ASTM E1440-23

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: E1440 − 23
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
Calculation of Results 12
Acceptability of the Test 13
1.1 This guide describes procedures for obtaining laboratory
Report 14
data concerning the acute toxicity of chemicals and aqueous Keywords 15
effluents released into fresh, estuarine or marine waters. 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. This guide can also be used to investigate the effects
trolled conditions. This guide describes a test for using B.
on rotifer survival of pH, hardness, and salinity and on
calyciflorus, a freshwater rotifer, and the Appendix describes
materials such as aqueous effluents, leachates, oils, particulate
modifications of this test for estuarine and marine waters using
matter, sediments, and surface waters. This guide might not be
B. plicatilis. These procedures lead to an estimation of acute
appropriate for materials with high oxygen demand, with high
toxicity, including the concentration expected to kill 50 % of
volatility, subject to rapid biological or chemical transforma-
the test rotifers (LC50) in 24 h. Procedures not specifically
tion or those 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, health, and environmental practices and deter-
procedures is more important than following prescribed
mine the applicability of regulatory limitations prior to use.
procedures, the results of tests conducted using modified
For specific hazards statements, see Section 8.
procedures might not be comparable to rotifer acute tests that
1.6 This international standard was developed in accor-
follow the protocol described here. Comparison of the results
dance with internationally recognized principles on standard-
using modified procedures might provide useful information
ization established in the Decision on Principles for the
concerning new concepts and procedures for conducting acute
Development of International Standards, Guides and Recom-
toxicity tests on chemicals and aqueous effluents.
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.3 This guide is organized as follows:
Section
2. Referenced Documents
Scope 1
2.1 ASTM Standards:
Referenced Documents 2
D1129 Terminology Relating to Water
Terminology 3
Summary of Guide 4
D4447 Guide for Disposal of Laboratory Chemicals and
Significance and Use 5
Samples
Apparatus 6
E729 Guide for Conducting Acute Toxicity Tests on Test
Dilution Water 7
Hazards 8
Materials with Fishes, Macroinvertebrates, and Amphib-
Test Material 9
ians
Test Organisms 10
E943 Terminology Relating to Biological Effects and Envi-
Test Procedure 11
ronmental Fate
E1192 Guide for Conducting Acute Toxicity Tests on Aque-
ous Ambient Samples and Effluents with Fishes,
Macroinvertebrates, and Amphibians
This guide is under the jurisdiction of ASTM Committee E50 on Environmental
Assessment, Risk Management and Corrective Action and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Sept. 1, 2023. Published October 2023. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 1991. Last previous edition approved in 2012 as E1440 – 91 (2012) contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
which was withdrawn January 2021 and reinstated in September 2023. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E1440-23. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1440 − 23
E1733 Guide for Use of Lighting in Laboratory Testing 5. Significance and Use
E1847 Practice for Statistical Analysis of Toxicity Tests
5.1 An important goal of aquatic toxicology is to determine
Conducted Under ASTM Guidelines (Withdrawn 2022)
the effects of toxic compounds on species that play a central
IEEE/ASTM SI 10 American National Standard for Use of
role in aquatic communities. Rotifers have a major impact on
the International System of Units (SI): The Modern Metric
several important ecological processes in freshwater and
System
coastal marine environments. As filter-feeders on phytoplank-
ton and bacteria, rotifers exert substantial grazing pressure that
3. Terminology
at times exceeds that of the larger crustacean zooplankton (1,
2). Rotifer grazing on phytoplankton is highly selective (2-4)
3.1 Definitions of Terms Specific to This Standard:
and can influence phytoplankton composition, the coexistence
3.1.1 rotifer cyst, n—a rotifer embryo arrested at an early
of competitors, and overall water quality (5). The contribution
stage in development, enclosed in an envelope and resistant to
of rotifers to the secondary production of many aquatic
desiccation and temperature extremes. Rotifer cysts are often
communities is substantial (6-9). In fresh water, rotifers often
incorrectly referred to as resting eggs. Upon hydration, embry-
account for the major fraction of zooplankton biomass at
onic development resumes until a neonate female emerges
certain times of the year (10, 11). Rotifers and other zooplank-
from the cyst.
ton are a significant food source for many larval fish, plank-
3.1.2 rotifer neonate, n—a newly hatched, freely swimming
tivorous adult fish (12, 13), and several invertebrate predators
rotifer. All neonates hatched from cysts are females.
(14-16). The high metabolic rates of rotifers contribute to their
3.1.3 strain, n—a geographically identified population of a
role in nutrient cycling, which might make rotifers more
single species. Strains are usually separated by considerable
important than crustaceans in certain communities (17, 18).
distances and can be characterized genetically through isozyme
5.2 In addition to their important ecological role in aquatic
analysis or physiologically by their population dynamics and
communities, rotifers are attractive organisms for toxicological
sensitivity to toxicants.
studies because an extensive database exists on the basic
3.2 For definitions of other terms used in this guide, refer to biology of this group. Techniques have been published for the
Terminology E943 and D1129, and Guides E729 and E1192. culture of many rotifer species (3, 19). The rotifer life cycle is
For an explanation of units and symbols, refer to Practice well defined (20, 21), and the factors regulating it are reason-
IEEE/ASTM SI 10. ably well understood (22-25). Several aspects of rotifer behav-
ior have been examined closely (26-29). The biogeography of
3.3 The words “must,” “should,” “may,” “can,” and “might”
many rotifer species has been characterized (30, 31), and the
have very specific meanings in this guide. “Must” is used to
systematics of the group are well described (32, 33).
express an absolute requirement, that is, to state that the test
5.3 Toxicity tests with rotifers of the genus Brachionus are
ought to be designed to satisfy the specified condition, unless
more easily performed than with many other aquatic animals
the purpose of the test requires a different design. “Must” is
because of their rapid reproduction, short generation times,
used only in connection with factors directly relating to the
sensitivity (34), and the commercial availability of rotifer
acceptability of the test (see 13.1). “Should” is used to state
cysts. Brachionus spp. have a cosmopolitan distribution that
that the specified condition is recommended and ought to be
spans six continents (31), and they are ecologically important
met if possible. Although violation of one “should” statement
members of many aquatic communities impacted by pollution.
is rarely a serious matter, violation of several will often render
The use of B. plicatilis in an acute toxicity test for estuarine
the results questionable. Terms such as “is desirable,” “is often
and marine environments and B. rubens in fresh water has been
desirable,” and “might be desirable” are used in connection
described, as well as their sensitivity to several toxicants (35,
with less important factors. “May” is used to mean “is (are)
36, 37, 38).
allowed to,” “can” is used to mean “is (are) able to,” and
5.3.1 High correlations were found between the no observed
“might” is used to mean “could possibly.” Thus, the classic
effect concentrations (NOECs) or 10 percent effect concentra-
distinction between “may” and “can” is preserved, and “might”
tions (EC10s) for Pseudokirchneriella sp. after 72-hour expo-
is never used as a synonym for either “may” or “can.”
sures; for 2-day Brachionus NOECs/EC10s, and for 21-day
Daphnia magna NOECs among 16 chemicals (37). The toxi-
4. Summary of Guide
cological response of rotifers and microalgae were within the
4.1 Rotifer cysts are induced to hatch in 16 h to 22 h by
same order of magnitude as the response of Daphnia in 80 %
incubating them at 25 °C in standard dilution water. These
of the cases (that is, 13/16 chemicals).
neonates are then exposed immediately to two or more
5.4 The test described here is fast, easy to execute, sensitive
concentrations of test material plus a control in covered dishes.
and cost-effective. Obtaining test animals from cysts greatly
After 24 h, the percent of dead animals in each dish is recorded.
reduces some of the major problems in routine aquatic toxico-
An appropriate statistical method is used to calculate an LC50
logical testing, such as the limited availability of test animals
or some other appropriate endpoint.
and the inconsistency of sensitivity over time. Rotifers hatched
3 4
The last approved version of this historical standard is referenced on The boldface numbers in parentheses refer to the list of references at the end of
www.astm.org. this standard.
E1440 − 23
from cysts are of similar age and are physiologically uniform, tive gloves, especially when washing equipment or putting
thus eliminating pre-test conditions as a source of variability in hands in test solutions. Laboratory coats, aprons, and protec-
the toxicity test. Cysts can be shipped inexpensively world- tive glasses should always be worn, and pipets should be used
wide, allowing all laboratories to use standard, genetically to remove organisms from test solutions. Special precautions,
defined strains that have been calibrated with reference toxi- such as covering test chambers and ventilating the area
cants. The convenience of an off-the-shelf source of test surrounding the chambers, should be taken when conducting
animals that require no pre-conditioning is likely to permit new tests on volatile materials. Information on toxicity to humans
applications of aquatic toxicity tests. (43-47), recommended handling procedures (48-51), and
chemical and physical properties of the test material, as
5.5 Sensitivity to toxicants is compound and species
available on the safety data sheet, should be studied before a
specific, but the sensitivity of B. calyciflorus is generally
test is begun. Special procedures might be necessary with
comparable to that of Daphnia (39).
radiolabeled test materials (52, 53) and with test materials that
5.6 Rotifer cysts are commercially available, but these can
are, or are suspected of being, carcinogenic (54).
also be obtained from natural populations and from laboratory
8.2 Although the disposal of stock solutions, test solutions,
cultures. Techniques for rotifer cyst production in laboratory
and test organisms poses no special problems in most cases,
populations have been described (24, 25, 40, 41). However,
health and safety precautions and applicable regulations should
using a well-characterized rotifer strain is best, since strains are
be considered before beginning a test. Removal or degradation
known to have differing toxicant sensitivities.
of the test material might be desirable before disposal of the
stock and test solutions.
6. Apparatus
8.3 Cleaning of equipment with a volatile solvent such as
6.1 Laboratory Facilities—Preparation of the test, storage
acetone should be performed only in a well-ventilated area in
of the dilution water, and all the stages of the test procedure
which no smoking is allowed and no open flame, such as a pilot
should take place in an atmosphere free from dust and toxic
light, is present.
vapors.
8.4 An acidic solution should not be mixed with a hypo-
6.2 Equipment—The equipment required for this test in-
chlorite solution because hazardous fumes might be produced.
cludes: a constant temperature bath or environmental chamber
capable of maintaining 25 °C, petri dishes with covers or
8.5 To prepare dilute acid solutions, concentrated acid
multiwell tissue culture plates, micropipettes with smoothed
should be added to water, not vice versa. Opening a bottle of
openings, test tubes or petri dishes for hatching cysts, a
concentrated acid and adding concentrated acid to water should
stereomicroscope capable of 10× to 15× magnification, and a
be performed only in a well-ventilated area.
20 W to 40 W fluorescent light (see Guide E1733).
8.6 Because water is such a good conductor of electricity,
ground fault systems and leak detectors should be used to help
7. Dilution Water
avoid electrical shocks.
7.1 Reconstituted fresh water is prepared with high-quality
deionized or distilled water to which 96 mg of NaHCO , 60 mg
9. Test Material
CaSO ·2H O, 60 mg MgSO ·7H O, and 4 mg KCl are added
4 2 4 2
9.1 Single Chemical—Guide E729, sections on stock
per litre (42). This moderately hard dilution water (with a
solutions, solvents, solvent controls, and test concentrations
hardness of 80 mg to 100 mg CaCO per litre and alkalinity of
apply to this test.
60 mg per litre to 70 mg per litre) is stirred for 24 h, and
adjusted to pH 7.5 using concentrated hydrochloric acid or
9.2 Effluents—Guide E1192, sections on collection,
sodium hydroxide. This dilution water may be used for up to
preservation, treatment and test concentrations of effluents,
seven days, but then it should be discarded. The dissolved
apply to this test.
oxygen content should be at least 90 % of saturation at the
beginning of the test. Unexpected and inconsistent results can
10. Test Organisms
often be traced to problems with the dilution water, so it should
10.1 Test animals are obtained by hatching cysts. Rotifer
be prepared and stored very carefully.
cyst hatching should be initiated approximately 16 h before the
7.2 Other reconstituted dilution waters may be used as
start of the toxicity test. Hatching is initiated by placing B.
described in Guide E729. In addition, natural dilution water
calyciflorus cysts in the dilution water (see 7.1) and incubating
sometimes might be desirable (Guide E729). Cyst hatching and
at 25 °C and at an illumination level of 1000 lux to 3000 lux.
LC50s in these dilution waters might differ from those previ-
Hatching should begin after approximately 15 h, and by 20 h
ously reported (39).
approximately 50 % of the cysts should have hatched. A
hatching percent of 50 % is common. Cooler temperatures, low
8. Hazards
or high pH, low light, elevated hardness, and alkalinity can all
8.1 Many materials can affect humans adversely if precau- delay hatching. If hatching is delayed, the cysts should be
tions are inadequate. Therefore, guidelines for the handling and checked hourly to ensure collection of the test animals within
disposal of hazardous materials should be strictly followed 0 to 2 h of hatching. It is important to obtain 0 to 2-h-old
(Guide D4447). Skin contact with all test materials and animals for the test because there is no feeding during the
solutions should be minimized by wearing appropriate protec- toxicity test. Consequently, food deprivation begins to cause
E1440 − 23
mortality after about 32 h at 25 °C. If rotifers are older than 32 11.5 Plates containing rotifers should be incubated at 25 °C
h at the end of the test, excessive control mortality might result. 6 1 °C for 24 h. Incubation should be conducted in darkness
unless light is necessary to meet study objectives such as
11. Test Procedure activating toxicity of the test material or to assess a photope-
riod set to mimic environmental circumstances to which the
11.1 Experimental Design:
microorganisms are naturally exposed. Containers of water
11.1.1 Decisions concerning aspects of the experimental
should be placed in the incubator to maintain high humidity
design, such as the dilution factor, number of treatments and
and prevent desiccation of the test wells. A summary of
number of test chambers per treatment, should be based on the
recommended test conditions is given in Table 1.
purpose of the test and the type of procedure that is used to
11.6 After 24 h, the live and dead rotifers in each well
calculate the results (see Practice E1847). One of the following
should be counted at 10× to 15× magnification and recorded.
types of experimental designs will probably be appropriate in
With experience, it is easy to determine dead rotifers based on
most cases.
lack of movement, clearing of internal tissues and retraction of
11.1.2 If it is necessary to determine only whether a specific
the corona. If it is questionable whether an individual is dead,
concentration affects survival, then a pass/fail type of test
observe the rotifer for 5 s. Lack of movement, including
consisting of a single concentration and controls is useful. An
mastax and foot movement, indicates death. Mortality in the
example of this design would be a test in which a control is
controls must be 10 % or less; otherwise the test is considered
compared to a 100 % effluent concentration (42).
invalid.
11.1.3 To determine the LC50 for a test material, a concen-
tration series including a control should be prepared according
11.7 Range Finding Test—This test is to determine the
to Guide E1192. Tissue culture plates containing 24 wells are “critical range” within which mortality changes from 0 % at
convenient for LC50 determination because these permit a
the low nominal concentration to 100 % at the high nominal
control and five test material concentrations on a single plate.
concentration. A series of logarithmically spaced concentra-
However, other containers may be used. Tests are conducted in
tions or dilutions of the test material should be prepared using
1 mL of test solution with ten animals per well. This design
reconstituted freshwater (see 7.1). For example, the following
might be modified to fit the question being asked more
concentration series might be used for a chemical: 0.01, 0.1, 1,
appropriately.
10, 100, 1000 mg/L. For effluents, the following concentrations
might be used: 0.01, 0.1, 1, 10, 100 %. If effluent characteris-
11.2 Brachionus calyciflorus, is a small animal approxi-
tics (that is, NH , NO , and so forth) are altered by aeration, the
3 2
mately 250 μm in length, which is one-fourth the size of
stabilization times for the effluents and controls must be
newborn Daphnia. Although they are small and require mag-
decreased. The range-finding test is conducted with only one
nification for transferring, these microorganisms swim slowly
test well per concentration. An additional well with ten rotifers
and are easy to catch with a micropipette. Newly hatched
in the dilution water must be included as a control. It should be
rotifers are white and are most visible against a dark back-
noted that a range-finding test with an effluent will require at
ground. A stereomicroscope with 10× to 15× magnification and
least 24 h storage of the effluent before a definitive test takes
dark field, substage illumination is ideal. Since they are
place. This could be a significant factor with an effluent
moderately phototactic, rotifers tend to congregate around the
containing easily degraded compounds.
edges of a dish.
11.8 Definitive Test—This test is conducted to determine the
11.3 Rotifers should be transferred using a micropipette
24 h LC50 for B. calyciflorus. From the critical concentration
with a bore large enough to allow animals to enter and exit
range obtained in the range-finding test, concentrations or
without injury. The volume of medium carried over with the
rotifers should be minimized.
TABLE 1 Recommended Test Conditions for the Definitive Acute
11.4 Several rotifers should be collected with a micropipette
Toxicity Test with the Rotifer B. calyciflorus
and transferred to a rinsing well containing the appropriate
Test Type Static Acute
concentration of toxicant. Rotifers can then be transferred to
Duration 24 h
the test wells, observing under the microscope their exit from
Endpoint LC50
the micropipette and entry into the test solutions. Rotifers must
Temperature 25 °C
Dilution water Reconstituted, moderately hard
be randomly assigned to the test chambers. This procedure
freshwater (see 7.1)
permits counting exactly ten animals per well and confirms
Photoperiod OL:24D (continuous darkness)
their arrival into the test well in good condition. This procedure Test chamber size 2.5 mL
Test solution volume 1.0 mL
should be repeated until all control and treatment wells are
Test concentrations 5 plus a control
loaded. A piece of parafilm should be stretched across the top
Total volume required for test about 125 mL
of the plate and the cover put on tightly. The temperature, pH,
Age of test animals 0–2 h
Number neonates per 3
and hardness of the test solutions must be recorded at the
concentration
beginning and at the end of a test. Dissolved oxygen must be
Number of neonates per 30
measured at the beginning of a test. Because test chambers concentration
Feeding none
contain only one (1) mL, it is technically difficult to measure
Aeration none
dissolved oxygen at the end of a test. However, brachionids are
Test acceptability <10 % control mortality
not sensitive to low oxygen levels (55).
E1440 − 23
dilutions of the test material should be selected from a situations, it may be necessary to use a non-parametric proce-
geometric scale. A control and five or more nominal test dure such as the Spearman-Karber method to obtain statisti-
concentrations are usua
...