ASTM D6081-98(2014)
(Practice)Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation
Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation
SIGNIFICANCE AND USE
5.1 This practice gives techniques to use in the preparation of lubricants or lubricant components for acute or chronic aquatic toxicity tests. Most lubricants and lubricant components are difficult to evaluate in toxicity tests because they are mixtures of chemical compounds with varying and usually poor solubility in water. Lubricants or lubricant component mixtures should not be added directly to aquatic systems for toxicity testing because the details of the addition procedure will have a large effect on the results of the toxicity test. Use of the techniques described in this practice will produce well-characterized test systems that will lead to tests with meaningful and reproducible results.
5.2 The toxicity of mixtures of poorly soluble components cannot be expressed in the usual terms of lethal concentration (or the similar terms of effect concentration or inhibition concentration) because the mixtures may not be completely soluble at treat levels that lead to toxic effects. The test material preparation techniques given in this practice lead to test results expressed in terms of loading rate, which is a practical and meaningful concept for expressing the toxicity of this type of material.
5.3 One of the recommended methods of material preparation for lubricants or their components is the mechanical dispersion technique. This particular technique generates turbulence, and thus, it should not be used for poorly swimming organisms.
SCOPE
1.1 This practice covers procedures to be used in the preparation of lubricants or their components for toxicity testing in aquatic systems and in the interpretation of the results of such tests.
1.2 This practice is suitable for use on fully-formulated lubricants or their components that are not completely soluble at the intended test treat rates. It is also suitable for use with additives, if the additive is tested after being blended into a carrier fluid at the approximate concentration as in the intended fully formulated lubricant. The carrier fluid shall meet the above solubility criterion, be known to be minimally toxic in the toxicity test in which the material will be tested, and be known to have a chemical composition similar to the rest of the intended fully formulated lubricant.
1.3 Samples prepared in accordance with this practice may be used in acute or chronic aquatic toxicity tests conducted in fresh water or salt water with fish, large invertebrates, or algae. This practice does not address preparation of samples for plant toxicity testing other than algae.
1.4 Standard acute and chronic aquatic toxicity procedures are more appropriate for lubricants with compositions that are completely soluble at the intended test treat rates (1, 2, 3, 4, 5).2
1.5 This practice is intended for use with lubricants or lubricant components of any volatility.
1.6 This practice does not address any questions regarding the effects of any lubricant or lubricant component on human health.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D6081 − 98 (Reapproved 2014)
Standard Practice for
Aquatic Toxicity Testing of Lubricants: Sample Preparation
and Results Interpretation
This standard is issued under the fixed designation D6081; 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 1.8 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice covers procedures to be used in the
responsibility of the user of this standard to establish appro-
preparation of lubricants or their components for toxicity
priate safety and health practices and determine the applica-
testinginaquaticsystemsandintheinterpretationoftheresults
bility of regulatory limitations prior to use.
of such tests.
2. Referenced Documents
1.2 This practice is suitable for use on fully-formulated
lubricants or their components that are not completely soluble
2.1 ASTM Standards:
at the intended test treat rates. It is also suitable for use with
D1129 Terminology Relating to Water
additives, if the additive is tested after being blended into a
D1193 Specification for Reagent Water
carrierfluidattheapproximateconcentrationasintheintended
E729 Guide for Conducting Acute Toxicity Tests on Test
fully formulated lubricant. The carrier fluid shall meet the
Materials with Fishes, Macroinvertebrates, and Amphib-
above solubility criterion, be known to be minimally toxic in
ians
the toxicity test in which the material will be tested, and be
E943 Terminology Relating to Biological Effects and Envi-
knowntohaveachemicalcompositionsimilartotherestofthe
ronmental Fate
intended fully formulated lubricant.
3. Terminology
1.3 Samples prepared in accordance with this practice may
3.1 Definitions:
be used in acute or chronic aquatic toxicity tests conducted in
3.1.1 acute toxicity test, n—a comparative toxicity test in
fresh water or salt water with fish, large invertebrates, or algae.
which a representative subpopulation of organisms is exposed
This practice does not address preparation of samples for plant
to different treat rates of a test material and is observed for a
toxicity testing other than algae.
short period, usually not constituting a substantial portion of
1.4 Standard acute and chronic aquatic toxicity procedures
their life span.
are more appropriate for lubricants with compositions that are
3.1.2 chronic toxicity test, n—a comparative toxicity test in
completelysolubleattheintendedtesttreatrates (1, 2, 3, 4, 5).
which a representative subpopulation of organisms is exposed
1.5 This practice is intended for use with lubricants or
to different treat rates of a test material and is observed for a
lubricant components of any volatility.
periodoftimethatconstitutesamajorportionoftheirlifespan.
1.6 This practice does not address any questions regarding
3.1.3 ecotoxicity, n—propensityofatestmaterialtoproduce
the effects of any lubricant or lubricant component on human
adverse behavioral, biochemical, or physiological effects in
health.
non-human organisms or populations.
1.7 The values stated in SI units are to be regarded as 3.1.4 effect load XX (ELXX), n—astatisticallyorgraphically
estimated loading rate of test material that is expected to cause
standard. No other units of measurement are included in this
standard. one or more specified effects in XX % of a representative
subpopulation of organisms under specified conditions.
3.1.4.1 Discussion—This terminology should be used in-
stead of the standard ECXX when the test material is not
This practice is under the jurisdiction ofASTM Committee D02 on Petroleum
completely soluble at the test treat rates.
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom-
mittee D02.12 on Environmental Standards for Lubricants.
Current edition approved Oct. 1, 2014. Published November 2014. Originally
approved in 1998. Last previous edition approved in 2009 as D6081–98(2009). For referenced ASTM standards, visit the ASTM website, www.astm.org, or
DOI: 10.1520/D6081-98R14. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers in parentheses refer to the list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6081 − 98 (2014)
3.1.5 inhibition load XX (ILXX), n—a statistically or graphi- 4. Summary of Practice
cally estimated loading rate of test material that is expected to
4.1 Thispracticediscussesvarioustechniquesfortheprepa-
cause a XX % inhibition of a biological process (such as
ration of test material for toxicity testing of poorly water-
growth or reproduction) that has an analog as opposed to a
soluble lubricants or their components under acute or chronic
digital measure.
exposure conditions with fish, large invertebrates, or algae.
3.1.5.1 Discussion—An example of a digital measure would
bealive/dead.Thisterminology(ILXX)shouldbeusedinstead
5. Significance and Use
of the standard ICXX when the test material is not completely
5.1 This practice gives techniques to use in the preparation
soluble at the test treat rates.
of lubricants or lubricant components for acute or chronic
3.1.6 lethal load XX (LLXX), n—a statistically or graphi-
aquatic toxicity tests. Most lubricants and lubricant compo-
cally estimated loading rate of test material that is expected to
nents are difficult to evaluate in toxicity tests because they are
be lethal to XX % of a representative subpopulation of organ-
mixtures of chemical compounds with varying and usually
isms under specified conditions.
poor solubility in water. Lubricants or lubricant component
3.1.6.1 Discussion—This terminology should be used in-
mixtures should not be added directly to aquatic systems for
stead of the standard LCXX when the material is not com-
toxicity testing because the details of the addition procedure
pletely soluble at the test treat rates.
will have a large effect on the results of the toxicity test. Use
3.1.7 loading rate, n—the ratio of test material to aqueous
of the techniques described in this practice will produce
medium used in the preparation of WAF, WSF, or mechanical
well-characterized test systems that will lead to tests with
dispersion and in the interpretation of the results of a toxicity
meaningful and reproducible results.
study with a poorly water-soluble lubricant or lubricant com-
5.2 The toxicity of mixtures of poorly soluble components
ponent.
cannot be expressed in the usual terms of lethal concentration
3.1.8 mechanical dispersion, n—a low energy aqueous me-
(or the similar terms of effect concentration or inhibition
dium produced by continuous stirring of the test solution and
concentration) because the mixtures may not be completely
containing both dissolved and undissolved components of the
solubleattreatlevelsthatleadtotoxiceffects.Thetestmaterial
test material.
preparation techniques given in this practice lead to test results
3.1.9 toxicity, n—thepropensityofatestmaterialtoproduce expressed in terms of loading rate, which is a practical and
adverse behavioral, biochemical, or physiological effects in a
meaningful concept for expressing the toxicity of this type of
living organism. material.
3.1.10 water accommodated fraction (WAF), n—the pre-
5.3 One of the recommended methods of material prepara-
dominantly aqueous portion of a mixture of water and a
tion for lubricants or their components is the mechanical
material poorly soluble in water, which separates in a specified
dispersion technique. This particular technique generates
period of time after the mixture has undergone a specified
turbulence, and thus, it should not be used for poorly swim-
degree of mixing and which includes water, dissolved
ming organisms.
components, and dispersed droplets of the poorly soluble
material.
6. Preparation of Exposure Matrix
3.1.10.1 Discussion—The composition of the WAF depends
6.1 Experimental design considerations for fish and inver-
on the ratio of poorly soluble material to water in the original
tebrateswilldeterminethetestspecies,numberofloadingrates
mixture as well as on the details of the mixing procedure.
tested, and number of organisms per treatment. Experimental
3.1.11 water soluble fraction (WSF), n—the filtrate or cen-
design considerations for algae will determine the test species,
trifugate of the water accommodated fraction, which includes
number of loading rates tested, and appropriate initial biomass
allpartsoftheWAF,exceptthedisperseddropletsofthepoorly
per treatment.
soluble material.
6.2 An acute or chronic test intended to allow calculation of
3.2 Definitions of Terms Specific to This Standard:
an LLXX, ILXX, or ELXX will typically consist of a dilution
3.2.1 dilution water, n—the water used in the aquatic test
water or test medium control and a series of at least five
under consideration, which may contain organic or inorganic
loading rates of test material.
components depending on the test requirements.
6.3 Test material should be added directly to the dilution
3.2.2 exposure matrix, n—the WAF, WSF, or mechanical
water to prepare the exposure matrix. Use of a solvent or
dispersion to which test organisms are exposed for toxicity
chemical dispersant is not recommended because these may
testing.
alter the nature of the exposure to the test material and lead to
3.2.3 test systems, n—the test organisms plus the exposure
non-representative ecotoxicity relationships.
matrix and the test chambers themselves.
6.4 Preparation of Water-Accommodated Fraction (WAF):
3.2.4 water, n—a reagent that is defined by Type II of
6.4.1 Components dissolved in the water phase or entrained
Specification D1193.
as stable droplets in the water phase are evaluated in a WAF
3.3 Definitions of additional terms used in this practice can study (6). Individual WAFs must be generated for each test
be found in Terminologies D1129 and E943. exposure loading. Serial dilutions of a single WAF are not
D6081 − 98 (2014)
appropriate due to differential solubility of constituents at low these dense fluids, a propeller-driven system that stirs the test
exposure loads (6, 7). A schematic of the WAF setup is solution from the top of the vessel may be preferable to a stir
provided in Fig. 1.
plate that stirs the test solution from the bottom of the vessel.
6.4.2 Vessels for WAF preparation should be of suitable
In all cases, care should be taken to ensure that non-dispersed
shape and size for the experimental design. The vessels should
test material is not collected with the WAF.
be of sufficient size to maintain the vortex intensity and
6.4.3 Vessels for WAF preparation should be filled with the
optimize surface area contact between the test material and the
appropriate volume of dilution water, and be prepared for
water. Height to diameter ratio for the WAF preparation at
stirring as described in 6.4.2. Test material should be slowly
initiation should be between 1:1 and 2:1, and solutions should
added to the top of each vessel after dilution water addition.
notexceed20Lperindividualpreparation.Iflargervolumesof
The vessel should be capped with foil or a non-reactive
test solution are required for experimental design, separate
covering and stirred at the test temperature. For test tempera-
WAF preparations of the same test load may be combined.
tures different than room temperature, the WAF preparation
Aspiratorbottlesorothervesselswithoutletportsatthebottom
vessels may need to be housed in water baths or incubators to
are preferable for WAF collection, if the test material is less
maintainthedesiredtemperature.Ifphotodecompositionofthe
dense than water. If a glass siphon is used to remove the WAF,
test material is likely, the stirring phase should be conducted in
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 that are denser
than water should be decanted from the top of the vessel. For to ensure an appropriate vortex is maintained.
FIG. 1 Water Accommodated Fraction (WAF) Schematic
D6081 − 98 (2014)
6.4.4 The amount of test material added to each vessel
should be determined by the desired nominal exposure load.
Nominal exposure loads may be based on direct weight of
material added per volume solution (wt/vol) or calculated on
the basis of test material density and added on a vol/vol basis.
6.4.5 The stir plates should be run at a sufficient speed to
ensure a vortex depth of 10 to 35 % of the test solution height
in the WAF preparation vessel. The vortex in all WAF
preparationvesselsforanyoneexperimentshouldbethesame.
Exercise care to ensure that test material is not pulled down to
the bottom of the vessel, and that the rate of stirring is not so
vigorous as to promote emulsification.
6.4.6 The duration of WAF preparation may depend on the
results of an optional equilibrium/stability study (see 6.7). In
the absence of this study, a standard 20 to 24-h mixing and 1
to 4-h settling period should be used (6, 8).
6.4.7 Upon completion of the WAF mixing and settling
period, the aqueous solution should be drained or decanted
from below the surface of each preparation vessel. The first 5
to 10 mL of solution decanted from the vessel should be
discarded. The remaining solution should be used undiluted in
the exposure vessels.
6.5 Preparation of Water-Soluble Fraction (WSF):
6.5.1 Only components dissolved in the water phase are
evaluated in a WSF study (6) . WSFs are prepared by filtration
of WAFs through a 0.45-µm nominal cellulose-acetate filter or
by centrifugation to remove undissolved material. Preliminary
testing may be necessary to ensure that the filter disc will not
removetestmaterialconstituentsthataredissolvedinthewater
FIG. 2 Water Soluble Fraction (WSF) Schematic
phase. Individual WSFs must be generated for each test
exposure load. Serial dilutions of a single WSF are not
appropriate due to differential solubility of constituents at low
exposure loads. A schematic of the WSF setup is provided in
Fig. 2.
6.5.2 The procedures described in 6.4.3 – 6.4.7 for theWAF
preparation are the initial steps of the WSF procedure.
6.5.3 Upon completion of the WAF mixing and settling
period, the aqueous solution should be decanted from below
the surface of ea
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D6081 − 98 (Reapproved 2009) D6081 − 98 (Reapproved 2014)
Standard Practice for
Aquatic Toxicity Testing of Lubricants: Sample Preparation
and Results Interpretation
This standard is issued under the fixed designation D6081; 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
1.1 This practice covers procedures to be used in the preparation of lubricants or their components for toxicity testing in aquatic
systems and in the interpretation of the results of such tests.
1.2 This practice is suitable for use on fully-formulated lubricants or their components that are not completely soluble at the
intended test treat rates. It is also suitable for use with additives, if the additive is tested after being blended into a carrier fluid
at the approximate concentration as in the intended fully formulated lubricant. The carrier fluid shall meet the above solubility
criterion, be known to be minimally toxic in the toxicity test in which the material will be tested, and be known to have a chemical
composition similar to the rest of the intended fully formulated lubricant.
1.3 Samples prepared in accordance with this practice may be used in acute or chronic aquatic toxicity tests conducted in fresh
water or salt water with fish, large invertebrates, or algae. This practice does not address preparation of samples for plant toxicity
testing other than algae.
1.4 Standard acute and chronic aquatic toxicity procedures are more appropriate for lubricants with compositions that are
completely soluble at the intended test treat rates (1, 2, 3, 4, 5).
1.5 This practice is intended for use with lubricants or lubricant components of any volatility.
1.6 This practice does not address any questions regarding the effects of any lubricant or lubricant component on human health.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
E729 Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
E943 Terminology Relating to Biological Effects and Environmental Fate
3. Terminology
3.1 Definitions:
3.1.1 acute toxicity test, n—a comparative toxicity test in which a representative subpopulation of organisms is exposed to
different treat rates of a test material and is observed for a short period, usually not constituting a substantial portion of their life
span.
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum Products Products, Liquid Fuels, and Lubricants and is the direct responsibility of
Subcommittee D02.12 on Environmental Standards for Lubricants.
Current edition approved Oct. 1, 2009Oct. 1, 2014. Published November 2009November 2014. Originally approved in 1998. Last previous edition approved in 20042009
as D6081–98(2004).D6081–98(2009). DOI: 10.1520/D6081-98R09.10.1520/D6081-98R14.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6081 − 98 (2014)
3.1.2 chronic toxicity test, n—a comparative toxicity test in which a representative subpopulation of organisms is exposed to
different treat rates of a test material and is observed for a period of time that constitutes a major portion of their life span.
3.1.3 ecotoxicity, n—propensity of a test material to produce adverse behavioral, biochemical, or physiological effects in
non-human organisms or populations.
3.1.4 effect load XX (ELXX), n—a statistically or graphically estimated loading rate of test material that is expected to cause one
or more specified effects in XX % of a representative subpopulation of organisms under specified conditions.
3.1.4.1 Discussion—
This terminology should be used instead of the standard ECXX when the test material is not completely soluble at the test treat
rates.
3.1.5 inhibition load XX (ILXX), n—a statistically or graphically estimated loading rate of test material that is expected to cause
a XX % inhibition of a biological process (such as growth or reproduction) that has an analog as opposed to a digital measure.
3.1.5.1 Discussion—
An example of a digital measure would be alive/dead. This terminology (ILXX) should be used instead of the standard ICXX when
the test material is not completely soluble at the test treat rates.
3.1.6 lethal load XX (LLXX), n—a statistically or graphically estimated loading rate of test material that is expected to be lethal
to XX % of a representative subpopulation of organisms under specified conditions.
3.1.6.1 Discussion—
This terminology should be used instead of the standard LCXX when the material is not completely soluble at the test treat rates.
3.1.7 loading rate, n—the ratio of test material to aqueous medium used in the preparation of WAF, WSF, or mechanical
dispersion and in the interpretation of the results of a toxicity study with a poorly water-soluble lubricant or lubricant component.
3.1.8 mechanical dispersion, n—a low energy aqueous medium produced by continuous stirring of the test solution and
containing both dissolved and undissolved components of the test material.
3.1.9 toxicity, n—the propensity of a test material to produce adverse behavioral, biochemical, or physiological effects in a living
organism.
3.1.10 water accommodated fraction (WAF), n—the predominantly aqueous portion of a mixture of water and a material poorly
soluble in water, which separates in a specified period of time after the mixture has undergone a specified degree of mixing and
which includes water, dissolved components, and dispersed droplets of the poorly soluble material.
3.1.10.1 Discussion—
The composition of the WAF depends on the ratio of poorly soluble material to water in the original mixture as well as on the
details of the mixing procedure.
3.1.11 water soluble fraction (WSF), n—the filtrate or centrifugate of the water accommodated fraction, which includes all parts
of the WAF, except the dispersed droplets of the poorly soluble material.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 dilution water, n—the water used in the aquatic test under consideration, which may contain organic or inorganic
components depending on the test requirements.
3.2.2 exposure matrix, n—the WAF, WSF, or mechanical dispersion to which test organisms are exposed for toxicity testing.
3.2.3 test systems, n—the test organisms plus the exposure matrix and the test chambers themselves.
3.2.4 water, n—a reagent that is defined by Type II of Specification D1193.
3.3 Definitions of additional terms used in this practice can be found in Terminologies D1129 and E943.
4. Summary of Practice
4.1 This practice discusses various techniques for the preparation of test material for toxicity testing of poorly water-soluble
lubricants or their components under acute or chronic exposure conditions with fish, large invertebrates, or algae.
D6081 − 98 (2014)
5. Significance and Use
5.1 This practice gives techniques to use in the preparation of lubricants or lubricant components for acute or chronic aquatic
toxicity tests. Most lubricants and lubricant components are difficult to evaluate in toxicity tests because they are mixtures of
chemical compounds with varying and usually poor solubility in water. Lubricants or lubricant component mixtures should not be
added directly to aquatic systems for toxicity testing because the details of the addition procedure will have a large effect on the
results of the toxicity test. Use of the techniques described in this practice will produce well-characterized test systems that will
lead to tests with meaningful and reproducible results.
5.2 The toxicity of mixtures of poorly soluble components cannot be expressed in the usual terms of lethal concentration (or
the similar terms of effect concentration or inhibition concentration) because the mixtures may not be completely soluble at treat
levels that lead to toxic effects. The test material preparation techniques given in this practice lead to test results expressed in terms
of loading rate, which is a practical and meaningful concept for expressing the toxicity of this type of material.
5.3 One of the recommended methods of material preparation for lubricants or their components is the mechanical dispersion
technique. This particular technique generates turbulence, and thus, it should not be used for poorly swimming organisms.
6. Preparation of Exposure Matrix
6.1 Experimental design considerations for fish and invertebrates will determine the test species, number of loading rates tested,
and number of organisms per treatment. Experimental design considerations for algae will determine the test species, number of
loading rates tested, and appropriate initial biomass per treatment.
6.2 An acute or chronic test intended to allow calculation of an LLXX, ILXX, or ELXX will typically consist of a dilution water
or test medium control and a series of at least five loading rates of test material.
6.3 Test material should be added directly to the dilution water to prepare the exposure matrix. Use of a solvent or chemical
dispersant is not recommended because these may alter the nature of the exposure to the test material and lead to non-representative
ecotoxicity relationships.
6.4 Preparation of Water-Accommodated Fraction (WAF):
6.4.1 Components dissolved in the water phase or entrained as stable droplets in the water phase are evaluated in a WAF study
(6). Individual WAFs must be generated for each test exposure loading. Serial dilutions of a single WAF are not appropriate due
to differential solubility of constituents at low exposure loads (6, 7). A schematic of the WAF setup is provided in Fig. 1.
6.4.2 Vessels for WAF preparation should be of suitable shape and size for the experimental design. The vessels should be of
sufficient size to maintain the vortex intensity and optimize surface area contact between the test material and the water. Height
to diameter ratio for the WAF preparation at initiation should be between 1:1 and 2:1, and solutions should not exceed 20 L per
individual preparation. If larger volumes of test solution are required for experimental design, separate WAF preparations of the
same test load may be combined. Aspirator bottles or other vessels with outlet ports at the bottom are preferable for WAF
collection, if the test material is less dense than water. If a glass siphon is used to remove the WAF, it should be in place prior to
the introduction of the test material. WAF solutions from test materials that are denser than water should be decanted from the top
of the vessel. For these dense fluids, a propeller-driven system that stirs the test solution from the top of the vessel may be
preferable to a stir plate that stirs the test solution from the bottom of the vessel. In all cases, care should be taken to ensure that
non-dispersed test material is not collected with the WAF.
6.4.3 Vessels for WAF preparation should be filled with the appropriate volume of dilution water, and be prepared for stirring
as described in 6.4.2. Test material should be slowly added to the top of each vessel after dilution water addition. The vessel should
be capped with foil or a non-reactive covering and stirred at the test temperature. For test temperatures different than room
temperature, the WAF preparation vessels may need to be housed in water baths or incubators to maintain the desired temperature.
If photodecomposition of the test material is likely, the stirring phase should be conducted in the dark. Preparation vessels should
be inspected occasionally to ensure an appropriate vortex is maintained.
6.4.4 The amount of test material added to each vessel should be determined by the desired nominal exposure load. Nominal
exposure loads may be based on direct weight of material added per volume solution (wt/vol) or calculated on the basis of test
material density and added on a vol/vol basis.
6.4.5 The stir plates should be run at a sufficient speed to ensure a vortex depth of 10 to 35 % of the test solution height in the
WAF preparation vessel. The vortex in all WAF preparation vessels for any one experiment should be the same. Exercise care to
ensure that test material is not pulled down to the bottom of the vessel, and that the rate of stirring is not so vigorous as to promote
emulsification.
6.4.6 The duration of WAF preparation may depend on the results of an optional equilibrium/stability study (see 6.7). In the
absence of this study, a standard 20 to 24-h mixing and 1 to 4-h settling period should be used (6, 8).
6.4.7 Upon completion of the WAF mixing and settling period, the aqueous solution should be drained or decanted from below
the surface of each preparation vessel. The first 5 to 10 mL of solution decanted from the vessel should be discarded. The remaining
solution should be used undiluted in the exposure vessels.
6.5 Preparation of Water-Soluble Fraction (WSF):
D6081 − 98 (2014)
FIG. 1 Water Accommodated Fraction (WAF) Schematic
6.5.1 Only components dissolved in the water phase are evaluated in a WSF study (6) . WSFs are prepared by filtration of WAFs
through a 0.45-μm nominal cellulose-acetate filter or by centrifugation to remove undissolved material. Preliminary testing may
be necessary to ensure that the filter disc will not remove test material constituents that are dissolved in the water phase. Individual
WSFs must be genera
...
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