SIST EN ISO 14852:2004
(Main)Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium - Method by analysis of evolved carbon dioxide (ISO 14852:1999)
Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium - Method by analysis of evolved carbon dioxide (ISO 14852:1999)
Bestimmung der vollständigen aeroben Bioabbaubarkeit von Kunststoff-Materialien in einem wässrigen Medium - Verfahren mittels Analyse des freigesetzten Kohlenstoffdioxides (ISO 14852:1999)
Evaluation de la biodégradabilité aérobie ultime des matériaux plastiques en milieu aqueux - Méthode par analyse du dioxyde de carbone libéré (ISO 14852:1999)
Določanje končne aerobne biorazgradljivosti polimernih materialov v vodnem mediju – Metoda z analizo sproščenega ogljikovega dioksida (ISO 14852:1999)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14852:2004
01-oktober-2004
'RORþDQMHNRQþQHDHUREQHELRUD]JUDGOMLYRVWLSROLPHUQLKPDWHULDORYYYRGQHP
PHGLMX±0HWRGD]DQDOL]RVSURãþHQHJDRJOMLNRYHJDGLRNVLGD ,62
Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous
medium - Method by analysis of evolved carbon dioxide (ISO 14852:1999)
Bestimmung der vollständigen aeroben Bioabbaubarkeit von Kunststoff-Materialien in
einem wässrigen Medium - Verfahren mittels Analyse des freigesetzten
Kohlenstoffdioxides (ISO 14852:1999)
Evaluation de la biodégradabilité aérobie ultime des matériaux plastiques en milieu
aqueux - Méthode par analyse du dioxyde de carbone libéré (ISO 14852:1999)
Ta slovenski standard je istoveten z: EN ISO 14852:2004
ICS:
83.080.01 Polimerni materiali na Plastics in general
splošno
SIST EN ISO 14852:2004 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 14852:2004
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SIST EN ISO 14852:2004
EUROPEAN STANDARD
EN ISO 14852
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2004
ICS 83.080.01
English version
Determination of the ultimate aerobic biodegradability of plastic
materials in an aqueous medium - Method by analysis of
evolved carbon dioxide (ISO 14852:1999)
Evaluation de la biodégradabilité aérobie ultime des
matériaux plastiques en milieu aqueux - Méthode par
analyse du dioxyde de carbone libéré (ISO 14852:1999)
This European Standard was approved by CEN on 21 June 2004.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14852:2004: E
worldwide for CEN national Members.
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SIST EN ISO 14852:2004
EN ISO 14852:2004 (E)
Foreword
The text of ISO 14852:1999 has been prepared by Technical Committee ISO/TC 61 "Plastics” of
the International Organization for Standardization (ISO) and has been taken over as EN ISO
14852:2004 by Technical Committee CEN/TC 249 "Plastics", the secretariat of which is held by
IBN.
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 January 2005, and conflicting national
standards shall be withdrawn at the latest by January 2005.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Endorsement notice
The text of ISO 14852:1999 has been approved by CEN as EN ISO 14852:2004 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
2
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SIST EN ISO 14852:2004
EN ISO 14852:2004 (E)
Annex ZA
(normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions of
any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications, indicated
by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 10634 1995 Water quality - Guidance for the EN ISO 10634 1995
preparation and treatment of poorly
water-soluble organic compounds
for the subsequent evaluation of
their biodegradability in an aqueous
medium
3
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SIST EN ISO 14852:2004
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SIST EN ISO 14852:2004
INTERNATIONAL ISO
STANDARD 14852
First edition
1999-05-15
Determination of the ultimate aerobic
biodegradability of plastic materials in an
aqueous medium — Method by analysis of
evolved carbon dioxide
Évaluation de la biodégradabilité aérobie ultime des matériaux plastiques
en milieu aqueux — Méthode par analyse du dioxyde de carbone libéré
A
Reference number
ISO 14852:1999(E)
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SIST EN ISO 14852:2004
ISO 14852:1999(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.
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.
International Standard ISO 14852 was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 5,
Physical-chemical properties.
Annexes A to E of this International Standard are for information only.
© ISO 1999
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 the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
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SIST EN ISO 14852:2004
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ISO 14852:1999(E)
Introduction
With the increasing use of plastics, their recovery and disposal have become a major issue. As a first priority,
recovery should be promoted. Complete recovery of plastics, however, is difficult. For example, plastic litter, which
comes mainly from consumers, is difficult to recover completely. Additional examples of plastics which are difficult to
recover are fishing tackle, agricultural mulches and water-soluble polymers. These plastic materials tend to leak
from closed waste-management cycles into the environment. Biodegradable plastics are now emerging as one of
the options available to solve such environmental problems. Plastic materials, such as products or packaging, which
are sent to composting facilities should be potentially biodegradable. Therefore it is very important to determine the
potential biodegradability of such materials and to obtain an indication of their biodegradability in natural
environments.
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SIST EN ISO 14852:2004
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SIST EN ISO 14852:2004
INTERNATIONAL STANDARD © ISO ISO 14852:1999(E)
Determination of the ultimate aerobic biodegradability of plastic
materials in an aqueous medium — Method by analysis of evolved
carbon dioxide
WARNING — Sewage, activated sludge, soil and compost may contain potentially pathogenic organisms.
Therefore appropriate precautions should be taken when handling them. Toxic test compounds and those
whose properties are unknown should be handled with care.
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 conditions used in this International Standard do not necessarily correspond to the optimum conditions allowing
maximum biodegradation to occur, but the standard is designed to determine the potential biodegradability of plastic
materials or give an indication of their biodegradability in natural environments.
The method enables the assessment of the biodegradability to be improved by calculating a carbon balance
(optional, see annex C).
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
[2]
effects can be determined using an inhibition control or by another appropriate method (see e.g. ISO 8192 ). If
the test material is inhibitory to the inoculum, a lower test concentration, another inoculum or a pre-exposed
inoculum can be used.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 8245:1999, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved
organic carbon (DOC).
1
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SIST EN ISO 14852:2004
© ISO
ISO 14852:1999(E)
1)
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 of their biodegradability in an aqueous medium.
ISO/TR 15462:1997, Water quality — Selection of tests 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, water
and mineral salts of any other elements present (mineralization) plus new biomass
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
o
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
-2
centrifugation at 40 000 m×s for 15 min or by membrane filtration using membranes with pores of 0,2 mm to
0,45 mm diameter
3.5
theoretical amount of evolved carbon dioxide
ThCO
2
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
-2
centrifugation at 40 000 m⋅s for 15 min or by membrane filtration using membranes with pores of 0,2 mm to
0,45 mm diameter
1)
To be published. (Revision of ISO 9439:1990)
2
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SIST EN ISO 14852:2004
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ISO 14852:1999(E)
3.8
lag phase
the 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
3.9
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 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
3.11
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 adaptation and/or selection of 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 of 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/l and 2 000 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-free 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.
The level of biodegradation is determined by comparing the amount of carbon dioxide evolved with the theoretical
amount (ThCO ) and expressed in per cent. The test result is the maximum level of biodegradation, determined
2
from the plateau phase of the biodegradation curve. Optionally, a carbon balance may be calculated to give
additional information on the biodegradation (see annex C).
Unlike ISO 9439, which is used for a variety of organic compounds, this International Standard is specially designed
for the determination of the biodegradability of plastic materials. The special requirements necessary affect the
choice of the inoculum and the test medium, and there is the possibility of improving the evaluation of the
biodegradability by calculating a carbon balance.
5 Test environment
Incubation shall take place in the dark or in diffuse light in an enclosure which is free from vapours 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.
NOTE With a compost inoculum, higher temperatures may be appropriate.
3
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SIST EN ISO 14852:2004
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ISO 14852:1999(E)
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
Dissolve
anhydrous potassium dihydrogen phosphate (KH PO ) 8,5 g
2 4
anhydrous dipotassium hydrogen phosphate (K HPO ) 21,75 g
2 4
disodium hydrogen phosphate dihydrate (Na HPO ×2H O) 33,4 g
2 4 2
ammonium chloride (NH Cl) 0,5 g
4
in water (6.1) and make up to 1000 ml.
NOTE The correct composition of the solution can be checked by measuring the pH, which should be 7,4.
6.2.1.2 Solution B
Dissolve 22,5 g of magnesium sulfate heptahydrate (MgSO ×7H O) in water (6.1) and make up to 1000 ml.
4 2
6.2.1.3 Solution C
Dissolve 36,4 g of calcium chloride dihydrate (CaCl ×2H O) in water (6.1) and make up to 1000 ml.
2 2
6.2.1.4 Solution D
Dissolve 0,25 g of iron(III) chloride hexahydrate (FeCl ×6H O) in water (6.1) and make up to 1000 ml.
3 2
Prepare this solution freshly before use to avoid precipitation, or add a drop of concentrated hydrochloric acid (HCl)
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 ml of solution A;
1 ml of each of solutions B to D.
Make up to 1000 ml 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 mg/l of phosphorus and 50 mg/l of nitrogen and is therefore suitable for concentrations in the test material of
4
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SIST EN ISO 14852:2004
© ISO
ISO 14852:1999(E)
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 C:N ratio at about 40:1.
6.2.2.1 Solution A
Dissolve
anhydrous potassium dihydrogen phosphate (KH PO ) 37,5 g
2 4
disodium hydrogen phosphate dihydrate (Na HPO ×2H O) 87,3 g
2 4 2
ammonium chloride (NH Cl) 2,0 g
4
in water (6.1) and make up to 1000 ml.
6.2.2.2 Solution B
Dissolve 22,5 g of magnesium sulfate heptahydrate (MgSO ×7H O) in water (6.1) and make up to 1000 ml.
4 2
6.2.2.3 Solution C
Dissolve 36,4 g of calcium chloride dihydrate (CaCl ×2H O) in water (6.1) and make up to 1000 ml.
2 2
6.2.2.4 Solution D
Dissolve 0,25 g of iron(III) chloride hexahydrate (FeCl ×6H O) in water (6.1) and make up to 1000 ml (see second
3 2
paragraph of 6.2.1.4).
6.2.2.5 Solution E (trace-element solution, optional)
Dissolve in 10 ml of aqueous HCl solution (25 %, 7,7 mol/l), in the following sequence:
70 mg of ZnCl , 100 mg of MnCl ×4H O, 6 mg of H BO , 190 mg of CoCl ×6H O, 3 mg of CuCl ×2H O, 240 mg of
2 2 2 3 3 2 2 2 2
NiCl ×6H O, 36 mg of Na MoO ×2H O, 33 mg of Na WO ×2H O and 26 mg of Na SeO ×5H O
2 2 2 4 2 2 4 2 2 3 2
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 mg of yeast extract in
100 ml of water (6.1). Filter the solution for sterilization using membrane filters (see 7.6).
NOTE Solutions E and F are optional and are not required if a sufficient concentration of the inoculum is used, e.g.
activated sludge, soil or compost. It is recommended that 1 ml portions be prepared and kept refrigerated until use.
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.
NOTE The correct composition of the test medium can be checked by measuring the pH, which should be 7,0 ± 0,2.
6.3 Pyrophosphate solution.
Dissolve 2,66 g of sodium pyrophosphate (Na P O ) in water (6.1) and make up to 1000 ml.
4 2 7
5
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7 Apparatus
Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter.
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 . The vessels shall be located in a constant-temperature room or in a
2
thermostatted apparatus (e.g. water-bath).
7.2 CO -free-air production system, capable of supplying CO -free air at a flow rate between 50 ml/min and
2 2
100 ml/min to each test flask, held constant to within ± 10 % (see example of system, including test vessels, in
annex A).
7.3 Analytical instrument for determining carbon dioxide, consisting of any suitable apparatus with sufficient
accuracy, e.g. a CO or DIC analyser or apparatus for titrimetric determination after complete absorption in a basic
2
solution (see examples in annex B). Note that, if an analyser with an IR detector, for instance, is used, CO -free air
2
is not necessary.
7.4 Analytical equipment for measuring total organic carbon (TOC) and dissolved organic carbon (DOC)
(see ISO 8245).
7.5 Analytical balance (usual laboratory equipment).
or with membrane filters (0,45 mm pore size) which neither adsorb nor release
7.6 Centrifuge, filtration device
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 material
The test material shall be of known mass and contain sufficient carbon to yield CO in a quantity that can be
2
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 ThCO . Use a concentration of test material such that the TOC content is at least 100 mg/l. The
2
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 2 000 mg/l, 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.
NOTE The test material should preferably be used in powder form, but it may also be introduced as films, pieces,
fragments or shaped articles. The form and shape of the test material may influence its biodegradability. Similar shapes should
preferably be used if different kinds of plastic material are to be compared. If the test material is used in the form of a powder,
particles of known, narrow size distribution should be used. A particle-size distribution with the maximum at 250 mm diameter is
recommended. Also, the size of the test equipment used may depend on the form of the test material. It should be ascertained
that no substantial mechanical aberrations occur due to the test conditions, for example due to the type of stirring mechanism
used. Processing of the test material (e.g. the use of powder in the case of composites) should not influence significantly the
degradation behaviour of the material. Optionally, record the hydrogen, oxygen, nitrogen, phosphorus and sulfur contents and
the molecular mass of a polymeric test material, using for example liquid exclusion chromatography (see e.g.
[1]
ASTM D 3536-91 or any other applicable standard method). 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 poorly water-soluble compounds, see ISO 10634.
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8.2 Reference material
Use aniline and/or a well defined biodegradable polymer (for example microcrystalline cellulose powder, ashless
cellulose filters or poly-ß-hydroxybutyrate) as a reference material. If possible, the TOC, form and size should be
comparable to that of the test material.
As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same form as the test material can
optionally be used.
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 (see note to clause 5).
NOTE It may be useful to determine the colony-forming units (cfu) of the inoculum used. The test mixture should preferably
-6
contain about 10 cfu/ml.
8.3.1 Inoculum from wastewater-treatment plants
Take a sample of activated sludge 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).
[3]
Before use, determine the concentration of suspended solids (use e.g. ISO 11923 ). 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/l to 1000 mg/l in the final mixture.
NOTE 1 When biodegradation processes in a natural environment are to be simulated or when a carbon balance
determination (see annex C) is to be carried out, an inoculum concentration of 30 mg/l suspended solids is recommended. As
solid matter can interfere with the carbon balance determination, the following procedure for preparing the inoculum is
recommended. Take 500 ml of the activated sludge and homogenize for 2 min at medium speed in a blender or in a suitable
high-speed mixer. Allow to settle until the supernatant liquid contains no significant amounts of suspended matter, but in any
case for at least 30 min. Decant a sufficient volume of the supernatant liquid and add it to the test flasks to obtain a
concentration of 1 % (V/V) to 5 % (V/V) in the test medium. Avoid carrying over sludge particles.
NOTE 2 An inoculum may be pre-conditioned, but normally no pre-exposed inoculum should be used, especially in the case
of standard tests simulating biodegradation behaviour in natural environments. Depending on the purpose of the test, a pre-
exposed inoculum may also be used, provided this is clearly stated in the test report (e.g. per cent biodegradation = x %, using
pre-exposed inocula) and the method of pre-exposure detailed in the test report. Pre-exposed inocula can be obtained from
suitable laboratory biodegradation tests (see ISO/TR 15462) 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).
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 ml of the test medium (6.2.1 or 6.2.2) or in a pyrophosphate solution (6.3) which is commonly
...
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