ASTM D6731-01(2005)
(Test Method)Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer
Standard Test Method for Determining the Aerobic, Aquatic Biodegradability of Lubricants or Lubricant Components in a Closed Respirometer
SCOPE
1.1 This test method covers a procedure for determining the degree of biodegradability of lubricants or their components in an aerobic aqueous medium on exposure to an inoculum under controlled laboratory conditions. This test method is an ultimate biodegradation test that measures oxygen demand in a closed respirometer.
1.2 This test method is suitable for evaluating the biodegradation of volatile as well as nonvolatile lubricants or lubricant components.
1.3 This test method is applicable to lubricants and lubricant components which are not toxic and not inhibitory to the test microorganisms at the test concentration.
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
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 given in Section .
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Designation:D6731–01 (Reapproved 2005)
Standard Test Method for
Determining the Aerobic, Aquatic Biodegradability of
Lubricants or Lubricant Components in a Closed
Respirometer
This standard is issued under the fixed designation D6731; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope D6384 Terminology Relating to Biodegradability and Eco-
toxicity of Lubricants
1.1 This test method covers a procedure for determining the
E943 Terminology Relating to Biological Effects and Envi-
degree of biodegradability of lubricants or their components in
ronmental Fate
an aerobic aqueous medium on exposure to an inoculum under
2.2 ISO Standards:
controlled laboratory conditions. This test method is an ulti-
ISO 4259:1992(E) Petroleum Products–Determination and
mate biodegradation test that measures oxygen demand in a
Application of Precision Data in Relation to Methods of
closed respirometer.
Test
1.2 This test method is suitable for evaluating the biodeg-
ISO 6107-2:1997 Water Quality–Vocabulary–Part 2
radation of volatile as well as nonvolatile lubricants or lubri-
ISO 8192:1986 Water Quality–Test for Inhibition of Oxy-
cant components.
gen Consumption by Activated Sludge
1.3 Thistestmethodisapplicabletolubricantsandlubricant
ISO 9408:1999 Water Quality–Evaluation of Ultimate
components which are not toxic and not inhibitory to the test
Aerobic Biodegradability of Organic Compounds in
microorganisms at the test concentration.
Aqueous Medium by Determination of Oxygen Demand
1.4 The values stated in SI units are to be regarded as
in a Closed Respirometer
standard. No other units of measurement are included in this
2.3 OECD Standards:
standard.
OECD 301F:1992 Ready Biodegradability-Manometric Re-
1.5 This standard does not purport to address all of the
spirometry
safety concerns, if any, associated with its use. It is the
2.4 APHA Standards:
responsibility of the user of this standard to establish appro-
2540B Total Solids Dried at 103-105°C
priate safety and health practices and determine the applica-
9215 Heterotrophic Plate Count
bility of regulatory limitations prior to use.Specifichazardsare
given in Section 10.
3. Terminology
2. Referenced Documents 3.1 Definitions:
3.1.1 Definitions of terms applicable to this test method
2.1 ASTM Standards:
appear in the Compilation of ASTM Standard Definitions and
D1129 Terminology Relating to Water
the following terminology standards: D1129, D4175, D6384,
D1193 Specification for Reagent Water
E943, and ISO 6107-2:1997.
D1293 Test Methods for pH of Water
3.1.2 activated sludge, n—the precipitated solid matter,
D4175 Terminology Relating to Petroleum, Petroleum
consisting mainly of bacteria and other aquatic microorgan-
Products, and Lubricants
isms, that is produced at a domestic wastewater treatment plant
D4447 Guide for Disposal of Laboratory Chemicals and
and is used primarily in secondary sewage treatment to
Samples
microbially oxidize dissolved organic matter in the effluent.
This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products and Lubricants and is the direct responsibility of Subcommittee
D02.12 on Environmental Standards for Lubricants. Available from American National Standards Institute, 25 W. 43rd St., 4th
Current edition approved Nov. 1, 2005. Published November 2005. Originally Floor, New York, NY 10036.
approved in 2001. Last previous edition approved in 2001 as D6731–01. DOI: Available from the Head of Publications Service, Organization for Economic
10.1520/D6731-01R05. CooperationandDevelopment(OECD),2,rueAndre-Pascal,75775ParisCedex16,
For referenced ASTM standards, visit the ASTM website, www.astm.org, or France.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM From Standard Methods for the Examination of Water and Wastewater, latest
Standards volume information, refer to the standard’s Document Summary page on edition. Available from the American Public Health Assoc., 1015 18th St., NW,
the ASTM website. Washington, DC 20036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6731–01 (2005)
3.1.3 aerobic, adj—(a) taking place in the presence of alongside the test material to confirm that the inoculum is
oxygen; (b) living or active in the presence of oxygen. viableandcapableofbiodegradingsuitablematerialsunderthe
3.1.4 biochemical oxygen demand (BOD), n—the mass test conditions. The test material or reference material concen-
concentration of dissolved oxygen consumed under specified tration is normally 50 to 100 mg/L, providing a theoretical
conditions by the biological oxidation of organic or inorganic oxygen demand of at least 50 mg O /L but no more than 200
matter, or both. mg O /L. The ThO of the test and reference materials will be
2 2
3.1.4.1 Discussion—BODdeterminationisperformedusing determined from measured elemental compositional analysis
empirical tests employing standardized laboratory procedures. and will be calculated as in 13.1.
These tests measure oxygen utilization during a specified 4.3 The inoculated medium is stirred in a closed flask and
incubation period for the biochemical degradation of organic the consumption of oxygen is determined either by measuring
material (carbonaceous demand) in water. the amount of oxygen required to maintain a constant gas
3.1.5 biodegradation, n—the process of chemical break- volume in the respirometer flask, or by measuring the change
down or transformation of a test material caused by microor- in volume or pressure (or a combination of the two) in the
ganisms or their enzymes. apparatus.
3.1.5.1 Discussion—Biodegradation is only one mechanism 4.4 EvolvedCO (carbondioxide)isabsorbedinanalkaline
by which materials are removed, transformed, or both, in the trap solution (for example, 10 M NaOH or KOH) or other
environment. CO -absorbing system suspended within the test vessel, typi-
3.1.6 lag phase, n—the period of diminished physiological cally in the headspace of the test vessel.
activity and cell division following the addition of microorgan- 4.5 Biodegradation is followed over a specified period by
isms to a new culture medium. determining the consumption of oxygen. The amount of
3.1.7 log phase, n—the period of growth of microorganisms oxygen utilized in oxidation of the test and reference material
during which cells divide at a positive constant rate. is corrected for oxygen uptake by the inoculum in the blank
3.1.8 pre-adaptation, n—the incubation of an inoculum in controls and is expressed as a percentage of the theoretical
the presence of the test material which is done prior to the oxygen demand (ThO ) calculated from the empirical formula
initiation of the test and under conditions similar to the test of the material. Evaluation of the biodegradability of the test
conditions. material is made on the basis of these data. Normally the test
3.1.8.1 Discussion—The aim of pre-adaptation is to im- duration is 28 days; however, the test may be terminated if
prove the precision of the test method by decreasing variability oxygen consumption has plateaued. The test may be extended
in the rate of biodegradation produced by the inoculum. as long as the systems’ integrity is maintained and the
Pre-adaptation may mimic the natural processes which cause inoculum in the blank systems is viable. The duration of the
changes in the microbial population of the inoculum leading to testwillbedependentonthelengthoftimerequiredfortherate
more rapid biodegradation of the test material but is not of test material biodegradation to achieve a plateau. A graphi-
expected to change the overall extent of biodegradation of the cal illustration of the test results for a biodegradable material is
test material. presented in Fig. 1.
3.1.9 pre-condition, n—the pre-incubation of an inoculum
5. Significance and Use
under the conditions of the test in the absence of the test
5.1 Results from this test method suggest the degree of
material.
aerobic, aquatic biodegradation of a lubricant or lubricant
3.1.10 sludge, n—a water-formed sedimentary deposit.
component. The rate and extent of oxygen consumption is
3.1.11 suspended solids (of an activated sludge or other
measured upon exposure of the test material to an inoculum
inoculum samples), n—solids present in activated sludge or
within the confines of a controlled laboratory setting. Test
other inoculum samples that are not removed by settling under
materialswhichachieveahighdegreeofbiodegradationinthis
specified conditions.
test may be assumed to easily biodegrade in many aerobic
4. Summary of Test Method
aquatic environments.
4.1 Biodegradation of a lubricant or the component(s) of a 5.2 Because of the stringency of this test method, low
lubricant is determined by measuring the oxygen consumed results do not necessarily mean that the test material is not
whenthelubricantorcomponentisexposedtomicroorganisms biodegradable under environmental conditions, but indicate
under controlled aerobic aquatic conditions. This value is then that further testing is necessary to establish biodegradability.
compared to the theoretical amount of oxygen (ThO ) which is 5.3 If the pH value at the end of the test is outside the range
required to oxidize all of the elements (that is, carbon, from 6 to 8 and if the percentage degradation of the test
hydrogen, nitrogen, and so forth) in the test material. This test material is less than 50 %, it is advisable to repeat the test with
method mixes the test material (lubricant or component) with a lower concentration of the test material or a higher concen-
aerobic microorganisms in a closed respirometer containing a tration of the buffer solution, or both.
defined aquatic medium and measures the biodegradation of 5.4 A reference or control material known to biodegrade
the test material by following the decrease in oxygen in the under the conditions of this test method is necessary in order to
respirometer. verify the activity of the inoculum. The test must be regarded
4.2 The test material is the sole source of carbon and energy as invalid and shall be repeated using a fresh inoculum if the
inthemedium.Areferencematerialknowntobiodegrade,such reference material does not demonstrate biodegradation to the
as low erucic acid rapeseed oil (LEAR or canola oil) is run extent of >60 % of the ThO within 28 days.
D6731–01 (2005)
FIG. 1 Respirometric Test–Biodegradation Curve
5.5 Information on the toxicity of the test material to the isms consume oxygen and produce carbon dioxide. Oxygen
inoculum may be useful in the interpretation of low biodegra- fromtheheadspaceisthendissolvedintheliquidtoreestablish
dationresults.Toxicityofthetestmaterialtotheinoculummay chemical equilibrium. The carbon dioxide produced by the
be evaluated by testing the test material in combination with microorganisms diffuses into the headspace where it is trapped
the reference material in inhibition control systems. If an inanabsorbentsolutionormaterialandthetotalpressureinthe
inhibition control is included, the test material is assumed to be flask then decreases.
inhibiting if the degradation percentage of the reference 6.1.3 This pressure drop is detected by a manometer, which
material is lower than 40 % (ISO 8192:1986). In this case, it is produces a signal that results in the electrolytic generation of
advisabletorepeatthetestwithlowerconcentrationsofthetest oxygen.Whentheoriginalpressureisre-established,thesignal
material. is stopped and the quantity of electricity used is measured.The
5.6 Total oxygen utilization in the blank at the end of the amount of electricity used is proportional to the amount of
test exceeding 60 mg O /L invalidates the test. consumed oxygen.This is indicated on a plotter or a printer, or
5.7 The water solubility or dispersibility of the lubricant or the data are collected using an appropriate software program.
component may influence the results obtained and hence 6.2 Water-Bath or Constant Temperature Room, to comply
comparison of test results may be limited to lubricants or with 11.2.
components with similar solubilities. 6.3 Centrifuge.
5.8 The behaviors of complex mixtures are not always 6.4 pH-meter.
consistent with the individual properties of the components. 6.5 Analytical Balance, capable of weighing to appropriate
Test results for individual lubricant components may be sug- precision and accuracy (for example, 60.0001 g).
gestiveofwhetheramixturecontainingthesecomponents(that
7. Reagents and Materials
is, fully formulated lubricants) is biodegradable, but such
7.1 Purity of Reagents—Reagent grade chemicals shall be
information should be used judiciously.
used in all tests. Unless otherwise indicated, it is intended that
6. Apparatus all reagents conform to the specifications of the Committee on
Analytical Reagents of the American Chemical Society where
6.1 Closed Respirometer:
such specifications are available. Other grades may be used,
6.1.1 The principle of a closed respirometer is given in Fig.
provided it is first ascertained that the reagent is of sufficiently
2. When testing volatile compounds, the apparatus used shall
high purity to permit its use without decreasing the accuracy of
be appropriate or adapted to this particular purpose in accor-
the determination.
dance with the manufacturer’s instructions. Exercise care that
7.2 Purity of Water—Unless otherwise indicated, references
the closed respirometer apparatus is well sealed to prevent any
to water shall be understood to mean reagent water as defined
loss (for example, leakage) of volatile compounds from the
by Type II of Specification D1193.
system or of oxygen into the system.
6.1.2 The test mixture is stirred by a magnetic stirrer in the
test flask, which is filled with sufficient volume to minimize
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
headspace and prevent delay of O and CO diffusion through
2 2
listed by the American Chemical Society, see Analar Standards for Laboratory
the air-water phases. This volume is dependent on the selected
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
flask size, and is normally specified by the manufacturer of the
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
respirometer. If biodegradation takes place, the microorgan- MD.
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