ASTM E1611-21

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: E1611 − 21
Standard Guide for
Conducting Sediment Toxicity Tests with Polychaetous
Annelids
This standard is issued under the fixed designation E1611; 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* formulations, commercial products, and known or unknown
mixtures. These procedures can be used with appropriate
1.1 This guide covers procedures for obtaining laboratory
modificationstoconductsedimenttoxicitytestsonfactorssuch
dataconcerningtheadverseeffectsofpotentiallycontaminated
as temperature, salinity, dissolved oxygen (DO), and natural
sediment, or of a test material added experimentally to con-
sedimentcharacteristics(forexample,particlesizedistribution,
taminated or uncontaminated sediment, on marine or estuarine
organiccarboncontent,andtotalsolids).Theseprocedurescan
infaunal polychaetes during 10-day or 20 to 28-day exposures.
also be used to conduct bioconcentration tests and in situ tests,
These procedures are useful for testing the effects of various
and to assess the toxicity of potentially contaminated field
geochemical characteristics of sediments on marine and estua-
sediments,orofmaterialssuchassewagesludge,oils,particu-
rine polychaetes and could be used to assess sediment toxicity
late matter, and solutions of toxicants added to sediments. A
tootherinfaunaltaxa,althoughmodificationsoftheprocedures
median lethal concentration (LC50) or median sublethal effect
appropriate to the test species might be necessary. Procedures
concentration (EC50) of toxicants or of highly contaminated
for the 10-day static test are described for Neanthes arenaceo-
sediment mixed into uncontaminated sediment can be deter-
dentata and Alitta virens (formerly Nereis virens and Nean-
mined.Materialsadheringtosedimentparticlesordissolvedin
thes virens) and for the 20 to 28-day static-renewal sediment
interstitial water can be tested.
toxicity for N. arenaceodentata.
1.5 The results of 10-day toxicity tests with contaminated
1.2 Modifications of these procedures might be appropriate
sediments can be reported as a LC50 if a series of concentra-
for other sediment toxicity test procedures, such as flow-
tions is tested or as a percent mortality relative to a control or
through or partial life-cycle tests.The methods outlined in this
reference sediment. The results of 20 to 28-day toxicity tests
guide should also be useful for conducting sediment toxicity
with contaminated sediments can be reported as a LC50 if a
tests with other aquatic taxa, although modifications might be
series of concentrations is tested or as a percent mortality or
necessary. Other test organisms might include other species of
growth relative to a control or reference sediment.
polychaetes, crustaceans, and bivalves.
1.6 This guide is arranged as follows:
1.3 Other modifications of these procedures might be ap-
Section
propriate for special needs or circumstances. Although using
Referenced Documents 2
appropriate procedures is more important than following pre-
Terminology 3
scribedprocedures,theresultsoftestsconductedusingunusual Summary of Guide 4
Significance and Use 5
procedures are not likely to be comparable to those of many
Interferences 6
othertests.Comparisonsoftheresultsobtainedusingmodified
Apparatus 7
and unmodified versions of these procedures might provide Facilities 7.1
Construction Materials 7.2
useful information concerning new concepts and procedures
Test Chambers 7.3
for conducting sediment tests with infaunal organisms.
Cleaning 7.4
Acceptability 7.5
1.4 These procedures are applicable to sediments contami-
Hazards 8
nated with most chemicals, either individually or in
Test Water 9
General Requirements 9.1
Source 9.2
Preparation 9.3
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Characterization 9.4
Assessment, Risk Management and CorrectiveAction and is the direct responsibil- Test and Control Sediments 10
General 10.1
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Characterization 10.2
Current edition approved Nov. 1, 2021. Published December 2021. Originally
Control Sediment 10.3
approved in 1994. Last previous edition approved in 2013 as E1611-00(2013).
Field-Collected Test Sediment 10.4
DOI: 10.1520/E1611-21.
Reference Sediment 10.5
World Register of Marine Species (WoRMS) at
Laboratory-Spiked Test Sediment 10.6
https://www.marinespecies.org/aphia.php?p=taxdetails&id=234851
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1611 − 21
Aquatic Organisms and Their Uses
Section
Test Concentration(s) 10.7
E1192Guide for ConductingAcute Toxicity Tests onAque-
Addition of Toxicant to Sediment 10.8
ous Ambient Samples and Effluents with Fishes,
Test Organisms 11
Macroinvertebrates, and Amphibians
Species 11.1
Age 11.2
E1241GuideforConductingEarlyLife-StageToxicityTests
Feeding 11.3 4
with Fishes (Withdrawn 2022)
Source 11.4
E1367TestMethodforMeasuringtheToxicityofSediment-
Collection and Handling 11.5
Quality 11.6
Associated Contaminants with Estuarine and Marine In-
Experimental Design 12
vertebrates
Controls 12.2
Field Survey Design 12.3 E1391Guide for Collection, Storage, Characterization, and
Laboratory Experiments 12.4
Manipulation of Sediments for Toxicological Testing and
Procedure 13
for Selection of Samplers Used to Collect Benthic Inver-
Dissolved Oxygen 13.1
Temperature 13.2 tebrates
Salinity 13.3
E1525GuideforDesigningBiologicalTestswithSediments
Light 13.4
E1706TestMethodforMeasuringtheToxicityofSediment-
Feeding 13.5
Associated Contaminants with Freshwater Invertebrates
Beginning of Test 13.6
Duration of Test 13.7
E1733Guide for Use of Lighting in Laboratory Testing
Biological Data 13.8
E1847Practice for Statistical Analysis of Toxicity Tests
Other Measurements 13.9
Analytical Methodology 14 Conducted Under ASTM Guidelines (Withdrawn 2022)
Acceptability of Test 15
SI10-02IEEE/ASTMSI10AmericanNationalStandardfor
Interpretation of Results 16
UseoftheInternationalSystemofUnits(SI):TheModern
Report 17
Keywords 18 Metric System
Annexes
Neanthes arenaceodentata Annex A1
3. Terminology
Alitta virens Annex A2
3.1 Definitions:
1.7 The values stated in SI units are to be regarded as
3.1.1 The words “must,” “should,” “may,” “can,” and
standard. No other units of measurement are included in this
“might” have very specific meanings in this guide. “Must” is
standard.
used to express the strongest possible recommendation, just
1.8 This standard does not purport to address all of the
short of an absolute requirement, that is, to state that this test
safety concerns, if any, associated with its use. It is the
oughttobedesignedtosatisfythespecificcondition,unlessthe
responsibility of the user of this standard to establish appro-
purpose of the test requires a different design. “Must” is used
priate safety, health, and environmental practices and deter-
only in connection with factors that relate directly to the
mine the applicability of regulatory limitations prior to use.
acceptability of the test (see Section 14). “Should” is used to
Specific hazards statements are given in Section 8.
state that the specific condition is recommended and ought to
1.9 This international standard was developed in accor-
be met if possible. Although the violation of one “should” is
dance with internationally recognized principles on standard-
rarelyaseriousmatter,theviolationofseveralwilloftenrender
ization established in the Decision on Principles for the
theresultsquestionable.Termssuchas“isdesirable,”“isoften
Development of International Standards, Guides and Recom-
desirable,” and “might be desirable” are used in connection
mendations issued by the World Trade Organization Technical
with less important factors. “May” is used to mean “is (are)
Barriers to Trade (TBT) Committee.
allowed to,” “can” is used to mean “is (are) able to,” and
“might” is used to mean “could possibly.” Thus the classic
2. Referenced Documents
distinctionbetween“may”and“can”ispreserved,and“might”
2.1 ASTM Standards:
is never used as a synonym for either “may” or “can.”
D1129Terminology Relating to Water 3.1.2 For definitions of other terms used in this guide, refer
D3976Practice for Preparation of Sediment Samples for
to Terminologies D1129 and E943, Guides E729, E1023,
Chemical Analysis E1192, E1367, and E1525. For an explanation of units and
D4447Guide for Disposal of Laboratory Chemicals and
symbols, refer to SI10-02 IEEE/ASTM SI 10.
Samples 3.2 Definitions of Terms Specific to This Standard:
E729Guide for Conducting Acute Toxicity Tests on Test
3.2.1 ash-free dry weight—Organism weight determined by
Materials with Fishes, Macroinvertebrates, and Amphib- subtracting the standard dry weight from the ashed (550°C
ians
dried) weight to remove the inorganic contribution.
E943Terminology Relating to Biological Effects and Envi-
3.2.1.1 Discussion—AFDW is therefore the weight of the
ronmental Fate
organic content of the organism.
E1023Guide for Assessing the Hazard of a Material to
3.2.2 clean sediment, n—sediment that does not contain
concentrationsoftoxicantsthatcauseapparentstresstothetest
organism or reduce their survival.
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.
E1611 − 21
3.2.3 control sediment, n—asedimentthatisessentiallyfree survival; and (2) the 20-day to 28-day test, which determines
of contaminants and is used routinely to assess the acceptabil- the effect of contaminated sediment on survival and growth. If
ity of a test. smaller worms are used, such as N. arenaceodentata,a
minimum of five worms are placed in a 1-Lglass test chamber
3.2.4 estimated individual dry weight, n—a value that is
with a minimum sediment depth of 2cm to 3 cm and the
calculated by dividing the total dry weight by the number of
overlying water aerated. The survival of the worm exposed to
surviving worms within a replicate.
the test sediment is compared with the survival in a negative
3.2.5 exposure, n—contact with a chemical or physical
control or reference sediment in the 10-day test. The same
agent (see Terminology E943).
procedure is used in the 20-day to 28-day test, except for the
3.2.6 interstitial water, n—water occupying the space be-
testduration(seeAnnexA1).Iflargerwormsareused,suchas
tween sediment or soil particles; a synonym for pore water.
A. virens, a minimum of ten worms are placed in beakers or
glass aquaria (4Lto 37 L) with a minimum sediment depth of
3.2.7 overlying water, n—the water added to the test cham-
5 cm and the overlying water aerated. A negative control or
ber over the solid phase of the sediment in a toxicity test.
referencesedimentisusedtogiveameasureoftheacceptabil-
3.2.8 pore water, n—water occupying the space between
ity of the test by (1) providing evidence of the health and
sediment particles; a synonym for interstitial water.
relative quality of the test organisms, suitability of the overly-
3.2.9 reference sediment, n—awholesedimentnearthearea
ing water, test conditions, and handling procedures, etc.; and
of concern used to assess sediment conditions exclusive of
(2)providingabasisforinterpretingdataobtainedfromthetest
material(s) of interest.
sediments.
3.2.10 sediment, n—particulate material that usually lies 4.1.1 The percent survival of polychaetes exposed to field-
belowwater.Formulatedparticulatematerialthatisintendedto
collected sediment is compared to those exposed to a negative
lie below water in a test. control or reference sediment in 10-day tests.The survival and
body weight of the animals surviving in field-collected sedi-
3.2.11 short-term toxicity tests, n—generally used to deter-
ment is compared to those exposed to negative control or
mine the concentration of test material that produces a specific
reference sediment in 20 to 28-day tests. The toxicity of field
adverseeffectonaspecificpercentageoftestorganismsduring
sediments may also be assessed by testing dilutions of highly
a short exposure (for example, 10 days).
toxictestsedimentswithcleansedimentstoobtaininformation
3.2.11.1 Discussion—Because death is obviously an impor-
on the toxicity of proportions of that sediment.
tant adverse effect and is detected easily for many species, the
4.1.2 The toxicity of a material added experimentally to
most common end point is survival. Both survival and growth
sediments can be expressed by analyzing the survival and
are used as end points in the 20 to 28-day test. Effect on 50%
growth data to determine a LC50 for the material for the
of a group of test organisms is the most experimentally
duration of exposure.
reproducibleandeasilydeterminedmeasureoftoxicity,and96
hisoftenaconvenient,usefulexposureduration.Themeasure
4.2 The annexes at the end of this guide outline the
usedmostofteninacutetestsisthereforethe96-hLC50value.
techniques for collecting, identifying, holding, and testing N.
In tests with contaminated sediment, however, the exposure
arenaceodentata and A. virens and culturing N. arenaceoden-
period is generally 10 days or 20 to 28 days. Death is used as
tata.
the measure of toxicity in the 10-day test; the results are
reported as a 10-day LC50 or response relative to a control or
5. Significance and Use
referencesediment.Drybodyweightisusedasthemeasureof
5.1 The test procedure covered in this guide is not intended
effect in the 20 to 28-day test or the 20 to 28-day LC50 if
to simulate exactly the exposure of benthic polychaetes to
dilutionsaretested.Ash-freedryweightmayalsobemeasured
chemicals under natural conditions, but rather to provide a
to differentiate the influence of the gut contents from final
conveniently rapid, standard toxicity test procedure yielding a
tissue mass.
reasonably sensitive indication of the toxicity of materials in
3.2.12 spiked sediments, n—a sediment to which a material
marine and estuarine sediments.
has been added for experimental purposes.
5.2 The protection of a community of organisms requires
3.2.13 toxicity, n—thepropertyofamaterialorcombination
avertingdetrimentalcontaminant-relatedeffectsonthenumber
of materials that affects organisms adversely (seeTerminology
and health of individuals and species within that population.
E943).
Sediment toxicity tests provide information on the toxicity of
3.2.14 whole sediment, n—sediment that has not had mate-
test materials in sediments. Theoretically, projection of the
rial extracted or removed. most sensitive species within a community will protect the
community as a whole.
4. Summary of Guide
5.3 Polychaetes are an important component of the benthic
4.1 Twoproceduresareusedtomeasuretherelativetoxicity community. They are preyed upon by many species of fish,
ofmarineorestuarinesedimentstopolychaetes:(1)the10-day birds,andlargerinvertebratespecies,andtheyarepredatorsof
test, which measures the effect of contaminated sediment on smaller invertebrates, larval stages of invertebrates, and, in
E1611 − 21
some cases, algae, as well as organic material associated with 6.2 The results of sediment toxicity tests will depend partly
sediment. Polychaetes are sensitive to both organic and inor- on the temperature, water quality, physical and chemical
ganic chemicals (1, 2). The ecological importance of propertiesofthetestsediment,conditionofthetestorganisms,
polychaetes, their wide geographical distribution and ability to exposure technique, and other factors. Factors potentially
be cultured in the laboratory, and sensitivity to chemicals, affecting the results from static sediment toxicity tests might
make them appropriate toxicity test organisms. include the following:
6.2.1 The alteration of field sediments in preparation for
5.4 An acute or 10-day toxicity test is conducted to obtain
laboratory testing.
informationconcerningtheimmediateeffectstoatestmaterial
6.2.1.1 Maintaining the integrity of the sediment environ-
on a test organism under specified experimental conditions for
ment during its removal, transport, and testing in the
a short period of time. An acute toxicity test does not
laboratory, which is extremely difficult (Guide E1391). The
necessarily provide information concerning whether delayed
sediment environment is composed of a myriad of
effectswilloccur,althoughapost-exposureobservationperiod,
microenvironments, redox gradients, and other interacting
with appropriate feeding, if necessary, could provide such
physiochemical and biological processes. Many of these char-
information.
acteristicsinfluencethesedimenttoxicityandbioavailabilityto
5.5 The results of acute sediment toxicity tests can be used
benthic and planktonic organisms, microbial degradation, and
to predict acute effects likely to occur on aquatic organisms in
chemical sorption.Any disruption of this environment compli-
field situations as a result of exposure under comparable
cates the interpretations of treatment effects, causative factors,
conditions, except that (1) motile organisms might avoid
and in situ comparisons.
exposure when possible and (2) toxicity to benthic organisms
6.2.1.2 Testing of sediments at temperatures or salinities
can be dependent on sediment characteristics, the dynamics of
other than those at which they were collected might affect
equilibrium partitioning, and the route of exposure to the
chemicalsolubility,partitioningcoefficients,andotherphysical
benthic organisms.
and chemical characteristics.
5.6 The polychaete sediment toxicity test might be used to
6.2.2 Interactions among the sediment particles, overlying
determine the temporal or spatial distribution of sediment
water, interstitial water, humic substances, and the sediment to
toxicity. Test methods can be used to detect horizontal and overlying water ratio.
vertical gradients to toxicity. Mortality data can be used to
6.2.3 Interactions among chemicals that might be present in
indicate the relative toxicity of field-collected sediments.
the test sediment.
6.2.4 Therealismofusingspikedsediment(thatis,whether
5.7 The results of acute tests with toxicants added experi-
the spiked sediment is at equilibrium and mixed evenly or
mentally to sediments can be used to compare the acute
representsthebioavailabilityofnaturallyoccurringchemicals).
sensitivitiesofdifferentspeciesandacutetoxicitiesofdifferent
6.2.5 Photolysis and other processes degrading the test
test materials, and to define the effects of various environmen-
chemicals.
tal factors on the results of such tests.
6.2.6 Maintaining an acceptable quality of the overlying
5.8 Theresultsofacutesedimenttoxicitytestsareusefulfor
water.
studying the biological availability of, and structure-activity
6.2.7 Excess food might change the sediment partitioning
relationships between, test materials in sediment.
and water quality parameters.
5.9 The results of acute sediment toxicity tests might be an
6.2.8 Resuspension of sediment during the toxicity test.
important consideration when assessing the hazards of materi-
6.2.9 A limited opportunity for biological observation dur-
als to aquatic organisms (see Guide E1023) or when deriving
ing the test because organisms bury in the sediment.
the sediment quality for aquatic organisms (3). Sediment
6.2.10 The natural geochemical properties of test sediment
toxicity tests might be useful for making decisions regarding
collected from the field that might not be within the tolerance
the extent of remedial action necessary for contaminated sites.
limits of the test organisms.
5.10 A 10-day test provides data on the short-term effects 6.2.11 It may be difficult to recover the worms from
that are useful for comparisons to other species but does not sediment if growth is stunted.
provide information on delayed effects. Results of the 20-day 6.2.12 Endemic organisms that might be present in field-
to 28-day sediment toxicity test, which measures growth in collected sediments, including (1) predators; (2) species that
addition to survival, can be useful indicators of the effects of might be the same as or closely related to the test species; (3)
contaminated sediments over a longer time period. microorganisms (for example, bacteria and molds); and (4)
algae colonizing sediment and test chamber surfaces.
6. Interferences
6.3 Static tests might not be applicable to materials that are
6.1 The methodology continues to develop and evolve with
highly volatile or are rapidly transformed biologically or
time and research needs. There are limitations to the methods
chemically. Furthermore, the overlying water quality might
described in this guide because of the developmental nature of
change considerably from the initial overlying water. The
sediment toxicity testing.
procedurescanusuallybeappliedtomaterialsthathaveahigh
oxygen demand because the experimental chambers are aer-
ated. Materials dissolved in interstitial waters might be re-
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. moved from solution in substantial quantities by adsorption to
E1611 − 21
sediment particles and to the test chamber during the test. The 7.3.1 Testchambersaredefinedasthesmallestphysicalunit
dynamicsofchemicalpartitioningbetweensolidanddissolved betweenwhichtherearenowaterconnectionsinatoxicitytest
phases at the initiation of the test should therefore be
with aquatic organisms.The test chambers for both the 10-day
considered, especially in relation to assumptions of chemical
and 20 to 28-day sediment toxicity tests are 1-Lglass contain-
equilibrium. ers with an inside diameter of approximately 10 cm. An
appropriate number of worms, as specified by the scope of the
7. Apparatus
study (13.6.7), are placed within each test chamber, which
gives each juvenile worm approximately 15 cm of surface
7.1 Facilities—Beakers, aquaria, or tanks containing either
area. The chambers are covered with glass lids to reduce
clean (uncontaminated), natural sea water, or reconstituted sea
contaminationofthecontentsandminimizeevaporationofthe
water should be used for holding the polychaetes before a test.
water or test material. The test chambers are maintained at
The holding tanks and any area used for manipulating live
20°C 6 1°C in either a shallow water bath or a constant
polychaetes should be located in a room or space separated
temperatureroom.Thetestchambersshouldbeaeratedwithair
from that in which the toxicity tests are to be conducted, stock
free of fumes, oil, and water. The air is delivered to the test
solutions or test materials are to be prepared, or equipment is
chamberbynon-toxictubingconnectedtoa1mLglasspipette
to be cleaned. The sea water should be monitored periodically
that is suspended 3 to 4 cm below the water surface.Aeration
to ensure a constant salinity. The holding tanks, water supply,
should be bubbled into the test chambers at <100 bubbles/min
or room in which they are kept should be equipped with
or at a rate that maintains a ≥90% dissolved oxygen (DO)
temperature control. Aeration can be provided to ensure that
concentration (see 13.1). Larger-sized specimens are used in
the DO is greater than 60% saturation and that there is
some sediment toxicity tests, which requires a larger-sized test
adequate water circulation in the tanks.
2 2
chamber.Asurfaceareaofapproximately30cm to40cm per
7.2 Construction Materials—Equipment and facilities that
specimen is necessary when larger-sized worms are used.
contact stock solutions, test solutions, or any water into which
7.4 Cleaning—The test chambers and other glassware and
thetestorganismswillbeplacedshouldnotcontainsubstances
equipment used to store and prepare test sea water, stock
that can be leached or dissolved by aqueous solutions in
solutions, and test sediments should be cleaned before use.
amounts that affect the test organisms adversely. In addition,
Newitemsshouldbecleanedbeforeeachusebywashingwith
equipment and facilities that contact stock or test solutions
laboratory detergent; rinsing with water, a weak-miscible
should be chosen to minimize the sorption of test materials
organic solvent, water, and acid (10% nitric or hydrochloric
from water. Glass, Type 316 stainless steel, nylon, and
acid); and rinsing twice with distilled, deionized, or dilution
polypropylene, or polyethylene should be used whenever
possible to minimize dissolutions, leaching, and sorption, water. Metals, sulfides, and carbonate deposits are removed by
theacidrinse,andorganicsareremovedbytheorganicsolvent
except that stainless steel should not be used in tests on metals
in salt water. Fluorocarbon plastics should be avoided espe- rinse. A dichromate-sulfuric acid cleaning solution may be
cially in studies evaluating these types of chemicals with used in place of both the organic solvent and acid rinses, but it
increasing environmental concern (Per- and polyfluoroalkyl
might attack silicone adhesives. At the end of each test, all
substances (PFAS)). Concrete and rigid plastics may be used
items to be used again should immediately be (1) emptied; (2)
for holding tanks and in the water-supply system, but they
rinsed with water; (3) cleaned by a procedure appropriate for
should be soaked, preferably in flowing dilution water, for a
removingthetestmaterial(forexample,acidtoremovemetals
weekormorebeforeuse (4).Brass,copper,lead,castironpipe,
and solvents to remove organics); and (4) rinsed at least twice
galvanized metal, and natural rubber must not contact the test
withdeionized,distilled,ordilutionwater.Acidisoftenusedto
−
seawater,stocksolutions,ortestsedimentbeforeorduringthe
remove mineral deposits, and 200 mg of hypochlorite (ClO )
test.Aspeciallydesignedsystemisusuallynecessarytoobtain
per litre is often used to remove organic matter and for
salt water from a natural water source (5). Tubing used in
disinfection. (A solution containing approximately 200 mg of
−
making up test sea water and in aerating the test chambers
ClO /L may be prepared by adding 6 mL of liquid household
−
should be nontoxic vinyl. New tubing should be aged in sea
chlorinebleachto1Lofwater.However,ClO isquitetoxicto
water at least one week prior to use. Separate sieves, dishes,
many aquatic animals (6) and is difficult to remove from some
containers, and other equipment should be used to handle the
construction materials. It is often removed by soaking in a
testsedimentorothertoxicmaterials,andtheseshouldbekept
sodiumthiosulfate,sodiumsulfite,orsodiumbisulfitesolution,
and stored separately from those used to handle the live
by autoclaving in distilled water for 20 min, or by drying the
animals prior to testing. Items made from other materials not
item and letting it sit for at least 24 h before use. An item
mentioned previously should not be used unless it has been
cleaned or disinfected with hypochlorite should not be used
shown that their use will not affect either the survival, growth,
unless it has been demonstrated at least once that the test
or reproduction of the polychaetes adversely.
polychaete species do not show signs of apparent stress, such
as discoloration, unusual behavior, or death, when held for at
7.3 Test Chambers—Species-specific information on test
least 48 h in static dilution water in which the item is soaking
chambersisgivenintheannexes.Thetestchambersshouldbe
placed in either a temperature-controlled room or a water bath than when held in static dilution water containing a similar
−
item that was not treated with ClO .) Glassware used only for
to minimize temperature fluctuations, and they should be
aerated. Aeration can be provided as described in 13.1. liveanimals,notexposedtotestmaterial,maybecleanedusing
E1611 − 21
only clean distilled or dilution water, since the use of deter- in which no smoking is allowed and no open flame, such as a
gents is sometimes detrimental to live organisms. pilot light, is present. Cleaning of the equipment with acids
should be performed only in a well-ventilated area, and
7.5 Acceptability—The acceptability of new holding or
protective gloves and safety goggles should be worn. Hexane
testing facilities should be demonstrated by conducting a
mightalsobeusedasasolventforremovingnon-ionicorganic
non-toxicant test in which all test chambers contain sediment
compounds.However,acetoneispreferableifonlyoneorganic
and clean sea water. The survival of the test species will
solvent is used to clean equipment.
demonstratewhetherthefacilities,water,controlsediment,and
handling techniques are adequate to result in acceptable 8.7 Anacidicsolutionshouldnotbemixedwithahypochlo-
(≥90%) control level survival in the absence of toxicants.
rite solution because hazardous fumes might be produced.
8.8 Concentrated acid should be added to water, not vice
8. Hazards
versa, to prepare dilute acid solutions. Opening a bottle of
8.1 Many materials can affect humans adversely if precau-
concentratedacidandaddingconcentratedacidtowatershould
tions are inadequate. Skin contact with all test materials and
be performed only in a well-ventilated room or chemical fume
theirsolutionsshouldthereforebeminimizedbysuchmeansas
hood.
wearing appropriate protective gloves (especially when wash-
ing equipment or putting hands into the test solutions), labo-
9. Test Water
ratory coats, aprons, and glasses. Special precautions, such as
coveringthetestchambersandventilatingtheareasurrounding
9.1 GeneralRequirements—Inadditiontobeingavailablein
the chambers, should be taken when conducting tests on
adequate supply, water used in sediment toxicity tests should
volatile materials. Information on toxicity to humans (7),
beacceptabletothetestorganismsandpurposeofthetest.The
recommended handling procedures (8), and chemical and
minimumrequirementforacceptablewaterforuseinsediment
physicalpropertiesofthetestmaterialshouldbestudiedbefore
toxicity tests is that healthy test organisms survive (≥90%) in
a test is begun. Special precautions might be necessary with
thewaterwithsedimentforthedurationofholdingandtesting
radiolabeledtestmaterials (9)andtestmaterialsthatare,orare
without showing signs of disease or apparent stress such as
suspected of being, carcinogenic.
unusual behavior, changes in appearance, or death. The water
in which the test organisms are held prior to the test should be
8.2 Field sediments to be tested, especially those from
uniform in quality in that the concentration of chemicals and
effluent areas, might contain organisms that can be pathogenic
the range of temperature and salinity encountered during the
to humans. When dealing with these sediments, special pre-
holding period do not adversely affect the survival of the test
cautions might include immunizations prior to sampling and
organismsintheholdingtanksorcontroltreatmentsduringthe
the use of bactericidal soaps after working with the sediments.
test. A better criterion for an acceptable sea water is that in
8.3 Sediments collected from the field might be contami-
which the test species will survive and grow.
nated with unknown concentrations of many potentially toxic
9.2 Source:
materials, and laboratory-prepared sediments might be spiked
9.2.1 Natural Sea Water—If natural salt water is used, it
withhighconcentrationsoftoxicants.Anypotentiallycontami-
should be obtained from an uncontaminated area known to
natedsedimentsshouldbehandledinamannertominimizethe
support a healthy, naturally reproducing population of the test
exposure of researchers to toxic compounds. Mixing of toxic
organism or a comparably sensitive species. The water intake
sediments in open containers, spiking of laboratory-prepared
shouldbepositionedtominimizefluctuationsinqualityandthe
sediments, and loading of toxic sediments into the test cham-
possibility of contamination, and to maximize the concentra-
bers should be performed in a well-ventilated area, preferably
tion of DO to help ensure low sulfide and iron concentrations.
a chemical fume hood. Face shields or protective goggles
Aspecially designed system might be necessary to obtain salt
should be worn during any operations that might involve the
waterfromanaturalwatersource(seeGuideE729).Thewater
accidentalsplashingofsediments,suchassieving,mixing,and
should be monitored in accordance with 9.4 to ensure uniform
loading into test chambers.
quality. These precautions ensure that the test organisms are
8.4 Healthandsafetyprecautionsandapplicableregulations
not stressed by the water quality during holding, acclimation,
for the disposal of stock solutions, overlying water from test
and testing and that the water quality does not affect the test
chambers, test organisms, and sediments should be considered
results unnecessarily.
before beginning a test (see Guide D4447). Consideration of
9.2.2 Reconstituted Salt Water—Reconstituted salt water
cost as well as detailed regulatory requirements might be
canbepreparedbyaddingacommerciallyavailableseasaltor
necessary. Removal or degradation of the toxicants before
specified amounts (see Guide E729 and Table 1) of reagent
disposal of the stock solutions, test sediments, and water is
grade chemicals (10-12) to high-quality water with (1)a
sometimes desirable for tests involving spiked sediments, with
conductivity below 1 µS/cm and (2) either a total organic
known toxicants.
carbon (TOC) below 2 mg/L or a chemical oxygen demand
8.5 The use of ground fault systems and leak detectors is
(COD) below 5 mg/L. Commercial sea salt mixes should be
recommended strongly in order to help prevent electrical
“bioassay grade” without the addition of the metal chelating
shocks because salt water is a good conductor of electricity.
agent ethylenediaminetetraacetic acid (EDTA) or dechlorina-
8.6 Cleaning of the equipment with a volatile solvent such tion chemicals such as sodium thioosulfate. These compounds
as acetone should be performed only in a well-ventilated area are present in hobbyist salts and may mask the toxicity of
E1611 − 21
TABLE 1 Reconstituted Salt Water (from Guide E729)
water might be required for sieving control sediment to adjust
the salinity or for holding the test worms prior to the test.
NOTE 1—Add the following reagent grade (10) chemicals in the
amounts and order listed to 890 mL of water. Each chemical must be 9.3.3 The experimental design might require the use of sea
A
dissolved before the next is added.
water from the test sediment collection site for certain appli-
Chemical Amount
cations. Experimental treatments might involve manipulation
NaF 3 mg
of the test water conditions in other instances.
SrCl ·6HO20mg
2 2
H BO 30 mg
3 3
9.4 Characterization—The following items should be mea-
KBr 100 mg
sured at least once each year, and more often if such measure-
KCl 700 mg
mentshavenotbeenmadesemiannuallyforatleasttwoyears:
CaCl ·2HO1.47g
2 2
Na SO 4.00 g
2 4
9.4.1 Salinity,pH,particulatematter,TOC,organophospho-
MgCl ·6H O 10.78 g
2 2
rus pesticides, organic chlorine (or organochlorine pesticides
NaCl 23.50 g
Na SiO ·9HO20mg and polychlorinated biphenyls (PCBs)), chlorinated phenoxy
2 3 2
NaHCO 200 mg
herbicides, ammonia, cyanide, sulfide, fluoride, iodide, nitrate,
A
If the resulting solution is diluted to 1 L, the salinity should be 34 g/kg ± 0.5 g/kg
phosphate, sulfate, calcium, chromium, cobalt, copper, iron,
and the pH 8.0 ± 0.2.The desired test salinity is attained by dilution at the time of
lead, manganese, mercury, molybdenum, nickel, selenium,
use. The reconstituted salt water should be stripped of trace metals.
silver, tributyltin, and zinc.
9.4.2 More frequent monitoring might be necessary in
estuarine areas, in which large diurnal, tidal, and seasonal
environmental or spiked sediment samples. Acceptable water
variations in the concentrations of organics, heavy metals, and
can usually be prepared using properly operated deionization
waterqualitymightoccur.Inparticular,dailymeasurementsof
or distillation units. Conductivity should be measured on each
salinity, temperature, and pH, and quarterly monitoring of
batch, and TOC or COD should be measured at least once per
other parameters over a tidal cycle, might be desirable.
year and whenever significant changes might be expected.The
9.4.3 The methods used (see 14.2) should either (1)be
TOCorCODshouldbemeasuredoneachbatchifthewateris
accurate and precise enough to characterize the toxicity test
prepared from a surface water. The reconstituted water should
water adequately or (2) have detection limits below concen-
be aerated intensively before use. The solution should be
trations that have been shown to affect the test species
filtered if a residue or precipitate is present. Problems have
adversely (14).
been encountered with some species in some salt reconstituted
waters, but these problems have sometimes been overcome by
10. Test and Control Sediments
aging the reconstituted water for one or more weeks. The salt
10.1 General—Before the preparation or collection of test
water should meet the criteria given in 9.1.
sediment, an approved written procedure should be prepared
9.2.3 Chlorinated water must never be used in the prepara-
for the handling of sediment that might contain unknown
tion of salt water for toxicity tests because residual chlorine
quantities of many potentially toxic chemicals (see Section 8).
and chlorine-produced oxidants are highly toxic to many
Sediments are spatially and temporally variable. Replicate
aquatic animals (6). The use of dechlorinated water should be
samples should be collected to determine variance in sediment
avoidedbecausedechlorinationisoftenincomplete.Municipal
characteristics. Sediment should be collected with as little
drinking water is not recommended for use because it often
disruption as possible; however, subsampling, compositing, or
containsunacceptablehighconcentrationsofmetalsinaddition
homogenization of sediment samples may be necessary for
to residual chlorine, and the quality is often highly variable
some experimental designs. Sampling may cause loss of
(see Guide E729).
sedimentintegrity,changeinchemicalspeciation,ordisruption
9.3 Preparation:
of chemical equilibrium (Guide E1391).Abethic grab or core
9.3.1 Sea water used in the sediment toxicity test should be
should be used rather than a dredge to minimize disruption of
passed through a filter effective to 5 µm or less to remove
the sediment sample. Sediment should be collected from a
suspended particles and organisms from the water. Water that
depth that will represent expected exposure.
might be contaminated with facultative pathogens should be
passed through a properly maintained ultraviolet sterilizer (13) 10.2 Characterization—Sedimentschosenforuseshouldbe
or filter with a pore size of 0.45 µm or less. characterized,andatleastthefollowingshouldbedetermined:
9.3.1.1 The salinity should be reduced by diluting the sea salinity, pH, ammonia, hydrogen sulfide, organic carbon con-
water with a high-quality deionized distilled water (see 9.2.2) tent (TOC or total volatile solids), particle size distribution
if necessary. Salinity can be raised by the addition of clean (percent sand, silt, and clay), and percent water content. Other
filtered oceanic water, brine, or reagent grade chemicals in analyses on sediments might include biological oxygen
accordance with 9.2.2. demand, chemical oxygen demand, Eh or pE, total inorganic
9.3.2 Fresh sea water used in the test should be prepared carbon, metals, synthetic organic compounds, oil and grease,
within two days of the test and stored in clean, covered organosilicones,andpetroleumhydrocarbons.Interstitialwater
containers until sediment and water are added to the test mightalsobeanalyzedasdescribedin14.4andinTestMethod
chambers. It might be necessary to age reconstituted sea water E1706. Toxicological results can identify samples that should
for one to two weeks before use. Sufficient water should be besubjectedtomoreintensivephysical,chemical,orbiological
prepared at one time for all of the test chambers. Additional testing.
E1611 − 21
10.3 Control Sediment: hold the sediments after collection in the dark at 4°C.
Traditional convention has held that sediment tests should be
10.3.1 Collection—Control sediment should be collected
started as soon as possible following collection from the field,
from the polychaete collection site or from another area that is
although actual recommended storage times range from two
withinthegeochemicalrequirementsofthetestspeciesandcan
provide a nontoxic reference sediment for evaluation of the weeks (Guide E1391) to less than eight weeks (USEPA-
USACE, 1998) (15). Discrepancies in recommended storage
condition of the test population subject to laboratory
procedures, and for statistical comparison with the test sedi- times reflected a lack of data concerning the effects of
ment (see Guide E1847). Control sediment should be brought long-term storage on the physical, chemical, and toxicological
tothesievingareainacleancontainer.Anywateroverlyingthe characteristics of the sediment. However, numerous studies
sediment or used to wash the sediment into the container
have recently been conducted to address issues related to
shouldbesavedsothatanyfineparticlescontainedinthewater sediment storage (see Refs (16-22). The conclusions and
can be recombined into the sediment. Any sediment showing
recommendations offered by these studies vary substantially
evidence of contamination (for example, oil sheen) should be and appear to depend primarily upon the type or class of
discarded. As the sediment is collected, the bottom
chemical(s) present. Considered collectively, these studies
temperatures, salinity, and sediment temperature should be suggest that the recommended guidance that sediments be
recorded,andacompositesedimentsamplefromallshovelful,
tested sometime between the time of collection and 8 weeks
dredge hauls, or grabs should be collected for analysis of the
storage is appropriate. Additional guidance is provided below
water content, particle size distribution, and organic content.
and in Guide E1391 and Test Method E1706.
10.3.2 Control or reference sediment should be character-
10.4.3 Extended storage of sediments that contain high
ized empirically as described in 10.2 at least annually.
concentrations of labile chemicals (for example, ammonia,
10.3.3 Sieving—Aseparate clean container should be set up
volatile organic compounds) may lead to a loss of these
to sieve and contain the control sediment. Control sediment
chemicals and a corresponding reduction in toxicity or bio-
should be sieved twice: first to remove individuals of the test
availability.Underthesecircumstances,thesedimentshouldbe
species and other macrobenthos, and second to adjust intersti-
tested as soon as possible after collection, but not later than
tial water to the test salinity if necessary. Water for sieving
within 2 weeks (20). Sediments that exhibit low-level to
should be clean sea water prepared in accordance with Section
moderate toxicity or contamination can exhibit considerable
9. The entire contents of the collecting basin, including water
temporal variability in toxicity or contamination, although the
and suspended particles, should be sieved (for example,
directionofchangeisoftenunpredictable (18,19,22).Forthese
through a 0.5mm screen) without allowing overflow from the
typesofsediments,therecommendedstoragetimeof<8weeks
sievingcontainer.Afterthefirstsieving,thesedimentshouldbe
may be most appropriate. In some situations, a minimum
leftundisturbedforasufficienttimetoallowthesettlingoffine
storage period for low-to-moderately contaminated sediments
particles (usually overnight). The overlying water should then
may help reduce variability. For example, DeFoe and Ankley
be decanted and the sediment resieved (for example, through a
(1998) (22) observed high variability in survival during early
0.5mm screen) into water of a salinity calculated to bring the
testing periods (for exa
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