1、Designation: D6691 09Standard Test Method forDetermining Aerobic Biodegradation of Plastic Materials inthe Marine Environment by a Defined Microbial Consortiumor Natural Sea Water Inoculum1This standard is issued under the fixed designation D6691; the number immediately following the designation ind
2、icates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is used to determine the
3、 degree and rateof aerobic biodegradation of plastic materials (including for-mulation additives) exposed to pre-grown population of at leastten aerobic marine microorganisms of known genera or theindigenous population existing in natural seawater. The testmethod is conducted under controlled labora
4、tory conditions.1.2 This test method is designed to index polymer materialsthat are possibly biodegradable, relative to a positive referencematerial, in an aerobic environment.1.3 This test method is applicable to all polymer materialscontaining at least 20 % carbon that are not inhibitory to themic
5、roorganisms present in a marine environment.1.4 The values stated in SI units are to be regarded as thestandard.1.5 There is no similar or equivalent ISO standard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the
6、user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D618 Practice for Conditioning Plastics for TestingD883 Terminology Relating to PlasticsD1193 Specification f
7、or Reagent WaterD2593 Test Method for Butadiene Purity and HydrocarbonImpurities by Gas ChromatographyD4129 Test Method for Total and Organic Carbon in Waterby High Temperature Oxidation and by Coulometric De-tection3. Terminology3.1 Definitions of Terms Specific to This StandardDefinitions of terms
8、 applying to this test method appear inTerminology D883.4. Summary of Test Method4.1 This test method consists of the following:4.1.1 Selecting and characterizing (carbon content, molecu-lar weight) plastic materials for testing,4.1.2 Preparing a uniform inoculum of various isolatedmarine microorgan
9、isms, or obtaining a natural sea watersample (with added inorganic nutrients) for the test relying onthe microbes present as the inoculum.4.1.3 Exposing the test materials to the inoculum,4.1.4 Using a respirometer to measure the total biogas(CO2) produced as a function of time, and4.1.5 Assessing t
10、he degree of biodegradability.4.2 Biodegradability is assessed by determining the propor-tion of polymer-C converted to biogas-C. The percent oftheoretical gas production, expressed as a fraction of themeasured or theoretical carbon content of the test material, isreported as a function of time.5. S
11、ignificance and Use5.1 The use of plastics aboard ships is on the rise and the useof the sea as a trash dumping site is no longer a possibility;consequently, the disposal of plastic materials while at searemains a major issue. It is possible that biodegradable plasticswill help to allay public conce
12、rn by allowing for the safedisposal of plastic materials at sea. This test method has beendeveloped to assess the rate and degree of aerobic biodegra-dation of plastics exposed to marine microorganisms. Aerobicbiodegradation is determined by measuring the amount ofbiogas (carbon dioxide) produced du
13、ring such an exposure.5.2 It is acceptable to use the degree and rate of aerobicbiodegradability of a plastic under the conditions of this testmethod to estimate the persistence of that plastic in biologi-cally active marine environments, for example, seashore andopen-ocean. However, it shall be rec
14、ognized that predictinglong-term environmental fate and effects from the results ofshort-term exposure to a simulated marine environment is1This test method is under the jurisdiction of ASTM Committee D20 on Plasticsand is the direct responsibility of Subcommittee D20.96 on EnvironmentallyDegradable
15、 Plastics and Biobased Products.Current edition approved Nov. 15, 2009. Published December 2009. Originallyapproved in 2001. Last previous edition approved in 2001 as D6691 - 01. DOI:10.1520/D6691-09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Servic
16、e at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.difficult. Thus, caution shall be exercised wh
17、en extrapolatingthe results obtained from this or any other controlled-environment test to disposal in the natural environment.6. Apparatus6.1 Aerobic Digestion and Gas Measuring Apparatus:6.1.1 Biogas production can be monitored through the useof any number of respirometry systems. The respirometry
18、system must be able to detect low levels of carbon dioxideproduction. A carbon dioxide sensor consisting of a singlebeam, nondispersive infrared device with a maximum mea-surement capability of 1 % carbon dioxide is recommended.6.1.2 Sample Bottles125-mL autoclave bottles with plas-tic, screw-on lid
19、s. The lids shall contain three entry ports forbiogas collection as well as a tetrafluorethylene seal ring. Theseflasks as well as their lids are supplied by the variousrespirometry companies.6.1.3 All components of the gas-volume measuring andcollection system must be of sufficient quality to preve
20、nt gasdiffusion between the system and the surrounding atmosphere.6.2 Water Bath or Controlled-Environment Shaker/Incubator, capable of maintaining the temperature of thedigestion flasks at 30 6 2C.6.3 Analytical Balance,(60.1 mg), to weigh the test mate-rials.7. Reagents and Materials7.1 All chemic
21、als shall be of American Chemical Society(ACS) reagent-grade quality.7.2 Type IV distilled water shall be prepared in accordancewith Specification D1193.7.3 Marine agar per litre consists of the following:Bacto tryptone 5.0 6 0.1 gBacto yeast extract 2.5 6 0.1 gBacto dextrose (glucose) 1.0 6 0.1 gBa
22、cto agar 15.0 6 0.1 g7.4 Marine broth per litre consists of the following:Peptone 5.0 6 0.1 gYeast extract 1.0 6 0.1 gFerric citrate 0.1 6 0.1 gSodium chloride 19.4 6 0.1 gMagnesium chloride, dried 5.9 6 0.1 gSodium sulfate 3.24 6 0.1 gCalcium chloride 1.8 6 0.1 gPotassium bromide 0.08 6 0.1 gStront
23、ium chloride 34.0 6 0.1 mgBoric acid 4.0 6 0.1 mgSodium silicate 4.0 6 0.1 mgSodium fluoride 2.4 6 0.1 mgAmmonium nitrate 1.6 6 0.1 mgDisodium phosphate 8.0 6 0.1 mg7.5 Marine SolutionShall be either 7.5.1 or 7.5.2.7.5.1 Refer to Table 1.All of the components must be mixedwith 1 L of Type IV distill
24、ed water, until all of the salts havedissolved and then sterilized.7.5.2 Natural sea water with inorganic nutrients (0.5 g/L ofNH4Cl and 0.1g/L of KH2(PO4).7.6 Reference MaterialsCellulose, chitin and Kraft paper,or all three, can act as the positive control and solitaryinoculum as the negative cont
25、rol. Reference materials shall beprovided in the same form as the test specimens, that is,powders, films, foams, and so forth. Sodium bicarbonate (100mg) and sodium sulfite (100 mg) in an acidic water solution(100 mL) shall be tested also to ensure that the CO2sensors ofthe respirometry apparatus ar
26、e functioning properly.7.7 Microorganisms shall be selected on the basis of abilityto degrade various biodegradable polymers, starches, cellulo-sics, and bacterial polyesters. Table 2 shows the composition ofthe synthetic sea salt solution.7.8 It is important that sampling for the natural sea water
27、befrom a site not influenced by sewage outflow, chemicaldumping, waste water discharge areas or oil slicks in the water.Also, do not take the samples from a river estuary havingsignificant tidal flow characteristics as it is possible that thiswill not be representative of natural sea water.8. Hazard
28、s8.1 All microorganisms present the possibility of diseaseand shall be handled with due caution. Hands shall be washedbefore and after exposure. Latex gloves and safety glasses shallbe used along with a mouth cover. All spills containingTABLE 1 Components of Minimal Marine SolutionSubstance Formula
29、MW, g/mol Concentration, g/LAmmonium chloride NH4Cl 53.49 2.00 6 0.05Synthetic sea salt . . . . . . 17.50 6 0.05Magnesium sulfate, 7-hydrate MgSO47H2O 246.48 2.0 6 0.05Potassium nitrate KNO3101.1 0.5 6 0.05Potassium phosphate K2HPO4 3H2O 228.2 0.1 6 0.05TABLE 2 Composition of Synthetic Sea Salt Solu
30、tion atApproximate Salinity of 34 ppt, Production Variance of 65%Ion Concentration, mg/LChloride 19251Sodium 10757Sulfate 2659Magnesium 1317Potassium 402Calcium 398Carbonate/bicarbonate 192Strontium 8.6Boron 5.6Bromide 2.3Fluoride 1.0Iodide 0.22Lithium 0.18Copper trace (0.03)Iron trace (0.03)Nickel
31、trace (0.04)Zinc trace (0.02)Manganese trace (0.01)Molybdenum trace (0.01)Cobalt trace (0.05)Vanadium trace (0.04)Selenium traceLead trace (0.005)Arsenic trace (0.0002)Cadmium trace (0.02)Chromium trace (0.0006)Aluminum trace (0.04)Tin traceAntimony traceRubidium traceBarium trace (0.05)Mercury none
32、Nitrate nonePhosphate noneD6691 092organisms shall be cleaned with germicidal/antibacterialagents, and all old cultures shall be autoclaved before beingdiscarded.8.2 This test method requires the use of hazardous chemi-cals. Avoid contact with chemicals and follow the manufactur-ers instructions and
33、 Material Safety Data Sheets.8.3 All purchased media also can be hazardous. Read allsafety instructions.9. Inoculum Test Organisms9.1 The inoculum shall consist of the compositions found in9.1.1 or 9.1.2.9.1.1 A minimum of ten test organisms. The microorgan-isms were identified by using bacterial id
34、entification tests (thatis, Biolog system, gram stains). Their identifications to at leastgenus are the following: Alteromonas haloplanktis, Xanthomo-nas campestri, Vibrio alginolyticus, Vibrio proteolyticus, Acti-nomycete sp., Bacillus megaterium, Bacillus sp., Zooster sp.and Pseudomonas sp. Pseudo
35、monas sp has multiple species.9.1.2 Natural sea water collected in the local area that doesnot have any hydrocarbon residue present on the surface, andincludes the addition of inorganic nutrients (.5 g/L of NH4Cland .1 g/L of KH2(PO4) and maintains aeration during therespirometry.10. Test Specimen10
36、.1 Weigh test samples to the nearest 0.1 mg and havesufficient carbon content (minimum 20 %) to yield carbondioxide volumes that can be measured accurately by therespirometer. A method for determining carbon content of thetest material is determined by calculation or elemental analysisin accordance
37、with Test Method D4129.10.2 Acceptable forms in which it is possible for the testspecimen to be included are powders, films, pieces, fragments,formed articles, or aqueous solutions. The test materials shallbe conditioned in accordance with Practice D618. Test speci-mens in the form of powders shall
38、be characterized as toparticle size distribution. Mean particle size less than 25 mm isrecommended.10.3 If the specimens are solids, grinding is recommendedto obtain a powder form of the sample to maximize surfacearea. Cryogenically milling with liquid nitrogen by means ofimpact is a recommended met
39、hod to obtain powders.10.4 OptionalUse Test Method D2593 to measure andrecord the molecular weight of the test material.11. Procedure11.1 Inoculum Buildup and Preparation:11.1.1 All media shall be prepared in accordance with thedirections described on the label. Forty mL portions of themarine broth
40、shall be placed in up to thirteen 125-mL Erlen-meyer flasks (one for every organism) and autoclaved for 20min at 121C at 15 lb of pressure.11.1.2 Each flask shall be inoculated with 250-L stockcultures of the microorganisms that were grown overnight or toan optical density of approximately 2.0 at a
41、wavelength of 660and placed in a shaker/incubator at 30C.11.1.3 When the cultures have reached the state of growthwhere the cells are in late logarithmic growth or earlystationary phase (24 h), each separate culture must be centri-fuged at a relative centrifugal force (RCF) of approximately26890 (g)
42、 for 8 min to obtain pellets.11.1.4 After centrifuging, decant the media away from thepellets and resuspend the pellets in 20 mL of minimal marinesolution. Repeat the centrifuging and decanting to remove anycarbon source from the microorganisms. After decanting forthe second time, resuspend the pell
43、ets in 10 mL of minimalmarine solution. Repeat the centrifuging and decanting of thesolution and then resuspend the pellets in 4 mL of minimalmarine solution.11.1.5 Place the 4-mL resuspensions of all the microorgan-isms into a single sterile flask with cap. Mix the variousresuspensions together by
44、the use of a vortex on the flask.11.2 Preparation of Respirometry Flasks:11.2.1 A stock solution containing enough minimal marinesolution for the number of flasks to be used shall be autoclavedat 121C for 20 min prior to the day of the experiment. A tubewith attached air sparger, as well as all of t
45、he empty respirom-eter flasks and lids shall also be autoclaved.11.2.2 Seventyfive mL of the minimal marine stock solu-tion or natural sea water with inorganic nutrients shall beplaced aseptically into each respirometry 125-mL bottle in asterile environment.11.3 Test and Reference (Control) Specimen
46、:11.3.1 The test and control specimens must be sterilizedbefore placement into the respirometry bottles. Specimen sizeis usually 20 mg depending on the carbon content of the sample(pre-weighed, 60.1 mg). Sterilization of polymers usuallycannot be performed through the use of an autoclave due thelimi
47、ted thermal stability of the polymers and the effect of hightemperatures on a polymers structure. For this reason, ethyl-ene oxide sterilization of samples is recommended.11.3.2 Within a sterile environment constituted by a laminarflow hood, the samples shall be added to the respirometry flaskand th
48、e flask lid screwed on. The sampled reference materialsand blanks shall be prepared in triplicate.11.4 Inoculating the Respirometry Flasks:11.4.1 Inoculate each of the respirometry flasks containingthe minimal marine stock solution with 100 6 L of theinoculum established in 11.1.5. If natural seawat
49、er with inor-ganic nutrients is used, then no inoculation of pre-grownmicroorganisms is done.11.4.2 Reserve one of the bottles for the sodium bicarbonatecontrol, and do not inoculate it.11.5 System Start-Up and Maintenance:11.5.1 After attaching all of the respirometry bottles to therespirometer, perform standard diagnostic tests to find anysystem leaks. The respirometry bottles shall be placed in eithera water bath or shaker apparatus set to 30 6 1C and a rotationof 175 6 5 rpm.11.5.2 Before starting the experiment, place the sample ofsodium bicarbonate and sodium su
