ASTM D6081-98

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An American National Standard
Designation: D 6081 – 98
Standard Practice for
Aquatic Toxicity Testing of Lubricants: Sample Preparation
and Results Interpretation
This standard is issued under the fixed designation D 6081; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 This practice covers procedures to be used in the 2.1 ASTM Standards:
preparation of lubricants or their components for toxicity D 1129 Terminology Relating to Water
testing in aquatic systems and in the interpretation of the results D 1193 Specification for Reagent Water
of such tests. E 729 Guide for Conducting Acute Tests on Test Materials
1.2 This practice is suitable for use on fully-formulated with Fishes, Macroinvertebrates, and Amphibians
lubricants or their components which are not completely E 943 Terminology Relating to Biological Effects and En-
soluble at the intended test treat rates. It is also suitable for use vironmental Fate
with additives, if the additive is tested after being blended into
3. Terminology
a carrier fluid at the approximate concentration as in the
3.1 Definitions:
intended fully-formulated lubricant. The carrier fluid shall
meet the above solubility criterion, be known to be minimally 3.1.1 acute toxicity test, n—a comparative toxicity test in
which a representative subpopulation of organisms is exposed
toxic in the toxicity test in which the material will be tested,
and be known to have a chemical composition similar to the to different treat rates of a test material and is observed for a
short period usually not constituting a substantial portion of
rest of the intended fully-formulated lubricant.
1.3 Samples prepared in accordance with this practice may their life span.
3.1.2 chronic toxicity test, n—a comparative toxicity test in
be used in acute or chronic aquatic toxicity tests conducted in
fresh water or salt water with fish, large invertebrates, or algae. which a representative subpopulation of organisms are exposed
to different treat rates of a test material and is observed for a
This practice does not address preparation of samples for plant
period of time which constitutes a major portion of their life
toxicity testing other than algae.
1.4 Standard acute and chronic aquatic toxicity procedures span.
3.1.3 ecotoxicity, n—propensity of a test material to pro-
are more appropriate for lubricants with compositions which
are completely soluble at the intended test treat rates duce adverse behavioral, biochemical, or physiological effects
in non-human organisms or populations.
(1,2,3,4,5).
1.5 This practice is intended for use with lubricants or 3.1.4 effect load XX (ELXX), n—a statistically or graphi-
cally estimated loading rate of test material that is expected to
lubricant components of any volatility.
1.6 This practice does not address any questions regarding cause one or more specified effects in XX % of a representative
subpopulation of organisms under specified conditions.
the effects of any lubricant or lubricant component on human
health. 3.1.4.1 Discussion—This terminology should be used in-
stead of the standard ECXX when the test material is not
1.7 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the completely soluble at the test treat rates.
3.1.5 inhibition load XX (ILXX), n—a statistically or graphi-
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- cally estimated loading rate of test material that is expected to
cause a XX % inhibition of a biological process (such as
bility of regulatory limitations prior to use.
growth or reproduction) which has an analog as opposed to a
digital measure.
3.1.5.1 Discussion—An example of a digital measure would
This practice is under the jurisdiction of ASTM Committee D-2 on Petroleum
be alive/dead. This terminology (ILXX) should be used instead
Products and Lubricants and is the direct responsibility of Subcommittee D02.12 on
Environmental Standards for Lubricants.
Current edition approved May 10, 1998. Published September 1998.
2 3
The boldface numbers in parentheses refer to the list of references at the end of Annual Book of ASTM Standards, Vol 11.01.
this standard. Annual Book of ASTM Standards, Vol 11.05.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 6081
of the standard ICXX when the test material is not completely mixtures of chemical compounds with varying and usually
soluble at the test treat rates. poor solubility in water. Lubricants or lubricant component
3.1.6 lethal load XX (LLXX), n—a statistically or graphi- mixtures should not be added directly to aquatic systems for
cally estimated loading rate of test material that is expected to toxicity testing because the details of the addition procedure
be lethal to XX % of a representative subpopulation of organ- will have a large effect on the results of the toxicity test. Use
isms under specified conditions. of the techniques described in this practice will produce well
3.1.6.1 Discussion—This terminology should be used in- characterized test systems which will lead to tests with
stead of the standard LCXX when the material is not com- meaningful and reproducible results.
pletely soluble at the test treat rates. 5.2 The toxicity of mixtures of poorly soluble components
3.1.7 loading rate, n—the ratio of test material to aqueous cannot be expressed in the usual terms of lethal concentration
medium used in the preparation of WAF, WSF, or mechanical (or the similar terms of effect concentration or inhibition
dispersion and in the interpretation of the results of a toxicity concentration) because the mixtures may not be completely
study with a poorly water-soluble lubricant or lubricant com- soluble at treat levels which lead to toxic effects. The test
ponent. material preparation techniques given in this practice lead to
3.1.8 mechanical dispersion, n—a low energy aqueous test results expressed in terms of loading rate, which is a
medium produced by continuous stirring of the test solution practical and meaningful concept for expressing the toxicity of
and containing both dissolved and undissolved components of this type of material.
the test material. 5.3 One of the recommended methods of material prepara-
3.1.9 toxicity, n—the propensity of a test material to pro- tion for lubricants or their components is the mechanical
duce adverse behavioral, biochemical, or physiological effects dispersion technique. This particular technique generates tur-
in a living organism. bulence, and thus, it should not be used for poorly-swimming
3.1.10 water accommodated fraction (WAF), n—the pre- organisms.
dominantly aqueous portion of a mixture of water and a
6. Preparation of Exposure Matrix
material poorly soluble in water which separates in a specified
6.1 Experimental design considerations for fish and inver-
period of time after the mixture has undergone a specified
tebrates will determine the test species, number of loading rates
degree of mixing and which includes water, dissolved compo-
tested, and number of organisms per treatment. Experimental
nents, and dispersed droplets of the poorly soluble material.
design considerations for algae will determine the test species,
3.1.10.1 Discussion—The composition of the WAF depends
number of loading rates tested, and appropriate initial biomass
on the ratio of poorly soluble material to water in the original
per treatment.
mixture as well as on the details of the mixing procedure.
6.2 An acute or chronic test intended to allow calculation of
3.1.11 water soluble fraction (WSF), n—the filtrate or
an LLXX, ILXX, or ELXX will typically consist of a dilution
centrifugate of the water accommodated fraction which in-
water or test medium control and a series of at least five
cludes all parts of the WAF, except the dispersed droplets of the
loading rates of test material.
poorly soluble material.
6.3 Test material should be added directly to the dilution
3.2 Definitions of Terms Specific to This Standard:
water to prepare the exposure matrix. Use of a solvent or
3.2.1 dilution water, n—the water used in the aquatic test
chemical dispersant is not recommended because these may
under consideration which may contain organic or inorganic
alter the nature of the exposure to the test material and lead to
components depending on the test requirements.
non-representative ecotoxicity relationships.
3.2.2 exposure matrix, n—the WAF, WSF, or mechanical
6.4 Preparation of Water-Accommodated Fraction (WAF):
dispersion to which test organisms are exposed for toxicity
6.4.1 Components dissolved in the water phase or entrained
testing.
as stable droplets in the water phase are evaluated in a WAF
3.2.3 test systems, n—the test organisms plus the exposure
study (6). Individual WAFs must be generated for each test
matrix and the test chambers themselves.
exposure loading. Serial dilutions of a single WAF are not
3.2.4 water, n—a reagent which is defined by Type II of
appropriate due to differential solubility of constituents at low
Specification D 1193.
exposure loads (6,7). A schematic of the WAF setup is provided
3.3 Definitions of additional terms used in this practice can
in Fig. 1.
be found in Terminologies D 1129 and E 943.
6.4.2 Vessels for WAF preparation should be of suitable
4. Summary of Practice
shape and size for the experimental design. The vessels should
be of sufficient size to maintain the vortex intensity and
4.1 This practice discusses various techniques for the prepa-
optimize surface area contact between the test material and the
ration of test material for toxicity testing of poorly water-
water. Height to diameter ratio for the WAF preparation at
soluble lubricants or their components under acute or chronic
initiation should be between 1:1 and 2:1, and solutions should
exposure conditions with fish, large invertebrates, or algae.
not exceed 20 L per individual preparation. If larger volumes of
5. Significance and Use
test solution are required for experimental design, separate
5.1 This practice gives techniques to use in the preparation WAF preparations of the same test load may be combined.
of lubricants or lubricant components for acute or chronic Aspirator bottles or other vessels with outlet ports at the bottom
aquatic toxicity tests. Most lubricants and lubricant compo- are preferable for WAF collection, if the test material is less
nents are difficult to evaluate in toxicity tests because they are dense than water. If a glass siphon is used to remove the WAF,
D 6081
FIG. 1 Water Accommodated Fraction (WAF) Schematic
it should be in place prior to the introduction of the test the dark. Preparation vessels should be inspected occasionally
material. WAF solutions from test materials which are denser to ensure an appropriate vortex is maintained.
than water should be decanted from the top of the vessel. For 6.4.4 The amount of test material added to each vessel
these dense fluids, a propeller-driven system which stirs the test should be determined by the desired nominal exposure load.
solution from the top of the vessel may be preferable to a stir Nominal exposure loads may be based on direct weight of
plate which stirs the test solution from the bottom of the vessel. material added per volume solution (wt/vol) or calculated on
In all cases, care should be taken to ensure that non-dispersed the basis of test material density and added on a vol/vol basis.
test material is not collected with the WAF. 6.4.5 The stir plates should be run at a sufficient speed to
6.4.3 Vessels for WAF preparation should be filled with the ensure a vortex depth of 10 to 35 % of the test solution height
appropriate volume of dilution water, and be prepared for in the WAF preparation vessel. The vortex in all WAF
stirring as described in 6.4.2. Test material should be slowly preparation vessels for any one experiment should be the same.
added to the top of each vessel after dilution water addition. Exercise care to ensure that test material is not pulled down to
The vessel should be capped with foil or a non-reactive the bottom of the vessel, and that the rate of stirring is not so
covering and stirred at the test temperature. For test tempera- vigorous as to promote emulsification.
tures different than room temperature, the WAF preparation 6.4.6 The duration of WAF preparation may depend on the
vessels may need to be housed in water baths or incubators to results of an optional equilibrium/stability study (see 6.7). In
maintain the desired temperature. If photodecomposition of the the absence of this study, a standard 20 to 24-h mixing and 1
test material is likely, the stirring phase should be conducted in to 4-h settling period should be used (6,8).
D 6081
6.4.7 Upon completion of the WAF mixing and settling supernatant is the WSF exposure matrix and should be used
period, the aqueous solution should be drained or decanted undiluted in the exposure vessels.
from below the surface of each preparation vessel. The first 5
6.6 Preparation of a Mechanical Dispersion:
to 10 mL of solution decanted from the vessel should be
6.6.1 In the mechanical dispersion technique, a motor-
discarded. The remaining solution should be used undiluted in
driven propeller assembly is used to continually move test
the exposure vessels.
material and dilution water in the test vessel during the study
6.5 Preparation of Water-Soluble Fraction (WSF):
(6,7,10). This procedure should not be used for small-sized,
6.5.1 Only components dissolved in the water phase are
slowly, or poorly-swimming organisms. It is most appropriate
evaluated in a WSF study (6). WSFs are prepared by filtration
for juvenile fish evaluated in acute studies. A schematic of the
of WAFs through a 0.45 μm nominal cellulose-acetate filter or
mechanical dispersion setup is provided in Fig. 3.
by centrifugation to remove undissolved material. Preliminary
6.6.2 Each test vessel is equipped with a central cylinder
testing may be necessary to ensure that the filter disc will not
equipped with a three-bladed propeller. The cylinder should be
remove test material constituents which are dissolved in the
of appropriate configuration to allow water and test material to
water phase. Individual WSFs must be generated for each test
spill in
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