications of these procedures might be justi-
Summary of Guide 4
fied by special needs or circumstances.Although using appro- Significance and Use 5
Hazards 7
priate procedures is more important than following prescribed
Apparatus 6
procedures,resultsoftestsconductedusingunusualprocedures
Facilities 6.1
are not likely to be comparable to results of many other tests. Equipment 6.2
Test Vessels 6.3
Comparisonofresultsobtainedusingmodifiedandunmodified
Cleaning 6.4
versions of these procedures might provide useful information
Acceptability 6.5
concerning new concepts and procedures for conducting tox- Growth Medium 8
Test Material 9
icity tests with microalgae.
General 9.1
Stock Solution 9.2
1.3 These procedures are applicable to many chemicals,
Test Concentration(s) 9.3
either individually or in formulations, commercial products, or
Test Organisms 10
known mixtures. With appropriate modifications, these proce-
Species 10.1
Source 10.2
dures can be used to conduct tests on temperature, and pH and
Culture 10.3
on such materials as aqueous effluents (see Guide E1192),
Quality 10.4
leachates, oils, particulate matter, sediments, and surface wa-
Procedure 11
Experimental Design 11.1
ters. Static tests might not be applicable to materials that are
Temperature 11.2
highly volatile, are rapidly biologically or chemically trans-
Illumination 11.3
formed in aqueous solutions, or are removed from test solu-
Beginning the Test 11.4
Gas Exchange 11.5
tions in substantial quantities by the test vessels or organisms
Duration of Test 11.6
Biological Data 11.7
Other Measurements 11.8
Determination of Algistatic and Algicidal Effects 11.8.5
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Analytical Methodology 12
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
Acceptability of Test 13
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Calculation 14
Current edition approved Nov. 1, 2021. Published December 2021. Originally
Report 15
approvedin1990.Lastpreviouseditionapprovedin2012asE1218-04(2012)which Keywords 16
was withdrawn January 2021 and reinstated in November 2021. DOI: 10.1520/
E1218-21.
This standard guide is a document, developed using the consensus mechanisms
of ASTM, that provides guidance for the selection of procedures to accomplish a The boldface numbers given in parentheses refer to a list of references at the
specific test, but which does not stipulate specific procedures. end of the text.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1218 − 21
1.6 This standard does not purport to address all of the 3.1.1.3 should—is used to state that the specified condition
safety concerns, if any, associated with its use. It is the is recommended and ought to be met if possible. Although
responsibility of the user of this standard to establish appro- violationofone“should”israrelyaseriousmatter,violationof
priate safety, health, and environmental practices and deter- several will often render the results questionable. Terms such
mine the applicability of regulatory limitations prior to use. as“isdesirable,”“isoftendesirable,”and“mightbedesirable”
Specific hazard statements are given in Section 7. are used in connection with less important factors.
1.7 This international standard was developed in accor- 3.2 Definitions of Terms Specific to This Standard:
dance with internationally recognized principles on standard- 3.2.1 algicidal—having the property of killing algae.
ization established in the Decision on Principles for the
3.2.2 algistatic—having the property of inhibiting algal
Development of International Standards, Guides and Recom-
growth.
mendations issued by the World Trade Organization Technical
3.2.3 biomass—the dry weight of living matter present in a
Barriers to Trade (TBT) Committee.
population and expressed in terms of a given area or volume,
for example, mg algae per liter. Because biomass is difficult to
2. Referenced Documents
measure accurately, surrogate measures of biomass, such as
2.1 ASTM Standards:
cell counts, are typically used in this test.
D1129Terminology Relating to Water
3.2.4 growth rate—the increase in biomass per unit of time.
D3731PracticesforMeasurementofChlorophyllContentof
Algae in Surface Waters 3.2.5 IC50—a statistically or graphically estimated concen-
D3978Practice for Algal Growth Potential Testing with tration that is expected to cause a 50% inhibition of one or
Pseudokirchneriella subcapitata more specified biological processes (such as growth or repro-
D4447Guide for Disposal of Laboratory Chemicals and duction) for which the data are not dichotomous, under
Samples specified conditions. Alternative values for inhibition, such as
E380Practice for Use of the International System of Units 10% or 20%, are referred to as IC10 or IC20.
(SI) (the Modernized Metric System) (Withdrawn 1997)
3.2.6 standing crop—thealgalbiomassattheendofthetest.
E729Guide for Conducting Acute Toxicity Tests on Test
3.2.7 yield—the algal biomass at the end of the test minus
Materials with Fishes, Macroinvertebrates, and Amphib-
the algal biomass at the beginning of the test.
ians
3.3 Fordefinitionsofothertermsusedinthisstandard,refer
E943Terminology Relating to Biological Effects and Envi-
to Guides E729 and E1023 and Terminology E943. For
ronmental Fate
explanation of units and symbols, refer to Practice E380.
E1023Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
4. Summary of Guide
E1192Guide for ConductingAcute Toxicity Tests onAque-
ous Ambient Samples and Effluents with Fishes,
4.1 In each of two or more treatments, organisms of one
Macroinvertebrates, and Amphibians
species of microalgae are maintained in replicate test vessels
E1733Guide for Use of Lighting in Laboratory Testing
using the static technique. The test duration is typically 96 h,
E1847Practice for Statistical Analysis of Toxicity Tests
but shorter periods (for example, 72 h) have been used for
Conducted Under ASTM Guidelines
fast-growing algae and longer periods (for example, 120 h)
may be necessary for slower-growing algae. In each of the one
3. Terminology
or more control treatments, the algae are maintained in growth
3.1 Definitions:
medium to which no test material has been added in order to
3.1.1 The words “must,” “should,” “may,”“ can,” and
providethefollowing:ameasureoftheacceptabilityofthetest
“might” have very specific meanings in this guide.
by giving an indication of the quality of the algae and the
3.1.1.1 may—is used to mean “is (are) allowed to,” “can” is
suitability of the growth medium, test conditions, handling
used to mean“ is (are) able to,” and “might” is used to mean
procedures, and so forth, and the basis for interpreting data
“could possibly”. Therefore the classic distinction between
obtained from the other treatments. In each of the one or more
“may” and “can” is preserved, and “might” is never used as a
othertreatments,thealgaearemaintainedingrowthmediumto
synonym for either “may” or “can”.
which test material has been added to achieve a selected
3.1.1.2 must—is used to express an absolute requirement,
concentration. Specified data on population growth are ob-
that is, to state that the test ought to be designed to satisfy the
tainedduringthetestandareusuallyanalyzedtodeterminethe
specified condition, unless the purpose of the test requires a
IC50 based on reduction in growth.
differentdesign.“Must”isonlyusedinconnectionwithfactors
that directly relate to the acceptability of the test (see 13.1).
5. Significance and Use
5.1 Tests with algae provide information on the toxicity of
test materials to an important component of the aquatic biota
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
and might indicate whether additional testing (2) is desirable.
Standards volume information, refer to the standard’s Document Summary page on
Specific testing procedures under various regulatory jurisdic-
the ASTM website.
tionsfollowproceduressimilartothosedescribedinthisGuide
The last approved version of this historical standard is referenced on
www.astm.org. (3, 4). Users should consult with any specific regulatory
E1218 − 21
requirements to determine the applicability and consistency of 6.2.9 Analytical Balance,
this standard with such requirements.
6.2.10 Autoclave or Microwave Oven,
6.2.11 pH Meter,
5.2 Algae are ubiquitous in aquatic ecosystems, where they
–2 –1
6.2.12 Calibrated Light Meter, reading in µmol m s or
incorporate solar energy into biomass, produce oxygen, func-
lumens,
tion in nutrient cycling and serve as food for animals. Because
6.2.13 Microscope, capable of 100× to 400× magnification,
of their ecological importance, sensitivity to many toxicants,
ready availability, ease of culture, and fast growth rates 6.2.14 Hemacytometer Counting Chamber or Plankton
(rendering it possible to conduct a multi-generation test in a Counting Chamber and Ocular Micrometer,
short period of time), algae are often used in toxicity testing.
6.2.15 Particle Counter, with 70µm or 100µm aperture
tube, and (optional) mean cell volume computer, or
5.3 Results of algal toxicity tests might be used to compare
6.2.16 Fluorometer, equipped to measure chlorophyll a,or
the sensitivities of different species of algae and the toxicities
6.2.17 Spectrophotometer, to measure cell densities in log
ofdifferentmaterialstoalgaeandtostudytheeffectsofvarious
phase cultures.
environmental factors on results of such tests.
6.3 Test Vessels:
5.4 Results of algal toxicity tests might be an important
consideration when assessing the hazards of materials to 6.3.1 Inatoxicitytestwithaquaticorganisms,testchambers
aquaticorganisms(SeeGuideE1023)orderivingwaterquality (also referred to as test vessels) are defined as the smallest
criteria for aquatic organisms (5). physical units between which there are no water connections.
Vessels should be covered to keep out extraneous
5.5 Results of algal toxicity tests might be useful for
contaminants, especially bacteria and undesirable algae. Be-
studying biological availability of, and structure-activity rela-
cause algae consume carbon dioxide, the covers used for algal
tionships between, test materials.
testsmustnotpreventthepassageofair.Allvesselsandcovers
5.6 Results of algal toxicity tests will depend on the
in a test must be identical.
temperature, composition of the growth medium, and other
6.3.2 Sterile Erlenmeyer flasks of borosilicate glass or
factors. These tests are conducted in solutions that contain
polycarbonate are usually used as test and culture vessels.Any
concentrations of salts, minerals, and nutrients that greatly
size flask can be used as long as the test solution volume does
exceed those in most surface waters. These conditions may
not exceed 50% of the flask volume for tests conducted on a
over- or under-estimate the effects of the test material if
shaker, and not more than 20% of the flask volume for tests
discharged to surface waters.
not conducted on a shaker. The proper solution/volume ratio
should be determined for each test species in each laboratory
6. Apparatus
because the ratio is dependent on the species and conditions.
6.1 Facilities—Cultures and test vessels should be main-
6.4 Cleaning:
tained in rooms, incubators, or environmental chambers with
6.4.1 Test vessels and equipment used to prepare and store
constant temperatures (see 11.2) and appropriate illumination
growth medium, stock solutions, and test solutions should be
(see 11.3). A water bath is generally not acceptable because it
cleaned before use. New items should be washed with deter-
prevents proper illumination of the test vessels. The facility
gent and rinsed with water, a water-miscible organic solvent,
should be well ventilated and free of fumes. To further reduce
water, acid (such as 10% concentrated hydrochloric acid), and
the possibility of contamination by test materials and other
at least twice with deionized or distilled water. (Some lots of
substances, especially volatile ones, algae should not be
some organic solvents might leave a film that is insoluble in
cultured in a room in which toxicity tests are conducted, stock
water.)Attheendofthetest,allitemsthataretobeusedagain
solutions or test solutions are prepared, or equipment is
should be immediately emptied, rinsed with water, cleaned by
cleaned.
a procedure appropriate for removing the test material (for
6.2 Equipment—Some or all of the following will be example, acid to remove metals and bases; detergent, organic
needed:
solvent, or activated carbon to remove organic chemicals),
6.2.1 Centrifuge, cleaned with a non-phosphate detergent using a stiff bristle
6.2.2 Centrifuge Tubes, glass or polycarbonate with screw- brush to loosenanyattachedmaterials and rinsedatleasttwice
cap lids, with deionized or distilled water.Acid is often used to remove
6.2.3 Rotary or Oscillation Shaker, with variable speed mineral deposits.
control capable of 100 r/min (or oscillations per minute),
6.4.2 If an electronic particle counter is to be used to count
6.2.4 Erlenmeyer Flasks, borosilicate glass, or polycarbon- algal cells, the final rinse should be with water that has been
ate filtered through a 0.22-µm membrane filter.
6.2.5 Stainless Steel Caps, Shimatsu Enclosures, Foam
6.4.3 Test vessels may be dried in an oven at 50°C to
Plugs, Glass Caps, or Standard Screw Caps, (plastic/bakelite)
100°C and capped with either stainless steel, foam or glass
(all closures should be loose-fitting),
caps, or Shimatsu closures. Glassware should be sterilized by
6.2.6 Pipets, Eppendorf or equivalent,
autoclaving for 20 min at 121°C and 1.1 kg/cm or by
6.2.7 Filtration Apparatus, microwaving (6). Hand-made cotton plugs should not be used.
6.2.8 Membrane Filters, with 0.45µm and 0.22µm pore The acceptability of foam plugs should be investigated prior to
size, use because some brands have been reported to be toxic.
E1218 − 21
6.5 Acceptability—Before a toxicity test is conducted with medium)ortoASTMTypeIwater(seeAnnexA3;“Complete”
algae in new test facilities, it is desirable to conduct a medium).Avariety of salt waters are acceptable, for example,
“non-toxicant” test, in which all test vessels contain growth various reconstituted waters prepared with salts or filtered
medium with no added test material, to determine before the (0.22-µm membrane filter) natural seawater. Salinity should be
first test whether algae will grow acceptably in the new between 24 and 35 g/kg. Salinity may be adjusted by adding
facilities, whether the growth medium, handling procedures, ASTM Type I water, NaCl, or sea salts.
and so forth, are acceptable, whether there are any location
8.3 If using a natural water to prepare the medium, indig-
effects on growth, and the magnitude of the between vessel
enous plankton should be removed from the water before use
variance.
especially if the test is used to determine the quality of a
surface water. Two methods are available. First, the water can
7. Hazards
be filtered through a 0.22µm membrane filter. Second, the
7.1 Many materials can affect humans adversely if precau- water can be filtered through a 0.45µm membrane filter and
tions are inadequate. Therefore, skin contact with all test then for saltwater algae the water should be heated for4hat
materials and solutions of them should be minimized by such 60°C, and for freshwater algae, the water should be sterilized
means as wearing appropriate protective gloves (especially by microwaving or autoclaving at 1.1 kg/cm and 121°C for
20 min. (Autoclaving might cause the formation of a precipi-
when washing equipment or putting hands in test solutions),
laboratorycoats,aprons,andglasses.Specialprecautions,such tate.)Filtrationthrougha0.22µmmembranefilterwillremove
indigenous organisms. After filtration, microwaving, or
ascoveringtestvesselsandventilatingtheareasurroundingthe
vessels, should be taken when conducting tests on volatile autoclaving, the water should be equilibrated by letting the
water sit in a loosely capped vessel or aerating with sterile air
materials. Information on toxicity to humans (7), recom-
mended handling procedures (8), and chemical and physical for 1 to 2 h. Air used for aeration should be sterile (filtered
through a 0.22 µm bacterial filter) and free of fumes, oil and
properties of the test material, as available on the material
safety data sheet, should be studied before a test is begun. water; filters to remove oil and water are desirable.
Special procedures might be necessary with radiolabeled test
8.4 It may be desirable to reduce the amount or omit EDTA
materials (9) and with test materials that are, or are suspected
from the medium in toxicity testing if it is suspected that the
of being, carcinogenic (10).
chelator will interact with the test material. However, the
7.2 Although disposal of stock solutions, test solutions, and amount of EDTA in the freshwater medium is minimal and is
test organisms poses no special problems in most cases, health necessary to obtain consistent and acceptable algal growth. If
and safety precautions and applicable regulations should be EDTAis reduced or omitted from the freshwater medium, two
considered before beginning a test. Removal or degradation of sets of controls should be used, one of medium with the
test material might be desirable before disposal of stock and recommendedconcentrationofEDTAandoneofmediumwith
test solutions. the reduced concentration or without EDTA. These controls
should be evaluated according to the discussion in 9.2.4.3 and
7.3 Cleaning of equipment with a volatile solvent such as
9.2.4.4
acetone should be performed only in a well-ventilated area in
whichnosmokingisallowedandnoopenflame,suchasapilot
9. Test Material
light, is present.
9.1 General—The test material should be reagent-grade or
7.4 Anacidicsolutionshouldnotbemixedwithahypochlo-
better, unless a test on a formulation, commercial product, or
rite solution because hazardous fumes might be produced.
technical-grade or use-grade material is specifically needed.
7.5 To prepare dilute acid solutions, concentrated acid
Before a test is begun, the following should be known about
should be added to water, not vice versa. Opening a bottle of
the test material:
concentratedacidandaddingconcentratedacidtowatershould
9.1.1 Identities and concentrations of major ingredients and
be performed only in a fume hood.
major impurities, for example, impurities constituting more
than about 1% of the material.
7.6 Because growth medium and test solutions are usually
9.1.2 Solubility, stability, and volatility in the growth me-
good conductors of electricity, use of ground fault systems and
leak detectors should be considered to help prevent electrical dium.
9.1.3 Measured or estimated toxicity to the test species or a
shocks.
similar species. (If nothing is known about the toxicity to the
test species, a range-finding test is suggested).
8. Growth Medium
8.1 Growth medium for tests with freshwater algae is
preparedbyaddingappropriateamountsofspecifiedchemicals
Salts such as Instant Ocean, available from Aquarium Systems, 8141 Tyler
(see AnnexA1) toASTM Type I water.Alternatively, depend-
Blvd., Mentor, OH have been found suitable for this purpose.
inguponthepurposeofthetoxicitytest,anaturalwatercanbe
Reagent Chemicals, American Chemical Society Specifications . American
used as the basis for preparing the medium.
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
8.2 Growth medium water for tests with saltwater algae is
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeial
preparedbyaddingappropriateamountsofspecifiedchemicals
and National Formulary. U.S. Pharamceutical Convention, Inc. (USPC), Rockville,
to natural or artificial saltwater (see Annex A2; “Enriched” MD.
E1218 − 21
9.1.4 Precision and bias of the analytical method at the 9.2.4.1 If a solvent control is required and the concentration
planned concentration(s) of the test material, if the test con- of solvent is the same in all test solutions that contain test
centration(s) are to be measured. material, the solvent control must contain the same concentra-
tion of solvent.
9.1.5 Estimate of toxicity to humans.
9.2.4.2 If a solvent control is required and the concentration
9.1.6 Recommended handling procedures (see 7.1).
of solvent is not the same in all test solutions that contain test
9.2 Stock Solution:
material, either a solvent test must be conducted to determine
9.2.1 Insomecasesthetestmaterialcanbeaddeddirectlyto
whether growth of the test species is related to the concentra-
the growth medium, but usually it is dissolved in a solvent to
tion of solvent over the range used in the toxicity test, or such
form a stock solution that is then added to the growth medium.
asolventtestmusthavealreadybeenconductedusingthesame
Ifastocksolutionisused,theconcentrationandstabilityofthe
growth medium and test species. If growth is found to be
testmaterialinitshouldbedeterminedbeforethebeginningof
related to the concentration of solvent, an algal test with that
the test. If the test material is subject to photolysis, the stock
species in that medium is unacceptable if any treatment
solution should be shielded from light. If the test material is
contained a concentration of solvent in that range. If growth is
subjecttohydrolysis,preservationbyadjustmentofpHmaybe
not found to be related to the concentration of solvent, an algal
necessary, and, in this instance, it may be desirable to investi-
test with that species in that medium may contain solvent
gate the influence of any pH adjustments on the medium or the
concentrations within the tested range, but the solvent control
test organism.
must contain the highest concentration of solvent present in
9.2.2 Except possibly for tests on hydrolyzable, oxidizable, any of the other treatments.
and reducible materials, the preferred solvent is growth me-
9.2.4.3 If the test contains both a growth medium control
dium. ASTM Type I water may also be used as a solvent, but
and a solvent control, the growth in the two controls should be
the amount of water added to growth medium to prepare the
compared using a t-test. The use of a large alpha level (for
test solutions should be kept to less than 10 % of the total
example, 0.25) will make it more difficult to accept the null
volume to avoid dilution of the growth medium. Several
hypothesiswhenitshouldnotbeaccepted.Theteststatistic,its
techniques have been specifically developed for preparing
significance level, the minimum detectable difference, and the
aqueous stock solutions of slightly soluble materials (11). The
power of the test should be reported.
minimum necessary amount of a strong acid or base, or both,
9.2.4.4 If a statistical difference in growth is detected
may be used in the preparation of an aqueous stock solution,
betweenthetwocontrols,onlythesolventcontrolmaybeused
but such reagents might affect the pH of test solutions
for meeting the requirements of 13.1.7 and as the basis for
appreciably. Use of a more soluble form of the test material,
calculation of results. If a t-test indicates that the solvent
such as chloride or sulfate salts of organic amines, sodium or
control and the control are different and if the concentration of
potassium salts of phenols and organic acids, and chloride or
solvent at each test material concentration is not equal, the use
nitrate salts of metals, might affect the pH less than the use of
of hypothesis testing statistics for comparison of treatments to
the minimum necessary amount of a strong acid or base.
each other or to the solvent control is not appropriate. If no
9.2.3 If a solvent other than growth medium is used, its
statistically significant difference is detected, the data from
concentration in test solutions should be kept to a minimum
both controls should be used for meeting the requirements of
and should be low enough that it does not affect growth of the
13.1.7 and as the basis for calculation of results.(12 )
test species. Because of its low volatility and high ability to
9.2.5 If an organic solvent is used to prepare a stock
dissolve many organic chemicals, N,N-dimethylformamide is
solution, it might be desirable to conduct simultaneous tests
often a good organic solvent for preparing stock solutions.
using two chemically unrelated solvents or two different
Other water-miscible organic solvents such as ethanol and
concentrations of the same solvent to obtain information
acetonemayalsobeused,buttheymightstimulateundesirable
concerning possible effects of the solvent on the results of the
growthofmicroorganisms,andacetoneisalsoquitevolatile.If
test.
anorganicsolventisused,itshouldbereagent-grade orbetter
9.3 Test Concentration(s):
and its concentration in any test solution should not exceed
0.5mL⁄L,andpreferably0.1mL⁄L.Asurfactantshouldnotbe
9.3.1 Depending on the nature of the test material, test
used in the preparation of a stock solution because it might
solutions are prepared by one of two methods. In the first
affect the form and toxicity of the test material in the test
method, technical materials (solids or liquids) are tested by
solutions. (These limitations do not apply to any ingredients of
weight/volume concentration. The material may be added
a mixture, formulation, or commercial product unless an extra
directly by weight to the growth medium or a stock solution
amount of solvent is used in the preparation of the stock
may be prepared (see 9.2) and aliquots added to each test
solution.)
solution or test vessel. If it is not possible to prepare a
9.2.4 If a solvent other than growth medium is used, at least homogeneous solution of the test material, it must be added
one solvent control, using solvent from the same batch used to directly to each test solution or test vessel. In the second
make the stock solution, must be included in the test, and a method, aqueous effluents are tested by percent volume
growth medium control should be included in the test. If a (volume/volume).Nutrientsareaddedinthesamequantitiesas
solvent control is not required, a growth medium control must in the medium (see Annex A1 – Annex A3)to1Lofthe
be included in the test. effluent. This mixture, without sterilization by filtration or any
E1218 − 21
other alteration, is used as the 100% test material concentra- laboratory, and have been successfully used; however, these
tion.The other test solutions are prepared on a volume-percent species might not be the most sensitive. Their use is encour-
basis by diluting the 100% effluent solution with growth aged to increase comparability of results.
medium. If the effluent itself contained nutrient(s), the treat- 10.1.1 Fresh Water—The species most widely used for
ments that contain test material will contain more of the testing is the green alga Raphidocelis subcapitata formerly
nutrient(s) than will the control treatment. However, in most known asPseudokirchneriella subcapitataSelenastrum capri-
cases this is not critical for short-term tests. USEPA 2002, (3) cornutumPrintz andthecultureandtestproceduresdescribed
describes a method for testing the effects of effluents on algae. herearemostapplicabletoit (13).Otherspeciesthathavebeen
successfully used include Desmodesmus subspicatus (formerly
9.3.2 If the test is intended to allow calculation of the IC50,
Scenedesmus subspicatus) Chodat and Chlorella vulgaris Bei-
the test concentrations (see 11.1.1.1) should bracket the pre-
jerinck.Freshwateralgaefromotherphylacanbeused,suchas
dicted IC50. The prediction might be based on the results of a
thediatom, Navicula pelliculosaBrebissonHilse.Otherorgan-
test on the same or a similar test material with the same or a
isms that have been used in this test that were formerly
similaralgalspecies.Ifausefulpredictionisnotavailable,itis
classified as blue-green algae are now considered
usually desirable to conduct a range-finding test in which the
cyanobacteria, including Microcystis aeruginosa Kutzing and
test species is exposed to a control and three to five concen-
Anabaena flos-aquae (Lyngb.) De Brebisson.
trations of the test material that differ by a factor of ten. The
10.1.2 Salt Water—Thespeciesmostwidelyusedfortesting
greater the similarity between the range-finding test and the
is the diatom, Skeletonema costatum, (Greville) Clevel., (14)
actual test, the more useful the range-finding test will be.
also known as the newly classified species Skeletonema mari-
9.3.2.1 Testing materials at levels above their water solubil-
noi(15). Other species that have been successfully used are the
ity presents several difficulties.At test material loadings above
centricdiatom, Thalassiosira pseudonana,HasleandHeimdal,
the solubility (note a true concentration cannot exist above
the flagellate, Dunaliella tertiolecta, Butcher (16) and the
solubility and a term such as “loading” is used), test materials
goldenbrown alga, Phaeodactylum tricornutum.
existinavarietyofaggregateforms(forexample,particulates,
10.1.3 Because the sensitivities of algal species often differ
crystals, liquid crystals, etc.) Relatively little is known about
substantially,itisusuallydesirabletoconducttestswithtwoor
the uptake of aggregated compounds into biological mem-
morespeciesfromdifferentphyla.Differentstrainsofthesame
branes and the expression of toxicity as a result. In fact,
species may differ also in sensitivity.
toxicity may be due to certain physical effects (reduction in
light penetration and interference with nutrient uptake by test
10.2 Source—Many species can be obtained from the
material particulates, or flocculation of algae onto test material
American Type Culture Collection, the University of Texas
particles). For materials tested at loadings in excess of
Collection, the Bigelow Laboratory for Ocean Sciencesand
solubility,theuseofdatainriskassessmentsorforcomparison
Carolina Biological Supply.
with other test materials, is complicated by the lack of
10.3 Culture—Key references for culturing algae are those
knowledgeastowhethertheeffectisduetoaphysicaleffector
of Stein and Provasoli (17). Aseptic stock transfer should be
true toxicity. These difficulties suggest that toxicity testing at
performed weekly to maintain a continuous supply of cells in
loadingsabovesolubilityshouldbediscouraged.Toensurethat
or near logarithmic growth phase. The volume transferred is
solubility has been achieved in the toxicity test, it may be
not critical, but enough cells should be transferred to ensure a
appropriate to test up to approximately twice the solubility
minimum visual lag period in growth (for example, 1.0mL
limit in the medium being used. However, in this case toxicity
culture added to 50mL medium in a 125mL flask). Extreme
should be expressed as the solubility limit (that is, no effect at
care should be exercised to avoid contamination of stock
the solubility limit, or 20% reduction in cell numbers at the
cultures. If tests are conducted infrequently, long-term main-
solubility limit). Analytical verification of the solubility under
tenance of the test species using a solid medium containing
the test conditions can be critical for test materials of limited
1% agar in sterile Petri plates or test tubes might be desirable.
aqueous solubility.
10.4 Quality—A culture should not be used for starting a
9.3.3 In some (usually regulatory) situations, it is only
test if it is not in log growth phase, if visual examination at
necessary to determine whether a specific concentration of test
400×showsitiscontaminatedbyfungiorotheralgae,orifthe
material affects growth of the test species or whether the IC50
health of the culture is doubtful in any respect. In order to
is above or below a specific concentration. For example, the
assessculturehealth,sufficientexperiencewiththetestspecies
specific concentration might be the concentration occurring in
should be developed prior to use in testing. When a testing
a surface water, the concentration resulting from the direct
facility receives a culture of a species that has not been
application of the material to a body of water, or the solubility
limit of the material in water. When there is only interest in a
specific concentration, it is often only necessary to test that
Renamed by Gunnar Nygaard, Jirf Komárek, Jørgen Kristiansen and Olav M.
concentration (see 11.1.1.2), and it is not necessary to deter- Skulberg, 1986. Taxonomic designations of the bioassay alga NIVA-CHL1 (“Sel-
enastrum capricornutum”) and some related strains. Opera Botanica 90:5-46.
mine the IC50.
Algal species are available from American Type Culture Collection 12301
ParklawnDr.,Rockville,MD20852,fromtheUniversityofTexasAlgalCollection,
10. Test Organisms
Botany Department, Austin, TX 78712, from the Bigelow Laboratory for Ocean
Sciences , National Center for Marine Algae and Microbiota, 60 Bigelow Drive,
10.1 Species—The suggested test species were selected
East Boothbay, Maine 04544, and from Carolina Biological Supply Company, 2700
because they are readily available, easy to culture in the York Road, Burlington, NC, 27215.
E1218 − 21
previously maintained in that facility, the species should be in all test vessels should be as similar as possible unless the
cultured over a period of at least six weeks to establish the purpose of the test is to study the effect of temperature. Test
ability to successfully maintain a healthy, reproducibly- vessels are usually arranged in one or more rows. Treatments
growing culture. Once experience has been obtained with a must be randomly assigned to individual test vessel locations
particular species from a particular source, subsequent cultures and may be randomly reassigned during testing.Arandomized
should be maintained for at least two weeks after receipt prior block design (with each treatment being present in each block,
to use in testing. which may be a row or a rectangle) is preferable to a
completely randomized design.
11.1.3 The minimum desirable number of test vessels per
11. Procedure
treatment should be calculated from the expected variance
11.1 Experimental Design:
between test vessels within a treatment, and either the maxi-
11.1.1 For detailed guidance of experimental design and
mum acceptable confidence interval on a point estimate or the
statisticalanalyses,refertoPracticeE1847.Decisionsconcern-
minimum difference that is desired to be detectable using
ing such aspects of experimental design as the dilution factor,
hypothesis testing (18). If such calculations are not made, at
numberoftreatments,andnumberoftestvesselspertreatment,
least three test vessels must be used for each treatment (test
should be based on the purpose of the test and the type of
concentration and control). If each test concentration is more
procedure that is to be used to calculate results (see Section
than 60% of the next higher one and the results are to be
14). One of the following two types of experimental design
analyzed using regression analysis, proportionately fewer test
will probably be appropriate in most cases.
vessels may be used for each treatment that contains test
11.1.1.1 An algal test intended to allow calculation of an
material, but not for the control treatment(s). Replicate test
IC50 usually consists of one or more control treatments and a
vessels (that is, experimental units) within a treatment are
geometric series of at least five concentrations of test material.
necessary in order to allow estimation of experimental error
In the growth medium or solvent control(s), or both, (see
(19). Because of the importance of the controls, it might be
9.2.3), algae are exposed to growth medium to which no test
desirable to use more test vessels for the control treatment(s)
material has been added. Except for the control(s) and the
than for each of the other treatments, such that if a control
highest concentration, each concentration should be at least
replicate is lost, sufficient replicates remain for statistical
60%ofthenexthigherone,unlessinformationconcerningthe
analyses.
concentration-effect curve indicates that a different dilution
11.2 Temperature—Tests with the recommended freshwater
factor is more appropriate. At a dilution factor of 0.6, five
microalgaeshouldbeconductedat24 62°Candtestswiththe
properly chosen concentrations are a reasonable compromise
recommended saltwater microalgae should be conducted at 20
between cost and the risk of all concentrations being either too
6 2°C.
high or too low. If the estimate of toxicity is particularly
uncertain, six or seven concentrations might be desirable. 11.3 Illumination—Continuous “cool-white” fluorescent
–2 –1
lighting should be used to provide 60 µmol m s (4300
11.1.1.2 If it is only necessary to determine whether a
lm/m ) for the recommended freshwater diatoms and green
specific concentration unacceptably affects growth or whether
–2 –1 2
algae and 30 µmol m s (2150 lm/m ) for the recommended
theIC50isaboveorbelowaspecificconcentration(see9.3.3),
–2 –1
freshwatercyanobacteria.ForThalassiosira,80µmolm s to
only that concentration and the control(s) are necessary. Two
–2 –1 2 2
90 µmol m s (5900 lm/m to 6500 lm/m ) is recommended
additional concentrations at about one half and two times the
–2 –1 2
and 60 µmol m s (4300 lm/m ) for Skeletonema (14). The
specified concentration are desirable to increase confidence in
light fluence rate at each test vessel position should be
the results.
measured and should not differ by more than 15% from the
11.1.1.3 If an IC near the extremes of toxicity, such as an
selected fluence rate. For further information on the use of
IC5 or IC95, is to be calculated, at least one concentration of
lighting in laboratory testing, especially with regard to using
test material should have reduced growth by a percentage,
other spectral regions (such as ultraviolet radiation), see Guide
other than 0 or 100%, near the percentage for which the IC is
E1733. A photoperiod should be used with Skeletonema (that
to be calculated. This requirement might be met in a test
is, 14 h light: 10 h dark, or 16 h light: 8 h dark).
designed to determine an IC50, but a special test with appro-
priate concentrations of test material will usually be necessary. 11.4 Beginning the Test:
11.1.2 The primary focus of the physical and experimental 11.4.1 A large enough batch of the growth medium should
design of the test and the statistical analysis of the data is the be prepared so that the desired volume can be placed in each
experimentalunit,thatisdefinedasthesmallestphysicalentity control test vessel, the necessary volume of each test solution
to which treatments can be independently assigned. Thus, the can be prepared, and the desired analyses can be performed
test vessel is the experimental unit. As the number of test (see 11.8). Enough test solution should be prepared for each
vessels(thatis,experimentalunits)pertreatmentincreases,the treatment so that the desired volume can be placed in each test
number of degrees of freedom increases, and therefore, the vessel and any desired analyses of water quality, test material,
width of the confidence interval on a point estimate decreases etc. (see 11.8) can be performed.
and the power of a significance test increases. With respect to 11.4.2 Each test vessel should be inoculated at an initial
factors that might affect results within test vessels and, population density to allow sufficient growth under the test
therefore,theresultsofthetest,allvesselsinthetestshouldbe conditions without resulting in nutrient or carbon dioxide
treated as similarly as possible. For example, the temperature limitation. Recommended concentrations are as follows:
E1218 − 21
cells/mL. For microscopic counting, two samples should be
Raphidocelis subcapitata and other 1-2×10 cells ⁄mL
freshwater green algae
taken from each flask and two counts made of each sample.
Navicula pelliculosa 1-2×10 cells ⁄mL
4 Whenever feasible, at least 400 cells per flask should be
Microcystis aeruginosa 5×10 cells ⁄mL
Anabaena flos-aquae 1-2×10
cells ⁄mL counted in order to obtain 6 10 % accuracy at the 95 %
Saltwater Species 1-2 × 10 cells ⁄mL
confidence level. Counting is relatively time-consuming, but
The primary criterion for an inoculum concentration is that
less so than determining dry weight.
accurate biomass estimates can be obtained with the chosen
11.7.2.1 An alternative method that is able to enumerate
methodofmeasurementduringthetest,ifnecessary,andatthe
large numbers of cells very rapidly is to use an electronic
endofthetest.Lowerconcentrationsmaybepracticalforsome
particle counter (13). If a particle counter is used, representa-
laboratories. Higher concentrations might be desirable if there
tive samples of cells should be also microscopically examined
are indications that logarithmic growth will not be attained
formorphologicalabnormalitiesattesttermination.Automated
within 4 days or if results are to be based on measurements
particle counting, although the most rapid and sensitive
madeduringthetest.However,theupperlimitoftheinoculum
method, has limitations (21), some related to particle interfer-
should be no more than 10 cells/mL. After determining the
ences. If the test solution does not have a low background in
concentration of algal cells in a logarithmically-growing stock
the particle size range of the test species, masking errors will
culture, the following equation can be used to calculate the
result.Anextratestvesselateachconcentrationcontainingtest
total volume of culture required for inoculation of all test
material and growth medium, but no algae, will allow mea-
vessels:
surement of particle interferences. The other methods are not
A 5 B 3C 3D 3E /F (1)
~ !
affected by particulate material in the growth medium.
where:
11.7.2.2 Cell chains of filamentous species, such as Ana-
baena flos-aquae, must be broken up prior to counting,
A = volume of stock culture required to prepare the
inoculum, mL, regardless of the counting method selected. An effective
B = desired initial concentration of cells in test vessel,
method for reducing the length of the filaments without
cells/mL,
rupturing the cells is sonication. Subsamples from the test
C = volume of solution in one test chamber, mL,
vessels should be sonicated for a sufficient duration to reduce
D = number of test vessels,
the filame
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