ASTM D6006-23

Standard Guide for Assessing Biodegradability of Hydraulic Fluids

SIGNIFICANCE AND USE
5.1 This guide discusses ways to assess the likelihood that a hydraulic fluid will undergo biodegradation if it enters an environment that is known to support biodegradation of some substances, for example the material used as the positive control in the test. The information can be used in making or assessing claims of biodegradability of a fluid formula.  
5.2 Biodegradation occurs when a fluid interacts with the environment, and so the extent of biodegradation is a function of both the chemical composition of the hydraulic fluid and the physical, chemical, and biological status of the environment at the time the fluid enters it. This guide cannot assist in judging the status of a particular environment, so it is not meant to provide standards for judging the persistence of a hydraulic fluid in any specific environment either natural or man-made.  
5.3 If any of the tests discussed in this guide gives a high result, it implies that the hydraulic fluid will biodegrade and will not persist in the environmental compartment being considered. If a low result is obtained, it does not mean necessarily that the substance will not biodegrade in the environment, but does mean that further testing is required if a claim of biodegradability is to be made. Such testing may include, but is not limited to, other tests mentioned in this guide or simulation tests for a particular environmental compartment.
SCOPE
1.1 This guide covers and provides information to assist in planning a laboratory test or series of tests from which may be inferred information about the biodegradability of an unused fully formulated hydraulic fluid in its original form. Biodegradability is one of three characteristics which are assessed when judging the environmental impact of a hydraulic fluid. The other two characteristics are ecotoxicity and bioaccumulation.  
1.2 Biodegradability may be considered by type of environmental compartment: aerobic fresh water, aerobic marine, aerobic soil, and anaerobic media. Test methods for aerobic fresh water, aerobic soil and anaerobic media have been developed that are appropriate for the concerns and needs of testing in each compartment.  
1.3 This guide addresses releases to the environment that are incidental to the use of a hydraulic fluid but is not intended to cover situations of major, accidental release. The tests discussed in this guide take a minimum of three to four weeks. Therefore, issues relating to the biodegradability of hydraulic fluid are more effectively addressed before the fluid is used, and thus before incidental release may occur. Nothing in this guide should be taken to relieve the user of the responsibility to properly use and dispose of hydraulic fluids.  
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.  
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: 30-Jun-2023
• ICS: 75.120 - Hydraulic fluids
• 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 D5864-23 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 01-Nov-2023
• Refers: ASTM D4175-23e1 - Standard Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• Effective Date: 01-Jul-2023
• Refers: ASTM D5864-18 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 01-Jun-2018
• Refers: ASTM D5864-17 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 01-Jan-2017
• Refers: ASTM D5291-10(2015) - Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
• Effective Date: 01-Apr-2015
• Refers: ASTM D5864-11 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 01-Mar-2011
• Refers: ASTM D5291-09 - Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
• Effective Date: 01-Dec-2009
• Refers: ASTM D5210-92(2007) - Standard Test Method for Determining the Anaerobic Biodegradation of Plastic Materials in the Presence of Municipal Sewage Sludge (Withdrawn 2016)
• Effective Date: 01-Oct-2007
• Refers: ASTM D5291-02(2007) - Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
• Effective Date: 01-May-2007
• Refers: ASTM D5291-02 - Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
• Effective Date: 10-Apr-2002
• Refers: ASTM D5291-96 - Standard Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
• Effective Date: 10-Nov-2001
• Refers: ASTM D5864-00 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 10-Dec-2000
• Refers: ASTM D5864-05 - Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
• Effective Date: 10-Dec-2000
• Refers: ASTM D5210-92 - Standard Test Method for Determining the Anaerobic Biodegradation of Plastic Materials in the Presence of Municipal Sewage Sludge
• Effective Date: 01-Jan-2000

Overview

ASTM D6006-23: Standard Guide for Assessing Biodegradability of Hydraulic Fluids provides methodologies and guidance for evaluating the biodegradability of unused, fully formulated hydraulic fluids under laboratory conditions. Developed by ASTM Committee D02, this guide supports responsible product development and regulatory compliance by helping users plan and execute tests that estimate the environmental persistence of hydraulic fluids if released into ecosystems.

Biodegradability is a critical factor in assessing the environmental impact of hydraulic fluids, alongside ecotoxicity and bioaccumulation. The guide covers the selection and application of laboratory test methods relevant to different environmental compartments, including aerobic fresh water, aerobic marine, aerobic soil, and anaerobic conditions. It does not address the impact of major accidental releases, focusing instead on incidental environmental exposure that could occur through normal use.

Key Topics

• Purpose and Scope

  • Guides planning and interpretation of laboratory biodegradability tests for hydraulic fluids.
  • Focuses on unused, fully formulated fluids, not field-aged samples.
  • Addresses only incidental releases, not large spills.

• Environmental Compartments

  • Test methods differentiated for:
    • Aerobic fresh water
    • Aerobic marine water
    • Aerobic soil
    • Anaerobic media

• Test Method Selection

  • Recommends suitable, internationally recognized protocols such as OECD 301B and 301F, ISO 9439, and ASTM D5864.
  • Describes both ultimate biodegradation (e.g., CO₂ evolution, oxygen consumption) and primary biodegradation (e.g., substance disappearance) tests.
  • Emphasizes proper selection and reporting of inocula, reference substances, and sample handling methods, especially for water-insoluble fluids.

• Interpretation of Test Results

  • High biodegradation results suggest that a fluid is non-persistent in the tested compartment.
  • Lower results indicate the need for additional testing before making biodegradability claims.
  • Results are specific to laboratory conditions and cannot be directly extrapolated to every real-world environment.

• Testing Protocols

  • Guidance on gravimetric dosing, use of replicates, statistical analysis, and test-specific factors such as volatility and solubility.
  • Importance of Good Laboratory Practice (GLP) for regulatory or marketing uses.

Applications

The standard guide is practically valuable for:

• Product Development

  • Formulators and manufacturers can use test planning guidance to design more environmentally friendly hydraulic fluids.
  • Supports claims of “readily biodegradable” products in line with regulatory or green procurement requirements.

• Environmental Compliance

  • Helps meet regulatory and customer requirements for environmental risk assessment in sectors like construction, agriculture, forestry, and marine industries.

• Testing Laboratories

  • Provides a structured approach to select, adapt, and validate appropriate laboratory biodegradability methods for different fluid chemistries and environmental conditions.
  • Assists laboratories in reporting results transparently and reproducibly.

• Environmental Impact Assessments

  • Supports risk assessments for incidental leaks or losses during regular hydraulic equipment operation.

Related Standards

ASTM D6006-23 is closely linked with various international and sector-specific tests and terminology standards, including:

• ASTM D4175 - Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
• ASTM D5864 - Determining Aerobic Aquatic Biodegradation of Lubricants or Components
• ASTM D5291 - Instrumental Determination of Carbon, Hydrogen, and Nitrogen
• ISO 9439 - Water Quality - Evaluation of Ultimate Biodegradability
• OECD 301B/301F/306/304A - Guidelines for Testing Biodegradability in Different Media

These referenced standards ensure global harmonization and comparability of results for hydraulic fluid biodegradation testing.

---

Keywords: ASTM D6006, biodegradability assessment, hydraulic fluids, environmental testing, laboratory methods, persistence, water-insoluble lubricants, OECD guidelines, ISO standards, environmental compliance.

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: D6006 − 23
Standard Guide for
Assessing Biodegradability of Hydraulic Fluids
This standard is issued under the fixed designation D6006; 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* mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This guide covers and provides information to assist in
planning a laboratory test or series of tests from which may be
2. Referenced Documents
inferred information about the biodegradability of an unused
fully formulated hydraulic fluid in its original form. Biode-
2.1 ASTM Standards:
gradability is one of three characteristics which are assessed
D4175 Terminology Relating to Petroleum Products, Liquid
when judging the environmental impact of a hydraulic fluid.
Fuels, and Lubricants
The other two characteristics are ecotoxicity and bioaccumu-
D5210 Test Method for Determining the Anaerobic Biodeg-
lation.
radation of Plastic Materials in the Presence of Municipal
Sewage Sludge (Withdrawn 2016)
1.2 Biodegradability may be considered by type of environ-
D5291 Test Methods for Instrumental Determination of
mental compartment: aerobic fresh water, aerobic marine,
Carbon, Hydrogen, and Nitrogen in Petroleum Products
aerobic soil, and anaerobic media. Test methods for aerobic
and Lubricants
fresh water, aerobic soil and anaerobic media have been
D5480 Test Method for Engine Oil Volatility by Gas Chro-
developed that are appropriate for the concerns and needs of
matography (Withdrawn 2003)
testing in each compartment.
D5864 Test Method for Determining Aerobic Aquatic Bio-
1.3 This guide addresses releases to the environment that
degradation of Lubricants or Their Components
are incidental to the use of a hydraulic fluid but is not intended
E1196 Test Method for Determining the Anaerobic Biodeg-
to cover situations of major, accidental release. The tests
radation Potential of Organic Chemicals (Withdrawn
discussed in this guide take a minimum of three to four weeks. 3
1998)
Therefore, issues relating to the biodegradability of hydraulic
2.2 ISO Standards:
fluid are more effectively addressed before the fluid is used,
ISO 9439:1990 Technical Corrigendum I, Water Quali-
and thus before incidental release may occur. Nothing in this
ty–Evaluation in an Aqueous Medium of the Ultimate
guide should be taken to relieve the user of the responsibility
Biodegradability of Organic Compounds
to properly use and dispose of hydraulic fluids.
ISO 4259:1992(E) Petroleum Products–Determination and
1.4 The values stated in SI units are to be regarded as
Application of Precision Data in Relation to Methods of
standard. No other units of measurement are included in this
Test
standard.
2.3 OECD Standards:
1.5 This standard does not purport to address all of the
OECD 301B (the Modified Sturm Test) Guidelines for
safety concerns, if any, associated with its use. It is the
Testing Chemicals
responsibility of the user of this standard to establish appro-
OECD 301F (the Manometric Respirometry Test) Guide-
priate safety, health, and environmental practices and deter-
lines for Testing of Chemicals
mine the applicability of regulatory limitations prior to use.
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
ization established in the Decision on Principles for the
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Development of International Standards, Guides and Recom-
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
The last approved version of this historical standard is referenced on
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum www.astm.org.
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom- Available from International Organization for Standardization (ISO), ISO
mittee D02.12 on Environmental Standards for Lubricants. Central Secretariat, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
Current edition approved July 1, 2023. Published August 2023. Originally Switzerland, https://www.iso.org.
approved in 1996. Last previous edition approved in 2017 as D6006 – 17. DOI: Available from Organisation for Economic Co-Operation and Development
10.1520/D6006-23. (OECD), 2, rue André Pascal, F-75775 Paris Cedex 16, France.
*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
D6006 − 23
3. Terminology 3.1.11 primary biodegradation test, n—a test which follows
the disappearance of a test substance by measuring some
3.1 Definitions:
attribute of the substance.
3.1.1 For definitions of terms used in this guide, refer to
3.1.11.1 Discussion—The extent to which the results of a
Terminology D4175.
primary biodegradation test correspond to the biological con-
3.1.2 aerobic, adj—1. taking place in the presence of
version of the test substance will depend on the attribute which
oxygen; 2. living or active in the presence of oxygen.
is being measured.
3.1.3 anaerobic, adj—1. taking place in the absence of
3.1.12 sonication, n—the act of subjecting a material to the
oxygen; 2. living or active in the absence of oxygen.
shearing forces of high-frequency sound waves.
3.1.4 biodegradation, n—the process of chemical break-
3.1.12.1 Discussion—Sonication of a two phase liquid sys-
down or transformation of a material caused by organisms or
tem may result in the dispersal of one phase as fine droplets in
their enzymes.
the other phase.
3.1.4.1 Discussion—Biodegradation is only one mechanism
3.1.13 ultimate biodegradation, n—degradation achieved
by which materials are transformed in the environment.
when a material is totally utilized by microorganisms resulting
3.1.5 biomass, n—biological material including any mate-
in the production of carbon dioxide (and possibly methane in
rial other than fossil fuels which is or was a living organism or
the case of anaerobic biodegradation), water, inorganic
component or product of a living organism.
compounds, and new microbial cellular constituents (biomass
3.1.5.1 Discussion—In biology and environmental science,
or secretions, or both).
biomass is typically expressed as density of biological material
3.1.14 ultimate biodegradation test, n—a protocol which
per unit sample volume, area, or mass (g biomass / g (or / mL
estimates the extent to which the carbon in a material is
or / cm ) sample); when used for products derived from
converted to CO or methane, either directly, by measuring the
organisms biomass is typically expressed in terms of mass (kg,
production of CO or methane, or, in the case of aerobic
MT, etc.) or volume (L, m , bbl, etc.).
biodegradation, indirectly by measuring the consumption of
3.1.5.2 Discussion—Products of living organisms include
O .
those materials produced directly by living organisms as
3.1.14.1 Discussion—The measurement of new biomass
metabolites (for example, ethanol, various carbohydrates and
usually is not attempted.
fatty acids), materials manufactured by processing living
organisms (for example: pellets manufactured by shredding
4. Summary of Guide
and pelletizing plant material) and materials produced by
4.1 This guide gives two kinds of information which relate
processing living organisms, their components or metabolites
to testing of hydraulic fluids for biodegradability. First, it gives
(for example, transesterified oil; also called biodiesel).
information of a general nature relating to biodegradability. For
3.1.6 blank, n—in biodegradability testing, a test system
example, it includes definitions of terms not traditionally used
containing all system components with the exception of the test
by users or producers of hydraulic fluids (Section 3) and a brief
material.
discussion of some of the technical issues which are common
3.1.7 environmental compartment, n—a subdivision of the
to most biodegradability tests when they are applied to
environment based on physical or chemical properties, or both.
hydraulic fluids (Section 7). Second, the guide gives more
3.1.7.1 Discussion—Examples of environmental compart- specific information on the methods, advantages, and disad-
ments are aerobic fresh water, aerobic marine, and aerobic soil. vantages of several of the biodegradation tests frequently used
The results of test procedures may be applied to environmental for hydraulic fluids (Section 6).
compartments but the test systems do not constitute an envi-
5. Significance and Use
ronmental compartment.
5.1 This guide discusses ways to assess the likelihood that a
3.1.8 inoculum, n—spores, bacteria, single celled
hydraulic fluid will undergo biodegradation if it enters an
organisms, or other live materials that are introduced into a test
environment that is known to support biodegradation of some
medium.
substances, for example the material used as the positive
3.1.9 pre-adaptation, n—the pre-incubation of an inoculum
control in the test. The information can be used in making or
in the presence of the test material under conditions similar to
assessing claims of biodegradability of a fluid formula.
the test conditions.
3.1.9.1 Discussion—The aim of pre-adaptation is to im- 5.2 Biodegradation occurs when a fluid interacts with the
environment, and so the extent of biodegradation is a function
prove the precision of the test method by decreasing variability
in the rate of biodegradation produced by the inoculum. of both the chemical composition of the hydraulic fluid and the
physical, chemical, and biological status of the environment at
Pre-adaptation may mimic the natural processes which cause
changes in the microbial population of the inoculum leading to the time the fluid enters it. This guide cannot assist in judging
the status of a particular environment, so it is not meant to
more rapid biodegradation of the test material, but not to a
change in the final degree of biodegradation. provide standards for judging the persistence of a hydraulic
fluid in any specific environment either natural or man-made.
3.1.10 primary biodegradation, n—degradation of the test
substance resulting in a change in its physical or chemical 5.3 If any of the tests discussed in this guide gives a high
properties, or both. result, it implies that the hydraulic fluid will biodegrade and
D6006 − 23
will not persist in the environmental compartment being by the Coordinating European Council in the early 1980s and
considered. If a low result is obtained, it does not mean approved by the CEC in 1993. This test, which was called the
necessarily that the substance will not biodegrade in the CEC L-33-T-82 test prior to approval, measures the IR
environment, but does mean that further testing is required if a absorption spectrum of saturated carbons found in the test
claim of biodegradability is to be made. Such testing may materials. It was designed specifically for two-stroke outboard
include, but is not limited to, other tests mentioned in this guide engine lubricants; however, it is frequently used for measuring
or simulation tests for a particular environmental compartment. the biodegradability of other lubricants. It is suitable for
measuring the primary biodegradation of hydraulic fluids if
6. Test Methods
they have methylene hydrogens (-CH -) in their chemical
structures. CEC results for some materials have been found to
6.1 Aerobic Fresh Water Environment—The most com-
correlate with the results of ultimate biodegradation tests, but
monly performed tests cover aerobic biodegradation in fresh
for some substances results from the CEC L-33-A-94 test
water. The tests conducted for this compartment may be
over-predict ultimate biodegradability results (2, 3, 4, 5). The
ultimate biodegradation tests measuring CO production or
test is not suitable for either volatile or water soluble test
primary biodegradation tests measuring the disappearance of
materials.
the test fluid. The test medium is based on high-grade,
carbon-free water. Some salts will be included as necessary for
6.2 Aerobic Marine Environment—Tests for oil biodegrada-
maintenance of solution pH and provision of trace minerals
tion in a marine environment are cited in the literature and
necessary for microbial life.
OECD has published a standardized method, OECD 306 (6).
6.1.1 The majority of ultimate biodegradation tests measure
At the present time the standardized method has not been
produced CO . Examples of test procedures for ultimate
2 widely used, and there are significant uncertainties regarding
biodegradability in an aerobic aquatic environment are: the
the test methods cited in literature. Each case must be exam-
Modified Sturm Test (OECD 301B); the Manometric Re-
ined individually.
spirometry Test (OECD 301F); the U.S. EPA Aerobic Aquatic
6.2.1 The OECD 306 test method has two options for the
Biodegradation Test, which also is called the EPA Shake Flask
test procedure: the Shake Flask Method (which is not the same
Test and was derived from the Gledhill Test (1); Test Method
as the U.S. EPA Shake Flask Test) and the Closed Bottle
D5864; and the ISO Test 9439:1990. With the exception of Test
Method. The Shake Flask Method measures dissolved organic
Method D5864, these tests were originally designed for water-
carbon (DOC) and is not suitable for substances with low water
soluble pure compounds and so the test procedures allow some
solubility (less the 2 mg/L). If poorly soluble substances are
procedural options that are not suitable for water-insoluble
tested with this test method, unchanged and undissolved
substances, such as addition of the test substance in an aqueous
carbon will be removed from the test system during a filtration
solution or calculation, rather than measurement, of carbon
step and the test substance will appear to have biodegraded
content. In other tests, such as the Manometric Respirometry
when it has not. The Closed Bottle Method measures oxygen
Test, oxygen consumption is measured as a surrogate for CO
2 content of the test system and OECD states in the method that
production. Oxygen consumption is not a direct measure of
it is not recommended for substances with low water solubility.
ultimate biodegradation but is expected to correlate closely
6.3 Aerobic Soil Environment—Test procedures in aerobic
with it. The procedures listed are screening tests suitable for
soils are not as well developed as test procedures for aerobic
laboratory evaluation of the hydraulic fluid. Although all the
aqueous environments (7). It is not possible to sterilize soil
tests referred to above specify that the length of the test is 28
without drastically changing its physical properties, so a sterile
days, a high level of biodegradation in longer time frames can
starting soil similar in function to high-grade, carbon-free
be taken as evidence that the hydraulic fluid is ultimately
water, is not possible. The inoculum for these test procedures
biodegradable and nonpersistent in fresh water.
is typically the microbial community that naturally resides in
6.1.1.1 If the biodegradability of a hydraulic fluid with a
the soil sample used for the test. No further augmentation
nonnegligible vapor pressure is measured in any one of these
generally is required. The sources of the soil samples should be
tests, except the OECD 301F, a false negative may result. The
reported with test results. A low result in any soil test may not
hydraulic fluid could vaporize from the test solution before
mean that the hydraulic fluid will persist in an aerobic soil
conversion to CO . In this case a biodegradable fluid would
environment, but does mean that more testing is required.
have a low measured percent theoretical CO . If the aerobic
6.3.1 With modifications, published tests for aerobic bio-
aquatic biodegradability of a volatile hydraulic fluid is to be
degradation in soils could be suitable for hydraulic fluids, but
measured, the OECD 301F test should be used.
none of the available standardized tests can be used as written.
6.1.2 Tests for primary biodegradation must be designed for
In some cases only minor changes are necessary, such as
specific classes of test substances. The results of a primary
development of a method for introduction of a water insoluble
biodegradation test should not be considered equivalent to or
substance. Tests of soil biodegradation that currently are
substitutable for the results of an ultimate biodegradation test.
available fall into three categories.
6.1.2.1 The most commonly performed primary biodegra-
6.3.1.1 First are those tests that follow CO production by
dation test for lubricants is the CEC L-33-A-94 test, developed
chemical means. An example of this kind of test has been
published by the U.S. FDA (8). These tests are suitable for
adaptation to assess the biodegradability of a hydraulic fluid in
The boldface numbers in parentheses refer to the list of ref
...

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: D6006 − 17 D6006 − 23
Standard Guide for
Assessing Biodegradability of Hydraulic Fluids
This standard is issued under the fixed designation D6006; 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 guide covers and provides information to assist in planning a laboratory test or series of tests from which may be inferred
information about the biodegradability of an unused fully formulated hydraulic fluid in its original form. Biodegradability is one
of three characteristics which are assessed when judging the environmental impact of a hydraulic fluid. The other two
characteristics are ecotoxicity and bioaccumulation.
1.2 Biodegradability may be considered by type of environmental compartment: aerobic fresh water, aerobic marine, aerobic soil,
and anaerobic media. Test methods for aerobic fresh water, aerobic soil and anaerobic media have been developed that are
appropriate for the concerns and needs of testing in each compartment.
1.3 This guide addresses releases to the environment that are incidental to the use of a hydraulic fluid but is not intended to cover
situations of major, accidental release. The tests discussed in this guide take a minimum of three to four weeks. Therefore, issues
relating to the biodegradability of hydraulic fluid are more effectively addressed before the fluid is used, and thus before incidental
release may occur. Nothing in this guide should be taken to relieve the user of the responsibility to properly use and dispose of
hydraulic fluids.
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 and healthsafety, health, and environmental practices and determine
the applicability of regulatory limitations prior to use.
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:
D4175 Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D5210 Test Method for Determining the Anaerobic Biodegradation of Plastic Materials in the Presence of Municipal Sewage
Sludge (Withdrawn 2016)
This guide 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 Jan. 1, 2017July 1, 2023. Published February 2017August 2023. Originally approved in 1996. Last previous edition approved in 20112017 as
D6006 – 11.D6006 – 17. DOI: 10.1520/D6006-17.10.1520/D6006-23.
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.
The last approved version of this historical standard is referenced on www.astm.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
D6006 − 23
D5291 Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
D5480 Test Method for Engine Oil Volatility by Gas Chromatography (Withdrawn 2003)
D5864 Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
E1196 Test Method for Determining the Anaerobic Biodegradation Potential of Organic Chemicals (Withdrawn 1998)
2.2 ISO Standards:
ISO 9439:1990 Technical Corrigendum I, Water Quality–Evaluation in an Aqueous Medium of the Ultimate Biodegradability
of Organic Compounds
ISO 4259:1992(E) Petroleum Products–Determination and Application of Precision Data in Relation to Methods of Test
2.3 OECD Standards:
OECD 301B (the Modified Sturm Test) Guidelines for Testing Chemicals
OECD 301F (the Manometric Respirometry Test) Guidelines for Testing of Chemicals
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this guide, refer to Terminology D4175.
3.1.2 aerobic, adj—1. taking place in the presence of oxygen; 2. living or active in the presence of oxygen.
3.1.3 anaerobic, adj—1. taking place in the absence of oxygen; 2. living or active in the absence of oxygen.
3.1.4 biodegradation, n—the process of chemical breakdown or transformation of a material caused by organisms or their
enzymes.
3.1.4.1 Discussion—
Biodegradation is only one mechanism by which materials are transformed in the environment.
3.1.5 biomass, n—biological material including any material other than fossil fuels which is or was a living organism or
component or product of a living organism.
3.1.5.1 Discussion—
In biology and environmental science, biomass is typically expressed as density of biological material per unit sample volume,
area, or mass (g biomass / g (or / mL or / cm ) sample); when used for products derived from organisms biomass is typically
expressed in terms of mass (kg, MT, etc.) or volume (L, m , bbl, etc.).
3.1.5.2 Discussion—
Products of living organisms include those materials produced directly by living organisms as metabolites (for example, ethanol,
various carbohydrates and fatty acids), materials manufactured by processing living organisms (for example: pellets manufactured
by shredding and pelletizing plant material) and materials produced by processing living organisms, their components or
metabolites (for example, transesterified oil; also called biodiesel).
3.1.6 blank, n—in biodegradability testing, a test system containing all system components with the exception of the test
substance.material.
3.1.7 environmental compartment, n—a subdivision of the environment based on physical or chemical properties, or both.
3.1.7.1 Discussion—
Examples of environmental compartments are aerobic fresh water, aerobic marine, and aerobic soil. The results of test procedures
may be applied to environmental compartments but the test systems do not constitute an environmental compartment.
3.1.8 inoculum, n—spores, bacteria, single celled organisms, or other live materials that are introduced into a test medium.
3.1.9 pre-adaptation, n—the pre-incubation of an inoculum in the presence of the test material under conditions similar to the test
conditions.
3.1.9.1 Discussion—
The aim of pre-adaptation is to improve the precision of the test method by decreasing variability in the rate of biodegradation
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.International Organization for
Standardization (ISO), ISO Central Secretariat, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva, Switzerland, https://www.iso.org.
Available from Organisation for Economic Co-Operation and Development (OECD), 2, rue André Pascal, F-75775 Paris Cedex 16, France.
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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 not to a change in the final degree of biodegradation.
3.1.10 primary biodegradation, n—degradation of the test substance resulting in a change in its physical or chemical properties,
or both.
3.1.11 primary biodegradation test, n—a test which follows the disappearance of a test substance by measuring some attribute of
the substance.
3.1.11.1 Discussion—
The extent to which the results of a primary biodegradation test correspond to the biological conversion of the test substance will
depend on the attribute which is being measured.
3.1.12 sonication, n—the act of subjecting a material to the shearing forces of high-frequency sound waves.
3.1.12.1 Discussion—
Sonication of a two phase liquid system may result in the dispersal of one phase as fine droplets in the other phase.
3.1.13 ultimate biodegradation, n—degradation achieved when a substancematerial is totally utilized by microorganisms resulting
in the production of carbon dioxide (and possibly methane in the case of anaerobic biodegradation), water, inorganic compounds,
and new microbial cellular constituents (biomass or secretions, or both).
3.1.14 ultimate biodegradation test, n—a testprotocol which estimates the extent to which the carbon in a productmaterial is
converted to CO or methane, either directly, by measuring the production of CO or methane, or, in the case of aerobic
2 2
biodegradation, indirectly by measuring the consumption of O .
3.1.14.1 Discussion—
The measurement of new biomass usually is not attempted.
4. Summary of Guide
4.1 This guide gives two kinds of information which relate to testing of hydraulic fluids for biodegradability. First, it gives
information of a general nature relating to biodegradability. For example, it includes definitions of terms not traditionally used by
users or producers of hydraulic fluids (Section 3) and a brief discussion of some of the technical issues which are common to most
biodegradability tests when they are applied to hydraulic fluids (Section 7). Second, the guide gives more specific information on
the methods, advantages, and disadvantages of several of the biodegradation tests frequently used for hydraulic fluids (Section 6).
5. Significance and Use
5.1 This guide discusses ways to assess the likelihood that a hydraulic fluid will undergo biodegradation if it enters an environment
that is known to support biodegradation of some substances, for example the material used as the positive control in the test. The
information can be used in making or assessing claims of biodegradability of a fluid formula.
5.2 Biodegradation occurs when a fluid interacts with the environment, and so the extent of biodegradation is a function of both
the chemical composition of the hydraulic fluid and the physical, chemical, and biological status of the environment at the time
the fluid enters it. This guide cannot assist in judging the status of a particular environment, so it is not meant to provide standards
for judging the persistence of a hydraulic fluid in any specific environment either natural or man-made.
5.3 If any of the tests discussed in this guide gives a high result, it implies that the hydraulic fluid will biodegrade and will not
persist in the environmental compartment being considered. If a low result is obtained, it does not mean necessarily that the
substance will not biodegrade in the environment, but does mean that further testing is required if a claim of biodegradability is
to be made. Such testing may include, but is not limited to, other tests mentioned in this guide or simulation tests for a particular
environmental compartment.
6. Test Methods
6.1 Aerobic Fresh Water Environment—The most commonly performed tests cover aerobic biodegradation in fresh water. The
tests conducted for this compartment may be ultimate biodegradation tests measuring CO production or primary biodegradation
tests measuring the disappearance of the test fluid. The test medium is based on high-grade, carbon-free water. Some salts will be
included as necessary for maintenance of solution pH and provision of trace minerals necessary for microbial life.
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6.1.1 The majority of ultimate biodegradation tests measure produced CO . Examples of test procedures for ultimate
biodegradability in an aerobic aquatic environment are: the Modified Sturm Test (OECD 301B); the Manometric Respirometry Test
(OECD 301F); the U.S. EPA Aerobic Aquatic Biodegradation Test, which also is called the EPA Shake Flask Test and was derived
from the Gledhill Test (1); Test Method D5864; and the ISO Test 9439:1990. With the exception of Test Method D5864, these
tests were originally designed for water-soluble pure compounds and so the test procedures allow some procedural options that are
not suitable for water-insoluble substances, such as addition of the test substance in an aqueous solution or calculation, rather than
measurement, of carbon content. In other tests, such as the Manometric Respirometry Test, oxygen consumption is measured as
a surrogate for CO production. Oxygen consumption is not a direct measure of ultimate biodegradation but is expected to correlate
closely with it. The procedures listed are screening tests suitable for laboratory evaluation of the hydraulic fluid. Although all the
tests referred to above specify that the length of the test is 28 days, a high level of biodegradation in longer time frames can be
taken as evidence that the hydraulic fluid is ultimately biodegradable and nonpersistent in fresh water.
6.1.1.1 If the biodegradability of a hydraulic fluid with a nonnegligible vapor pressure is measured in any one of these tests, except
the OECD 301F, a false negative may result. The hydraulic fluid could vaporize from the test solution before conversion to CO .
In this case a biodegradable fluid would have a low measured percent theoretical CO . If the aerobic aquatic biodegradability of
a volatile hydraulic fluid is to be measured, the OECD 301F test should be used.
6.1.2 Tests for primary biodegradation must be designed for specific classes of test substances. The results of a primary
biodegradation test should not be considered equivalent to or substitutable for the results of an ultimate biodegradation test.
6.1.2.1 The most commonly performed primary biodegradation test for lubricants is the CEC L-33-A-94 test, developed by the
Coordinating European Council in the early 1980s and approved by the CEC in 1993. This test, which was called the CEC
L-33-T-82 test prior to approval, measures the IR absorption spectrum of saturated carbons found in the test materials. It was
designed specifically for two-stroke outboard engine lubricants; however, it is frequently used for measuring the biodegradability
of other lubricants. It is suitable for measuring the primary biodegradation of hydraulic fluids if they have methylene hydrogens
(-CH -) in their chemical structures. CEC results for some materials have been found to correlate with the results of ultimate
biodegradation tests, but for some substances results from the CEC L-33-A-94 test over-predict ultimate biodegradability results
(2, 3, 4, 5). The test is not suitable for either volatile or water soluble test materials.
6.2 Aerobic Marine Environment—Tests for oil biodegradation in a marine environment are cited in the literature and OECD has
published a standardized method, OECD 306 (6). At the present time the standardized method has not been widely used, and there
are significant uncertainties regarding the test methods cited in literature. Each case must be examined individually.
6.2.1 The OECD 306 test method has two options for the test procedure: the Shake Flask Method (which is not the same as the
U.S. EPA Shake Flask Test) and the Closed Bottle Method. The Shake Flask Method measures dissolved organic carbon (DOC)
and is not suitable for substances with low water solubility (less the 2 mg/L). If poorly soluble substances are tested with this test
method, unchanged and undissolved carbon will be removed from the test system during a filtration step and the test substance
will appear to have biodegraded when it has not. The Closed Bottle Method measures oxygen content of the test system and OECD
states in the method that it is not recommended for substances with low water solubility.
6.3 Aerobic Soil Environment—Test procedures in aerobic soils are not as well developed as test procedures for aerobic aqueous
environments (7)). . It is not possible to sterilize soil without drastically changing its physical properties, so a sterile starting soil
similar in function to high-grade, carbon-free water, is not possible. The inoculum for these test procedures is typically the
microbial community that naturally resides in the soil sample used for the test. No further augmentation generally is required. The
sources of the soil samples should be reported with test results. A low result in any soil test may not mean that the hydraulic fluid
will persist in an aerobic soil environment, but does mean that more testing is required.
6.3.1 With modifications, published tests for aerobic biodegradation in soils could be suitable for hydraulic fluids, but none of the
available standardized tests can be used as written. In some cases only minor changes are necessary, such as development of a
method for introduction of a water insoluble substance. Tests of soil biodegradation that currently are available fall into three
categories.
6.3.1.1 First are those tests that follow CO production by chemical means. An example of this kind of test has been published
The boldface numbers in parentheses refer to the list of references at
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