Leather - Determination of degradability by micro-organisms (ISO 20136:2020)

This document specifies a test method to determine the degree and rate of aerobic biodegradation
of hides and skins of different animal origin, whether they are tanned or not, through the indirect
determination of CO2 produced by the degradation of collagen.
The test material is exposed to an inoculum (activated sludge from tannery wastewater) in an aqueous
medium. If there is not a tannery nearby then urban wastewater can be used as the inoculum.
The conditions established in this document correspond to optimum laboratory conditions to achieve
the maximum level of biodegradation. However, they might not necessarily correspond to the optimum
conditions or maximum level of biodegradation in the natural medium.
In general, the experimental procedure covers the determination of the degradation degree and
rate of the material under controlled conditions, which allows the analysis of the evolved carbon
dioxide produced throughout the test. For this purpose, the testing equipment complies with strict
requirements with regard to flow, temperature and agitation control.
This method applies to the following materials:
— natural polymers of animal stroma (animal tissue/skins);
— animal hides and skins tanned (leather) using organic or inorganic tanning agents;
— leathers that, under testing conditions, do not inhibit the activity of microorganisms present in the
inoculum.

Leder - Bestimmung der Abbaubarkeit durch Mikroorganismen (ISO 20136:2020)

Dieses Dokument legt ein Prüfverfahren fest, mit dem durch die indirekte Bestimmung von CO2, das durch den Abbau von Kollagen entsteht, der Grad und die Geschwindigkeit des aeroben Bioabbaus von entweder gegerbten oder ungegerbten Fellen und Häuten unterschiedlichen tierischen Ursprungs bestimmt wird.
Die Prüfsubstanz wird einem Inokulum (Belebtschlamm aus Gerbereiabwässern) in einem wässrigen Medium ausgesetzt. Wenn es keine Gerberei in der Nähe gibt, kann kommunales Abwasser als Inokulum verwendet werden.
Die in diesem Dokument festgelegten Bedingungen entsprechen den optimalen Laborbedingungen zum Erreichen des maximalen Grades des Bioabbaus. Sie entsprechen jedoch möglicherweise nicht unbedingt den optimalen Bedingungen oder dem maximalen Grad des Bioabbaus im natürlichen Medium.
Im Allgemeinen deckt das experimentelle Verfahren die Bestimmung des Grades und der Geschwindigkeit des Bioabbaus des Materials unter kontrollierten Bedingungen ab, welches die Analyse des freigesetzten Kohlenstoffdioxids, das während der gesamten Prüfung gebildet wurde, ermöglicht. Für diesen Zweck entspricht die Prüfvorrichtung den strikten Anforderungen in Bezug auf die Regelung des Durchflusses, der Temperatur sowie der Bewegung.
Dieses Verfahren gilt für die folgenden Materialien:
- natürliche Polymere von tierischem Stroma (tierisches Gewebe/tierische Häute);
- tierische Felle und Häute, gegerbt (Leder) mit organischen oder anorganischen Gerbstoffen;
- Leder, die unter Prüfbedingungen die Aktivität der im Inokulum vorhandenen Mikroorganismen nicht hemmen.

Cuir - Détermination de la dégradabilité par les micro-organismes (ISO 20136:2020)

Le présent document spécifie une méthode d'essai pour déterminer le degré et la vitesse de biodégradation aérobie de peaux de différents animaux, tannées ou non, par la détermination indirecte du CO2 produit par la dégradation du collagène.
Le matériau d'essai est exposé à un inoculum (boues activées d'eaux résiduaires de tannage) dans un milieu aqueux. En l'absence de tannerie à proximité, des eaux usées urbaines peuvent servir d'inoculum.
Les conditions établies dans le présent document correspondent aux conditions de laboratoire optimales pour obtenir le niveau maximal de biodégradation. Il se pourrait toutefois qu'elles ne correspondent pas aux conditions optimales ou au niveau maximal de biodégradation dans le milieu naturel.
De manière générale, le mode opératoire expérimental inclut la détermination du degré et de la vitesse de dégradation du matériau dans des conditions contrôlées, ce qui permet d'analyser le dégagement de dioxyde de carbone tout au long de l'essai. À cet effet, l'équipement d'essai répond à des exigences strictes concernant le contrôle du débit, de la température et de l'agitation.
La présente méthode s'applique aux matériaux suivants:
— les polymères naturels de stromas animaux (tissus/peaux d'animaux);
— les peaux d'animaux qui ont été tannées (cuir) en utilisant des agents de tannage organiques ou inorganiques;
— les cuirs qui, dans les conditions d'essai, n'ont pas d'effet inhibiteur sur l'activité des micro-organismes présents dans l'inoculum.

Usnje - Ugotavljanje razgradljivosti z mikroorganizmi (ISO 20136:2020)

General Information

Status
Published
Public Enquiry End Date
31-Mar-2019
Publication Date
10-Aug-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
03-Aug-2020
Due Date
08-Oct-2020
Completion Date
11-Aug-2020

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SLOVENSKI STANDARD
SIST EN ISO 20136:2020
01-september-2020
Nadomešča:
SIST EN ISO 20136:2017
Usnje - Ugotavljanje razgradljivosti z mikroorganizmi (ISO 20136:2020)
Leather - Determination of degradability by micro-organisms (ISO 20136:2020)
Leder - Bestimmung der Abbaubarkeit durch Mikroorganismen (ISO 20136:2020)

Cuir - Détermination de la dégradabilité par les micro-organismes (ISO 20136:2020)

Ta slovenski standard je istoveten z: EN ISO 20136:2020
ICS:
59.140.30 Usnje in krzno Leather and furs
SIST EN ISO 20136:2020 en,fr,de

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

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SIST EN ISO 20136:2020
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SIST EN ISO 20136:2020
EN ISO 20136
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2020
EUROPÄISCHE NORM
ICS 59.140.30 Supersedes EN ISO 20136:2017
English Version
Leather - Determination of degradability by micro-
organisms (ISO 20136:2020)

Cuir - Détermination de la dégradabilité par les micro- Leder - Bestimmung der Abbaubarkeit durch

organismes (ISO 20136:2020) Mikroorganismen (ISO 20136:2020)
This European Standard was approved by CEN on 8 June 2020.

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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20136:2020 E

worldwide for CEN national Members.
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SIST EN ISO 20136:2020
EN ISO 20136:2020 (E)
Contents Page

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

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SIST EN ISO 20136:2020
EN ISO 20136:2020 (E)
European foreword

This document (EN ISO 20136:2020) has been prepared by Technical Committee ISO/IULTCS

"International Union of Leather Technologists and Chemists Societies" in collaboration with Technical

Committee CEN/TC 289 “Leather” the secretariat of which is held by UNI.

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

identical text or by endorsement, at the latest by January 2021, and conflicting national standards shall

be withdrawn at the latest by January 2021.

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.

This document supersedes EN ISO 20136:2017.

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 20136:2020 has been approved by CEN as EN ISO 20136:2020 without any modification.

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SIST EN ISO 20136:2020
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SIST EN ISO 20136:2020
INTERNATIONAL ISO
STANDARD 20136
IULTCS/IUC 37
Second edition
2020-06
Leather — Determination of
degradability by micro-organisms
Cuir — Détermination de la dégradabilité par les micro-organismes
Reference numbers
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
ISO 2020
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
Contents Page

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

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

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

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

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

4 Symbols and abbreviated terms ........................................................................................................................................................... 2

5 Principle ........................................................................................................................................................................................................................ 2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Assessment of biodegr adation by manual titration; method A .................................................................... 2

5.3 Assessment of biodegr adation by infrared (IR) detection; method B..................................................... 3

6 Chemicals ..................................................................................................................................................................................................................... 3

7 Apparatus and materials.............................................................................................................................................................................. 4

8 Procedure..................................................................................................................................................................................................................... 7

8.1 Collection and preparation of the inoculum .................................................................................................................. 7

8.2 Preparation of the test material and reference material .................................................................................... 7

8.3 Test conditions and incubation period............................................................................................................................... 7

8.4 Termination of the test ..................................................................................................................................................................... 7

9 Quantification .......................................................................................................................................................................................................... 8

9.1 Assessment of biodegr adation by manual titration (method A) ................................................................. 8

9.1.1 Determination of the organic carbon content ........................................................................................ 8

9.1.2 Determination of the amount of CO produced .................................................................................... 8

9.1.3 Correcting for normality of HCl ........................................................................................................................... 8

9.1.4 Percentage of biodegradation from CO evolved ................................................................................. 9

9.2 Assessment of biodegr adation by IR (method B) ..................................................................................................... 9

9.2.1 Determination of the organic carbon content ........................................................................................ 9

9.2.2 Determination of the amount of CO produced .................................................................................10

9.2.3 Percentage of biodegradation from CO data ......................................................................................10

10 Expression of results .....................................................................................................................................................................................15

11 Validity of results ..............................................................................................................................................................................................15

12 Test report ................................................................................................................................................................................................................15

Annex A (informative) Determination of the degree and rate of degradation of the material .............16

Annex B (informative) Quantitative determination of leather biodegradation ...................................................19

Annex C (informative) Comparative biodegradability using different waste waters ......................................23

Bibliography .............................................................................................................................................................................................................................24

© ISO 2020 – All rights reserved iii
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(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 the Chemical Tests Commission of the International Union of Leather

Technologists and Chemists Societies (IUC Commission, IULTCS) in collaboration with the European

Committee for Standardization (CEN) Technical Committee CEN/TC 289, Leather, the secretariat of

which is held by UNI, in accordance with the agreement on technical cooperation between ISO and CEN

(Vienna Agreement).

IULTCS, originally formed in 1897, is a world-wide organization of professional leather societies to

further the advancement of leather science and technology. IULTCS has three Commissions, which

are responsible for establishing international methods for the sampling and testing of leather. ISO

recognizes IULTCS as an international standardizing body for the preparation of test methods for

leather.

This second edition cancels and replaces the first edition (ISO 20136:2017), which has been technically

revised. The main changes to the previous edition are as follows:

— Method B in the first edition described a closed O circuit system. This system had the inconvenience

that, over time, the O concentration decreased and, therefore, so did the activity of the

microorganism. Now an open O circuit system has been developed where there is no O limitation

2 2
and, therefore, the activity of the microorganism is always optimal.

— An explanation about the results calculation method has been added to method B. The CO

accumulated in the test (area under the CO moles curve vs time) is calculated.

— The possibility of using municipal wastewater instead of tannery wastewater as an inoculum has

been included.

— A new Annex C has been added which compares the biodegradability with different inoculum

sources.

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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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
Introduction

One of the main issues faced by the footwear industry is waste treatment. Such wastes, and especially

leather, even though they are considered non-hazardous by the regulations in force, are generated in

vast quantities and mostly end up in landfills, where natural degradation time is much longer than the

product’s useful life.

Faced with this problem, there is a growing search for alternative tanning agents that confer the same

properties on leather as those provided by the agents currently employed, but which in turn reduce the

time to biodegrade in nature.

This document allows the measurement of leather biodegradability in a liquid system by using

aerobic microorganisms as an inoculum. The test is considered valid when collagen (positive control)

degrades by at least 70 % in a maximum period of 50 days. In order to determine how biodegradable

a leather sample (test material) is, its percentage degradability value is compared with the percentage

degradability value obtained in collagen, in the same test and period of time. The closer the percentage

degradability values, the shorter the time to biodegrade in nature. Therefore, those test materials

showing percentage degradability values well below the collagen value will require a longer time for

biodegradation in nature.
© ISO 2020 – All rights reserved v
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SIST EN ISO 20136:2020
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
INTERNATIONAL STANDARD
IULTCS/IUC 37:2020(E)
Leather — Determination of degradability by micro-
organisms
1 Scope

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

of hides and skins of different animal origin, whether they are tanned or not, through the indirect

determination of CO produced by the degradation of collagen.

The test material is exposed to an inoculum (activated sludge from tannery wastewater) in an aqueous

medium. If there is not a tannery nearby then urban wastewater can be used as the inoculum.

The conditions established in this document correspond to optimum laboratory conditions to achieve

the maximum level of biodegradation. However, they might not necessarily correspond to the optimum

conditions or maximum level of biodegradation in the natural medium.

In general, the experimental procedure covers the determination of the degradation degree and

rate of the material under controlled conditions, which allows the analysis of the evolved carbon

dioxide produced throughout the test. For this purpose, the testing equipment complies with strict

requirements with regard to flow, temperature and agitation control.
This method applies to the following materials:
— natural polymers of animal stroma (animal tissue/skins);

— animal hides and skins tanned (leather) using organic or inorganic tanning agents;

— leathers that, under testing conditions, do not inhibit the activity of microorganisms present in the

inoculum.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

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
filter pore No. 1
diffuser with pore size from 100 µm to 160 µm
Note 1 to entry: This measurement is standard.
3.2
inoculum
activated sludge from tannery wastewater

Note 1 to entry: If there is not a tannery nearby then urban wastewater can be used as the inoculum.

© ISO 2020 – All rights reserved 1
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
4 Symbols and abbreviated terms
atm the standard atmosphere, a unit of pressure defined as 101 325 Pa
[Ba(OH) ] barium hydroxide
C carbon
CO carbon dioxide
GL18 threads are used with H-SA V40/45 Erlenmeyer flasks (5 000 ml volume)
GL14 threads are used with H-SA V29/32 Erlenmeyer flasks (2 000 ml volume)

H-SA V 29/32 inner and outer measures in millimetres of the orifice of the mouth of the

Erlenmeyer flasks

H-SA V H40/45 inner and outer measures in millimetres of the orifice of the mouth of the

Erlenmeyer flasks
IR infrared
ppm parts-per-million (10 ), e.g. 1 mg per kilogram (mg/kg)
PSA pressure swing adsorption
Q the air flow, in mol, passing through the system per hour (mol/h)
nar
Q the CO air flow, in mol, passing through the system per hour (mol/h)
nCO2 2
5 Principle
5.1 General

The procedure consists of the quantification of the CO evolved during the degradation of the

polymerised amino acids making up the collagen polymer by the action of microorganisms present

in the sludge of tannery biological tanks. The CO evolved is stoichiometrically proportional to the

amount of carbon (C) present in said polymer. The initial carbon percentage present in each of the

tested samples is determined by elemental analysis. The CO accumulated during the test is converted

into biodegradation percentage by means of mathematical equations. The tests shall be conducted

in duplicate in the presence of a positive control, comprising minimum test medium (6.3), inoculum

(activated sludge from tannery wastewater) and collagen, and a negative control, comprising minimum

test medium and inoculum only. The test shall be regarded as valid if the degree of biodegradation of

the positive control (pure collagen) is equal to or higher than 70 %.

The tests shall be carried out using equipment able to provide the conditions needed to carry out the

test. Agitation, temperature and CO -free air inflow should be controlled.

The initial carbon (C) percentage present in the collagen under study is determined by the elemental

analysis of the test specimen. The biodegradation percentage does not include the amount of carbon

transformed into new cellular biomass that has not been metabolised to carbon dioxide throughout

the test.
5.2 Assessment of biod egradation by manual titration; method A

This test method determines the biodegradation percentage of tanned or untanned hides and skins

through the indirect measurement of CO evolved during the degradation of collagen, which is the

major constituent of the skin, by the action of the microorganisms present in tannery wastewater.

2 © ISO 2020 – All rights reserved
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)

The CO evolved during the test is indirectly determined through the reaction of [Ba(OH) ] with CO ,

2 2 2

which is precipitated as barium carbonate (BaCO ). The amount of CO evolved is determined by

3 2

titrating the remaining non-precipitated [Ba(OH)2] with a 0,05 mol/l hydrochloric acid solution. These

measurements are taken on a daily basis throughout the test.

Biodegradability is assessed by indirectly measuring the CO evolved as a function of time and

calculating the biodegradation degree by the difference between the initial carbon percentage present

in collagen and the remaining soluble organic carbon content that has not been transformed into CO in

the course of the process (see Figures A.1 to A.3, Annex A).
5.3 Assessment of biod egradation by infrared (IR) detection; method B

With this method, biodegradation is determined through the quantification of the CO evolved

throughout the degradation of collagen by means of the direct IR detection and continuous monitoring

Erlenmeyer

of the CO concentration using equipment capable of evaluating 12 flasks simultaneously (see

Figure B.1 to B.5, Annex B).

The equipment (see Figure B.1, Annex B) is ready to measure the CO value of several samples contained

in different Erlenmeyer flasks. CO evolved during the degradation of the sample by the action of

microorganisms is measured by an IR detector. Said detector is managed by a multiplexer system

comprising a rotating drum with 12 inlet channels in such a way that every air outlet of the Erlenmeyer

flasks is connected to an air inlet of the multiplexer system. The drum is provided with an outlet directly

connected to an air flow meter measuring the air flow (l/h) and subsequently to an airtight tank where

the CO sensor is located. Annex B (see Table B.1) summarizes the parameters, units of measure and

range of detection values. Air flow and CO concentration values are saved in a data-capturing system

connected to a computer.
6 Chemicals
®1)

6.1 Deionised or ultrapure (Milli Q ) water, free from toxic materials with resistivity > 18 MΩ/cm.

6.2 Stock solutions, use only analytical grade reagents. The stock solutions employed in the tests are

the same for the two methods used in this document. Prepare synthetic stock solutions by dissolving

each of the following in distilled water (6.1) and made up to 1 l in separate flasks.

6.2.1 Ferric chloride (FeCl ·6H O), 1,00 g.
3 2
6.2.2 Magnesium sulfate (MgSO ·7H O), 22,50 g.
4 2
6.2.3 Calcium chloride (CaCl ·2H O), 36,43 g.
2 2
6.2.4 Phosphate buffer:
— Potassium dihydrogen phosphate (KH PO ), 8,50 g;
2 4
— Potassium phosphate dibasic trihydrate (K HPO ·3H O), 28,50 g;
2 4 2
— Sodium hydrogen phosphate (Na HPO ), 17,68 g;
2 4
— Ammonium chloride (NH Cl), 1,70 g.
6.2.5 Ammonium sulfate [(NH ) SO ], 40,00 g.
4 2 4

1) Milli Q 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.

© ISO 2020 – All rights reserved 3
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
6.3 Minimum test medium

The minimum test medium shall contain the following stock solutions diluted to 1 l with deionised water:

6.3.1 Ferric chloride stock solution (6.2.1), 2 ml.
6.3.2 Magnesium sulfate stock solution (6.2.2), 2 ml.
6.3.3 Calcium chloride stock solution (6.2.3), 2 ml.
6.3.4 Phosphate buffer stock solution (6.2.4), 4 ml.
6.3.5 Ammonium sulfate stock solution (6.2.5), 2 ml.
®2)

6.4 Test specimens: use collagen type I (Sigma or similar) as a positive control. Test specimens

shall be basically natural polymers or leather from the tanning industry used for the production of

leather clothing.

6.5 Only for method A: a [Ba(OH)2] solution, 0,025 mol/l, is prepared by dissolving 4,0 g [Ba(OH) ]

per litre of distilled water. Filter free of solid material, confirm molarity by titration with standard acid

and store sealed as a clear solution to prevent absorption of CO from the air. It is recommended that 5 l

be prepared at a time when running a series of tests.
6.6 Hydrochloric acid, 0,05 mol/l.
7 Apparatus and materials
The usual laboratory equipment and, in particular, the following:
7.1 Analytical balance, capable of reading to 0,000 1 g.
7.2 Pipettes, 5 ml to 25 ml capacity.
7.3 Micro-pipettes, 500 μl and 1 000 μl.
7.4 Pre-treatment flasks and flasks (only for Method A), various sizes.
7.5 Burettes, 100 ml.

7.6 Autonomous CO -free air source, consisting of a noiseless compressor connected to a pressure

swing adsorption (PSA) system provided with a molecular sieve.
7.7 Sepiolite to filter impurities and humidity from the ventilation system.
7.8 Stoppers, flexible non-permeable to CO plastic tubing.
7.9 Test vessels

7.9.1 Method A: eight 5 l Erlenmeyer flasks (reaction flasks) for each test (two controls and two test

specimens per test). 5 000 ml H-SA V H40/50 Erlenmeyer flasks shall be used, as well as V2 distilling heads

2) Sigma 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.

4 © ISO 2020 – All rights reserved
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SIST EN ISO 20136:2020
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)

with GL18 threads and filter pore No. 1 diffuser. The volume of the liquid (culture medium + inoculum)

shall be 2,5 l in total.

7.9.2 Method B: 12 flasks with a test volume of 1 l (reaction flasks) incorporating a distilling head

and an air diffuser which are used to conduct the tests (two controls and four samples in duplicate).

The Erlenmeyer flasks shall have a capacity of 2 000 ml with three notches and be of the H-SA V 29/32

(SQ13) model type. They shall incorporate V2 distilling heads with GL14 threads (6 mm air intake and

8 mm air outlet) and filter pore No. 1 diffuser. The volume of the liquid (culture medium + inoculum)

shall be 1 l in total.
7.10 Test equipment
7.10.1 Assessment of biodegradation by manual titration (equipment A)

Equipment A operates in such a way that the CO -free air is bubbled through a series of seven Erlenmeyer

flasks (pre-treatment flasks) that trap residual carbon dioxide in the air flow coming from the PSA

device (7.6). The system is then divided into eight lines controlled by eight valves that allow the flow

to be independently controlled, which in turn supply eight Erlenmeyer flasks (reaction flasks) located

inside the tank. The outlet of each one of the eight Erlenmeyer flasks is directly connected to a series

of three glass Erlenmeyer flasks (analysis bottles) connected, each one containing 100 ml of [Ba(OH) ]

0,025 mol/l, from which the results will be obtained (see Figures A.2 and A.3, Annex A).

The equipment also features a thermostat that allows the regulation of the temperature of the reaction

flasks through the recirculation of water in a closed circuit. The test is carried out at 23 °C ± 1 °C.

The reaction flasks are constantly agitated at 24
...

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