EN ISO 20136:2017

SLOVENSKI STANDARD
01-junij-2017
Usnje - Ugotavljanje razgradljivosti z mikroorganizmi (ISO 20136:2017)
Leather - Determination of degradability by micro-organisms (ISO 20136:2017)
Leder - Bestimmung der Abbaubarkeit durch Mikroorganismen (ISO 20136:2017)
Cuir - Détermination de la dégradabilité par les micro-organismes (ISO 20136:2017)
Ta slovenski standard je istoveten z: EN ISO 20136:2017
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
March 2017
EUROPÄISCHE NORM
ICS 59.140.30
English Version
Leather - Determination of degradability by micro-
organisms (ISO 20136:2017)
Cuir - Détermination de la dégradabilité par les micro- Leder - Bestimmung der Abbaubarkeit durch
organismes (ISO 20136:2017) Mikroorganismen (ISO 20136:2017)
This European Standard was approved by CEN on 14 February 2017.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20136:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 20136:2017) has been prepared by Technical Committee 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 September 2017, and conflicting national standards
shall be withdrawn at the latest by September 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 20136:2017 has been approved by CEN as EN ISO 20136:2017 without any modification.
INTERNATIONAL ISO
STANDARD 20136
IULTCS/IUC 37
First edition
2017-03
Leather — Determination of
degradability by micro-organisms
Cuir — Détermination de la dégradabilité par les micro-organismes
Reference numbers
ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
©
ISO 2017
ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

ISO 20136:2017(E)
IULTCS/IUC 37:2017(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 Method A: assessment of biodegradation by manual titration . 2
5.2 Method B: assessment of biodegradation by infrared detection . 2
6 Chemicals . 3
7 Apparatus and materials. 4
8 Procedure. 6
8.1 Collection and preparation of the inoculum . 6
8.2 Preparation of the test material and reference material . 6
8.3 Test conditions and incubation period. 6
8.4 Test equipment . 6
8.4.1 Equipment for the assessment of biodegradation by manual titration
(equipment A) . 6
8.4.2 Equipment for the assessment of biodegradation by IR detection (equipment B) 7
8.5 End of the test . 7
9 Quantification . 8
9.1 Equipment for the assessment of biodegradation by manual titration (equipment A). 8
9.1.1 Determination of the organic carbon content . 8
9.1.2 Determination of the amount of carbon dioxide produced (Method A) . 8
9.1.3 Correcting for normality of HCl . 8
9.1.4 Percentage of biodegradation from carbon dioxide evolved . 8
9.2 Equipment for the assessment of biodegradation by IR detection (Method B) . 8
9.2.1 Determination of the organic carbon content . 8
9.2.2 Determination of the amount of carbon dioxide (CO produced) . 9
9.2.3 Percentage of biodegradation from CO data . 9
10 Expression of results .10
11 Validity of results .10
12 Test report .10
Annex A (informative) Determination of the degree and rate of degradation of the material .11
Annex B (informative) Quantitative determination of leather biodegradation .16
Bibliography .20
ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .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 method for the sampling and testing of leather. ISO recognizes
IULTCS as an international standardizing body for the preparation of test methods for leather.
iv © ISO 2017 – All rights reserved

ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
Introduction
One of the big problems faced by the footwear industry is waste treatment. Although this waste,
especially in the case of leather, is not considered hazardous by current legislation, it is however
produced in large quantities which present a problem for municipal landfill sites.
The aim of the tanning process is to avoid skin putrefaction and increase the resistence of the obtained
leather. For this purpose, chemical and biological agents are used which are involved in the denaturation
and hardening of the main stromal protein, collagen, thus also producing physicochemical changes in
the skin.
There is a wide range of different agents used for leather tanning, which can be based on organic
products, vegetable extracts or inorganic products, mostly metals.
The most used tanning agent in the footwear industry is Chromium (III), which gives the skin desirable
characteristics, such as elasticity, easy buffing and a good breathability and vapour permeability.
However, the traditional tanning industry, and especially chrome tanning, generates wastes that pose
an environmental threat. Also, chrome-tanned hides and skins have too long a lifespan, much larger
than the useful life of the final products. Therefore, the use of additives that are less harmful to the
environment and which generate products that have a certain ease of degradation, once the material
has achieved its purpose, would be preferred, thus minimising waste products.
Within this sector, the development of fast biodegradability quantification methods for leather that has
been treated with alternative tanning agents is needed in order to predict whether these materials are
more biodegradable than their predecessors. The methodology described in this document attempts to
allow the completion of this form of analysis in a test time of no more than 35 days.
ISO 20136:2017(E)
INTERNATIONAL STANDARD
IULTCS/IUC 37:2017(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.
The conditions established in this document correspond to optimum laboratory conditions to achieve
the maximum level of biodegradation. However, they may 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:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
filter pore no. 1
diffuser with pore size from 100 microns to 160 microns
Note 1 to entry: This measurement is standard.
3.2
inoculum
activated sludge from tannery wastewater
ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
4 Symbols and abbreviated terms
[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
PSA pressure swing adsorption
5 Principle
5.1 Method A: assessment of biodegradation by manual titration
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.
The CO evolved during the test is indirectly determined through the reaction of [Ba(OH) ] with
2 2
CO , which is precipitated as barium carbonate (BaCO ). The amount of CO evolved is determined
2 3 2
by titrating the remaining barium hydroxide 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.
The initial carbon percentage (C) 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 a new cellular biomass that has not been metabolized to carbon dioxide throughout
the test.
The tests shall be carried out using equipment able to provide the conditions needed to carry out the
test. Agitation, experiment temperature and CO -free air inflow should be controlled.
The test shall be carried out in duplicate in the presence of a positive control, which is made up of a
synthetic medium, microorganisms and collagen, and a negative control, which is made up only of a
synthetic medium and inoculum (activated sludge from tannery wastewater), allowing the assessment
of two different leather samples that can be evaluated in duplicate.
5.2 Method B: assessment of biodegradation by infrared detection
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
of the CO concentration. The equipment comprises a reaction unit made up of a closed set of
2 © ISO 2017 – All rights reserved

ISO 20136:2017(E)
IULTCS/IUC 37:2017(E)
unidirectional gas flow recirculation tubes, an aerator immersed in the reaction fluid contained in
the reaction flask, a membrane pump for unidirectional flow that makes the gas go through the CO
concentration detector area, an IR sensor, and a data capture system connected to a computer.
This system is in its final development stage. The methodology will be added to this document
application at a later stage.
The initial percentage of carbon (C) present in the collagen under study is determined through the
elemental analysis of each sample. The percentage of biodegradation does not include the quantity of
carbon converted into a new cellular biomass which is not metabolised into carbon dioxide during the
course of the test.
The tests shall be carried out using equipment able to provide the conditions needed to carry out the
test. Agitation, experiment temperature and CO -free air inflow should be controlled.
The tests are conducted in duplicate in the presence of a positive control, composed of a synthetic
medium, microorganisms and collagen, and a negative control, composed only of a synthetic medium
and an inoculum, allowing the assessment of five different leather samples that can be evaluated in
duplicate.
6 Chemicals
The reagents employed in the tests are the same for the two methods used in this document (Method
A and Method B) only with some adjustments in the volume of the reaction flasks specific of each
methodology (method A: a final liquid volume of 2,68 l; method B: a final liquid volume of 1 l).
1)
6.1 Deionised or ultrapure (Milli Q® ) water, free from toxic materials with resistivity >18 MΩ/cm.
6.2 Test medium: Use only analytical grade reagents.
6.2.1 Prepare synthetic stock solutions by dissolving each of the following in distilled water to 1 l:
6.2.1.1 Ferric chloride (FeCl •6H O), 1,00 g.
3 2
6.2.1.2 Magnesium sulfate (MgSO •7H O), 22,5 g.
4 2
6.2.1.3 Calcium chloride (CaCl •2H O), 36,43 g.
2 2
6.2.1.4 Phosphate buffer KH HPO 8,5 g, K HPO •3H O 28,5 g, Na HPO 17,68 g, and NH Cl 1,7 g, for
2 4 2 4 2 2 4 4
a total of 56,38 g.
6.2.1.5 Ammonium sulfate [(NH ) SO ], 40 g.
4 2 4
6.2.2 The test medium shall contain the following reagents diluted to 1 l with high-quality distilled water:
6.2.2.1 Magnesium sulfate solution, 2 ml.
6.2.2.2 Calcium chloride solution, 2 ml.
6.2.2.3 Phosphate buffer solution, 4 ml.
6.2.2.4 Ferric chloride solution, 2 ml.
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 20136:2017(E)
IULTCS/IUC 37:2017(E)
6.2.2.5 Ammonium sulfate solution, 2 ml.
6.3 Test specimens: Use collagen type I (Sigma or similar) as a positive control. Test specimens shall
be basically leather from the tanning industry used for the production of leather clothing.
6.4 Only for Method A: Barium hydroxide solution, 0,025 mol/l, is prepared 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.
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 Volumetric flask, 1 l.
For each method, the following materials should be employed:
7.5 Method A: Assessment of biodegradation by manual titration
7.5.1 Biodegradation test equipment
The procedure is partially automated thanks to
...