1、Designation: D 6691 01Standard Test Method forDetermining Aerobic Biodegradation of Plastic Materials inthe Marine Environment by a Defined Microbial Consortium1This standard is issued under the fixed designation D 6691; the number immediately following the designation indicates the year oforiginal
2、adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is used to determine the degree and rateof aerobic
3、 biodegradation of plastic materials (including for-mulation additives) exposed to pre-grown population of at leastten aerobic marine microorganisms of known genera. The testmethod is conducted under controlled laboratory conditions.1.2 This test method is designed to index polymer materialsthat are
4、 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 themicroorganisms present in a marine environment.1.4 The values stated in SI units are t
5、o 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 user of this standard to establish appro-priate safety and health practices and det
6、ermine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:D 618 Practice for Conditioning Plastics and ElectricalInsulating Materials for Testing2D 883 Terminology Relating to Plastics2D 1193 Specification for Reagent Water3D 3593 Test Method for Mole
7、cular Weight Averages/Distribution of Certain Polymers by Liquid Size-ExclusionChromatography (Gel Permeation Chromatography GPC)Using Universal Calibration4D 4129 Test Method for Total and Organic Carbon in Waterby High-Temperature Oxidation and Coulometric Detec-tion53. Terminology3.1 Definitions
8、of Terms Specific to This StandardDefinitions of terms applying to this test method appear inTerminology D 883.4. 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
9、a uniform inoculum of various isolatedmarine microorganisms,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 the degree of biodegradability.4.2 Biodegradability is assessed by determining
10、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. Significance and Use5.1 The use of plastics aboard ships is on the rise and the
11、 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. Biodegradable plastics may help to allaypublic concern by allowing for the safe disposal of plasticmaterials at sea. This test method has been develope
12、d to assessthe rate and degree of aerobic biodegradation of plasticsexposed to marine microorganisms. Aerobic biodegradation isdetermined by measuring the amount of biogas (carbon diox-ide) produced during such an exposure.5.2 The degree and rate of aerobic biodegradability of aplastic under the con
13、ditions of this test method may be used toestimate the persistence of that plastic in biologically activemarine environments, for example, seashore and open-ocean.However, it shall be recognized that predicting long-termenvironmental fate and effects from the results of short-termexposure to a simul
14、ated marine environment is difficult. Thus,caution shall be exercised when extrapolating the resultsobtained from this or any other controlled-environment test todisposal in the natural environment.1This test method is under the jurisdiction of ASTM Committee D20 on Plasticsand is the direct respons
15、ibility of Subcommittee D20.96 on EnvironmentallyDegradable Plastics.Current edition approved June 10, 2001. Published August 2001.2Annual Book of ASTM Standards, Vol 08.01.3Annual Book of ASTM Standards, Vol 11.01.4Discontinued 1993; Replaced by Test Method D 5296.5Annual Book of ASTM Standards, Vo
16、l 11.02.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.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 respirometrysyst
17、em 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 lids. T
18、he 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 prevent g
19、asdiffusion 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 chemicals
20、shall be of American Chemical Society(ACS) reagent-grade quality.7.2 Type IV distilled water shall be prepared in accordancewith Specification D 1193.7.3 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 gBacto
21、 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 gStrontium
22、 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 SolutionRefer to Table 1. All of the compo-nents must be mixed with 1 L of reagent grade, sterile water,until all of the salts
23、have disappeared.7.6 Reference MaterialsCellulose, chitin and Kraft paper,or all three, can act as the positive control and solitaryinoculum as the negative control. Reference materials shall beprovided in the same form as the test specimens, that is,powders, films, foams, and so forth. Sodium bicar
24、bonate (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 are functioning properly.7.7 Microorganisms shall be selected on the basis of abilityto degrade various biodegradable polymers, starches, cel
25、lulo-sics, and bacterial polyesters. Table 2 shows the composition ofthe synthetic sea salt.8. Hazards8.1 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 wit
26、h a mouth cover. All spills containingorganisms 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
27、and Material Safety Data Sheets.8.3 All purchased media also can be hazardous. Read allsafety instructions.9. Inoculum Test Organisms9.1 The inoculum consists of a minimum of nine testorganisms. The microorganisms were identified by using bac-terial identification test (that is, Biolog system, gram
28、stains).TABLE 1 Components of Minimal Marine SolutionSubstance Formula 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 K2H
29、PO43H2O 228.2 0.1 6 0.05TABLE 2 Composition of Synthetic Sea Salt Solution 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
30、 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 (0.02)Chromium trace (0.0006)Aluminum trace (0.0
31、4)Tin traceAntimony traceRubidium traceBarium trace (0.05)Mercury noneNitrate nonePhosphate noneD 66912Their identifications to at least genus are the following:Alteromonas haloplanktis, Xanthomonas campestri, Vibrio al-ginolyticus, Vibrio proteolyticus, Actinomycete sp., Bacillusmegaterium, Bacillu
32、s sp., Zooster sp. and Pseudomonas sp.Pseudomonas sp has multiple species.10. Test Specimen10.1 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. The carbon content of the test
33、material may bedetermined by calculation or elemental analysis in accordancewith Test Method D 4129.10.2 The test specimen may be in the form of powders,films, pieces, fragments, formed articles, or aqueous solutions.The test materials shall be conditioned in accordance withPractice D 618. Test spec
34、imens in the form of powders shall becharacterized as to particle size distribution. Mean particle sizeless than 25 mm is recommended.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
35、means ofimpact is a recommended method to obtain powders.10.4 OptionalThe molecular weight of the test materialmay be measured (Test Method D 3593) and recorded.11. Procedure11.1 Inoculum Buildup and Preparation:11.1.1 All media shall be prepared in accordance with thedirections described on the lab
36、el. Forty mL portions of themarine broth shall be placed into thirteen 125-mL Erlenmeyerflasks (one for every organism) and autoclaved for 20 min at121C 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 outside
37、 diameter of approximately 2.0 at a wavelength of660 and place in a shaker/incubator at 30C.11.1.3 When the cultures have reached the state of growthwhere the cells are late logarithmic growth or early stationaryphase (24 h), each separate culture must be centrifuged at arelative centrifugal force (
38、RCF) of approximately 26890 (g) for8 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 s
39、econd time, resuspend the pellets in 10 mL of minimalmarine solution. Repeat the centrifuging and decanting of thesolution then resuspend the pellets in 4 mL of minimal marinesolution.11.1.5 Place the 4-mL resuspensions of all the microorgan-isms into a single sterile flask with cap. Mix the various
40、resuspensions together by 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 sp
41、arger, as well as all of the empty respirom-eter flasks and lids shall also be autoclaved.11.2.2 Seventyfive mL of the minimal marine stock solu-tion shall be placed aseptically into each respirometry 125-mLbottle in a sterile environment.11.3 Test and Reference (Control) Specimen:11.3.1 The test an
42、d 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 thelimited thermal stabili
43、ty 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 the flask lid screwed
44、 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 with 100 6L of the inoculum established in 11.1.5.11.4.2 Reserve one of the bottles for the sodium bicarbonatecontrol, and do not i
45、noculate 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 arotation of 17
46、5 6 5 rpm.11.5.2 Before starting the experiment, place the sample ofsodium bicarbonate and sodium sulfite into its assigned flaskand close the lid immediately to avoid letting any of theevolved CO2escape.11.5.3 Once the system has started, continuously monitorCO2production and the condition of the s
47、ystem as a whole.Follow the manufacturers instructions for maintenance, moni-toring, and parts replacement.11.5.4 Typical respirometer runs last anywhere from 10-90days in duration; although, if the specimens are extremelybiodegradable, then the experiment may be terminated whencumulative CO2product
48、ion plateaus or extended if no biodeg-radation has occurred in a specific time.12. Calculation12.1 Theoretical Maximum CO2Production:12.1.1 Determine the total organic carbon content of the testmaterial (by elemental analysis or, if the chemical compositionis well-established, by calculation).12.1.2
49、 Calculate the maximum amount CO2that theoreti-cally can be evolved during the aerobic biodegradation of thetest materials. The relevant chemical reactions are:Cpolymer1 O2 CO2Each mmole (12 mg) of polymer-C can be converted to 1mmole of gaseous CO2. The theoretical maximum amount ofCO2that can be produced is calculated as follows:Maxbiogas 2 C 5W%CT!10031 mmol gaseous C12 mg Cwhere:W = total weight (mg) of the test material, andD 66913%CT= percent organic carbon in the test material.12.1.3 Calculate the percentage of mineralization (that is,aerobic biodegradation) of th
