EN ISO 20136:2020

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
• Publication Date: 14-Jul-2020
• Withdrawal Date: 30-Jan-2021
• ICS: 59.140.30 - Leather and furs
• Technical Committee: CEN/TC 289 - Leather
• Drafting Committee: CEN/TC 289/WG 1 - Chemical test methods
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Jul-2020
• Completion Date: 15-Jul-2020
• Ref Project: SIST EN ISO 20136:2020 - Leather - Determination of degradability by micro-organisms (ISO 20136:2020)

Relations

• Replaces: EN ISO 20136:2017 - Leather - Determination of degradability by micro-organisms (ISO 20136:2017)
• Effective Date: 23-May-2018
• Consolidated By: ISO 20136:2020 - Leather — Determination of degradability by micro-organisms
• Effective Date: 12-Feb-2026

Overview

EN ISO 20136:2020 - Leather: Determination of degradability by micro‑organisms defines a laboratory test method to quantify the aerobic biodegradability of hides and skins (tanned or untanned) by indirectly measuring the CO2 evolved during microbial degradation of collagen. The method exposes leather test pieces to an inoculum (activated sludge from tannery wastewater or, if unavailable, municipal/urban wastewater) in a controlled aqueous system and records the degree and rate of biodegradation under optimum laboratory conditions.

Key topics and technical requirements

• Measurement principle: Indirect determination of biodegradation by monitoring CO2 production from collagen decomposition.
• Test scope: Applies to natural animal stroma (skins/hides), leathers tanned with organic or inorganic agents, and leathers that do not inhibit microbial activity.
• Inoculum: Activated sludge from tannery wastewater is preferred; municipal wastewater is an accepted alternative when tannery effluent is not available.
• Controlled conditions: Strict control of flow, temperature and agitation to maintain reproducible, optimal microbial activity.
• Analytical methods: Two assessment routes are included:
  • Method A - manual titration (acid/base titration to quantify CO2 capture).
  • Method B - infrared (IR) detection of CO2 (note: the 2020 edition uses an open O2 circuit to avoid oxygen limitation and maintain microbial activity).
• Validity criterion: The test includes a positive control (collagen). A valid run requires collagen biodegradation ≥ 70% within a maximum of 50 days; leather samples are compared against this reference.
• Data and reporting: Quantification of biodegradation percent and rate (CO2 moles over time), with requirements for equipment calibration and test-report content.

Practical applications and users

EN ISO 20136:2020 is relevant for:

• Tanneries and leather manufacturers assessing environmental performance of tanning agents and processes.
• R&D laboratories developing biodegradable leather treatments or alternative tanning chemistries.
• Environmental testing labs performing standardized biodegradability assays for compliance, labeling, or life‑cycle analysis.
• Regulatory bodies and sustainability auditors evaluating waste management, end‑of‑life behavior and claims related to biodegradability. Practical uses include screening of new tanning agents, comparative biodegradability testing, supporting product declarations, and informing waste‑treatment planning.

Related standards

• EN ISO 20136:2020 is produced by ISO in cooperation with IULTCS and CEN/TC 289 (Leather). Laboratories should align test execution and reporting with national accreditation requirements (e.g., ISO/IEC 17025) and consider complementary standards for leather sampling and characterization.

Keywords: EN ISO 20136:2020, leather biodegradability, CO2 monitoring, tannery wastewater, activated sludge, leather testing, collagen biodegradation.

Standards Content (Sample)

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

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.

Contents Page
European foreword . 3

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.

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
ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)
© 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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

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 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

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 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 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
−6
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

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 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

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 rpm (to-and-fro motion) throughout the entire test
duration.
The inoculum volume of each flask varies depending on its degree of activity, ranging between 10 % and
20 % of the total volume (inoculum + minimum test medium), which is 2,5 l. If the inoculum is from urban
wastewater the total volume (inoculum + minimum medium) can increase up to 40 % of the total volume.
The air needs to leave the generator through the PSA system which shall have been working for 16 h
(overnight) before the start of the test in order to ensure that a stable CO concentration of less than
1 ppm is achieved in the air flow.
During the test, a constant CO -free air flow of 150 ml/min is supplied to each reaction flask. The air
flow is regularly checked at each outlet by means of scaled flow meters in order to ensure that there are
not any leaks in the system.
The quantification of the CO evolved by aerobic digestion of the specimen by microorganisms is carried
out by measuring the level of carbonation of 0,025 mol/l [Ba(OH )] contained in the three analysis
flasks connected to each reaction flask. The analysis flasks are replaced every 24 h with others with
the same initial amount of 0,025 mol/l [Ba(OH )].
The daily quantification values of the carbonation of [Ba(OH )] are entered into a spreadsheet that
converts them into biodegradation percentages (Clause 10).
7.10.2 Assessment of biodegradation by IR detection (equipment B)
7.10.2.1 Gen
...