EN ISO 17556:2012

SLOVENSKI STANDARD
01-oktober-2012
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SIST EN ISO 17556:2005
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Plastics - Determination of the ultimate aerobic biodegradability of plastic materials in soil
by measuring the oxygen demand in a respirometer or the amount of carbon dioxide
evolved (ISO 17556:2012)
Kunststoffe - Bestimmung der vollständigen aeroben biologischen Abbaubarkeit von
Kunststoffmaterialien im Boden durch Messung des Sauerstoffbedarfs in einem
Respirometer oder der Menge des entstandenen Kohlendioxids (ISO 17556:2012)
Plastiques - Détermination de la biodégradabilité aérobie ultime des matériaux plastiques
dans le sol par mesure de la demande en oxygène dans un respiromètre ou de la teneur
en dioxyde de carbone libéré (ISO 17556:2012)
Ta slovenski standard je istoveten z: EN ISO 17556:2012
ICS:
83.080.01 Polimerni materiali na Plastics in general
splošno
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 17556
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2012
ICS 83.080.01 Supersedes EN ISO 17556:2004
English Version
Plastics - Determination of the ultimate aerobic biodegradability
of plastic materials in soil by measuring the oxygen demand in a
respirometer or the amount of carbon dioxide evolved (ISO
17556:2012)
Plastiques - Détermination de la biodégradabilité aérobie Kunststoffe - Bestimmung der vollständigen aeroben
ultime des matériaux plastiques dans le sol par mesure de biologischen Abbaubarkeit von Kunststoffmaterialien im
la demande en oxygène dans un respiromètre ou de la Boden durch Messung des Sauerstoffbedarfs in einem
teneur en dioxyde de carbone libéré (ISO 17556:2012) Respirometer oder der Menge des entstandenen
Kohlendioxids (ISO 17556:2012)
This European Standard was approved by CEN on 20 July 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17556:2012: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 17556:2012) has been prepared by Technical Committee ISO/TC 61 “Plastics” in
collaboration with Technical Committee CEN/TC 249 “Plastics” the secretariat of which is held by NBN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by February 2013, and conflicting national standards shall be withdrawn
at the latest by February 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 17556:2004.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 17556:2012 has been approved by CEN as a EN ISO 17556:2012 without any modification.

INTERNATIONAL ISO
STANDARD 17556
Second edition
2012-08-15
Plastics — Determination of the ultimate
aerobic biodegradability of plastic
materials in soil by measuring the oxygen
demand in a respirometer or the amount
of carbon dioxide evolved
Plastiques — Détermination de la biodégradabilité aérobie ultime des
matériaux plastiques dans le sol par mesure de la demande en oxygène
dans un respiromètre ou de la teneur en dioxyde de carbone libéré
Reference number
ISO 17556:2012(E)
©
ISO 2012
ISO 17556:2012(E)
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 17556:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Test environment . 4
6 Materials . 4
7 Apparatus . 4
8 Procedure . 4
8.1 Preparation of the test material . 4
8.2 Preparation of the reference material . 5
8.3 Preparation of the test soil . 5
8.4 Start-up and execution of the test . 7
9 Calculation and expression of results . 9
9.1 Calculation . 9
9.2 Expression and interpretation of results .10
10 Validity of results .10
11 Test report .10
Annex A (informative) Principle of a manometric respirometer (example) .12
Annex B (informative) Example of a system for measuring the amount of carbon dioxide evolved .13
Annex C (informative) Examples of methods for the determination of evolved carbon dioxide .14
Annex D (informative) Theoretical oxygen demand (ThOD) .16
Annex E (informative) Example of a determination of the amount and the molecular mass of water-
insoluble polymer remaining at the end of a biodegradation test .17
Annex F (informative) Examples of long-term tests .18
Annex G (informative) Round-robin testing .22
Bibliography .26
ISO 17556:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 17556 was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 5, Physical-
chemical properties.
This second edition cancels and replaces the first edition (ISO 17556:2003), which has been technically revised.
The main changes are as follows:
a) the introduction has been revised;
b) a definition of the term “total organic carbon” has been added (see 3.14);
c) the temperature of the test environment has been changed (see Clause 5);
d) the specifications for the analytical instrument for determining the amount of carbon dioxide evolved have
been revised (see 7.2.3);
e) Subclause 8.1 describing the preparation of the test material has been revised;
f) Subclause 8.3.1 describing the collection and sieving of soil has been revised;
g) the use of a standard soil is now permitted as an alternative to natural soil (see 8.3.2);
h) Subclause 8.4 describing the start-up and execution of the test has been revised;
i) the test report has been extended (see Clause 11);
j) a new annex (Annex F) giving examples of long-term tests has been added;
k) a new annex (Annex G) giving the results of round-robin testing has been added.
iv © ISO 2012 – All rights reserved

ISO 17556:2012(E)
Introduction
A number of plastic materials and products have been designed for applications ending up in or on soil. They
have been developed for applications where biodegradation is beneficial from a technical, environmental, social
or economic standpoint. Examples can be found in agriculture (e.g. mulching film), horticulture (e.g. twines and
clips, flower pots, pins), funeral items (e.g. body bags), recreation (e.g. plastic “clay” pigeons for shooting,
hunting cartridges), etc. In many cases, recovery and/or recycling of these plastic items is either difficult or not
economically viable. Various types of biodegradable plastics have been developed which have been designed
to biodegrade and disappear in situ at the end of their useful life. Several International Standards specify test
methods for determining the ultimate aerobic or anaerobic biodegradation of plastic materials in aqueous or
compost conditions. Considering the use and disposal of biodegradable plastics, it is important to establish a
test method to determine the ultimate aerobic biodegradation of such plastic materials in soil.
INTERNATIONAL STANDARD ISO 17556:2012(E)
Plastics — Determination of the ultimate aerobic
biodegradability of plastic materials in soil by measuring the
oxygen demand in a respirometer or the amount of carbon
dioxide evolved
WARNING — Appropriate precautions should be taken when handling soil because it might contain
potentially pathogenic organisms. Toxic test compounds and those whose properties are unknown
should be handled with care.
1 Scope
This International Standard specifies a method for determining the ultimate aerobic biodegradability of plastic
materials in soil by measuring the oxygen demand in a closed respirometer or the amount of carbon dioxide
evolved. The method is designed to yield an optimum degree of biodegradation by adjusting the humidity of
the test soil.
If a non-adapted soil is used as an inoculum, the test simulates the biodegradation processes which take place
in a natural environment; if a pre-exposed soil is used, the method can be used to investigate the potential
biodegradability of a test material.
This method applies to the following materials:
— natural and/or synthetic polymers, copolymers or mixtures of these;
— plastic materials which contain additives such as plasticizers or colorants;
— water-soluble polymers.
It does not necessarily apply to materials which, under the test conditions, inhibit the activity of the microorganisms
present in the soil. Inhibitory effects can be measured using an inhibition control or by another suitable method.
If the test material inhibits the microorganisms in the soil, a lower test material concentration, another type of
soil or a pre-exposed soil can be used.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10634, Water quality — Guidance for the preparation and treatment of poorly water-soluble organic
compounds for the subsequent evaluation of their biodegradability in an aqueous medium
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11274, Soil quality — Determination of the water-retention characteristic — Laboratory methods
ISO 17556:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
ultimate aerobic biodegradation
breakdown of an organic compound by microorganisms in the presence of oxygen into carbon dioxide, water
and mineral salts of any other elements present (mineralization) plus new biomass
3.2
biochemical oxygen demand
BOD
mass concentration of dissolved oxygen consumed under specified conditions by the aerobic biological
oxidation of a chemical compound or organic matter
NOTE It is expressed as milligrams of oxygen uptake per milligram or gram of test compound.
3.3
dissolved organic carbon
DOC
that part of the organic carbon in water which cannot be removed by specified phase separation
NOTE 1 It is expressed as milligrams of carbon per 100 milligrams of test compound.
–2
NOTE 2 Typical means of separation are centrifugation at 40 000 m⋅s for 15 min or membrane filtration using
membranes with pores of diameter 0,2 µm to 0,45 µm.
3.4
theoretical oxygen demand
ThOD
maximum theoretical amount of oxygen required to oxidize a chemical compound completely, calculated from
the molecular formula
NOTE It is expressed as milligrams of oxygen uptake per milligram or gram of test compound.
3.5
theoretical amount of evolved carbon dioxide
ThCO
maximum theoretical amount of carbon dioxide evolved after completely oxidizing a chemical compound,
calculated from the molecular formula
NOTE It is expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound.
3.6
lag phase
time, measured in days, from the start of a test until adaptation and/or selection of 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
3.7
biodegradation phase
time, measured in days, from the end of the lag phase of a test until about 90 % of the maximum level of
biodegradation has been reached
3.8
maximum level of biodegradation
degree of biodegradation of a chemical compound or organic matter in a test, above which no further
biodegradation takes place during the test
2 © ISO 2012 – All rights reserved

ISO 17556:2012(E)
3.9
plateau phase
time from the end of the biodegradation phase until the end of the test
NOTE It is measured in days.
3.10
pre-conditioning
pre-incubation of soil 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 performance of the test by acclimatization of the
microorganisms to the test conditions
3.11
pre-exposure
pre-incubation of soil in the presence of the chemical compound or organic matter under test, with the aim
of enhancing the ability of the soil to biodegrade the test material by adaptation and/or selection of the
microorganisms
3.12
water content
mass of water which evaporates from the soil when the soil is dried to constant mass at 105 °C, divided by the
dry mass of the soil
NOTE This is simply the ratio between the mass of the water and that of the soil particles in a soil sample.
3.13
total water-holding capacity
mass of water which evaporates from soil saturated with water when the soil is dried to constant mass at
105 °C, divided by the dry mass of the soil
3.14
total organic carbon
TOC
amount of carbon bound in an organic compound
NOTE It is expressed as milligrams of carbon per 100 milligrams of the compound.
4 Principle
This method is designed to yield the optimum rate of biodegradation of a plastic material in a test soil by
controlling the humidity of the soil, and to determine the ultimate biodegradability of the material.
The plastic material, which is the sole source of carbon and energy, is mixed with the soil. The mixture is
allowed to stand in a flask over a period of time during which the amount of oxygen consumed (BOD) or the
amount of carbon dioxide evolved is determined. Provided the CO evolved is absorbed, the BOD can be
determined, for example, by measuring the amount of oxygen required to maintain a constant gas volume in
a respirometer flask, or by measuring either automatically or manually the change in volume or pressure (or
a combination of the two). An example of a suitable respirometer is shown in Annex A. The amount of carbon
dioxide evolved is measured at intervals dependent on the biodegradation kinetics of the test substance by
passing carbon-dioxide-free air over the soil and then determining the carbon dioxide content of the air by a
suitable method. Examples of suitable methods are given in Annexes B and C.
The level of biodegradation, expressed as a percentage, is determined by comparing the BOD with the
theoretical oxygen demand (ThOD) or by comparing the amount of carbon dioxide evolved with the theoretical
amount (ThCO ). The influence of possible nitrification processes on the BOD has to be considered. The test
is terminated when a constant level of biodegradation has been attained or, at the latest, after six months.
Unlike ISO 11266, which is used for a variety of organic compounds, this International Standard is specially
designed to determine the biodegradability of plastic materials.
ISO 17556:2012(E)
5 Test environment
Incubation shall take place in the dark or in diffused light in an enclosure which is free from vapours toxic to
microorganisms and is maintained at a temperature constant to within ±2 °C in the range between 20 °C and
28 °C, preferably 25 °C.
6 Materials
6.1 Distilled water, containing less than 2 mg of DOC per litre.
6.2 Carbon dioxide absorber, preferably soda lime pellets.
7 Apparatus
Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter.
7.1 Closed respirometer, including test flasks and all other necessary equipment, located in a constant-
temperature enclosure or in a thermostatically controlled apparatus (e.g. a water-bath). An example is
described in Annex A.
Any respirometer capable of determining with sufficient accuracy the biochemical oxygen demand is suitable,
preferably an apparatus which measures and automatically replaces the oxygen consumed so that no oxygen
deficiency and no inhibition of the microbial activity occurs during the degradation process.
7.2 Apparatus for measuring the amount of carbon dioxide evolved
7.2.1 Test flasks: glass vessels (e.g. conical flasks or bottles), fitted with tubing impermeable to carbon
dioxide to allow purging with gas, and located in a constant-temperature enclosure or in a thermostatically
controlled apparatus (e.g. a water-bath).
7.2.2 CO -free-air production system, capable of supplying CO -free air at a flow rate of several ml/min
2 2
to each test flask, held constant to within ±10 % (see example of system, including test vessels, in Annex B).
Alternatively, the incubation apparatus shown in ASTM D5988 may be used.
7.2.3 Analytical equipment for accurately determining carbon dioxide. Typical examples are a carbon
dioxide IR analyser, a dissolved inorganic carbon (DIC) analyser, apparatus for titrimetric determination after
complete absorption in a basic solution (see Annex C), and apparatus for the gravimetric determination of
carbon dioxide in accordance with ISO 14855-2.
7.3 Analytical balance.
7.4 pH-meter.
8 Procedure
8.1 Preparation of the test material
The test material shall be of known mass and contain sufficient carbon to yield a BOD or a quantity of carbon
dioxide that can be adequately measured by the analytical equipment 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 ThOD or ThCO (see Annexes C and D).
NOTE Although elemental analysis is generally less accurate for macromolecules than for low-molecular-mass
compounds, the accuracy is usually acceptable for the purposes of calculating the ThOD or ThCO .
4 © ISO 2012 – All rights reserved

ISO 17556:2012(E)
The amount of test material shall be sufficient to outweigh any variations in the background oxygen consumption
or any carbon dioxide evolved from the test soil: 100 mg to 300 mg of test material to 100 g to 300 g of soil is
usually adequate. The maximum amount of test material is limited by the oxygen supply to the test system. The
use of 200 mg of test material with 200 g of soil is recommended unless the soil contains an excessively large
amount of organic matter.
When using test systems based on the determination of the carbon dioxide evolved, higher test material
amounts can be used (e.g. 2 500 mg for 200 g of soil) in order to increase the difference between the test
material CO production and the blank control CO production. Furthermore, a greater amount of test material
2 2
will be required if a final mass balance determination is to be carried out (see Annex E).
Pre-aeration of the test material or the addition of inert material is recommended, if necessary, to reduce the
respiration of the soil in the blank flasks.
The test material should preferably be used in powder form, but it may also be introduced in the form of films,
fragments or shaped articles.
Test samples may be reduced in size by means of cryogenic milling.
Experiments have shown that the ultimate degree of biodegradation is almost independent of the form and
shape of the test material. The speed of biodegradation, however, does depend on the form and shape of the
material. Test materials of similar form and shape should therefore be used if different kinds of plastic material
are to be compared in tests of the same duration. If the test material is in the form of a powder, small particles
of known size distribution should be used. A particle-size distribution with its maximum at 250 µm diameter is
recommended. If the test material is not in powder form, the size of the pieces of material should not be greater
than 5 mm × 5 mm. Also, the size of the test equipment used might depend on the form of the test material.
It should be ascertained that no undesired changes are caused in the test material due to the design of the
equipment, such as grinders, used. Normally, processing of the test material will not significantly influence the
degradation behaviour of the material (e.g. the use of powder in the case of composites).
Optionally, determine the hydrogen, oxygen, nitrogen, phosphorus and sulfur contents, as well as the molecular
mass of the test material, using, for example, size exclusion chromatography. Preferably, plastic materials without
additives such as plasticizers should be tested. When the material does contain such additives, information on
their biodegradability will be needed to assess the biodegradability of the polymeric material itself.
For details on how to handle compounds with limited solubility in water, see ISO 10634.
8.2 Preparation of the reference material
Use as reference material a well-defined biodegradable polymer [microcrystalline-cellulose powder, ashless
cellulose filters or poly(β-hydroxybutyrate)]. If possible, the physical form and size of the reference material
should be comparable to that of the test material.
As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same physical form as the test
material may be used.
8.3 Preparation of the test soil
8.3.1 Collection and sieving of soil
Use natural soil collected from the surface layer of fields and/or forests. If the potential biodegradability of the test
material is to be assessed, this soil may be pre-exposed to the test material. Sieve the soil to give particles of less
than 5 mm, preferably less than 2 mm, in size and remove obvious plant material, stones and other inert materials.
It is important to remove organic solids, such as straw, as far as practicable because they can decompose
during the test and influence the results.
The soil may be pre-conditioned but, normally, pre-exposed soil should not be used, especially when
biodegradation behaviour in natural environments is being simulated. Depending on the purpose of the test,
however, pre-exposed soil may be used, provided that this is clearly stated in the test report (e.g. percent
ISO 17556:2012(E)
biodegradation = x %, using pre-exposed soil) and the method of pre-exposure detailed. Pre-exposed soil
can be obtained from suitable laboratory biodegradation tests conducted under a variety of conditions or from
samples collected from locations where relevant environmental conditions exist (e.g. contaminated areas or
industrial treatment plants).
Record the sampling site, its location, the presence of plants or previous crops, the sampling date, the sampling
depth and, if possible, the soil history, such as details of fertilizer and pesticide application.
8.3.2 Preparation of standard soil
As an alternative to the natural soil described in 8.3.1, a standard soil may be used. The composition of the
standard soil is shown in Table 1. The use of standard soil is very useful in determining the biodegradability of
plastic materials in bulky soils (loamy or clayey soils), reducing handling and aeration problems.
Table 1 — Standard-soil composition
Percentage
Constituent Remarks expressed on dry
mass basis
Industrial quartz sand Predominantly fine sand in which the size of more than 50 % of 70
the particles lies in the range 0,05 mm to 0,2 mm
Clay Kaolinite clay (containing not less than 30 % kaolinite) or 10
calcium bentonite
Natural soil See 8.3.1 16
Mature compost Use well-aerated compost from an aerobic composting plant. In 4
order to stabilize the microbial activity in the standard soil, it is
recommended that 1-year-matured compost be used. If this is
not possible, use a compost which has matured for a minimum
of 2-3 months. The compost shall be homogeneous and free
from large, inert objects, such as pieces of glass, stones or
pieces of metal. Remove them manually and then sieve the
compost through a screen of mesh size about 2 cm to 5 cm.
To the soil specified in Table 1 are added the salts listed in Table 2, preferably dissolved in water and preferably
at the moment of adjustment of the water content (see 8.3.4).
Table 2 — Added salts
Constituent g/kg of soil
KH PO 0,2
2 4
MgSO 0,1
NaNO 0,4
Urea 0,2
NH Cl 0,4
A round-robin test was carried out to validate the standard soil (see Annex G).
6 © ISO 2012 – All rights reserved

ISO 17556:2012(E)
8.3.3 Measurement of soil characteristics
Knowledge of the soil characteristics is essential for full interpretation of the results of the study. It is therefore
recommended that at least the following tests be performed on the soil selected:
a) total water-holding capacity, in accordance with ISO 11274;
b) pH of the soil, in accordance with ISO 10390;
c) organic-matter content, in accordance with ISO 10694.
8.3.4 Adjustment of the water content and the pH of the soil
Adjust the water content of the soil to a suitable value for the test material by adding an appropriate amount of
water to the soil, or by drying the soil in the air in a shaded place followed by addition of an appropriate amount
of water. Adjust the pH of the soil to between 6,0 and 8,0 if it is not already within this range.
NOTE The optimum water content of the test soil is dependent on the test material. It is usually between 40 % and
60 % of the total water-holding capacity.
It is recommended that the ratio of organic carbon in the test or reference material to nitrogen in the soil
(C:N ratio) be adjusted to at least 40:1, if it is not already at this level, so as to ensure good biodegradation. This
may be done by adding nitrogen as an aqueous solution of ammonium chloride or by using an aqueous solution
containing the salts listed in Table 2.
8.3.5 Handling and storage of the soil
Store the soil in a sealed container at 4 °C ± 2 °C until it is used in the test. Do not handle the soil in any way
that could inhibit the activity of the microorganisms in it.
It is important that ISO 10381-6 be followed to ensure that the microbial activity of the soil is not affected by sampling.
8.4 Start-up and execution of the test
Prepare the following numbers of flasks:
a) three test flasks for the test material (symbol F );
T
b) three test flasks for the blank control (symbol F );
B
c) three test flasks for checking the soil activity using a reference material (symbol F );
C
and, if required:
d) one flask for checking for possible abiotic degradation or non-biological changes in the test material
(symbol F );
S
e) one flask for checking for any possible inhibiting effect of the test material (symbol F ).
l
Place the soil (see 8.3) at the bottom of each flask and add test material (see 8.1) or reference material (see
8.2), as indicated in Table 3, to the soil. Record the mass of each flask containing this test mixture. When two
replicates are used, this shall be stated in the test report.
It is important that the test material be homogeneously mixed with the soil, in the case of powder, and as
widely spread as possible in the soil, in the case of film, to improve the contact of the test material with the
microorganisms in the soil. Also, it is recommended that the surface of the test mixture be pressed with a
spatula to improve the contact between the test material and the microorganisms in the soil.
If the abiotic-degradation check is carried out, details of the procedures used to inhibit microbial activity at the
start of the test and maintain aseptic conditions during the test shall be provided in the test report.
ISO 17556:2012(E)
Place the flasks in a constant-temperature environment (see Clause 5) and allow all the flasks to reach the
desired temperature. Make all necessary connections with the respirometer or CO -free-air production system
and start the incubation.
If measuring the oxygen consumption, take the necessary readings on the manometers (if manual) or verify
that the recorder of oxygen consumption is functioning correctly (automatic respirometer) (see Annex A).
If measuring the carbon dioxide evolved, measure (at regular intervals depending on the carbon dioxide
evolution rate) the amount of carbon dioxide evolved from each flask, using a suitable and sufficiently accurate
method (see Annexes B and C).
Table 3 — Final distribution of test and reference materials
Reference
Flask Test material Test soil
material
F Test + − +
T
F Test + − +
T
F Test + − +
T
F Blank − − +
B
F Blank − − +
B
F Blank − − +
B
F Soil activity check − + +
C
− + +
F Soil activity check
C
− + +
F Soil activity check
C
+ − −
F Abiotic-degradation check (optional)
S
+ + +
F Inhibition check (optional)
I
+ = present;  − = not present.
If the biodegradation rate is considered to have slowed down because the test soil has dried out during the
test, stop the measurements and remove the flasks from the respirometer or CO -free-air production system.
Weigh the flasks and add a suitable amount of water to the test soil to bring its water content back to its initial
value. Reconnect the flasks to the system and restart measurement of the oxygen consumed or carbon dioxide
evolved. These operations shall be conducted without inhibiting the activity of the soil microorganisms and
without influencing the measurement of oxygen consumption or carbon dioxide evolution, and the fact that they
have been carried out shall be clearly stated in the test report.
When a constant level of BOD or carbon dioxide evolution is attained (plateau phase reached) and no further
biodegradation is expected, the test is considered to be completed.
The test period should typically not exceed six months. However, if significant biodegradation is still observed
and the plateau phase has not been reached after this length of time, then the test may be extended, but not
to longer than 2 years. If the test is run for longer than six months, check periodically for possible leaks. Any
extension and any special measures taken, e.g. to ensure microbial diversity or to provide sufficient nutrients,
shall be detailed in the test report.
At the end of the test, remove the flasks and weigh them to check for any decrease in the water content of the
test soil. Optionally, the residual test material may be extracted from the soil with a suitable solvent (if this is
possible) and weighed.
8 © ISO 2012 – All rights reserved

ISO 17556:2012(E)
9 Calculation and expression of results
9.1 Calculation
9.1.1 Percentage biodegradation from oxygen consumption values
Read the oxygen consumption value for each flask, using the method given by the manufacturer for the type of
respirometer concerned. Calculate the specific biochemical oxygen demand (BOD ) of the test material using
S
Equation (1):
BODB− OD
ttB
BOD = (1)
S
ρ
T
where
BOD is the specific BOD, in milligrams per gram of test material;
S
BOD is the BOD of the flask F containing test material at time t, in milligrams per kilogram of test
t T
soil, calculated by dividing the measured oxygen consumption, in milligrams, by the amount of
test soil, in kilograms;
BOD is the BOD of the blank control flask F at time t, in milligrams per kilogram of test soil;
Bt B
ρ is the concentration of the test material in the reaction mixture of flask F , in milligrams per
T T
kilogram of test soil;
Calculate the percentage biodegradation D as the ratio of the specific biochemical oxygen demand to the
t
theoretical oxygen demand (ThOD, in milligrams per gram of test material) using Equation (2):
BOD
S
D =×100 (2)
t
ThOD
Calculate in the same way the BOD and percentage biodegradation of the reference material F and, if included,
C
the abiotic-degradation check F and the inhibition check F . For calculation of the ThOD, see Annex D.
S I
9.1.2 Percentage biodegradation from carbon dioxide evolved
9.1.2.1 Theoretical amount of carbon dioxide evolved by test material
The theoretical amount of carbon dioxide evolved by the test material (ThCO ) is given, in milligrams, by
Equation (3):
ThCO =×mw× (3)
2C
where
m is the mass of test material, in milligrams, introduced into the test system;
w is the carbon content of the test material, determined from the chemical formula or from elemental
C
analysis, expressed as a mass fraction;
44 and 12 are the relative molecular and atomic masses of carbon dioxide and carbon, respectively.
Calculate in the same way the theoretical amount of carbon dioxide evolved by the reference material and by
the mixture of test and reference material in flask F .
I
ISO 17556:2012(E)
9.1.2.2 Percentage biodegradation
Calculate the percentage biodegradation D for each test flask F from the amount of carbon dioxide evolved
t T
during each measurement interval using Equation (4):
ΣΣmm−
TB
D = ×100 (4)
t
ThCO
where
is the amount of carbon dioxide, in milligrams, evolved in the test flask F between the start of
T
Σm
T
the test and time t;
Σm is the amount of carbon dioxide, in milligrams, evolved in the blank control flask F between
B
B
the start of the test and time t;
ThCO is the theoretical amount of carbon dioxide, in milligrams, evolved by the test material.
Calculate in the same way the percentage biodegradation of the reference material in the soil activity check flask F .
C
9.2 Expression and interpretation of results
Compile a table of the BOD values or amounts of carbon dioxide measured and the percentage biodegradation
values for each point in time when measurements were made. For each flask, plot a curve of BOD or carbon
dioxide evolved as a function of time and a curve of percentage biodegradation as a function of time. If
comparable results are obtained for the duplicate flasks, the mean curve may be plotted.
The maximum level of biodegradation determined as the mean value of the plateau phase of the biodegradation
curve characterizes the degree of biodegradation of the test material.
The wettability of the test material and the shape of the pieces of test material might influence the result obtained,
and this should be taken into consideration when comparing the results obtained with different test materials.
Information on the toxicity of the test material might 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 is more than 60 % at the plateau phase or at the
end of the test;
and
b) the BOD values of, or amount of carbon dioxide evolved from, the three blanks F are within 20 % of the
B
mean at the plateau phase or at the end of the test.
If these criteria are not fulfilled, repeat the test using another pre-conditioned or pre-exposed soil.
11 Test report
The test report shall contain at least the following information:
a) a reference to this International Standard;
b) all information necessary to identify the test and reference material, including name, chemical composition
and formula (if known), ThOD, ThCO (including the method of calculation), form, particle shape,
amount/concentration in the samples tested, and content of additives (if possible);
10 © ISO 2012 – All rights reserved

ISO 17556:2012(E)
c) the history of the test material (virgin granules, converted final plastic product or aged samples), detailing
the pre-treatment conditions, if pre-treatment was carried out;
d) complete information on the soil, including source, date of collection, characteristics, amount used in the
test, storage conditions, handling and details of any pre-exposure;
e) the main test conditions, including the amount of test material used, the incubation temperature and the
duration of incubation;
f) the analytical techniques used, including the principle of the respirometer and the method used to measure
the amount of carbon dioxide evolved;
g) all other operations carried out, including any addition of water to the test mixture during the test, and the
results of analyses of the test mixture, including the water
...