Plastics - Determination of the aerobic biodegradation of non-floating materials exposed to marine sediment - Method by analysis of evolved carbon dioxide (ISO 22404:2019)

This document specifies a laboratory test method to determine the degree and rate of aerobic biodegradation level of plastic materials. This test method can also be applied to other materials.
Biodegradation is determined by measuring the CO2 evolved by the plastic material when exposed to marine sediments sampled from a sandy tidal zone and kept wet with salt-water under laboratory conditions.
This test method is a simulation under laboratory conditions of the habitat found in sandy tidal zone that, in marine science, is called eulittoral zone.
The conditions described in this document might not always correspond to the optimum conditions for the maximum degree of biodegradation to occur.
Deviations from the test conditions described in this document are justified in the test report.

Kunststoffe - Bestimmung des aeroben Bioabbaus von nicht-schwimmenden Materialien, die marinem Sediment ausgesetzt sind - Verfahren mittels Analyse des freigesetzten Kohlenstoffdioxids (ISO 22404:2019)

Dieses Dokument legt ein Laborprüfverfahren für die Bestimmung des Grads und der Geschindigkeit des aeroben Bioabbaus von Kunststoffmaterialien fest. Dieses Prüfverfahren kann darüber hinaus auch auf andere Materialien angewendet werden.
Bioabbau wird durch die Messung des durch das Kunststoffmaterial erzeugten CO2 bestimmt, wenn das Material Meeressediment ausgesetzt war, das einer sandigen Gezeitenzone entnommen und mit Salzwasser unter Laborbedingungen aufbewahrt wurde.
Dieses Prüfverfahren ist eine Simulation unter Laborbedingungen des in der sandigen Gezeitenzone ange¬troffenen Habitats, das in der Meereswissenschaft als eulitorale Zone bezeichnet wird.
Die in diesem Dokument beschriebenen Bedingungen entsprechen möglicherweise nicht immer den Optimal¬bedingungen, unter denen der höchste Grad an biologischem Abbau möglich ist.
Abweichungen von den in diesem Dokument beschriebenen Prüfbedingungen werden in dem Prüfbericht begründet.

Plastiques - Détermination de la biodégradation aérobie des matériaux non flottants exposés aux sédiments marins - Méthode par analyse du dioxyde de carbone libéré (ISO 22404:2019)

Polimerni materiali - Določanje aerobne biorazgradljivosti neplavajočih materialov, izpostavljenih morskim sedimentom - Metoda z analizo sproščenega ogljikovega dioksida (ISO 22404:2019)

General Information

Status
Published
Public Enquiry End Date
30-Sep-2021
Publication Date
24-Nov-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Nov-2021
Due Date
22-Jan-2022
Completion Date
25-Nov-2021

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SLOVENSKI STANDARD
SIST EN ISO 22404:2022
01-januar-2022

Polimerni materiali - Določanje aerobne biorazgradljivosti neplavajočih materialov,

izpostavljenih morskim sedimentom - Metoda z analizo sproščenega ogljikovega
dioksida (ISO 22404:2019)

Plastics - Determination of the aerobic biodegradation of non-floating materials exposed

to marine sediment - Method by analysis of evolved carbon dioxide (ISO 22404:2019)

Kunststoffe - Bestimmung des aeroben Bioabbaus von nicht-schwimmenden Materialien,

die marinem Sediment ausgesetzt sind - Verfahren mittels Analyse des freigesetzten

Kohlenstoffdioxids (ISO 22404:2019)

Plastiques - Détermination de la biodégradation aérobie des matériaux non flottants

exposés aux sédiments marins - Méthode par analyse du dioxyde de carbone libéré
(ISO 22404:2019)
Ta slovenski standard je istoveten z: EN ISO 22404:2021
ICS:
13.020.40 Onesnaževanje, nadzor nad Pollution, pollution control
onesnaževanjem in and conservation
ohranjanje
83.080.01 Polimerni materiali na Plastics in general
splošno
SIST EN ISO 22404:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 22404:2022
---------------------- Page: 2 ----------------------
SIST EN ISO 22404:2022
EN ISO 22404
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 83.080.01
English Version
Plastics - Determination of the aerobic biodegradation of
non-floating materials exposed to marine sediment -
Method by analysis of evolved carbon dioxide (ISO
22404:2019)

Plastiques - Détermination de la biodégradation Kunststoffe - Bestimmung des aeroben Bioabbaus von

aérobie des matériaux non flottants exposés aux nicht-schwimmenden Materialien, die marinem

sédiments marins - Méthode par analyse du dioxyde de Sediment ausgesetzt sind - Verfahren mittels Analyse

carbone libéré (ISO 22404:2019) des freigesetzten Kohlenstoffdioxids (ISO 22404:2019)

This European Standard was approved by CEN on 8 November 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22404:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 22404:2022
EN ISO 22404:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 22404:2022
EN ISO 22404:2021 (E)
European foreword

The text of ISO 22404:2019 has been prepared by Technical Committee ISO/TC 61 "Plastics” of the

International Organization for Standardization (ISO) and has been taken over as EN ISO 22404:2021 by

Technical Committee CEN/TC 249 “Plastics” the secretariat of which is held by NBN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be

withdrawn at the latest by May 2022.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 22404:2019 has been approved by CEN as EN ISO 22404:2021 without any modification.

---------------------- Page: 5 ----------------------
SIST EN ISO 22404:2022
---------------------- Page: 6 ----------------------
SIST EN ISO 22404:2022
INTERNATIONAL ISO
STANDARD 22404
First edition
2019-09
Plastics — Determination of the
aerobic biodegradation of non-
floating materials exposed to marine
sediment — Method by analysis of
evolved carbon dioxide
Plastiques — Détermination de la biodégradation aérobie des
matériaux non flottants exposés aux sédiments marins — Méthode
par analyse du dioxyde de carbone libéré
Reference number
ISO 22404:2019(E)
ISO 2019
---------------------- Page: 7 ----------------------
SIST EN ISO 22404:2022
ISO 22404:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 22404:2022
ISO 22404:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Test environment ................................................................................................................................................................................................. 2

6 Reagents ........................................................................................................................................................................................................................ 2

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Procedure..................................................................................................................................................................................................................... 3

8.1 Test material ............................................................................................................................................................................................. 3

8.2 Reference material ............................................................................................................................................................................... 4

8.3 Preparation of the sediment ........................................................................................................................................................ 4

8.4 Test setup ..................................................................................................................................................................................................... 4

8.5 Pre-conditioning phase .................................................................................................................................................................... 4

8.6 Start of the test ........................................................................................................................................................................................ 5

8.7 Carbon dioxide measurement .................................................................................................................................................... 5

8.8 End of the test .......................................................................................................................................................................................... 6

9 Calculation and expression of results ............................................................................................................................................. 6

9.1 Calculation .................................................................................................................................................................................................. 6

9.1.1 Amount of CO produced .......................................................................................................................................... 6

9.1.2 Percentage of biodegradation............................................................................................................................... 8

9.2 Expression and interpretation of results ......................................................................................................................... 8

10 Validity of results ................................................................................................................................................................................................. 9

11 Test report ................................................................................................................................................................................................................... 9

Bibliography .............................................................................................................................................................................................................................10

© ISO 2019 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 22404:2022
ISO 22404:2019(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 voluntary nature of standards, 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.

This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14,

Environmental aspects.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 22404:2022
ISO 22404:2019(E)
Introduction

Products made with biodegradable plastics and other biodegradable materials are designed to

be recoverable by means of organic recycling in composting plants or in anaerobic digesters. The

uncontrolled dispersion of biodegradable plastics in natural environments is not desirable. The

biodegradability of products cannot be considered as an excuse to spread wastes that should be

recovered and recycled. However, test methods to measure rate and level of biodegradation in natural

environments (such as soil or the marine environment) are of interest in order to better characterize

the behaviour of plastics in these very particular environments. As a matter of fact, some plastics are

used in products that are applied in the sea (for example, fishing gear) and sometimes they can get lost

or put willingly in marine environment. The characterization of biodegradable plastic materials can be

enlarged by applying specific test methods that enable the quantitative assessment of biodegradation

of plastics exposed to marine sediment and seawater. In order to carry out a proper product design, it

is important to know whether a plastic material is inherently biodegradable when exposed to marine

inocula.

This document provides a test method for calculating and reporting biodegradation level obtained

under laboratory conditions using a marine inoculum. The marine inoculum is sediment sampled at the

tidal zone. The plastic material is exposed to this environmental matrix and biodegradation is followed

by measuring the evolved CO .
© ISO 2019 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 22404:2022
---------------------- Page: 12 ----------------------
SIST EN ISO 22404:2022
INTERNATIONAL STANDARD ISO 22404:2019(E)
Plastics — Determination of the aerobic biodegradation
of non-floating materials exposed to marine sediment —
Method by analysis of evolved carbon dioxide
1 Scope

This document specifies a laboratory test method to determine the degree and rate of aerobic

biodegradation level of plastic materials. This test method can also be applied to other materials.

Biodegradation is determined by measuring the CO evolved by the plastic material when exposed

to marine sediments sampled from a sandy tidal zone and kept wet with salt-water under laboratory

conditions.

This test method is a simulation under laboratory conditions of the habitat found in sandy tidal zone

that, in marine science, is called eulittoral zone.

The conditions described in this document might not always correspond to the optimum conditions for

the maximum degree of biodegradation to occur.

Deviations from the test conditions described in this document are justified in the test report.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10210, Plastics — Methods for the preparation of samples for biodegradation testing of plastic

materials
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
tidal zone

borderline between sea and land that extends from the high tide line, which is rarely inundated with

water, to the low tide line, which is typically always covered with water

Note 1 to entry: The tidal zone is frequently a sandy area that is kept constantly damp by the lapping of the waves.

Note 2 to entry: Stony and rocky shorelines also exist.

Note 3 to entry: They are also known as eulittoral zone, midlittoral zone, mediolittoral zone, intertidal zone,

foreshore.
© ISO 2019 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 22404:2022
ISO 22404:2019(E)
3.2
theoretical amount of evolved carbon dioxide
ThCO

maximum theoretical amount of carbon dioxide evolved after completely oxidising a chemical

compound, calculated from the molecular formula or from determination of total organic carbon (TOC)

(3.3) and expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound

3.3
total organic carbon
TOC
amount of carbon bound in an organic compound

Note 1 to entry: Total organic carbon is expressed as milligrams of carbon per 100 milligrams of the compound.

3.4
dissolved organic carbon
DOC

part of the organic carbon in water which cannot be removed by specified phase separation methods,

for example by centrifugation at 40 000 ms for 15 min or by membranes with pores of 0,2 µm to

0,45 µm diameter
3.5
pre-conditioning phase

pre-incubation of an inoculum under the conditions of the subsequent test in the absence of test material,

with the aim to consume potential organic matter present in excess that could disturb biodegradation

measurement and to improve the acclimatization of the microorganisms to the test conditions

4 Principle

This test method is based on the determination of the evolved carbon dioxide and derives from

ISO 19679. The testing medium is based on a sandy marine sediment laid in the bottom of a closed flask;

the sediment is kept wet with natural sea water. The test material is preferably in the form of a powder.

The carbon dioxide evolved during the microbial degradation is determined by a suitable anal

...

SLOVENSKI STANDARD
oSIST prEN ISO 22404:2021
01-september-2021

Polimerni materiali - Določanje aerobne biorazgradljivosti neplavajočih materialov,

izpostavljenih morskim sedimentom - Metoda z analizo sproščenega ogljikovega
dioksida (ISO 22404:2019)

Plastics - Determination of the aerobic biodegradation of non-floating materials exposed

to marine sediment - Method by analysis of evolved carbon dioxide (ISO 22404:2019)

Kunststoffe - Bestimmung des aeroben Bioabbaus von nicht-schwimmenden Materialien,

die marinem Sediment ausgesetzt sind - Verfahren mittels Analyse des freigesetzten

Kohlenstoffdioxids (ISO 22404:2019)

Plastiques - Détermination de la biodégradation aérobie des matériaux non flottants

exposés aux sédiments marins - Méthode par analyse du dioxyde de carbone libéré
(ISO 22404:2019)
Ta slovenski standard je istoveten z: prEN ISO 22404
ICS:
13.020.40 Onesnaževanje, nadzor nad Pollution, pollution control
onesnaževanjem in and conservation
ohranjanje
83.080.01 Polimerni materiali na Plastics in general
splošno
oSIST prEN ISO 22404:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 22404:2021
---------------------- Page: 2 ----------------------
oSIST prEN ISO 22404:2021
INTERNATIONAL ISO
STANDARD 22404
First edition
2019-09
Plastics — Determination of the
aerobic biodegradation of non-
floating materials exposed to marine
sediment — Method by analysis of
evolved carbon dioxide
Plastiques — Détermination de la biodégradation aérobie des
matériaux non flottants exposés aux sédiments marins — Méthode
par analyse du dioxyde de carbone libéré
Reference number
ISO 22404:2019(E)
ISO 2019
---------------------- Page: 3 ----------------------
oSIST prEN ISO 22404:2021
ISO 22404:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 22404:2021
ISO 22404:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Test environment ................................................................................................................................................................................................. 2

6 Reagents ........................................................................................................................................................................................................................ 2

7 Apparatus ..................................................................................................................................................................................................................... 3

8 Procedure..................................................................................................................................................................................................................... 3

8.1 Test material ............................................................................................................................................................................................. 3

8.2 Reference material ............................................................................................................................................................................... 4

8.3 Preparation of the sediment ........................................................................................................................................................ 4

8.4 Test setup ..................................................................................................................................................................................................... 4

8.5 Pre-conditioning phase .................................................................................................................................................................... 4

8.6 Start of the test ........................................................................................................................................................................................ 5

8.7 Carbon dioxide measurement .................................................................................................................................................... 5

8.8 End of the test .......................................................................................................................................................................................... 6

9 Calculation and expression of results ............................................................................................................................................. 6

9.1 Calculation .................................................................................................................................................................................................. 6

9.1.1 Amount of CO produced .......................................................................................................................................... 6

9.1.2 Percentage of biodegradation............................................................................................................................... 8

9.2 Expression and interpretation of results ......................................................................................................................... 8

10 Validity of results ................................................................................................................................................................................................. 9

11 Test report ................................................................................................................................................................................................................... 9

Bibliography .............................................................................................................................................................................................................................10

© ISO 2019 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN ISO 22404:2021
ISO 22404:2019(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 voluntary nature of standards, 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.

This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14,

Environmental aspects.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 22404:2021
ISO 22404:2019(E)
Introduction

Products made with biodegradable plastics and other biodegradable materials are designed to

be recoverable by means of organic recycling in composting plants or in anaerobic digesters. The

uncontrolled dispersion of biodegradable plastics in natural environments is not desirable. The

biodegradability of products cannot be considered as an excuse to spread wastes that should be

recovered and recycled. However, test methods to measure rate and level of biodegradation in natural

environments (such as soil or the marine environment) are of interest in order to better characterize

the behaviour of plastics in these very particular environments. As a matter of fact, some plastics are

used in products that are applied in the sea (for example, fishing gear) and sometimes they can get lost

or put willingly in marine environment. The characterization of biodegradable plastic materials can be

enlarged by applying specific test methods that enable the quantitative assessment of biodegradation

of plastics exposed to marine sediment and seawater. In order to carry out a proper product design, it

is important to know whether a plastic material is inherently biodegradable when exposed to marine

inocula.

This document provides a test method for calculating and reporting biodegradation level obtained

under laboratory conditions using a marine inoculum. The marine inoculum is sediment sampled at the

tidal zone. The plastic material is exposed to this environmental matrix and biodegradation is followed

by measuring the evolved CO .
© ISO 2019 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST prEN ISO 22404:2021
---------------------- Page: 8 ----------------------
oSIST prEN ISO 22404:2021
INTERNATIONAL STANDARD ISO 22404:2019(E)
Plastics — Determination of the aerobic biodegradation
of non-floating materials exposed to marine sediment —
Method by analysis of evolved carbon dioxide
1 Scope

This document specifies a laboratory test method to determine the degree and rate of aerobic

biodegradation level of plastic materials. This test method can also be applied to other materials.

Biodegradation is determined by measuring the CO evolved by the plastic material when exposed

to marine sediments sampled from a sandy tidal zone and kept wet with salt-water under laboratory

conditions.

This test method is a simulation under laboratory conditions of the habitat found in sandy tidal zone

that, in marine science, is called eulittoral zone.

The conditions described in this document might not always correspond to the optimum conditions for

the maximum degree of biodegradation to occur.

Deviations from the test conditions described in this document are justified in the test report.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10210, Plastics — Methods for the preparation of samples for biodegradation testing of plastic

materials
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
tidal zone

borderline between sea and land that extends from the high tide line, which is rarely inundated with

water, to the low tide line, which is typically always covered with water

Note 1 to entry: The tidal zone is frequently a sandy area that is kept constantly damp by the lapping of the waves.

Note 2 to entry: Stony and rocky shorelines also exist.

Note 3 to entry: They are also known as eulittoral zone, midlittoral zone, mediolittoral zone, intertidal zone,

foreshore.
© ISO 2019 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST prEN ISO 22404:2021
ISO 22404:2019(E)
3.2
theoretical amount of evolved carbon dioxide
ThCO

maximum theoretical amount of carbon dioxide evolved after completely oxidising a chemical

compound, calculated from the molecular formula or from determination of total organic carbon (TOC)

(3.3) and expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound

3.3
total organic carbon
TOC
amount of carbon bound in an organic compound

Note 1 to entry: Total organic carbon is expressed as milligrams of carbon per 100 milligrams of the compound.

3.4
dissolved organic carbon
DOC

part of the organic carbon in water which cannot be removed by specified phase separation methods,

for example by centrifugation at 40 000 ms for 15 min or by membranes with pores of 0,2 µm to

0,45 µm diameter
3.5
pre-conditioning phase

pre-incubation of an inoculum under the conditions of the subsequent test in the absence of test material,

with the aim to consume potential organic matter present in excess that could disturb biodegradation

measurement and to improve the acclimatization of the microorganisms to the test conditions

4 Principle

This test method is based on the determination of the evolved carbon dioxide and derives from

ISO 19679. The testing medium is based on a sandy marine sediment laid in the bottom of a closed flask;

the sediment is kept wet with natural sea water. The test material is preferably in the form of a powder.

The carbon dioxide evolved during the microbial degradation is determined by a suitable analytical

method. The level of biodegradation is determined by comparing the amount of carbon dioxide evolved

with the theoretical amount (ThCO ) and expressed in per cent. The test result is the maximum level

of biodegradation, determined from the plateau phase of the biodegradation curve. The principle of a

system for measuring evolved carbon dioxide is given in ISO 14852:2018, Annex A.
5 Test environment

Incubation shall take place in the dark in an enclosure which is free from vapours inhibitory to

microorganisms and which is maintained at a constant temperature, preferably between 15 °C to 25 °C,

but not exceeding 28 °C, to an accuracy of ±2 °C. Any change in temperature shall be justified and clearly

indicated in the test report.

NOTE Test results are obtained for temperature that can be different from real conditions in marine

environment.
6 Reagents

6.1 Water, distilled or deionized water, free of toxic substances (copper in particular) and containing

less than 2 mg/l of DOC.
2 © ISO 2019 – All rights reserved
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oSIST prEN ISO 22404:2021
ISO 22404:2019(E)
6.2 Natural seawater/sediment.

Take a sample of a sandy sediment and seawater with a shovel beneath the low-water line into a bucket.

Transfer the wet sediment together with seawater into sealed containers for transport and fast deliver

it to the laboratory. After delivery conserve the sediment at low temperature (approximately 4 °C) until

use. The seawater/sediment sample should be preferably used within 4 weeks after sampling. Record

storage time and conditions. Before use remove any obvious large material (such as plant material,

shells).

NOTE 1 It is possible to obtain sediment from multiple samples collected from different locations to increase

microbial variability.

NOTE 2 Seawater and sediment can also be sampled from large, well running public marine aquaria.

Measure the TOC, pH and nitrogen content of the sediment. The total organic carbon content of sediment

should be in the range 0,1 % to 2 %.
A preliminary oxidation can be applied to the sediment in order to decr
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