1、Designation: D6691 09D6691 17Standard 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 designa
2、tion indicates 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. Scope Scope*1.1 This test method is used t
3、o determine the degree and rate of aerobic biodegradation of plastic materials (includingformulation additives) exposed to pre-grown population of at least ten aerobic marine microorganisms of known genera or theindigenous population existing in natural seawater. The test method is conducted under c
4、ontrolled laboratory conditions.1.2 This test method is designed to index polymer materials that are possibly biodegradable, relative to a positive referencematerial, in an aerobic environment.1.3 This test method is applicable to all polymer materials containing at least 20 % carbon that are not in
5、hibitory to themicroorganisms 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. known ISO equivalent to this standard.1.6 This standard does not purport to address all of the safety concerns, if an
6、y, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.7 This international standard was developed in accordance with in
7、ternationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D618
8、 Practice for Conditioning Plastics for TestingD883 Terminology Relating to PlasticsD1193 Specification for Reagent WaterD2593 Test Method for Butadiene Purity and Hydrocarbon Impurities by Gas ChromatographyD4129 Test Method for Total and Organic Carbon in Water by High Temperature Oxidation and by
9、 Coulometric Detection3. Terminology3.1 Definitions of Terms Specific to This StandardDefinitions of terms applying to this test method appear in TerminologyD883.4. Summary of Test Method4.1 This test method consists of the following:4.1.1 Selecting and characterizing (carbon content, molecular weig
10、ht) plastic materials for testing,4.1.2 Preparing a uniform inoculum of various isolated marine microorganisms, or obtaining a natural sea water sample (withadded inorganic nutrients) for the test relying on the microbes present as the inoculum.1 This test method is under the jurisdiction of ASTM Co
11、mmittee D20 on Plastics and is the direct responsibility of Subcommittee D20.96 on Environmentally DegradablePlastics and Biobased Products.Current edition approved Nov. 15, 2009Dec. 1, 2017. Published December 2009January 2018. Originally approved in 2001. Last previous edition approved in 20012009
12、as D6691 - 01.D6691 - 09. DOI: 10.1520/D6691-09.10.1520/D6691-17.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM webs
13、ite.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. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior
14、 editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428
15、-2959. United States14.1.3 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 the degree of biodegradability.4.2 Biodegradability is assessed by determining the proportion of polymer-C converted
16、to biogas-C. The percent of theoreticalgas production, expressed as a fraction of the measured or theoretical carbon content of the test material, is reported as a functionof time.5. Significance and Use5.1 The use of plastics aboard ships is on the rise and the use of the sea as a trash dumping sit
17、e is no longer a possibility;consequently, the disposal of plastic materials while at sea remains a major issue. It is possible that biodegradable plastics will helpto allay public concern by allowing for the safe disposal of plastic materials at sea. This test method has been developed to assessthe
18、 rate and degree of aerobic biodegradation of plastics exposed to marine microorganisms.Aerobic biodegradation is determinedby measuring the amount of biogas (carbon dioxide) produced during such an exposure.5.2 It is acceptable to use the degree and rate of aerobic biodegradability of a plastic und
19、er the conditions of this test methodto 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 exposureto a simul
20、ated marine environment is difficult. Thus, caution shall be exercised when extrapolating the results obtained from thisor 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 monitor
21、ed through the use of any number of respirometry systems. The respirometry systemmust be able to detect low levels of carbon dioxide production.Acarbon dioxide sensor consisting of a single beam, nondispersiveinfrared device with a maximum measurement capability of 1 % carbon dioxide is recommended.
22、6.1.2 Sample Bottles125-mL autoclave bottles with plastic, screw-on lids. The lids shall contain three entry ports for biogascollection as well as a tetrafluorethylene seal ring. These flasks as well as their lids are supplied by the various respirometrycompanies.6.1.3 All components of the gas-volu
23、me measuring and collection system must be of sufficient quality to prevent gas diffusionbetween the system and the surrounding atmosphere.6.2 Water Bath or Controlled-Environment Shaker/Incubator, capable of maintaining the temperature of the digestion flasks at30 6 2C.6.3 Analytical Balance, (60.1
24、 mg), to weigh the test materials.7. Reagents and Materials7.1 All chemicals shall be of American Chemical Society (ACS) reagent-grade quality.7.2 Type IV distilled water shall be prepared in accordance with Specification D1193.7.3 Marine agar per litre consists of the following:Bacto tryptone 5.0 0
25、.1 gBacto yeast extract 2.5 0.1 gBacto dextrose (glucose) 1.0 0.1 gBacto agar 15.0 0.1 g7.4 Marine broth per litre consists of the following:Peptone 5.0 0.1 gYeast extract 1.0 0.1 gFerric citrate 0.1 0.1 gSodium chloride 19.4 0.1 gMagnesium chloride, dried 5.9 0.1 gSodium sulfate 3.24 0.1 gCalcium c
26、hloride 1.8 0.1 gPotassium bromide 0.08 0.1 gStrontium chloride 34.0 0.1 mgBoric acid 4.0 0.1 mgSodium silicate 4.0 0.1 mgSodium fluoride 2.4 0.1 mgAmmonium nitrate 1.6 0.1 mgDisodium phosphate 8.0 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
27、must be mixed with 1 L of Type IV distilled water, until all of the salts havedissolved and then sterilized.D6691 1727.5.2 Natural sea water with inorganic nutrients (0.5 g/L of NH4Cl and 0.1g/L of KH2(PO4).7.6 Reference MaterialsCellulose, chitin and Kraft paper, or all three, can act as the positi
28、ve control and solitary inoculumas the negative control. Reference materials shall be provided in the same form as the test specimens, that is, powders, films, foams,and so forth. Sodium bicarbonate (100 mg) and sodium sulfite (100 mg) in an acidic water solution (100 mL) shall be tested alsoto ensu
29、re that the CO2 sensors of the respirometry apparatus are functioning properly.7.7 Microorganisms shall be selected on the basis of ability to degrade various biodegradable polymers, starches, cellulosics,and bacterial polyesters. Table 2 shows the composition of the synthetic sea salt solution.7.8
30、It is important that sampling for the natural sea water be from a site not influenced by sewage outflow, chemical dumping,waste water discharge areas or oil slicks in the water. Also, do not take the samples from a river estuary having significant tidalflow characteristics as it is possible that thi
31、s will not be representative of natural sea water.8. Hazards8.1 All microorganisms present the possibility of disease and shall be handled with due caution. Hands shall be washed beforeand after exposure. Latex gloves and safety glasses shall be used along with a mouth cover. All spills containing o
32、rganisms shallbe cleaned with germicidal/antibacterial agents, and all old cultures shall be autoclaved before being discarded.8.2 This test method requires the use of hazardous chemicals. Avoid contact with chemicals and follow the manufacturersinstructions and Material Safety Data Sheets.8.3 All p
33、urchased media also can be hazardous. Read all safety instructions.TABLE 1 Components of Minimal Marine SolutionSubstance Formula MW,g/molConcentration,g/LAmmonium chloride NH4Cl 53.49 2.00 0.05Synthetic sea salt . . . . . . 17.50 0.05Magnesium sulfate, 7-hydrate MgSO47H2O 246.48 2.0 0.05Potassium n
34、itrate KNO3 101.1 0.5 0.05Potassium phosphate K2HPO4 3H2O 228.2 0.1 0.05TABLE 2 Composition of Synthetic Sea Salt Solution atApproximate Salinity of 34 ppt, Production Variance of 5 %Ion Concentration, mg/LChloride 19251Sodium 10757Sulfate 2659Magnesium 1317Potassium 402Calcium 398Carbonate/bicarbon
35、ate 192Strontium 8.6Boron 5.6Bromide 2.3Fluoride 1.0Iodide 0.22Lithium 0.18Copper trace (0.03)Iron trace (0.03)Nickel 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
36、(0.02)Chromium trace (0.0006)Aluminum trace (0.04)Tin traceAntimony traceRubidium traceBarium trace (0.05)Mercury noneNitrate nonePhosphate noneD6691 1739. Inoculum Test Organisms9.1 The inoculum shall consist of the compositions found in 9.1.1 or 9.1.2.9.1.1 A minimum of ten test organisms. The mic
37、roorganisms were identified by using bacterial identification tests (that is,Biolog system, gram stains). Their identifications to at least genus are the following: Alteromonas haloplanktis,Xanthomonascampestri,Vibrio alginolyticus,Vibrio proteolyticus,Actinomycete sp., Bacillus megaterium,Bacillus
38、sp., Zooster sp. and Pseudomo-nas sp. Pseudomonas sp has multiple species.9.1.2 Natural sea water collected in the local area that does not have any hydrocarbon residue present on the surface, andincludes the addition of inorganic nutrients (.5 g/L of NH4Cl and .1 g/L of KH2(PO4) and maintains aerat
39、ion during therespirometry.10. Test Specimen10.1 Weigh test samples to the nearest 0.1 mg and have sufficient carbon content (minimum 20 %) to yield carbon dioxidevolumes that can be measured accurately by the respirometer. A method for determining carbon content of the test material isdetermined by
40、 calculation or elemental analysis in accordance with Test Method D4129.10.2 Acceptable forms in which it is possible for the test specimen to be included are powders, films, pieces, fragments, formedarticles, or aqueous solutions. The test materials shall be conditioned in accordance with Practice
41、D618. Test specimens in the formof powders shall be characterized as to particle size distribution. Mean particle size less than 25 mm is recommended.10.3 If the specimens are solids, grinding is recommended to obtain a powder form of the sample to maximize surface area.Cryogenically milling with li
42、quid nitrogen by means of impact is a recommended method to obtain powders.10.4 OptionalUse Test Method D2593 to measure and record 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 the directions descri
43、bed on the label. Forty mL portions of the marinebroth shall be placed in up to thirteen 125-mL Erlenmeyer flasks (one for every organism) and autoclaved for 20 min at 121Cat 15 lb of pressure.11.1.2 Each flask shall be inoculated with 250-L stock cultures of the microorganisms that were grown overn
44、ight or to anoptical density of approximately 2.0 at a wavelength of 660 and placed in a shaker/incubator at 30C.11.1.3 When the cultures have reached the state of growth where the cells are in late logarithmic growth or early stationaryphase (24 h), each separate culture must be centrifuged at a re
45、lative centrifugal force (RCF) of approximately 26890 (g) for 8 minto obtain pellets.11.1.4 After centrifuging, decant the media away from the pellets and resuspend the pellets in 20 mL of minimal marinesolution. Repeat the centrifuging and decanting to remove any carbon source from the microorganis
46、ms. After decanting for thesecond time, resuspend the pellets in 10 mL of minimal marine solution. Repeat the centrifuging and decanting of the solution andthen resuspend the pellets in 4 mL of minimal marine solution.11.1.5 Place the 4-mLresuspensions of all the microorganisms into a single sterile
47、 flask with cap. Mix the various resuspensionstogether by the use of a vortex on the flask.11.2 Preparation of Respirometry Flasks:11.2.1 A stock solution containing enough minimal marine solution for the number of flasks to be used shall be autoclaved at121C for 20 min prior to the day of the exper
48、iment.Atube with attached air sparger, as well as all of the empty respirometer flasksand lids shall also be autoclaved.11.2.2 Seventyfive mL of the minimal marine stock solution or natural sea water with inorganic nutrients shall be placedaseptically into each respirometry 125-mL bottle in a steril
49、e environment.11.3 Test and Reference (Control) Specimen:11.3.1 The test and control specimens must be sterilized before placement into the respirometry bottles. Specimen size is usually20 mg depending on the carbon content of the sample (pre-weighed, 60.1 mg). Sterilization of polymers usually cannot beperformed through the use of an autoclave due the limited thermal stability of the polymers and the effect of high temperatureson a polymers structure. For this reason, ethylene oxide sterilization of samples is recommen
