ASTM D5271-93
(Test Method)Standard Test Method for Determining the Aerobic Biodegradation of Plastic Materials in an Activated-Sludge-Wastewater-Treatment System
Standard Test Method for Determining the Aerobic Biodegradation of Plastic Materials in an Activated-Sludge-Wastewater-Treatment System
SCOPE
1.1 This test method is designed to index plastic materials which are more or less biodegradable relative to a standard in aerobic activated-sludge-treatment systems.
1.2 This test method is designed to be applicable to all plastic materials that are not inhibitory to the bacteria present in the activated sludge. Compounds with toxic properties may delay or inhibit the degradation process.
1.3 This test method measures the degree and rate of aerobic biodegradation of plastic materials (including formulation additives which may be biodegradable) on exposure to activated-sludge biomass in the concentration range from 0.1 to 2.5 g/L mixed-liquor volatile suspended solids (MLVSS) under laboratory conditions.
1.4 The high MLVSS concentration relative to other biodegradation tests has the advantage of improved repeatability and increased likelihood of more rapid adaptation or acclimation of the biomass.
1.5 This test method allows for the determination of biological nitrification and the oxidation of other non-carbon components of the plastic.
1.6 This test method does not purport to determine whether or not a plastic material will pass through primary treatment to the aeration basin of an activated-sludge wastewater-treatment plant. The size or density of the plastic material may exclude it from the secondary-treatment stage of a treatment facility.
1.7 This test method is equivalent to ISO 14851.
1.8 This standard does not purport to address all of the safety problems, 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. For a specific hazards statement, see Section 8.
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Designation: D 5271 – 93 An American National Standard
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
Standard Test Method for
Determining the Aerobic Biodegradation of Plastic Materials
in an Activated-Sludge-Wastewater-Treatment System
This standard is issued under the fixed designation D 5271; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope D 1193 Specification for Reagent Water
D 1898 Practice for Sampling of Plastics
1.1 This test method is designed to index plastic materials
D 2579 Test Methods for Total and Organic Carbon in
which are more or less biodegradable relative to a standard in
Water
aerobic activated-sludge-treatment systems.
D 3593 Test Method for Molecular Weight Averages and
1.2 This test method is designed to be applicable to all
Molecular Weight Distribution of Certain Polymers by
plastic materials that are not inhibitory to the bacteria present
Liquid Size-Exclusion Chromatography (Gel Permeation
in the activated sludge. Compounds with toxic properties may
Chromatography GPC) Using Universal Calibration
delay or inhibit the degradation process.
D 5209 Test Method for Determining the Aerobic Biodeg-
1.3 This test method measures the degree and rate of aerobic
radation of Plastic Materials in the Presence of Municipal
biodegradation of plastic materials (including formulation
Sewer Sludge
additives which may be biodegradable) on exposure to
2.2 APHA-AWWA-WPCF Standards:
activated-sludge biomass in the concentration range from 0.1
2540D Total Suspended Solids Dried at 103°–105°C
to 2.5 g/L mixed-liquor volatile suspended solids (MLVSS)
2540E Fixed and Volatile Solids Ignited at 550°C
under laboratory conditions.
1.4 The high MLVSS concentration relative to other bio-
3. Terminology
degradation tests has the advantage of improved repeatability
3.1 Definitions:
and increased likelihood of more rapid adaptation or acclima-
3.1.1 Definitions of terms applying to this test method
tion of the biomass.
appear in Terminology D 883.
1.5 This test method allows for the determination of bio-
3.1.2 biological nitrification—the process by which organic
logical nitrification and the oxidation of other non-carbon
nitrogen or ammonia salts are oxidized to nitrite (NO )or
components of the plastic.
nitrate (NO ), or both, by means of the metabolic pathways of
1.6 This test method does not purport to determine whether
microorganisms.
or not a plastic material will pass through primary treatment to
3.2 Definitions of Terms Specific to This Standard:
the aeration basin of an activated-sludge wastewater-treatment
3.2.1 mixed liquid volatile suspended solids (MLVSS)—the
plant. The size or density of the plastic material may exclude it
VSS in a completely mixed activated sludge reactor.
from the secondary-treatment stage of a treatment facility.
3.2.2 soluble organic carbon (SOC)—the TOC that is ca-
1.7 There is no similar or equivalent ISO standard.
pable of passing through a 0.45-μm pore-size filter.
1.8 This standard does not purport to address all of the
3.2.3 theoretical biochemical oxygen demand (BODT)—the
safety problems, if any, associated with its use. It is the
amount of oxygen required for complete biochemical oxidation
responsibility of the user of this standard to establish appro-
of a compound.
priate safety and health practices and determine the applica-
3.2.4 total organic carbon (TOC)—the concentration ex-
bility of regulatory limitations prior to use. For a specific
pressed in milligrams per litre of carbon atoms in solution as
hazards statement, see Section 8.
determined by Test Methods D 2579.
2. Referenced Documents 3.2.5 volatile suspended solids (VSS)—the concentration of
solids expressed in milligrams per litre in solution as defined
2.1 ASTM Standards:
by APHA-AWWA-WPCF Standard Method 2540D.
D 883 Terminology Relating to Plastics
Annual Book of ASTM Standards, Vol 11.01.
Annual Book of ASTM Standards, Vol 08.02.
This test method is under the jurisdiction of ASTM Committee D-20 on Plastics
Annual Book of ASTM Standards, Vol 11.02.
and is the direct responsibility of Subcommittee D20.96 on Environmentally
Annual Book of ASTM Standards, Vol 08.02.
Degradable Plastics. Annual Book of ASTM Standards, Vol 08.03.
Current edition approved July 15, 1993. Published September 1993. Originally Standard Methods for the Examination of Water and Wastewater, American
published as D 5271 – 92. Last previous edition D 5271 – 92. Public Health Association-American Water Works Association-Water Pollution
Annual Book of ASTM Standards, Vol 08.01. Control Federation, 17th Edition, 1989.
D 5271
3.3 Abbreviation:
3.3.1 TSS—total suspended solids.
4. Summary of Test Method
4.1 This test method consists of (1) selection of plastic
material for the determination of aerobic biodegradability, (2)
obtaining activated sludge from a wastewater-treatment plant
and preparing inoculum, (3) exposing plastic material to the
aerated inoculum, (4) measuring oxygen consumed as a result
of metabolism of the substrate, soluble organic carbon (SOC)
consumption, nitrate and nitrite determinations (when appli-
cable) and residual-polymer weight, and (5) assessing the
degree of biodegradability.
4.2 Report the percent of theoretical aerobic biodegradation
based on measured or calculated carbon, hydrogen, oxygen,
nitrogen, phosphorus, and sulfur content with respect to time
and the ultimate amount of biodegradation obtained.
5. Significance and Use
5.1 The degree and rate of aerobic biodegradability of a
plastic material in the environment determines to what extent
and in what time period that plastic may be eliminated from
certain environments. With increasing use of plastics, disposal
is becoming a major issue. This procedure estimates the degree
and time required to biodegrade plastics in an activated-sludge-
wastewater-treatment aeration basin. This test method deter-
FIG. 1 Electrolytic Respirometer
mines the degree of aerobic biodegradation by measuring the
consumption of oxygen due to respiration of the microbial
population, as a function of time when the plastic is exposed to
an inoculum of activated sewer sludge in the concentration
range from 0.1 to 1.0 g/L MLVSS. This test method is designed
to measure the oxidation of plastics containing carbon, hydro-
gen, oxygen, nitrogen, phosphorus, sulfur, chlorine, and so-
dium. Changes in the molecular weight and physical charac-
teristics of the polymer after exposure to activated-sludge
inoculum can be assessed by other ASTM test methods, such as
Test Method D 5209.
5.2 Activated sludge from a sewage treatment plant that
treats principally municipal waste is considered to be an
acceptable active aerobic inoculum available over a wide
geographical area in which to test a broad range of plastic
materials. When biodegradation in a specific activated-sludge-
wastewater-treatment system is to be determined seed should
be collected from that environment.
6. Apparatus
6.1 Respirometry Apparatus (see Figs. 1 and 2).
6.1.1 Cylindrical Glass Vessels, with a volume of 0.5 to 4.0
L, capable of forming an airtight seal.
6.1.2 Oxygen-Supply or Generation Device, that forms an
airtight seal with the cylindrical glass vessel. This may be an
electrolytic cell that generates oxygen by hydrolysis, or a
gaseous-oxygen supply in the form of pure oxygen.
6.1.3 Carbon Dioxide-(CO ) Absorbing Material, placed in
the headspace of the sealed cylindrical glass vessel. This may
FIG. 2 Pneumatic Computerized Respirometer
be KOH pellets or a KOH solution sufficient to adsorb all CO
generated due to substrate conversion and endogenous respi-
ration. The CO produced by 0.1 g of carbon will be adsorbed solution may be added as an indicator (the KOH solution will
by approximately1gof KOH. One drop of phenolphthalein turn from pink to clear when the KOH is consumed). If the
D 5271
KOH is consumed prior to the end of the test, the test should g/L; Na HPO 7H O, 33.4 g/L; and NH Cl, 1.7 g/L. The pH
2 4 2 4
be repeated with either a smaller sample size or additional should be 7.2 without further adjustment.
carbon dioxide-scrubbing capacity.
7.3.8 Sodium Hydroxide,1 N, NaOH, 40 g/L.
6.1.4 Oxygen Sensor/Regulator, that measures the oxygen 7.3.9 Sulfuric Acid,1 N,H SO , 28 mL/L.
2 4
demand in the cylindrical glass vessel and regulates the amount
7.4 Prepare the test medium so that it contains the following
of oxygen supplied to the respirometer.
stock solutions in 1 L of high-quality water: 1 mL ferric
6.1.5 Recorder, that measures and records the amount of
chloride solution, 1 mL magnesium sulfate solution, 1 mL
oxygen generated or supplied to the respirometer. This may be
calcium chloride solution, 20 mL phosphate buffer solution,
an analog- or a digital-recording device such as a strip-chart
and 10 mL ammonium bicarbonate solution.
recorder or a computer.
7.4.1 If nitrification inhibition is desired add 20 mL allylth-
6.1.6 A means of stirring the solution to maintain the
iourea solution.
MLVSS in suspension. This may be a magnetic stir bar,
mechanical stirrer, or agitation by circulating the gaseous or
8. Hazards
liquid phases.
8.1 This test method includes the use of hazardous chemi-
6.2 Analytical Equipment, to measure soluble organic car-
cals. Avoid contact with chemicals and follow manufacturers’
bon before and after the experiment is concluded (Test Meth-
instructions and Material Safety Data Sheets.
ods D 2579). Optionally, analytical equipment such as an ion
8.2 This test method includes the use of hazardous material
chromatograph to measure the nitrite and nitrate concentration
from a waste-treatment plant. Avoid contact with the sludge by
at the beginning and end of the run may be used.
using gloves and other appropriate protective equipment.
6.3 pH Meter:
Follow good laboratory practices and use good personal
6.4 Analytical Balance, to weigh the test specimen before
hygiene to minimize exposure to harmful microbial agents.
and after exposure.
6.5 Membrane Filters (0.45-μm pore size) that do not
9. Inoculum Test Organisms
generate or absorb SOC.
9.1 The source of the test organisms is activated sludge
6.5.1 The filter can be shown to not generate SOC by
freshly sampled from a well-operated municipal-sewage treat-
comparing the TOC of high-quality water before and after it
ment plant. This sewage-treatment plant should receive no
passes through the filter.
toxicants that inhibit or show adverse effects to the biomass.
6.5.2 The filter can be shown not to absorb SOC by
comparing the TOC of a centrifuged sample to the TOC of a
NOTE 1—If biodegradation in a specific treatment system is to be tested,
filtered sample. activated sludge from that municipal or industrial sewage treatment plant
preferably should be used and referenced in the results.
6.6 Centrifuge, capable of at least 1 000 g but less than
100 000 g at the tip of the centrifuge tube.
9.2 Aerate sludge in the laboratory for 4 h. Determine the
VSS in accordance with APHA-AWWA-WPCF Standard
v r
g 5 (1)
Methods 2540D and 2540E.
9.3 Adaptation of the biomass may be desirable to provide
where:
increased reproducibility. The adaptation conditions and period
g 5 acceleration, cm/s ,
must be reported in 14.1.1.
v5 angular velocity, rad/s,
9.4 Calculate the amount of seed-bacteria MLVSS to be
r/min
v5
added to each vessel:
3 2p rad, and
60 s/min
desired VSS 3 media volume
r 5 radius to tip of centrifuge tubes, cm.
volume of MLVSS,mL 5 (2)
measured VSS ~see 9.2 !
7. Reagents and Materials
NOTE 2—Desired VSS must fall within the range from 100 to 1000
7.1 High-Quality Water (see Specification D 1193), free of mg/L. (For example, if the desired VSS 5 500 mg/L and the media
volume was to be 1000 mL, if 2500 mg/L was the measured VSS, then 200
toxic substances (copper, in particular), with low-carbon con-
mL of MLVSS would be required.)
tent (<2.0 mg/L SOC) and with resistivity >18 MV/cm. The
water must never contain more than 10 % of organic carbon
10. Test Specimen
introduced by the test material.
10.1 The test specimen should be of known mass and
7.2 Phenophthalein Solution, 1 % phenolphthalein in etha-
contain at least 50 mg of carbon for each gram of VSS used.
nol.
The maximum sample weight is limited by nutrients added and
7.3 Stock Solutions, for test medium, each made up in
should not exceed 500 mg/L TOC.
high-quality water.
7.3.1 Allylthiourea,CH CHCH NHCSNH , 2.3 g/L. 10.2 The test specimens may be in the form of films, pieces,
2 2 2
fragments, or formed articles.
7.3.2 Ammonium Bicarbonate,NH HCO , 7.9 g/L.
4 3
7.3.3 Calcium Chloride, CaCl , 27.5 g/L. 10.3 Determine the carbon, hydrogen, oxygen, nitrogen,
phosphorus, and sulfur content of the sample by an appropriate
7.3.4 2-Chloro-6-(Trichloromethyl)Pyridine, 10 mg/L.
7.3.5 Ferric Chloride, FeCl 6H O, 0.25 g/L. analytical technique if it is not known from the formulation.
3 2
7.3.6 Magnesium Sulfate, MgSO 7H O, 22.5 g/L. 10.4 Optionally, record molecular weight of the polymer in
4 2
7.3.7 Phosphate Buffer,KH HPO , 8.5 g/L; K HPO , 21.75 the appropriate section.
2 4 2 4
D 5271
11. Procedure optional nitrite/nitrate analysis. Immediately filter samples
through a 0.45-μm pore-size membrane.
NOTE 3—This procedure is written for a 1000-mL media volume in a
11.12 Filter the contents of each vessel through a 50 to
1200-mL vessel. The procedure should be scaled proportionally to the
100-mesh pore-size membrane to remove remaining insoluble
volume desired. A control blank that includes all components except the
test specimen and a positive control such as starch or cellulose shall be run plastic, which is washed, dried and weighed. Molecular weight
with each set. A negative control such as polyethylene is optional. It is
may also be measured.
recommended that all specimens and controls be run at least in triplicate.
11.1 Add approximately 600 mL of high-quality water to 12. Calculation
each vessel. Allow the water to warm to room temperature (23
12.1 If not known determine the chemical composition (C
c
6 2°C) with gentle mixing.
H O N P S Na X , where X 5 chlorine, bromine, and
h o n p s na x
11.2 To each vessel add: 1 mL ferric chloride solution, 1 mL
fluorine) of the test material from elemental analysis. This
magnesium sulfate solution, 1 mL calcium chloride solution,
allo
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