EN ISO 14853:2017
(Main)Plastics - Determination of the ultimate anaerobic biodegradation of plastic materials in an aqueous system - Method by measurement of biogas production (ISO 14853:2016)
Plastics - Determination of the ultimate anaerobic biodegradation of plastic materials in an aqueous system - Method by measurement of biogas production (ISO 14853:2016)
ISO 14853:2016 specifies a method for the determination of the ultimate anaerobic biodegradability of plastics by anaerobic microorganisms. The conditions described in ISO 14853 do not necessarily correspond to the optimum conditions for the maximum degree of biodegradation to occur. The test calls for exposure of the test material to sludge for a period of up to 90 d, which is longer than the normal sludge retention time (25 to 30 d) in anaerobic digesters, although digesters at industrial sites can have much longer retention times.
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 13641). If the test material is inhibitory to the inoculum, a lower test concentration, another inoculum or a pre-exposed inoculum can be used.
Kunststoffe - Bestimmung des vollständigen anaeroben Bioabbaus von Kunststoff-Materialien in einem wässrigen Medium - Verfahren mittels Analyse der Biogasentwicklung (ISO 14853:2016)
Plastiques - Évaluation de la biodégradabilité anaérobie ultime des matériaux plastiques en milieu aqueux - Méthode par détermination de la production de biogaz (ISO 14853:2016)
L'ISO 14853:2016 spécifie une méthode pour la détermination de la biodégradabilité anaérobie ultime des plastiques par des micro-organismes anaérobies. Les conditions décrites dans l'ISO 14853:2016 ne correspondent pas nécessairement aux conditions optimales permettant d'obtenir le taux maximal de biodégradation. L'essai exige que le matériau d'essai soit exposé aux boues pendant une période allant jusqu'à 90 j, ce qui est plus long que le temps de rétention normal de la boue (25 j à 30 j) dans les digesteurs anaérobies, bien que les digesteurs sur les sites industriels puissent avoir des temps de rétention beaucoup plus longs.
La présente méthode s'applique aux matériaux suivants:
- polymères naturels et/ou synthétiques, copolymères ou mélanges de ceux-ci;
- matériaux plastiques contenant des additifs, tels que plastifiants, colorants ou autres composés;
- polymères hydrosolubles;
- matériaux qui, dans les conditions d'essai, n'ont pas d'effet inhibiteur sur les micro-organismes présents dans l'inoculum. Les effets inhibiteurs peuvent être déterminés en utilisant une substance de contrôle de l'effet inhibiteur ou par toute autre méthode appropriée (voir, par exemple, l'ISO 13641). Si le matériau d'essai a un effet inhibiteur vis-à-vis de l'inoculum, il est possible d'utiliser une plus faible concentration, un autre inoculum ou un inoculum pré-exposé.
Polimerni materiali - Ugotavljanje dokončne anaerobne biodegradacije plastičnih materialov v vodnem sistemu - Metoda z merjenjem proizvodnje bioplina (ISO 14853:2016)
Standard ISO 14853:2016 določa metodo za ugotavljanje končne anaerobne biorazgradljivosti polimernih materialov, ki jo povzročajo anaerobni mikroorganizmi. Pogoji, ki so opisani v standardu ISO 14853, niso nujno enaki idealnim pogojem, pri katerih pride do največje stopnje biorazgradljivosti. Preskus zahteva izpostavljenost preskusnega materiala blatu do 90 d, kar je dlje od običajnega časa hranjenja blata (25 do 30 d) v anaerobnih gniliščih, čeprav imajo lahko gnilišča na industrijskih površinah veliko daljši čas hranjenja.
Ta metoda se uporablja za naslednje materiale:
– naravne in/ali sintetične polimere, kopolimere ali njihove mešanice,
– polimerne materiale, ki vsebujejo dodatke, kot so mehčala, barvila ali druge spojine,
– polimere, topne v vodi,
– materiale, ki v preskusnih pogojih ne zavirajo delovanja mikroorganizmov, prisotnih v inokulumu. Zaviralne učinke je mogoče določiti na podlagi nadzora zaviranja ali z drugo ustrezno metodo (glej npr. standard ISO 13641). Če preskusni material zavira inokulum, se lahko uporabi manjša preskusna koncentracija, drug inokulum ali predhodno izpostavljeni inokulum.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2018
3ROLPHUQLPDWHULDOL8JRWDYOMDQMHGRNRQþQHDQDHUREQHELRGHJUDGDFLMHSODVWLþQLK
PDWHULDORYYYRGQHPVLVWHPX0HWRGD]PHUMHQMHPSURL]YRGQMHELRSOLQD ,62
Plastics - Determination of the ultimate anaerobic biodegradation of plastic materials in
an aqueous system - Method by measurement of biogas production (ISO 14853:2016)
Kunststoffe - Bestimmung der vollständigen anaeroben Bioabbaubarkeit von Kunststoff-
Materialien in einem wässrigen Medium - Verfahren mittels Analyse der
Biogasentwicklung (ISO 14853:2016)
Plastiques - Évaluation de la biodégradabilité anaérobie ultime des matériaux plastiques
en milieu aqueux - Méthode par détermination de la production de biogaz (ISO
14853:2016)
Ta slovenski standard je istoveten z: EN ISO 14853:2017
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.
EN ISO 14853
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2017
EUROPÄISCHE NORM
ICS 83.080.01
English Version
Plastics - Determination of the ultimate anaerobic
biodegradation of plastic materials in an aqueous system -
Method by measurement of biogas production (ISO
14853:2016)
Plastiques - Évaluation de la biodégradabilité Kunststoffe - Bestimmung der vollständigen anaeroben
anaérobie ultime des matériaux plastiques en milieu Bioabbaubarkeit von Kunststoff-Materialien in einem
aqueux - Méthode par détermination de la production wässrigen Medium - Verfahren mittels Analyse der
de biogaz (ISO 14853:2016) Biogasentwicklung (ISO 14853:2016)
This European Standard was approved by CEN on 17 October 2017.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14853:2017 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
The text of ISO 14853:2016 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 14853:2017 by
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 June 2018, and conflicting national standards shall be
withdrawn at the latest by June 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 14853:2016 has been approved by CEN as EN ISO 14853:2017 without any modification.
INTERNATIONAL ISO
STANDARD 14853
Second edition
2016-07-15
Plastics — Determination of the
ultimate anaerobic biodegradation of
plastic materials in an aqueous system
— Method by measurement of biogas
production
Plastiques — Évaluation de la biodégradabilité anaérobie ultime des
matériaux plastiques en milieu aqueux — Méthode par détermination
de la production de biogaz
Reference number
ISO 14853:2016(E)
©
ISO 2016
ISO 14853:2016(E)
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
ISO 14853:2016(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Reagents and materials . 3
6 Apparatus . 5
6.1 Laboratory equipment . 5
6.2 Apparatus for use when biogas is measured by a manometric method . 6
6.3 Apparatus for use when biogas is measured by a volumetric method . 6
7 Procedure. 6
7.1 General . 6
7.2 Digested sludge . 6
7.3 Preparation of the inoculum . 7
7.4 Preparation of test suspensions and controls . 7
7.5 Incubation and gas measurement . 8
7.6 Test duration . 9
7.7 Measurement of inorganic carbon . 9
7.8 Specific analyses . 9
8 Calculation and expression of results . 9
8.1 Amount of carbon in headspace . 9
8.2 Calculation of amount of carbon in headspace when manometric measurement
method is used .10
8.3 Calculation of amount of carbon in headspace when volumetric measurement
method is used .11
8.4 Amount of inorganic carbon in the liquid .11
8.5 Total amount of carbon converted to gas .11
8.6 Amount of carbon in test material .12
8.7 Calculation of percentage biodegradation .12
9 Validity of results .12
9.1 Maintenance of anaerobic conditions .12
9.2 Inhibition of degradation .12
9.3 Validity of the test .12
10 Test report .13
Annex A (informative) Example of apparatus for determining the amount of biogas
produced by measuring the increase in gas pressure .14
Annex B (informative) Example of apparatus for determining volumetrically the amount of
biogas produced .15
Annex C (informative) Example of a biodegradation curve .17
Annex D (informative) Examples of data sheets for anaerobic biodegradability tests .18
Annex E (informative) Table of water vapour pressures at various temperatures .21
Annex F (informative) Calculation of theoretical carbon dioxide (ThCO ) and theoretical
methane (ThCH ) production .22
Annex G (informative) Example of determination of recovery rate .23
Annex H (informative) Example of a workflow scheme .26
ISO 14853:2016(E)
Bibliography .28
iv © ISO 2016 – All rights reserved
ISO 14853:2016(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 61, Plastics, Subcommittee SC 5, Physical-
chemical properties.
This second edition cancels and replaces the first edition (ISO 14853:2005), which has been technically
revised. It also incorporates the Technical Corrigendum ISO 14853:2005/Cor.1:2009.
ISO 14853:2016(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. For example, plastic litter, which originates mainly from
consumers, is difficult to recover completely. Additional examples of materials difficult to recover
are found in the disposal of fishing tackle, agricultural mulch films and water-soluble polymers.
These plastic materials tend to leak from closed waste management infrastructures into natural
environments. Biodegradable plastics are now emerging as one of the available options to solve such
environmental issues. Plastic materials, such as products or packaging, which are sent to anaerobic
treatment facilities should be potentially biodegradable. Therefore, it is very important to determine
the potential biodegradability of such materials and to obtain a quantitative measure of their
biodegradability in anaerobic environments.
vi © ISO 2016 – All rights reserved
INTERNATIONAL STANDARD ISO 14853:2016(E)
Plastics — Determination of the ultimate anaerobic
biodegradation of plastic materials in an aqueous system
— Method by measurement of biogas production
WARNING — Sewage and activated sludge may contain potentially pathogenic organisms.
Therefore, appropriate precautions should be taken when handling them. Digesting sewage
sludge produces flammable gases which present fire and explosion risks. Care should be taken
when transporting and storing quantities of digesting sludge. Toxic test chemicals and those
whose properties are not known should be handled with care and in accordance with safety
instructions. The pressure meter and microsyringes should be handled carefully to avoid needle
stick injuries. Contaminated syringe needles should be disposed of in a safe manner.
1 Scope
This International Standard specifies a method for the determination of the ultimate anaerobic
biodegradability of plastics by anaerobic microorganisms. The conditions described in this
International Standard do not necessarily correspond to the optimum conditions for the maximum
degree of biodegradation to occur. The test calls for exposure of the test material to sludge for a period
of up to 90 d, which is longer than the normal sludge retention time (25 to 30 d) in anaerobic digesters,
although digesters at industrial sites can have much longer retention times.
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 13641). 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
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
ultimate anaerobic biodegradation
breakdown of an organic compound by microorganisms in the absence of oxygen to carbon dioxide,
methane, water and mineral salts of any other elements present (mineralization) plus new biomass
3.2
primary anaerobic biodegradation
structural change (transformation) of a chemical compound by microorganisms, resulting in the loss of
a specific property
ISO 14853:2016(E)
3.3
digested sludge
mixture of settled sewage and activated sludge which have been incubated in an anaerobic digester at
about 35 °C to reduce the biomass and odour and to improve the dewaterability of the sludge
Note 1 to entry: Digested sludge contains an association of anaerobic fermentative and methanogenic bacteria
producing carbon dioxide and methane.
3.4
concentration of suspended solids in digested sludge
amount of solids obtained by filtration or centrifugation of a known volume of activated sludge and
drying at about 105 °C to constant mass
3.5
dissolved organic carbon
DOC
organic carbon in the water phase which cannot be removed by specified phase separation, for example,
–2
by centrifugation at 40 000 m⋅s for 15 min or by membrane filtration using membranes with pores of
0,2 µm to 0,45 µm diameter
3.6
inorganic carbon
IC
inorganic carbon which is dissolved or dispersed in the aqueous phase of a liquid and is recoverable
from the supernatant liquid after the sludge has been allowed to settle
3.7
total dry solids
amount of solids obtained by taking a known volume of test material or inoculum and drying at about
105 °C to constant mass
3.8
theoretical amount of evolved biogas
Thbiogas
maximum theoretical amount of biogas (CH + CO ) evolved after complete biodegradation of an
4 2
organic material under anaerobic conditions, calculated from the molecular formula and expressed as
millilitres of biogas evolved per milligram of test material under standard conditions
3.9
theoretical amount of evolved carbon dioxide
ThCO
maximum theoretical amount of carbon dioxide evolved after complete oxidation of an organic material,
calculated from the molecular formula and expressed as milligrams of carbon dioxide per milligram of
test material
3.10
theoretical amount of evolved methane
ThCH
maximum theoretical amount of methane evolved after complete reduction of an organic material,
calculated from the molecular formula and expressed as milligrams of methane evolved per milligram
of test material
3.11
lag phase
lag period
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
2 © ISO 2016 – All rights reserved
ISO 14853:2016(E)
3.12
plateau phase
time, measured in days, from the end of the biodegradation phase until the end of the test
3.13
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
3.14
maximum level of biodegradation
degree of biodegradation, measured in percent, of a chemical compound or organic matter in a test,
above which no further biodegradation takes place during the test
4 Principle
The biodegradability of a plastic material is determined using anaerobic conditions in an aqueous
system. Test material with a concentration of 20 mg/l to 200 mg/l organic carbon (OC) is incubated
at (35 ± 2) °C in sealed vessels together with digested sludge for a period normally not exceeding 90 d.
Before use, the digested sludge is washed so that it contains very low amounts of inorganic carbon
(IC) and diluted to 1 g/l to 3 g/l total solids concentration. The increase in headspace pressure or the
volumetric increase (depending on the method used for measuring biogas evolution) in the test vessels
resulting from the production of carbon dioxide (CO ) and methane (CH ) is measured. A considerable
2 4
amount of CO will be dissolved in water or transformed to bicarbonate or carbonate under the
conditions of the test. This inorganic carbon (IC) is measured at the end of the test. The amount of
microbiologically produced biogas carbon is calculated from the net biogas production and the net IC
formation in excess of blank values. The percentage biodegradation is calculated from the total amount
of carbon transformed to biogas and IC and the measured or calculated amount of carbon added as
test material. The course of biodegradation can be followed by making intermediate measurements
of biogas production. As additional information, the primary biodegradability can be determined by
specific analyses at the beginning and end of the test.
This test method is designed to determine the biodegradability of plastic materials under anaerobic
conditions. Optionally, the assessment of the recovery rate may also be of interest (see Annex G).
5 Reagents and materials
5.1 Distilled or deionized water, free of toxic substances, containing less than 2 mg/l of DOC.
5.2 Test medium, prepared using only reagents of recognized analytical grade.
Prepare the test medium to contain the following constituents in the stated amounts:
Anhydrous potassium dihydrogen phosphate KH PO 0,27 g
2 4
Disodium hydrogen phosphate dodecahydrate Na HPO ⋅12H O 1,12 g
2 4 2
Ammonium chloride NH Cl 0,53 g
Calcium chloride dihydrate CaCl ⋅2H O 0,075 g
2 2
Magnesium chloride hexahydrate MgCl ⋅6H O 0,10 g
2 2
Iron (II) chloride tetrahydrate FeCl ⋅4H O 0,02 g
2 2
Resazurin (oxygen indicator) 0,001 g
a
Disodium sulfide nonahydrate Na S⋅9H O 0,1 g
2 2
Stock solution of trace elements (optional) 10 ml
Stock solutions of vitamins (optional) Vitamin solution No. 1 0,5 ml
ISO 14853:2016(E)
Anhydrous potassium dihydrogen phosphate KH PO 0,27 g
2 4
Vitamin solution No. 2 0,5 ml
Add water (5.1) (oxygen-free) to 1 l
a
Use freshly prepared sodium sulfide, or wash and dry it before use, to ensure sufficient reductive capacity.
In order to ensure strictly anaerobic conditions, it is recommended that a small amount of sodium dithionite be
added to the medium after it has been prepared until it becomes colourless. Do not use more than 10 mg/l because
higher concentrations may produce inhibitory effects.
Adjust the pH of the medium with dilute mineral acid or alkali, if necessary, to 7 ± 0,2.
To ensure oxygen-free conditions, purge the water with nitrogen for about 20 min immediately before use.
5.3 Trace-element solution (optional).
It is recommended that the test medium be supplemented with the following trace elements to improve
the anaerobic degradation process, especially if low inoculum concentrations are used:
Manganese chloride tetrahydrate MnCl ⋅4H O 0,05 g
2 2
Boric acid H BO 0,005 g
3 3
Zinc chloride ZnCl 0,005 g
Copper (II) chloride CuCl 0,003 g
Disodium molybdate dihydrate Na MoO ⋅2H O 0,001 g
2 4 2
Cobalt chloride hexahydrate CoCl ⋅6H O 0,1 g
2 2
Nickel chloride hexahydrate NiCl ⋅6H O 0,01 g
2 2
Disodium selenite Na SeO 0,005 g
2 3
Disodium tungstate dihydrate Na WO ⋅2H O 0,002 g
2 4 2
Add water (5.1) (oxygen free) to 1 l
Use 10 ml of trace-element solution per litre of test medium.
5.4 Vitamin solutions (optional).
5.4.1 Vitamin solution No. 1
4-Aminobenzoic acid 40 mg
d-Biotin 10 mg
Dissolve in hot water (5.1) 500 ml
Allow to cool and add:
d-Pantothenic acid, calcium salt 50 mg
Pyridoxamine dihydrochloride 150 mg
Thiamine dichloride 100 mg
Filter the solution through a membrane filter (pore size 0,45 µm) that neither adsorbs nor releases
organic carbon in significant amounts, and store in the dark at 4 °C.
Use 0,5 ml of vitamin solution per litre of test medium.
5.4.2 Vitamin solution No. 2
Cyanocobalamin (vitamin B12) 10 mg
Dissolve in water (5.1) 100 ml
4 © ISO 2016 – All rights reserved
ISO 14853:2016(E)
Filter the solution through a membrane filter (pore size 0,45 µm) that neither adsorbs nor releases
organic carbon in significant amounts, and store in the dark at 4 °C.
Use 0,5 ml of vitamin solution per litre of test medium.
5.5 Barrier solution.
NaCl 200 g
Dissolve in water (5.1) 1 000 ml
Acidify with citric acid 5 g
Add a pH indicator such as bromophenol blue or methyl orange in order to be able to verify that the
solution remains acid during the test.
5.6 Test material.
The test material is usually added directly as solid to give a concentration of 20 mg/l to 200 mg/l
organic carbon. The test material (plastic) should be used in powdered form, if possible.
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 µm diameter is
recommended. Also, the size of the test equipment used may depend on the form of the test material.
The biodegradability of plastic materials which are not inhibitory to microorganisms can be determined
using concentrations higher than 200 mg/l organic carbon. In this case, ensure that the buffer capacity
and mineral-salt content of the medium are sufficient.
5.7 Reference material.
Use a well-defined anaerobically biodegradable polymer, e.g. poly-β-hydroxybutyrate, cellulose or
poly(ethylene glycol) 400 as a reference material. If possible, the form, size, solubility and concentration
of the reference material should be comparable with that of the test material.
Prepare the reference material in the same way as the test material.
5.8 Inhibition control (optional).
Add both the test material and the reference material to a vessel containing test medium (5.2) to give
the concentrations specified in 5.6 and 5.7, respectively.
6 Apparatus
6.1 Laboratory equipment
Required is usual laboratory equipment, plus the following:
6.1.1 Incubator or water or sand bath, thermostatically controlled at (35 ± 2) °C.
6.1.2 Carbon analyser (optional), suitable for the direct determination of inorganic carbon in the
range 1 mg/l to 200 mg/l IC. Alternatively, the IC in the supernatant may be determined indirectly by
release of the dissolved IC as carbon dioxide that can be measured in the headspace, as described in 7.7.
ISO 14853:2016(E)
6.2 Apparatus for use when biogas is measured by a manometric method
6.2.1 Pressure-resistant glass test vessels, nominal size 0,1 l to 1 l, each fitted with a gastight septum
capable of withstanding about 2 000 hPa (for an example, see Annex A). The headspace volume shall be
about 10 % to 30 % of the total volume. If gas is released at regular intervals, about 10 % headspace
volume is adequate, but if gas is released only at the end of the test, 30 % is more appropriate.
From a practical point of view, the use of serum bottles sealed with butyl rubber serum caps and
crimped aluminium rings is recommended.
6.2.2 Pressure-measuring device, e.g. a manometer connected to a suitable syringe needle, with
a gastight three-way valve to facilitate the release of excess pressure. Use and calibrate the device in
accordance with the manufacturer’s instructions.
It is necessary to keep the internal volume of the tubing and the valve as low as possible so that errors
introduced by neglecting the volume of the device are not significant.
6.3 Apparatus for use when biogas is measured by a volumetric method
6.3.1 Glass test vessels (e.g. conical flasks or bottles), nominal size 0,1 l to 1 l, preferably 300 ml for
every 250 ml of medium. If foaming is not expected to occur, a headspace volume of 10 % to 20 % is
recommended. The vessels shall be equipped with a septum for gas sampling (see Annex B) and shall be
connected via gastight tubing to a graduated glass gas-collection tube which is filled with acidified salt
solution (barrier solution 5.5). This graduated glass tube shall be connected to an expansion tank which
can be moved up and down to bring the surface of the acidified solution in the expansion tank to the
same level as that in the gas-collection tube.
7 Procedure
7.1 General
Carry out the following initial operations using techniques which will ensure that the digested sludge
comes into contact with oxygen as little as practicable, e.g. work in a glove-box in an atmosphere of
nitrogen or purge the test vessels with nitrogen.
7.2 Digested sludge
Collect digested sludge from a digester at a sewage treatment plant treating predominantly domestic
sewage. Be sure to collect active sludge. Use wide-necked bottles made of high-density polyethylene or
a similar material which can expand. Glass is not recommended for safety reasons. Fill the bottles to
within 1 cm of the top and seal. After transport to the laboratory, use directly or place in a laboratory-
scale digester. Release excess biogas.
Alternatively, use a laboratory-grown anaerobic sludge as a source of the inoculum.
Consider pre-incubation of the sludge to reduce background gas production and to decrease the
influence of the blanks. Allow the sludge to digest, without the addition of any nutrients or substrates,
at (35 ± 2) °C for up to 7 d.
It has been shown that pre-incubation for about 5 d gives an optimum decrease in gas production by the
blank without an unacceptable increase in either lag period or incubation period during the test. For
test materials which are expected to be poorly biodegradable, consider pre-incubating the sludge with
the test material to get a better adapted inoculum. In such a case, add test material with a concentration
of 5 mg/l to 20 mg/l OC to the digested sludge. Wash the pre-incubated sludge carefully before use.
Indicate in the test report that pre-incubation was carried out.
6 © ISO 2016 – All rights reserved
ISO 14853:2016(E)
7.3 Preparation of the inoculum
Wash the sludge just prior to use to reduce the IC content to less than 20 mg/l in the final test suspension.
If the IC has not been sufficiently lowered, wash the sludge an additional two times. Finally, suspend the
sludge in the requisite volume of test medium (5.2) and determine the concentration of total solids (see
3.7). The final concentration of total solids in the test vessels shall be in the range 1 g/l to 3 g/l. Conduct
the above operations in such a way that the sludge has minimal contact with oxygen (e.g. use a nitrogen
atmosphere).
7.4 Preparation of test suspensions and controls
At least three test vessels, F , shall be prepared for the test material, at least three for the blanks,
T
F , and, F , for the positive control (reference material). One or more vessels, F , may optionally be
B P I
prepared for each test material as an inhibition control (see Table 1). The same blanks and controls
can be used for several different test materials which are being tested together. Into all the vessels,
introduce aliquots of the diluted inoculum prepared in 7.3 so that the concentration of total solids is
the same in all the vessels (between 1 g/l and 3 g/l). Add the test material (5.6) and the reference
material (5.7) to the appropriate vessels. The OC concentration in the test suspensions shall normally
be 100 mg/l. In the case of toxic test materials, it may be reduced to 20 mg/l OC or even less if only the
primary biodegradability is to be determined with specific analyses.
NOTE Using lower test concentrations may result in a greater scatter of the test results.
In the case of the blank vessels, add equivalent amounts of oxygen-free water (5.1) instead of the test
material. An extra (replicate) test vessel containing test suspension may also be prepared for analyses,
carried out at the beginning of the test, to determine the pH and, if required, the total solids and IC.
Adjust the pH to 7 ± 0,2, if necessary, with small amounts of dilute mineral acid or alkali. Add the same
amount of neutralizing agent to all the test vessels. If the primary degradability is to be measured,
take a suitable sample from the extra test vessel and measure the test material concentration using
a suitable method. Place magnetic stirrer bars in the vessels if the test suspensions are to be stirred
(optional). Ensure that the total volume of liquid, V , and the volume of the headspace, V , are the same
L H
in all vessels (see 6.2.1). Note V and V (see Clause 8). If necessary, add additional oxygen-free test
L H
medium (5.2). Seal each vessel with a gastight septum and put them into the incubator (6.1.1).
Table 1 — Scheme of test and control assays
Vessel Test material Reference material Inoculum
(biodegradable)
F Test + +
T1
F Test + +
T2
F Test + +
T3
F Blank +
B1
F Blank +
B2
F Blank +
B3
F Positive control + +
P1
F Positive control + +
P2
F Positive control + +
P3
Extra replicate for analysis at
+ +
beginning of test
F Inhibition control (optional) + + +
I
ISO 14853:2016(E)
7.5 Incubation and gas measurement
7.5.1 General
Incubation shall take place in sealed vessels at a constant temperature of (35 ± 2) °C, a normal
temperature for an anaerobic digester, in the absence of oxygen, initially in an atmosphere of pure
nitrogen.
7.5.2 Gas measurement using a manometer (see Annex A)
Incubate the prepared vessels at (35 ± 2) °C for about 1 h to allow equilibration, and vent excess gas
to the atmosphere, for example, by shaking each vessel in turn, inserting the needle of the manometer
through the seal and opening the valve until the manometer reads zero. If at this stage, or when making
intermediate measurements, the headspace pressure is less than atmospheric, introduce nitrogen gas
to re-establish atmospheric pressure. Close the valve and continue to incubate in the dark, ensuring
that all parts of the vessels are maintained at the incubation temperature.
Observe the vessels after incubation for 24 h to 48 h. Reject vessels if their contents show a distinct
pink colouration in the supernatant liquid. This is due to a change in colour of the resazurin, indicating
the presence of oxygen. While small amounts of oxygen can be tolerated in the system, higher
concentrations can seriously inhibit the course of anaerobic biodegradation.
Carefully mix the contents of each vessel by stirring or shaking for a few minutes at least two or three
times per week and before each pressure measurement. Measure the gas pressure, for example, by
inserting, through the septum, the syringe needle connected to the manometer. Record the pressure in
hectopascals.
Shaking resuspends the inoculum and ensures gas equilibrium. While measuring pressure, maintain
the gas in the headspace at the incubation temperature. Take care to prevent water entering the syringe
needle. Should this occur, dry the wetted parts and fit a new needle.
Either measure the gas pressure in the vessels weekly, venting excess gas to the atmosphere, or
measure the pressure only at the end of the test to detect the total amount of biogas produced. It is
strongly recommended, however, that intermediate readings of gas pressure be made, since the
pressure increase provides guidance as to when the test may be terminated and allows the kinetics to
be followed.
7.5.3 Gas measurement using a volumetric device (see Annex B)
The biogas produced can be collected in a graduated glass tube separated from the atmosphere by a
barrier solution in such a way that the pressure remains nearly constant (except for atmospheric
pressure changes) during the test. After incubation of the prepared vessels at (35 ± 2) °C for about 1 h,
vent excess gas to the atmosphere, for example, by shaking each vessel in turn, inserting a syringe needle
through the septum seal and allowing gas to escape until the surface of the barrier solution in the gas-
collection tube reaches zero. Make sure that the surface of the barrier solution in the expansion tank is
at the same level as in the gas-collection tube. Remove the syringe needle and continue to incubate in
the dark, ensuring that all parts of all the vessels are maintained at the incubation temperature.
Readings of the gas volume can be made directly from the gas-collection tube. Before taking a reading,
bring the surface of the liquid in the expansion tank to the same level as the surface of the liquid in
the collection tube so that the gas volume is read at atmospheric pressure (see Annex B for operating
instructions). Make a sufficient number of measurements of gas volume, pressure and temperature
(normally every day) to determine the rate of gas production. More frequent readings in the early
stages may be required, with less frequent readings needed as time progresses.
8 © ISO 2016 – All rights reserved
ISO 14853:2016(E)
7.6 Test duration
The normal test duration is 60 d. The test may be terminated earlier if the biodegradation curve
obtained from the pressure or volume measurements has reached a plateau phase (see 3.12). If, at the
end of the normal incubation period, an obvious plateau phase has not been reached, the test can be
extended until such time as a plateau phase is reached. However, the test duration shall not exceed 90 d.
7.7 Measurement of inorganic carbon
At the end of the test, after the last measurement of gas pressure or increase in gas volume, allow the
sludge to settle, open each vessel and immediately determine the concentration of inorganic carbon (IC)
(in mg/l) in the supernatant liquid. The supernatant liquid shall not be centrifuged or filtered at this
stage (see note). After IC measurement, record the pH. Carry out similar measurements on the blanks,
the reference material and any optional controls.
Alternatively, the IC in the supernatant may be determined indirectly by release of the dissolved IC as
carbon dioxide that can be measured in the headspace. Following the last measurement of gas pressure,
adjust the pressure in each of the test vessels to atmospheric pressure. Acidify the contents of each vessel
to approximately pH 1 by adding of concentrated mineral acid (e.g. 1M H PO or 1M H SO ) through
3 4 2 4
the septum of the sealed vessels. Incubate the shaken vessels at (35 ± 2) °C for approximately 24 h and
measure the gas pressure resulting from the evolved carbon dioxide by using the pressure meter.
NOTE Centrifugation or filtration would result in an unacceptable loss of dissolved carbon dioxide. If the
sample of supernatant liquid cannot be analysed immediately, it may be stored in a suitable sealed vial, without
headspace, at about 4 °C for up to 2 d.
In some cases, especially if the same blanks or controls are used for several different test materials,
give consideration to measuring intermediate IC concentrations in the test and control vessels. In this
case, use the following procedure.
After measuring the gas pressure or the volume increase without releasing excess gas, take an aliquot
of the supernatant liquid which is as small as possible with a syringe through the septum without
opening the vessel and determine the IC in the sample. After having taken the sample, excess gas may
be vented from the vessel (see 7.5).
Note that even a small decrease in th
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