Plastics — Determination of the aerobic biodegradation of plastic materials exposed to seawater — Part 1: Method by analysis of evolved carbon dioxide

This document specifies a laboratory test method for determining the degree and rate of the aerobic biodegradation level of plastic materials. Biodegradation is determined by measuring the CO2 evolved from plastic materials when exposed to seawater sampled from coastal areas under laboratory conditions. The conditions described in this document might not always correspond to the optimum conditions for the maximum degree of biodegradation, however this test method is designed to give an indication of the potential biodegradability of plastic materials. NOTE This document addresses plastic materials but can also be used for other materials.

Plastiques — Détermination de la biodégradation aérobie des matériaux plastiques exposés à l'eau de mer — Partie 1: Méthode par analyse du dioxyde de carbone libéré

Le présent document spécifie une méthode d’essai en laboratoire permettant de déterminer le taux et le niveau de biodégradation aérobie des matériaux plastiques. La biodégradation est déterminée en mesurant le CO2 libéré par des matériaux plastiques exposés à de l’eau de mer prélevée dans des zones côtières, dans des conditions de laboratoire. Les conditions décrites dans le présent document ne correspondent pas nécessairement aux conditions optimales permettant d’obtenir le taux maximal de biodégradation; cependant, cette méthode d’essai est conçue pour donner une indication de la biodégradabilité potentielle des matériaux plastiques. NOTE Le présent document concerne les matériaux plastiques, mais il peut aussi être utilisé pour d’autres matériaux.

General Information

Status
Published
Publication Date
09-Nov-2020
Current Stage
6060 - International Standard published
Start Date
10-Nov-2020
Completion Date
10-Nov-2020
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INTERNATIONAL ISO
STANDARD 23977-1
First edition
2020-11
Plastics — Determination of the
aerobic biodegradation of plastic
materials exposed to seawater —
Part 1:
Method by analysis of evolved carbon
dioxide
Plastiques — Détermination de la biodégradation aérobie des
matières plastiques exposées à l'eau de mer —
Partie 1: Méthode par analyse du dioxyde de carbone dégagé
Reference number
ISO 23977-1:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 23977-1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23977-1:2020(E)
Contents Page

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

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 3

5 Test environment ................................................................................................................................................................................................. 3

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Test material ............................................................................................................................................................................................. 5

8.2 Reference materials ............................................................................................................................................................................ 5

8.3 Test set up ................................................................................................................................................................................................... 6

8.4 Pre-conditioning phase .................................................................................................................................................................... 6

8.5 Start of the test ........................................................................................................................................................................................ 6

8.6 Carbon dioxide measurement .................................................................................................................................................... 7

8.7 End of the test .......................................................................................................................................................................................... 7

9 Calculation and expression of results ............................................................................................................................................. 8

9.1 Calculation .................................................................................................................................................................................................. 8

9.1.1 Amount of CO produced .......................................................................................................................................... 8

9.1.2 Percentage of biodegradation............................................................................................................................10

9.2 Visual inspection ................................................................................................................................................................................11

9.3 Expression and interpretation of results ......................................................................................................................11

10 Validity of results ..............................................................................................................................................................................................11

11 Test report ................................................................................................................................................................................................................12

Annex A (informative) Example of a respirometric system .......................................................................................................13

Bibliography .............................................................................................................................................................................................................................15

© ISO 2020 – All rights reserved iii
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ISO 23977-1:2020(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 of 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

www .iso .org/ iso/ foreword .html.

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

Environmental aspects.
A list of all parts in the ISO 23997 series can be found on the ISO website.

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 2020 – All rights reserved
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ISO 23977-1:2020(E)
Introduction

According to the United Nations Environment Program (UNEP), one of the most notable properties of

synthetic polymers and plastics is their durability which, combined with their accidental loss, deliberate

release and poor waste management has resulted in the ubiquitous presence of plastic in oceans (UNEP,

[16]
2015 ).

It is well known and documented that marine litter can pose risks and a negative impact on living

marine organisms and on human beings. Degradability of plastic materials exposed to the marine

environment is one of the factors affecting impact and strength of effects. The uncontrolled dispersion

of biodegradables 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 are of interest in

order to better characterize the behaviour of plastics in these very particular environments. Thus, the

degree and rate of biodegradation is of major interest in order to obtain an indication of the potential

biodegradability of plastic materials when exposed to different marine habitats.

ISO/TC 61/SC 14 has established several test methods for biodegradation testing of plastic materials

under laboratory conditions covering different environmental compartments and test conditions, as

shown in Table 1.
Table 1 — Test methods for biodegradation testing of plastics
Conditions
Test methods
Environmental compartment Presence/absence of oxygen
ISO 14855-1
Controlled composting conditions Aerobic conditions
ISO 14855-2
High-solids anaerobic-digestion
Anaerobic conditions ISO 15985
conditions
Controlled anaerobic slurry system Anaerobic conditions ISO 13975
Soil Aerobic conditions ISO 17556
ISO 14851
Aerobic conditions
Aqueous medium ISO 14852
Anaerobic conditions ISO 14853
ISO 18830
Seawater/sandy sediment interface Aerobic conditions
ISO 19679
Marine sediment Aerobic conditions ISO 22404
ISO 23977-1
Seawater Aerobic conditions
ISO 23977-2

Test method for measuring biodegradation of plastic materials when exposed to marine microbes.

All marine biodegradation test methods are based on exposure of plastic materials to marine samples

(seawater and/or sediment) taken from shoreline areas. By a quantitative viewpoint, these methods

are not equivalent, because, for example, the microbial density in seawater is generally lower compared

to the density determined in sediment. In addition, the microbial composition and diversity can be

different. Moreover, as a rule, the nutrient concentration found in sediment is normally higher compared

to the concentration in seawater.

This document provides a test method for determining the biodegradation level of plastic materials

exposed to the microbial population present in seawater from a pelagic zone under laboratory

conditions. The biodegradation is followed by measuring the evolved CO .

The test is performed with either seawater only (“pelagic seawater test”) or with seawater to which

little sediment was added (“suspended sediment seawater test”).
© ISO 2020 – All rights reserved v
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ISO 23977-1:2020(E)

The pelagic seawater test simulates the conditions found in offshore areas with low water currents and

low tidal movements, whereas the suspended sediment seawater test simulates conditions which might

be found in coastal areas with stronger water currents and tidal movements.
vi © ISO 2020 – All rights reserved
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INTERNATIONAL STANDARD ISO 23977-1:2020(E)
Plastics — Determination of the aerobic biodegradation of
plastic materials exposed to seawater —
Part 1:
Method by analysis of evolved carbon dioxide
1 Scope

This document specifies a laboratory test method for determining the degree and rate of the aerobic

biodegradation level of plastic materials. Biodegradation is determined by measuring the CO evolved

from plastic materials when exposed to seawater sampled from coastal areas under laboratory

conditions.

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

the maximum degree of biodegradation, however this test method is designed to give an indication of

the potential biodegradability of plastic materials.

NOTE This document addresses plastic materials but can also be used for other materials.

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 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples

ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved

organic carbon (DOC)

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

materials
ISO 10523, Water quality — Determination of pH

ISO 11261, Soil quality — Determination of total nitrogen — Modified Kjeldahl method

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
pelagic zone
water body above the seafloor
Note 1 to entry: It is also referred to as the open water or the water column.
© ISO 2020 – All rights reserved 1
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ISO 23977-1:2020(E)

Note 2 to entry: The surface of the pelagic zone is moved by wind-driven waves, is in contact with the atmosphere

and exposed to sunlight. With increasing depth pressure increases, temperature decreases, and light and surface

wave energy are attenuated.
[SOURCE: ISO 22766:2020, 3.4]
3.2
dissolved inorganic carbon
DIC

part of the inorganic carbon in water which cannot be removed by specified phase separation

Note 1 to entry: Phase separation can be achieved 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.
[SOURCE: ISO 14852:—, 3.4]
3.3
theoretical amount of evolved carbon dioxide
ThCO

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

compound, calculated from the molecular formula

Note 1 to entry: It is expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound.

[SOURCE: ISO 14852:—, 3.5]
3.4
total organic carbon
TOC
amount of carbon bound in an organic compound

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

[SOURCE: ISO 17556:2019, 3.14]
3.5
dissolved organic carbon
DOC

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

Note 1 to entry: Phase separation can be achieved 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.
[SOURCE: ISO 14852:—, 3.7]
3.6
lag phase

time from the start of a test until adaptation and/or selection of the degrading microorganisms is

achieved and the degree of biodegradation of a chemical compound or organic matter has increased to

about 10 % of the maximum level of biodegradation (3.8)
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.8]
3.7
biodegradation phase

time from the end of the lag phase (3.6) of a test until the plateau phase has been reached

Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.10]
2 © ISO 2020 – All rights reserved
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ISO 23977-1:2020(E)
3.8
maximum level of biodegradation

degree of biodegradation of a chemical compound or organic matter in a test, above which no further

biodegradation takes place during the test
Note 1 to entry: It is measured in per cent.
[SOURCE: ISO 14852:—, 3.9]
3.9
plateau phase
time from the end of the biodegradation phase (3.7) until the end of a test
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.11]
3.10
pre-conditioning

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

compound or organic matter under test, with the aim of improving the test by acclimatization of the

microorganisms to the test conditions
[SOURCE: ISO 14852:—, 3.13]
4 Principle

This document describes two variations of a test method for determining the biodegradability of plastic

materials by the indigenous population of microorganisms in natural seawater using a static aqueous

test system. The test is performed under mesophilic test conditions for up to two years by incubating

plastic materials with either seawater only (“pelagic seawater test”) or with seawater to which low

amount of sediment has been added (“suspended sediment seawater test”), coming from the same site

as that from which the seawater was taken.

Biodegradation is followed by measuring the evolution of carbon dioxide using a suitable, analytical

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

with the theoretical amount [theoretical amount of evolved carbon dioxide (ThCO )] and expressed in

percentage. 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:—, Annex A.
5 Test environment

Incubation shall take place in the dark or in diffused light, in an enclosure which is free from vapours

inhibitory to marine microorganisms and which is maintained at a constant mesophilic temperature.

It should preferably be between 15 °C to 25 °C, but not exceeding 28 °C, to an accuracy of ±1 °C. Any

change in temperature shall be justified and clearly indicated in the test report.

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

environment.
6 Reagents
Use only reagents of recognized analytical grade.
6.1 Water

Distilled or deionized water, free of toxic substances (copper in particular) and containing less than

2 mg/l of TOC.
© ISO 2020 – All rights reserved 3
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ISO 23977-1:2020(E)
6.2 Natural seawater/sediment

Sampling, preservation, handling, transport and storage of natural seawater, and, if applicable,

sediment collected from the same site as that from which the seawater is taken, shall be in accordance

with ISO 5667-3.

Prior to use, remove coarse particles from the seawater and, if applicable, from the sediment by

appropriate means. The procedure used shall be reported.

Seawater can be filtered using a paper filter in order to remove coarse particles. It is recommended to

reduce the amount of coarse particles in sediment by means of at least two washing steps using filtered

seawater without coarse particles.

Measure TOC, pH and nitrogen content of seawater and, if applicable, of sediment samples according to

ISO 8245, ISO 10523 and ISO 11261, respectively.

If the TOC content of the seawater sample is found to be high, the seawater should be pre-conditioned

for about a week prior to use. If, for instance, the background concentration of TOC exceeds about 20 %

of the total TOC after addition of the test item, then pre-condition the seawater and, if applicable, the

sediment by stirring under aerobic conditions at the test temperature and in the dark or in diffuse light

in order to reduce the content of easily degradable organic material.

Provide the following information on the seawater, and, if applicable, on the sediment sample itself:

— date of collection;
— depth of collection (m);
— appearance of sample - turbid, clear, etc.;
— temperature at the time of collection (°C);
— salinity (PSU);
— total organic carbon (TOC; mg/l);
— nitrogen (total-N; mg/l);
— pH;
— description of the pre-conditioning process, if applicable.
7 Apparatus

Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter.

Required is usual laboratory equipment, plus the following.

7.1 Test flasks. Biometric flasks of the volume of about 300 ml are appropriate. The vessels shall be

located in a constant temperature room or in an apparatus fitted with a thermostat (e.g. water-bath).

Reactors with higher or lower volumes can be used, if environmental conditions are not affected.

7.2 Container for the CO absorber, (e.g. glass beaker) to be located in the headspace of a test flask

and filled with 10 ml of Ba(OH) 0,012 5 mol/l or 3 ml of KOH 0,5 mol/l.

As an alternative to Ba(OH) and KOH 4 ml of NaOH 1 mol/l can be used as a CO -absorber.

2 2
A suitable apparatus is shown in Annex A, Figure A.1.
4 © ISO 2020 – All rights reserved
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ISO 23977-1:2020(E)

7.3 Analytical equipment for determining carbon dioxide, consisting of any suitable apparatus

with sufficient accuracy, such as a CO or dissolved inorganic carbon (DIC) analyser or apparatus for

titrimetric determination after complete absorption in a basic solution, shall be used.

7.4 Analytical balance, which shall have a sensitivity of at least 0,1 mg.
7.5 Magnetic stirrer.
7.6 pH meter.
8 Procedure
8.1 Test material

The sample shall be of known mass and contain enough carbon to yield CO that can be adequately

measured by the chosen system. Use a test material concentration of at least 100 mg per litre of seawater.

This mass of the sample should correspond to TOC of about 60 mg/l. The maximum mass of sample per

flask is limited by the oxygen supply in the glass flask. The recommended amount per litre seawater

is 150 mg to 300 mg of test material per litre seawater. Calculate the TOC from the chemical formula

or determine it by means of a suitable analytical technique (e.g. elemental analysis or measurement in

accordance with ISO 8245) and calculate the ThCO .

The test material is added to a test flask, either as powder or in the form of a film. If the test material

is used in the form of powder, particles of known, narrow size distribution should be used. A particle-

size distribution with a maximum diameter of 250 µm is recommended. The preparation of powder

shall be performed in accordance with ISO 10210. If the test material is used in the form of a film, it

can be added either as pieces in the range of 0,2 cm × 0,2 cm to 0,5 cm × 0,5 cm or as a single plastic

strip (width: approximately 1,0 cm, length: depending on weight of the polymer and thickness of the

film). It is recommended that the plastic strip is fixed in, for example, a Polytetrafluoroethylene (PTFE)

coated fibre net (size: approximately 4 cm × 9 cm, mesh size: 5 mm × 5 mm). The fibre net is folded

into 2 layers (approximately 2 cm × 9 cm) with the plastic strip test material fixed in between. Then, the

two ends of the fibre net are attached together. The test material fixed between the fibre net is placed

upright on the ground of a bottle base in the form of a cylinder (see Annex A, Figure A.2).

The form and shape of the test material can influence its biodegradability. Similar particle sizes of power

should preferably be used in the test. Similar shapes and thicknesses of the films should preferably be

used if different kinds of plastic materials are to be compared.

When powder or pieces of films are used in the test, particles or film pieces can stick on the inner

wall of the testing bottle above the seawater. In such cases, a slight manual shaking of the bottle is

recommended to regain the powder or film pieces back to the seawater sample. If the material is added

as a cylindrical plastic strip fixed between, such as a Polytetrafluoroethylene (PTFE) coated fibre net

(see Annex A, Figure A.2), it is immersed in the seawater most of the time.
8.2 Reference materials

Use microcrystalline cellulose or ashless cellulose filters as a reference material . If possible, the TOC,

form, and size should be comparable to that of the test material.

1) PTFE Glass Fabric (product no 9002) produced by Fiberflon (https:// www .fiberflon .de/ Products/ PTFE -Coated

-Open -Mesh -Fabrics/ Page -307 -17 .aspx) has been found satisfactory for this purpose and is an example of a suitable

product available commercially. This information is given for the convenience of users of this document and does

not constitute an endorsement by ISO of this product.

2) Microcrystalline Cellulose "Avicel" produced by Merck or laboratory filter paper Whatman n° 42 has been found

satisfactory for this purpose and are examples of suitable products available commercially. This information is given

for the convenience of users of this document and does not constitute an endorsement by ISO of these products.

© ISO 2020 – All rights reserved 5
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ISO 23977-1:2020(E)

As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same form as the test

material can be used.
8.3 Test set up
Provide several flasks, so that the test includes at least the following:
a) three flasks for the test material (symbol F );
b) three flasks for the blank (symbol F );
c) three flasks for reference material (symbol F ).

In addition, if biodegradation is expected to take longer than 6 months, it is recommended that a

negative control is included:
d) three flasks for negative control (symbol F ).

Two flasks for test material, blank, reference material, and negative control may be used instead of

three for screening purposes.
8.4 Pre-conditioning phase
As a rule, use a test flask with a volume of 300 ml.

The test is performed in batch by incubating the test materials with either 90 ml of natural seawater

only (“pelagic seawater test”) or with 90 ml of natural seawater to which sediment of 0,1 g/l to 1,0 g/l

(wet weight) has added (“suspended sediment seawater test”).

Add carbon dioxide absorber to the absorber compartments of the test flask, as a rule 10 ml Ba(OH)

0,012 5 mol/l, 3 ml of KOH 0,5 mol/l or 4 ml of NaOH 1,0 mol/l. Place the sealed flasks on a magnetic stirrer

(7.5) in a constant-temperature environment and allow all vessels to reach the desired temperature.

Agitation shall be continuous (e.g. 100 r/min agitation) in order to maintain microorganisms and, if

applicable, sediment in suspension.

The abrasion of sediment in coastal areas is a natural phenomenon caused by water currents and tidal

movements. Nevertheless, if a magnetic stirring bar is used to mix the seawater to which sediment has

added (“suspended sediment seawater test”), it is recommended that either a PTFE-coated dumbbell

shaped magnetic stirring bar be used or a PTFE-coated magnetic bar equipped with a pivot ring in

order to reduce excessive abrasion of sediment during the test period. Other stirring systems can be

[17]

used, too. Examples of suitable set-ups are given in Briassoulis D. et.al. and OECD TG 308:2002,

[14]
Annex 4 .

Take the necessary readings and monitor the CO evolution. This phase is carried out to verify that the

endogenous respiration is similar in the different vessels. In addition, the background concentration of

easily degradable organic material in natural seawater and, if applicable, in sediment is reduced in this

phase, following the pre-conditioning procedure given in 6.2.
8.5 Start of the test

After the pre-conditioning phase open the flasks and add the test material either as powder or in the

form of a film to the test flasks (7.1). The mass of samples shall be about 20 mg test material when using

a flask with a volume of 300 ml corresponding to an initial test item concentration specified in 8.1.

Repeat the procedure for the reference material and, if applicable, for the material of the negative

control. Record the mass of the test sample, the volume of seawater and, if applicable, the mass of the

sediment which has been added to each flask.

It is recommended to add KH PO (0,1 g/l) and NH Cl (0,05 g/l) to seawater samples at the beginning

2 4 4
of a test.
6 © ISO 2020 – All rights reserved
------------
...

NORME ISO
INTERNATIONALE 23977-1
Première édition
2020-11
Plastiques — Détermination de la
biodégradation aérobie des matériaux
plastiques exposés à l'eau de mer —
Partie 1:
Méthode par analyse du dioxyde de
carbone libéré
Plastics — Determination of the aerobic biodegradation of plastic
materials exposed to seawater —
Part 1: Method by analysis of evolved carbon dioxide
Numéro de référence
ISO 23977-1:2020(F)
© ISO 2020
---------------------- Page: 1 ----------------------
ISO 23977-1:2020(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2020

Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, 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, ou la diffusion sur l’internet ou sur un intranet, sans autorisation écrite préalable. Une autorisation peut

être demandée 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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Publié en Suisse
© ISO 2020 – Tous droits réservés
---------------------- Page: 2 ----------------------
ISO 23977-1:2020(F)
Sommaire Page

Avant-propos .............................................................................................................................................................................................................................iv

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

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

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

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

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

5 Environnement d’essai ..................................................................................................................................................................................3

6 Réactifs ........................................................................................................................................................................................................................... 4

7 Appareillage .............................................................................................................................................................................................................. 5

8 Mode opératoire ................................................................................................................................................................................................... 5

8.1 Matériau d’essai ..................................................................................................................................................................................... 5

8.2 Matériaux de référence ................................................................................................................................................................... 6

8.3 Configuration d’essai ........................................................................................................................................................................ 6

8.4 Phase de préconditionnement .................................................................................................................................................. 6

8.5 Début de l’essai ....................................................................................................................................................................................... 7

8.6 Mesurage du dioxyde de carbone .......................................................................................................................................... 8

8.7 Fin de l’essai .............................................................................................................................................................................................. 8

9 Calcul et expression des résultats .....................................................................................................................................................8

9.1 Calcul ............................................................................................................................................................................................................... 8

9.1.1 Quantité de CO produit ............................................................................................................................................... 8

9.1.2 Pourcentage de biodégradation ......................................................................................................................... 11

9.2 Inspection visuelle ........................................................................................................................................................................... 11

9.3 Expression et interprétation des résultats ................................................................................................................ 11

10 Validité des résultats....................................................................................................................................................................................12

11 Rapport d’essai ...................................................................................................................................................................................................12

Annexe A (informative) Exemple de système respirométrique ..........................................................................................14

Bibliographie ...........................................................................................................................................................................................................................16

iii
© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(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 attirée 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 nature volontaire des normes, 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 www.iso.org/avant-propos.

Le présent document a été élaboré par le comité technique ISO/TC 61, Plastiques, sous-comité SC 14,

Aspects liés à l’environnement.

Une liste de toutes les parties de la série ISO 23997 se trouve sur le site web de l’ISO.

Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent

document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l’adresse www.iso.org/fr/members.html.
© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)
Introduction

Selon le Programme des Nations Unies pour l’environnement (PNUE), l’une des propriétés les plus

notables des polymères synthétiques et des plastiques est leur durabilité qui, combinée à leur perte

accidentelle, leur déversement délibéré ou une mauvaise gestion des déchets, a conduit à l’omniprésence

[16]
des plastiques dans les océans (PNUE, 2015 ).

On sait, et cela a été documenté, que les déchets marins peuvent présenter des risques et avoir un

impact négatif sur les organismes marins vivants et les êtres humains. La dégradabilité des matériaux

plastiques exposés à l’environnement marin est l’un des facteurs ayant une incidence sur l’impact et

la force des effets. La dispersion incontrôlée des plastiques biodégradables dans les environnements

naturels n’est pas souhaitable. La biodégradabilité des produits ne peut pas être considérée comme

une excuse pour épandre des déchets qui devraient être valorisés et recyclés. Cependant, les méthodes

d’essai permettant de mesurer le taux et le niveau de biodégradation dans les environnements naturels

présentent un intérêt, car elles permettent de mieux caractériser le comportement des plastiques dans

ces environnements très particuliers. Ainsi, le taux et le niveau de biodégradation présentent un intérêt

majeur pour obtenir une indication de la biodégradabilité potentielle des matériaux plastiques lorsqu’ils

sont exposés à différents habitats marins.

L’ISO/TC 61/SC 14 a mis au point plusieurs méthodes d’essai portant sur la biodégradation des matériaux

plastiques dans des conditions de laboratoire couvrant différents compartiments environnementaux et

différentes conditions d’essai, comme indiqué dans le Tableau 1.
Tableau 1 — Méthodes d’essai sur la biodégradation des plastiques
Conditions
Méthodes d’essai
Compartiment environnemental Présence/absence d’oxygène
ISO 14855-1
Conditions de compostage contrô-
Conditions aérobies
lées
ISO 14855-2
Conditions de digestion anaérobie à
Conditions anaérobies ISO 15985
teneur élevée en solides
Système de boue anaérobie contrô- ISO 13975
Conditions anaérobies
lée
Sol Conditions aérobies ISO 17556
ISO 14851
Conditions aérobies
Milieu aqueux ISO 14852
Conditions anaérobies ISO 14853
ISO 18830
Interface eau de mer/sédiment
Conditions aérobies
sableux a
ISO 19679
Sédiment marin Conditions aérobies ISO 22404
ISO 23977-1
Eau de mer Conditions aérobies
ISO 23977-2

Méthode d’essai pour mesurer la biodégradation des matériaux plastiques exposés à des microbes marins.

Toutes les méthodes d’essai de biodégradation marine reposent sur une exposition des matériaux

plastiques à des échantillons marins (eau de mer et/ou sédiment) prélevés dans des zones côtières.

D’un point de vue quantitatif, ces méthodes ne sont pas équivalentes, car, par exemple, la densité

microbienne dans l’eau de mer est généralement plus faible que la densité déterminée dans le sédiment.

En outre, la composition et la diversité microbiennes peuvent être différentes. De plus, en règle générale,

la concentration en nutriments trouvée dans le sédiment est normalement plus élevée que celle dans

l’eau de mer.

Le présent document fournit une méthode d’essai permettant de déterminer le niveau de biodégradation

des matériaux plastiques exposés à la population microbienne présente dans l’eau de mer provenant

© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)

d’une zone pélagique, dans des conditions de laboratoire. La biodégradation est obtenue en mesurant le

CO libéré.

L’essai est réalisé uniquement avec de l’eau de mer («essai avec eau de mer pélagique») ou avec de l’eau

de mer à laquelle un peu de sédiment a été ajouté («essai avec eau de mer et sédiment en suspension»).

L’essai avec eau de mer pélagique simule les conditions présentes dans des zones au large des côtes avec

de faibles courants et de faibles mouvements de marées, tandis que l’essai avec eau de mer et sédiment

en suspension simule des conditions pouvant être présentes dans des zones côtières avec de plus forts

courants et mouvements de marées.
© ISO 2020 – Tous droits réservés
---------------------- Page: 6 ----------------------
NORME INTERNATIONALE ISO 23977-1:2020(F)
Plastiques — Détermination de la biodégradation aérobie
des matériaux plastiques exposés à l'eau de mer —
Partie 1:
Méthode par analyse du dioxyde de carbone libéré
1 Domaine d’application

Le présent document spécifie une méthode d’essai en laboratoire permettant de déterminer le taux et

le niveau de biodégradation aérobie des matériaux plastiques. La biodégradation est déterminée en

mesurant le CO libéré par des matériaux plastiques exposés à de l’eau de mer prélevée dans des zones

côtières, dans des conditions de laboratoire.

Les conditions décrites dans le présent document ne correspondent pas nécessairement aux conditions

optimales permettant d’obtenir le taux maximal de biodégradation; cependant, cette méthode d’essai

est conçue pour donner une indication de la biodégradabilité potentielle des matériaux plastiques.

NOTE Le présent document concerne les matériaux plastiques, mais il peut aussi être utilisé pour d’autres

matériaux.
2 Références normatives

Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur

contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.

Pour les références non datées, la dernière édition du document de référence s’applique (y compris les

éventuels amendements).

ISO 5667-3, Qualité de l'eau — Échantillonnage — Partie 3: Conservation et manipulation des échantillons

d'eau

ISO 8245, Qualité de l'eau — Lignes directrices pour le dosage du carbone organique total (COT) et du

carbone organique dissous (COD)

ISO 10210, Plastiques — Méthodes de préparation des échantillons pour les essais de biodégradation des

matériaux plastiques
ISO 10523, Qualité de l'eau — Détermination du pH

ISO 11261, Qualité du sol — Dosage de l'azote total — Méthode de Kjeldahl modifiée

3 Termes et définitions

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

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp

— IEC Electropedia: disponible à l’adresse https:// www .electropedia .org/
© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)
3.1
zone pélagique
masse d’eau au-dessus du fond océanique
Note 1 à l'article: Est aussi appelée eau libre ou colonne d’eau.

Note 2 à l'article: La surface de la zone pélagique est mue par des vagues poussées par le vent, est en contact avec

l’atmosphère et est exposée aux rayons du soleil. Au fur et mesure que la pression augmente en profondeur, la

température diminue et la lumière et l’énergie des vagues en surface sont atténuées.

[SOURCE: ISO 22766:2020, 3.4]
3.2
carbone inorganique dissous
CID

proportion du carbone inorganique contenu dans l’eau qui ne peut pas être éliminée par une séparation

de phase spécifique

Note 1 à l'article: La séparation de phase peut être obtenue par exemple par centrifugation à 40 000 m⋅s pendant

15 min ou par une filtration sur membrane au moyen de membranes ayant des pores de 0,2 µm à 0,45 µm de

diamètre.
[SOURCE: ISO 14852:—, 3.4]
3.3
quantité théorique de dioxyde de carbone libéré
ThCO

quantité théorique maximale de dioxyde de carbone libéré après l’oxydation complète d’un composé

chimique, calculée d’après la formule moléculaire

Note 1 à l'article: Elle est exprimée en milligrammes de dioxyde de carbone libéré par milligramme ou par

gramme de composé soumis à essai.
[SOURCE: ISO 14852:—, 3.5]
3.4
carbone organique total
COT
quantité de carbone incluse dans un composé organique

Note 1 à l'article: Il est exprimé en milligrammes de carbone par 100 mg de composé.

[SOURCE: ISO 17556:2019, 3.14]
3.5
carbone organique dissous
COD

proportion du carbone organique contenu dans l’eau qui ne peut pas être éliminée par une séparation

de phase spécifique

Note 1 à l'article: La séparation de phase peut être obtenue par exemple par centrifugation à 40 000 m⋅s pendant

15 min ou par une filtration sur membrane au moyen de membranes ayant des pores de 0,2 µm à 0,45 µm de

diamètre.
[SOURCE: ISO 14852:—, 3.7]
3.6
phase de latence

durée 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 (3.8)

Note 1 à l'article: Elle est mesurée en jours.
© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)
[SOURCE: ISO 14852:—, 3.8]
3.7
phase de biodégradation

durée depuis la fin de la phase de latence (3.6) de l’essai jusqu’à ce que l’on ait obtenu la phase stationnaire

Note 1 à l'article: Elle est mesurée en jours.
[SOURCE: ISO 14852:—, 3.10]
3.8
niveau maximal de biodégradation

degré de biodégradation d’un composé chimique ou d’une matière organique lors d’un essai, au-dessus

duquel la biodégradation ne se poursuit pas
Note 1 à l'article: Il est mesuré en pourcentage.
[SOURCE: ISO 14852:—, 3.9]
3.9
phase stationnaire

durée écoulée entre la fin de la phase de biodégradation (3.7) et la fin de l’essai

Note 1 à l'article: Elle est mesurée en jours.
[SOURCE: ISO 14852:—, 3.11]
3.10
préconditionnement

pré-incubation d’un inoculum dans les conditions de l’essai effectué ultérieurement, en l’absence de la

matière organique ou du composé chimique à analyser, dans le but d’améliorer l’essai par acclimatation

des micro-organismes aux conditions d’essai
[SOURCE: ISO 14852:—, 3.13]
4 Principe

Le présent document décrit deux variantes d’une méthode d’essai permettant de déterminer la

biodégradabilité des matériaux plastiques par la population indigène de micro-organismes dans l’eau

de mer naturelle à l’aide d’un système d’essai aqueux statique. L’essai est réalisé dans des conditions

d’essai mésophiles, pour une durée allant jusqu’à deux ans, en incubant des matériaux plastiques

uniquement avec de l’eau de mer («essai avec eau de mer pélagique») ou avec de l’eau de mer à laquelle

une petite quantité de sédiment a été ajoutée («essai avec eau de mer et sédiment en suspension»),

provenant du même site que l’eau de mer prélevée.

La biodégradation est obtenue en mesurant la libération de dioxyde de carbone à l’aide d’une méthode

analytique adaptée. Le niveau de biodégradation est déterminé en comparant la quantité de dioxyde de

carbone libéré avec la quantité théorique [quantité théorique de dioxyde de carbone libéré (ThCO )] et

en l’exprimant en pourcentage. Le résultat d’essai est le niveau maximal de biodégradation, déterminé

à partir du plateau de la courbe de biodégradation. Le principe de fonctionnement d’un système d’essai

permettant de mesurer le dioxyde de carbone libéré est présenté dans l’ISO 14852:—, Annexe A.

5 Environnement d’essai

L’incubation doit avoir lieu dans l’obscurité ou sous une lumière diffuse dans une enceinte exempte de

vapeurs susceptibles d’inhiber les micro-organismes marins et qui est maintenue à une température

mésophile constante. Il convient de préférence qu’elle soit comprise entre 15 °C et 25 °C, sans toutefois

© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)

dépasser 28 °C, avec une précision de ±1 °C. Tout changement de température doit être justifié et

clairement indiqué dans le rapport d’essai.

NOTE Les résultats d’essai sont obtenus pour des températures pouvant être différentes des conditions

réelles en environnement marin.
6 Réactifs
Utiliser uniquement des réactifs de qualité analytique reconnue.
6.1 Eau

Eau distillée ou déionisée, exempte de substances toxiques (en particulier, le cuivre) et contenant moins

de 2 mg/l de COT.
6.2 Eau de mer naturelle/sédiment

L’échantillonnage, la préservation, la manipulation, le transport et le stockage de l’eau de mer naturelle

et, le cas échéant, du sédiment recueilli sur le même site que celui de l’eau de mer prélevée, doivent être

conformes à l’ISO 5667-3.

Avant utilisation, retirer les particules grossières de l’eau de mer et, le cas échéant, du sédiment par des

moyens appropriés. Le mode opératoire utilisé doit figurer dans le rapport d’essai.

L’eau de mer peut être filtrée sur un papier-filtre pour éliminer les particules grossières. Il est

recommandé de réduire la quantité de particules grossières dans le sédiment en réalisant au moins

deux étapes de lavage avec de l’eau de mer filtrée sans particules grossières.

Mesurer le COT, le pH et la teneur en azote des échantillons d’eau de mer et, le cas échéant, de sédiment

conformément à l’ISO 8245, l’ISO 10523 et l’ISO 11261, respectivement.

Si la teneur en COT de l’échantillon d’eau de mer est trop élevée, il convient de préconditionner l’eau de

mer pendant environ une semaine avant utilisation. Si, par exemple, la concentration de fond du COT

dépasse d’environ 20 % le COT total après ajout de l’élément d’essai, alors préconditionner l’eau de mer

et, le cas échéant, le sédiment par agitation dans des conditions aérobies à la température d’essai et

dans l’obscurité ou sous une lumière diffuse, afin de réduire la teneur en matière organique facilement

dégradable.

Fournir les informations suivantes sur l’échantillon d’eau de mer et, le cas échéant, de sédiment:

— date de collecte;
— profondeur de collecte (m);
— aspect de l’échantillon - trouble, clair, etc.;
— température au moment de la collecte (°C);
— salinité (PSU);
— carbone organique total (COT; mg/l);
— azote (N total; mg/l);
— pH;
— description du processus de préconditionnement, le cas échéant.
© ISO 2020 – Tous droits réservés
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ISO 23977-1:2020(F)
7 Appareillage

S’assurer que toute la verrerie de laboratoire a été soigneusement nettoyée et, en particulier, qu’elle

est exempte de toute trace de substances organiques ou toxiques. Est nécessaire le matériel courant de

laboratoire, et ce qui suit.

7.1 Fioles d’essai. Des fioles biométriques d’un volume d’environ 300 ml conviennent. Les récipients

doivent être placés dans une salle à température constante ou dans un appareil muni d’un thermostat

(par exemple bain-marie).

Des réacteurs de volumes supérieurs ou inférieurs peuvent être utilisés si cela n’influe pas sur les

conditions environnementales.

7.2 Récipient pour l’absorbeur de CO (par exemple bécher en verre) à placer dans l’espace de tête

de la fiole d’essai et à remplir avec 10 ml de Ba(OH) à 0,012 5 mol/l ou avec 3 ml de KOH à 0,5 mol/l.

Autrement, il est possible d’utiliser 4 ml de NaOH à 1 mol/l comme absorbeur de CO à la place du

Ba(OH) et du KOH.
Un appareillage adapté est illustré dans l’Annexe A, Figure A.1.

7.3 Équipement analytique pour la détermination du dioxyde de carbone, c’est-à-dire tout

appareil adapté ayant une exactitude suffisante, tel qu’un analyseur de CO ou de carbone inorganique

dissous (CID) ou un appareil pour la détermination titrimétrique après absorption complète dans une

solution basique.
7.4 Balance analytique, devant avoir une sensibilité d’au moins 0,1 mg.
7.5 Agitateur magnétique.
7.6 pH-mètre.
8 Mode opératoire
8.1 Matériau d’essai

L’échantillon doit avoir une masse connue et contenir suffisamment de carbone pour donner une

quantité de CO susceptible d’être mesurée de manière adéquate par le système choisi. Utiliser une

concentration de matériau d’essai d’au moins 100 mg/l d’eau de mer. Il convient que cette masse

d’échantillon corresponde à un COT d’environ 60 mg/l. La masse maximale d’échantillon par fiole est

limitée par l’alimentation en oxygène de la fiole en verre. La quantité recommandée est de 150 mg à

300 mg de matériau d’essai par litre d’eau de mer. Calculer le COT d’après la formule chimique ou le

déterminer par une technique d’analyse appropriée (par exemple analyse élémentaire ou mesurage

conformément à l’ISO 8245) et calculer la ThCO .

Introduire le matériau d’essai dans une fiole d’essai sous forme de poudre ou de film. 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 maximal de

250 µm est recommandée. La préparation de la poudre doit être réalisée conformément à l’ISO 10210.

Si le matériau d’essai est utilisé sous forme de film, il peut être ajouté soit sous forme de morceaux

mesurant de 0,2 cm × 0,2 cm à 0,5 cm × 0,5 cm, soit sous la forme d’une bande de plastique (largeur:

environ 1,0 cm, longueur: en fonction du poids du polymère et de l’épaisseur du film). Il est recommandé

de fixer la bande de plastique, par exemple dans un filet en fibres revêtues de polytétrafluoroéthylène

© ISO 2020 – Tous droits réservés
---------------------- Page: 11 ----------------------
ISO 23977-1:2020(F)

(PTFE) (dimension: environ 4 cm × 9 cm, taille de maille: 5 mm × 5 mm). Le filet en fibres est plié en

2 couches (d’environ 2 cm × 9 cm) et la bande de plastique est fixée entre les deux. Ensuite, les deux

bords du filet en fibres sont attachés ensemble. Le matériau d’essai fixé dans le filet en fibres est placé à

la verticale dans un fond de bouteille de forme cylindrique (voir l’Annexe A, Figure A.2).

La consistance et la forme du matériau d’essai peuvent influer sur sa biodégradabilité. Pour l’essai, il

convient d’utiliser de préférence des particules de poudre de grosseurs similaires. Il convient d’utiliser,

de préférence, des formes et épaisseurs similaires de films si l’on doit comparer différents types de

matériaux plastiques.

Lorsque de la poudre ou des morceaux de film sont utilisés pour l’essai, les particules ou morceaux de

film peuvent adhérer à la paroi intérieure de la bouteille d’essai au-dessus du niveau de l’eau de mer.

Dans ce cas, une légère agitation manuelle de la bouteille est recommandée pour ramener la poudre ou

les morceaux de film dans l’échantillon d’eau de mer. Si le matériau est ajouté sous la forme d’une bande

de plastique cylindrique fixée par exemple dans un filet en fibres revêtues de polytétrafluoroéthylène

(PTFE) (voir l’Annexe A, Figure A.2), il est immergé dans l’eau de mer la plupart du temps.

8.2 Matériaux de référence

Utiliser des filtres de cellulose microcristalline ou de cellulose exempte de cendres comme matériau de

référence . Il convient, si possible, que le COT, la consistance et la taille du matériau de référence soient

comparables à ceux du matériau d’essai.

Comme témoin négatif, il est possible d’utiliser un polymère non biodégradable (par exemple du

polyéthylène) sous la même forme que le matériau d’essai.
8.3 Configuration d’essai
Prévoir plusieurs fioles pour que l’essai comprenne au moins ce qui suit:
a) trois fioles pour le matériau d’essai (symbole F );
b) trois fioles pour le blanc (symbole F );
c) trois fioles pour le matériau de référence (symbole F ).

En outre, si la biodégradation est censée durer plus de 6 mois, il est recommandé qu’un témoin négatif

soit inclus:
d) trois fioles pour le témoin négatif (symbole F ).

Il est possible d’utiliser deux fioles au lieu de trois pour le matériau d’essai, le blanc, le matériau de

référence et le témoin négatif, à des fins de sélection.
8.4 Phase de préconditionnement
En règle générale, utiliser une fiole d’essai d’un volume de 300 ml.

L’essai est réalisé par lots en incubant les matériaux d’essai avec 90 ml d’eau de mer naturelle

uniquement («essai avec eau de mer pélagique») ou avec 90 ml d’eau de mer naturelle à laquelle une

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FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 23977-1
ISO/TC 61/SC 14
Plastics — Determination of the
Secretariat: DIN
aerobic biodegradation of plastic
Voting begins on:
2020-08-17 materials exposed to seawater —
Voting terminates on:
Part 1:
2020-10-12
Method by analysis of evolved carbon
dioxide
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 23977-1:2020(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 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 23977-1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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ISO/FDIS 23977-1:2020(E)
Contents Page

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

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 3

5 Test environment ................................................................................................................................................................................................. 3

6 Reagents ........................................................................................................................................................................................................................ 3

7 Apparatus ..................................................................................................................................................................................................................... 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Test material ............................................................................................................................................................................................. 5

8.2 Reference materials ............................................................................................................................................................................ 5

8.3 Test set up ................................................................................................................................................................................................... 6

8.4 Pre-conditioning phase .................................................................................................................................................................... 6

8.5 Start of the test ........................................................................................................................................................................................ 6

8.6 Carbon dioxide measurement .................................................................................................................................................... 7

8.7 End of the test .......................................................................................................................................................................................... 7

9 Calculation and expression of results ............................................................................................................................................. 8

9.1 Calculation .................................................................................................................................................................................................. 8

9.1.1 Amount of CO produced .......................................................................................................................................... 8

9.1.2 Percentage of biodegradation............................................................................................................................10

9.2 Visual inspection ................................................................................................................................................................................11

9.3 Expression and interpretation of results ......................................................................................................................11

10 Validity of results ..............................................................................................................................................................................................11

11 Test report ................................................................................................................................................................................................................12

Annex A (informative) Example of a respirometric system .......................................................................................................13

Bibliography .............................................................................................................................................................................................................................15

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ISO/FDIS 23977-1:2020(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 of 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

www .iso .org/ iso/ foreword .html.

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

Environmental aspects.
A list of all parts in the ISO 23997 series can be found on the ISO website.

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.
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ISO/FDIS 23977-1:2020(E)
Introduction

According to the United Nations Environment Program (UNEP), one of the most notable properties of

synthetic polymers and plastics is their durability which, combined with their accidental loss, deliberate

release and poor waste management has resulted in the ubiquitous presence of plastic in oceans (UNEP,

[16]
2015 ).

It is well known and documented that marine litter can pose risks and a negative impact on living

marine organisms and on human beings. Degradability of plastic materials exposed to the marine

environment is one of the factors affecting impact and strength of effects. The uncontrolled dispersion

of biodegradables 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 are of interest in

order to better characterize the behaviour of plastics in these very particular environments. Thus, the

degree and rate of biodegradation is of major interest in order to obtain an indication of the potential

biodegradability of plastic materials when exposed to different marine habitats.

ISO/TC 61/SC 14 has established several test methods for biodegradation testing of plastic materials

under laboratory conditions covering different environmental compartments and test conditions, as

shown in Table 1.
Table 1 — Test methods for biodegradation testing of plastics
Conditions
Test methods
Environmental compartment Presence/absence of oxygen
ISO 14855-1
Controlled composting conditions Aerobic conditions
ISO 14855-2
High-solids anaerobic-digestion
Anaerobic conditions ISO 15985
conditions
Controlled anaerobic slurry system Anaerobic conditions ISO 13975
Soil Aerobic conditions ISO 17556
ISO 14851
Aerobic conditions
Aqueous medium ISO 14852
Anaerobic conditions ISO 14853
ISO 18830
Seawater/sandy sediment interface Aerobic conditions
ISO 19679
Marine sediment Aerobic conditions ISO 22404
ISO 23977-1
Seawater Aerobic conditions
ISO 23977-2

Test method for measuring biodegradation of plastic materials when exposed to marine microbes.

All marine biodegradation test methods are based on exposure of plastic materials to marine samples

(seawater and/or sediment) taken from shoreline areas. By a quantitative viewpoint, these methods

are not equivalent, because, for example, the microbial density in seawater is generally lower compared

to the density determined in sediment. In addition, the microbial composition and diversity can be

different. Moreover, as a rule, the nutrient concentration found in sediment is normally higher compared

to the concentration in seawater.

This document provides a test method for determining the biodegradation level of plastic materials

exposed to the microbial population present in seawater from a pelagic zone under laboratory

conditions. The biodegradation is followed by measuring the evolved CO .

The test is performed with either seawater only (“pelagic seawater test”) or with seawater to which

little sediment was added (“suspended sediment seawater test”).
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ISO/FDIS 23977-1:2020(E)

The pelagic seawater test simulates the conditions found in offshore areas with low water currents and

low tidal movements, whereas the suspended sediment seawater test simulates conditions which might

be found in coastal areas with stronger water currents and tidal movements.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 23977-1:2020(E)
Plastics — Determination of the aerobic biodegradation of
plastic materials exposed to seawater —
Part 1:
Method by analysis of evolved carbon dioxide
1 Scope

This document specifies a laboratory test method for determining the degree and rate of the aerobic

biodegradation level of plastic materials. Biodegradation is determined by measuring the CO evolved

from plastic materials when exposed to seawater sampled from coastal areas under laboratory

conditions.

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

the maximum degree of biodegradation, however this test method is designed to give an indication of

the potential biodegradability of plastic materials.

NOTE This document addresses plastic materials but can also be used for other materials.

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 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples

ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved

organic carbon (DOC)

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

materials
ISO 10523, Water quality — Determination of pH

ISO 11261, Soil quality — Determination of total nitrogen — Modified Kjeldahl method

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
pelagic zone
water body above the seafloor
Note 1 to entry: It is also referred to as the open water or the water column.
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ISO/FDIS 23977-1:2020(E)

Note 2 to entry: The surface of the pelagic zone is moved by wind-driven waves, is in contact with the atmosphere

and exposed to sunlight. With increasing depth pressure increases, temperature decreases, and light and surface

wave energy are attenuated.
[SOURCE: ISO 22766:2020, 3.4]
3.2
dissolved inorganic carbon
DIC

part of the inorganic carbon in water which cannot be removed by specified phase separation

Note 1 to entry: Phase separation can be achieved 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.
[SOURCE: ISO 14852:—, 3.4]
3.3
theoretical amount of evolved carbon dioxide
ThCO

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

compound, calculated from the molecular formula

Note 1 to entry: It is expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound.

[SOURCE: ISO 14852:—, 3.5]
3.4
total organic carbon
TOC
amount of carbon bound in an organic compound

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

[SOURCE: ISO 17556:2019, 3.14]
3.5
dissolved organic carbon
DOC

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

Note 1 to entry: Phase separation can be achieved 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.
[SOURCE: ISO 14852:—, 3.7]
3.6
lag phase

time from the start of a test until adaptation and/or selection of the degrading microorganisms is

achieved and the degree of biodegradation of a chemical compound or organic matter has increased to

about 10 % of the maximum level of biodegradation (3.8)
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.8]
3.7
biodegradation phase

time from the end of the lag phase (3.6) of a test until the plateau phase has been reached

Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.10]
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ISO/FDIS 23977-1:2020(E)
3.8
maximum level of biodegradation

degree of biodegradation of a chemical compound or organic matter in a test, above which no further

biodegradation takes place during the test
Note 1 to entry: It is measured in per cent.
[SOURCE: ISO 14852:—, 3.9]
3.9
plateau phase
time from the end of the biodegradation phase (3.7) until the end of a test
Note 1 to entry: It is measured in days.
[SOURCE: ISO 14852:—, 3.11]
3.10
pre-conditioning

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

compound or organic matter under test, with the aim of improving the test by acclimatization of the

microorganisms to the test conditions
[SOURCE: ISO 14852:—, 3.13]
4 Principle

This document describes two variations of a test method for determining the biodegradability of plastic

materials by the indigenous population of microorganisms in natural seawater using a static aqueous

test system. The test is performed under mesophilic test conditions for up to two years by incubating

plastic materials with either seawater only (“pelagic seawater test”) or with seawater to which low

amount of sediment has been added (“suspended sediment seawater test”), coming from the same site

as that from which the seawater was taken.

Biodegradation is followed by measuring the evolution of carbon dioxide using a suitable, analytical

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

with the theoretical amount [theoretical amount of evolved carbon dioxide (ThCO )] and expressed in

percentage. 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:—, Annex A.
5 Test environment

Incubation shall take place in the dark or in diffused light, in an enclosure which is free from vapours

inhibitory to marine microorganisms and which is maintained at a constant mesophilic temperature.

It should preferably be between 15 °C to 25 °C, but not exceeding 28 °C, to an accuracy of ±1 °C. Any

change in temperature shall be justified and clearly indicated in the test report.

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

environment.
6 Reagents
Use only reagents of recognized analytical grade.
6.1 Water

Distilled or deionized water, free of toxic substances (copper in particular) and containing less than

2 mg/l of TOC.
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6.2 Natural seawater/sediment

Sampling, preservation, handling, transport and storage of natural seawater, and, if applicable,

sediment collected from the same site as that from which the seawater is taken, shall be in accordance

with ISO 5667-3.

Prior to use, remove coarse particles from the seawater and, if applicable, from the sediment by

appropriate means. The procedure used shall be reported.

Seawater can be filtered using a paper filter in order to remove coarse particles. It is recommended to

reduce the amount of coarse particles in sediment by means of at least two washing steps using filtered

seawater without coarse particles.

Measure TOC, pH and nitrogen content of seawater and, if applicable, of sediment samples according to

ISO 8245, ISO 10523 and ISO 11261, respectively.

If the TOC content of the seawater sample is found to be high, the seawater should be pre-conditioned

for about a week prior to use. If, for instance, the background concentration of TOC exceeds about 20 %

of the total TOC after addition of the test item, then pre-condition the seawater and, if applicable, the

sediment by stirring under aerobic conditions at the test temperature and in the dark or in diffuse light

in order to reduce the content of easily degradable organic material.

Provide the following information on the seawater, and, if applicable, on the sediment sample itself:

— date of collection;
— depth of collection (m);
— appearance of sample - turbid, clear, etc.;
— temperature at the time of collection (°C);
— salinity (PSU);
— total organic carbon (TOC; mg/l);
— nitrogen (total-N; mg/l);
— pH;
— description of the pre-conditioning process, if applicable.
7 Apparatus

Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or toxic matter.

Required is usual laboratory equipment, plus the following.

7.1 Test flasks. Biometric flasks of the volume of about 300 ml are appropriate. The vessels shall be

located in a constant temperature room or in an apparatus fitted with a thermostat (e.g. water-bath).

Reactors with higher or lower volumes can be used, if environmental conditions are not affected.

7.2 Container for the CO absorber, (e.g. glass beaker) to be located in the headspace of a test flask

and filled with 10 ml of Ba(OH) 0,012 5 mol/l or 3 ml of KOH 0,5 mol/l.

As an alternative to Ba(OH) and KOH 4 ml of NaOH 1 mol/l can be used as a CO -absorber.

2 2
A suitable apparatus is shown in Annex A, Figure A.1.
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7.3 Analytical equipment for determining carbon dioxide, consisting of any suitable apparatus

with sufficient accuracy, such as a CO or dissolved inorganic carbon (DIC) analyser or apparatus for

titrimetric determination after complete absorption in a basic solution, shall be used.

7.4 Analytical balance, which shall have a sensitivity of at least 0,1 mg.
7.5 Magnetic stirrer.
7.6 pH meter.
8 Procedure
8.1 Test material

The sample shall be of known mass and contain enough carbon to yield CO that can be adequately

measured by the chosen system. Use a test material concentration of at least 100 mg per litre of seawater.

This mass of the sample should correspond to TOC of about 60 mg/l. The maximum mass of sample per

flask is limited by the oxygen supply in the glass flask. The recommended amount per litre seawater

is 150 mg to 300 mg of test material per litre seawater. Calculate the TOC from the chemical formula

or determine it by means of a suitable analytical technique (e.g. elemental analysis or measurement in

accordance with ISO 8245) and calculate the ThCO .

The test material is added to a test flask, either as powder or in the form of a film. If the test material

is used in the form of powder, particles of known, narrow size distribution should be used. A particle-

size distribution with a maximum diameter of 250 µm is recommended. The preparation of powder

shall be performed in accordance with ISO 10210. If the test material is used in the form of a film, it

can be added either as pieces in the range of 0,2 cm × 0,2 cm to 0,5 cm × 0,5 cm or as a single plastic

strip (width: approximately 1,0 cm, length: depending on weight of the polymer and thickness of the

film). It is recommended that the plastic strip is fixed in, for example, a Polytetrafluoroethylene (PTFE)

coated fibre net (size: approximately 4 cm × 9 cm, mesh size: 5 mm × 5 mm). The fibre net is folded

into 2 layers (approximately 2 cm × 9 cm) with the plastic strip test material fixed in between. Then, the

two ends of the fibre net are attached together. The test material fixed between the fibre net is placed

upright on the ground of a bottle base in the form of a cylinder (see Annex A, Figure A.2).

The form and shape of the test material can influence its biodegradability. Similar particle sizes of power

should preferably be used in the test. Similar shapes and thicknesses of the films should preferably be

used if different kinds of plastic materials are to be compared.

When powder or pieces of films are used in the test, particles or film pieces can stick on the inner

wall of the testing bottle above the seawater. In such cases, a slight manual shaking of the bottle is

recommended to regain the powder or film pieces back to the seawater sample. If the material is added

as a cylindrical plastic strip fixed between, such as a Polytetrafluoroethylene (PTFE) coated fibre net

(see Annex A, Figure A.2), it is immersed in the seawater most of the time.
8.2 Reference materials

Use microcrystalline cellulose or ashless cellulose filters as a reference material . If possible, the TOC,

form, and size should be comparable to that of the test material.

1) PTFE Glass Fabric (product no 9002) produced by Fiberflon (https:// www .fiberflon .de/ Products/ PTFE -Coated

-Open -Mesh -Fabrics/ Page -307 -17 .aspx) has been found satisfactory for this purpose and is an example of a suitable

product available commercially. This information is given for the convenience of users of this document and does

not constitute an endorsement by ISO of this product.

2) Microcrystalline Cellulose "Avicel" produced by Merck or laboratory filter paper Whatman n° 42 has been found

satisfactory for this purpose and are examples of suitable products available commercially. This information is given

for the convenience of users of this document and does not constitute an endorsement by ISO of these products.

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As a negative control, a non-biodegradable polymer (e.g. polyethylene) in the same form as the test

material can be used.
8.3 Test set up
Provide several flasks, so that the test includes at least the following:
a) three flasks for the test material (symbol F );
b) three flasks for the blank (symbol F );
c) three flasks for reference material (symbol F ).

In addition, if biodegradation is expected to take longer than 6 months, it is recommended that a

negative control is included:
d) three flasks for negative control (symbol F ).

Two flasks for test material, blank, reference material, and negative control may be used instead of

three for screening purposes.
8.4 Pre-conditioning phase
As a rule, use a test flask with a volume of 300 ml.

The test is performed in batch by incubating the test materials with either 90 ml of natural seawater

only (“pelagic seawater test”) or with 90 ml of natural seawater to which sediment of 0,1 g/l to 1,0 g/l

(wet weight) has added (“suspended sediment seawater test”).

Add carbon dioxide absorber to the absorber compartments of the test flask, as a rule 10 ml Ba(OH)

0,012 5 mol/l, 3 ml of KOH 0,5 mol/l or 4 ml of NaOH 1,0 mol/l. Place the sealed flasks on a magnetic stirrer

(7.5) in a constant-temperature environment and allow all vessels to reach the desired temperature.

Agitation shall be continuous (e.g. 100 r/min agitation) in order to maintain microorganisms and, if

applicable, sediment in suspension.

The abrasion of sediment in coastal areas is a natural phenomenon caused by water currents and tidal

movements. Nevertheless, if a magnetic stirring bar is used to mix the seawater to which sediment has

added (“suspended sediment seawater test”), it is recommended that either a PTFE-coated dumbbell

shaped magnetic stirring bar be used or a PTFE-coated magnetic bar equipped with a pivot ring in

order to reduce excessive abrasion of sediment during the test period. Other stirring systems can be

[17]

used, too. Examples of suitable set-ups are given in Briassoulis D. et.al. and OECD TG 308:2002,

[14]
Annex 4 .

Take the necessary readings and monitor the CO evolution. This phase is carried out to verify that the

endogenous respiration is similar in the different vessels. In addition, the background concentration of

easily degradable organic material in natural seawater and, if applicable, in sediment is reduced in this

phase, following the pre-conditioning procedure given in 6.2.
8.5 Start of the test

After the pre-conditioning phase open the flasks and add the test material either as powder or in the

form of a film to the test flasks (7.1). The mass of samples shall be about 20 mg test material when using

a flask with a volume of 300 ml correspondin
...

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