ASTM D6691-01
(Test Method)Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in the Marine Environment by a Defined Microbial Consortium
Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in the Marine Environment by a Defined Microbial Consortium
SIGNIFICANCE AND USE
The use of plastics aboard ships is on the rise and the use of the sea as a trash dumping site is no longer a possibility; consequently, the disposal of plastic materials while at sea remains a major issue. Biodegradable plastics may help to allay public concern by allowing for the safe disposal of plastic materials at sea. This test method has been developed to assess the rate and degree of aerobic biodegradation of plastics exposed to marine microorganisms. Aerobic biodegradation is determined by measuring the amount of biogas (carbon dioxide) produced during such an exposure.
The degree and rate of aerobic biodegradability of a plastic under the conditions of this test method may be used to estimate the persistence of that plastic in biologically active marine environments, for example, seashore and open-ocean. However, it shall be recognized that predicting long-term environmental fate and effects from the results of short-term exposure to a simulated marine environment is difficult. Thus, caution shall be exercised when extrapolating the results obtained from this or any other controlled-environment test to disposal in the natural environment.
SCOPE
1.1 This test method is used to determine the degree and rate of aerobic biodegradation of plastic materials (including formulation additives) exposed to pre-grown population of at least ten aerobic marine microorganisms of known genera. The test method is conducted under controlled laboratory conditions.
1.2 This test method is designed to index polymer materials that are possibly biodegradable, relative to a positive reference material, in an aerobic environment.
1.3 This test method is applicable to all polymer materials containing at least 20 % carbon that are not inhibitory to the microorganisms present in a marine environment.
1.4 The values stated in SI units are to be regarded as the standard.
1.5 There is no similar or equivalent ISO standard.
1.6 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.
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Standards Content (Sample)
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Designation: D6691 – 01
Standard Test Method for
Determining Aerobic Biodegradation of Plastic Materials in
the Marine Environment by a Defined Microbial Consortium
This standard is issued under the fixed designation D6691; 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 4. Summary of Test Method
4.1 This test method consists of the following:
1.1 Thistestmethodisusedtodeterminethedegreeandrate
of aerobic biodegradation of plastic materials (including for- 4.1.1 Selecting and characterizing (carbon content, molecu-
mulationadditives)exposedtopre-grownpopulationofatleast lar weight) plastic materials for testing,
ten aerobic marine microorganisms of known genera. The test 4.1.2 Preparing a uniform inoculum of various isolated
method is conducted under controlled laboratory conditions. marine microorganisms,
1.2 This test method is designed to index polymer materials 4.1.3 Exposing the test materials to the inoculum,
that are possibly biodegradable, relative to a positive reference 4.1.4 Using a respirometer to measure the total biogas
material, in an aerobic environment. (CO ) produced as a function of time, and
1.3 This test method is applicable to all polymer materials 4.1.5 Assessing the degree of biodegradability.
containing at least 20 % carbon that are not inhibitory to the 4.2 Biodegradability is assessed by determining the propor-
microorganisms present in a marine environment. tion of polymer-C converted to biogas-C. The percent of
1.4 The values stated in SI units are to be regarded as the theoretical gas production, expressed as a fraction of the
standard. measured or theoretical carbon content of the test material, is
1.5 There is no similar or equivalent ISO standard. reported as a function of time.
1.6 This standard does not purport to address all of the
5. Significance and Use
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 5.1 Theuseofplasticsaboardshipsisontheriseandtheuse
of the sea as a trash dumping site is no longer a possibility;
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. consequently, the disposal of plastic materials while at sea
remainsamajorissue.Biodegradableplasticsmayhelptoallay
2. Referenced Documents
public concern by allowing for the safe disposal of plastic
2.1 ASTM Standards: materials at sea. This test method has been developed to assess
D618 Practice for Conditioning Plastics for Testing the rate and degree of aerobic biodegradation of plastics
D883 Terminology Relating to Plastics exposed to marine microorganisms. Aerobic biodegradation is
D1193 Specification for Reagent Water determined by measuring the amount of biogas (carbon diox-
D3593 NO TITLE ide) produced during such an exposure.
D4129 Test Method for Total and Organic Carbon in Water 5.2 The degree and rate of aerobic biodegradability of a
by High Temperature Oxidation and by Coulometric De- plastic under the conditions of this test method may be used to
tection estimate the persistence of that plastic in biologically active
marine environments, for example, seashore and open-ocean.
3. Terminology
However, it shall be recognized that predicting long-term
3.1 Definitions of Terms Specific to This Standard—
environmental fate and effects from the results of short-term
Definitions of terms applying to this test method appear in exposure to a simulated marine environment is difficult. Thus,
Terminology D883.
caution shall be exercised when extrapolating the results
obtained from this or any other controlled-environment test to
disposal in the natural environment.
This test method is under the jurisdiction ofASTM Committee D20 on Plastics
and is the direct responsibility of Subcommittee D20.96 on Environmentally
Degradable Plastics. 6. Apparatus
Current edition approved June 10, 2001. PublishedAugust 2001. DOI: 10.1520/
6.1 Aerobic Digestion and Gas Measuring Apparatus:
D6691-01.
6.1.1 Biogas production can be monitored through the use
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
of any number of respirometry systems. The respirometry
Standards volume information, refer to the standard’s Document Summary page on
system must be able to detect low levels of carbon dioxide
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6691 – 01
production. A carbon dioxide sensor consisting of a single 7.7 Microorganisms shall be selected on the basis of ability
beam, nondispersive infrared device with a maximum mea- to degrade various biodegradable polymers, starches, cellulo-
surement capability of 1 % carbon dioxide is recommended. sics,andbacterialpolyesters.Table2showsthecompositionof
6.1.2 Sample Bottles—125-mL autoclave bottles with plas- the synthetic sea salt.
tic, screw-on lids. The lids shall contain three entry ports for
8. Hazards
biogascollectionaswellasatetrafluorethylenesealring.These
flasks as well as their lids are supplied by the various
8.1 All microorganisms present the possibility of disease
respirometry companies. and shall be handled with due caution. Hands shall be washed
6.1.3 All components of the gas-volume measuring and
beforeandafterexposure.Latexglovesandsafetyglassesshall
collection system must be of sufficient quality to prevent gas be used along with a mouth cover. All spills containing
diffusion between the system and the surrounding atmosphere.
organisms shall be cleaned with germicidal/antibacterial
6.2 Water Bath or Controlled-Environment Shaker/ agents, and all old cultures shall be autoclaved before being
Incubator, capable of maintaining the temperature of the
discarded.
digestion flasks at 30 6 2°C. 8.2 This test method requires the use of hazardous chemi-
6.3 Analytical Balance,(60.1 mg), to weigh the test mate- cals.Avoid contact with chemicals and follow the manufactur-
rials.
er’s instructions and Material Safety Data Sheets.
8.3 All purchased media also can be hazardous. Read all
7. Reagents and Materials
safety instructions.
7.1 All chemicals shall be of American Chemical Society
9. Inoculum Test Organisms
(ACS) reagent-grade quality.
9.1 The inoculum consists of a minimum of nine test
7.2 Type IV distilled water shall be prepared in accordance
organisms. The microorganisms were identified by using bac-
with Specification D1193.
terial identification test (that is, Biolog system, gram stains).
7.3 Marine agar per litre consists of the following:
Their identifications to at least genus are the following:
Bacto tryptone 5.0 6 0.1 g
Bacto yeast extract 2.5 6 0.1 g
Alteromonas haloplanktis, Xanthomonas campestri, Vibrio al-
Bacto dextrose (glucose) 1.0 6 0.1 g
ginolyticus, Vibrio proteolyticus, Actinomycete sp., Bacillus
Bacto agar 15.0 6 0.1 g
megaterium, Bacillus sp., Zooster sp. and Pseudomonas sp.
7.4 Marine broth per litre consists of the following:
Pseudomonas sp has multiple species.
Peptone 5.0 6 0.1 g
Yeast extract 1.0 6 0.1 g
TABLE 2 Composition of Synthetic Sea Salt Solution at
Ferric citrate 0.1 6 0.1 g
Sodium chloride 19.4 6 0.1 g Approximate Salinity of 34 ppt, Production Variance of 65%
Magnesium chloride, dried 5.9 6 0.1 g
Ion Concentration, mg/L
Sodium sulfate 3.24 6 0.1 g
Calcium chloride 1.8 6 0.1 g Chloride 19251
Potassium bromide 0.08 6 0.1 g Sodium 10757
Strontium chloride 34.0 6 0.1 mg Sulfate 2659
Boric acid 4.0 6 0.1 mg Magnesium 1317
Sodium silicate 4.0 6 0.1 mg Potassium 402
Sodium fluoride 2.4 6 0.1 mg Calcium 398
Ammonium nitrate 1.6 6 0.1 mg Carbonate/bicarbonate 192
Disodium phosphate 8.0 6 0.1 mg Strontium 8.6
Boron 5.6
7.5 Marine Solution—Refer to Table 1. All of the compo-
Bromide 2.3
Fluoride 1.0
nents must be mixed with 1 L of reagent grade, sterile water,
Iodide 0.22
until all of the salts have disappeared.
Lithium 0.18
7.6 Reference Materials—Cellulose, chitin and Kraft paper,
Copper trace (<0.03)
Iron trace (<0.03)
or all three, can act as the positive control and solitary
Nickel trace (<0.04)
inoculum as the negative control. Reference materials shall be
Zinc trace (<0.02)
provided in the same form as the test specimens, that is,
Manganese trace (<0.01)
Molybdenum trace (<0.01)
powders, films, foams, and so forth. Sodium bicarbonate (100
Cobalt trace (<0.05)
mg) and sodium sulfite (100 mg) in an acidic water solution
Vanadium trace (<0.04)
(100 mL) shall be tested also to ensure that the CO sensors of
Selenium trace
Lead trace (<0.005)
the respirometry apparatus are functioning properly.
Arsenic trace (<0.0002)
Cadmium trace (<0.02)
Chromium trace (<0.0006)
TABLE 1 Components of Minimal Marine Solution Aluminum trace (<0.04)
Tin trace
Substance Formula MW, g/mol Concentration, g/L
Antimony trace
Ammonium chloride NH Cl 53.49 2.00 6 0.05 Rubidium trace
Synthetic sea salt . . . . . . 17.50 6 0.05 Barium trace (<0.05)
Magnesium sulfate, 7-hydrate MgSO 7H2O 246.48 2.0 6 0.05 Mercury none
Potassium nitrate KNO 101.1 0.5 6 0.05 Nitrate none
Potassium phosphate K HPO4 H2O 228.2 0.1 6 0.05 Phosphate none
2 3
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