ASTM D6139-00
(Test Method)Standard Test Method for Determining the Aerobic Aquatic Biodegradation of Lubricants or Their Components Using the Gledhill Shake Flask
Standard Test Method for Determining the Aerobic Aquatic Biodegradation of Lubricants or Their Components Using the Gledhill Shake Flask
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 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 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 health practices and determine the applicability of regulatory limitations prior to use. Specific hazards are discussed in Section 10.
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An American National Standard
Designation:D6139–00
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 D 6139; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope E 943 Terminology Relating to Biological Effects and En-
vironmental Fate
1.1 This test method covers the determination of the degree
2.2 ISO Standard:
of aerobic aquatic biodegradation of fully formulated lubri-
4259:1992(E) Petroleum Products—Determination and ap-
cants or their components on exposure to an inoculum under
plication of precision data in relation to methods of test
controlled laboratory conditions. This test method is an ulti-
2.3 APHA Standards:
mate biodegradation test that measures carbon dioxide (CO )
2540B Total Solids Dried at 103–105°C
evolution.
9215 Heterotrophic Plate Count
1.2 This test method is intended to specifically address the
difficulties associated with testing water insoluble materials
3. Terminology
and complex mixtures such as are found in many lubricants.
3.1 Definitions:
1.3 This test method is designed to be applicable to all non
3.1.1 Definitions of terms applicable to this test method
volatile lubricants or lubricant components that are not toxic
which are not described herein, appear in the Compilation of
and not inhibitory at the test concentration to the organisms
ASTM Standard Definitions (1990) or Terminology E 943.
present in the inoculum.
3.1.2 activated sludge, n—the precipitated solid matter,
1.4 This standard does not purport to address all the safety
consisting mainly of bacteria and other aquatic microorgan-
concerns, if any, associated with its use. It is the responsibility
isms, that is produced at a domestic wastewater treatment
of the user of this standard to establish appropriate safety and
plant; activated sludge is used primarily in secondary sewage
health practices and to determine the applicability of regula-
treatmenttomicrobiallyoxidizedissolvedorganicmatterinthe
tory limitations prior to use. Specific hazards are discussed in
effluent.
Section 10.
3.1.3 aerobic, adj.—(1) taking place in the presence of
2. Referenced Documents oxygen; ( 2) living or active in the presence of oxygen.
3.1.4 biodegradation, n—the process of chemical break-
2.1 ASTM Standards:
2 down or transformation of a test material caused by organisms
D 1129 Terminology Relating to Water
or their enzymes.
D 1193 Specification for Reagent Water
2 3.1.4.1 Discussion—Biodegradation is only one mechanism
D 1293 Test Methods for pH of Water
by which substances are removed from the environment.
D 4447 Guide for Disposal of Laboratory Chemicals and
3 3.1.5 biomass, n—any material, excluding fossil fuels,
Samples
which is or was a living organism or component of a living
D 5291 Test Methods for Instrumental Determination of
organism.
Carbon, Hydrogen, and Nitrogen in Petroleum Products
3.1.6 blank, n—in biodegradability testing, a test system
and Lubricants
containingallsystemcomponentswiththeexceptionofthetest
D 5864 Test Method for DeterminingAerobicAquatic Bio-
material.
degradation of Lubricants or Their Components
3.1.7 inoculum, n—spores, bacteria, single celled organ-
isms, or other live materials, that are introduced into a test
medium.
This test method is under the jurisdiction of ASTM Committee D02 on
PetroleumProductsandLubricants andisthedirectresponsibilityofSubcommittee
D02.12 on Environmental Standards for Lubricants.
Current edition approved April 10, 2000. Published July 2000. Originally
published as D 6139 - 97. Last previous edition D 6139 - 97. Available from American National Standards Institute, 11 West 42nd St., 13th
Annual Book of ASTM Standards, Vol. 11.01. Floor, New York, NY 10036
3 7
Annual Book of ASTM Standards, Vol. 11.04. Methods from Standard Methods for the Examination of Water and Wastewa-
Annual Book of ASTM Standards, Vol. 05.02. ter, latest edition.Available from theAmerican Public HealthAssoc. (APHA), 1015
Annual Book of ASTM Standards, Vol. 11.05. 18th St., N.W., Washington, D.C. 20036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6139–00
3.1.8 lag phase, n—the period of diminished physiological 4.2 The carbon content of the test material is determined by
activity and cell division following the addition of microorgan- Test Method D 5291 or another appropriate method and the
isms to a new culture medium. theoretical CO is calculated from that measurement. It is
3.1.9 log phase, n—the period of growth of microorganisms necessary to directly measure the carbon content of the test
during which cells divide at a positive constant rate. material instead of calculating this number, because of the
3.1.10 mixed liquor, n—insewagetreatment,thecontentsof complexity of the mixture of compounds present in lubricants.
an aeration tank including the activated sludge mixed with 4.3 Biodegradability is expressed as a percentage of theo-
primary effluent or the raw wastewater and return sludge. retical CO production.
3.1.11 pre-adaptation, n—the incubation of an inoculum in
5. Significance and Use
the presence of the test material which is done prior to the
initiation of the test and under conditions similar to the test
5.1 Results from this CO evolution test method suggest,
conditions.
within the confines of a controlled laboratory setting, the
3.1.11.1 Discussion—The aim of pre adaptation is to im-
degree of ultimate aerobic aquatic biodegradability of a lubri-
prove the precision of the test method by decreasing variability
cantorcomponentsofalubricant.Testmaterialswhichachieve
in the rate of biodegradation produced by the inoculum. Pre
a high degree of biodegradation in this test method may be
adaptation may mimic the natural processes which cause
assumed to easily biodegrade in many aerobic aquatic envi-
changes in the microbial population of the inoculum leading to
ronments.
a more rapid rate of biodegradation of the test material but is
5.2 Because of the stringency of this test method, a low
not expected to change the overall extent of biodegradation of
yield of CO does not necessarily mean that the test material is
the test material.
not biodegradable under environmental conditions, but indi-
3.1.12 pre-condition, n—the pre-incubation of an inoculum
cates that further testing needs to be carried out in order to
under the conditions of the test in the absence of the test
establish biodegradability.
material.
5.3 Information on the toxicity of the test material to the
3.1.13 supernatant, n—the liquid above settled solids.
inoculum may be useful in the interpretation of low biodegra-
3.1.14 suspended solids (of activated sludge or other inocu-
dation results.
lum samples), n—solids present in activated sludge or inocu-
5.4 Activated sewage-sludge from a sewage treatment plant
lum samples that are not removed by settling under specified
that principally treats domestic waste may be used as an
conditions.
aerobic inoculum. An inoculum derived from soil or natural
3.1.15 theoretical carbon dioxide (ThCO ), n—the amount
2 surface waters, or any combination of the three sources, may
of CO which could theoretically be produced from the
2 also be used in this test method.
complete biological oxidation of all of the carbon in a test
NOTE 1—Allowance for various and multiple inoculum sources pro-
material.
vides access to a greater diversity of biochemical competency and
3.1.16 ultimate biodegradation, n—degradation achieved
potentially represents more accurately the capacity for biodegradation.
when the test material is totally utilized by microorganisms
5.5 A reference or control material known to biodegrade
resulting in the production of CO , (and possibly methane in
under the conditions of this test method is necessary in order to
the case of anaerobic biodegradation), water, inorganic com-
verify the activity of the inoculum. The test method must be
pounds, and new microbial cellular constituents (biomass and
regarded as invalid and should be repeated using a fresh
secretions).
inoculum if the reference does not demonstrate biodegradation
3.1.17 ultimate biodegradation test, n—a test which esti-
to the extent of >60 % of the theoretical CO within 28 days.
mates the extent to which the carbon in a product has been
5.6 The water solubility or dispersibility of the lubricant or
converted to CO or methane, either directly by measuring the
components may influence the results obtained and hence the
production of CO or methane, or indirectly by measuring the
procedure may be limited to comparing lubricants or compo-
consumption of O .
nents with similar solubilities.
3.1.17.1 Discussion—The measurement of new biomass is
5.7 The ratio of carbon incorporated into cellular material to
not attempted.
carbon metabolized to CO will vary depending on the organic
4. Summary of Test Method
substrate, on the particular microorganisms carrying out the
conversion, and on the environmental conditions under which
4.1 Biodegradation of a lubricant or the component(s) of a
the conversion takes place. In principle, this variability com-
lubricant is estimated by collecting and measuring the CO
plicates the interpretation of the results from this test method.
produced when the lubricant or component is exposed to
5.8 The behavior of complex mixtures may not always be
microorganisms under controlled aerobic aquatic conditions.
consistent with the individual properties of the components.
This value is then compared to the theoretical amount of CO
The biodegradability of the components may be suggestive of
whichcouldbegeneratedifallofthecarboninthetestmaterial
whether a mixture containing these components (that is, a fully
were converted to CO . Carbon dioxide is a product of aerobic
formulated lubricant) is biodegradable but such information
microbial metabolism of carbon-containing materials and so is
should be used judiciously.
a direct measure of the test material’s ultimate biodegradation.
The evolved CO is trapped in a Ba(OH) or other alkaline
2 2
6. Apparatus
solution and the amount of CO absorbed is determined by
titrating the remaining hydroxide in solution. 6.1 Carbon Dioxide Scrubbing Apparatus (see Fig. 1):
D6139–00
FIG. 1 NaOH Scrubber – Flask Trap Assembly for Providing CO -Free Air
6.1.1 The following are required to produce a stream of equivalent) welded to a glass support rod, or an equivalent
CO -free air for aeration and for sparging aqueous solutions apparatus, will be used to hold the Ba(OH) solution for
2 2
and mixtures (for example, test medium, sewage inoculum): trapping the evolved CO from aerobic biodegradation. The
6.1.1.1 Erlenmeyer flask, One 1-Lwith side arm containing opening in the alkaline trap tube is large enough to permit CO
500 mL of 10 M sodium hydroxide (NaOH), and fitted with a diffusion into the barium hydroxide solution. The support rod
rubber stopper and an inlet tube that extends below the level of of the conical trap shall fit tightly in the stopper.
the NaOH solution or an equivalent apparatus or system. 6.2.4 Inlet and Outlet Vent Tubes—The inlet vent tube
6.1.1.2 Erlenmeyer flask, One 1-L with side arm containing attached to the stopper extends down into the flask so that it
500 mL of distilled water and fitted with a stopper and inlet will be immersed below the surface of the aqueous medium
tube, or an equivalent apparatus or system. and will be used for sparging. The outlet vent tube will be
6.1.1.3 It is optional to add an empty 1-L Erlenmeyer flask situated significantly above the level of the aqueous medium
in series with the flasks to prevent liquid carryover. andwillbeusedforventing.Thetwoventtubesshallfittightly
6.1.1.4 It is optional to add a 1-L Erlenmeyer flask contain- in the stopper.
ing 500 mL of 0.1M barium hydroxide [Ba(OH) ] solution to 6.2.5 Flexible tubing which is non-permeable to CO will
monitor for possible breakthrough CO . be used to connect the tops of inlet and outlet vent tubes to
6.1.2 Connect the flasks in series as shown in Fig. 1, using form a closed system.
vinyl or other suitable non -gas-permeable tubing, to a pres-
6.2.6 Agitators—Incubator-shaker table unit or equivalent,
surized air system and purge air through the scrubbing solu- or stirrers may be used to agitate the aqueous mixture in the
tion.
Erlenmeyer flasks.
6.1.3 The CO scrubbing apparatus upstream of the Erlen- 6.3 Analytical Balance, to weigh out test material or refer-
meyerflaskcontainingtheBa(OH) maybesubstitutedwithan
ence material to be added to the test flask (capable of weighing
alternative system which effectively and consistently produces toappropriateprecisionandaccuracy,forexample, 60.0001g)
CO -free air (that is, containing <1 ppm CO ).
6.4 Titration Apparatus for Measuring the Production of
2 2
6.2 Incubation/Biodegradation Apparatus – Gledhill-type CO :
Shake Flask Units (see Fig. 2)—Each test material, reference,
6.4.1 Appropriate graduated burette filled with standard
or blank control requires the following: HCl solution.
6.2.1 Erlenmeyer Flasks, 2-L—2-L Erlenmeyer flasks are
6.4.2 Alternatively, an automatic titration apparatus in
used to hold the 1 L of total final aqueous volume but larger which the burette dispenser is filled with standard HCl solu-
volume Erlenmeyer flasks (as large as 3 to 4-L) may be used if
tion. Automatic titrations are carried out to a potentiometric
2 to 3-L final aqueous volumes are required. The amounts end point of pH 8.3 (that is, phenolphthalein end point
described here are for 1-Lfinal aqueous volumes carried out in
equivalent)
2-L Erlenmeyer flasks; scale procedure accordingly if larger 6.5 Glass Wool, for filtering the inoculum.
final aqueous volumes and larger Erlenmeyer flasks are neces-
sary.
7. Reagents and Materials
6.2.2 Stoppers—Each stopper is fitted with a conical alka-
7.1 Purity of Reagents—Reagent grade chemicals shall be
line trap, an outlet and an inlet vent tube (see Fig. 2). Ensure
used in all tests. Unless otherwise indicated, it is intended that
that the stopper fits tightly in the Erlenmeyer flask to prevent
all reagents conform to the specifications of the Committee on
any leaks.
Analytical Reagents of the American Chemical Society where
6.2.3 Conical Alkaline Trap Tube or Unit—Glass, 40- mL
such specifications are available. Other grades ma
...
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