Plastics — Determination of the ultimate anaerobic biodegradation of plastic materials in an aqueous system — Method by measurement of biogas production

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.

Plastiques — Évaluation de la biodégradabilité anaérobie ultime des matériaux plastiques en milieu aqueux — Méthode par détermination de la production de biogaz

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.

General Information

Status
Published
Publication Date
13-Jul-2016
Current Stage
9093 - International Standard confirmed
Start Date
21-Sep-2021
Completion Date
21-Sep-2021
Ref Project

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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
---------------------- Page: 1 ----------------------
ISO 14853:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
---------------------- Page: 2 ----------------------
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 2016 – All rights reserved iii
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ISO 14853:2016(E)

Bibliography .............................................................................................................................................................................................................................28

iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
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 2016 – All rights reserved v
---------------------- Page: 5 ----------------------
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
---------------------- Page: 6 ----------------------
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 2016 – All rights reserved 1
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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,

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

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
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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
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 2016 – All rights reserved 3
---------------------- Page: 9 ----------------------
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

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
---------------------- Page: 10 ----------------------
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 2016 – All rights reserved 5
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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
---------------------- Page: 12 ----------------------
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
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 14853
ISO/TC 61/SC 5
Plastics — Determination of the
Secretariat: DIN
ultimate anaerobic biodegradation of
Voting begins on:
2016­04-12 plastic materials in an aqueous system
— Method by measurement of biogas
Voting terminates on:
2016­06-12
production
Plastiques — Évaluation de la biodégradabilité anaérobie ultime des
matériaux plastiques en milieu aqueux — Méthode par détermination
de la production de biogaz
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 14853:2016(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2016
---------------------- Page: 1 ----------------------
ISO/FDIS 14853:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
CH­1214 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
---------------------- Page: 2 ----------------------
ISO/FDIS 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 .................................................................................................................................................................................10

8 Calculation and expression of results ..........................................................................................................................................10

8.1 Amount of carbon in headspace ............................................................................................................................................10

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 .....................................................................................................................12

8.5 Total amount of carbon converted to gas ......................................................................................................................12

8.6 Amount of carbon in test material ......................................................................................................................................12

8.7 Calculation of percentage biodegradation ...................................................................................................................13

9 Validity of results ..............................................................................................................................................................................................13

9.1 Maintenance of anaerobic conditions ..............................................................................................................................13

9.2 Inhibition of degradation ............................................................................................................................................................13

9.3 Validity of the test ..............................................................................................................................................................................13

10 Test report ................................................................................................................................................................................................................13

Annex A (informative) Example of apparatus for determining the amount of biogas

produced by measuring the increase in gas pressure ................................................................................................15

Annex B (informative) Example of apparatus for determining volumetrically the amount of

biogas produced .................................................................................................................................................................................................16

Annex C (informative) Example of a biodegradation curve .......................................................................................................18

Annex D (informative) Examples of data sheets for anaerobic biodegradability tests .................................19

Annex E (informative) Table of water vapour pressures at various temperatures ...........................................22

Annex F (informative) Calculation of theoretical carbon dioxide (ThCO ) and theoretical

methane (ThCH ) production ..............................................................................................................................................................23

Annex G (informative) Example of determination of recovery rate ..................................................................................24

Annex H (informative) Example of a workflow scheme .................................................................................................................27

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ISO/FDIS 14853:2016(E)

Bibliography .............................................................................................................................................................................................................................29

iv © ISO 2016 – All rights reserved
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ISO/FDIS 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 non­governmental, 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 2016 – All rights reserved v
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ISO/FDIS 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
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 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 pre­exposed 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
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ISO/FDIS 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,

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

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
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ISO/FDIS 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
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
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ISO/FDIS 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

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 trace­element solution per litre of test medium.
5.4 Vitamin solutions (optional).
5.4.1 Vitamin solution No. 1
4­Aminobenzoic acid 40 mg
d­Biotin 10 mg
Dissolve in hot water (5.1) 500 ml
Allow to cool and add:
d­Pantothenic 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
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ISO/FDIS 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 particle­size 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.

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ISO/FDIS 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 gas­collection 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 pre­incubation 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 po
...

NORME ISO
INTERNATIONALE 14853
Deuxième édition
2016-07-15
Plastiques — Évaluation de la
biodégradabilité anaérobie ultime
des matériaux plastiques en milieu
aqueux — Méthode par détermination
de la production de biogaz
Plastics — Determination of the ultimate anaerobic biodegradation
of plastic materials in an aqueous system — Method by measurement
of biogas production
Numéro de référence
ISO 14853:2016(F)
ISO 2016
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ISO 14853:2016(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2016, Publié en Suisse

Droits de reproduction réservés. Sauf indication contraire, aucune partie de cette publication ne peut être reproduite ni utilisée

sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie, l’affichage sur

l’internet ou sur un Intranet, sans autorisation écrite préalable. Les demandes d’autorisation peuvent être adressées à l’ISO à

l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.
ISO copyright office
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Tel. +41 22 749 01 11
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copyright@iso.org
www.iso.org
ii © ISO 2016 – Tous droits réservés
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ISO 14853:2016(F)
Sommaire Page

Avant-propos ................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 1

3 Termes et définitions ....................................................................................................................................................................................... 1

4 Principe .......................................................................................................................................................................................................................... 3

5 Réactifs et matériaux ....................................................................................................................................................................................... 3

6 Appareillage .............................................................................................................................................................................................................. 6

6.1 Matériel de laboratoire .................................................................................................................................................................... 6

6.2 Appareillage à utiliser lorsque le biogaz est mesuré par une méthode manométrique.......... 6

6.3 Appareillage à utiliser lorsque le biogaz est mesuré par une méthode volumétrique ............. 7

7 Mode opératoire.................................................................................................................................................................................................... 7

7.1 Généralités .................................................................................................................................................................................................. 7

7.2 Boue digérée ............................................................................................................................................................................................. 7

7.3 Préparation de l’inoculum ............................................................................................................................................................. 7

7.4 Préparation des suspensions d’essai et des substances de contrôle........................................................ 7

7.5 Incubation et mesurage du gaz ................................................................................................................................................. 8

7.6 Durée de l’essai ....................................................................................................................................................................................... 9

7.7 Mesurage du carbone inorganique .....................................................................................................................................10

7.8 Analyses spécifiques .......................................................................................................................................................................10

8 Calcul et expression des résultats ...................................................................................................................................................10

8.1 Quantité de carbone dans l’espace de tête ...................................................................................................................10

8.2 Calcul de la quantité de carbone dans l’espace de tête avec une méthode de

mesure manométrique .................. ................................................................................................................................................11

8.3 Calcul de la quantité de carbone dans l’espace de tête avec une méthode de

mesure volumétrique .....................................................................................................................................................................11

8.4 Quantité de carbone inorganique dans le liquide ..................................................................................................12

8.5 Quantité totale de carbone converti en gaz .................................................................................................................12

8.6 Quantité de carbone dans le matériau d’essai ..........................................................................................................13

8.7 Calcul du pourcentage de biodégradation....................................................................................................................13

9 Validité des résultats .....................................................................................................................................................................................13

9.1 Maintien des conditions anaérobies..................................................................................................................................13

9.2 Inhibition de la dégradation .....................................................................................................................................................13

9.3 Validité de l’essai ................................................................................................................................................................................13

10 Rapport d’essai ....................................................................................................................................................................................................14

Annexe A (informative) Exemple d’appareillage pour déterminer la quantité de biogaz

produit en mesurant l’augmentation de la pression du gaz ................................................................................15

Annexe B (informative) Exemple d’appareillage pour déterminer de manière volumétrique

la quantité de biogaz produit ...............................................................................................................................................................16

Annexe C (informative) Exemple de courbe de biodégradation ...........................................................................................18

Annexe D (informative) Exemples de fiches techniques pour les essais de

biodégradabilité anaérobie ...................................................................................................................................................................19

Annexe E (informative) Table des pressions de vapeur d’eau à différentes températures ......................24

Annexe F (informative) Calcul de la production théorique de dioxyde de carbone (ThCO )

et de méthane (ThCH ) ...............................................................................................................................................................................25

Annexe G (informative) Exemple de détermination du taux de récupération .......................................................26

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ISO 14853:2016(F)

Annexe H (informative) Exemple de diagramme de flux ..............................................................................................................29

Bibliographie ...........................................................................................................................................................................................................................31

iv © ISO 2016 – Tous droits réservés
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ISO 14853:2016(F)
Avant-propos

L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes

nationaux de normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est

en général confiée aux comités techniques de l’ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l’ISO participent également aux travaux.

L’ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d’approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www.

iso.org/directives).

L’attention est appelée sur le fait que certains des éléments du présent document peuvent faire l’objet de

droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l’élaboration du document sont indiqués dans l’Introduction et/ou dans la liste des déclarations de

brevets reçues par l’ISO (voir www.iso.org/brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la signification des termes et expressions spécifiques de l’ISO liés à l’évaluation

de la conformité, ou pour toute information au sujet de l’adhésion de l’ISO aux principes de l’Organisation

mondiale du commerce (OMC) concernant les obstacles techniques au commerce (OTC), voir le lien

suivant: http ://www.iso.org/iso/fr/foreword.html.

Le comité chargé de l’élaboration du présent document est l’ISO/TC 61, Plastiques, sous-comité SC 5,

Propriétés physicochimiques.

Cette deuxième édition annule et remplace la première édition (ISO 14853:2005), qui a fait l’objet d’une

révision technique. Elle intègre également le Corrigendum technique ISO 14853:2005/Cor.1:2009.

© ISO 2016 – Tous droits réservés v
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ISO 14853:2016(F)
Introduction

Les plastiques étant de plus en plus utilisés, leur valorisation et leur élimination sont devenues un enjeu

majeur. Il convient de favoriser en priorité leur valorisation. Par exemple, un déchet plastique, venant

principalement des consommateurs, est difficile à valoriser complètement. Autres exemples de produits

difficiles à valoriser: les articles de pêche, les paillages agricoles et les polymères hydrosolubles. Ces

matériaux plastiques tendent à migrer des infrastructures fermées de gestion des déchets vers le milieu

naturel. Désormais, les plastiques biodégradables apparaissent comme l’une des options possibles

pour résoudre ce genre de problème environnemental. Il convient que les matériaux plastiques, sous

forme de produits ou d’emballages, qui sont envoyés dans les installations de traitement anaérobie

soient potentiellement biodégradables. Il est donc très important de déterminer la biodégradabilité

potentielle de ce type de matériaux et d’obtenir une mesure quantitative de leur biodégradabilité en

milieu anaérobie.
vi © ISO 2016 – Tous droits réservés
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NORME INTERNATIONALE ISO 14853:2016(F)
Plastiques — Évaluation de la biodégradabilité anaérobie
ultime des matériaux plastiques en milieu aqueux —
Méthode par détermination de la production de biogaz

AVERTISSEMENT — Les eaux usées et les boues activées peuvent contenir des organismes

potentiellement pathogènes. Il convient donc de prendre les précautions appropriées pour les

manipuler. Les boues d’eaux usées digérées produisent des gaz inflammables qui présentent des

risques d’incendie et d’explosion. Il convient de prendre des précautions lors du transport et

du stockage de grandes quantités de boues digérées. Il convient de manipuler avec précaution

et en respectant les instructions de sécurité les produits chimiques toxiques et ceux dont les

propriétés sont inconnues. Il convient de manipuler avec précaution le pressiomètre et les

microseringues pour éviter les piqûres d’aiguilles. Il convient d’éliminer de manière appropriée

les aiguilles de seringues contaminées.
1 Domaine d’application

La présente Norme internationale spécifie une méthode pour la détermination de la biodégradabilité

anaérobie ultime des plastiques par des micro-organismes anaérobies. Les conditions décrites dans

la présente Norme internationale 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.
2 Références normatives
Il n’y a pas de références normatives dans le présent document.
3 Termes et définitions

Pour les besoins du présent document, les termes et définitions suivants s’appliquent.

3.1
biodégradation anaérobie ultime

décomposition d’un composé organique par des micro-organismes en l’absence d’oxygène, en dioxyde

de carbone, méthane, eau et sels minéraux de tous les autres éléments présents (minéralisation) et

production d’une nouvelle biomasse
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3.2
biodégradation anaérobie primaire

modification structurelle (transformation) d’un composé chimique par des micro-organismes, résultant

en la perte d’une propriété spécifique
3.3
boue digérée

mélange d’eaux usées décantées et de boues activées qui ont été incubées dans un digesteur anaérobie à

environ 35 °C pour réduire la biomasse et l’odeur et pour améliorer la déshydratation de la boue

Note 1 à l’article: Les boues digérées contiennent un ensemble de bactéries fermentatives et méthanogènes

anaérobies qui produisent du dioxyde de carbone et du méthane.
3.4
concentration de la boue digérée en matières solides en suspension

quantité de matières solides obtenue par filtration ou centrifugation d’un volume connu de boue activée

et séchage à environ 105 °C jusqu’à l’obtention d’une masse constante
3.5
carbone organique dissous
COD

carbone organique contenu dans la phase aqueuse, qui ne peut pas être éliminé par une séparation de

phase spécifique, par exemple par centrifugation à 40 000 m⋅s pendant 15 min ou par filtration sur

des membranes ayant des pores de 0,2 µm à 0,45 µm de diamètre
3.6
carbone inorganique

carbone inorganique qui est dissous ou dispersé dans la phase aqueuse d’un liquide et qui est

récupérable dans le liquide surnageant une fois que la boue a décanté
3.7
matières sèches totales

quantité de matières solides obtenue par prélèvement d’un volume connu de matériau d’essai ou

d’inoculum et séchage à environ 105 °C jusqu’à l’obtention d’une masse constante
3.8
quantité théorique de biogaz libéré
Thbiogaz

quantité théorique maximale de biogaz (CH + CO ) libéré après la biodégradation complète d’une

4 2

matière organique dans des conditions anaérobies, calculée d’après la formule moléculaire et exprimée

en millilitres de biogaz libéré par milligramme de matériau d’essai dans les conditions normales

3.9
quantité théorique de dioxyde de carbone libéré
ThCO

quantité théorique maximale de dioxyde de carbone libéré après oxydation complète d’une matière

organique, calculée d’après la formule moléculaire et exprimée en milligrammes de dioxyde de carbone

par milligramme de matériau d’essai
3.10
quantité théorique de méthane libéré
ThCH

quantité théorique maximale de méthane libéré après réduction complète d’une matière organique,

calculée d’après la formule moléculaire et exprimée en milligrammes de méthane libéré par

milligramme de matériau d’essai
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ISO 14853:2016(F)
3.11
phase de latence
période de latence

durée, mesurée en jours, écoulée à partir du début de l’essai jusqu’à l’obtention de l’adaptation

et/ou de la sélection des micro-organismes qui provoquent la dégradation, et jusqu’à ce que le taux de

biodégradation du composé chimique ou de la matière organique ait atteint environ 10 % du niveau

maximal de biodégradation
3.12
phase stationnaire

durée, mesurée en jours, écoulée entre la fin de la phase de biodégradation et la fin de l’essai

3.13
phase de biodégradation

durée, mesurée en jours, depuis la fin de la phase de latence de l’essai jusqu’à ce que l’on ait obtenu

environ 90 % du niveau maximal de biodégradation
3.14
niveau maximal de biodégradation

taux de biodégradation, mesuré en pourcentage, d’un composé chimique ou d’une matière organique

lors d’un essai, au-dessus duquel la biodégradation ne se poursuit pas
4 Principe

La biodégradabilité d’un matériau plastique est déterminée dans des conditions anaérobies en milieu

aqueux. Le matériau d’essai d’une concentration de 20 mg/l à 200 mg/l de carbone organique (CO) est

incubé à (35 ± 2) °C dans des récipients fermés avec la boue digérée pendant une durée ne dépassant

normalement pas 90 j. Avant utilisation, la boue digérée est lavée afin qu’elle contienne de très petites

quantités de carbone inorganique (CI) et elle est diluée à une concentration comprise entre 1 g/l et

3 g/l de matières solides totales. L’augmentation de pression dans l’espace de tête ou l’augmentation

volumétrique (en fonction de la méthode utilisée pour mesurer la libération de biogaz) dans les

récipients d’essai, résultant de la production de dioxyde de carbone (CO ) et de méthane (CH ), est

2 4

mesurée. Une quantité considérable de CO sera dissoute dans l’eau ou transformée en bicarbonate ou

en carbonate dans les conditions de l’essai. Ce carbone inorganique (CI) est mesuré à la fin de l’essai.

La quantité de carbone de biogaz produit de manière microbiologique est calculée à partir de la

production nette de biogaz et de la formation nette de CI dépassant les valeurs du blanc. Le pourcentage

de biodégradation est calculé à partir de la quantité totale de carbone transformé en biogaz et en CI

et de la quantité mesurée ou calculée de carbone ajouté en tant que matériau d’essai. L’évolution de la

biodégradation peut être suivie en réalisant des mesurages intermédiaires de la production de biogaz.

À titre d’informations complémentaires, la biodégradabilité primaire peut être déterminée par des

analyses spécifiques réalisées au début et à la fin de l’essai.

La présente méthode d’essai est conçue pour déterminer la biodégradabilité des matériaux plastiques

dans des conditions anaérobies. À titre facultatif, l’évaluation du taux de récupération peut également

présenter un intérêt (voir l’Annexe G).
5 Réactifs et matériaux

5.1 Eau distillée ou déionisée, exempte de substances toxiques et contenant moins de 2 mg/l de COD.

5.2 Milieu d’essai, préparé uniquement avec des réactifs de qualité analytique reconnue.

Préparer le milieu d’essai en utilisant les constituants suivants dans les quantités indiquées:

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Dihydrogénophosphate de potassium anhydre KH PO 0,27 g
2 4
Hydrogénophosphate disodique Na HPO ⋅12H O 1,12 g
2 4 2
dodécahydraté
Chlorure d’ammonium NH Cl 0,53 g
Chlorure de calcium dihydraté CaCl ⋅2H O 0,075 g
2 2
Chlorure de magnésium hexahydraté MgCl ⋅6H O 0,10 g
2 2
Chlorure de fer(II) tétrahydraté FeCl ⋅4H O 0,02 g
2 2
Résazurine (indicateur d’oxygène) 0,001 g
Sulfure disodique nonahydraté Na S⋅9H O 0,1 g
2 2
Solution mère d’éléments traces (facultative) 10 ml
Solutions mères de vitamines (facultatives) Solution de vitamines n° 1 0,5 ml
Solution de vitamines n° 2 0,5 ml
Ajouter de l’eau (5.1) (exempte d’oxygène) 1 l

Utiliser du sulfure de sodium fraichement préparé ou le laver et le sécher avant utilisation pour

garantir une capacité réductrice suffisante. Pour garantir des conditions strictement anaérobies, il

est recommandé d’ajouter une petite quantité de dithionite de sodium dans le milieu préparé, jusqu’à

ce qu’il devienne incolore. Ne pas utiliser plus de 10 mg/l car des concentrations plus élevées peuvent

produire des effets inhibiteurs.

Ajuster le pH du milieu avec de l’acide ou de la base minéral(e) dilué(e), si nécessaire, à pH 7 ± 0,2.

Pour garantir des conditions exemptes d’oxygène, purger l’eau avec de l’azote pendant environ 20 min

juste avant utilisation.
5.3 Solution d’éléments traces (facultative).

Il est recommandé d’ajouter les éléments traces suivants dans le milieu d’essai pour améliorer le

processus de dégradation anaérobie, en particulier si de faibles concentrations d’inoculum sont

utilisées:
Chlorure de manganèse tétrahydraté MnCl ⋅4H O 0,05 g
2 2
Acide borique H BO 0,005 g
3 3
Chlorure de zinc ZnCl 0,005 g
Chlorure de cuivre(II) CuCl 0,003 g
Molybdate disodique dihydraté Na MoO ⋅2H O 0,001 g
2 4 2
Chlorure de cobalt hexahydraté CoCl ⋅6H O 0,1 g
2 2
Chlorure de nickel hexahydraté NiCl ⋅6H O 0,01 g
2 2
Sélénite disodique Na SeO 0,005 g
2 3
Tungstate disodique dihydraté Na WO ⋅2H O 0,002 g
2 4 2
Ajouter de l’eau (5.1) (exempte d’oxygène) 1 l
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ISO 14853:2016(F)
Utiliser 10 ml de solution d’éléments traces par litre de milieu d’essai.
5.4 Solutions de vitamines (facultatives).
5.4.1 Solution de vitamines n° 1
Acide 4-aminobenzoïque 40 mg
d-biotine 10 mg
Dissoudre dans de l’eau chaude (5.1) 500 ml
Laisser refroidir et ajouter:
Acide d-pantothénique, sel de calcium 50 mg
Dihydrochlorure de pyridoxamine 150 mg
Dichlorure de thiamine 100 mg

Filtrer la solution sur une membrane (ouverture de pore de 0,45 µm) qui n’adsorbe pas et ne libère pas

de carbone organique en quantités significatives, et la conserver dans l’obscurité à 4 °C.

Utiliser 0,5 ml de solution de vitamines par litre de milieu d’essai.
5.4.2 Solution de vitamines n° 2
Cyanocobalamine (vitamine B12) 10 mg
Dissoudre dans de l’eau (5.1) 100 ml

Filtrer la solution sur une membrane (ouverture de pore de 0,45 µm) qui n’adsorbe pas et ne libère pas

de carbone organique en quantités significatives, et la conserver dans l’obscurité à 4 °C.

Utiliser 0,5 ml de solution de vitamines par litre de milieu d’essai.
5.5 Solution barrière.
NaCl 200 g
Dissoudre dans de l’eau (5.1) 1 000 ml
Acidifier avec de l’acide citrique 5 g

Ajouter un indicateur de pH, par exemple du bleu de bromophénol ou de l’orange de méthyle, pour

pouvoir vérifier que la solution reste acide lors de l’essai.
5.6 Matériau d’essai.

Le matériau d’essai est généralement ajouté directement sous forme solide pour donner une

concentration de 20 mg/l à 200 mg/l de carbone organique. Il convient que le matériau d’essai

(plastique) soit utilisé sous forme de poudre, dans la mesure du possible.

Il convient d’utiliser le matériau d’essai de préférence sous forme de poudre; toutefois, ce dernier peut

également être employé sous forme de film, de morceaux, de fragments ou d’articles façonnés. La

consistance et la forme du matériau d’essai peuvent influer sur sa biodégradabilité. Il convient d’utiliser,

de préférence, des formes similaires si l’on doit comparer différents types de matériaux plastiques. Si

le matériau d’essai est utilisé sous forme de poudre, il est recommandé d’utiliser des particules ayant

une distribution granulométrique étroite connue. Une distribution granulométrique avec un diamètre

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ISO 14853:2016(F)

maximal de 250 µm est recommandée. D’autre part, la forme du matériau d’essai peut également avoir

une influence sur la taille du dispositif d’essai utilisé.

La biodégradabilité des matériaux plastiques qui ne sont pas inhibiteurs de micro-organismes peut

être déterminée à l’aide de concentrations supérieures à 200 mg/l de carbone organique. Dans ce cas,

s’assurer que le pouvoir tampon et la teneur en sels minéraux du milieu sont suffisants.

5.7 Matériau de référence.

Utiliser un polymère biodégradable anaérobie bien défini, par exemple du poly-β-hydroxybutyrate, de

la cellulose ou du poly(éthylène glycol) 400 comme matériau de référence. Si possible, il convient que la

forme, la taille, la solubilité et la concentration du matériau de référence soient comparables à celles du

matériau d’essai.
Préparer le matériau de référence de la même façon que le matériau d’essai.
5.8 Substance de contrôle de l’effet inhibiteur (facultative).

Ajouter à la fois le matériau d’essai et le matériau de référence dans un récipient contenant le milieu

d’essai (5.2) pour obtenir les concentrations spécifiées en 5.6 et 5.7, respectivement.

6 Appareillage
6.1 Matériel de laboratoire
Est nécessaire le matériel courant de laboratoire, et ce qui suit:
6.1.1 Incubateur, bain-marie ou bain de sable, thermostaté à (35 ± 2) °C.

6.1.2 Analyseur de carbone (facultatif), adapté pour la détermination directe du carbone inorganique

dans la plage de 1 mg/l à 200 mg/l de CI. Autrement, le CI présent dans le liquide surnageant peut être

déterminé indirectement par la libération de CI dissous sous forme de dioxyde de carbone qui peut être

mesurée dans l’espace de tête, comme décrit en 7.7.
6.2 Appareillage à utiliser lorsque le biogaz est mesuré par une méthode
manométrique

6.2.1 Récipients d’essai en verre résistants à la pression, d’une taille nominale de 0,1 l à 1 l, munis

d’un septum étanche au gaz capable de résister à environ 2 000 hPa (pour un exemple, voir l’Annexe A).

Le volume de l’espace de tête doit représenter environ 10 % à 30 % du volume total. Si du gaz est libéré

à intervalles réguliers, un volume d’espace de tête d’environ 10 % est adéquat, mais si le gaz est libéré

uniquement à la fin de l’essai, une valeur de 30 % est plus appropriée.

D’un point de vue pratique, l’utilisation de flacons à sérum fermés par des bouchons en caoutchouc

butyle et des anneaux sertis en aluminium est recommandée.

6.2.2 Dispositif de mesure de la pression, par exemple un manomètre raccordé à une aiguille de

seringue adaptée, avec un robinet à trois voies étanche au gaz pour faciliter la libération de la pression en

excès. Utiliser et étalonner ce dispositif conformément aux instructions du fabricant.

Il est nécessaire de faire en sorte que le volume interne du tube et du robinet soit le plus petit possible

pour éviter que les erreurs introduites du fait que le volume du dispositif a été ignoré ne soient pas

significatives.
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ISO 14853:2016(F)
6.3 Appareillage à utiliser lorsq
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