ASTM E1563-21a

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: E1563 − 21a
Standard Guide for
Conducting Short-Term Chronic Toxicity Tests with Echinoid
1,2
Embryos
This standard is issued under the fixed designation E1563; 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* also Guide E1192), leachates, oils, particulate matter, surface
waters, effluents, and sediments (Annex A1). Renewal tests
1.1 This guide covers procedures for obtaining laboratory
mightbepreferabletostatictestsformaterialsthathaveahigh
dataconcerningtheshort-termchroniceffectsofatestmaterial
oxygen demand, are highly volatile, are rapidly transformed
on echinoderm embryos and the resulting larvae (sea urchins
biologically or chemically in aqueous solution, or are removed
and sand dollars) during static 48- to 96-h exposures. These
from test solutions in substantial quantities by the test cham-
procedures have generally been used with U.S. East Coast
bers or organisms during the test.
(Arbacia punctulata and Strongylocentrotus droebachiensis )
(1) andWestCoastspecies(Strongylocentrotus purpuratus, S. 1.4 Results of short-term chronic toxicity tests with echi-
noid embryos should usually be reported as the 50% effect
droebachiensis, and Dendraster excentricus) (2). The basic
procedures described in this guide first originated in Japan and concentration (EC50) based on the total abnormally developed
embryos and larvae. In some situations, it might only be
Scandanavia (3), and parallel procedures have been used with
foreign species, especially in Japan and the Mediterranean (4). necessary to determine whether a specific concentration is
toxic to embryos or whether the EC50 is above or below a
These procedures will probably be useful for conducting static
toxicity tests with embryos of other echinoid species, although specific concentration.
modifications might be necessary.
1.5 This guide is arranged as follows:
1.2 Other modifications of these procedures might be justi-
fied by special needs or circumstances.Although using proce-
dures appropriate to a particular species or special needs and
Section
Scope 1
circumstances is more important than following prescribed
Referenced Documents 2
procedures, the results of tests conducted by using unusual
Terminology 3
procedures are not likely to be comparable with those of many
Summary of Guide 4
Significance and Use 5
other tests. The comparison of results obtained by using
Apparatus 6
modified and unmodified versions of these procedures might
Facilities 6.1
provide useful information concerning new concepts and
Construction Materials 6.2
Test Chambers 6.3
procedures for conducting tests starting with embryos of
Cleaning 6.4
echinoids.
Acceptability 6.5
Safety Precautions 7
1.3 These procedures are applicable to most chemicals,
Dilution Water 8
either individually or in formulations, commercial products, or
Requirements 8.1
known mixtures. With appropriate modifications, these proce- Source 8.2
Treatment 8.3
dures can be used to conduct tests on temperature, dissolved
Characterization 8.4
oxygen,andpHandonsuchmaterialsasaqueouseffluents(see
Test Material 9
General 9.1
Stock Solution 9.2
Test Concentration(s) 9.3
A Standard Guide is a document, developed using the consensus mechanisms
Test Organisms 10
of ASTM that provides guidance for the selection of procedures to accomplish a
Species 10.1
specific test, but which does not stipulate specific procedures.
Age 10.2
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Source of Embryos 10.3
Assessment, Risk Management and CorrectiveAction and is the direct responsibil- Handling 10.4
Test Animal Source and Condition 10.5
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Spawning and Fertilization 10.6
Current edition approved Nov. 1, 2021. Published January 2022. Originally
Quality 10.7
approved in 1995. Last previous edition approved in 2021 as E1563–21. DOI:
Procedure 11
10.1520/E1563-21A.
Experimental Design 11.1
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
Dissolved Oxygen 11.2
this standard.
*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
E1563 − 21a
E1525GuideforDesigningBiologicalTestswithSediments
Temperature 11.3
Beginning the Test 11.4
E1706TestMethodforMeasuringtheToxicityofSediment-
Feeding 11.5
Associated Contaminants with Freshwater Invertebrates
Duration of Test 11.6
E1733Guide for Use of Lighting in Laboratory Testing
Biological Data 11.7
Control Performance 11.8
Other Measurements 11.9
3. Terminology
Analytical Methods 12
Acceptability of Test 13 3.1 Definitions:
Calculation of Results 14
3.1.1 The words “must,” “should,” “may,” “can,” and
Report 15
“might” have very specific meanings in this guide. “Must” is
Keywords 16
Annex
usedtoexpressanabsoluterequirement,thatis,tostatethatthe
Sediment Tests Annex A1
test ought to be designed to satisfy the specified condition,
unless the purpose of the test requires a different design.
“Must” is only used in connection with factors that directly
1.6 The values stated in SI units are to be regarded as the
relate to the acceptability of the test (see 13.1). “Should” is
standard.
used to state that the specified condition is recommended and
oughttobemetifpossible.Althoughviolationofone“should”
1.7 This standard does not purport to address all of the
statement is rarely a serious matter, violation of several will
safety concerns, if any, associated with its use. It is the
often render the results questionable. Terms such as “is
responsibility of the user of this standard to establish appro-
desirable,” “is often desirable,” and “might be desirable” are
priate safety, health, and environmental practices and deter-
used in connection with less important factors. “May” is used
mine the applicability of regulatory limitations prior to use.
to mean “is (are) allowed to,” “can” is used to mean “is (are)
Specific precautionary statements are given in Section 7.
able to,” and “might” is used to mean “could possibly.” Thus,
1.8 This international standard was developed in accor-
the classic distinction between “may” and “can” is preserved,
dance with internationally recognized principles on standard-
and “might” is never used as a synonym for either “may” or
ization established in the Decision on Principles for the
“can.”
Development of International Standards, Guides and Recom-
3.1.2 For definitions of other terms used in this guide, refer
mendations issued by the World Trade Organization Technical
to Guide E729 and Terminology E943. For sediment tests
Barriers to Trade (TBT) Committee.
(Annex A1), refer to Guides E1391 and E1525. For an
2. Referenced Documents
explanation of units and symbols, refer to Practice E380.
3.2 Definitions of Terms Specific to This Standard:
2.1 ASTM Standards:
3.2.1 embryo, n—the stages of a multicellular organism’s
E380Practice for Use of the International System of Units
developmentthatoccurbetweenthefertilizationoftheeggand
(SI) (the Modernized Metric System) (Withdrawn 1997)
the pluteus larva.
E724Guide for Conducting Static Short-Term ChronicTox-
3.2.2 pluteus larva, n—a juvenile lifecycle stage character-
icity Tests Starting with Embryos of Four Species of
Saltwater Bivalve Molluscs istic of all echinoids The term “embryo” is used herein to
denote the stages between the fertilization of the egg and the
E729Guide for Conducting Acute Toxicity Tests on Test
Materials with Fishes, Macroinvertebrates, and Amphib- pluteus larva. The term “larva” is used herein to refer to the
ians pluteus larva characteristic of all echinoids (5) (Fig. 1 and Fig.
E943Terminology Relating to Biological Effects and Envi- 2).
ronmental Fate
4. Summary of Guide
E1023Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
4.1 Adult sea urchins and sand dollars are brought into the
E1192Guide for ConductingAcute Toxicity Tests onAque-
laboratory and identified to species. If the gonads are not ripe,
ous Ambient Samples and Effluents with Fishes,
the sea urchins or sand dollars should be held and fed until the
Macroinvertebrates, and Amphibians
gonadsarebroughtintoasuitablereproductivestate.Echinoids
E1367TestMethodforMeasuringtheToxicityofSediment-
withripegonadsaremaintainedunderconditionsthatkeepthe
Associated Contaminants with Estuarine and Marine In-
gonads ripe without inducing undesired spontaneous spawning
vertebrates
or resorption of gametes. In order to start a test, spawning is
E1391Guide for Collection, Storage, Characterization, and
induced by using one or more stimuli, which may be physical
Manipulation of Sediments for Toxicological Testing and
(for example, heat or electrical current) or chemical (for
for Selection of Samplers Used to Collect Benthic Inver-
example, potassium chloride).
tebrates
4.2 In each of two or more treatments, embryos and the
resulting larvae of one species are maintained for 48 to 96 h,
depending on the species and test temperature. In each of one
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
or more control treatments, the embryos and resulting larvae
Standards volume information, refer to the standard’s Document Summary page on
are maintained in dilution water to which no test material has
the ASTM website.
been added in order to provide (1) a measure of the accept-
The last approved version of this historical standard is referenced on
www.astm.org. ability of the test by giving an indication of the quality of the
E1563 − 21a
FIG. 1 Drawings Exemplifying Key Developmental Stages of Normal Echinoid Larvae Occurring During the First 48 to 96 h of Develop-
ment and Examples of Abnormal or Arrested Development (6)
organisms and the suitability of the dilution water, test development test includes a sublethal endpoint, but is also
conditions, handling procedures, etc.; and (2) the basis for short in duration, these tests are considered to be short-term
interpretingdataobtainedfromtheothertreatments.Ineachof chronic tests, consistent with EPA guidance.
oneormoreothertreatments,theembryosandresultinglarvae
5.2 Because embryos and larvae are usually assumed to be
are maintained in dilution water to which a selected concen-
the most sensitive life stages of these echinoid species, and
tration of test material has been added.The EC50 is calculated
because some of these species are commercially and recre-
based on the proportion of larvae that develop into normal
ationally important, the results of these tests are often consid-
pluteus larvae in chambers containing the test material relative
ered to be a good indication of the acceptability of pollutant
to normal larvae in the controls at the termination of the test.
concentrations to saltwater species in general. The results of
these toxicity tests are often assumed to be an important
5. Significance and Use
consideration when assessing the hazard of materials to other
5.1 An acute toxicity test is conducted to assess effects of a
saltwater organisms (see Guides E724 and E1023) or when
short-term exposure of organisms to a test material under
deriving water quality criteria for saltwater organisms (7).
specific experimental conditions. An acute toxicity test does
not provide information concerning whether delayed effects 5.3 The results of short-term chronic toxicity tests might be
willoccurandtypicallyevaluateseffectsonsurvival.Achronic used to predict effects likely to occur to aquatic organisms in
test is typically longer in duration and includes a sublethal field situations as a result of exposure under comparable
endpoint to assess effects on a population that might occur conditions,exceptthattoxicitytobenthicspeciesmightdepend
beyond the exposure period. Because the echinoderm embryo on sorption or settling of the test material onto the substrate.
E1563 − 21a
FIG. 2 (a) Examples of Normal and Abnormal Development of
FIG. 2 (b) (continued)
Purple Sea Urchin (Strongylocentrotus purpuratus) Embryos at
the Conclusion of a 72 to 96-h Toxicity Test. Figs. 2a and 2b
Show Examples of Normal Echinopluteus’ with Four Distinct
Arms and Good Symmetrical Development. Fig. 2c Shows a Bor-
derline Abnormal Pluteus with Poor Symmetrical Form and One
5.8 Results of short-term chronic toxicity tests might be
Missing Arm. Figs. 2d through 2j Show Examples of Increasing
used to predict effects likely to occur to aquatic organisms
Abnormal and/or Retarded (Considered Abnormal) Development.
exposed to bedded whole sediments.
Other Species of Sea Urchins and Sand Dollars Will Have the
Same General Larval Form, But Will Vary in Size, Conformation,
and Number of Larval Arms. Each Investigator Using a Different
6. Apparatus
Species Should Carefully Compare Well-Developed Embryos
6.1 Facilities:
from Controls with Gradations of Abnormal Development in a
Toxicant to Identify Distinctions between Normal and Abnormal 6.1.1 Flow-throughtroughswithappropriatetraysshouldbe
for Their Given Species
available for holding and conditioning test animals (8). The
water-supply system should be equipped for temperature con-
trol and aeration (see 8.3) and should contain strainers and air
traps. Air used for aeration should be free of fumes, oil, and
5.4 The results of short-term chronic tests might be used to water; filters to remove oil and water are desirable. Test
compare the sensitivities of different species and the acute
chambersshouldbeinaconstant-temperatureroom,incubator,
toxicitiesofdifferenttestmaterials,andtodeterminetheeffects or recirculating water bath. A dilution-water tank or headbox,
of various environmental factors on the results of such tests.
which may be used to prepare reconstituted water, is often
elevated so that dilution water can be gravity-fed into holding
5.5 The results of short-term chronic toxicity tests might be
and conditioning troughs and test chambers. The facility
usefulforstudyingthebiologicalavailabilityof,andstructure-
should be well ventilated and free of fumes. To further reduce
activity relationships between, test materials.
the possibility of contamination of the test organisms by test
5.6 The results of any toxicity tests will depend on
materials and other substances, especially volatile ones, hold-
temperature,compositionofthedilutionwater,conditionofthe
ing and conditioning troughs should not be in a room in which
test organisms, and other factors.
thetoxicitytestsareconducted,stocksolutionsortestsolutions
5.7 Results of short-term chronic toxicity tests might be are prepared, or equipment is cleaned. Organisms should be
used to predict effects likely to occur to aquatic organisms shielded from disturbances, with curtains or partitions, to
exposed to suspended particulates of dredged sediments dis- prevent unnecessary stress during holding, conditioning, and
posed through the water column. testing.
E1563 − 21a
FIG. 2 (d) (continued)
FIG. 2 (c) (continued)
6.1.2 It is desirable, if feasible, to include some safeguards
in the system that supplies water to holding and conditioning
troughs. Monitors, possibly connected to auxiliary power
supplies, might be designed to initiate aeration, sound alarms,
or activate telephone autodialing alarms if the water flow or
temperature deviates from preset limits. If the temperature
becomestoohighorlow,correctiveactionshouldnotcausethe
temperatureofthewaterinholdingandconditioningtroughsto
increase or decrease more than 2°C/day to reduce the chances
of spontaneous spawning.
6.2 Construction Materials—Equipment and facilities that
contact stock solutions, test solutions, or any water into which
thetestorganismswillbeplacedshouldnotcontainsubstances
that can be leached or dissolved by aqueous solutions in
amounts that affect the test organisms adversely. In addition,
equipment and facilities that contact the stock solutions or test
solutions should be chosen to minimize the sorption of test
FIG. 2 (e) (continued)
materials from water. Glass, Type 316 stainless steel, nylon,
and fluorocarbon plastics should be used whenever possible to
of neoprene rubber and other materials not mentioned above
minimize dissolution, leaching, and sorption, except that stain-
should not be used unless it has been shown that the embryos
lesssteelshouldnotbeusedwhentestingmetals.Concreteand
and resulting larvae of the test species do not show more signs
rigid plastics may be used for holding and conditioning tanks
of stress, such as discoloration, abnormal development, or
and in the water-supply system, but they should be soaked,
death, when held for 48 to 96 h in the static dilution water in
preferablyinflowingdilutionwater,foraweekormorebefore
which the item is soaking than when held in static dilution
use (9). Brass, copper, lead, galvanized metal, cast-iron pipe,
water that does not contain the item.
and natural rubber should not contact the dilution water, stock
solutions,ortestsolutionsbeforeorduringthetest.Itemsmade 6.3 Test Chambers:
E1563 − 21a
FIG. 2 (f) (continued)
FIG. 2 (h) (continued)
should be cleaned before use. New items should be washed
with detergent and rinsed with water, a water-miscible organic
solvent, water, acid (such as 10% concentrated hydrochloric
acid), and at least twice with deionized, distilled, or dilution
water. (Some lots of some organic solvents might leave a film
that is insoluble in water.)At the end of the test, all items that
are to be used again should immediately be (1) emptied; (2)
rinsed with water; (3) cleaned by a procedure appropriate for
FIG. 2 (g) (continued)
removingthetestmaterialfromtheitem(forexample,acidfor
removingmetalsandbasesanddetergentororganicsolventfor
removingorganicchemicals);and(4)rinsedatleasttwicewith
6.3.1 Inatoxicitytestwithaquaticorganisms,testchambers
are defined as the smallest physical units between which there deionized, distilled, or dilution water. Acid is often used to
remove mineral deposits. A hypochlorite solution, often rec-
are no water connections. The chambers should be covered to
keepoutextraneouscontaminantsandbacteriaandtominimize ommendedasadisinfectionagentortoremoveorganicmatter,
should not be used due to the extreme toxicity of chlorine-
the evaporation of test solution and material. Substantial
concentrationsofbacteriainthetestsolutionsmightreducethe produced oxidants to echinoid larvae (11). The test chambers
survival of the embryos and resulting larvae severely, whereas should be rinsed with dilution water just before use.
differences in the amount of evaporation among test chambers
6.5 Acceptability— Before a test is begun with echinoid
will contribute directly to between-chamber variation in sur-
embryos in new test facilities, it is desirable to conduct a
vival. All chambers in a test must be identical.
“non-toxicant” test in which all test chambers contain dilution
6.3.2 Tests are usually conducted in glass chambers that are
water with no added test material to determine (1) whether
250mLto1Lincapacity.Verysmalltestchambers,containing
embryos will survive and develop acceptably (see 11.8); (2)
as little as 10 to 30 mL (10), and sealed test chambers may be
whether the dilution water, handling procedures, etc., are
used if the survival and development of the embryos and
acceptable; (3) whether there are any location effects on either
resulting larvae in the control(s) are acceptable (see 11.8).
survival or development; and (4) the magnitude of between-
6.4 Cleaning—Test chambers and equipment used to pre- chamber variance in the percentage of embryos that develop
pareandstoredilutionwater,stocksolutions,andtestsolutions into normal larvae. It is also highly recommended that each
E1563 − 21a
when washing equipment or putting hands in test solutions),
laboratorycoats,aprons,andglasses.Specialprecautions,such
as covering the test chambers and ventilating the area sur-
rounding the chambers, should be taken when conducting tests
on volatile materials. Information concerning toxicity to hu-
mans (12), recommended handling procedures (13), and
chemical and physical properties of the test material should be
studiedbeforeatestisbegun.Specialproceduresarenecessary
with radiolabeled test materials (14) and with materials that
are, or are suspected of being, carcinogenic (15).
7.2 Although in most cases the disposal of stock solutions,
test solutions, and test organisms poses no special problems,
healthandsafetyprecautionsandapplicableregulationsshould
be considered before beginning a test. Removal or degradation
of the test material might be desirable before disposal of the
stock and test solutions.
7.3 Cleaning of equipment with a volatile solvent such as
acetone should be performed only in a well-ventilated area in
whichnosmokingisallowedandnoopenflame,suchasapilot
light, is present.
7.4 Warning—Anacidicsolutionshouldnotbemixedwith
a hypochlorite solution because hazardous fumes might be
produced.
7.5 To prepare dilute acid solutions, concentrated acid
should be added to water, not vice versa. Opening a bottle of
concentratedacidandaddingconcentratedacidtowatershould
be performed only in a fume hood.
FIG. 2 (i) (continued)
7.6 Precaution—The use of ground-fault systems and leak
detectors is recommended strongly to help prevent electrical
shocks because salt water is a good conductor of electricity.
7.7 Care should be exercised when collecting and handling
sea urchins to avoid puncture wounds from spines. Where
possible, species with blunt spines should be selected over
those with long, sharp spines.
7.8 Preservation of larvae to facilitate microscopic enu-
meration will be performed with a fixative agent such as
buffered formalin, and biological stains (that is, Rose Bengal).
Appropriatesafetyprecautionsshouldbetakenwhenhandling.
8. Dilution Water
8.1 Requirements:
8.1.1 In addition to being available in adequate supply, the
dilution water should (1) be acceptable to adult echinoids and
theirembryosandlarvae,(2)beofuniformquality,and(3)not
affect the test results unnecessarily.
8.1.2 The minimal requirement for an acceptable dilution
FIG. 2 (j) (continued)
water for toxicity tests starting with embryos of sea urchins or
sand dollars is that at least 70% of the embryos resulting from
laboratorydevelopandmaintaina“controlchart”oftheresults
eggs and sperm produced by appropriately conditioned adults
of routine reference toxicant testing and control responses.
result in normal larvae while being maintained in the dilution
waterfor48to96h.Naturalsaltwatervariesinqualityenough
7. Safety Precautions
that even though it is usually acceptable, it might occasionally
7.1 Many materials can affect humans adversely if precau- be toxic to embryos or larvae if, for example, certain toxic
tions are inadequate. Therefore, skin contact with all test algae species are present such as some dinoflagellates (16).
materials and their solutions should be minimized by such 8.1.3 Thequalityofthedilutionwatershouldbesufficiently
means as wearing appropriate protective gloves (especially uniform that the test animals are held and conditioned and that
E1563 − 21a
TABLE 1 Reconstituted Salt Water
thetestisconductedinwaterofthesamequality.Inparticular,
the salinity should always be between 27 and 36 g/kg or parts
NOTE 1—Add the following reagent-grade chemicals (18) in the
per thousand (ppt) (17) and within a test should not vary by
amountsandorderlistedto890mLoffreshwater.Eachchemicalmustbe
A
dissolved before the next is added.
more than 1 ppt among treatments or any renewals during a
Chemical Amount
test. When a test is conducted on an effluent, brine, drilling
B
NaF 3 mg
mud, or other material that affects salinity when mixed with
SrClO20mg
2·6H
dilutionwater,itmightbedesirabletoadjustsalinitybyadding
H BO 30 mg
3 3
artificialseasaltsornaturalsalinebrinestoraisethesalinityor
KBr 100 mg
KCl 700 mg
by adding distilled or deionized water to lower the salinity. If
CaCl ·2H O 1470 mg
2 2
saltsareadded,theadjustedmaterialshouldbewellmixedand
Na SO 4000 mg
2 4
allowed to equilibrate for minimum of 2 h with gentle aeration
MgCl ·6H O 10 780 mg
2 2
NaCl 23 500 mg
(24 h preferred) and salinity confirmed as salts dissolve. The
Na SiO ·HO20mg
2 3 2
additionofartificialsaltscanproduceartifactualtoxicitythatis
NaHCO 200 mg
ameliorated with aging.
A
If the resulting solution is diluted to 1 L, the salinity should be 34 g/kg ± 0.2.The
8.1.4 The dilution water should not affect the test unneces-
reconstitutedwatershouldbestrippedoftracemetals(33).Ifnecessary,thewater
should be diluted to the desired salinity at the time of use.
sarily because of such things as sorption or complexation of
B
It is presently unknown if NaF affects the development of echinoid embryos and
test material. Therefore, concentrations of both total organic
larvae.InitialtestsshouldbeconductedwithandwithoutNaFifreconstitutedwater
carbon (TOC) and particulate matter should be less than 5
is used for test animal holding or test water.
mg/L in the dilution water. The concentrations of both TOC
and particulate matter can be greater than 5 mg/L in the water
in which the test animals are held and conditioned, since food
or COD should be measured at least twice per year and
will normally be present in the holding tanks.
whenever substantial changes might be expected. If the water
8.1.5 If it is desired to study the effect of an environmental
is prepared from a surface water, TOC or COD should be
factor such asTOC, particulate matter, or dissolved oxygen on
measuredoneachbatch.Problemshavebeenencounteredwith
theresultsofatest,itisnecessarytousewaterthatisnaturally
some species in reconstituted salt water, but sometimes these
or artificially high in TOC or particulate matter or low in
problems have been overcome by conditioning (aging) and
dissolved oxygen. If such water is used, it is important that
aerating the reconstituted water.
adequate analyses be performed to characterize the water and
8.2.3 Natural Dilution Water—If natural salt water is used,
that a comparable test be available or be conducted in a more
itshouldbeobtainedfromanuncontaminated,uniformquality
usual dilution water to facilitate interpretation of the results
source. The quality of saline well water is usually more
obtained in the special water.
uniform than that of saline surface water, but acceptability
based on embryo and larval survival and normal development
8.2 Source:
should be assessed. If surface water is used, it should be
8.2.1 Reconstituted Water—Use of reconstituted water is
obtained from an area known to support a healthy, naturally
often not worth the effort for tests with echinoid embryos
reproducing population of echinoids. The water intake should
because of (1) the large volume needed for holding and
bepositioned(forexample,approximately5to10mbelowthe
conditioning the test animals, (2) the necessity of providing
surface) to minimize fluctuations in quality and the possibility
adequate food for the test animals (see 10.5.5), and (3)
of contamination and to maximize the concentration of dis-
occasionally poor survival and development of the embryos
solved oxygen and healthy phytoplankton (see 10.5.5). A
and resulting larvae. Commercially available sea salts have
specially designed system is usually necessary to obtain salt
been used successfully for echinoid embryo testing given
water from a natural source (see Guide E729). Chlorinated
proper conditioning of the water. Be sure to use only salts that
water should not be used as, or in the preparation of, saline
are fast-dissolving and closely match the ion mix found in
dilution water because chlorine-produced oxidants are quite
naturalseawater.Atrialtestwithanynewbrandorbatchofsalt
toxictotheembryosandlarvaeofseaurchinsandsanddollars
is recommended prior to any testing of samples when feasible.
(15). Dechlorinated water should be used only as a last resort
Additionally,commerciallyavailableseasaltsshouldbefreeof
because dechlorination is often incomplete. Sodium bisulfite is
ethylenediaminetetraacetic acid (EDTA) or other chelating
probably better for dechlorinating water than sodium sulfite,
agents that may remove or mask toxicity in a sample that is
and both are more reliable than carbon filters, especially for
being tested.
removing chloramines (19). Some organic chloramines,
8.2.2 Reconstituted water is prepared by adding a commer-
however,reactslowlywithsodiumbisulfite (20).Inadditionto
cially available sea salt or specified amounts of reagent-grade
residualchlorine,municipaldrinkingwateroftencontainshigh
chemicals (18) to high-quality water with (1) conductivity less
concentrations of copper, lead, zinc, and fluoride, and the
than 1 µS/cm and (2) eitherTOC less than 2 mg/Lor chemical
quality is often rather variable. The concentrations of most
oxygen demand (COD) less than 5 mg/L. A formula for
metals can usually be reduced by using a chelating resin (21),
reconstituted water that may be acceptable for use with
but the use of different-dilution water might be preferable.
echinoids is given in Table 1. Acceptable water for the
dissolution of sea salts can usually be prepared by using a 8.3 Treatment:
properly operated deionization, distillation, or reverse osmosis 8.3.1 Unless natural seawater is used, dilution water should
unit.Conductivityshouldbemeasuredoneachbatch,andTOC be aerated intensively for 24 to 48 h by such means as air
E1563 − 21a
stones, surface aerators, or column aerators (22) before addi- 8.4.2 For each method used (see Section 12), the detection
tion of the test material. To prevent contamination with limit should be below either (1) the concentration in the
undesirable bacterial species during aeration, the air used dilution water or (2) the lowest concentration that has been
should be filtered through a 0.22-µm bacterial filter, the shown to affect embryos, larvae, or adults of sea urchins and
container should be covered, and aeration should not last for sand dollars unacceptably (26).
more than 48 h. Adequate aeration will bring the pH and
concentrations of dissolved oxygen and other gases into
9. Test Material
equilibrium with air and minimize the oxygen demand and
9.1 General—Test materials may include a range of sample
concentrations of volatiles. The concentration of dissolved
types such as effluents, materials or products, specific
oxygen in dilution water should be between 90 and 100%
chemicals, solvents, oils, surface waters, drilling fluids,
saturation (23) to help ensure that dissolved oxygen concen-
stormwater, and sediments. For chemical or product testing
trationsareacceptableinthetestchambers.Supersaturationby 6
studiesthetestmaterialshouldbereagent-grade (18) orbetter,
dissolved gases, which can be caused by heating the dilution
unless a test on a formulation, commercial product, or
water,shouldbeavoidedtopreventpossiblesymptomssimilar
technical-grade or use-grade material is specifically needed.
to gas-bubble disease in fish (22, 24).
Before a test is begun, the following should be known about
8.3.2 The salinity and pH of dilution water may be adjusted
the test material:
by the addition of appropriate reagent-grade chemicals (18),
9.1.1 Identities and concentrations of major ingredients and
sea salts or brine (especially to prevent excessive decreases in
major impurities, that is, impurities that constitute more than
salinity,see8.2),acid,base,anddeionizedordistilledwater,if
approximately 1% of the material.
it has been shown that the addition does not cause adverse
9.1.2 Solubility and stability in the dilution water.
effects on embryos, larvae, and adults of the test species at the
9.1.3 Measured or estimated toxicity to an aquatic species,
concentration used.
preferablythetestspeciesorlarvalstageofanotherechinoidor
8.3.3 Except possibly when holding and conditioning adult
marine invertebrate.
echinoids (see 10.5.5), filtration through bag, sand, sock, or
9.1.4 Precision and bias of the analytical method at the
depth-type (honeycomb) cartridge filters may be used to keep
planned concentration(s) of the test material, if the test con-
the concentration of particulate matter acceptably low (see
centrations are to be measured.
8.1.4) and as a pretreatment before ultraviolet sterilization or
9.1.5 Estimate of toxicity to humans.
filtration through a finer filter.
9.1.6 Recommended handling procedures (see 7.1).
8.3.4 Water that might be contaminated with facultative
9.2 Stock Solution:
pathogens may be passed through a properly maintained
9.2.1 In some cases, the test material can be added directly
ultraviolet sterilizer (25) equipped with an intensity meter and
to the dilution water, but often it is dissolved in a solvent to
flow controls or passed through a filter effective to 0.45 µm or
formastocksolutionthatisthenaddedtothedilutionwater.If
less.
a stock solution is used, the concentration and stability of the
8.3.5 Water from a surface water source should be passed
test material in it should be determined before beginning the
throughagradedseriesoffilters,withthefinesteffectiveto1.0
test. If the test material is subject to photolysis, the stock
µm or less to remove the embryos and larvae of marine
solution should be shielded from light.
animals, parasites, and predators. If bacteria are to be removed
9.2.2 Except possibly for tests on hydrolyzable, oxidizable,
by filtration, a filter effective to 0.45 µm or less must be used.
and reducible materials, the preferred solvent is dilution water,
Filtration through activated carbon may be used to remove
although filtration or sterilization (or both) of the water might
toxic algal exocrines and other organic chemicals.
be necessary. If the salinity of the test treatments will not be
8.4 Characterization:
affected, deionized or distilled water may be used. Several
8.4.1 Thefollowingitemsshouldbemeasuredatleasttwice
techniques have been developed specifically for preparing
per year and more often if such measurements have not been
aqueous stock solutions of slightly soluble materials (27). The
made semiannually for at least two years or if surface water is
minimum necessary amount of a strong acid or base may be
used: salinity (or chlorinity), pH, particulate matter, TOC,
used in the preparation of an aqueous stock solution, but such
organophosphorus pesticides, organic chlorine (or organochlo-
reagents might affect the pH of test solutions appreciably. Use
rine pesticides plus PCBs), chlorinated phenoxy herbicides,
of a more soluble form of the test material, such as chloride or
ammonia, cyanide, sulfide, bromide, fluoride, iodide, nitrate,
sulfate salts of organic amines, sodium or potassium salts of
phosphate, sulfate, calcium, magnesium, potassium,
phenols and organic acids, and chloride or nitrate salts of
aluminum, arsenic, beryllium, boron, cadmium, chromium,
metals, might affect the pH more than use of the minimum
cobalt, copper, iron, lead, manganese, mercury, molybdenum,
necessary amount of a strong acid or base.
nickel, selenium, silver, and zinc.
9.2.3 If a solvent other than dilution water is used, its
concentration in test solutions should be kept to a minimum
and should be low enough that it does not affect the test
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
species. Because of its low toxicity to aquatic animals, low
listed by the American Chemical Society, see Analar Standards for Laboratory
volatility, and high ability to dissolve many organic chemicals,
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
triethylene glycol is often a good organic solvent for preparing
and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
MD. stock solutions (28). Other water-miscible organic solvents
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such as methanol, ethanol, and acetone may also be used, but exposed to concentrations above solubility.The use of concen-
they might stimulate undesirable growths of microorganisms, trations that are more than ten times greater than solubility is
and acetone is also quite volatile. If an organic solvent is used, probably not worthwhile.With some test materials, it might be
itshouldbereagent-grade (18)orbetter, anditsconcentration found that concentrations above solubility do not kill or affect
in any test solution should not exceed 0.5 mL/L. A surfactant a greater percentage of test organisms than does the concen-
should not be used in the preparation of a stock solution tration that is the solubility limit; such information is certainly
becauseitmightaffecttheformandtoxicityofthetestmaterial worth knowing.
in the test solutions. (These limitations do not apply to any
9.3.3 In some situations, usually related to regulatory
ingredient of a mixture, formulation, or commercial product
activities, it is necessary to determine only whether (1)a
unless an extra amount of solvent is used in preparation of the
specificconcentrationoftestmaterialistoxictotheembryosor
stock solution.)
larvae of the test species or (2) the EC50 is above or below a
9.2.4 If no solvent other than water is used, only a dilution-
specific concentration. For example, the specific concentration
water control must be included in the test. For echinoids, at
might be the concentration occurring in a surface water, the
least70%oftheembryosintroducedintothecontroltreatment
concentration resulting from the direct application of the
must result in normal larvae at the end of the test. These
material to a body of water, or the solubility limit of the
stipulationsmaybespecies-specificandmaybetoohighortoo
material in water. When there is only interest in a specific
low for some lesser tested echinoid species.
concentration, it is only necessary to test only that concentra-
9.2.5 If a solvent other than water is used and the concen- tion plus a control (see 11.1), and it is not necessary to
tration of solvent is the same in all test solutions that contain
determine an EC50.
test material, a solvent control, containing the same concen-
tration of solvent as the test solutions and using solvent from
10. Test Organisms
the same batch used to make the stock solution, must be
10.1 Species—Whenever possible, either Atlantic sea ur-
includedinthetest.Inaddition,adilution-watercontrolshould
chins (Arbacia punctulata), green sea urchins (Strongylocen-
be included in the test. The number of embryos that result in
trotus droebachiensis), available on the northern Atlantic and
normallarvaeattheendofthetestmustbeatleast70%ofthe
Pacific coasts, Pacific purple sea urchins (S. purpuratus), or
initialnumberinthesolventcontrol.Ifadilution-watercontrol
Pacific eccentric sand dollars (Dendraster excentricus) should
is included in the test, the number of embryos that result in
be used as the test species. These species were selected on the
normal larvae at the end of the test should be at least 70% of
basis of availability, commercial importance of several of the
the initial number in the dilution-water control.
species, past successful use, and ease of handling in the
9.2.6 If a solvent other than water is used and the concen-
laboratory. Their use is encouraged to increase the compara-
tration of solvent is not the same in all test solutions that
bility of results and availability of much information on a few
contain test material, both a solvent control, containing the
species rather than little information on many species. The
highest concentration of solvent present in any other treatment
species used should be identified by using an appropriate
and using solvent from the same batch used to make the stock
taxonomickey.Successfultoxicitytestscanbeconductedwith
solution, and a dilution-water control must be included in the
embryosofotherechinoidspecies,butthecomparabilityofthe
test.The number of embryos that result in normal larvae at the
results will be less.
endofthetestmustbeatleast70%oftheinitialnumberinthe
solvent control and in the dilution-water control. 10.2 Age—The test must be begun with embryos within 4 h
afterfertilization,whentheembryosareinthe2-,4-,and8-cell
9.2.7 If a solvent other than water is used to prepare a stock
stages.
solution,itmightbedesirabletoconductsimultaneoustestson
the test material using two chemically unrelated solvents or
10.3 Source of Embryos—Embryos used to begin a test can
two different concentrations of the same solvent to obtain
be obtained from females and males that have been collected
information concerning possible effects of the solvent on the
freshly from the field or that had been maintained in the
results of the test.
dilution water in the laboratory before they were stimulated to
9.3 Test Concentration(s): spawn.
9.3.1 If the test is intended to allow the calculation of an
10.4 Handling—The organisms should be handled as little
EC50, the test concentrations should bracket the predicted
as possible. When handling is necessary, it should be done
EC50. The prediction might be based on the results of a test
carefully, gently, and quickly, so that the organisms are not
with the same or a similar test material and the same or a
stressed unnecessarily.Adults that are injured during handling
similar species. If a useful prediction is not available, it is
should be discarded. Equipment used to transfer embryos (but
usually desirable to conduct a range-finding test in which
not exposed to toxicants) should be cleaned between uses by
embryosandresultinglarvaeareexposedforatotalof48to96
washing with clean sea water or distilled or deionized water.
h to a control and three to five concentrations of the test
Handsshouldbewashedbeforeandafterhandlingbroodstock.
materialthatdifferbyafactoroften.Thegreaterthesimilarity
10.5 Test Animal Source and Condition:
between the range-finding test and the actual test, the more
useful the range-finding test will be.
10.5.1 For any one test or a series of related tests, all test
9.3.2 Ifnecessary,concentrationsabovesolubilityshouldbe animals should be collected from the same location, which
used because organisms in the real world are sometimes should be known precisely. The test animals may be obtained
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from a commercial source only if the original specific collec- availabilityofgoodqualityeggs;spermforeggfertilizationare
tion location of the sea urchins or sand dollars can be almost always available.
identified.Tominimizethepossibilityofgeneticorphysiologi-
10.5.4 When the test animals are first brought into the
cal adaptation to chemicals or aberrant water quality, the
laboratory, they should be acclimated to dilution water over a
organisms should be collected from a location that is not
period of two or more days to prevent stress due to abrupt
subject to obvious point or non-point source pollution and has
changes in water quality. The temperature may generally be
water that is comparable in quality to that which will be used
changed at a rate not to exceed 2°C⁄day, and the salinity at a
for the holding and testing. Mature individuals should be
rate not to exceed 1 g/kg/day. An abrupt increase in tempera-
obtained. Sex ratios are generally 1:1, although sea urchins
ture or salinity might not only induce spawning, especially of
maysometimesaggregatebysexinthefield.Hermaphroditism
males, but also harm the gametes seriously (30) and kill the
is rare but can occur. The echinoid species, like bivalve
adults. Following proper acclimation to the laboratory holding
mollusks,areseasonallygravid,butconoftenbemaintainedin
conditions, some test animals can be test-spawned to check
a gravid condition with proper laboratory manipulation of the
gamete quality if enough animals are available. The animals
holdingconditions(thatis,temperature,light,andsalinity)and
can be spawned by the method of choice (see 10.6.2) and the
foodsupply(bothquantityandquali
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