ASTM D6731-22

Standard Test Method for<!--ask about discussion in biodegradation def (keep?)--> Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer

SIGNIFICANCE AND USE
5.1 Results from this test method suggest the degree of aerobic, aquatic biodegradation of a lubricant or lubricant component. The rate and extent of oxygen consumption is measured upon exposure of the test material to an inoculum within the confines of a controlled laboratory setting. Test materials which achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in many aerobic aquatic environments.  
5.2 Because of the stringency of this test method, low results do not necessarily mean that the test material is not biodegradable under environmental conditions, but indicate that further testing is necessary to establish biodegradability.  
5.3 If the pH value at the end of the test is outside the range from 6 to 8 and if the percentage degradation of the test material is less than 50 %, it is advisable to repeat the test with a lower concentration of the test material or a higher concentration of the buffer solution, or both.  
5.4 A reference or control material known to biodegrade under the conditions of this test method is necessary in order to verify the activity of the inoculum. The test must be regarded as invalid and shall be repeated using a fresh inoculum if the reference material does not demonstrate biodegradation to the extent of >60 % of the ThO2 within 28 days.  
5.5 Information on the toxicity of the test material to the inoculum may be useful in the interpretation of low biodegradation results. Toxicity of the test material to the inoculum may be evaluated by testing the test material in combination with the reference material in inhibition control systems. If an inhibition control is included, the test material is assumed to be inhibiting if the degradation percentage of the reference material is lower than 40 % (ISO 8192). In this case, it is advisable to repeat the test with lower concentrations of the test material.  
5.6 Total oxygen utilization in the blank at the end of the test exceeding 60 mg O2/L invalid...
SCOPE
1.1 This test method covers a procedure for determining the degree of biodegradability of lubricants or their components in an aerobic aqueous medium on exposure to an inoculum under controlled laboratory conditions. This test method is an ultimate biodegradation test that measures oxygen demand in a closed respirometer.  
1.2 This test method is suitable for evaluating the biodegradation of volatile as well as nonvolatile lubricants or lubricant components.  
1.3 This test method is applicable to lubricants and lubricant components which are not toxic and not inhibitory to the test microorganisms at the test concentration.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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. Specific hazards are given in Section 10.  
1.6 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: 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 D6384-18 - Standard Terminology Relating to Biodegradability and Ecotoxicity of Lubricants
• Effective Date: 01-Jun-2018
• Refers: ASTM D6384-17 - Standard Terminology Relating to Biodegradability and Ecotoxicity of Lubricants
• Effective Date: 01-Apr-2017
• Refers: ASTM D1293-12 - Standard Test Methods for pH of Water
• Effective Date: 01-Jan-2012
• Refers: ASTM D6384-11 - Standard Terminology Relating to Biodegradability and Ecotoxicity of Lubricants
• Effective Date: 01-Jan-2011
• 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 D4447-06 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• Refers: ASTM D4447-10 - Standard Guide for Disposal of Laboratory Chemicals and Samples
• Effective Date: 15-Nov-2006
• 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

Overview

ASTM D6731-22, titled Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer, is a key international standard developed by ASTM International. This method provides a procedure for evaluating the biodegradability of lubricants and lubricant components when exposed to aerobic aquatic environments under controlled laboratory conditions. By measuring oxygen demand in a closed respirometer, this test method offers a reliable means to predict the degree and rate of biodegradation for both volatile and nonvolatile lubricants, supporting environmental performance claims and compliance with regulatory requirements.

Key Topics

• Biodegradability Assessment: The standard measures the oxygen consumed by microorganisms as they biodegrade the test lubricant or component in water, using a closed respirometer system.
• Test Applicability: Suitable for a broad range of lubricants-including water-soluble and insoluble types-that are not toxic or inhibitory to test microorganisms at specified concentrations.
• Stringent Laboratory Conditions: Results from this method are considered indicative of a lubricant’s potential biodegradation in the environment. A high degree of degradation in this test suggests ready biodegradability in aerobic aquatic settings.
• Controls and Validity:
  • Requires concurrent testing of a reference material known to be biodegradable to verify inoculum activity.
  • Includes blank controls to adjust for background oxygen consumption.
  • Lays out procedures for managing test validity, including actions if pH, degradation, or oxygen utilization values fall outside acceptable ranges.
• Interpretation Guidance: Low test values may not definitively indicate non-biodegradability in the environment but could suggest the need for further testing, taking into account factors such as solubility, toxicity, or inhibition of the test system.

Applications

Adoption of ASTM D6731-22 offers practical value in several areas:

• Product Environmental Assessment: Enables manufacturers to assess the aerobic, aquatic biodegradability of lubricants and lubricant components, supporting the development of more environmentally friendly products.
• Regulatory Compliance: Assists companies in meeting environmental regulations and eco-labeling requirements by providing standardized data on lubricant biodegradability.
• R&D and Innovation: Facilitates research and comparison of alternative lubricant formulations aimed at reducing environmental impact.
• Risk Management: Offers supporting evidence for claims around lubricant decomposition in the environment, helping mitigate liability related to the release of persistent substances.
• Sustainability Certification: Enhances green credentials for lubricants through reliable, third-party-validated biodegradability testing.

Related Standards

ASTM D6731-22 references and is complemented by multiple international standards for biodegradability, water quality, and laboratory practices:

• ASTM Standards:
  • ASTM D1129: Terminology Relating to Water
  • ASTM D1193: Specification for Reagent Water
  • ASTM D1293: Test Methods for pH of Water
  • ASTM D4447: Guide for Disposal of Laboratory Chemicals and Samples
  • ASTM D6384: Terminology Relating to Biodegradability and Ecotoxicity of Lubricants
• ISO Standards:
  • ISO 8192: Water Quality - Test for Inhibition of Oxygen Consumption
  • ISO 9408: Evaluation of Ultimate Aerobic Biodegradability by Oxygen Demand in Closed Respirometer
• OECD Guidelines:
  • OECD 301F: Ready Biodegradability-Manometric Respirometry Test

Implementing ASTM D6731-22 alongside these related standards ensures thorough, credible evaluation of lubricant biodegradability and supports global harmonization of environmental testing in the lubricants industry.

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: D6731 − 22
Standard Test Method for
Determining the Aerobic, Aquatic Biodegradability of
Lubricants or Lubricant Components in a Closed
Respirometer
This standard is issued under the fixed designation D6731; 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* 2. Referenced Documents
1.1 This test method covers a procedure for determining the 2.1 ASTM Standards:
degree of biodegradability of lubricants or their components in
D1129 Terminology Relating to Water
an aerobic aqueous medium on exposure to an inoculum under
D1193 Specification for Reagent Water
controlled laboratory conditions. This test method is an ulti- D1293 Test Methods for pH of Water
mate biodegradation test that measures oxygen demand in a D4175 Terminology Relating to Petroleum Products, Liquid
closed respirometer. Fuels, and Lubricants
D4447 Guide for Disposal of Laboratory Chemicals and
1.2 This test method is suitable for evaluating the biodeg-
Samples
radation of volatile as well as nonvolatile lubricants or lubri-
D6384 Terminology Relating to Biodegradability and Eco-
cant components.
toxicity of Lubricants
1.3 Thistestmethodisapplicabletolubricantsandlubricant E943 Terminology Relating to Biological Effects and Envi-
components which are not toxic and not inhibitory to the test
ronmental Fate
microorganisms at the test concentration. 3
2.2 ISO Standards:
ISO 4259–1,2 Petroleum products–Determination and appli-
1.4 The values stated in SI units are to be regarded as
cation of precision data in relation to methods of test
standard. No other units of measurement are included in this
ISO 6107-2 Water quality–Vocabulary–Part 2
standard.
ISO 8192 Water quality–Test for inhibition of oxygen con-
1.5 This standard does not purport to address all of the
sumption by activated sludge for carbonaceous and am-
safety concerns, if any, associated with its use. It is the
monium oxidation
responsibility of the user of this standard to establish appro-
ISO 9408 Water quality–Evaluation of ultimate aerobic bio-
priate safety, health, and environmental practices and deter-
degradability of organic compounds in aqueous medium
mine the applicability of regulatory limitations prior to use.
by determination of oxygen demand in a closed respirom-
Specific hazards are given in Section 10.
eter
1.6 This international standard was developed in accor-
2.3 OECD Standards:
dance with internationally recognized principles on standard-
OECD 301F Ready Biodegradability-Manometric Re-
ization established in the Decision on Principles for the
spirometry
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
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
This test method is under the jurisdiction of ASTM Committee D02 on the ASTM website.
Petroleum Products, Liquid Fuels, and Lubricantsand is the direct responsibility of Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
Subcommittee D02.12 on Environmental Standards for Lubricants. 4th Floor, New York, NY 10036, http://www.ansi.org.
Current edition approved April 1, 2022. Published April 2022. Originally Available from Organisation for Economic Cooperation and Development
approved in 2001. Last previous edition approved in 2018 as D6731 – 18. DOI: (OECD), 2 rue André Pascal, F-75775, Paris Cedex 16, France, http://
10.1520/D6731-22. www.oecd.org.
*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
D6731 − 22
2.4 APHA Standards: 3.1.11 suspended solids (of an activated sludge or other
2540B Total Solids Dried at 103-105°C inoculum samples), n—solids present in activated sludge or
9215 Heterotrophic Plate Count
other inoculum samples that are not removed by settling under
specified conditions.
3. Terminology
4. Summary of Test Method
3.1 Definitions:
3.1.1 Definitions of terms applicable to this test method
4.1 Biodegradation of a lubricant or the component(s) of a
appear in the Compilation of ASTM Standard Definitions and
lubricant is determined by measuring the oxygen consumed
the following terminology standards: D1129, D4175, D6384,
whenthelubricantorcomponentisexposedtomicroorganisms
E943, and ISO 6107-2.
under controlled aerobic aquatic conditions. This value is then
3.1.2 activated sludge, n—the precipitated solid matter,
compared to the theoretical amount of oxygen (ThO ) which is
consisting mainly of bacteria and other aquatic
required to oxidize all of the elements (that is, carbon,
microorganisms, that is produced at a domestic wastewater
hydrogen, nitrogen, and so forth) in the test material. This test
treatment plant and is used primarily in secondary sewage
method mixes the test material (lubricant or component) with
treatmenttomicrobiallyoxidizedissolvedorganicmatterinthe
aerobic microorganisms in a closed respirometer containing a
effluent.
defined aquatic medium and measures the biodegradation of
3.1.3 aerobic, adj—(a) taking place in the presence of
the test material by following the decrease in oxygen in the
oxygen; (b) living or active in the presence of oxygen.
respirometer.
3.1.4 biochemical oxygen demand (BOD), n—the mass
4.2 The test material is the sole source of carbon and energy
concentration of dissolved oxygen consumed under specified
inthemedium.Areferencematerialknowntobiodegrade,such
conditions by the biological oxidation of organic or inorganic
as low erucic acid rapeseed oil (LEAR or canola oil) is run
matter, or both.
alongside the test material to confirm that the inoculum is
3.1.4.1 Discussion—BOD determination is performed using
viableandcapableofbiodegradingsuitablematerialsunderthe
empirical tests employing standardized laboratory procedures.
test conditions. The test material or reference material concen-
These tests measure oxygen utilization during a specified
tration is normally 50 mg⁄Lto 100 mg⁄L, providing a theoreti-
incubation period for the biochemical degradation of organic
material (carbonaceous demand) in water. cal oxygen demand of at least 50 mg O /L but no more than
200 mg O /L.TheThO of the test and reference materials will
2 2
3.1.5 biodegradation, n—the process of chemical break-
be determined from measured elemental compositional analy-
down or transformation of a material caused by microorgan-
sis and will be calculated as in 13.1.
isms or their enzymes.
3.1.5.1 Discussion—Biodegradation is only one mechanism
4.3 The inoculated medium is stirred in a closed flask and
by which materials are removed, transformed, or both, in the
the consumption of oxygen is determined either by measuring
environment.
the amount of oxygen required to maintain a constant gas
3.1.6 lag phase, n—the period of diminished physiological
volume in the respirometer flask, or by measuring the change
activity and cell division following the addition of microorgan-
in volume or pressure (or a combination of the two) in the
isms to a new culture medium.
apparatus.
3.1.7 log phase, n—the period of growth of microorganisms
4.4 EvolvedCO (carbondioxide)isabsorbedinanalkaline
during which cells divide at a positive constant rate.
trap solution (for example, 10 M NaOH or KOH) or other
3.1.8 pre-adaptation, n—the pre-incubation of an inoculum
CO -absorbing system suspended within the test vessel, typi-
in the presence of the test material under conditions similar to
cally in the headspace of the test vessel.
the test conditions.
4.5 Biodegradation is followed over a specified period by
3.1.8.1 Discussion—The aim of pre-adaptation is to im-
determining the consumption of oxygen. The amount of
prove the precision of the test method by decreasing variability
oxygen utilized in oxidation of the test and reference material
in the rate of biodegradation produced by the inoculum.
is corrected for oxygen uptake by the inoculum in the blank
Pre-adaptation may mimic the natural processes which cause
controls and is expressed as a percentage of the theoretical
changes in the microbial population of the inoculum leading to
oxygen demand (ThO ) calculated from the empirical formula
more rapid biodegradation of the test material but not to a
of the material. Evaluation of the biodegradability of the test
changeinthefinalextentofbiodegradationofthetestmaterial.
material is made on the basis of these data. Normally the test
3.1.9 pre-condition, n—the pre-incubation of an inoculum
duration is 28 days; however, the test may be terminated if
under the conditions of the test in the absence of the test
oxygen consumption has plateaued. The test may be extended
material.
as long as the systems’ integrity is maintained and the
3.1.10 sludge, n—a water-formed sedimentary deposit.
inoculum in the blank systems is viable. The duration of the
testwillbedependentonthelengthoftimerequiredfortherate
of test material biodegradation to achieve a plateau. A graphi-
From Standard Methods for the Examination of Water and Wastewater, latest
cal illustration of the test results for a biodegradable material is
edition. Available from the American Public Health Assoc., 1015 18th St., NW,
Washington, DC 20036. presented in Fig. 1.
D6731 − 22
FIG. 1 Respirometric Test—Biodegradation Curve
5. Significance and Use 5.7 The water solubility or dispersibility of the lubricant or
component may influence the results obtained and hence
5.1 Results from this test method suggest the degree of
comparison of test results may be limited to lubricants or
aerobic, aquatic biodegradation of a lubricant or lubricant
components with similar solubilities.
component. The rate and extent of oxygen consumption is
measured upon exposure of the test material to an inoculum
5.8 The behaviors of complex mixtures are not always
within the confines of a controlled laboratory setting. Test
consistent with the individual properties of the components.
materialswhichachieveahighdegreeofbiodegradationinthis
Test results for individual lubricant components may be sug-
test may be assumed to easily biodegrade in many aerobic
gestiveofwhetheramixturecontainingthesecomponents(that
aquatic environments.
is, fully formulated lubricants) is biodegradable, but such
information should be used judiciously.
5.2 Because of the stringency of this test method, low
results do not necessarily mean that the test material is not
6. Apparatus
biodegradable under environmental conditions, but indicate
that further testing is necessary to establish biodegradability.
6.1 Closed Respirometer:
6.1.1 The principle of a closed respirometer is given in Fig.
5.3 If the pH value at the end of the test is outside the range
2. When testing volatile compounds, the apparatus used shall
from 6 to 8 and if the percentage degradation of the test
be appropriate or adapted to this particular purpose in accor-
material is less than 50 %, it is advisable to repeat the test with
dance with the manufacturer’s instructions. Exercise care that
a lower concentration of the test material or a higher concen-
the closed respirometer apparatus is well sealed to prevent any
tration of the buffer solution, or both.
loss (for example, leakage) of volatile compounds from the
5.4 A reference or control material known to biodegrade
system or of oxygen into the system.
under the conditions of this test method is necessary in order to
6.1.2 The test mixture is stirred by a magnetic stirrer in the
verify the activity of the inoculum. The test must be regarded
test flask, which is filled with sufficient volume to minimize
as invalid and shall be repeated using a fresh inoculum if the
headspace and prevent delay of O and CO diffusion through
2 2
reference material does not demonstrate biodegradation to the
the air-water phases. This volume is dependent on the selected
extent of >60 % of the ThO within 28 days.
flask size, and is normally specified by the manufacturer of the
5.5 Information on the toxicity of the test material to the
respirometer. If biodegradation takes place, the microorgan-
inoculum may be useful in the interpretation of low biodegra-
isms consume oxygen and produce carbon dioxide. Oxygen
dationresults.Toxicityofthetestmaterialtotheinoculummay
fromtheheadspaceisthendissolvedintheliquidtoreestablish
be evaluated by testing the test material in combination with
chemical equilibrium. The carbon dioxide produced by the
the reference material in inhibition control systems. If an
microorganisms diffuses into the headspace where it is trapped
inhibition control is included, the test material is assumed to be
inanabsorbentsolutionormaterialandthetotalpressureinthe
inhibiting if the degradation percentage of the reference
flask then decreases.
material is lower than 40 % (ISO 8192). In this case, it is
6.1.3 This pressure drop is detected by a manometer, which
advisabletorepeatthetestwithlowerconcentrationsofthetest
produces a signal that results in the electrolytic generation of
material.
oxygen.Whentheoriginalpressureisre-established,thesignal
5.6 Total oxygen utilization in the blank at the end of the is stopped and the quantity of electricity used is measured.The
test exceeding 60 mg O /L invalidates the test. amount of electricity used is proportional to the amount of
D6731 − 22
FIG. 2 Principle of a Closed Respirometer
consumed oxygen. This is indicated on a plotter or a printer, or 7.3.1 Calcium Chloride Solution—Dissolve 27.5 g of anhy-
the data are collected using an appropriate software program. drous calcium chloride (CaCl ) or 36.4 g of calcium chloride
dihydrate (CaCl ·2H O) in water and dilute to 1 L.
2 2
6.2 Water-Bath or Constant Temperature Room, to comply
7.3.2 Ferric Chloride Solution—Dissolve 0.25 g of iron
with 11.2.
(III) chloride hexahydrate (FeCl ·6H O) in water and dilute to
3 2
6.3 Centrifuge.
1 L. Prepare this solution just before use or add a drop of
6.4 pH-meter.
concentrated hydrochloric acid (HCl) (Warning—Corrosive,
6.5 Analytical Balance, capable of weighing to appropriate fumes cause irritation.) or 0.4 g⁄L of ethylenediamine-
precision and accuracy (for example, 60.0001 g). tetraacetic acid (EDTA).
7.3.3 Magnesium Sulfate Solution—Dissolve 22.5 g of mag-
7. Reagents and Materials
nesium sulfate heptahydrate (MgSO ·7H O) in water and
4 2
7.1 Purity of Reagents—Reagent grade chemicals shall be
dilute to 1 L.
used in all tests. Unless otherwise indicated, it is intended that
7.3.4 Phosphate Buffer Solution—Dissolve 8.5 g of anhy-
all reagents conform to the specifications of the Committee on
drous potassium dihydrogen phosphate (KH PO ), 21.75 g
2 4
Analytical Reagents of the American Chemical Society where
anhydrous potassium monohydrogen phosphate (K HPO ),
6 2 4
such specifications are available. Other grades may be used,
33.4 g disodium hydrogen phosphate dihydrate
provided it is first ascertained that the reagent is of sufficiently
(Na HPO ·2H O), and 0.5 g ammonium chloride (NH Cl) in
2 4 2 4
high purity to permit its use without decreasing the accuracy of
water and dilute to 1 L. Alternatively, 50.3 g of disodium
the determination.
hydrogen phosphate, heptahydrate (Na HPO ·7H O) may be
2 4 2
7.2 Purity of Water—Unless otherwise indicated, references
used in place of Na HPO ·2H O. The pH of this solution shall
2 4 2
to water shall be understood to mean reagent water as defined
be about 7.4.
by Type II of Specification D1193.
7.3.5 Sodium Hydroxide 10 M Solution—(Warning—
7.3 Prepare the following stock solutions: Causes severe burns.) Cautiously dissolve 400 g NaOH
(Warning—Causes severe burns.) in distilled water to a final
volume of 1 L. Filter the solution to free it of solids.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
7.3.6 Potassium Hydroxide 10 M Solution—(Warning—
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Causes severe burns.) Cautiously dissolve 561 g KOH
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
(Warning—Causes severe burns.) in distilled water to a final
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
copeial Convention, Inc. (USPC), Rockville, MD. volume of 1 L. Filter the solution to free it of solids.
D6731 − 22
8. Inoculum Test Organisms point to the test systems (12.2). Up to 100 mL of this type of
inoculum may be used per litre of medium.
8.1 Sources of the Inoculum—Activated sewage-sludge
8.1.3 Inoculum from Surface Water—A further source for
from a sewage-treatment plant that treats principally domestic
the inoculum is surface water. In this case, collect a sample of
waste may be considered as an aerobic inoculum.An inoculum
an appropriate surface water (for example, river or lake) and
derived from soil or natural surface waters, or any combination
keep aerobic until required.
of the three sources, may also be used in this test method.
8.1.3.1 Filter the surface water through a coarse filter paper
Allowance for various and multiple inoculum sources provides
or glass wool plug, and discard the first 200 mL. Aerate the
access to a greater diversity of biochemical competency and
remaining filtered sample using CO -free air in the laboratory
potentially represents more accurately the capacity for biodeg-
for 4 h. The inoculum may be used to prepare a composite
radation.The following provides several options for where and
inoculum (8.1.5), pre-adapted to the test material (8.3.1), or
how to obtain an appropriate inoculum:
added directly to the test systems (12.2). Up to 100 mL of this
8.1.1 Inoculum from Activated Sludge—Activated sludge
type of inoculum may be used per litre of medium.
freshly sampled (that is, less than 24 h old) from a well-
8.1.4 Inoculum from Soil:
operated predominantly domestic sewage treatment plant (that
8.1.4.1 Suspend 100 g of soil in 1000 mL of water.
is, one with no recent upsets and operating within its design
8.1.4.2 Allow the suspension to settle for 30 min.
parameters) may be used. This sewage treatment plant should
8.1.4.3 Filter the supernatant through a coarse filter paper or
receive minimal or no effluent from industry.
glass wool plug, and discard the first 200 mL. The filtrate is
8.1.1.1 Using CO -free air, aerate sludge in the laboratory
2 aerated immediately and continuously until used. The soil
for 4 h. Depending on the number of test systems, sufficient
inoculum may be used to prepare a composite inoculum
volume of the mixed liquor is sampled and homogenized for
(8.1.5), pre-adapted to the test material (8.3.1), or added
2 min at medium speed using a high-sheer/high-speed blender.
directly to the test systems (12.2). Up to 100 mLof this type of
Withdraw a sample for the determination of the dry weight of
inoculum may be used per litre of medium.
the suspended solids (8.2.2). Keep the inoculum continuously
8.1.5 Composite Inoculum—The four inoculum sources
well mixed until all sample preparation is completed to avoid
may be combined in any proportion and mixed well.
solids settling.
8.2 Determination of Microorganisms:
8.1.1.2 Calculate the volume of homogenized mixed liquor
8.2.1 APHATest Method 9215, or equivalent, shall be used
necessary to achieve a final sludge dry-weight concentration in
to enumerate the microorganisms in the inoculum. The inocu-
the test medium of 30 mg⁄L (suspended solids, 8.2.2). The 6 7
lumshallcontain10 to10 colony-formingunitspermillilitre.
inoculumpreparedfromthehomogenizedmixedliquormaybe
It is optional to measure the total bacterial count of the
used to prepare a composite inoculum (8.1.5), pre-a
...

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: D6731 − 18 D6731 − 22
Standard Test Method for
Determining the Aerobic, Aquatic Biodegradability of
Lubricants or Lubricant Components in a Closed
Respirometer
This standard is issued under the fixed designation D6731; 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 test method covers a procedure for determining the degree of biodegradability of lubricants or their components in an
aerobic aqueous medium on exposure to an inoculum under controlled laboratory conditions. This test method is an ultimate
biodegradation test that measures oxygen demand in a closed respirometer.
1.2 This test method is suitable for evaluating the biodegradation of volatile as well as nonvolatile lubricants or lubricant
components.
1.3 This test method is applicable to lubricants and lubricant components which are not toxic and not inhibitory to the test
microorganisms at the test concentration.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 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. Specific hazards are given in Section 10.
1.6 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.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D1293 Test Methods for pH of Water
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D4447 Guide for Disposal of Laboratory Chemicals and Samples
D6384 Terminology Relating to Biodegradability and Ecotoxicity of Lubricants
This test method is under the jurisdiction of ASTM Committee D02 on Petroleum Products, Liquid Fuels, and Lubricantsand is the direct responsibility of Subcommittee
D02.12 on Environmental Standards for Lubricants.
Current edition approved Oct. 1, 2018April 1, 2022. Published October 2018April 2022. Originally approved in 2001. Last previous edition approved in 20112018 as
D6731 – 01 (2011).D6731 – 18. DOI: 10.1520/D6731-18.10.1520/D6731-22.
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.
*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
D6731 − 22
E943 Terminology Relating to Biological Effects and Environmental Fate
2.2 ISO Standards:
ISO 4259–1,2 Petroleum products–Determination and application of precision data in relation to methods of test
ISO 6107-2 Water quality–Vocabulary–Part 2
ISO 8192 Water quality–Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium
oxidation
ISO 9408 Water quality–Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by
determination of oxygen demand in a closed respirometer
2.3 OECD Standards:
OECD 301F Ready Biodegradability-Manometric Respirometry
2.4 APHA Standards:
2540B Total Solids Dried at 103-105°C
9215 Heterotrophic Plate Count
3. Terminology
3.1 Definitions:
3.1.1 Definitions of terms applicable to this test method appear in the Compilation of ASTM Standard Definitions and the
following terminology standards: D1129, D4175, D6384, E943, and ISO 6107-2.
3.1.2 activated sludge, n—the precipitated solid matter, consisting mainly of bacteria and other aquatic microorganisms, that is
produced at a domestic wastewater treatment plant and is used primarily in secondary sewage treatment to microbially oxidize
dissolved organic matter in the effluent.
3.1.3 aerobic, adj—(a) taking place in the presence of oxygen; (b) living or active in the presence of oxygen.
3.1.4 biochemical oxygen demand (BOD), n—the mass concentration of dissolved oxygen consumed under specified conditions
by the biological oxidation of organic or inorganic matter, or both.
3.1.4.1 Discussion—
BOD determination is performed using empirical tests employing standardized laboratory procedures. These tests measure oxygen
utilization during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand) in
water.
3.1.5 biodegradation, n—the process of chemical breakdown or transformation of a test material caused by microorganisms or
their enzymes.
3.1.5.1 Discussion—
Biodegradation is only one mechanism by which materials are removed, transformed, or both, in the environment.
3.1.6 lag phase, n—the period of diminished physiological activity and cell division following the addition of microorganisms to
a new culture medium.
3.1.7 log phase, n—the period of growth of microorganisms during which cells divide at a positive constant rate.
3.1.8 pre-adaptation, n—the incubationpre-incubation of an inoculum in the presence of the test material which is done prior to
the initiation of the test and under conditions similar to the test conditions.
3.1.8.1 Discussion—
The aim of pre-adaptation is to improve the precision of the test method by decreasing variability in the rate of biodegradation
produced by the inoculum. Pre-adaptation may mimic the natural processes which cause changes in the microbial population of
the inoculum leading to more rapid biodegradation of the test material but is not expected to a change in the overallfinal extent
of biodegradation of the test material.
3.1.9 pre-condition, n—the pre-incubation of an inoculum under the conditions of the test in the absence of the test material.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Available from Organisation for Economic Cooperation and Development (OECD), 2 rue André Pascal, F-75775, Paris Cedex 16, France, http://www.oecd.org.
From Standard Methods for the Examination of Water and Wastewater, latest edition. Available from the American Public Health Assoc., 1015 18th St., NW, Washington,
DC 20036.
D6731 − 22
3.1.10 sludge, n—a water-formed sedimentary deposit.
3.1.11 suspended solids (of an activated sludge or other inoculum samples), n—solids present in activated sludge or other
inoculum samples that are not removed by settling under specified conditions.
4. Summary of Test Method
4.1 Biodegradation of a lubricant or the component(s) of a lubricant is determined by measuring the oxygen consumed when the
lubricant or component is exposed to microorganisms under controlled aerobic aquatic conditions. This value is then compared to
the theoretical amount of oxygen (ThO ) which is required to oxidize all of the elements (that is, carbon, hydrogen, nitrogen, and
so forth) in the test material. This test method mixes the test material (lubricant or component) with aerobic microorganisms in
a closed respirometer containing a defined aquatic medium and measures the biodegradation of the test material by following the
decrease in oxygen in the respirometer.
4.2 The test material is the sole source of carbon and energy in the medium. A reference material known to biodegrade, such as
low erucic acid rapeseed oil (LEAR or canola oil) is run alongside the test material to confirm that the inoculum is viable and
capable of biodegrading suitable materials under the test conditions. The test material or reference material concentration is
normally 50 mg ⁄L to 100 mg ⁄L, providing a theoretical oxygen demand of at least 50 mg O /L but no more than 200 mg O /L.
2 2
The ThO of the test and reference materials will be determined from measured elemental compositional analysis and will be
calculated as in 13.1.
4.3 The inoculated medium is stirred in a closed flask and the consumption of oxygen is determined either by measuring the
amount of oxygen required to maintain a constant gas volume in the respirometer flask, or by measuring the change in volume or
pressure (or a combination of the two) in the apparatus.
4.4 Evolved CO (carbon dioxide) is absorbed in an alkaline trap solution (for example, 10 M NaOH or KOH) or other
CO -absorbing system suspended within the test vessel, typically in the headspace of the test vessel.
4.5 Biodegradation is followed over a specified period by determining the consumption of oxygen. The amount of oxygen utilized
in oxidation of the test and reference material is corrected for oxygen uptake by the inoculum in the blank controls and is expressed
as a percentage of the theoretical oxygen demand (ThO ) calculated from the empirical formula of the material. Evaluation of the
biodegradability of the test material is made on the basis of these data. Normally the test duration is 28 days; however, the test
may be terminated if oxygen consumption has plateaued. The test may be extended as long as the systems’ integrity is maintained
and the inoculum in the blank systems is viable. The duration of the test will be dependent on the length of time required for the
rate of test material biodegradation to achieve a plateau. A graphical illustration of the test results for a biodegradable material is
presented in Fig. 1.
5. Significance and Use
5.1 Results from this test method suggest the degree of aerobic, aquatic biodegradation of a lubricant or lubricant component. The
rate and extent of oxygen consumption is measured upon exposure of the test material to an inoculum within the confines of a
controlled laboratory setting. Test materials which achieve a high degree of biodegradation in this test may be assumed to easily
biodegrade in many aerobic aquatic environments.
5.2 Because of the stringency of this test method, low results do not necessarily mean that the test material is not biodegradable
under environmental conditions, but indicate that further testing is necessary to establish biodegradability.
5.3 If the pH value at the end of the test is outside the range from 6 to 8 and if the percentage degradation of the test material
is less than 50 %, it is advisable to repeat the test with a lower concentration of the test material or a higher concentration of the
buffer solution, or both.
5.4 A reference or control material known to biodegrade under the conditions of this test method is necessary in order to verify
the activity of the inoculum. The test must be regarded as invalid and shall be repeated using a fresh inoculum if the reference
material does not demonstrate biodegradation to the extent of >60 % of the ThO within 28 days.
D6731 − 22
FIG. 1 Respirometric Test—Biodegradation Curve
5.5 Information on the toxicity of the test material to the inoculum may be useful in the interpretation of low biodegradation
results. Toxicity of the test material to the inoculum may be evaluated by testing the test material in combination with the reference
material in inhibition control systems. If an inhibition control is included, the test material is assumed to be inhibiting if the
degradation percentage of the reference material is lower than 40 % (ISO 8192). In this case, it is advisable to repeat the test with
lower concentrations of the test material.
5.6 Total oxygen utilization in the blank at the end of the test exceeding 60 mg O /L invalidates the test.
5.7 The water solubility or dispersibility of the lubricant or component may influence the results obtained and hence comparison
of test results may be limited to lubricants or components with similar solubilities.
5.8 The behaviors of complex mixtures are not always consistent with the individual properties of the components. Test results
for individual lubricant components may be suggestive of whether a mixture containing these components (that is, fully formulated
lubricants) is biodegradable, but such information should be used judiciously.
6. Apparatus
6.1 Closed Respirometer:
6.1.1 The principle of a closed respirometer is given in Fig. 2. When testing volatile compounds, the apparatus used shall be
appropriate or adapted to this particular purpose in accordance with the manufacturer’s instructions. Exercise care that the closed
respirometer apparatus is well sealed to prevent any loss (for example, leakage) of volatile compounds from the system or of
oxygen into the system.
6.1.2 The test mixture is stirred by a magnetic stirrer in the test flask, which is filled with sufficient volume to minimize headspace
and prevent delay of O and CO diffusion through the air-water phases. This volume is dependent on the selected flask size, and
2 2
is normally specified by the manufacturer of the respirometer. If biodegradation takes place, the microorganisms consume oxygen
and produce carbon dioxide. Oxygen from the headspace is then dissolved in the liquid to reestablish chemical equilibrium. The
carbon dioxide produced by the microorganisms diffuses into the headspace where it is trapped in an absorbent solution or material
and the total pressure in the flask then decreases.
6.1.3 This pressure drop is detected by a manometer, which produces a signal that results in the electrolytic generation of oxygen.
When the original pressure is re-established, the signal is stopped and the quantity of electricity used is measured. The amount of
electricity used is proportional to the amount of consumed oxygen. This is indicated on a plotter or a printer, or the data are
collected using an appropriate software program.
D6731 − 22
FIG. 2 Principle of a Closed Respirometer
6.2 Water-Bath or Constant Temperature Room, to comply with 11.2.
6.3 Centrifuge.
6.4 pH-meter.
6.5 Analytical Balance, capable of weighing to appropriate precision and accuracy (for example, 60.0001 g).
7. Reagents and Materials
7.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where such
specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without decreasing the accuracy of the determination.
7.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water as defined by Type
II of Specification D1193.
7.3 Prepare the following stock solutions:
7.3.1 Calcium Chloride Solution—Dissolve 27.5 g of anhydrous calcium chloride (CaCl ) or 36.4 g of calcium chloride dihydrate
(CaCl ·2H O) in water and dilute to 1 L.
2 2
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by the American Chemical Society, see Analar Standards for
Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC),
Rockville, MD.
D6731 − 22
7.3.2 Ferric Chloride Solution—Dissolve 0.25 g of iron (III) chloride hexahydrate (FeCl ·6H O) in water and dilute to 1 L.
3 2
Prepare this solution just before use or add a drop of concentrated hydrochloric acid (HCl) (Warning—Corrosive, fumes cause
irritation.) or 0.4 g ⁄L of ethylenediamine-tetraacetic acid (EDTA).
7.3.3 Magnesium Sulfate Solution—Dissolve 22.5 g of magnesium sulfate heptahydrate (MgSO ·7H O) in water and dilute to 1 L.
4 2
7.3.4 Phosphate Buffer Solution—Dissolve 8.5 g of anhydrous potassium dihydrogen phosphate (KH PO ), 21.75 g anhydrous
2 4
potassium monohydrogen phosphate (K HPO ), 33.4 g disodium hydrogen phosphate dihydrate (Na HPO ·2H O), and 0.5 g
2 4 2 4 2
ammonium chloride (NH Cl) in water and dilute to 1 L. Alternatively, 50.3 g of disodium hydrogen phosphate, heptahydrate
(Na HPO ·7H O) may be used in place of Na HPO ·2H O. The pH of this solution shall be about 7.4.
2 4 2 2 4 2
7.3.5 Sodium Hydroxide 10 M Solution—(Warning—Causes severe burns.) Cautiously dissolve 400 g NaOH (Warning—Causes
severe burns.) in distilled water to a final volume of 1 L. Filter the solution to free it of solids.
7.3.6 Potassium Hydroxide 10 M Solution—(Warning—Causes severe burns.) Cautiously dissolve 561 g KOH (Warning—
Causes severe burns.) in distilled water to a final volume of 1 L. Filter the solution to free it of solids.
8. Inoculum Test Organisms
8.1 Sources of the Inoculum—Activated sewage-sludge from a sewage-treatment plant that treats principally domestic waste may
be considered as an aerobic inoculum. An inoculum derived from soil or natural surface waters, or any combination of the three
sources, may also be used in this test method. Allowance for various and multiple inoculum sources provides access to a greater
diversity of biochemical competency and potentially represents more accurately the capacity for biodegradation. The following
provides several options for where and how to obtain an appropriate inoculum:
8.1.1 Inoculum from Activated Sludge—Activated sludge freshly sampled (that is, less than 24 h old) from a well-operated
predominantly domestic sewage treatment plant (that is, one with no recent upsets and operating within its design parameters) may
be used. This sewage treatment plant should receive minimal or no effluent from industry.
8.1.1.1 Using CO -free air, aerate sludge in the laboratory for 4 h. Depending on the number of test systems, sufficient volume
of the mixed liquor is sampled and homogenized for 2 min at medium speed using a high-sheer/high-speed blender. Withdraw a
sample for the determination of the dry weight of the suspended solids (8.2.2). Keep the inoculum continuously well mixed until
all sample preparation is completed to avoid solids settling.
8.1.1.2 Calculate the volume of homogenized mixed liquor necessary to achieve a final sludge dry-weight concentration in the test
medium of 30 mg ⁄L (suspended solids, 8.2.2). The inoculum prepared from the homogenized mixed liquor may be used to prepare
a composite inoculum (8.1.5), pre-adapted to the test material (8.3.1), or added directly to the test systems (12.2).
8.1.1.3 Alternatively, settle the homogenized sludge for 30 min or longer (if required) and decant the liquid supernatant for use
as inoculum. The inoculum prepared from the supernatant may be used to prepare a composite inoculum (8.1.5), pre-adapted to
the test material (8.3.2), or added directly to the test systems (12.2).
8.1.1.4 It is optional to pre-condition the inoculum. Pre-conditioning consists of aerating the activated sludge for up to seven days.
Sometimes pre-conditioning improves the precision of the test method by reducing the amount of oxygen consumption in the blank
controls.
NOTE 1—Exercise care in pre-conditioning because of the sensitivity of inocula to prolonged aeration and starvation conditions. Pre-conditioning should
be applied in situations where it is known that the inoculum source consistently shows a high internal respiration rate.
8.1.2 Inoculum from Secondary Effluent—Alternatively, the inoculum can be derived from the secondary effluent of a treatment
plant or laboratory-scale unit receiving domestic sewage.
8.1.2.1 Allow the secondary effluent to settle for 1 h and collect the supernatant or filter the effluent through a coarse filter paper.
After supernatant collection or effluent filtration, aerate the sample using CO -free air in the laboratory for 4 h. The inoculum may
be used to prepare a composite inoculum (8.1.5), pre-adapted to the test material (8.3.2), or added at this point to the test systems
(12.2). Up to 100 mL of this type of inoculum may be used per litre of medium.
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