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 − 17

Standard Guide for

Assessing Biodegradability of Hydraulic Fluids1
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* 2. Referenced Documents

1.1 This guide covers and provides information to assist in 2.1 ASTM Standards:2
planning a laboratory test or series of tests from which may be D5210 Test Method for Determining the Anaerobic Biodeg-
inferred information about the biodegradability of an unused radation of Plastic Materials in the Presence of Municipal
fully formulated hydraulic fluid in its original form. Biode- Sewage Sludge (Withdrawn 2016)3
gradability is one of three characteristics which are assessed D5291 Test Methods for Instrumental Determination of
when judging the environmental impact of a hydraulic fluid. Carbon, Hydrogen, and Nitrogen in Petroleum Products
The other two characteristics are ecotoxicity and bioaccumu- and Lubricants
lation. D5480 Test Method for Engine Oil Volatility by Gas Chro-
1.2 Biodegradability may be considered by type of environ- matography (Withdrawn 2003)3
mental compartment: aerobic fresh water, aerobic marine, D5864 Test Method for Determining Aerobic Aquatic Bio-
aerobic soil, and anaerobic media. Test methods for aerobic degradation of Lubricants or Their Components
fresh water, aerobic soil and anaerobic media have been E1196 Test Method for Determining the Anaerobic Biodeg-
developed that are appropriate for the concerns and needs of radation Potential of Organic Chemicals (Withdrawn
testing in each compartment. 1998)3
2.2 ISO Standards:
1.3 This guide addresses releases to the environment that ISO 9439:1990 Technical Corrigendum I, Water Quali-
are incidental to the use of a hydraulic fluid but is not intended ty–Evaluation in an Aqueous Medium of the Ultimate
to cover situations of major, accidental release. The tests Biodegradability of Organic Compounds4
discussed in this guide take a minimum of three to four weeks. ISO 4259:1992(E) Petroleum Products–Determination and
Therefore, issues relating to the biodegradability of hydraulic Application of Precision Data in Relation to Methods of
fluid are more effectively addressed before the fluid is used, Test4
and thus before incidental release may occur. Nothing in this 2.3 OECD Standards:
guide should be taken to relieve the user of the responsibility OECD 301B (the Modified Sturm Test) Guidelines for
to properly use and dispose of hydraulic fluids. Testing Chemicals5
1.4 The values stated in SI units are to be regarded as OECD 301F (the Manometric Respirometry Test) Guide-
standard. No other units of measurement are included in this lines for Testing of Chemicals5
standard.
3. Terminology
1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 3.1 Definitions:
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- 2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
bility of regulatory limitations prior to use. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This guide is under the jurisdiction of ASTM Committee D02 on Petroleum The last approved version of this historical standard is referenced on
Products, Liquid Fuels, and Lubricants and is the direct responsibility of Subcom- www.astm.org.
4
mittee D02.12 on Environmental Standards for Lubricants. Available from American National Standards Institute (ANSI), 25 W. 43rd St.,
Current edition approved Jan. 1, 2017. Published February 2017. Originally 4th Floor, New York, NY 10036, http://www.ansi.org.
5
approved in 1996. Last previous edition approved in 2011 as D6006 – 11. DOI: Available from Organisation for Economic Co-Operation and Development
10.1520/D6006-17. (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

1
 D6006 − 17
3.1.1 aerobic, adj—1. taking place in the presence of primary biodegradation test correspond to the biological con-
oxygen; 2. living or active in the presence of oxygen. version of the test substance will depend on the attribute which
3.1.2 anaerobic, adj—1. taking place in the absence of is being measured.
oxygen; 2. living or active in the absence of oxygen. 3.1.11 sonication, n—the act of subjecting a material to the
3.1.3 biodegradation, n—the process of chemical break- shearing forces of high-frequency sound waves.
down or transformation of a material caused by organisms or 3.1.11.1 Discussion—Sonication of a two phase liquid sys-
their enzymes. tem may result in the dispersal of one phase as fine droplets in
3.1.3.1 Discussion—Biodegradation is only one mechanism the other phase.
by which materials are transformed in the environment. 3.1.12 ultimate biodegradation, n—degradation achieved
3.1.4 biomass, n—biological material including any mate- when a substance is totally utilized by microorganisms result-
rial other than fossil fuels which is or was a living organism or ing in the production of carbon dioxide (and possibly methane
component or product of a living organism. in the case of anaerobic biodegradation), water, inorganic
3.1.4.1 Discussion—In biology and environmental science, compounds, and new microbial cellular constituents (biomass
biomass is typically expressed as density of biological material or secretions, or both).
per unit sample volume, area, or mass (g biomass / g (or / mL 3.1.13 ultimate biodegradation test, n—a test which esti-
or / cm2) sample); when used for products derived from mates the extent to which the carbon in a product is converted
organisms biomass is typically expressed in terms of mass (kg, to CO2 or methane, either directly, by measuring the produc-
MT, etc.) or volume (L, m3, bbl, etc.). tion of CO2 or methane, or, in the case of aerobic
3.1.4.2 Discussion—Products of living organisms include biodegradation, indirectly by measuring the consumption of
those materials produced directly by living organisms as O2.
metabolites (for example, ethanol, various carbohydrates and 3.1.13.1 Discussion—The measurement of new biomass
fatty acids), materials manufactured by processing living usually is not attempted.
organisms (for example: pellets manufactured by shredding
and pelletizing plant material) and materials produced by 4. Summary of Guide
processing living organisms, their components or metabolites 4.1 This guide gives two kinds of information which relate
(for example, transesterified oil; also called biodiesel). to testing of hydraulic fluids for biodegradability. First, it gives
3.1.5 blank, n—in biodegradability testing, a test system information of a general nature relating to biodegradability. For
containing all system components with the exception of the test example, it includes definitions of terms not traditionally used
substance. by users or producers of hydraulic fluids (Section 3) and a brief
discussion of some of the technical issues which are common
3.1.6 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
3.1.6.1 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-
ronmental compartment. 5. Significance and Use
3.1.7 inoculum, n—spores, bacteria, single celled 5.1 This guide discusses ways to assess the likelihood that a
organisms, or other live materials that are introduced into a test hydraulic fluid will undergo biodegradation if it enters an
medium. environment that is known to support biodegradation of some
3.1.8 pre-adaptation, n—the pre-incubation of an inoculum substances, for example the material used as the positive
in the presence of the test material under conditions similar to control in the test. The information can be used in making or
the test conditions. assessing claims of biodegradability of a fluid formula.
3.1.8.1 Discussion—The aim of pre-adaptation is to im- 5.2 Biodegradation occurs when a fluid interacts with the
prove the precision of the test method by decreasing variability environment, and so the extent of biodegradation is a function
in the rate of biodegradation produced by the inoculum. of both the chemical composition of the hydraulic fluid and the
Pre-adaptation may mimic the natural processes which cause physical, chemical, and biological status of the environment at
changes in the microbial population of the inoculum leading to the time the fluid enters it. This guide cannot assist in judging
more rapid biodegradation of the test material, but not to a the status of a particular environment, so it is not meant to
change in the final degree of biodegradation. provide standards for judging the persistence of a hydraulic
3.1.9 primary biodegradation, n—degradation of the test fluid in any specific environment either natural or man-made.
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
3.1.10 primary biodegradation test, n—a test which follows will not persist in the environmental compartment being
the disappearance of a test substance by measuring some considered. If a low result is obtained, it does not mean
attribute of the substance. necessarily that the substance will not biodegrade in the
3.1.10.1 Discussion—The extent to which the results of a environment, but does mean that further testing is required if a

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 D6006 − 17
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 (-CH2-) in their chemical
6.1 Aerobic Fresh Water Environment—The most com- structures. CEC results for some materials have been found to
monly performed tests cover aerobic biodegradation in fresh correlate with the results of ultimate biodegradation tests, but
water. The tests conducted for this compartment may be for some substances results from the CEC L-33-A-94 test
ultimate biodegradation tests measuring CO2 production or over-predict ultimate biodegradability results (2, 3, 4, 5). The
primary biodegradation tests measuring the disappearance of test is not suitable for either volatile or water soluble test
the test fluid. The test medium is based on high-grade, materials.
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 CO2. Examples of test procedures for ultimate
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
Biodegradation Test, which also is called the EPA Shake Flask 6.2.1 The OECD 306 test method has two options for the
Test and was derived from the Gledhill Test (1);6 Test Method test procedure: the Shake Flask Method (which is not the same
D5864; and the ISO Test 9439:1990. With the exception of Test as the U.S. EPA Shake Flask Test) and the Closed Bottle
Method D5864, these tests were originally designed for water- Method. The Shake Flask Method measures dissolved organic
soluble pure compounds and so the test procedures allow some carbon (DOC) and is not suitable for substances with low water
procedural options that are not suitable for water-insoluble solubility (less the 2 mg/L). If poorly soluble substances are
substances, such as addition of the test substance in an aqueous tested with this test method, unchanged and undissolved
solution or calculation, rather than measurement, of carbon carbon will be removed from the test system during a filtration
content. In other tests, such as the Manometric Respirometry step and the test substance will appear to have biodegraded
Test, oxygen consumption is measured as a surrogate for CO2 when it has not. The Closed Bottle Method measures oxygen
production. Oxygen consumption is not a direct measure of content of the test system and OECD states in the method that
ultimate biodegradation but is expected to correlate closely it is not recommended for substances with low water solubility.
with it. The procedures listed are screening tests suitable for 6.3 Aerobic Soil Environment—Test procedures in aerobic
laboratory evaluation of the hydraulic fluid. Although all the soils are not as well developed as test procedures for aerobic
tests referred to above specify that the length of the test is 28 aqueous environments (7) . It is not possible to sterilize soil
days, a high level of biodegradation in longer time frames can without drastically changing its physical properties, so a sterile
be taken as evidence that the hydraulic fluid is ultimately starting soil similar in function to high-grade, carbon-free
biodegradable and nonpersistent in fresh water. water, is not possible. The inoculum for these test procedures
6.1.1.1 If the biodegradability of a hydraulic fluid with a is typically the microbial community that naturally resides in
nonnegligible vapor pressure is measured in any one of these the soil sample used for the test. No further augmentation
tests, except the OECD 301F, a false negative may result. The generally is required. The sources of the soil samples should be
hydraulic fluid could vaporize from the test solution before reported with test results. A low result in any soil test may not
conversion to CO2. In this case a biodegradable fluid would mean that the hydraulic fluid will persist in an aerobic soil
have a low measured percent theoretical CO2. If the aerobic environment, but does mean that more testing is required.
aquatic biodegradability of a volatile hydraulic fluid is to be
6.3.1 With modifications, published tests for aerobic bio-
measured, the OECD 301F test should be used.
degradation in soils could be suitable for hydraulic fluids, but
6.1.2 Tests for primary biodegradation must be designed for
none of the available standardized tests can be used as written.
specific classes of test substances. The results of a primary
In some cases only minor changes are necessary, such as
biodegradation test should not be considered equivalent to or
development of a method for introduction of a water insoluble
substitutable for the results of an ultimate biodegradation test.
substance. Tests of soil biodegradation that currently are
6.1.2.1 The most commonly performed primary biodegra-
available fall into three categories.
dation test for lubricants is the CEC L-33-A-94 test, developed
by the Coordinating European Council in the early 1980s and 6.3.1.1 First are those tests that follow CO2 production by
approved by the CEC in 1993. This test, which was called the chemical means. An example of this kind of test has been
CEC L-33-T-82 test prior to approval, measures the IR published by the U.S. FDA (8). These tests are suitable for
absorption spectrum of saturated carbons found in the test adaptation to assess the biodegradability of a hydraulic fluid in
aerobic soils. Such adaptation may include different sample
handling procedures or changes in sample concentration. The
6
The boldface numbers in parentheses refer to the list of references at the end of U.S. FDA test is not suitable for testing volatile hydraulic
this standard. fluids.

3
 D6006 − 17
6.3.1.2 Second are those that use test substances labeled dures cited, a low result may not mean that the fluid is
with radioactive tracers and follow the production of radioac- anaerobically persistent, but does mean that further testing is
tive CO2, for example OECD 304A (6). These tests have the needed.
advantage of allowing the use of very low concentrations of 6.5 Table 1 summarizes the test methods discussed in
test substance, but are inappropriate for hydraulic fluids be- Section 6.
cause hydraulic fluids are complex mixtures of many chemical
compounds, so the selection of an appropriate site for the label 7. Discussion
is impossible (7). 7.1 Hydraulic fluids are difficult to test for biodegradability
6.3.1.3 Third are soil slurry tests that make a well-mixed because usually they are insoluble in water and consist of
aerobic slurry of the soil and follow CO2 production. A complex mixtures of chemicals. With the exception of Test
consideration for soil slurry tests is that they are not a direct Method D5864, currently available tests for biodegradation
comparison to a soil system. The fungi normally present in soil have been developed for water-soluble pure chemicals. In some
will not be active in a slurry and the microbial activity may or cases, the tests contain procedural options not suitable for
may not be representative of that found in soil. testing hydraulic fluids, and in other cases the procedures are
6.3.2 A major reason for the difficulty of testing for biodeg- not specific enough to give good, reproducible results for
radation in soils is that soils are complex mixtures of living hydraulic fluids. Guidelines for the performance of test proce-
organisms and nonliving substances, some of which are dures for biodegradability testing of hydraulic fluids are given
sources of food for the organisms. Soils vary greatly between in this section. All test procedures for regulatory submission or
locations, and the range of variation is large enough to affect advertising purposes should be run under Good Laboratory
Practice (GLP) standards as outlined in the Toxic Substances
the biodegradation of hydraulic fluids in soil. Because of the
Control Act (12).
large variation in soil properties, demonstration of the biodeg-
radation of hydraulic fluids in soils should be done using at 7.2 Tests for aerobic biodegradation which measure CO2
least three different types of soil from three different locations. production are done frequently in systems that are swept
The characteristics of the soil should be reported with the test regularly with fresh, CO2-free air to maintain the aerobic
results. environment. Any test substance in the air space of the system
will be removed, so hydraulic fluids with high vapor pressure
6.3.2.1 Some soil characteristics that are important in bio-
above the test medium could give a false negative result.
degradation are pH, percent water holding capacity, moisture
7.2.1 The volatility of a petroleum-based hydraulic fluid
content, percent organic matter, percent silt and clay, nitrogen
above a test system can be estimated by its vapor pressure as
content and phosphorus content.
measured by Test Method D5480 or an equivalent method. If
6.3.2.2 Soil tests commonly are run using blank systems to the volatility exceeds 5 % at 371°C in accordance with Test
account for the metabolism and subsequent production of CO2 Method D5480, then the OECD 301F test should be used to
that would occur in the absence of the test substance. The blank measure aerobic aquatic biodegradability.
system contains everything the test system contains with the
7.3 The amount of hydraulic fluid to be added is determined
exception of the test substance. The amount of CO2 produced
by calculating the weight of the hydraulic fluid that will contain
by the blank is subtracted from the CO2 produced by the test
the amount of carbon specified in the selected test method.
system to obtain the CO2 produced during metabolism of the
Because of the complex nature of hydraulic fluids, the carbon
test substance.
6.3.2.3 Implicit in the use of a blank to account for intrinsic
TABLE 1 Biodegradation Test Methods
metabolism is the assumption that the addition of the test
Environmental
substance will not affect that metabolism. There have been Conditions
Test Method Section
some reports that some test substances, especially very easily Aerobic aquatic OECD 301B, the Modified Sturm Test 6.1 to
biodegraded substances, may stimulate the metabolic transfor- 6.1.1.1
mation of materials which occur naturally in the soil and so OECD 301F, the Manometric Respirometry Test 6.1 to
6.1.1.1
lead to measured biodegradabilities over 100 % theoretical U.S. EPA Aerobic Aquatic Biodegradation Test 6.1 to
CO2 (9). (EPA Shake Flask Test) 6.1.1.1
ISO Test 9439:1990 6.1 to
6.4 Anaerobic Environment—Testing for biodegradation in 6.1.1.1
Test Method D5864 6.1 to
an anaerobic environment may be done with the U.S. EPA 6.1.1.1
Anaerobic Biodegradation test procedure (10) or with the CEC L-33-A-94 (formerly CEC L-33-T-82) 6.1, 6.1.2
method recommended by ECETOC (11). Other test procedures Aerobic marine OECD 306, Biodegradability in Seawater 6.1.2
Aerobic soil U.S. FDA, Aerobic Biodegradation in Soil 6.3 to
may be modeled after Test Methods D5210 or E1196. The test 6.3.2.3
procedure and inoculum source, if not the naturally occurring OECD 304A 6.3 to
6.3.2.3
microbial community, should be reported with the test results. Anaerobic U.S. EPA Anaerobic Biodegradation 6.4
A high result indicates that the fluid will be biodegradable in ECETOC Evaluation of Anaerobic Biodegrada- 6.4
many natural anaerobic environments, such as swamps and tion
Test Method E1196 6.4
sediments and in manmade anaerobic environments, such as Test Method D5210 6.4
anaerobic waste water digesters. As with the other test proce-

4
 D6006 − 17
content of the fluid should be measured, not calculated. Test 7.8 Nonvolatile reference substances must be used in all test
Method D5291 or an equivalent procedure may be used. methods. The successful biodegradation of a substance known
to be biodegradable demonstrates the viability of the inoculum.
7.4 The hydraulic fluid should be added gravimetrically and
It is advisable to use a reference substance with water solubility
not volumetrically to the test medium. Volumetric measure-
characteristics and, if possible, viscosity similar to the test
ment of the hydraulic fluid will lead to large variability in the
substance. The suggested reference substance for a water-
added amount and thus large variability in test results. This
insoluble hydraulic fluid with low viscosity is low erucic acid
error will be prevalent particularly for hydraulic fluids with rapeseed (LEAR) oil, for example canola oil. The fatty acids in
high viscosity. Because of the small amount of fluid added to the low erucic acid rapeseed oil are a maximum of 2 % by
the test medium in these procedures, the gravimetric addition weight erucic acid. The reference substance for a water-soluble
may be done in two steps. In other words, the fluid may be hydraulic fluid may be glucose, phenol, or another reference
weighed into or onto a small container, and then the hydraulic substance that is cited in the standard tests referenced in 6.1.1.
fluid and the container itself added to the much larger test The reference substance and the test substance should be
medium container. dispersed in the test medium with the same procedures. The
7.5 Inocula for aerobic aquatic test methods may be ob- results of the reference substance should be reported with the
tained from a well-operated waste water treatment plant that results of the hydraulic fluid. The results obtained with the
receives predominantly domestic sewage. It also may be reference substance should not be used to generate a correction
obtained from surface waters or from soils after preparation as factor for interpretation of test results because the test sub-
may be outlined in an appropriate test method. It should be stance does not necessarily respond in a test procedure in the
used within 24 h either to initiate preadaptation of the culture same way as the reference substance.
or to start the test method itself. 7.9 In order to interpret tests that measure produced CO2,
7.6 The inoculum may be preadapted, which is expected to the ratio between the CO2 and the biomass produced by
decrease test to test variability. Preadaptation procedures can metabolism of the hydraulic fluid under the conditions of the
be found in the U.S. EPA Aerobic Aquatic Biodegradation Test test must be known or assumed. The Modified Sturm Test
(1) and Ref (13). (OECD 301B) referenced in 6.1 mentions 60 % of theoretical
CO2 in 28 days as being required for a passing result for a test
7.7 It may be advantageous to disperse poorly water soluble substance. The U.S. EPA Aerobic Aquatic Biodegradation Test
hydraulic fluids in addition to that dispersal that would be says that if the reference substance produces 60 % of theoreti-
achieved by the mixing prescribed in each test method. In the cal CO2 in 28 days, the inoculum is acceptable, and the ISO
event that additional dispersal of the hydraulic fluid is desired, test 9439:1990 requires that the reference reach 50 % of
physical means of dispersal are preferred. The applicability of theoretical CO2 in 14 days. An inference that could be drawn
various dispersal methods to hydraulic fluids is a matter still from these three test procedures is that good biodegradability
under investigation. Special care should be taken with the has been demonstrated if the test substance reaches the level set
dispersal of viscous fluids. The method of dispersal should be by the Modified Sturm Test, 60 % of theoretical CO2 in 28
reported with the test results. Examples of some dispersal days. The 60 % level assumes that the carbon mass ratio of
methods follow. CO2 to biomass produced during the biodegradation is greater
7.7.1 Sonication of the hydraulic fluid in water may create a than or equal to 60:40. If this ratio is exactly 60:40, a result of
dispersion or emulsion of very fine droplets. If this technique is 60 % theoretical CO2 means that all of the test substance has
used, the container holding the sonicated mixture during been biodegraded. If the ratio is 50:50, however, the test
sonication should be added to the preadaptation mixture or test substance will not pass even though it has completely
mixture. Addition of the container in which sonication is done biodegraded, and so will give a false negative. Conversely, if
to the test flask will ensure that all the hydraulic fluid is added the true ratio is 80:20, a result of 60 % means that the test
to the test flask. substance has not completely biodegraded, and in these time-
7.7.2 Coating of the hydraulic fluid onto glass beads, or limited tests, gives a false positive. The ratio of produced CO2
other fine inert substrate, will present a large surface area to the to biomass is under study (14,15,16).
inoculum. If this test method is used, the container holding the 7.9.1 The biodegradation tests published by OECD as tests
test material and the inert carrier should be added to the in the 301 series have as part of their pass criteria a requirement
mixture. commonly referred to as the 10-day window. This requirement
7.7.3 It may be acceptable to introduce a hydraulic fluid to states that the percent theoretical CO2 must reach 60 % within
the test flask in a volatile nonbiodegradable carrier solvent and 10 days after reaching the 10 % level. The OECD currently is
then allow the solvent to evaporate. In order for this procedure studying this requirement. It was based on data for water
to be acceptable, the blank system must be treated in exactly soluble substances, and there are questions as to its applicabil-
the same manner. It is advisable to conduct separate experi- ity to substances that are poorly soluble in water.
ments to determine that the carrier solvent does evaporate 7.10 At least duplicate test mixtures of all hydraulic fluids,
under the conditions used for this experiment. It also is reference substances and blanks, should be made and run.
advisable to have available data that demonstrates that the use Triplicates are preferred. The average of all replicates and their
of the carrier solvent does not affect the biodegradation of standard deviation are to be reported. In the case that one or
substances with known biodegradability. more replicates are excluded on statistical grounds as given in

5
 D6006 − 17
ISO 4259:1992(E), the excluded data and the reason for 8. Keywords
exclusion should be reported. More elaborate analysis of the 8.1 biodegradation; hydraulic fluids; persistence; water-
statistics of test results is part of test method development. insoluble

REFERENCES

(1) U.S. EPA Aerobic Aquatic Biodegradation Test, 40 CFR 796.3260 (8) Report No. FDA/CFSAN-87/30, Environmental Assessment Techni-
(also available as Federal Register, September 27, 1985, p. 39277, cal Assistance Handbook, “Aerobic Biodegradation in Soils,” U.S.
Section 796.3100 or in EPA publication 560/6-82-003, number FDA, March 1987.
CG-2000). Also see W. E. Gledhill, Appl. Microb., 30(b), 922–929, (9) Sharabi, N. E., and Bartha, R., Appl. Environ. Microbiol., 59(4), 1201
1975. (1993 ).
(2) Battersby, N. S., Pack, S. E., and Watkinson, R. J., CHEMOSPHERE, (10) “Anaerobic Biodegradability of Organic Chemicals,” U.S. EPA
“A Correlation Between the Biodegradability of Oil Products in the Chemical Fate Test Guidelines, EPA Report No. 560/6-82-003,
CEC L-33-T-82 and Modified Sturm Tests,” 24, 1989 (1992). August 1982, or CFR 40, paragraph 796.3140, p. 165, July 1, 1992
(3) Novick, N. J., Mehta, P. G., and McGoldrick, P. B., Fourth Interna- .
tional Symposium on the Performance Evaluation of Automotive (11) ECETOC Technical Report No. 28, Evaluation of Anaerobic
Fuels and Lubricants, “Assessment of the Biodegradability of Mineral Biodegradation, 1988.
Oil and Synthetic Ester Base Stocks, Using CO2 Ultimate Biodegrad- (12) “Toxic Substances Control Act (TSCA); Good Laboratory Practice
ability Tests and CEC-L-33-T-82,” Birmingham, UK, May 5–7, 1993. Standards, Final Rule,” U.S. Federal Register, 40 CFR Part 792,
(4) Battersby, N. S., Fieldwick, P. G., Ablitt, T., Lee, S. A., and Moys, G. August 17, 1989.
R., “The Interpretation of CEC L-33-T-82 Biodegradability Test (13) Sturm, R. N., J. Am. Oil Chemists Soc., Vol 50, 159–167, 1973.
Data,” Chemosphere, 28, 787–800 (1994). (14) OECD Test Guidelines Programme Periodical Review, Detailed
(5) “Biodegradability of Two-Stroke Cycle Outboard Engine Oils in Review Paper on Biodegradability Testing, H. A. Painter, OECD,
Water,” CEC L-33-A-94, Coordinating European Council, 1994. July 1992.
(6) OECD 306, “Biodegradability in Seawater,” OECD Guidelines for (15) Battersby, N. S., Girling, A. E., Stephenson, R. R., Watkinson, R. J.,
Testing of Chemicals, Organization for Economic Cooperation and Proc. 3rd CESIO International Surfactants Congress and Exhibition,
Development, Paris, France, 1993. June 1992.
(7) OECD 304A, “Inherent Biodegradability in Soil,” OECD Guide-lines (16) McGill, W. B., Rowell, M. J., and Westlake, D. W. S., Chap. 6 in
for Testing of Chemicals, Organization for Economic Cooperation and SOIL BIOCHEMISTRY, Ed. E. A. Paul, J. N. Ladd, Vol 4, Dekkar,
Development, Paris, France, 1993. 1981.

SUMMARY OF CHANGES

Subcommittee D02.12 has identified the location of selected changes to this standard since the last issue
(D6006 – 11) that may impact the use of this standard. (Approved Jan. 1, 2017.)

(1) Revised subsections 3.1.3 and 3.1.8.

ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned
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