ASTM D6081-22

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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2022
• ICS: 13.020.40 - Pollution, pollution control and conservation
• ICS: 75.100 - Lubricants, industrial oils and related products
• Technical Committee: D02 - Petroleum Products, Liquid Fuels, and Lubricants
• Drafting Committee: D02.12 - Environmental Standards for Lubricants

Relations

• Refers: ASTM D4175-23a - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 15-Dec-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM E943-08 - Standard Terminology Relating to Biological Effects and Environmental Fate
• Effective Date: 01-Mar-2008
• Refers: ASTM E729-96(2007) - Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• Effective Date: 01-Oct-2007
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D1129-04 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-03a - Standard Terminology Relating to Water
• Effective Date: 10-Aug-2003
• Refers: ASTM D1129-03 - Standard Terminology Relating to Water
• Effective Date: 10-Mar-2003
• Refers: ASTM D1129-01 - Standard Terminology Relating to Water
• Effective Date: 10-Jul-2002

Overview

ASTM D6081-22: Standard Practice for Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation provides comprehensive guidelines for preparing lubricant samples and interpreting results in aquatic toxicity tests. Developed by ASTM International, this standard addresses the unique challenges presented by lubricants and lubricant components, which are often mixtures of chemical compounds with limited water solubility. The standard outlines best practices to ensure repeatability, accuracy, and relevance in evaluating the aquatic toxicity of these substances under both acute and chronic testing scenarios.

Key Topics

• Sample Preparation: Guidance on preparing lubricants and lubricant components for toxicity testing using methods like Water Accommodated Fraction (WAF), Water Soluble Fraction (WSF), and mechanical dispersion. The procedures aim to account for variable solubility and the complex nature of many lubricant formulations.
• Exposure Matrix Creation: Detailed protocols for generating test matrices that are suitable for exposing fish, large invertebrates, and algae to lubricant samples in both freshwater and saltwater systems.
• Interpretation of Results: Introduction of metrics such as loading rate, lethal load (LLXX), effect load (ELXX), and inhibition load (ILXX), which are more practical for poorly soluble mixtures than traditional concentration-based measures.
• Limitations and Interferences: Identification of potential challenges, such as changes in test material composition due to volatility, storage, vessel interaction, or material partitioning, which can affect both test outcomes and data interpretation.
• Test Organisms: Recommendations for selecting appropriate aquatic organisms for toxicity testing, with consideration for the test method’s compatibility (e.g., avoiding mechanical dispersion for poorly swimming species).
• Safety and Environmental Considerations: The standard underscores the need for appropriate health and safety practices, without addressing human health impacts directly.

Applications

ASTM D6081-22 serves industries and laboratories that conduct environmental impact assessments and regulatory compliance evaluations for lubricants and lubricant additives. Typical applications include:

• Environmental Risk Assessment: Reliable methodologies for predicting the ecological impact of lubricant spills or releases to aquatic environments.
• Product Stewardship: Supporting lubricant manufacturers in demonstrating environmental responsibility and compliance with international regulatory frameworks.
• Regulatory Compliance: Satisfies requirements for aquatic toxicity testing under national and international regulations, especially where test substances are insoluble or only partially soluble in water.
• Research and Development: Facilitates consistent and credible aquatic toxicity data generation during the design and evaluation of new lubricants or additive packages.

Related Standards

ASTM D6081-22 references several related standards and guides that support and complement its procedures:

• ASTM D1129: Terminology Relating to Water
• ASTM D1193: Specification for Reagent Water
• ASTM D4175: Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• ASTM E729: Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
• ASTM E943: Terminology Relating to Biological Effects and Environmental Fate
• OECD 203, EPA Guidelines, and applicable Environment Canada methods for aquatic toxicity

By following ASTM D6081-22, organizations can ensure scientifically robust, reproducible aquatic toxicity testing with lubricants, yielding results that are meaningful for both regulatory decision-making and product safety assessments. Incorporating keywords like "aquatic toxicity," "lubricant testing," "sample preparation," and "environmental compliance" ensures the standard’s relevance within the environmental testing and lubricant manufacturing sectors.

Standards Content (Sample)

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: D6081 − 22
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, health, and environmental practices and deter-
testinginaquaticsystemsandintheinterpretationoftheresults
mine the applicability of regulatory limitations prior to use.
of such tests.
1.9 This international standard was developed in accor-
1.2 This practice is suitable for use on fully-formulated
dance with internationally recognized principles on standard-
lubricants or their components that are not completely soluble
ization established in the Decision on Principles for the
at the intended test treat rates. It is also suitable for use with
Development of International Standards, Guides and Recom-
additives, if the additive is tested after being blended into a
mendations issued by the World Trade Organization Technical
carrierfluidattheapproximateconcentrationasintheintended
Barriers to Trade (TBT) Committee.
fully formulated lubricant. The carrier fluid shall meet the
2. Referenced Documents
above solubility criterion, be known to be minimally toxic in
the toxicity test in which the material will be tested, and be
2.1 ASTM Standards:
knowntohaveachemicalcompositionsimilartotherestofthe
D1129 Terminology Relating to Water
intended fully formulated lubricant.
D1193 Specification for Reagent Water
D4175 Terminology Relating to Petroleum Products, Liquid
1.3 Samples prepared in accordance with this practice may
Fuels, and Lubricants
be used in acute or chronic aquatic toxicity tests conducted in
E729 Guide for Conducting Acute Toxicity Tests on Test
fresh water or salt water with fish, large invertebrates, or algae.
Materials with Fishes, Macroinvertebrates, and Amphib-
This practice does not address preparation of samples for plant
ians
toxicity testing other than algae.
E943 Terminology Relating to Biological Effects and Envi-
1.4 Standard acute and chronic aquatic toxicity procedures
ronmental Fate
are more appropriate for lubricants with compositions that are
completelysolubleattheintendedtesttreatrates (1, 2, 3, 4, 5).
3. Terminology
1.5 This practice is intended for use with lubricants or
3.1 For definitions of terms used in this practice, see
lubricant components of any volatility.
Terminologies D1129, D4175, and E943.
1.6 This practice does not address any questions regarding 3.2 Definitions:
the effects of any lubricant or lubricant component on human
3.2.1 acute toxicity test, n—a comparative test in which a
health. representative subpopulation of organisms is exposed to dif-
ferent treat rates of a test material and is observed for a short
1.7 The values stated in SI units are to be regarded as
period, usually not constituting a substantial portion of their
standard. No other units of measurement are included in this
life span.
standard.
3.2.2 chronic toxicity test, n—a comparative test in which a
representative subpopulation of organisms is exposed to dif-
ferent treat rates of a test material and is observed for a period
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum
of time that constitutes a major portion of their life span.
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom-
mittee D02.12 on Environmental Standards for Lubricants.
Current edition approved April 1, 2022. Published April 2022. Originally
approved in 1998. Last previous edition approved in 2020 as D6081 – 20. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/D6081-22. contact ASTM Customer Service at [e-mail]. 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.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6081 − 22
3.2.3 ecotoxicity, n—propensityofatestmaterialtoproduce 3.3.1 dilution water, n—the water used in the aquatic test
adverse behavioral, biochemical, or physiological effects in under consideration, which may contain organic or inorganic
non-human organisms or populations. components depending on the test requirements.
3.3.2 exposure matrix, n—the water accommodated fraction
3.2.4 effect load XX (ELXX), n—astatisticallyorgraphically
(WAF), water soluble fraction (WSF), or mechanical disper-
estimated loading rate of test material that is expected to cause
sion to which test organisms are exposed for toxicity testing.
one or more specified effects in XX % of a representative
subpopulation of organisms under specified conditions. 3.3.3 test systems, n—the test organisms plus the exposure
matrix and the test chambers themselves.
3.2.4.1 Discussion—This terminology should be used in-
stead of the standard ECXX when the test material is not
4. Summary of Practice
completely soluble at the test treat rates.
4.1 This practice discusses various techniques for the prepa-
3.2.5 inhibition load XX (ILXX), n—a statistically or graphi-
ration of test material for toxicity testing of poorly water-
cally estimated loading rate of test material that is expected to
soluble lubricants or their components under acute or chronic
cause a XX % inhibition of a biological process (such as
exposure conditions with fish, large invertebrates, or algae.
growth or reproduction) that has an analog as opposed to a
digital measure.
5. Significance and Use
3.2.5.1 Discussion—An example of a digital measure would
5.1 This practice gives techniques to use in the preparation
be alive/dead.This terminology (ILXX) should be used instead
of lubricants or lubricant components for acute or chronic
of the standard ICXX when the test material is not completely
aquatic toxicity tests. Most lubricants and lubricant compo-
soluble at the test treat rates.
nents are difficult to evaluate in toxicity tests because they are
mixtures of chemical compounds with varying and usually
3.2.6 lethal load XX (LLXX), n—a statistically or graphi-
poor solubility in water. Lubricants or lubricant component
cally estimated loading rate of test material that is expected to
mixtures should not be added directly to aquatic systems for
be lethal to XX % of a representative subpopulation of organ-
toxicity testing because the details of the addition procedure
isms under specified conditions.
will have a large effect on the results of the toxicity test. Use
3.2.6.1 Discussion—This terminology should be used in-
of the techniques described in this practice will produce
stead of the standard LCXX when the material is not com-
well-characterized test systems that will lead to tests with
pletely soluble at the test treat rates.
meaningful and reproducible results.
3.2.7 loading rate, n—the ratio of test material to aqueous
5.2 The toxicity of mixtures of poorly soluble components
medium used in the preparation of a water accommodated
cannot be expressed in the usual terms of lethal concentration
fraction (WAF), water soluble fraction (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-
solubleattreatlevelsthatleadtotoxiceffects.Thetestmaterial
ponent.
preparation techniques given in this practice lead to test results
3.2.8 mechanical dispersion, n—a low energy aqueous me-
expressed in terms of loading rate, which is a practical and
dium produced by continuous stirring of the test solution and
meaningful concept for expressing the toxicity of this type of
containing both dissolved and undissolved components of the material.
test material.
5.3 One of the recommended methods of material prepara-
tion for lubricants or their components is the mechanical
3.2.9 toxicity, n—thepropensityofatestmaterialtoproduce
adverse behavioral, biochemical, or physiological effects in a dispersion technique. This particular technique generates
turbulence, and thus, it should not be used for poorly swim-
living organism.
ming organisms.
3.2.10 water accommodated fraction (WAF), n—the pre-
dominantly aqueous portion of a mixture of water and a
6. Apparatus
material poorly soluble in water, which separates in a specified
6.1 Stirrer, magnetic, size sufficient to accommodate vessel
period of time after the mixture has undergone a specified
(6.2).
degree of mixing and which includes water, dissolved
6.2 Vessel, glass, bottom drain, capacity 10 L to 20 L.
components, and dispersed droplets of the poorly soluble
NOTE 1—The vessels shall be of sufficient size to maintain the vortex
material.
intensity and optimize surface area contact between the test material and
3.2.10.1 Discussion—The composition of the WAF depends
the water.
on the ratio of poorly soluble material to water in the original
NOTE 2—Height to diameter ratio for the WAF preparation at initiation
mixture as well as on the details of the mixing procedure. should be between 1:1 and 2:1, and solutions shall not exceed 20 L per
individual preparation. If larger volumes of test solution are required for
3.2.11 water soluble fraction (WSF), n—the filtrate or cen-
experimentaldesign,separateWAFpreparationsofthesametestloadmay
trifugate of the water accommodated fraction, which includes be combined.
NOTE 3—Aspirator bottles or other vessels with outlet ports at the
allpartsoftheWAF,exceptthedisperseddropletsofthepoorly
bottom are preferable for WAF collection, if the test material is less dense
soluble material.
thanwater.IfaglasssiphonisusedtoremovetheWAF,itshallbeinplace
3.3 Definitions of Terms Specific to This Standard: prior to the introduction of the test material.
D6081 − 22
NOTE 4—WAF solutions from test materials that are denser than water
8.4.4 Operate stir plates at a sufficient speed to ensure a
shall be decanted from the top of the vessel. For these dense fluids, a
vortex depth of 10 % to 35 % of the test solution height in the
propeller-driven system that stirs the test solution from the top of the
WAF preparation vessel.
vessel may be preferable to a stir plate that stirs the test solution from the
8.4.4.1 Ensure that the vortexes in all WAF preparation
bottom of the vessel.
vessels for any one experiment are the same length (that is,
NOTE 5—In all cases, take precautions to ensure that non-dispersed test
material is not collected with the WAF.
depth from fluid surface).
8.4.4.2 Exercise care to ensure that test material is not
7. Reagents
pulled down to the bottom of the vessel, and that the rate of
stirring is not so vigorous as to promote emulsification.
7.1 Test Material, lubricant from which water-soluble com-
8.4.5 The duration of WAF preparation will depend on the
ponent(s) is or are to be extracted.
results of an optional equilibrium/stability study (see 8.7). In
7.2 Water, Type II per Specification D1193.
the absence of this study, use a standard 20 h to 24 h mixing
and 1 h to 4 h settling period (6, 8).
8. Preparation of Exposure Matrix
8.4.6 Upon completion of the WAF mixing and settling
8.1 Experimental design considerations for fish and inver-
period, drain or decant the aqueous solution from below the
tebrateswilldeterminethetestspecies,numberofloadingrates surface of each preparation vessel.
tested, and number of organisms per treatment. Experimental
8.4.6.1 Discard the first 5 mLto 10 mLof solution decanted
design considerations for algae will determine the test species,
from the vessel.
number of loading rates tested, and appropriate initial biomass
8.4.6.2 Usetheremainingsolutionundilutedintheexposure
per treatment.
vessels.
8.2 An acute or chronic test intended to allow calculation of
8.5 Preparation of Water-Soluble Fraction (WSF):
an LLXX, ILXX, or ELXX will typically consist of a dilution
8.5.1 Evaluate only components dissolved in the water
water or test medium control and a series of at least five
phase in a WSF study (6).
loading rates of test material.
8.5.1.1 Prepare WSFs by filtering WAFs through a 0.45 µm
8.3 Test material shall be added directly to the dilution nominal cellulose-acetate filter or by centrifuging to remove
water to prepare the exposure matrix. Use of a solvent or undissolved material.
chemical dispersant is not recommended because these may
NOTE 6—Preliminary testing may be necessary to ensure that the filter
alter the nature of the exposure to the test material and lead to
disc will not remove test material constituents that are dissolved in the
non-representative ecotoxicity relationships.
water phase.
8.4 Preparation of Water-Accommodated Fraction (WAF):
8.5.1.2 Individual WSFs must be generated for each test
8.4.1 Components dissolved in the water phase or entrained exposure load. Serial dilutions of a single WSF are not
as stable droplets in the water phase are evaluated in a WAF appropriate due to differential solubility of constituents at low
study (6). Individual WAFs must be generated for each test exposure loads. A schematic of the WSF setup is provided in
exposure loading. Serial dilutions of a single WAF are not Fig. 2.
appropriate due to differential solubility of constituents at low
8.5.2 The procedures described in 8.4.2 – 8.4.6 for theWAF
exposure loads (6, 7). A schematic of the WAF setup is
preparation are the initial steps of the WSF procedure.
provided in Fig. 1.
8.5.3 Upon completion of the WAF mixing and settling
8.4.2 Fill vessels for WAF preparation with the appropriate
period, decant the aqueous solution from below the surface of
volume of dilution water, and prepare them for stirring as
each preparation vessel. The first 5 mL to 10 mL of solution
described in 6.2.
decanted from the vessel should be discarded, with the remain-
ing solution used to prepare the WSF. Filtration through a
8.4.2.1 Slowly add test material to the top of each vessel
after dilution water addition. 0.45 µm filter or low speed centrifugation (to remove droplets
but not to break an emulsion) is performed on the solution to
8.4.2.2 Cap the vessel with foil or a non-reactive covering
remove undissolved material. The filtrate or centrifugation
and stir at the test temperature.
supernatant is the WSF exposure matrix and should be used
8.4.2.3 If the test temperatures are different than room
undiluted in the exposure vessels.
temperature, house theWAF preparation vessels in water baths
or incubators to maintain the desired temperature.
8.6 Preparation of a Mechanical Dispersion:
8.4.2.4 If photo-decomposition of the test material is likely,
NOTE 7—In the mechanical dispersion technique, a motor-driven
conduct the stirring phase in the dark.
propeller assembly is used to continually move test material and dilution
8.4.2.5 Inspectpreparationvesselsoccasionallytoensurean
water in the test vessel during the study (6, 7, 9).
appropriate vortex is maintained.
NOTE 8—This procedure should not be used for small-sized, slowly, or
poorly swimming organisms. It is most appropriate for juvenile fish
8.4.3 Determine the amount of test material added to each
evaluated in acute studies.
vessel from the desired nominal exposure load.
8.6.1 Configuration:
8.4.3.1 Base nominal exposure loads on either direct weight
of material added per volume solution (wt/vol) or on the basis 8.6.1.1 A schematic of the mechanical dispersion setup is
of test material density and added on a vol/vol basis. provided in Fig. 3.
D6081 − 22
FIG. 1 Wa
...

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 − 20 D6081 − 22
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, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.9 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.
This practice is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcommittee
D02.12 on Environmental Standards for Lubricants.
Current edition approved Oct. 1, 2020April 1, 2022. Published October 2020April 2022. Originally approved in 1998. Last previous edition approved in 20192020 as
D6081 – 19.D6081 – 20. DOI: 10.1520/D6081-20.10.1520/D6081-22.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6081 − 22
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
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 For definitions of terms used in this practice, see Terminologies D1129, D4175, and E943.
3.2 Definitions:
3.2.1 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.
3.2.2 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.
3.2.3 ecotoxicity, n—propensity of a test material to produce adverse behavioral, biochemical, or physiological effects in
non-human organisms or populations.
3.2.4 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.
3.2.4.1 Discussion—
This terminology should be used instead of the standard ECXX when the test material is not completely soluble at the test treat
rates.
3.2.5 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.
3.2.5.1 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.
3.2.6 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.
3.2.6.1 Discussion—
This terminology should be used instead of the standard LCXX when the material is not completely soluble at the test treat rates.
3.2.7 loading rate, n—the ratio of test material to aqueous medium used in the preparation of WAF, WSF, a water accommodated
fraction (WAF), water soluble fraction (WSF), or mechanical dispersion and in the interpretation of the results of a toxicity study
with a poorly water-soluble lubricant or lubricant component.
3.2.8 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.
3.2.9 toxicity, n—the propensity of a test material to produce adverse behavioral, biochemical, or physiological effects in a living
organism.
3.2.10 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.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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3.2.10.1 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.
3.2.11 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.
3.3 Definitions of Terms Specific to This Standard:
3.3.1 dilution water, n—the water used in the aquatic test under consideration, which may contain organic or inorganic components
depending on the test requirements.
3.3.2 exposure matrix, n—the water accommodated fraction (WAF), water soluble fraction (WSF), or mechanical dispersion to
which test organisms are exposed for toxicity testing.
3.3.3 test systems, n—the test organisms plus the exposure matrix and the test chambers themselves.
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.
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. Apparatus
6.1 Stirrer, magnetic, size sufficient to accommodate vessel (6.2).
6.2 Vessel, glass, bottom drain, capacity 10 L to 20 L.
NOTE 1—The vessels shall be of sufficient size to maintain the vortex intensity and optimize surface area contact between the test material and the water.
NOTE 2—Height to diameter ratio for the WAF preparation at initiation should be between 1:1 and 2:1, and solutions shall 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.
NOTE 3—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 shall be in place prior to the introduction of the test material.
NOTE 4—WAF solutions from test materials that are denser than water shall 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.
NOTE 5—In all cases, take precautions to ensure that non-dispersed test material is not collected with the WAF.
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7. Reagents
7.1 Test Material, lubricant from which water-soluble component(s) is or are to be extracted.
7.2 Water, Type II per Specification D1193.
8. Preparation of Exposure Matrix
8.1 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.
8.2 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.
8.3 Test material shall 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.
8.4 Preparation of Water-Accommodated Fraction (WAF):
8.4.1 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.
8.4.2 Fill vessels for WAF preparation with the appropriate volume of dilution water, and prepare them for stirring as described
in 6.2.
8.4.2.1 Slowly add test material to the top of each vessel after dilution water addition.
8.4.2.2 Cap the vessel with foil or a non-reactive covering and stir at the test temperature.
8.4.2.3 If the test temperatures are different than room temperature, house the WAF preparation vessels in water baths or
incubators to maintain the desired temperature.
8.4.2.4 If photo-decomposition of the test material is likely, conduct the stirring phase in the dark.
8.4.2.5 Inspect preparation vessels occasionally to ensure an appropriate vortex is maintained.
8.4.3 Determine the amount of test material added to each vessel from the desired nominal exposure load.
8.4.3.1 Base nominal exposure loads on either direct weight of material added per volume solution (wt/vol) or on the basis of test
material density and added on a vol/vol basis.
8.4.4 Operate stir plates at a sufficient speed to ensure a vortex depth of 10 % to 35 % of the test solution height in the WAF
preparation vessel.
8.4.4.1 Ensure that the vortexes in all WAF preparation vessels for any one experiment are the same length (that is, depth from
fluid surface).
8.4.4.2 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.
8.4.5 The duration of WAF preparation will depend on the results of an optional equilibrium/stability study (see 8.7). In the
absence of this study, use a standard 20 h to 24 h mixing and 1 h to 4 h settling period (6, 8).
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FIG. 1 Water Accommodated Fraction (WAF) Schematic
8.4.6 Upon completion of the WAF mixing and settling period, drain or decant the aqueous solution from below the surface of
each preparation vessel.
8.4.6.1 Discard the first 5 mL to 10 mL of solution decanted from the vessel.
8.4.6.2 Use the remaining solution undiluted in the exposure vessels.
8.5 Preparation of Water-Soluble Fraction (WSF):
8.5.1 Evaluate only components dissolved in the water phase in a WSF study (6).
8.5.1.1 Prepare WSFs by filtering WAFs through a 0.45 μm nominal cellulose-acetate filter or by centrifuging to remove
undissolved material.
NOTE 6—Preliminary testing may be necessary to ensure that the filter disc will not remove test material constituents that are dissolved in the water phase.
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8.5.1.2 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.
8.5.2 The procedures described in 8.4.2 – 8.4.6 for the WAF preparation are the initial steps of the WSF procedure.
8.5.3 Upon completion of the WAF mixing and settling period, decant the aqueous solution from below the surface of each
preparation vessel. The first 5 mL to 10 mL of solution decanted from the vessel should be discarded, with the remaining solution
used to prepare the WSF. Filtration through a 0.45 μm filter or low speed centrifugation (to remove droplets but not to break an
emulsion) is performed on the solution to remove undissolved material. The filtrate or centrifugation supernatant is the WSF
exposure matrix and should be used undiluted in the exposure vessels.
8.6 Preparation of a Mechanical Dispersion:
NOTE 7—In the mechanical dispersion technique, a motor-driven propeller assembly is used to continually move test material and dilution water in the
test vessel during the study (6, 7, 9).
NOTE 8—This procedure should not be used for small-sized, slowly, or poorly swimming organisms. It is most appropriate for juvenile fish evaluated in
acute studies.
8.6.1 Configuration:
8.6.1.1 A schematic of the mechanical dispersion setup is provided in Fig. 3.
8.6.2 Ensure that each test vessel is equipped with a central cylinder equipped with a three
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