ASTM E1191-03A(2023)e1

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.
´1
Designation: E1191 − 03a (Reapproved 2023)
Standard Guide for
Conducting Life-Cycle Toxicity Tests with Saltwater Mysids
This standard is issued under the fixed designation E1191; 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.
ε NOTE—Section 13.1.11 and References were editorially corrected in January 2023.
1. Scope
Construction Materials 6.2
Metering System 6.3
1.1 Thisguidedescribesproceduresforobtaininglaboratory
Test Chambers 6.4
data concerning the adverse effects of a test material added to Cleaning 6.5
Acceptability 6.6
dilution water, but not to food, on certain species of saltwater
Dilution Water 8
mysids during continuous exposure from immediately after
Requirements 8.1
birth until after the beginning of reproduction using the Source 8.2
Treatment 8.3
flow-through technique. These procedures will probably be
Characterization 8.4
usefulforconductinglife-cycletoxicitytestswithotherspecies
Test Material 9
General 9.1
of mysids, although modifications might be necessary.
Stock Solution 9.2
1.2 Other modifications of these procedures might be justi-
Test Concentration(s) 9.3
Test Organisms 10
fied by special needs or circumstances.Although using appro-
Species 10.1
priate procedures is more important than following prescribed
Age 10.2
procedures,resultsoftestsconductedusingunusualprocedures
Source 10.3
Brood Stock 10.4
are not likely to be comparable to results of many other tests.
Food 10.5
Comparisonofresultsobtainedusingmodifiedandunmodified
Handling 10.6
versions of these procedures might provide useful information
Harvesting Young 10.7
Quality 10.8
on new concepts and procedures for conducting life-cycle
Procedure 11
toxicity tests with saltwater mysids.
Experimental Design 11.1
Dissolved Oxygen 11.2
1.3 These procedures are applicable to all chemicals, either
Temperature 11.3
individually or in formulations, commercial products, or
Beginning the Test 11.4
known mixtures, that can be measured accurately at the Feeding 11.5
Cleaning 11.6
necessary concentrations in water. With appropriate
Duration of Test 11.7
modifications,theseprocedurescanbeusedtoconducttestson
Biological Data 11.8
Other Measurements 11.9
temperature, dissolved oxygen, and pH and on such materials
Analytical Methodology 12
as aqueous effluents (see also Guide E1192), leachates, oils,
Acceptability of Test 13
particulate matter, sediments, and surface waters.
Calculation 14
Documentation 15
1.4 This guide is arranged as follows:
Keywords 16
Appendix
Section
X1. Statistical Guidance
Referenced Documents 2
1.5 This standard does not purport to address all of the
Terminology 3
safety concerns, if any, associated with its use. It is the
Summary of Guide 4
Significance and Use 5
responsibility of the user of this standard to establish appro-
Hazards 7
priate safety, health, and environmental practices and deter-
Apparatus 6
mine the applicability of regulatory limitations prior to use.
Facilities 6.1
Specific hazard statements are given in Section 7.
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Assessment,RiskManagementandCorrectiveActionandisthedirectresponsibility
ization established in the Decision on Principles for the
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Development of International Standards, Guides and Recom-
Current edition approved Jan. 1, 2023. Published January 2023. Originally
mendations issued by the World Trade Organization Technical
approved in 1987. Last previous edition approved in 2008 as E1191–03a(2008).
DOI: 10.1520/E1191-03AR23E01. Barriers to Trade (TBT) Committee.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E1191 − 03a (2023)
2. Referenced Documents and (2) the basis for interpreting data obtained from the other
2 treatments. In each of the one or more other treatments, the
2.1 ASTM Standards:
mysids are maintained in dilution water to which a selected
E729Guide for Conducting Acute Toxicity Tests on Test
concentrationoftestmaterialhasbeenadded.Specifieddataon
Materials with Fishes, Macroinvertebrates, and Amphib-
theconcentrationoftestmaterial,andthesurvival,growth,and
ians
reproduction of the mysids are obtained and analyzed to
E943Terminology Relating to Biological Effects and Envi-
3 determine the effect(s) of the test material on survival, growth,
ronmental Fate (Withdrawn 2023)
and reproduction of the test organisms.
E1023Guide for Assessing the Hazard of a Material to
Aquatic Organisms and Their Uses
5. Significance and Use
E1192Guide for ConductingAcute Toxicity Tests onAque-
5.1 Protection of a species requires prevention of unaccept-
ous Ambient Samples and Effluents with Fishes,
able effects on the number, weight, health, and uses of the
Macroinvertebrates, and Amphibians
individuals of that species. A life-cycle toxicity test is con-
E1203Practice for Using Brine Shrimp Nauplii as Food for
ducted to determine what changes in the numbers and weights
Test Animals in Aquatic Toxicology (Withdrawn 2013)
of individuals of the test species result from effects of the test
IEEE/ASTM SI 10American National Standard for Use of
material on survival, growth, and reproduction. Information
theInternationalSystemofUnits(SI):TheModernMetric
might also be obtained on effects of the material on the health
System
and uses of the species.
3. Terminology
5.2 Results of life-cycle tests with mysids might be used to
predict long-term effects likely to occur on mysids in field
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
have very specific meanings in this guide. situationsasaresultofexposureundercomparableconditions.
3.1.1 “Must” is used to express an absolute requirement,
5.3 Results of life-cycle tests with mysids might be used to
that is, to state that the test ought to be designed to satisfy the
compare the chronic sensitivities of different species and the
specified condition, unless the purpose of the test requires a
chronic toxicities of different materials, and also to study the
differentdesign.“Must”isonlyusedinconnectionwithfactors
effectsofvariousenvironmentalfactorsonresultsofsuchtests.
that directly relate to the acceptability of the test (see 13.1).
5.4 Results of life-cycle tests with mysids might be an
3.1.2 “Should”isusedtostatethatthespecifiedconditionis
important consideration when assessing the hazards of materi-
recommended and ought to be met if possible. Although
als to aquatic organisms (see Guide E1023) or when deriving
violationofone“should”israrelyaseriousmatter,violationof
water quality criteria for aquatic organisms (1).
several will often render the results questionable. Terms such
as“isdesirable,”“isoftendesirable,”and“mightbedesirable” 5.5 Results of a life-cycle test with mysids might be useful
are used in connection with less important factors. for predicting the results of chronic tests on the same test
3.1.3 “May” is used to mean “is (are) allowed to,” “can” is materialwiththesamespeciesinanotherwaterorwithanother
used to mean “is (are) able to,” and “might” is used to mean species in the same or a different water (2). Most such
“could possibly.” Therefore, the classic distinction between predictionstakeintoaccountresultsofacutetoxicitytests,and
may and can is preserved, and might is never used as a so the usefulness of the results from a life-cycle test with
synonym for either may or can. mysids is greatly increased by also reporting the results of an
acute toxicity test (see Guide E729) conducted under the same
3.2 Fordefinitionsofothertermsusedinthisguide,referto
conditions.
Guide E729, Terminology E943, and Guide E1023. For an
explanation of units and symbols, refer to IEEE/ASTM SI 10. 5.6 Results of life-cycle tests with mysids might be useful
for studying the biological availability of, and structure-
4. Summary of Guide
activity relationships between, test materials.
4.1 In each of two or more treatments, saltwater mysids of
5.7 Results of life-cycle tests with mysids might be useful
one species are maintained in two or more test chambers from
forpredictingpopulationeffectsonthesamespeciesinanother
immediately after birth until after the beginning of reproduc-
water or with another species in the same or a different water
tion in a flow-through system. In each of the one or more
(3).
controltreatments,themysidsaremaintainedindilutionwater,
to which no test material has been added, in order to provide 6. Apparatus
(1) a measure of the acceptability of the test by giving an
6.1 Facilities—Flow-through or recirculating brood-stock
indicationofthequalityofthemysidsandthesuitabilityofthe
tanks and flow-through, but not recirculating, test chambers
dilution water, food, test conditions, and handling procedures
should be maintained in constant-temperature areas or recircu-
lating water baths.An elevated headbox might be desirable so
dilutionwatercanbegravity-fedintobrood-stocktanksandthe
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
metering system (see 6.3), which mixes and delivers test
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 ASTM website.
3 4
The last approved version of this historical standard is referenced on Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
www.astm.org. this guide.
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E1191 − 03a (2023)
solutionstothetestchambers.Strainersandairtrapsshouldbe valves have been used successfully to control the concentra-
included in the water supply system. Headboxes and brood- tionsoftestmaterialin,andtheflowratesof,testsolutions(see
stock tanks should be equipped for temperature control and Guide E729).
aeration (see 8.3). Air used for aeration should be free of
6.3.2 The metering system should be calibrated before the
fumes, oil, and water; filters to remove oil and water are
testbydeterminingtheflowratethrougheachtestchamberand
desirable. Filtration of air through a 0.22-µm bacterial filter
measuring either the concentration of test material in each test
might be desirable. The facility should be well ventilated and
chamber or the volume of solution used in each portion of the
free of fumes. To further reduce the possibility of contamina-
meteringsystem.Thegeneraloperationofthemeteringsystem
tion by test materials and other substances, especially volatile
should be visually checked twice daily, in the morning and
ones, the brood-stock tanks should not be in a room in which
afternoon, throughout the test. The metering system should be
toxicitytestsareconducted,stocksolutionsortestsolutionsare
adjusted during the test if necessary and any malfunction or
prepared, or equipment is cleaned. During culture and testing, adjustment should be noted in the study records.
organisms should be shielded from disturbances with curtains
6.3.3 The flow rate through each test chamber should be at
or partitions to prevent unnecessary stress. A timing device
least five volume additions per 24 h. It is usually desirable to
should be used to provide either a 14-h light and 10-h dark or
construct the metering system to provide at least ten volume
a 16-h light and 8-h dark photoperiod. A 15 to 30-min
additionsper24hincasethereisrapidlossoftestmaterialdue
transition period (4) should be provided whenever lights go on
to microbial degradation, hydrolysis, oxidation, photolysis,
or off to reduce the possibility of mysids being stressed by
reduction, sorption, or volatilization (see 11.4.2). At any
instantaneous changes in light intensity. In the natural
particular time during the test, the flow rates through any two
environment, the normal vertical migration of mysids allows
test chambers should not differ by more than 10%. Flow rates
gradual acclimation to light intensity. Under artificial labora-
through all test chambers may be equally changed simultane-
tory conditions, some mysids exhibit an escape response to
ously during the test as long as the test temperature (see 11.3)
sudden increases or decreases in light intensity resulting in
and the concentrations of dissolved oxygen and test material
jumping and impingement on the sides of test chambers or
(see 11.4.1 and 11.9.3) remain acceptable (see 11.3, 11.9, and
compartments.
13).
6.2 Construction Materials—Equipment and facilities that
6.4 Test Chambers:
contact stock solutions, test solutions, or any water into which
6.4.1 Inatoxicitytestwithaquaticorganisms,testchambers
mysidswillbeplacedshouldnotcontainsubstancesthatcanbe
are defined as the smallest physical units between which there
leached or dissolved by aqueous solutions in amounts that
are no water connections. However, screens and cups may be
adversely affect mysids. In addition, equipment and facilities
usedtocreatetwoormorecompartmentswithineachchamber.
that contact stock solutions or test solutions should be chosen
Therefore, test solution can flow from one compartment to
to minimize sorption of test materials from water. Glass, Type
another within a test chamber, but, by definition, cannot flow
316 stainless steel, nylon, Teflon, and fluorocarbon plastics
from one chamber to another. Because solution can flow from
should be used whenever possible to minimize dissolution,
one compartment to another in the same test chamber, the
leaching, and sorption. Stainless steel should not be used for
temperature, concentration of test material, and levels of
tests on metals. Concrete and rigid plastics may be used for
pathogens and extraneous contaminants are likely to be more
brood-stock tanks and in the water supply, but they should be
similar between compartments in the same test chamber than
soaked, preferably in flowing dilution water, for a week or
between compartments in different test chambers in the same
morebeforeuse (5).Castironpipeshouldnotbeusedwithsalt
treatment.Chambersshouldbecoveredtokeepoutextraneous
water. Specially designed systems are usually necessary to
contaminantsandtoreduceevaporationoftestsolutionandtest
obtainsaltwaterfromanaturalwatersource(seeGuideE729).
material. All chambers and compartments in a test must be
Brass, copper, lead, galvanized metal, and natural rubber
identical.
should not contact dilution water, stock solutions, or test
6.4.2 Testchambersmaybeconstructedbywelding,butnot
solutions before or during the test. Items made of neoprene
soldering, stainless steel or by gluing double-strength or
rubber or other materials not mentioned previously should not
stronger window glass with clear silicone adhesive. Stoppers
be used unless it has been shown that their use will not
and silicone adhesive sorb some organochlorine and organo-
adversely affect either survival, growth, or reproduction of
phosphoruspesticidesthataredifficulttoremove.Therefore,as
mysids (see 13.1.9 and 13.1.10).
few stoppers and as little adhesive as possible should be in
6.3 Metering System: contactwithtestsolution.Ifextrabeadsofadhesiveareneeded
for strength, they should be on the outside of chambers rather
6.3.1 The metering system should be designed to accom-
than on the inside.
modate the type and concentration(s) of test material and the
necessary flow rates of test solutions. The system should 6.4.3 Mysids should be exposed in compartments that are
permit the mixing of the test material with dilution water placed within test chambers. Compartments that have been
immediatelybeforeentrancetothetestchambers(see11.9.3.4) used successfully include (1) 140-mm inside diameter glass
and permit the supply of selected concentration(s) of test Petri dish bottoms with collars made of 210 or 250-µm mesh
materialinareproduciblefashion(see9.3and11.1.1).Various nylon screen (6, 7), and (2) 110 by 180 by 200-mm deep glass
metering systems, using different combinations of syringes, rectangular chambers partitioned into compartments with a
dipping birds, siphons, pumps, saturators, solenoids, and 65-mmhigh,330-µmmeshnyloncollar (8).Thecompartments
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E1191 − 03a (2023)
may be removed to a light table (illuminated from the bottom, mysids from test solutions. Special precautions, such as cov-
such as used for viewing slides) for observation, or the test ering test chambers and ventilating the area surrounding the
chambers may be permanently located on a light table. To chambers, should be taken when conducting tests on volatile
ensure that test solution regularly flows into and out of each materials. Information on toxicity to humans (11), recom-
compartment, either (1) test solution should flow directly into mended handling procedures (12), and chemical and physical
the compartments, (2 ) the compartments should be oscillated properties of the test material should be studied before a test is
in the test solution by means of a rocker arm apparatus driven begun. Special procedures might be necessary with radiola-
bya1to6r/minelectricmotor (9),or(3)thewaterlevelinthe beled materials (13) and with test materials that are, or are
test chamber should be varied by means of a self-starting suspected of being, carcinogenic (14).
siphon (10).Themeteringsystem,testchambers,andcompart-
7.2 Although disposal of stock solutions, test solutions, and
ments should be constructed so that the mysids remain sub-
test organisms poses no special problems in most cases, health
merged and are not unacceptably stressed by crowding or
and safety precautions and applicable regulations should be
turbulence. Best survival and reproduction are obtained when
considered before beginning a test. Removal or degradation of
the compartment provides a surface area of at least 30 cm per
test material might be desirable before disposal of stock and
mysid and a solution depth of at least 25 mm (7) at all times.
test solutions.
6.4.4 Use of excessively large volumes of solution in test
chambers will probably unnecessarily increase the amount of 7.3 Cleaning of equipment with a volatile solvent, such as
dilutionwaterandtestmaterialused,andtheaverageretention
acetone, should be performed only in a well-ventilated area in
time.All glass chambers that are 300 by 450 by 150-mm deep whichnosmokingisallowedandnoopenflame,suchasapilot
containing a minimum test solution depth of 100 mm and
light, is present.
adequate compartments have been successfully used.
7.4 Anacidicsolutionshouldnotbemixedwithahypochlo-
6.5 Cleaning—The metering system, test chambers,
rite solution because hazardous fumes might be produced.
compartments, and equipment used to prepare and store
7.5 To prepare dilute acid solutions, concentrated acid
dilution water, stock solutions, and test solutions should be
should be added to water, not vice versa. Opening a bottle of
cleaned before use. New items should be washed with deter-
concentratedacidandaddingconcentratedacidtowatershould
gent and rinsed with water, a water-miscible organic solvent,
be performed only in a fume hood.
water,acid(suchas10%concentratedhydrochloricacid),and
at least twice with deionized, distilled, or dilution water. A
7.6 Use of ground fault systems and leak detectors is
dichromatesulfuricacidcleaningsolutionmaybeusedinplace
strongly recommended to help prevent electrical shocks be-
of both the organic solvent and the acid, but it might attack
cause salt water is a good conductor of electricity.
silicone adhesive. At the end of the test, all items that will be
used again should be immediately (1) emptied, (2) rinsed with
8. Dilution Water
water,(3)cleanedbyaprocedureappropriateforremovingthe
8.1 Requirements—The dilution water should (1)bein
test material (for example, acid to remove metals and bases;
adequate supply, (2) be acceptable to saltwater mysids, (3)be
detergent, organic solvent, or activated carbon to remove
of uniform quality, and (4) except as stated in 8.1.4, not
organic chemicals), and (4) rinsed at least twice with
unnecessarily affect results of the test.
deionized, distilled, or dilution water. Acid is often used to
remove mineral deposits. The metering system, test chambers, 8.1.1 The dilution water must allow satisfactory survival,
and compartments should be rinsed with dilution water just growth, and reproduction of saltwater mysids (see 13.1.9 and
before use. 13.1.10).
8.1.2 The quality of the dilution water should be uniform
6.6 Acceptability—Before a life-cycle test is conducted in
during the test. During the test each measured salinity should
new test facilities, it is desirable to conduct a nontoxicant test,
bebetween15g/kgand30g/kg,andthedifferencebetweenthe
inwhichalltestchamberscontaindilutionwaterwithnoadded
highest and lowest measured salinities should be less than 5
test material, to determine before the first test (1) whether
g/kg and must be less than 10 g/kg. Each measured pH should
mysids will survive, grow, and reproduce acceptably (see
be between 6.6 and 8.2.
13.1.9 and 13.1.10) in the new facilities, (2) whether the food,
water, and handling procedures are acceptable, (3) whether 8.1.3 The dilution water should not unnecessarily affect
results of a life-cycle test with mysids because of such things
there are any location effects on either survival, growth, or
reproduction, and (4) the magnitudes of the within-chamber as sorption or complexation of test material. Therefore, except
as stated in 8.1.4, concentrations of both total organic carbon
and between-chamber variances.
(TOC) and particulate matter should be less than 5 mg/L.
7. Hazards
8.1.4 If it is desired to study the effect of an environmental
7.1 Many materials can adversely affect humans if precau- factorsuchasTotalOrganicCarbon,(TOC),particulatematter,
tions are inadequate. Therefore, skin contact with all test or dissolved oxygen on the results of a life-cycle test with
materials and solutions should be minimized by wearing mysids, it will be necessary to use water that is naturally or
appropriate protective gloves (especially when washing equip- artificially high in TOC or particulate matter or low in
ment or putting hands into test solutions), laboratory coats, dissolved oxygen. If such water is used, it is important that
aprons, and glasses, and by using pipets or dip nets to remove adequate analyses be performed to characterize the water and
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E1191 − 03a (2023)
that a comparable test be available or be conducted in a more adverse effects on either survival, growth, or reproduction of
usual dilution water to facilitate interpretation of the results in saltwater mysids at the concentration used.
the special water.
8.4 Characterization:
8.2 Source: 8.4.1 Thefollowingitemsshouldbemeasuredatleasttwice
8.2.1 Some reconstituted salt waters prepared from either each year and more often if such measurements have not been
reagent-grade chemicals or sea salts have been shown to be madesemi-annuallyforatleasttwoyearsorifsurfacewateris
acceptable for life-cycle toxicity tests with saltwater mysids used: salinity (or chlorinity), pH, particulate matter, TOC,
(15). It might be desirable to condition (age) reconstituted salt organophosphorus pesticides, organic chlorine (or organochlo-
water by aerating it for two or more days. rine pesticides and polychlorinated biphenyls, (PCBs)), chlo-
8.2.2 Ifnaturalsaltwaterisused,itshouldbeobtainedfrom rinated phenoxy herbicides, ammonia, cyanide, sulfide,
anuncontaminated,uniformqualitysource.Thequalityofwell bromide, fluoride, iodide, nitrate, phosphate, sulfate, calcium,
water is usually more uniform than surface water. If surface magnesium, potassium, aluminum, arsenic, beryllium, boron,
water is used, the intake should be positioned (for example, cadmium, chromium, cobalt, copper, iron, lead, manganese,
mercury, molybdenum, nickel, selenium, silver, and zinc.
suspendedabout1mbelowafloat)tominimizefluctuationsin
quality and the possibility of contamination and to maximize 8.4.2 The methods used (see 12.3) should either (1)be
accurate and precise enough to adequately characterize the
the concentration of dissolved oxygen to help ensure low
concentrations of sulfide and iron. dilutionwateror(2)havedetectionlimitsbelowconcentrations
thathavebeenshowntoadverselyaffectsaltwatermysids (24).
8.2.3 Chlorinated water should not be used as, or in the
preparation of, dilution water because chlorine-produced oxi-
9. Test Material
dants are quite toxic to mysids. Dechlorinated water should be
used only as a last resort because dechlorination is often
9.1 General—The test material should be reagent-grade or
incomplete. Sodium bisulfite is probably better for dechlori-
better, unless a test on a formulation, commercial product, or
nating water than sodium sulfite and both are more reliable
technical-grade or use-grade material is specifically needed.
than carbon filters, especially for removing chloramines (16).
Before a test is begun, the following should be known about
Someorganicchloramines,however,reactslowlywithsodium
the test material:
bisulfite (17). In addition to residual chlorine, municipal
9.1.1 Identities and concentrations of major ingredients and
drinkingwateroftencontainsunacceptablyhighconcentrations
major impurities, for example, impurities constituting more
of copper, lead, zinc, and fluoride and the quality is often
than about 1% of the material.
variable. When necessary, excessive concentrations of most
9.1.2 Solubility and stability in dilution water.
metals can usually be removed by a chelating resin (18), but
9.1.3 Acute toxicity to the test species.
use of a different water might be preferable.
9.1.4 A measurement or estimate of chronic toxicity to the
test species.
8.3 Treatment:
9.1.5 Precision and bias of the analytical method at the
8.3.1 Dilution water should be aerated intensively by using
planned concentration(s) of the test material.
air stones, surface aerators, or column aerators (19, 20) before
9.1.6 Estimate of toxicity to humans.
addition of test material. Adequate aeration will bring the pH
9.1.7 Recommended handling procedures (see 7.1).
and concentrations of dissolved oxygen and other gases into
equilibrium with air and minimize oxygen demand and con-
9.2 Stock solution:
centrationsofvolatiles.Theconcentrationofdissolvedoxygen
9.2.1 In some cases, the test material can be added directly
indilutionwatershouldbebetween90and100%ofsaturation
to the dilution water in the metering system, but usually it is
(21)tohelpensurethatdissolvedoxygenconcentrationsinthe
dissolved in a solvent to form a stock solution that is then
test chambers are acceptable. Supersaturation by dissolved
added to the dilution water in the metering system. If a stock
gases, which can be caused by heating the dilution water,
solution is used, the concentration and stability of the test
should be avoided to prevent gas bubble disease (20, 22).
materialinitshouldbedeterminedbeforethebeginningofthe
8.3.2 Filtration through bag, sand, sock, or depth-type
test. If the test material is subject to photolysis, the stock
cartridge filters may be used to keep the concentration of
solution should be shielded from light.
particulate matter acceptably low (see 8.1.3) and as a pretreat-
9.2.2 Except possibly for tests on hydrolyzable, oxidizable,
ment before ultraviolet sterilization or filtration through a finer
and reducible materials, the preferred solvent is dilution water,
filter, or both.
although filtration or sterilization, or both, might be necessary.
8.3.3 Dilution water that might be contaminated with fac-
If the salinity of the dilution water will not be affected,
ultative pathogens may be passed through a properly main-
deionized or distilled water may be used. Several techniques
tained ultraviolet sterilizer (23) equipped with an intensity
have been specifically developed for preparing aqueous stock
meter and flow controls or passed through a filter with a pore
size of 0.45 µm or less.
8.3.4 Salt water from a surface water source should be
Reagent Chemicals, American Chemical Society Specifications , American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
passed through a filter effective to 15 µm or less to remove
listed by the American Chemical Society, see Analar Standards for Laboratory
parasites and larval stages of mysid predators.
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
8.3.5 When necessary, sea salt may be added to increase
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
salinity (see 8.1.2), if the salt has been shown to cause no MD.
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E1191 − 03a (2023)
solutions of slightly soluble materials (25). The minimum only the solvent control may be used for meeting the require-
necessary amount of a strong acid or base may be used in the ments of 13.1.9 and 13.1.10 and as the basis for calculation of
preparationofanaqueousstocksolution,butsuchacidorbase results. If no statistically significant difference is detected, the
mightaffectthepHoftestsolutionsappreciably.Useofamore data from both controls should be used for meeting the
soluble form of the test material, such as chloride or sulfate requirements of 13.1.9 and 13.1.10 and as the basis for
salts of organic amines, sodium or potassium salts of phenols calculation of results.
andorganicacids,andchlorideornitratesaltsofmetals,might 9.2.5 If a solvent other than water is used to prepare a stock
affect the pH more than the use of the minimum necessary solution,itmightbedesirabletoconductsimultaneoustestson
amounts of strong acids and bases. the test material using two chemically unrelated solvents or
two different concentrations of the same solvent to obtain
9.2.3 If a solvent other than dilution water is used, its
information concerning possible effects of solvent on the
concentration in test solutions should be kept to a minimum
results of the test.
andshouldbelowenoughthatitdoesnotaffecteithersurvival,
growth,orreproductionofsaltwatermysids.Triethyleneglycol
9.3 Test Concentration(s):
is often a good organic solvent for preparing stock solutions
9.3.1 Ifthetestisintendedtoprovideagoodestimateofthe
because of its low toxicity to aquatic animals (26), low
highest concentration of test material that will unacceptably
volatility,andhighabilitytodissolvemanyorganicchemicals.
affect neither survival, growth, nor reproduction of the test
Other water-miscible organic solvents such as methanol,
species, the test concentrations (see 11.1.1.1) should bracket
ethanol, dimethylformamide (DMF) (27), and acetone may
the best prediction of that concentration. Such a prediction is
also be used, but they might stimulate undesirable growths of
usually based on the results of a flow-through acute toxicity
microorganisms, and acetone is also quite volatile. If an
test (see Guide E729) on the test material using the same
organic solvent is used, it should be reagent-grade or better
dilution water and mysids of the same age as at the start of the
anditsconcentrationinanytestsolutionshouldnotexceed0.1
life-cycle test (less than 24-h post release from the brood sac).
mL/L. A surfactant should not be used in the preparation of a
If an acute to chronic ratio has been determined for the test
stock solution because it might affect the form and toxicity of
material with a species of comparable sensitivity, the result of
the test material in the test solutions. (These limitations do not
the acute test with the test species can be divided by the acute
apply to any ingredient of a mixture, formulation, or commer-
to chronic ratio. Except for a few materials (28), acute to
cial product unless an extra amount of solvent is used in the
chronic ratios determined with saltwater mysids are often less
preparation of the stock solution.)
thanfive.Therefore,ifnootherusefulinformationisavailable,
9.2.4 If a solvent other than water is used (1) at least one
the highest concentration of test material in a life-cycle test
solvent control, using solvent from the same batch used to with mysids is often selected to be equal to the lowest
make the stock solution must be included in the test and (2)a
concentrationthatcausedadverseeffectsinacomparableacute
dilution-water control should be included in the test. If no test.
solvent other than water is used, a dilution-water control must
9.3.2 In some (usually regulatory) situations, it is only
be included in the test. necessary to determine whether one specific concentration of
test material reduces survival, growth, or reproduction. For
9.2.4.1 If the concentration of solvent is the same in all test
example, the specific concentration might be the concentration
solutions that contain test material, the solvent control must
occurringinsurfacewater,theconcentrationresultingfromthe
contain the same concentration of solvent.
direct application of the material to a body of water, or the
9.2.4.2 If the concentration of solvent is not the same in all
solubility limit of the material in water. When there is only
test solutions that contain test material, either (1) a solvent test
interestinaspecificconcentration,itisoftenonlynecessaryto
must be conducted to determine whether either survival,
test that concentration (see 11.1.1.2).
growth, or reproduction of the test species is related to the
concentrationofsolventovertherangeusedinthetoxicitytest
10. Test Organisms
or (2) such a solvent test must have already been conducted
using the same dilution water and test species. If either
10.1 Species—The test species is usually selected on the
survival, growth, or reproduction is found to be related to the
basisofgeographicaldistribution,availability,easeofhandling
concentration of solvent, a life-cycle test with that species in
in the laboratory, and past successful use. Both Mysidopsis
that water is unacceptable if any treatment contained a con-
bahia (6, 7, 29, 30, 31) and Mysidopsis bigelowi (6) have been
centration of solvent in that range. If neither survival, growth,
successfully cultured and tested using these procedures. Other
nor reproduction is found to be related to the concentration of
species of mysids, such as Mysidopsis almyra (32), might also
solvent, a life-cycle toxicity test with that same species in that
be used satisfactorily. The species used should be identified
same water may contain solvent concentrations within the
using an appropriate taxonomic key (33).
tested range, but the solvent control must contain the highest
NOTE 1—Mysids are often incorrectly referred to as shrimp.
concentration of solvent present in any of the other treatments.
NOTE 2—Mysidopsis bahia has been redescribed as Americamysis
9.2.4.3 If the test contains both a dilution-water control and
bahia, Mysidopsis bigelowi has been redescribed as Americamysis
bigelowi, and Mysidopsis almyra has been redescribed as Americamysis
a solvent control, the survival, growth, and reproduction of the
almyra by Price et al., 1999 (33).
mysids in the two controls should be compared (seeAppendix
X1.4). If a statistically significant difference in either survival, 10.2 Age—Life-cycle tests with saltwater mysids must be
growth, or reproduction is detected between the two controls, started with individuals less than 24-h post release from the
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brood sac. Use of the youngest possible mysids of a consistent maintain live nauplii in the chambers at all times to prevent
ageisrecommendedtoensurethatdataondelaysinfirstbrood cannibalism of the young and (2) support adequate survival,
release are accurate. growth, and reproduction. The ration should be adjusted in
accordance with mysid density. A ration of 150 brine shrimp
10.3 Source—All mysids used in a test must be from the
nauplii per mysid per day has been used successfully (6).
same brood stock. The mysids used to start a test must have
Mysid growth and reproduction might be improved by feeding
been obtained from adults either (1) hatched and raised in the
twiceaday(75brineshrimppermysidperfeeding)ratherthan
laboratory or (2) brought into the laboratory before sexual
once a day. A batch of brine shrimp nauplii should not be fed
maturity and held for at least 14 days using the same food,
to mysids in a culture or test until it has been shown that the
water,temperature,andsalinityaswillbeusedinthelife-cycle
batch will support survival, growth, and reproduction of the
test. The first method is preferable because it will not only
species for at least three generations. It might be desirable to
acclimatethemysids,butwillalsodemonstratetheacceptabil-
supplement brine shrimp nauplii with an alga (for example,
ity of the food, water, and handling procedures before the test
Skeletonema costatum) a rotifer (for example, Brachionus
is begun.
plicatilus or SELCO, a commercial nutrient enrichment prod-
10.4 Brood Stock:
uct) (34). The food(s) used should be analyzed for the test
material, if it might be present in the environment.
10.4.1 Brood stock may be obtained from another
laboratory, a commercial source, or a wild population from an
10.6 Handling—Mysids should be handled as little as pos-
unpolluted area. When brood stock is brought into the
sible. When handling is necessary, it should be done gently,
laboratory,itshouldbeplacedinatankalongwiththewaterin
carefully, and quickly so that the mysids are not unnecessarily
which it was transported. The temperature should then be
stressed. Dip nets are best for removing gravid female mysids
changedataratenottoexceed3°Cwithin12handthesalinity
from brood-stock tanks. Such nets are commercially available
should be changed at a rate not to exceed 3 g/kg within 12 h.
or can be made from 350 µm mesh nylon netting, silk bolting
10.4.2 Mysidshavebeenculturedinreconstitutedsaltwater
cloth, plankton netting, or similar knotless material. Mysids
andfilterednaturalsaltwaterinrecirculatingandflow-through
that touch dry surfaces or are dropped or injured should be
systems (6, 7, 28, 29, 30, 31, 33). Cultures have been
discarded. Equipment used to handle mysids should be steril-
maintained for several generations in 76-L (20-gal) glass
ized between uses by autoclaving or by treatment with an
−
aquaria containing natural salt water (filtered through a 15-µm
iodophor (35) or with 200 mg of ClO /L for at least 1 h.
filter) at a flow rate of 100 mL/min. If outflows are at the top
Althoughiodophorsarenotacutelytoxictomysids,hypochlo-
of the tanks, no screen is needed to retain mysids and food
rite is.
washout is minimal. Under-gravel filters, with a 1-in. deep
10.7 Harvesting Young—Testorganismsmaybeobtainedby
dolomite substrate prewashed in deionized or distilled water,
using mysid generators (32) or by transferring gravid females
provide gentle aeration and a current conducive to feeding.
from brood-stock tanks to separate chambers and allowing an
10.4.3 The brood stock should be cared for properly so it is
overnightperiodforbroodrelease (29).Thenumberoffemales
not unnecessarily stressed. To maintain mysids in good condi-
needed varies with the size, age, food, temperature, and
tionandavoidunnecessarystress,theyshouldnotbesubjected
salinity.
to rapid changes in temperature, photoperiod, or water quality.
Mysids should not be subjected to more than a 3°C change in 10.8 Quality—Mysids less than 24h post-release from the
temperature or a 3 g/kg change in salinity in any 12h period. brood sac should be acceptable for starting a life-cycle test if
The concentration of dissolved oxygen should be maintained theywereobtainedfromabroodstockinwhichmorethanhalf
between 60% and 100% of saturation (21) and continuous of the adult females were producing young. Representative
gentle aeration is usually desirable. A 15min to 30min mysids from the brood stock should be analyzed for the test
transition period (4) should be provided when lights go on or material, if it might be present in the environment.
off.
11. Procedure
10.4.4 Reproduction might be depressed when culture den-
sityisabove20mysids/L (7).Therefore,whenculturesarenot
11.1 Experimental Design:
being used for supplying test organisms, enough adults should
11.1.1 Decisionsconcerningaspectsofexperimentaldesign,
be removed at least every 2 weeks to stimulate reproduction.
such as the dilution factor, number of treatments, and numbers
Mysidgeneratorsystems (32)maybeusedtoprovideconstant
of test chambers, compartments, and pairs of mysids per
croppingandtoobtainage-standardizedsubsamplesfortestsor
treatment, should be based on the purpose of the test and the
new cultures. Brood-stock tanks should be kept free of other
type of procedure that is to be used to calculate results (see
animals,suchashydroidsandworms,byscrapingthesidesand
Section 14). One of the following two types of experimental
siphoning the bottoms every one or two weeks. Salinity and
design will probably be appropriate in most cases.
temperature should be appropriate for the particular species
11.1.1.1 Alife-cycletestintendedtoallowcalculationofan
and consistent with the specified test conditions (see 8.1.2 and
endpoint (see X1.2) usually consists of one or more control
11.3).
treatmentsandageometricseriesofatleastfiveconcentrations
10.5 Food—At least once daily, saltwater mysids in brood- of test material. In the dilution water or solvent control(s), or
stock tanks and in test chambers should be fed live brine both,(see9.2.3)mysidsareexposedtodilutionwatertowhich
shrimp nauplii (see Practice E1203) in excess, in order to (1) no test material has been added. Except for the control(s) and
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E1191 − 03a (2023)
thehighestconcentration,eachconcentrationshouldbeatleast impairment is unacceptable. Selection of the proper number of
50%ofthenexthigherone,unlessinformationconcerningthe replicatesrequires(1)anestimateoftheexpectedcoefficientof
concentration-effect curve indicates that a different dilution variation of reproductive success for tests conducted at your
factor is more appropriate. At a dilution factor of 0.5, five laboratory or a default value; (2) a selection of the percentage
reduction in the number of young/female/reproductive day,
properly chosen concentrations are a reasonable compromise
betweencostandtheriskofallconcentrationsbeingeithertoo relative to controls, that you wish to be able to detect; and (3
) a selection of the frequency that you are willing to have tests
high or too low. If the estimate of chronic toxicity is particu-
larlynebulous(see9.3.1),sixorsevenconcentrationsmightbe fail because they lack the desired power.
11.1.4.1 An example of how this is accomplished uses a
desirable.
laboratory-specific coefficient of variation (CV) of 0.60 (7).
11.1.1.2 If it is necessary only to determine whether a
The appropriate number of replicates can be selected using
specific concentration reduces survival, growth, or reproduc-
Table 1 (or Fig. 1) after choosing the magnitude of the
tion (see 9.3.2), only that concentration and the control(s) are
percentage reduction relative to controls you wish to detect,
necessary.Twoadditionalconcentrationsataboutone-halfand
and the proportion of tests you wish to have this detection
twotimesthespecificconcentrationofconcernaredesirableto
potential. For example, with a CVof 0.60, if you wish the test
increase confidence in the results.
tostatisticallydetectan80%reductioninthenumberofyoung
11.1.2 The primary focus of the physical and experimental
80% of the time, eight replicates/treatment are required.
design of the test and the statistical analysis of the data is the
Alternatively, the goal might be to design tests that have the
experimental unit, which is defined as the smallest physical
power to detect reproductive impairments that population
entity to which treatments can be independently assigned (36).
models indicate would compromise population maintenance.
Because test solution can flow from one compartment to
In this case, application of the model described by Kuhn et al.
another, but not from one test chamber to another (see 6.4.1),
(3) to databases from 15 life-cycle tests (conducted at the U.S.
thetestchamberistheexperimentalunit.Asthenumberoftest
EPA Atlantic Ecology Division (AED) laboratory at
chambers (that is, experi
...