ASTM E1440-91(2012)
(Guide)Standard Guide for Acute Toxicity Test with the Rotifer Brachionus (Withdrawn 2021)
Standard Guide for Acute Toxicity Test with the Rotifer Brachionus (Withdrawn 2021)
SIGNIFICANCE AND USE
5.1 An important goal of aquatic toxicology is to determine the effects of toxic compounds on species that play a central role in aquatic communities. Rotifers have a major impact on several important ecological processes in freshwater and coastal marine environments. As filter-feeders on phytoplankton and bacteria, rotifers exert substantial grazing pressure that at times exceeds that of the larger crustacean zooplankton (1, 2).3 Rotifer grazing on phytoplankton is highly selective (2-4) and can influence phytoplankton composition, the coexistence of competitors, and overall water quality (5). The contribution of rotifers to the secondary production of many aquatic communities is substantial (6-9). In fresh water, rotifers often account for the major fraction of zooplankton biomass at certain times of the year (10, 11) . Rotifers and other zooplankton are a significant food source for many larval fish, planktivorous adult fish (12, 13), and several invertebrate predators (14-16) . The high metabolic rates of rotifers contribute to their role in nutrient cycling, which might make rotifers more important than crustaceans in certain communities (17, 18) .
5.2 In addition to their important ecological role in aquatic communities, rotifers are attractive organisms for toxicological studies because an extensive database exists on the basic biology of this group. Techniques have been published for the culture of many rotifer species (3, 19). The rotifer life cycle is well defined (20, 21), and the factors regulating it are reasonably well understood (22-25). Several aspects of rotifer behavior have been examined closely (26-29). The biogeography of many rotifer species has been characterized (30, 31), and the systematics of the group are well described (32, 33).
5.3 Toxicity tests with rotifers of the genus Brachionus are more easily performed than with many other aquatic animals because of their rapid reproduction, short generation times, sensitivity (34), and ...
SCOPE
1.1 This guide describes procedures for obtaining laboratory data concerning the acute toxicity of chemicals and aqueous effluents released into fresh, estuarine, or marine waters. Acute toxicity is measured by exposing Brachionus newly hatched from cysts to a series of toxicant concentrations under controlled conditions. This guide describes a test for using B. calyciflorus, a fresh water rotifer, and the Appendix describes modifications of this test for estuarine and marine waters using B. plicatilis. These procedures lead to an estimation of acute toxicity, including the concentration expected to kill 50 % of the test rotifers (LC50) in 24 h. Procedures not specifically stated in this guide should be conducted in accordance with Guide E729 and Guide E1192.
1.2 Modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using modified procedures might not be comparable to rotifer acute tests that follow the protocol described here. Comparison of the results using modified procedures might provide useful information concerning new concepts and procedures for conducting acute toxicity tests on chemicals and aqueous effluents.
1.3 This guide is organized as follows:
Section
Scope
1
Referenced Documents
2
Terminology
3
Summary of Guide
4
Significance and Use
5
Apparatus
6
Dilution Water
7
Hazards
8
Test Material
9
Test Organisms
10
Test Procedure
11
Calculation of Results
12
Acceptability of the Test
13
Report
14
Keywords
15
1.4 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or mixtures. This guide can also be used to conduct investigations of the...
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1440 − 91 (Reapproved 2012)
Standard Guide for
Acute Toxicity Test with the Rotifer Brachionus
This standard is issued under the fixed designation E1440; 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
Calculation of Results 12
Acceptability of the Test 13
1.1 Thisguidedescribesproceduresforobtaininglaboratory
Report 14
data concerning the acute toxicity of chemicals and aqueous Keywords 15
effluentsreleasedintofresh,estuarine,ormarinewaters.Acute
1.4 These procedures are applicable to most chemicals,
toxicity is measured by exposing Brachionus newly hatched
either individually or in formulations, commercial products, or
from cysts to a series of toxicant concentrations under con-
mixtures.Thisguidecanalsobeusedtoconductinvestigations
trolled conditions. This guide describes a test for using B.
of the effects on rotifer survival of pH, hardness, and salinity
calyciflorus, a fresh water rotifer, and the Appendix describes
and on materials such as aqueous effluents, leachates, oils,
modificationsofthistestforestuarineandmarinewatersusing
particulate matter, sediments, and surface waters. This guide
B. plicatilis. These procedures lead to an estimation of acute
might not be appropriate for materials with high oxygen
toxicity, including the concentration expected to kill 50% of
demand, with high volatility, subject to rapid biological or
the test rotifers (LC50) in 24 h. Procedures not specifically
chemical transformation, or that readily sorb to test chambers.
stated in this guide should be conducted in accordance with
1.5 This standard does not purport to address all of the
Guide E729 and Guide E1192.
safety concerns, if any, associated with its use. It is the
1.2 Modifications of these procedures might be justified by
responsibility of the user of this standard to establish appro-
special needs or circumstances. Although using appropriate
priate safety and health practices and determine the applica-
procedures is more important than following prescribed
bilityofregulatorylimitationspriortouse.Forspecifichazards
procedures, the results of tests conducted using modified
statements, see Section 8.
procedures might not be comparable to rotifer acute tests that
follow the protocol described here. Comparison of the results
2. Referenced Documents
using modified procedures might provide useful information
2.1 ASTM Standards:
concerning new concepts and procedures for conducting acute
E380Practice for Use of the International System of Units
toxicity tests on chemicals and aqueous effluents.
(SI) (the Modernized Metric System)
1.3 This guide is organized as follows:
E729Guide for Conducting Acute Toxicity Tests on Test
Section Materials with Fishes, Macroinvertebrates, and Amphib-
ians
Scope 1
E943Terminology Relating to Biological Effects and Envi-
Referenced Documents 2
Terminology 3 ronmental Fate
Summary of Guide 4
E1192Guide for ConductingAcute Toxicity Tests onAque-
Significance and Use 5
ous Ambient Samples and Effluents with Fishes,
Apparatus 6
Dilution Water 7 Macroinvertebrates, and Amphibians
Hazards 8
Test Material 9
3. Terminology
Test Organisms 10
Test Procedure 11
3.1 Definitions of Terms Specific to This Standard:
3.1.1 rotifer cyst—arotiferembryoarrestedatanearlystage
in development, enclosed in an envelope and resistant to
desiccation and temperature extremes. Rotifer cysts are often
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Dec. 1, 2012. Published December 2012. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1991. Last previous edition approved in 2004 as E1440–91 (2004). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/E1440-91R12. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1440 − 91 (2012)
incorrectlyreferredtoasrestingeggs.Uponhydration,embry- (14-16).The high metabolic rates of rotifers contribute to their
onic development resumes until a neonate female emerges role in nutrient cycling, which might make rotifers more
from the cyst. important than crustaceans in certain communities (17, 18).
3.1.2 rotifer neonate—a newly hatched, freely swimming
5.2 In addition to their important ecological role in aquatic
rotifer. All neonates hatched from cysts are females.
communities,rotifersareattractiveorganismsfortoxicological
studies because an extensive database exists on the basic
3.1.3 strain—a geographically identified population of a
biology of this group. Techniques have been published for the
single species. Strains are usually separated by considerable
culture of many rotifer species (3, 19). The rotifer life cycle is
distancesandcanbecharacterizedgeneticallythroughisozyme
well defined (20, 21), and the factors regulating it are reason-
analysis or physiologically by their population dynamics and
ably well understood (22-25). Several aspects of rotifer behav-
sensitivity to toxicants.
ior have been examined closely (26-29). The biogeography of
3.1.4 The words “must,” “should,” “may,” “can,” and
many rotifer species has been characterized (30, 31), and the
“might” have very specific meanings in this guide. “Must” is
systematics of the group are well described (32, 33).
usedtoexpressanabsoluterequirement,thatis,tostatethatthe
test ought to be designed to satisfy the specified condition, 5.3 Toxicity tests with rotifers of the genus Brachionus are
more easily performed than with many other aquatic animals
unless the purpose of the test requires a different design.
“Must”isusedonlyinconnectionwithfactorsdirectlyrelating because of their rapid reproduction, short generation times,
sensitivity (34), and the commercial availability of rotifer
to the acceptability of the test (see 13.1). “Should” is used to
state that the specified condition is recommended and ought to cysts. Brachionus spp. have a cosmopolitan distribution that
spans six continents (31), and they are ecologically important
be met if possible. Although violation of one “should” state-
ment is rarely a serious matter, violation of several will often members of many aquatic communities impacted by pollution.
The use of B. plicatilis in an acute toxicity test for estuarine
render the results questionable. Terms such as “is desirable,”
andmarineenvironmentsandB.rubensinfreshwaterhasbeen
“is often desirable,” and “might be desirable” are used in
described, as well as their sensitivity to several toxicants (35,
connection with less important factors. “May” is used to mean
36).
“is (are) allowed to,” “can” is used to mean “is (are) able to,”
and“might”isusedtomean“couldpossibly.”Thus,theclassic
5.4 Thetestdescribedhereisfast,easytoexecute,sensitive,
distinctionbetween“may”and“can”ispreserved,and“might”
and cost-effective. Obtaining test animals from cysts greatly
is never used as a synonym for either “may” or “can.”
reduces some of the major problems in routine aquatic toxico-
logical testing such as the limited availability of test animals
4. Summary of Guide
andtheinconsistencyofsensitivityovertime.Rotifershatched
4.1 Rotifer cysts are induced to hatch in 16 to 22 h by
from cysts are of similar age and are physiologically uniform,
incubating them at 25°C in standard dilution water. These
thuseliminatingpre-testconditionsasasourceofvariabilityin
neonates are then exposed immediately to two or more
the toxicity test. Cysts can be shipped inexpensively world-
concentrationsoftestmaterialplusacontrolincovereddishes.
wide, allowing all laboratories to use standard, genetically
After24h,thepercentofdeadanimalsineachdishisrecorded.
defined strains that have been calibrated with reference toxi-
An appropriate statistical method is used to calculate an LC50
cants. The convenience of an off-the-shelf source of test
or some other appropriate endpoint.
animalsthatrequirenopre-conditioningislikelytopermitnew
applications of aquatic toxicity tests.
5. Significance and Use
5.5 Sensitivity to toxicants is compound and species
5.1 An important goal of aquatic toxicology is to determine
specific, but the sensitivity of B. calyciflorus is generally
the effects of toxic compounds on species that play a central
comparable to that of Daphnia (37).
role in aquatic communities. Rotifers have a major impact on
5.6 Rotifer cysts are commercially available, but they can
several important ecological processes in freshwater and
also be obtained from natural populations and from laboratory
coastal marine environments. As filter-feeders on phytoplank-
cultures. Techniques for rotifer cyst production in laboratory
tonandbacteria,rotifersexertsubstantialgrazingpressurethat
populations have been described (24, 25, 38, 39). However,
at times exceeds that of the larger crustacean zooplankton (1,
usingawell-characterizedrotiferstrainisbestsincestrainsare
2). Rotifer grazing on phytoplankton is highly selective (2-4)
known to have differing toxicant sensitivities.
and can influence phytoplankton composition, the coexistence
of competitors, and overall water quality (5). The contribution
6. Apparatus
of rotifers to the secondary production of many aquatic
communities is substantial (6-9). In fresh water, rotifers often
6.1 Laboratory Facilities—Preparation of the test, storage
account for the major fraction of zooplankton biomass at
ofthedilutionwater,andallstagesofthetestprocedureshould
certain times of the year (10, 11). Rotifers and other zooplank-
take place in an atmosphere free from dust and toxic vapors.
ton are a significant food source for many larval fish, plank-
6.2 Equipment—The equipment required for this test in-
tivorous adult fish (12, 13), and several invertebrate predators
cludes: a constant temperature bath or environmental chamber
capable of maintaining 25°C, petri dishes with covers or
multiwell tissue culture plates, micropipets with smoothed
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. openings, test tubes or petri dishes for hatching cysts, a
E1440 − 91 (2012)
stereomicroscopecapableof10to15×magnification,anda20 8.6 Becuase water is such a good conductor of electricity,
to 40 W fluorescent light. ground fault systems and leak detectors should be used to help
avoid electrical shocks.
7. Dilution Water
9. Test Material
7.1 Reconstituted fresh water is prepared with high-quality
9.1 Single Chemical—Guide E729, sections on stock
deionizedordistilledwatertowhich96mgofNaHCO,60mg
CaSO ·2H O, 60 mg MgSO ·7H O, and 4 mg KCl are added solutions, solvents, solvent controls, and test concentrations
4 2 4 2
apply to this test.
per litre (40). This moderately hard dilution water (with a
hardness of 80 to 100 mg CaCO per litre and alkalinity of 60
9.2 Effluents—Guide E1192, sections on collection,
to 70 mg per litre) is stirred for 24 h and adjusted to pH 7.5
preservation, treatment, and test concentrations of effluents,
using concentrated hydrochloric acid or sodium hydroxide.
apply to this test.
This dilution water may be used for up to seven days, but then
itshouldbediscarded.Thedissolvedoxygencontentshouldbe
10. Test Organisms
at least 90% of saturation at the beginning of the test.
10.1 Test animals are obtained by hatching cysts. Rotifer
Unexpected and inconsistent results can often be traced to
cysthatchingshouldbeinitiatedapproximately16hbeforethe
problems with the dilution water, so it should be prepared and
start of the toxicity test. Hatching is initiated by placing B.
stored very carefully.
calycifloruscystsinthedilutionwater(see7.1)andincubating
7.2 Other reconstituted dilution waters may be used as
at 25°C at an illumination level of 1000 to 3000 lux. Hatching
described in Guide E729. In addition, natural dilution water
should begin after approximately 15 h, and by 20 h approxi-
sometimesmightbedesirable(GuideE729).Cysthatchingand
mately 50% of the cysts should have hatched. A hatching
LC50s in these dilution waters might differ from those previ-
percent of 50% is common. Cooler temperatures, low or high
ously reported (37).
pH, low light, elevated hardness, and alkalinity can all delay
hatching. If hatching is delayed, the cysts should be checked
8. Hazards
hourly to ensure collection of the test animals within 0 to 2 h
of hatching. It is important to obtain 0 to 2-h-old animals for
8.1 Many materials can affect humans adversely if precau-
the test because there is no feeding during the toxicity test.
tions are inadequate. Therefore, skin contact with all test
Consequently, food deprivation begins to cause mortality after
materials and solutions should be minimized by wearing
about 32 h at 25°C. If rotifers are older than 32 h at the end of
appropriate protective gloves, especially when washing equip-
the test, excessive control mortality might result.
ment or putting hands in test solutions. Laboratory coats,
aprons, and protective glasses should always be worn, and
11. Test Procedure
pipetsshouldbeusedtoremoveorganismsfromtestsolutions.
Special precautions, such as covering test chambers and
11.1 Experimental Design:
ventilating the area surrounding the chambers, should be taken
11.1.1 Decisions concerning aspects of the experimental
when conducting tests on volatile materials. Information on
design, such as the dilution factor, number of treatments, and
toxicitytohumans (41-45),recommendedhandlingprocedures
number of test chambers per treatment, should be based on the
(46-49), and chemical and physical properties of the test
purpose of the test and the type of procedure that is used to
material should be studied before a test is begun. Special
calculate the results. One of the following types of experimen-
procedures might be necessary with radiolabeled test materials
tal designs will probably be appropriate in most cases.
(50, 51) and with test materials that are, or are suspected of
11.1.2 Ifitisnecessarytodetermineonlywhetheraspecific
being, carcinogenic (52).
concentration affects survival, then a pass/fail type of test
consisting of a single concentration and controls is useful.An
8.2 Although the disposal of stock solutions, test solutions,
example of this design would be a test in which a control is
and test organisms poses no sp
...
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