ASTM E1562-22

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: E1562 − 22
Standard Guide for
Conducting Acute, Chronic, and Life-Cycle Aquatic Toxicity
Tests with Polychaetous Annelids
This standard is issued under the fixed designation E1562; 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 toxicant on life-cycle tests; data are analyzed statistically to
indicate that concentration at which a significant difference
1.1 This guide covers procedures for obtaining data con-
occurs between the test solutions and control(s).
cerning the adverse effects of a test material added to marine
and estuarine waters on certain species of polychaetes during
1.4 The results of dose-response acute or chronic toxicity
short- or long-term continuous exposure. The polychaete tests with toxicants added experimentally to salt water should
species used in these tests are either field collected or from
usually be reported in terms of an LC50 (mortality), or EC50
laboratory cultures and exposed to varying concentrations of a (medial effect concentration). The results of life-cycle toxicity
toxicant in static or static-renewal conditions. These proce-
tests with toxicants added experimentally to salt water should
dures may be useful for conducting toxicity tests with other be reported as that concentration at which a statistically
species of polychaetes, although modifications might be nec- significant difference in the number of offspring or growth
essary. (determined by weight) is produced with reference to the
control(s).
1.2 Modifications of these procedures might be justified by
special needs or circumstances. Although using appropriate 1.5 Where appropriate, this standard has been designed to
procedures is more important than following prescribed
be consistent with or complementary to other methods for
procedures, the results of tests conducted using unusual pro- assessing toxicity to invertebrates described in Test Methods
cedures are not likely to be comparable to those of many other
E1367 and E1706, and Guides E1391, E1525, E1611, and
tests. Comparisons of results obtained using modified and E1688.
unmodified versions of these procedures might provide useful
1.6 This guide is arranged as follows:
information concerning new concepts and procedures for
Section
conducting acute, chronic, or life-cycle tests with other species
Referenced Documents 2
of polychaetes.
Terminology 3
Summary of Guide 4
1.3 These procedures are applicable to most chemicals,
Significance and Use 5
either individually or in formulations, commercial products, Apparatus 6
Facilities 6.1
and known or unknown mixtures. With appropriate
Construction Materials 6.2
modifications, these procedures can be used to conduct these
Test Chambers 6.3
Cleaning 6.4
tests on factors such as temperature, salinity, and dissolved
Acceptability 6.5
oxygen. These procedures can also be used to assess the
Safety Precautions 7
toxicity of potentially toxic discharges such as municipal
Dilution Water 8
wastes,sediments/soils,oildrillingfluids,producedwaterfrom Requirements 8.1
Source 8.2
oil well production, and other types of industrial wastes. An
Treatment 8.3
LC50 (medial lethal concentration) may be calculated from the
Characterization 8.4
Test Material 9
data generated in each acute and chronic toxicity test when
General 9.1
multiple concentrations are tested. Growth, determined by a
Stock Solution 9.2
change in measured weight, and reproduction, as the change in
Test Concentrations 9.3
Test Organisms 10
total number of organisms, are used to measure the effect of a
Species 10.1
Age 10.2
Source 10.3
Feeding 10.4
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Holding 10.5
Assessment, Risk Management and CorrectiveActionand is the direct responsibility
Quality 10.6
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Procedure 11
Current edition approved Jan. 1, 2022. Published February 2022. Originally
Experimental Design 11.1
approved in 1994. Last previous edition approved in 2013 as E1562– 00(2013).
Acute Test 11.1.1
DOI: 10.1520/E1562-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1562 − 22
E1391 Guide for Collection, Storage, Characterization, and
Chronic Test 11.1.2
Life-Cycle Test 11.1.3
Manipulation of Sediments for Toxicological Testing and
Test Condition Specifications 11.2
for Selection of Samplers Used to Collect Benthic Inver-
Dissolved Oxygen 11.2.1
tebrates
Temperature 11.2.2
Loading 11.2.3
E1525 Guide for Designing BiologicalTests with Sediments
Salinity 11.2.4
E1611 Guide for Conducting Sediment Toxicity Tests with
Light 11.2.5
Polychaetous Annelids
Beginning the Test 11.3
Feeding 11.4
E1688 Guide for Determination of the Bioaccumulation of
Duration of Test 11.5
Sediment-Associated Contaminants by Benthic Inverte-
Biological Data 11.6
Other Measurements 11.7 brates
Hazards
E1706 Test Method for Measuring theToxicity of Sediment-
Analytical Methodology 13
Associated Contaminants with Freshwater Invertebrates
Acceptability of Test 14
Calculation of Results 15 E1733 Guide for Use of Lighting in Laboratory Testing
Report 16
E1847 Practice for Statistical Analysis of Toxicity Tests
Keywords 17
Conducted Under ASTM Guidelines (Withdrawn 2022)
Appendixes:
IEEE/ASTM SI 10 American National Standard for Use of
Neanthes arenaceodentata Appendix X1
Capitella capitata Appendix X2
theInternationalSystemofUnits(SI):TheModernMetric
Ophryotrocha diadema Appendix X3
System
Dinophilus gyrociliatus Appendix X4
1.7 The values stated in SI units are to be regarded as
3. Terminology
standard. No other units of measurement are included in this
3.1 Definitions:
standard.
3.1.1 The words “must,” “should,” “may,” “can,” and
1.8 This standard does not purport to address all of the
“might” have very specific meanings in this guide. “Must” is
safety concerns, if any, associated with its use. It is the
used to express the strongest possible recommendation, just
responsibility of the user of this standard to establish appro-
short of an absolute requirement, that is, to state that this test
priate safety, health, and environmental practices and deter-
oughttobedesignedtosatisfythespecificcondition,unlessthe
mine the applicability of regulatory limitations prior to use.
purpose of the test requires a different design. “Must” is used
Specific precautionary statements are given in Section 7.
only in connection with factors that relate directly to the
1.9 This international standard was developed in accor-
acceptability of the test (see Section 14). “Should” is used to
dance with internationally recognized principles on standard-
state that the specific condition is recommended and ought to
ization established in the Decision on Principles for the
bemet,ifpossible.Althoughviolationofone“should”israrely
Development of International Standards, Guides and Recom-
a serious matter, violation of several will often render the
mendations issued by the World Trade Organization Technical
results questionable. Terms such as “is desirable,” “is often
Barriers to Trade (TBT) Committee.
desirable,” and “might be desirable” are used in connection
with less important factors. “May” is used to mean “is (are)
2. Referenced Documents
allowed to,” “can” is used to mean “is (are) able to,” and
2.1 ASTM Standards:
“might” is used to mean “could possibly.” Thus the classic
D4447 Guide for Disposal of Laboratory Chemicals and
distinctionbetween“may”and“can”ispreserved,and“might”
Samples
is never used as a synonym for either “may” or “can.”
E177 Practice for Use of the Terms Precision and Bias in
3.1.2 For definitions of other terms used in this guide, refer
ASTM Test Methods
to Guide E729, Terminology E943, and Guide E1023. For an
E729 Guide for Conducting Acute Toxicity Tests on Test
explanation of units and symbols, refer to IEEE/ASTM SI 10.
Materials with Fishes, Macroinvertebrates, and Amphib-
ians
4. Summary of Guide
E943 Terminology Relating to Biological Effects and Envi-
4.1 Thetoxicityofasubstanceinmarineorestuarinewaters
ronmental Fate
can be determined through a 96 h acute or chronic toxicity test
E1023 Guide for Assessing the Hazard of a Material to
(see 11.1.2) in 100-mm diameter glass petri dishes for Nean-
Aquatic Organisms and Their Uses
thes arenaceodentata and Capitella capitata. Stender dishes
E1192 Guide for ConductingAcute Toxicity Tests onAque-
are recommended for acute, chronic, and life-cycle tests with
ous Ambient Samples and Effluents with Fishes,
the smaller species Ophryotrocha diadema and Dinophilus
Macroinvertebrates, and Amphibians
gyrociliatus. Petri dishes provide ample horizontal space to
E1367 Test Method for Measuring theToxicity of Sediment-
minimize cannibalism in the case of Neanthes arenaceoden-
Associated Contaminants with Estuarine and Marine In-
tata. Larger containers, such as ⁄2 gallon – 1 gallon jars, are
vertebrates
necessary for life-cycle tests using Neanthes arenaceodentata
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
Standards volume information, refer to the standard’s Document Summary page on The last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
E1562 − 22
and Capitella capitata. The static-renewal technique is recom- 6. Apparatus
mended for the chronic and life-cycle tests with these latter
6.1 Facilities—Aquaria (2 Lto 57 L) or gallon jars contain-
species; it is usually not necessary to renew the solutions in
ing either clean (uncontaminated), natural, or reconstituted sea
96-h tests. With either the static or static-renewal system, data
water should be used for culturing these four species of
on the concentration of test material are obtained and analyzed
polychaetes or holding them after field collection and prior to
to determine the effect(s) of the toxicant on survival and
a test.Aquaria provided with aeration have been used success-
reproduction.
fully for culturing and holding polychaetes. The holding tanks
and any area used for culturing or holding polychaetes should
5. Significance and Use
be located in a room or space separated from that in which
5.1 Polychaetes are an important component of the benthic
toxicity tests are to be conducted. The sea water should be
community,inwhichtheygenerallycomprise30to50 %ofthe
analyzed periodically to ensure a constant salinity.The holding
macroinvertebrate population. They are preyed upon by many
aquaria,watersupply,orroominwhichtheyarekeptshouldbe
species of fish, birds, and larger invertebrate species. Larger
equippedwithtemperaturecontrol.Aerationcanbeprovidedto
polychaetes feed on small invertebrates, larval stages of
ensure that dissolved oxygen is greater than 60 % saturation
invertebrates, and algae. Polychaetes are especially sensitive to
and that water circulation is adequate in the aquaria. A
inorganic toxicants and, to a lesser extent, to organic toxicants
consistent light cycle including 12 h -16 h of light should be
(1). The ecological importance of polychaetes and their wide
provided.
geographical distribution, ability to be cultured in the
6.2 Construction Materials—Equipment and facilities that
laboratory, and sensitivity to contaminants make them appro-
contact stock solutions, test solutions, or any water into which
priate acute and chronic toxicity test organisms. Their rela-
test organisms will be placed should not contain substances
tively short life cycle enables the investigator to measure the
that can be leached or dissolved by aqueous solutions in
effect of contaminants on reproduction.
amounts that affect the test organisms adversely. In addition,
5.2 An acute toxicity or chronic text is conducted to obtain
equipment and facilities that contact stock or test solutions
informationconcerningtheimmediateeffectsofanexposureto
should be chosen to minimize the sorption of test materials
a test material on a test organism under specified experimental
fromwater.Glass,Type316stainlesssteel,nylon,high-density
conditions. An acute toxicity test provides data on the short-
polyethylene, polycarbonate plastics should be used whenever
term effects, which are useful for comparisons to other species
possible to minimize dissolutions, leaching, and sorption,
but do not provide information on delayed effects. Chronic
except that stainless steel should not be used in tests on metals
toxicity tests provide data on long-term effects.
in salt water. Fluorocarbon plastics (that is, per- and polyfluo-
roalkyl substances or PFAS) should be avoided in studies
5.3 A life-cycle toxicity test is conducted to determine the
evaluatingthesetypesofchemicals.Concreteandrigidplastics
effects of the test material on survival, growth, and reproduc-
may be used for holding tanks and in the water-supply system,
tion of the test species. Additional sublethal endpoints (for
buttheyshouldbesoaked,preferablyinflowingdilutionwater,
example, biochemical, physiological, and histopathological)
for one week or more before use (3). Cast iron pipe should not
may be used to determine the health of the species under field
be used with salt water and probably should not be used in a
conditions.
fresh water-supply filter system because colloidal iron will be
5.4 Theresultsofacute,chronic,andlife-cycletoxicitytests
added to the dilution water and strainers will be necessary to
can be used to predict effects likely to occur on marine
remove rust particles. A specially designed system is usually
organisms under field conditions.
necessary to obtain salt water from a natural water source (4).
5.5 The results of acute, chronic, or life-cycle toxicity tests
Brass, copper, lead, galvanized metal, and natural rubber
might be used to compare the sensitivities of different species
should not contact the dilution water, stock solutions, or test
and the toxicities of different test materials, as well as to study
solutions before or during the test. Items made of neoprene
the effects of various environmental factors on the results of
rubber or other materials not mentioned previously should not
such tests.
be used unless it has been shown that their use will not affect
either the survival, growth, or reproduction of polychaetes
5.6 The results of acute, chronic, or life-cycle toxicity tests
adversely.
might be an important consideration when assessing the
hazardsofmaterialstomarineorganisms(seeGuideE1023)or 6.3 Test Chambers—In a toxicity test with marine
when deriving water quality criteria for aquatic organisms (2). organisms, test chambers are defined as the smallest physical
units between which there are no water connections. Glass
5.7 The results of acute, chronic, or life-cycle toxicity tests
petri dishes measuring 100 mm in diameter and 20 mm in
might be useful for studying the biological availability of, and
height are the most satisfactory chambers for use in acute and
structure activity relationships between, test materials.
chronic toxicity tests with Neanthes arenaceodentata and
5.8 Theresultsofacute,chronic,andlife-cycletoxicitytests
Capitella capitata.Onlyone Neanthes arenaceodentatashould
will depend partly on the temperature, quality of food, condi-
be placed in a single chamber since this species can be
tion of test organisms, test procedures, and other factors.
cannibalistic.Stenderdishesmeasuring40mmindiameterand
provided with a ground glass cover are the most satisfactory
chambers for acute, chronic, and life-cycle tests with Oph-
The boldface numbers in parentheses refer to the list of references at the end of
this guide. ryotrocha diadema and Dinophilus gyrociliatus. Capitella
E1562 − 22
capitata, Ophryotrocha diadema, and Dinophilus gyrociliatus 7.2 The use of ground fault systems and leak detectors is
are not cannibalistic, and 5 to 10 specimens may be placed recommended strongly to help prevent electrical shocks be-
within a single chamber. For reproductive tests, chambers cause salt water is a good conductor of electricity.
measuring 3.79 L and containing 2.5 L of test solution should
7.3 Although the disposal of stock solutions, test solutions,
be used with N. arenaceodentata and C. capitata. Aeration
and test organisms poses no special problems in most cases,
must be provided. Chambers the same size as that used in the
healthandsafetyprecautionsandapplicableregulationsshould
acute and chronic tests with O. diadema and D. gyrociliatus
be considered before beginning a test. The removal or degra-
can be used for the reproductive tests.The chambers should be
dationoftestmaterialmightbedesirablebeforethedisposalof
covered to keep out extraneous contaminants and to reduce the
stock and test solutions.
evaporation of test solution and test material. All chambers in
7.4 Cleaning of equipment with a volatile solvent such as
a test must be identical.
acetone should be performed only in a well-ventilated area in
6.4 Cleaning—Test chambers and other glassware, and
whichnosmokingisallowedandnoopenflame,suchasapilot
equipment used to store and prepare the test sea water, stock
light, is present.
solutions, and test sediments, should be cleaned before use.
7.5 Anacidicsolutionshouldnotbemixedwithahypochlo-
New items should be cleaned before each use by washing with
rite solution because hazardous fumes might be produced.
laboratory detergent and rinsing with water, a weak-miscible
7.6 To prepare dilute acid solutions, concentrated acid
organic solvent, and acid (10 % nitric or hydrochloric acid),
should be added to water, not vice versa. Opening a bottle of
and they should be rinsed twice with distilled, deionized, or
concentratedacidandaddingconcentratedacidtowatershould
dilution water. Metals, sulfides, and carbonate deposits are
be performed only in a fume hood.
removed by the acid rinse, and organics are removed by the
organic solvent rinse. A dichromate-sulfuric acid cleaning
8. Dilution Water
solution may be used in place of both the organic solvent and
acid rinses, but it might attack silicone adhesives.At the end of
8.1 Requirements—The dilution water, if needed, should (1)
eachtest,allitemsthataretobeusedagainshouldimmediately be available in adequate supply, (2) be acceptable to the test
be (1) emptied; (2) rinsed with water; (3) cleaned by a
organisms, (3) be uniform in quality, and (4) not affect the test
procedure appropriate for removing the test material (for results unnecessarily.
example, acid to remove metals and solvents to remove
8.1.1 The minimum requirement for acceptable water for
organics);and(4)rinsedatleasttwicewithdeionized,distilled, use in acute toxicity tests is that healthy test organisms survive
or dilution water. Acid is often used to remove mineral
in it for the duration of holding and testing without showing
deposits. The test chambers should be rinsed with dilution signs of stress such as unusual behavior, changes in
waterjustbeforeuse.Glasswareusedonlyforliveanimals,not
appearance,ordeath.Thewaterinwhichthetestorganismsare
exposed to toxicants, should be cleaned using only distilled or held prior to the test should be uniform. The range of
clean dilution water, since the use of detergents is sometimes
temperature and salinity encountered during the holding period
detrimental to live organisms. must not affect the survival of the test organisms adversely. A
better criterion for an acceptable dilution water is that the test
6.5 Acceptability—The acceptability of new holding or
species will survive, grow, and reproduce satisfactorily in it.
testing facilities should be demonstrated by conducting a
8.1.1.1 Salt Water—Water in which polychaetes will
“non-toxicant” test in which all test chambers contain dilution
survive,grow,andreproducesatisfactorilyinalife-cycletestis
water.Survivalofthetestspecieswilldemonstratewhetherthe
probably an acceptable dilution water for these tests.
facilities, water, control, and handling techniques are adequate
8.1.2 The quality of the dilution water should be uniform so
to result in acceptable (90 %) survival of control animals in the
that the test organisms are cultured or acclimated, and the test
absence of toxicants.
should be conducted in water of the same quality. In salt water,
the range of salinity should be less than 2 g/kg or 20 % of the
7. Safety Precautions
average salinity, whichever is higher.
7.1 Many materials can affect humans adversely if precau- 8.1.3 The dilution water should not affect the results of an
tions are inadequate. Therefore, skin contact with all test acute test unnecessarily because of such things as sorption or
materials and their solutions should be minimized by such complexation of the test material. Therefore, except as in
means as wearing appropriate protective gloves (especially accordance with 8.1.4, the concentration of both total organic
when washing equipment or putting hands into test solutions), carbon (TOC) and particulate matter should be below 5 mg/L.
laboratory coats, aprons, and glasses. Special precautions, such 8.1.4 If it is desired to study the effect of an environmental
as covering the test chambers and ventilating the area sur- factor such as TOC, particulate matter, or dissolved oxygen on
rounding the chambers, should be taken when conducting tests theresultsofanacutetest,itwillbenecessarytousewaterthat
on volatile materials. Information on toxicity to humans (5), is naturally or artificially high in TOC or particulate matter or
recommended handling procedures (6), and the chemical and low in dissolved oxygen. If such water is used, it is important
physical properties of test material should be studied before a that adequate analyses be performed to characterize the water
test is begun. Special precautions might be necessary with and that a comparable test be conducted in a more usual
radiolabeled test materials (7) and with test materials that are, dilution water in order to facilitate the interpretation of results
or are suspected of being, carcinogenic. in the special water.
E1562 − 22
8.2 Source: example, approximately 1 m below the surface) and the
8.2.1 Reconstituted Water—Some reconstituted salt waters possibility of contamination, as well as to maximize the
prepared from either reagent-grade chemicals or sea salts have concentration of dissolved oxygen to help ensure low concen-
been shown to be acceptable for life-cycle toxicity tests with trations of sulfide and iron.
polychaetes (8). It might be desirable to condition (age) 8.2.2.2 If desired, the hardness, salinity, pH, etc. of a water
reconstituted salt water by aerating it for two or more days. may be adjusted by the addition of appropriate reagent-grade
8.2.1.1 If reconstituted water is used for tests with saltwater chemicals, sea salt, acid, base, distilled or deionized water, etc.
species, the reconstituted water described in Table 1 should be When necessary, sea salt may be added to salt water to prevent
used whenever possible. If desired, reconstituted water may be excessive decreases in salinity (see 8.2.1.1) if the salt has been
prepared using a commercial sea salt preparation. Commercial shown to cause no adverse effects on the test species at the
sea salt mixes should be “bioassay grade” (such as Crystal concentration used.
Sea® Marinemix Bioassay Laboratory Formula) without the 8.2.3 Chlorinated water must never be used for dilution
addition of the metal chelating agent ethylenediaminetet- water in toxicity tests because residual chlorine and chlorine-
raacetic acid (EDTA) or dechlorinators such as sodium thiuo- produced oxidants are highly toxic to many aquatic animals
sulfate.Thesecompoundsarepresentinhobbyistsaltsandmay (9). Dechlorinated water should be used only as a last resort
mask the toxicity of environmental or spiked sediment
because dechlorination is often incomplete. Sodium bisulfite is
samples.Thereconstitutedwatershouldbeusedatasalinityof probably better for dechlorinating water than sodium sulfite,
34 g/kg and pH = 8.0 for tests with true marine stenohaline
and both are more reliable than carbon filters, especially for
species, and at a salinity of 17 g/kg and pH = 7.7 with removing chloramines (10). Some organic chloramines,
euryhaline species. Other salinities may be used for studying
however, react slowly with sodium bisulfite (11). In addition to
the effects of water quality on the results of toxicity tests. residual chlorine, municipal drinking water often contains
8.2.1.2 Reconstituted seawater is prepared by adding speci-
unacceptably high concentrations of metals, and its quality is
fied amounts of reagent-grade salts to high-quality water with highly variable. Excessive concentrations of most metals can
(1) conductivity approximately <1 µS⁄cm and (2) either TOC
usually be removed with chelating resin (12), but the use of a
below 5 mg/L or chemical oxygen demand (COD) less than different dilution water might be preferable. If dechlorinated
5mg/L. Acceptable water can usually be prepared using prop-
water is used as dilution water or in its preparation, during the
erly operated deionization, distillation, or reverse osmosis test it must be shown that either (1) a polychaete species will
units. Conductivity should be measured on each batch, and
survive, grow, and reproduce acceptably in it or (2) fresh
TOC or COD should be measured at least twice per year and samples of dilution water supplied at least three times each
wheneversignificantchangesmightbeexpected.Ifthewateris
week on nonconsecutive days do not cause either of the
prepared from a surface water, TOC or COD should be following:(1)thetestspeciesofpolychaetetoshowmoresigns
measured on each batch. The reconstituted water should be of stress, such as discoloration, unusual behavior, or death,
aerated intensively before use. Problems have been encoun- when held in a water that was not chlorinated and dechlori-
tered with some species in some fresh and salt reconstituted nated;or(2)theconcentrationofchlorine-producedoxidantsto
waters, but sometimes these problems have been overcome by
be below 7.5 µg/L (9).
aging the reconstituted water for one or more weeks.
8.3 Treatment:
8.2.2 Natural Dilution Water:
8.3.1 Dilution water should be well aerated by using air
8.2.2.1 If a natural dilution water is used, it should be
stones, surface aerators, or column aerators before the addition
obtained from an uncontaminated, uniform quality source. If
of test material.Adequate aeration will bring the concentration
surface water is used as a source of dilution water, the intake
of dissolved oxygen and other gases into equilibrium with air,
should be positioned to minimize fluctuations in quality (for
minimize oxygen demand and concentrations of volatiles, and
stabilize pH. The concentration of dissolved oxygen in the
dilution water should be between 90 and 100 % (13) to help
TABLE 1 Reconstituted Salt Water (from Practice E729)
ensure that dissolved oxygen concentrations in the test cham-
NOTE 1—Add the following reagent-grade chemicals in the amounts
bers are acceptable.
and order listed to 890 mL of water (see 8.2.1.2). Each chemical must be
A 8.3.2 Salt water from a surface water source should be
dissolved before the next is added.
passed through a filter effective to 15 µm or less to remove
Chemical Amount
parasites and larval stages of predators.
NaF 3 mg
8.3.3 When necessary, sea salt may be added to prevent
SrCl ·6HO20mg
2 2
H BO 30 mg
3 3
excessive decreases in salinity (see 8.2.1) if the salt has been
KBr 100 mg
shown to cause no adverse effects on the survival, growth, or
KCl 700 mg
reproduction of polychaetes at the concentration used.
CaCl ·2HO1.47g
2 2
Na SO 4.00 g
2 4
8.4 Characterization:
MgCl ·6H O 10.78 g
2 2
NaCl 23.50 g
8.4.1 The following items should be measured at least
Na SiO ·9HO20mg
2 3 2
annuallyor(1)moreoftenifsuchmeasurementshavenotbeen
NaHCO 200 mg
made for at least two years or (2) if surface water is used:
A
If the resulting solution is diluted to 1 L, the salinity should be 34 ± 0.5 g/kg and
salinity, pH, particulate matter, TOC, organophosphorus
the pH 8.0 ± 0.2. The desired test salinity is attained by dilution at the time of use.
pesticides, organic chlorine (or organochlorine pesticides and
E1562 − 22
polychlorinated biphenyls (PCBs)), chlorinated phenoxy before beginning the test. If the test material is subject to
herbicides, ammonia, cyanide, sulfide, fluoride, iodide, nitrate, photolysis, the stock solution should be shielded from light.
phosphate, sulfate, calcium, chromium, cobalt, copper, iron,
9.2.2 Except possibly for tests on hydrolyzable, oxidizable,
lead, manganese, mercury, molybdenum, nickel, selenium,
and reducible materials, the preferred solvent is dilution water,
silver, tributyltin, and zinc.
although filtration or sterilization, or both, might be necessary.
8.4.2 Forthepurposesof8.4.1,theterm“organophosphorus
Deionized or distilled water may be used if the salinity of the
pesticides” refers to chlorpyrifos, demeton, diazinon,
dilution water will not be affected. Several techniques have
disulfoton,fenitrothion,malathion,methylparathion,andpara-
been developed specifically for preparing aqueous stock solu-
thion; the term “organochlorine pesticides” refers to aldrin,
tions of slightly soluble materials (15). The minimum neces-
chlordane, DDD, DDE, DDT, dieldrin, endosulfan, endrin,
sary amount of strong acid or base may be used in the
heptachlor, heptachlor epoxide, lindane, methoxychlor, mirex,
preparation of an aqueous stock solution, but such acid or base
and toxaphene; and the term “chlorinated phenoxy herbicides”
might affect the pH of test solutions appreciably. The use of a
refers to the free acids, salts, and esters of 2,4-D, dicamba,
more soluble form of the test material, such as chloride or
silvex, and 2,4,5-T. The term “organic chlorine” refers to
sulfate salt of organic amines, sodium or potassium salts of
chlorine that would be detected if, when samples are prepared
phenols and organic acids, and chloride or nitrate salts of
forgaschromatographicanalysisforpolychlorinatedbiphenyls
metals, might affect the pH more than the use of the minimum
(PCBs) and the organochlorine pesticides listed above, a
necessary amounts of strong acids and bases.
chlorine detector is used instead of an electron capture detector
9.2.3 If a solvent other than dilution water is used, its
to measure compounds that elute from just before lindane to
concentrationinthetestsolutionsshouldbekepttoaminimum
just after mirex on the gas chromatograph being used. Organic
and should be low enough that it does not affect the survival of
chlorine does not refer only to chlorine associated with
the polychaetes. Triethylene glycol is often a good organic
organochlorine pesticides and PCBs; it also refers to all
solventforpreparingstocksolutionsbecauseofitslowtoxicity
chlorine that elutes within the specified period.
to aquatic animals (16), low volatility, and strong ability to
8.4.3 The methods used should either (1) be accurate and
dissolve many organic chemicals. Other water-miscible or-
precise enough to characterize the dilution water adequately or
ganicsolventssuchasmethanol,ethanol,andacetonemayalso
(2) have detection limits below concentrations that have been
be used, but they might stimulate undesirable growths of
shown to affect estuarine and saltwater polychaetes adversely
microorganisms, and acetone is also quite volatile. If an
(14). 5
organic solvent is used, it should be reagent-grade or better,
and its concentration in any test solution should not exceed 0.5
9. Test Material
mL/L. Higher concentrations may be used if shown through
9.1 General—The test material should be reagent-grade or
testingtonotcausesignificantnegativebiologicaleffectstothe
better unless a test of formulation, commercial product, or
test organisms. A surfactant should not be used in the prepa-
technical-grade or use-grade material is specifically needed.
ration of a stock solution because it might affect the form and
The following should be known about the test material before
toxicity of the test material in the test solutions. (These
a test is begun:
limitations do not apply to any ingredient of a mixture,
9.1.1 Identities and concentrations of major ingredients and
formulation, or commercial product unless an extra amount of
major impurities, for example, impurities constituting more
solvent is used in preparation of the stock solution.)
than approximately 1 % of the material. Certificate of analysis
9.2.4 If no solvent other than water is used, (1) a dilution
(COA) provided by the manufacturer should be obtained and
water control must be included in the test and (2) the
reviewed. The COA should remain on file.
percentage of organisms in the control that show signs of
9.1.2 Solubility and stability in dilution water.
disease or stress such as discoloration, unusual behavior, or
9.1.3 Precision and bias of the analytical method at the
death, must be 10 % or lower.
planned test concentration(s) of the material.
9.2.5 If a solvent other than water is used and the concen-
9.1.4 Estimate of toxicity to humans.
tration of solvent is the same in all test solutions that contain
9.1.5 Recommendedhandlingprocedures(seesection7.11).
test material, (1) at least one solvent control, containing the
9.1.6 Estimate of acute toxicity to test species.
same concentration of solvent and using solvent from the same
9.2 Stock Solution:
batch used to make the stock solution, must be included in the
9.2.1 In some cases, the test solution can be added directly
test, and (2) a dilution water control must be included in the
to the dilution water, but usually it is dissolved in a solvent to
test.The percentage of organisms that show signs of disease or
form a stock solution that is then added to the dilution water. If
stress,suchasdiscoloration,unusualbehavior,ordeath,should
a stock solution is used, the concentration and stability of the
be 10 % or lower in the solvent control and must be 10 % or
test material in it and the dilution water should be determined
lower in the dilution water control.
9.2.6 If a solvent other than water is used and the concen-
tration of solvent is not the same in all test solutions that
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
contain test material, both a solvent control, containing the
listed by the American Chemical Society, see Analar Standards for Laboratory
highest concentration of solvent present in any other treatment
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
and using solvent from the same batch used to make the stock
and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,
MD. solution, and a dilution water control must be included in the
E1562 − 22
test.The percentage of organisms that show signs of disease or Neanthes arenaceodentata, Capitella capitata, Ophryotrocha
stress,suchasdiscoloration,unusualbehavior,ordeath,should diadema, and Dinophilus gyrociliatus. Use of these species is
be 10 % or lower in the solvent control and must be 10 % or encouraged in order to increase the comparability of results.
lower in the dilution water control.
10.2 Age—The age of the test species suitable for each type
9.2.7 If a solvent other than water is used to prepare a stock
of toxicity test is indicated in Table 2.
solution, it might be desirable to conduct simultaneous tests on
10.3 Source—It is generally more convenient to obtain
the test material using two chemically unrelated solvents or
specimens from a supplier rather than to use field-collected
two different concentrations of the same solvent to obtain
specimens. Cultures of Neanthes arenaceodentata have been
information concerning the possible effects of solvent on the
maintained by Aquatic Toxicology Support, LLC, Bremerton,
test results.
Washington. Cultures of Dinophilus gyrociliatus have also
9.3 Test Concentration(s):
been maintained by the U.S. Geological Survey, Texas. If
9.3.1 If the test is intended to allow the calculation of an
field-collected specimens are to be used in testing, it is
LC50, the test concentrations (see section 11.1.1.1) should
desirable to establish fresh cultures of Capitella capitata,
bracket the predicted LC50. The prediction might be based on
Ophryotrocha diadema,or Dinophilus gyrociliatus because
the results of a test on the same or a similar test material with
their short life cycle precludes a 14-day laboratory acclimation
the same or a similar species. If a useful prediction is not
period. It is possible to hold Neanthes arenaceodentata in the
available, it is usually desirable to conduct a range-finding test
laboratory for 14 days before testing because of its longer life
inwhichgroupsoffiveormoreorganismsareexposedfor24 h
cycle. Food, water, temperature, and salinity conditions for the
to 96 h to a control and three to five concentrations of the test
laboratory stock and those held for an acclimation period must
material that differ by a factor of ten.The greater the similarity
be similar to those conditions used in the toxicity test.All four
between the range-finding test and the actual test, the more
species have been maintained and cultured successfully
useful the range-finding test will be.
throughmanygenerationsinthelaboratory (1).Theprocedures
9.3.1.1 If the test is intended to be a life-cycle test to
forcollecting,identifying,andculturingthesespeciesaregiven
determine that concentration at which there is a statistically
in Appendix X1 – Appendix X4.
significant suppression in the number of young produced
10.3.1 If test organisms are cultured or held for an extended
compared to the control, the results from an acute or chronic
periodoftimeinthelaboratory,theresponseoflaboratory-held
LC50 test can be used to predict the test concentration. The
organisms to contaminants should be compared to that of
concentration at which there is a significant suppression of
animals collected freshly from the field to ensure that apparent
reproduction is generally between a factor of 2 to 10 times less
laboratory stresses do not affect their sensitivity to toxicants.
than the 96 h LC50 for polychaetes (1).
10.4 Feeding—It is not necessary to feed these species of
9.3.1.2 If necessary, concentrations above solubility should
polychaetes during an acute (96 h) toxicity test. It is necessary
be used because organisms in the real world are sometimes
to feed these species during chronic and life-cycle tests.
exposed to concentrations above solubility and because solu-
Tetramin® or Tetramarine® fish flakes should be provided as
bility in dilution water is often not well known. The use of
the food source for maintaining and culturing these species
concentrations more than ten times greater than solubility are
(ground to <0.5 mm), as specified in the appendixes of this
probably not worthwhile. With some test materials, it might be
guide. During chronic or life-cycle tests, Neanthes arenaceo-
found that concentrations above solubility do not kill or affect
dentata should be fed ground fish food flakes on an every-
a greater percentage of test organisms than will the concentra-
other-day basis at a rate of 8 mg (dry weight) per animal.
tion that is the solubility limit; such information is certainly
Because Capitella capitata, Ophryotrocha diadema, and Dino-
worth knowing.
philus gyrociliatus are smaller than N. arenaceodentata, they
9.3.2 In some (usually regulatory) situations, it is necessary
are fed a mixture of 25 mg ground fish food flakes in 100-mL
to determine only (1) whether a specific concentration of test
sea water. One or more drops of this suspension is added to
material is acutely toxic to the test species or (2) whether the
each dish, as required. Ophryotrocha diadema and Dinophilus
LC50 is above or below a specific concentration. For example,
gyrociliatus can also be fed 50 µL of a 0.5 % spinach food
the specific concentration might be the concentration occurring
suspension (w/v).
in the surface water, the concentration resulting from direct
application of the material to a body of water, or the solubility 10.5 Holding—If the polychaetes are obtained from a
supplier,theyshouldbeacclimatedfullytothetesttemperature
limit of the material in water. When there is interest only in a
specific concentration, it is often necessary to test only that
TABLE 2 Age of Test Species Suitable for Each Type of
concentration (see 11.1.5), and it is not necessary to actually
Toxicity Test
determine the LC50.
Species Acute Chronic Life Cycle
A
Neanthes arenaceodentata 2 weeks to 3 2 weeks to 3 one month
10. Test Organisms
A A
months months
A
Capitella capitata 2 to 3 weeks 2 to 3 weeks 1 day
10.1 Species—Test species are usually selected on the basis
Ophryotrocha diadema 1 to 2 weeks 1 to 2 days 1 to 2 days
of geographical distribution, availability, ease of handling in
Dinophilus gyrociliatus 1 day 1 day 1 day
the laboratory, and past successful use. The appendixes of this
A
Age is determined from the time of emergence from the parent’s tube or egg
guideprovideinformationforthecollection,identification,and
capsule.
culture of four species of polychaetes used in toxicity testing:
E1562 − 22
and salinity conditions prior to their use in a toxicity test. On 11. Procedure
the other hand, if the worms are being cultured by the
11.1 Experimental Design—Decisions concerning aspects
laboratory conducting the test, the acclimation period is not
of experimental design, such as the dilution factor, number of
necessary if the test is being conducted under the same
treatments,andnumbersofspecimensandreplicates,shouldbe
environmental conditions.
based on the purpose of the test and the type of procedure that
is being used to calculate the results (see Section 15). One of
10.6 Quality—All polychaetes used in a test must be in
the following three types of experimental design will probably
healthy condition (see 10.6.1). A qualified polychaete taxono-
be appropriate in most cases.
mist must be consulted to ensure that the animals in the test
11.1.1 Acute Test—An acute test is generally conducted for
population are all of the same species.
a 96 h period and is intended to measure the toxicity, as
10.6.1 Polychaetes in holding containers should be checked
determined by survival or death, of a test material.
daily before initiation of the test. Individuals that appear
11.1.2 Chronic Test—A chronic test is conducted for a
unhealthy or dead should be discarded. If more than 5 % of the
period of time greater than 96 h. Survival, death, and growth
polychaetes appear to be unhealthy during the 48 h preceding
(as weight) is used as the measure of toxicity. However,
the test, the entire group should be discarded and not used in
because of the short life cycle of Dinophilus gyrociliatus, the
the test. It is necessary to examine Capitella capitata, Oph-
acute and chronic tests are the same. The chronic test with
ryotrocha diadema, and Dinophilus gyrociliatus under a dis-
Ophryotrocha diadema can run for 10 days, for 14 to 21 days
secting microscope to determine the health of these species.
with Capitella capitata, and for up to 28 days with Neanthes
10.6.2 It may be desirable to analyze field collected Nean-
arenaceodentata.
thes arenaceodentata prior to testing to determine if they have
11.1.3 Life-Cycle Test—A life-cycle test begins with juve-
been exposed to the chemical being tested, if the chemical
nile polychaetes and extends until they lay eggs. These four
might be present in the environment. Pre-exposure to a
species lay their eggs in either the parent’s mucoid tube or in
chemical may affect the test result by making the test organ-
a capsule, and all are fertilized at the time of laying (see
isms either more or less sensitive. Polychaetes may be used
Appendixes). The number of embryos laid per female is used
without chemical analysis if the specimens are obtained from
as the measure of toxicity of the test material. The life-cycl
...