This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles

for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

Designation: D5526 − 18

Standard Test Method for

Determining Anaerobic Biodegradation of Plastic Materials
Under Accelerated Landfill Conditions1
This standard is issued under the fixed designation D5526; 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 “biodegradable” claims. Reports shall clearly state the percent-

1.1 This test method covers determination of the degree and age of net gaseous carbon generation for both the test and
rate of anaerobic biodegradation of plastic materials in an reference samples at the completion of the test. Furthermore,
accelerated-landfill test environment. This test method is also results shall not be extrapolated past the actual duration of the
designed to produce mixtures of household waste and plastic test.
materials after different degrees of decomposition under con- 1.5 The values stated in SI units are to be regarded as the
ditions that resemble landfill conditions. The test materials are standard.
mixed with pretreated household waste and exposed to a 1.6 This standard does not purport to address all of the
methanogenic inoculum derived from anaerobic digesters op- safety concerns, if any, associated with its use. It is the
erating only on pretreated household waste. The anaerobic responsibility of the user of this standard to establish appro-
decomposition occurs under dry (more than 30 % total solids) priate safety, health, and environmental practices and deter-
and static nonmixed conditions. The mixtures obtained after mine the applicability of regulatory limitations prior to use.
this test method can be used to assess the environmental and Specific hazards statements are given in Section 8.
health risks of plastic materials that are degraded in a landfill.
NOTE 1—There is no known ISO equivalent to this standard.
1.2 This test method is designed to yield a percentage of
1.7 This international standard was developed in accor-
conversion of carbon in the sample to carbon in the gaseous
dance with internationally recognized principles on standard-
form under conditions that resemble landfill conditions. It is
ization established in the Decision on Principles for the
possible that this test method will not simulate all conditions
Development of International Standards, Guides and Recom-
found in landfills, especially biologically inactive landfills.
mendations issued by the World Trade Organization Technical
This test method more closely resembles those types of
Barriers to Trade (TBT) Committee.
landfills in which the gas generated is recovered or even
actively promoted, or both, for example, by inoculation (code- 2. Referenced Documents
position of anaerobic sewage sludge and anaerobic leachate
recirculation), moisture control in the landfill (leachate 2.1 ASTM Standards:3
recirculation), and temperature control (short-term injection of D618 Practice for Conditioning Plastics for Testing
oxygen and heating of recirculated leachate) (1-7).2 D883 Terminology Relating to Plastics
D1293 Test Methods for pH of Water
1.3 This test method is designed to produce partially de- D1888 Methods Of Test for Particulate and Dissolved Matter
graded mixtures of municipal solid waste and plastics that can in Water (Withdrawn 1989)4
be used to assess the ecotoxicological risks associated with the D2908 Practice for Measuring Volatile Organic Matter in
anaerobic degradation of plastics after various stages of an- Water by Aqueous-Injection Gas Chromatography
aerobic biodegradation in a landfill. D3590 Test Methods for Total Kjeldahl Nitrogen in Water
1.4 Claims of performance shall be limited to the numerical D4129 Test Method for Total and Organic Carbon in Water
result obtained in the test and not be used for unqualified by High Temperature Oxidation and by Coulometric
Detection
E260 Practice for Packed Column Gas Chromatography
1
This test method is under the jurisdiction of ASTM Committee D20 on Plastics
and is the direct responsibility of Subcommittee D20.96 on Environmentally
3
Degradable Plastics and Biobased Products. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Sept. 15, 2018. Published October 2018. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1994. Last previous edition approved in 2012 as D5526 – 12. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/D5526-18. the ASTM website.
2 4
The boldface numbers in parentheses refer to the list of references at the end of The last approved version of this historical standard is referenced on
this standard. www.astm.org.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States

1
 D5526 − 18
E355 Practice for Gas Chromatography Terms and Relation- the environmental hazards posed by the breakdown of plastics
ships to varying degrees in landfills. This test method has been
2.2 APHA-AWWA-WPCF Standards:5 designed to assess biodegradation under optimum and less-
2540D Total Suspended Solids Dried at 103°–105°C than-optimum conditions.
2540E Fixed and Volatile Solids Ignited at 550°C
5.3 Limitations—Because a wide variation exists in the
212 Nitrogen Ammonia
construction and operation of landfills, and because regulatory
3. Terminology requirements for landfills vary greatly, this procedure is not
intended to simulate the environment of all landfills. However,
3.1 Definitions—For definitions of terms used in this test it is expected to closely resemble the environment of a
method see Terminology D883. biologically active landfill. More specifically, the procedure is
3.2 Definitions of Terms Specific to This Standard: intended to create a standard laboratory environment that
3.2.1 methanogenic inoculum—anaerobically digested or- permits rapid and reproducible determination of the anaerobic
ganic waste containing a high concentration of anaerobic biodegradability under accelerated landfill conditions, while at
methane-producing microorganisms. the same time producing reproducible mixtures of fully and
partially decomposed household waste with plastic materials
4. Summary of Test Method for ecotoxicological assessment.
4.1 This test method described consists of the following: (1)
selecting and analyzing material for testing; (2) obtaining a 6. Apparatus
pretreated municipal-solid-waste fraction and a concentrated 6.1 Pressure-Resistant Glass Vessels—Twenty-seven, each
anaerobic inoculum from an anaerobic digester; (3) exposing with a volume of 4 to 6 L, which can be closed airtight and
the material to an anaerobic static batch fermentation at more capable of withstanding an overpressure of two atmospheres.
than 30 % solids; (4) measuring total carbon in the gas (CO2 The lids of the reactors are equipped with an overpressure
and CH4) evolved as a function of time; (5) removing the valve (to prevent the overpressure from becoming higher than
specimens for cleaning (optional), conditioning, testing, and 2 bars), a manometer that provides a rough indication of the
reporting; (6) assessing the degree of biodegradability; and (7) overpressure, a septum that allows one to take gas samples and
assessing the degree of biodegradability under less than opti- measure the exact overpressure, and, finally, a valve to release
mum conditions. the overpressure (Fig. 1).
4.2 The percentage of biodegradability is obtained by de-
termining the percent of conversion of carbon from the test 6.2 Incubators, sufficient to store the vessels in the dark at
material to carbon in the gaseous phase (CH4 and CO2). This 35 6 2°C for the duration of the test.
percentage of biodegradability will not include the amount of 6.3 Pressure Transducer, connected to a syringe needle to
carbon from the test substance that is converted to cell biomass measure the headspace pressure in the test vessel.
and that is not, in turn, metabolized to CO2 and CH4.
6.4 Gas Chromatograph, or other apparatus, equipped with
5. Significance and Use a suitable detector and column(s) for measuring methane and
carbon dioxide concentrations in the evolved gases.
5.1 Decomposition of a plastic within a landfill involves
biological processes that will affect the decomposition of other 6.5 pH Meter, precision balance (60.1 g), analytical balance
materials enclosed by, or in close proximity to, the plastic. (60.1 mg), thermometer, and barometer.
Rapid degradation of the plastic has the ability to increase the
6.6 Suitable Devices, for determining volatile fatty acids by
economic feasibility of landfill-gas recovery, minimize the
aqueous-injection chromatography, total Kjeldahl nitrogen,
duration of after-care of the landfill, and make possible the
ammonia nitrogen, dry solids (105°C), and volatile solids
recovery of the volume reduction of the waste due to biodeg-
(550°C) concentrations.
radation during the active life of the landfill. This procedure
has been developed to permit determination of the anaerobic
7. Reagents and Materials
biodegradability of plastic products when placed in biologi-
cally active environments simulating landfill conditions. 7.1 Pretreated-Household Waste, derived from mixed mu-
5.2 As degradation occurs inevitably in a landfill, it is of nicipal solid waste or the organic fraction thereof, after
immediate concern that the plastic materials do not produce homogenizing, screening over a screen with holes of a diam-
toxic metabolites or end products under the various conditions eter of 40 to 80 mm, and aerobically stabilized over a period of
that have the potential to occur in a landfill. The mixtures 2 to 4 weeks by blowing air into the material and maintaining
remaining after completion of the test method, containing fully a dry-matter content of 50 6 5 % and a temperature of 55 6
or partially degraded plastic materials or extracts, can be 10°C. (Optional: the pretreated household waste can be re-
submitted subsequently to ecotoxicity testing in order to assess placed by a similarly pretreated simulated solid waste.)
7.2 Anaerobic Inoculum, derived from a properly operating
5 th
Standard Methods for the Examination of Water and Wastewater, 20 ed.,
anaerobic digester with pretreated household waste as a sole
1999, available from American Public Health Association, 800 I Street, NW, substrate or a digester that treats predominantly household
Washington, D.C. 20001-3710, or http://www.standardmethods.org. waste.

2
 D5526 − 18
8.4 The methane produced during the procedure is explo-
sive and flammable. Upon release of the biogas from the
gas-collection system, take care in venting the biogas to the
outside or to a hood.
9. Inoculum
9.1 The inoculum can be derived either from a laboratory-
scale or full-scale continuous digester or batch digester, oper-
ating at 35°C and functioning with an organic fraction of
household waste as the predominant substrate. In case the
inoculum is derived from a continuous laboratory-scale or
full-scale digester, the digester must be operating for a period
of at least one month on the organic fraction of household
waste, with a maximum retention time of 30 days under
mesophilic conditions (35 6 2°C). Gas production yields must
be at least 15 mL at standard temperature and pressure of
biogas/gram of dry solids in the digester and per day for at least
7 days. In case the inoculum is derived from a batch digester,
the gas production rate must have exceeded 1 L/kg waste/day,
and the methane concentration of the biogas being produced
must be above 60 %.
9.2 The prepared inoculum needs to undergo a short meso-
philic post-fermentation of approximately 7 days at the same
dry-matter content as the digester from which it was derived.
This means that the inoculum is not fed but is allowed to
1 = Digester.
post-ferment anaerobically by itself. This is to ensure that
2 = Incubation chamber. large, easily biodegradable particles are degraded during this
3 = Overpressure valve. period and also to reduce the background level of degradation
4 = Manometer.
5 = Septum.
of the inoculum itself.
6 = Valve. 9.3 The biochemical characteristics of the inoculum shall be
FIG. 1 Setup of Accelerated Landfill as follows:
9.3.1 pH—Between 7.5 and 8.5 (in accordance with Test
Methods D1293);
9.3.2 Volatile Fatty Acids (VFA)—Below 1 g/kg wet weight
7.3 Cellulose, Analytical-Grade, for thin-layer chromatog-
(in accordance with Practice D2908); and
raphy as a positive control.6
9.3.3 NH+ 4- N—Between 0.5 and 2 g/kg (in accordance
7.4 Polyethylene (optional), as a negative control. It needs with APHA Test 212 and Test Method D3590).
to be in the same form as that in which the sample is tested:
9.4 Analyses are performed after dilution of the inoculum
film polyethylene for film samples, pellets of polyethylene in
with distilled water on a ratio of distilled water to inoculum of
case the sample is in the form of pellets, etc.
5 to 1 on a weight-over-weight basis.
8. Hazards 10. Test Specimen
8.1 This procedure involves the use of inoculum and mu- 10.1 The test specimen needs to be of sufficient carbon
nicipal solid waste containing biologically and possibly chemi- content, analyzed in accordance with Test Method D4129, to
cally active materials known to produce a variety of diseases. yield carbon dioxide and methane volumes that can be mea-
Avoid contact with these materials by wearing gloves and other sured accurately by the trapping devices described. Add more
appropriate protective equipment. Use good personal hygiene test specimen when low biodegradability is expected, up to 100
to minimize exposure. g of dry matter of the test specimen.
8.2 It is possible that the solid waste mixture will contain 10.2 It is acceptable for the test specimen to be in the form
sharp objects. Take extreme care when handling this mixture to of films, powder, pellets, or formed articles, or in the form of
avoid injury. a dog bone and in accordance with Practice D618.
8.3 This test method includes the use of hazardous chemi- 11. Procedure
cals. Avoid contact with the chemicals and follow the manu-
facturer’s instructions and material safety data sheets. 11.1 Preparation of the Mixtures:
11.1.1 Determine the volatile solids, dry solids, and nitrogen
content of the pretreated household waste and the inoculum in
6
Avicel®, available from EM Chemicals, Inc., Hawthorne, NY, was used for accordance with Test Methods D1888, D3590, and APHA
development of this test method. 2540D and 2540E.

3
 D5526 − 18
11.1.2 Determine the volatile solids, dry solids, and carbon 11.4.3 Remove sufficient residual material from the vessel
content of all test substances in accordance with APHA 2540D and submit to ecotoxicity testing, in accordance with appropri-
and 2540E and Test Method D4129. ate standard test methods and practices (optional).
11.1.3 Weigh and combine the components and adjust the
dry matter content of the final mixtures with water to reach the 12. Calculation
desired dry-matter content for each vessel. Roughly weigh out
12.1 By using the total carbon in the test specimen, calculate
600 g on a dry-weight basis of pretreated household waste, and
the maximum theoretical gas production (carbon dioxide plus
mix it with 100 g on a dry-weight basis of mesophilic
methane) originating from the anaerobic biodegradation of the
anaerobic inoculum from a continuously operating digester or
test specimen, based on the following biochemical transforma-
150 g on a dry-weight basis of anaerobic inoculum from a
tions:
batch digester. Add 60 to 100 g of dry matter of the test
substance. Add water until the appropriate final dry matter C12H 2 →CH4 (1)
content is reached. (In order to reach 60 % dry matter content
C1O 2 →CO2
in the mixture, it is necessary, in some cases, to have water
removed prior to combining the different components of the Each millimole (12 mg) of organic carbon from the test
mixture. This can be accomplished by drying the pretreated sample can be converted into 1 mmole of gaseous CH4 or CO2,
household waste or centrifuging the anaerobic inoculum.) Mix or both. One millimole of gas produced occupies 22.4 mL at
the required amounts of pretreated household waste, inoculum, standard temperature and pressure (STP).
and test substance in a small container for 2 to 3 min. Introduce
12.2 Temperature and Pressure—Measure the percentages
the mixture in the vessel, weigh the vessel with all of the
of CH4 and CO2, and transform the gas volumes to STP. Also
contents, and close it airtight. Prepare the pressure vessels in
correct for vapor pressure and atmospheric pressure variation
the triplicate at each of the following dry matter contents: 35,
during the test. Calculate the amount of gaseous carbon.
45, and 60 %, so nine vessels are necessary for each sample.
Determine the mean (of the three replicates) net gaseous
11.1.4 The blanks consist of 600 g dry matter of pretreated
carbon production by anaerobic biodegradation of the test
household waste and anaerobic inoculum at the respective total
substances by subtracting the mean gaseous carbon production
dry-matter contents. As references, thin-layer chromatography
of the control (three replicates) containing only the inoculum.
cellulose must be used as a positive control. The blank and
reference are performed in triplicate at the three different 12.3 Calculate the percent of biodegradation for each dry-
dry-matter contents. matter concentration by dividing the average net gaseous-
11.2 Start-Up Procedure—After all reactors are filled and carbon production of the test material by the original average
closed, place them in incubators at 35 6 2°C. Acclimate the amount of total carbon of the test compound and multiplying
reactors for approximately 1 h and release the pressure, which by 100.
originates from the temperature increase, to the atmosphere. mean C g ~ test! 2 mean C g ~ blank!
% biodegradation 5 3 100 (2)
Incubate the reactors in the dark for a period of four months. Ci
11.3 Operating Procedure: where:
11.3.1 Check the gas production (measured as a pressure Cg = amount of gaseous carbon produced, g, and
increase) at least weekly. When the overpressure reaches more Ci = amount of carbon in test compound added, g.
than 700 mbar, measure the pressure exactly with the pressure
Calculate the standard error, se, of the percentage of biodeg-
transducer and release to atmospheric pressure. Take care that
radation as follows:
the temperature decrease, due to the opening of the incubator
2
or incubation room, is not more than 1°C during measurement s e 5 SQRT ~~ s test /n1 ! 1 ~ s 2blank /n2 !! 3 100/C i (3)
of the overpressure. where:
11.3.2 Analyze the gas composition biweekly. Determine
n1 andn2 = number of replicate test and blank digesters,
the methane and carbon dioxide concentration by using ana-
respectively, and
lytical devices suitable for the detection and quantification of
s = standard deviation of the total gaseous carbon
these gases, such as a gas chromatograph with an appropriate
produced.
detector, conforming to Practices E260 and E355. Pay special
attention to the occurrence of leaks through the septum. Calculate the 95 % confidence limits as follows:
11.4 End of the Test: 95 % CL 5 % biodegradation6 ~ t 3 s e ! (4)
11.4.1 It is acceptable for the incubation time to be where:
extended, depending on the activity of the inoculum, until no
t = t-distribution value for 95 % probability with
significant gas production in excess of the blank has been
(n1 + n2 − 2) degrees of freedom; thus n = 3 + 3 − 2 = 4.
recorded during one week or until the positive reference has
degraded for more than 70 %.
11.4.2 At the end of the test, analyze the dry matter, volatile 13. Interpretation of Results
fatty acids, and pH for each of the reactor mixtures, in 13.1 Information on the toxicity of the plastic material is
accordance with APHA 2540E, Practice D2908, and Test potentially useful for interpreting inhibitory effects on the
Methods D1293. inoculum.

4
 D5526 − 18
13.2 This test method includes the use of thin-layer chro- 14.1.10 Temperature range of the test.
matography cellulose as a positive control. If sufficient biodeg- 14.1.11 Wet-weight loss (optional).
radation (a minimum of 70 % for cellulose) is not observed
within the duration of the test, the test must be regarded as 15. Precision and Bias
invalid and needs to be repeated with fresh inoculum. 15.1 The precision and bias of the procedure presented in
this test method for measuring the anaerobic biodegradation of
14. Report plastic materials under accelerated landfill conditions is being
14.1 Report the following data and information: determined.
14.1.1 Information on the inoculum, including source, pH, 15.2 Preliminary results at 40 % dry-matter content and
volatile fatty acids (in milligram per kilogram wet weight), 35°C are presented in Fig. 2. The curves in Fig. 2 represent the
NH4+ -N (in gram per kilogram wet weight), percent dry solids,
percent volatile solids, date of collection and use, storage time
and conditions, handling, and potential acclimation to the test
material.
14.1.2 Information on the pretreated household waste used
to produce the inoculum and used as a substance. In case a
simulated solid waste is used, report the composition of the
mix. For both pretreated household waste and simulated solid
waste, report the source, pH, type of pretreatment, NH4+ -N (in
gram per kilogram), percent dry solids, percent volatile solids,
date of collection, storage time and conditions, handling, and
transportation.
14.1.3 Carbon content of the plastic material and the posi-
tive control and maximum theoretical gas production (carbon
dioxide and methane) for each.
14.1.4 Record and display graphically the cumulative gas
evolution over time.
14.1.5 Analysis of gas as percent methane and percent
carbon dioxide for each reading at the end of the test, or each
time the gas is released to the atmosphere during the course of
the test. Concomitantly, report the barometric pressure and FIG. 2 Cumulative Biogas Production Over a Period of 300 Days
temperature in the incubator and in the gas-collection device. for 1 kg of Pretreated Household Waste Plus 100 g of Mesophilic
Anaerobic Inoculum Without Sample (Blank) and With 60 g of
14.1.6 Record the percent of carbon conversion, along with Cellulose (Plus Positive Control)
the form of plastic material, that is, sheet, powder, pellets, etc.
Record specific information on the size, shape, volume, and
thickness of the plastic materials and control substances tested. biogas production in litres from 1 kg of pretreated household
14.1.7 Percent of biodegradation relative to cellulose. waste plus 10 % mesophilic inoculum without sample (blank)
14.1.8 Standard deviation and 95 % confidence interval for and with 60 g of cellulose (plus positive control).
the percentage of biodegradation for each triplicate set.
14.1.9 In case biogas production has not reached a plateau 16. Keywords
for the vessels at 45 and 60 % dry matter, report total biogas 16.1 accelerated landfill; anaerobic biodegradation; biodeg-
production as percentage of total biogas production at 35 % dry radation; dry digestion; ecotoxicity; landfill; metabolites; plas-
matter. tics; test method

5
 D5526 − 18
REFERENCES

(1) Campbell, D. J. V., and Croft, B.,“Landfill Gas Enhancement: Management and Technology, Vol 18, No. 1, 1990, p. 40.
Brogborough Test Cell Programme,” Landfill Gas: Energy and (4) European Patent No. 84200801.3, 06.06.1984.
Environment 90, United Kingdom Department of Energy, 1990, p. (5) U.S. Patent No. 4 684 468, 03.31.1986.
281. (6) Westlake, K., “Landfill Microbiology,” Landfill Gas: Energy and
(2) De Baere, L. A., et al., “High-Rate Dry Anaerobic Composting Environment 90, United Kingdom Department of Energy, 1990, p.
Process for the Organic Fraction of Solid Wastes,” Biotechnology and 271.
Bioengineering Symposium No. 15, John Wiley & Sons, Inc., New (7) Suflita, J. M., et al., “The World’s Largest Landfill: A Multidisci-
York, NY, 1986, p. 321. plinary Investigation,” Environmental Science and Technology, Vol
(3) De Wilde, B., et al., “Dry Anaerobic Conversion of Source Separated 26, No. 8, 1992, p. 1486.
Household Waste to Biogas and Humotex,” Journal of Resource

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