ASTM D6139-22

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: D6139 − 18 D6139 − 22
Standard Test Method for
Determining the Aerobic Aquatic Biodegradation of
Lubricants or Their Components Using the Gledhill Shake
Flask
This standard is issued under the fixed designation D6139; 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 the determination of the degree of aerobic aquatic biodegradation of fully formulated lubricants or
their components on exposure to an inoculum under controlled laboratory conditions. This test method is an ultimate
biodegradation test that measures carbon dioxide (CO ) evolution.
1.2 This test method is intended to specifically address the difficulties associated with testing water insoluble materials and
complex mixtures such as are found in many lubricants.
1.3 This test method is designed to be applicable to all non-volatile lubricants or lubricant components that are not toxic and not
inhibitory at the test concentration to the organisms present in the inoculum.
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 discussed 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:
D1193 Specification for Reagent Water
D1293 Test Methods for pH of Water
D4447 Guide for Disposal of Laboratory Chemicals and Samples
D5291 Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and Lubricants
D5864 Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components
E943 Terminology Relating to Biological Effects and Environmental Fate
This test method 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 June 1, 2018April 1, 2022. Published June 2018April 2022. Originally approved in 1997. Last previous edition approved in 20172018 as
D6139 – 17.D6139 – 18. DOI: 10.1520/D6139-18.10.1520/D6139-22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*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
D6139 − 22
2.2 ISO Standard:
4259:1992(E) Petroleum Products—Determination and application of precision data in relation to methods of test
2.3 APHA Standards:
2540B Total Solids Dried at 103–105°C
9215 Heterotrophic Plate Count
3. Terminology
3.1 Definitions:
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
Methods from Standard Methods for the Examination of Water and Wastewater, latest edition. Available from the American Public Health Association (APHA), 800 I
Street, NW, Washington, DC 20001.
D6139 − 22
3.1.1 Definitions of terms applicable to this test method that are not described herein appear in the ASTM Online Dictionary of
Engineering Science and Technology or Terminology E943.
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—(1) taking place in the presence of oxygen; (2) living or active in the presence 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 material.
3.1.7 inoculum, n—spores, bacteria, single celled organisms, or other live materials, that are introduced into a test medium.
3.1.8 lag phase, n—the period of diminished physiological activity and cell division following the addition of microorganisms to
a new culture medium.
3.1.9 log phase, n—the period of growth of microorganisms during which cells divide at a positive constant rate.
3.1.10 mixed liquor, n—in sewage treatment, the contents of an aeration tank including the activated sludge mixed with primary
effluent or the raw wastewater and return sludge.
3.1.11 pre-adaptation, n—the pre-incubation of an inoculum in the presence of the test material under conditions similar to the
test conditions.
3.1.11.1 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 a more rapid rate of biodegradation of the test material but not to a change in the final degree of
biodegradation.
3.1.12 pre-condition, n—the pre-incubation of an inoculum under the conditions of the test in the absence of the test material.
3.1.13 supernatant, n—the liquid above settled solids.
3.1.14 suspended solids (of activated sludge or other inoculum samples), n—solids present in activated sludge or inoculum
samples that are not removed by settling under specified conditions.
3.1.15 theoretical CO , n—the amount of CO which could in theory be produced from the complete oxidation of all of the carbon
2 2
D6139 − 22
in a material.
3.1.16 ultimate biodegradation, n—degradation achieved when a material is totally utilized by microorganisms resulting in the
production of CO (and possibly methane in the case of anaerobic biodegradation), water, inorganic compounds, and new
microbial cellular constituents (biomass or secretions, or both).
3.2 Definitions of terms applicable to this test method that are not described herein appear in the ASTM Online Dictionary of
Engineering Science and Technology or Terminology E943.
3.3 activated sludge, n—the precipitated solid matter, consisting mainly of bacteria and other aquatic microorganisms, that is
produced at a domestic wastewater treatment plant; activated sludge is used primarily in secondary sewage treatment to microbially
oxidize dissolved organic matter in the effluent.
3.4 aerobic, adj—(1) taking place in the presence of oxygen; (2) living or active in the presence of oxygen.
3.5 biodegradation, n—the process of chemical breakdown or transformation of a material caused by organisms or their enzymes.
3.5.1 Discussion—
Biodegradation is only one mechanism by which materials are transformed in the environment.
3.6 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.
3.6.1 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.).
3.6.2 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).
3.7 blank, n—in biodegradability testing, a test system containing all system components with the exception of the test material.
3.8 inoculum, n—spores, bacteria, single celled organisms, or other live materials, that are introduced into a test medium.
3.9 lag phase, n—the period of diminished physiological activity and cell division following the addition of microorganisms to
a new culture medium.
3.10 log phase, n—the period of growth of microorganisms during which cells divide at a positive constant rate.
3.11 mixed liquor, n—in sewage treatment, the contents of an aeration tank including the activated sludge mixed with primary
effluent or the raw wastewater and return sludge.
3.12 pre-adaptation, n—the pre-incubation of an inoculum in the presence of the test material under conditions similar to the test
conditions.
3.12.1 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 a more rapid rate of biodegradation of the test material but not to a change in the final degree of
biodegradation.
3.13 pre-condition, n—the pre-incubation of an inoculum under the conditions of the test in the absence of the test material.
ASTM Online Dictionary of Engineering Science and Technology (Stock#DEFONLINE) is available on the ASTM website, www.astm.org, or contact ASTM Customer
Service at service@astm.org.
D6139 − 22
3.14 supernatant, n—the liquid above settled solids.
3.15 suspended solids (of activated sludge or other inoculum samples), n—solids present in activated sludge or inoculum samples
that are not removed by settling under specified conditions.
3.16 theoretical CO , n—the amount of CO which could in theory be produced from the complete oxidation of all of the carbon
2 2
in a material.
3.17 ultimate biodegradation, n—degradation achieved when a material is totally utilized by microorganisms resulting in the
production of CO (and possibly methane in the case of anaerobic biodegradation), water, inorganic compounds, and new
microbial cellular constituents (biomass and secretions).
4. Summary of Test Method
4.1 Biodegradation of a lubricant or the component(s) of a lubricant is estimated by collecting and measuring the CO produced
when the lubricant or component is exposed to microorganisms under controlled aerobic aquatic conditions. This value is then
compared to the theoretical amount of CO which could be generated if all of the carbon in the test material were converted to
CO . Carbon dioxide is a product of aerobic microbial metabolism of carbon-containing materials and so is a direct measure of
the test material’s ultimate biodegradation. The evolved CO is trapped in a Ba(OH) or other alkaline solution and the amount
2 2
of CO absorbed is determined by titrating the remaining hydroxide in solution.
4.2 The carbon content of the test material is determined by Test Methods D5291 or another appropriate method and the theoretical
CO is calculated from that measurement. It is necessary to directly measure the carbon content of the test material instead of
calculating this number, because of the complexity of the mixture of compounds present in lubricants.
4.3 Biodegradability is expressed as a percentage of theoretical CO production.
5. Significance and Use
5.1 Results from this CO evolution test method suggest, within the confines of a controlled laboratory setting, the degree of
ultimate aerobic aquatic biodegradability of a lubricant or components of a lubricant. Test materials which achieve a high degree
of biodegradation in this test method may be assumed to easily biodegrade in many aerobic aquatic environments. (See also Test
Method D5864.)
5.2 Because of the stringency of this test method, a low yield of CO does not necessarily mean that the test material is not
biodegradable under environmental conditions, but indicates that further testing needs to be carried out in order to establish
biodegradability.
5.3 Information on the toxicity of the test material to the inoculum may be useful in the interpretation of low biodegradation
results.
5.4 Activated sewage-sludge from a sewage treatment plant that principally treats domestic waste may be used 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.
NOTE 1—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.
5.5 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 method must be regarded as invalid and should be repeated using a fresh inoculum if the
reference does not demonstrate biodegradation to the extent of >60 % of the theoretical CO within 28 days.
5.6 The water solubility or dispersibility of the lubricant or components may influence the results obtained and hence the
procedure may be limited to comparing lubricants or components with similar solubilities.
D6139 − 22
FIG. 1 NaOH Scrubber – Flask Trap Assembly for Providing CO -Free Air
5.7 The ratio of carbon incorporated into cellular material to carbon metabolized to CO will vary depending on the organic
substrate, on the particular microorganisms carrying out the conversion, and on the environmental conditions under which the
conversion takes place. In principle, this variability complicates the interpretation of the results from this test method.
5.8 The behavior of complex mixtures may not always be consistent with the individual properties of the components. The
biodegradability of the components may be suggestive of whether a mixture containing these components (that is, a fully
formulated lubricant) is biodegradable but such information should be used judiciously.
6. Apparatus
6.1 Carbon Dioxide Scrubbing Apparatus (see Fig. 1):
6.1.1 The following are required to produce a stream of CO -free air for aeration and for sparging aqueous solutions and mixtures
(for example, test medium, sewage inoculum):
6.1.1.1 Erlenmeyer flask, one 1 L with side arm containing 500 mL of 10 M sodium hydroxide (NaOH), and fitted with a rubber
stopper and an inlet tube that extends below the level of the NaOH solution or an equivalent apparatus or system.
6.1.1.2 Erlenmeyer flask, one 1 L with side arm containing 500 mL of distilled water and fitted with a stopper and inlet tube, or
an equivalent apparatus or system.
6.1.1.3 It is optional to add an empty 1 L Erlenmeyer flask in series with the flasks to prevent liquid carryover.
6.1.1.4 It is optional to add a 1 L Erlenmeyer flask containing 500 mL of 0.1 M barium hydroxide [Ba(OH) ] solution to monitor
for possible breakthrough CO .
6.1.2 Connect the flasks in series as shown in Fig. 1, using vinyl or other suitable non-gas-permeable tubing, to a pressurized air
system and purge air through the scrubbing solution.
6.1.3 The CO scrubbing apparatus upstream of the Erlenmeyer flask containing the Ba(OH) may be substituted with an
2 2
alternative system which effectively and consistently produces CO -free air (that is, containing <1 ppm CO ).
2 2
6.2 Incubation/Biodegradation Apparatus—Gledhill-type Shake Flask Units (see Fig. 2)—Each test material, reference, or blank
control requires the following:
6.2.1 Erlenmeyer Flasks, 2 L—2 L Erlenmeyer flasks are used to hold the 1 L of total final aqueous volume but larger volume
Erlenmeyer flasks (as large as 3 L to 4 L) may be used if 2 L to 3 L final aqueous volumes are required. The amounts described
here are for 1 L final aqueous volumes carried out in 2 L Erlenmeyer flasks; scale procedure accordingly if larger final aqueous
volumes and larger Erlenmeyer flasks are necessary.
Gledhill, W. E., “Screening Test for Assessment of Ultimate Biodegradability: Linear Alkyl Benzene Sulfonate,” Applied Microbiology Vol 30, 1975, pp. 992–929. Also
see description of Gledhill shake flask unit in EPA Chemical Fate Testing Guidelines for Aerobic Aquatic Biodegradation, EPA Publication 560/6-82-003, No. CG-2000
(August 1982); Federal Register, September 27, 1985, p. 39277, Section 796.3100; 40 CFR 796.3100, 1994.
D6139 − 22
FIG. 2 Gledhill Shake Flask System for CO Evolution
6.2.2 Stoppers—Each stopper is fitted with a conical alkaline trap, an outlet and an inlet vent tube (see Fig. 2). Ensure that the
stopper fits tightly in the Erlenmeyer flask to prevent any leaks.
6.2.3 Conical Alkaline Trap Tube or Unit—Glass, 40 mL conical tube (borosilicate glass, No. 8120 centrifuge tube or equivalent)
welded to a glass support rod, or an equivalent apparatus, will be used to hold the Ba(OH) solution for trapping the evolved CO
2 2
from aerobic biodegradation. The opening in the alkaline trap tube is large enough to permit CO diffusion into the barium
hydroxide solution. The support rod of the conical trap shall fit tightly in the stopper.
6.2.4 Inlet and Outlet Vent Tubes—The inlet vent tube attached to the stopper extends down into the flask so that it will be
immersed below the surface of the aqueous medium and will be used for sparging. The outlet vent tube will be situated significantly
above the level of the aqueous medium and will be used for venting. The two vent tubes shall fit tightly in the stopper.
6.2.5 Flexible tubing which is non-permeable to CO will be used to connect the tops of inlet and outlet vent tubes to form a closed
system.
6.2.6 Agitators—Incubator-shaker table unit or equivalent, or stirrers may be used to agitate the aqueous mixture in the
Erlenmeyer flasks.
6.3 Analytical Balance, to weigh out test material or reference material to be added to the test flask (capable of weighing to
appropriate precision and accuracy, for example, 60.0001 g).
6.4 Titration Apparatus for Measuring the Production of CO :
6.4.1 Appropriate graduated burette filled with standard HCl solution.
6.4.2 Alternatively, an automatic titration apparatus in which the burette dispenser is filled with standard HCl solution. Automatic
titrations are carried out to a potentiometric end point of pH 8.3 (that is, phenolphthalein end point equivalent)
6.5 Glass Wool, for filtering the inoculum.
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 lessening 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.
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, D.C.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., Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial Convention, Inc.
(USPC), Rockville, MD.
D6139 − 22
7.3 Prepare the following stock solutions:
7.3.1 Ammonium Sulfate Solution (40 g ⁄L)—Dissolve 40.0 g ammonium sulfate [(NH ) SO ] in water and dilute to 1 L.
4 2 4
7.3.2 Calcium Chloride Solution (27.5 g ⁄L)—Dissolve 27.5 g anhydrous calcium chloride (CaCl ) in water and dilute to 1 L.
7.3.3 Ferric Chloride Solution (0.25 g ⁄L)—Dissolve 0.25 g ferric chloride hexahydrate (FeCl ·6 H O) in water and dilute to 1 L.
3 2
7.3.4 Magnesium Sulfate Solution (22.5 g ⁄L)—Dissolve 22.5 g magnesium sulfate heptahydrate (MgSO ·7 H O) in water and
4 2
dilute to 1 L.
7.3.5 Phosphate Buffer—Dissolve 8.5 g potassium dihydrogen phosphate (KH PO ), 21.7 g potassium monohydrogen phosphate
2 4
(K HPO ), 50.3 g sodium monohydrogen phosphate heptahydrate (Na HPO ·7 H O) [or alternatively, 33.4 g of sodium
2 4 2 4 2
monohydrogen phosphate dihydrate (Na HPO ·2 H·2 H O), the dihydrate equivalent form], and 1.7 g ammonium chloride
2 4 2
(NH Cl) in water and dilute to 1 L.
7.3.6 Trace Elements Solution—Dissolve 0.035 g manganous chloride tetrahydrate (MnCl ·4 H O), 0.057 g boric acid (H BO ),
2 2 3 3
0.043 g zinc sulfate heptahydrate (ZnSO ·7 H O), and 0.037 g ammonium molybdate tetrahydrate [(NH ) Mo O ·4 H O] in water
4 2 4 6 7 24 2
and dilute to 1 L.
7.4 Barium Hydroxide Solution, 0.1 M, is prepared by dissolving 32.0 g barium hydroxide octahydrate [Ba(OH) ·8H O] in
2 2
distilled water and diluting to 1 L. Filter free of solid material, confirm molarity by titration with standard acid, and store under
nitrogen sealed as a clear solution to prevent absorption of CO from the air. It is recommended that 2 L be prepared at a time when
running a series of tests.
7.5 Vitamin-free Casamino Acids.
7.6 Yeast Extract.
7.7 Phenolphthalein.
7.8 Standardized Hydrochloric Acid (0.190 M to 0.210 M).
8. Inoculum Test Organisms
8.1 Sources
...