IS/ISO-14852
DETERMINATION OF THE ULTIMATE
AEROBIC BIODEGRADABILITY OF PLASTIC
MATERIALS IN AN AQUEOUS MEDIUM -
METHOD BY ANALYSIS OF EVOLVED
CARBON DIOXIDE
INTEGRANTES:
MARLENY ROJAS SILVESTRE
KATHIA SANCHEZ MUÑOZ
LISBETH JOANNA CABRERA RUEDA
FRANKLIN OCHOA
OSCAR
1 SCOPE
This International Standard specifies a method, by measuring the
amount of carbon dioxide evolved, for the determination of the
degree of aerobic biodegradability of plastic materials, including
those containing formulation additives. The test material is
exposed in a synthetic medium under laboratory conditions to an
inoculum from activated sludge, compost or soil.
If an unadapted activated sludge is used as the inoculum, the test
simulates the biodegradation processes which occur in a natural
aqueous environment; if a mixed or pre-exposed inoculum is
used, the method can be used to investigate the potential
biodegradability of a test material.
The method applies to the following materials:
Natural and/or synthetic polymers. copolymers or mixtures
thereof.
Plastic materials which contain additives such as plasticizers,
colorants or other compounds.
Water-soluble polymers.
Materials which. under the test conditions. do not inhibit the
microorganisms present in the inoculum. Inhibitory effects can
be determined using an inhibition control or by another
appropriate method (see e.g. ISO 8192(21). If the test
material is inhibitory to the inoculum, a lower test
concentration, another inoculum or a pre-exposed inoculum
can be used.
2 NONNATIVE REFERENCES
ISO8245:1999. Water quality - Guidelines for the determination
of total organic carbon (TOC) and dissolved organic carbon
(DOC).
ISO 9439 _ ,), Water quality - EvaluatIon of ultimate aerobic
biodegradability of organic compounds in aqueous medium -
Carbon dioxide.evolution test.
ISO 10634:1995, Water quality - Guidance for the preparation
and treatment of poorly water-soluble organic compounds for
the subsequent evaluation oftheir biodegradability in an aqueous
medium.
ISO/TR 15462:1997, Water quality - Selection oftests for
biodegradability
3 DEFINITIONS
For the purposes of this International Standard, the following
definitions apply:
3.1 ultimate aerobic biodegradation.-the breakdown of an
organic compound by microorganisms in the presence of
oxygen into carbon dioxide
3.2 activated sludge.- biomass produced in the aerobic
treatment of waste water by the growth of bacteria and
other microorganisms in the presence of dissolved oxygen
3.3 concentration of suspended solids in an activated sludge.-
the amount of solids obtained by filtration or centrifugation
of a known volume of activated sludge and drying at about
105 ·C to constant mass
3.4 dissolved inorganic carbon DIC .- that part of the inorganic carbon
in water which cannot be removed by specified phase separation, for
example by centrifugation at 40 000 m S-2 for 15 min or by membrane
filtration using membranes with pores of 0,2 um to O,4511m diameter
3.5 theoretical amount of evolved carbon dioxide ThC02.- the
maximum theoretical amount of carbon dioxide evolved after
completely oxidizing a chemical compound, calculated from the
molecular formula and expressed as milligrams of carbon dioxide
evolved per milligram or gram of test compound
3.6 total organic carbon TOC .-all the carbon present In organic matter
which is dissolved or suspended in water
3.7 dissolved organic carbon DOC .-that part of the organic carbon in
water which cannot be removed by specified phase separation, for
example by centrifugation at 40000 ms-2 for 15 min or by membrane
filtration using membranes with pores of 0,2 um to 0,45 um diameter
3.8 lag phase .-the time, measured in days, from the start of a teat
until adaptation and/or selection 01 the degrading microorganisms is
achieved and the degree of biodegradation of a chemical compound
or organic matter has increased to about 10 % of the maximum level
of biodegradation
3.9 maximum level of biodegradation .-the degree of biodegradation,
measured in per cent, of a chemical compound or organic matter In a
test. above which no further biodegradation takes place during the
test
3.10 biodegradation phase.- the lime. measured in days , from the
end of the lag phase of a test until about 90 "10 of the maximum level
of biodegradation has been reached
3.11 plateau phase.- the time measured in days, from the end of the
biodegradation phase until the end of a test
3.12 pre-exposure .- the pre-incubation of an inoculum in
the presence of the chemical compound or organic matter
under test. with the aim of enhancing the ability of the
inoculum to biodegrade the test material by adaptalion
and/or selection 01 the microorganisms
3.13 pre-conditioning .-the pre-incubation of an inoculum
under the conditions of the subsequent test in the absence
of the chemical compound or organic matter under test.
with the aim of Improving the test by acclimatization 01
the microorganisms to the test conditions
4 PRINCIPLE
The biodegradability of a plastic material is determined using
aerobic microorganisms in an aqueous system . The test
mixture contains an inorganic medium, the organic test
material (the sole source of carbon and energy) with a
concentration between 100 mg/I and 2000 mg/l of organic
carbon , and activated sludge or a suspension of active soil or
compost as the inoculum. The mixture is agitated in test flasks
and aerated With carbon-dioxide-tree air over a period of time
depending on the biodegradation kinetics, but not exceeding 6
months. The carbon dioxide evolved during the microbial
degradation is determined by a suitable analytical method,
examples of which are given in annexes A and B.
5 TEST ENVIRONMENT
Incubation shall take place in the dar1l: or in diffuse light in
an enclosure which is free from vapors inhibitory to
microorganisms and which is maintained at a constant
temperature, preferably between 20 'C and 25 ·C. to an
accuracy of ± 1 ·C. or at any other appropriate temperature
depending on the inoculum used and the environment to be
assessed
6 REAGENTS
Use only reagents of recognized analytical grade .
6.1 Distilled or deionized water, free of toxic substances (copper in particular) and containing
less than 2 mg/l of DOC.
6.2 Test medium.
Depending on the purpose of the test, different test media may be used. For example, if
simulating a natural environment use the standard test medium (6.2.1). If a test material is
used at higher concentrations, use the optimized test medium (6.2.2) with higher buffering
capacity and nutrient concentrations.
6.2.1 Standard test medium
6.2.1.1 Solution A
anhydrous potassium dihydrogen phosphate (KH2P04)
anhydrous dipotassium hydrogen phosphate (K2HP04)
disodium hydrogen phosphate dihydrate (Na2HP04·2H20)
Ammonium chloride (NH4CI)
in water (6.1) and make up to 1000 ml.
6.2.1.2 Solution B
DissoIve 22.5 g. of magnesium sulfate heptahydrate (MgS04·7H20) in
water (6.1) and make up to 1000 ml.
6.2.1.3 Solution C
Dissolve 36.4 g. of calcium chloride dihydrate (CaCI2·2H20) in water
(6.1) and make up to 1000 ml.
6.2.1.4 Solution D
Dissolve 0,25 g. of iron(llI) chloride hexahydrate (FeCIJ6H20) in water
(6.1) and make up to 1000 ml.
Prepare this solution freshly before use to avoid precipitation, or add a
drop of concentrated hydrochloric acid (HCI) or a drop of 0,4 g/l aqueous
solution of ethylenediaminetetraacetic acid (EDTA).
6.2.1.5 Preparation
To prepare 1 litre of test medium. add, to about 500 ml of water (6.1),
10 mI of solution A;
1 mI of each of solutions 8 to D.
Make up to 1000 mI with water (6.1).
6.2.2 Optimized test medium
This optimized medium is highly buffered and contains more inorganic
nutrients. This is necessary to keep the pH constant in the system during
the test. even at high concentrations of the test material. The medium
contains about 2400 mgI1 of phosphorus and 50 mg/l of nitrogen and is
therefore suitable for concentrations in the test material of up to 2000
mg/l of organic carbon . If higher or lower test-material concentrations
are used . increase or decrease respectively the nitrogen content to keep
the CN ratio at about 40.
6.2.2.1 Solution A
Dissolve
anhydrous potassium dihydrogen phosphate (KH2P0 4 ) 37,5g
disodium hydrogen phosphate dihydrate (Na2HP0 4·2H20 ) 87,3 g
ammonium chloride (NH4CI)
in water (6.1) and make up to 1000 ml.
6.2.2.2 Solution
Dissolve 22,Sg of magnesium sulfate heptahydrate (MgS04·7H20) in water (6.1) and make up to 1000 ml.
6.2.2.3 Solution C
Dissolve 36,4 g of calcium chloride dihydrate (CaCI2·2H20) in water (6.1) and make up to 1000 ml.
6.2.2.4 Solution 0
Dissolve 0,25 g of iron{lIl) chloride hexahydrate (FeCI 3·6H20) in water (6.1) and make up to 1000 ml
(see second paragraph of 6.2.1.4).
6.2.2.5 Solution E (trace-element solution, optional)
Dissolve in 10 ml of aqueous HCI solution (25 %, 7,7 mol/l), in the following sequence:
70 mg of ZnCI2• 100 mg of MnCI2-4H20. 6 mg of H3B03, 190 mg of CoCI26H20. 3 mg of CuCI2 2~O. 240
mg of N,CI2 6H20. 36 mg of Na2Mo042H20, 33 rng of Na2W04·2H20 and 26 mg of Na2Se03 5H20
and make up to 1000 ml with water (6.1).
6.2.2.6 Solution F (vitamin solution, optional)
Dissolve in 100 ml of water (6.1) 0,6 mg of biotine. 2,0 mg of niacinamide, 2.0
mg of p-aminobenzoate. 1,0 mg of panthotenic acid, 10.0 mg of pyridoxal
hydrochloride, 5,0 mg of cyanocobalamine. 2.0 mg of folic acid, 5.0 mg of
riboflavin, 5.0 mg of DL-thioctic acid and 1,0 mg of thiamine dichloride or use a
solution of 15 rng of yeast extract in 100 ml of water (6.1). Filter the solution for
sterilization using membrane filters (see 7.6).
6.2.2.7 Preparation
To prepare 1 litre of test medium, add. to about 800 ml of water (6.1).
100 ml of solution A;
1 ml of each of solutions B to D and, optionally. E and F.
Make up to 1000 ml with water (6.1) and measure the pH.
6.3 Pyrophosphate solution
Dissolve 2.66 9 of sodium pyrophosphate (Na4P207) in water (6.1) arid make up
to 1000 mi.
7 APPARATUS
Ensure that all glassware is thoroughly cleaned and, in particular. free from organic or toxic malter.
Required is usual laboratory equipment, plus the following:
7.1 Test flasks: glass vessels (e.g. bottles or conical flasks) designed to allow gas purging and shaking or
stirring. and fitted with tubing impermeable to CO 2
7.2 CO2..free-air production system, capable of supplying CO2-free air at a flow rate between 50
ml/min and 100 ml/min to each test flask, held constant to within ± 10
7.3 Analytical instrument for determining carbon dioxide, consisting of any suitable apparatus with
sufficient accuracy. e.g. a CO 2 or DIC analyser or apparatus for titrimetric determination after
complete absorption In a basic solution Note that. if an analyser with an IR detector, for instance, IS
used, CO2-free air is not necessary.
7.4 Analytical equipment for measuring total organic carbon (TOC) and dissolved organic carbon (DOC)
7.5 Analytical balance (usual laboratory equipment).
7.6 Centrifuge, or filtration device with membrane tilters (0,45 urn pore size) which neither adsorb nor
release organic carbon significantly.
7.7 pH meter (usual laboratory equipment).
7.8 Magnetic stirrer or shaking device (usual laboratory equipment).
8 PROCEDURE
8.1 Test maternal.- The test material shall be of known mass and contain sufficient
carbon to yield CO2 in a quantity that can be adequately measured by the analytical
system used . Calculate the TOC from the chemical formula or determine it by a
suitable analytical technique (e.g. elemental analysis or measurement in accordance
with ISO 8245) and calculate the ThC02 Use a concentration of test material such that
the TOC content is at least 100 mg/l. The maximum amount of test material is
limited by the oxygen supply to the test system and the test medium used. When
using the optimized test medium (6.2.2) the test-material concentration shall be such
that the TOC does not exceed about 2000 mgI1. i.e. a C:N ratio of about 40:1. If
higher concentrations are to be tested. increase the nitrogen amount in the test
medium.
8.2 Reference material.- Use aniline and/or a well defined biodegradable polymer
(for example microcrystalline cellulose powder, ashless cellulose filters or poly-
hydroxybutyrate) as a reference material. If possible, the TOC . form and size should
be comparable to that of the test material.
8.3 Preparation of the Inoculum.- Activated sludge from a sewage-treatment plant
treating predominantly domestic sewage is a suitable source of the inoculum. It is
obtained from an active aerobic environment and is available over a wide geographical
area in which a broad range of plastic materials has to be tested. Alternatively, soil
and/or compost suspensions can be used for inoculation, as with some plastic materials
the activity of fungi IS important for biodegradation . When biodegradation in a specific
waste-treatment system is to be determined, collect the inoculum from that environment
The inoculum can be prepared from the sources described in 8.3.1 and 8.3.2. or from a
mixture of these sources In order to obtain a varied and concentrated microbial flora with
sufficient biodegradation activity. If the endogenous respiration of the inoculum is too
high . stabilize the inoculum by aeration before use. Harmonize the test temperature
with the inoculum used
8.3.1 Inoculum from wastewater-treatment plants .- Take a sample of activated sludqe
collected from a well-operated sewage-treatment plant or a laboratory plant handling
predominantly domestic sewage. MIX well . keep the sample under aerobic conditions and
use preferably on the day of collection (at least within 72 .h).
Before use, determine the concentration of suspended solids (use e.g. ISO 11923(3)). If
necessary. concentrate the sludge by settling so that the volume of sludge added to the
test assay is minimal. Add a suitable volume to obtain suspended solids in the range 30
mg/I to 1000 mg/l in the final mixture.
8.3.2 Inoculum from soil and/or compost
Suspend 10 g of non-sterile, fertile soil or compost from a composting plant
treating predominantly organic waste in 100 011 of the test medium (6.2.1
or 6.2 .2) or in a pyrophosphate solution (6.3) which is commonly used in
soil microbiology. Allow to settle for about 30 min . Decant and filter the
supernatant liquid through a coarse porous filter and add the inoculum to
the test flasks to obtain a concentration of 1 % (V/I/) to 5 % ( V/~1 in the
test medium . Higher amounts of inoculum can be used if necessary. but
this may cause problems in establishing carbon balances. The use of
compost can increase the number of fungi in the lest flasks and improve the
biodegradation of plastic materials. In this case, indicate the state of the
compost used in the test report (e.g. mature compost. compost from the
hot phase at about 50 ·C
8.4 Test.- Provide a number of flasks . so that the test Includes at least the following:
a) Two test flasks for the test material (symbol FT)·
b) Two flasks for the blank (symbol Fa)‘
c) One flask for checking the inoculum activity using a reference material (symbol Fc)
And , if required.
d) One flask for checking for possible abiotic degradation or non-biological change in
the tes.t material such as by hydrolysis (symbol Fs) The test solution in Fs snail be
sterilized. for example by autoclavinq or by the addition of a suitable inorganic toxic
compound to prevent microbial activity. Use, for example, 5 ml/l of a solution
containing 10 g/l of mercury(lI) chloride (HgCI2). Add the same amount of the toxic
substance during the test if required .
e) One flask as a negative control (symbol FN) using a non-biodegradable polymeric
substance (e.g. polyethylene) in the same form as the test material.
f) One flask for checking the possible Inhibiting effect of the test material on
microbial activity (symbol F,). Take care that the ratio of carbon in the test and
reference material to nitrogen in the medium is at least about C.N =40.1. Add
nitrogen if required.
9 CALCULATION AND EXPRESSION
OFRESULTS
9.1 Calculation
9.1.1 Theoretical amount of carbon dioxide evolved by the test material
Calculate the theoretical amount of carbon dioxide (ThC02) evolved, expressed
in milligrams. using equation (1)
ThC02 = m *Xc * 44/12
9.1.2 Percentage biodegradation from CO2 evolution
Calculate the percentage biodegradation D, for the test flasks FT from the
amount of carbon dioxide evolved for each measurement interval using equation
(2)
9.2 Expression and Interpretation of results
Compile a table of carbon dioxide released and the percentage biodegradation for
each measurement interval and each test flask For each vessel. plot a curve of the
carbon dioxide evolved and a curve of the percentage biodegradation as a function
of time if comparable results are obtained for the duplicate flasks, a mean curve
may be plotted
The maximum level of biodegradation determined as the mean value of the
plateau phase of the biodegradation curve Of the highest value. E. g. when the
curve decreases on further on, slowly Increases in the plateau phase, characterizes
the degree of biodegradation of the test material. If a carbon balance has been
determined, the result of this determination characterizes the total degree of
biodegradation .
The wettability and the shape of the test material may influence the result
obtained, and hence the test procedure may be limited comparing plastic materials
of similar chemical structure.
Information on the toxicity of the lest material may be useful in the interpretation
of test results showing a low biodegradability
10 VALIDITY OF RESULTS
The test IS considered valid if
a) the degree of biodegradation of the reference material (Inoculum check Fc) is > 60 % at
the end of the test,
b) the amount of carbon dioxide Which has evolved from the blank Fe at the end of the test
does not exceed an upper Iimrting value obtained by experience (this value depends on the
amount of inoculum and is, for example, in the case of 30 mg/I dry matter. about 90 mg/I
as interlaboratory tests have shown).
If in flask F, (inhibition check, If ,included) the percentage biodegradation is < 25 % and no
significant degradation of the test material is observed. It can be assumed that the test
material is Inhibitory.
If in flask Fs (abiotic degradation check . If Included) a Significant amount (> 10 %) of
evolved carbon dioxide is observed abiotic degradation process may have taken place.
If flask FN (negative control) was Included. no significant amount of evolved carbon dioxide
shall be observed.
If these criteria are not fulfilled . repeat the lest using another pre-conditioned or pre-
exposed inoculum.
11 TNT REPORT
The test report shall contain at least the following Information
a) A reference to his International Standard.
b) all Information necessary to identify the test and reference materials.
including their TOC. ThC02. chemical composition and formula (If
known). shape . form and amount/concentration in the samples tested;
c) the main test parameters. including test volume. test medium Used,
incubation temperature and final pH ;
d) the source and amount of the Inoculum used. including details of any
pre-exposure and the state of the ~used.
e) the analytical techniques used Including methods of carbon dioxide
detection and TOC. DOC and biomass dItIlrma18boI1.
