Leather — Determination of degradability by micro-organisms

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.

Cuir — Détermination de la dégradabilité par les micro-organismes

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.

General Information

Status
Published
Publication Date
24-Jun-2020
Current Stage
6060 - International Standard published
Start Date
08-May-2020
Completion Date
25-Jun-2020
Ref Project

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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
---------------------- Page: 1 ----------------------
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
---------------------- Page: 2 ----------------------
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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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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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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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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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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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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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
---------------------- Page: 9 ----------------------
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 General

The equipment works continuously in an open system in which the air free of CO (7.6) circulates

throughout the system impelled by a pump (see Figures B.1 to B.5, Annex B). To increase the amount of

oxygen dissolved in the liquid phase, the intake of air into the Erlenmeyer flask is made through the use

of an air diffuser incorporated into the distilling head that is in contact with the liquid medium.

The air flow that goes into each Erlenmeyer flask is controlled by a system of individual pressure

gauges. The system also features a digital air flow quantification system. Digital data for each

© ISO 2020 – All rights reserved 5
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ISO 20136:2020(E)
IULTCS/IUC 37:2020(E)

measurement and each Erlenmeyer flask are saved to a file and are subsequently converted into l/h

based on a calibration curve.

The equipment is provided with a thermostatic system capable of regulating the temperature of the

Erlenmeyer flasks by means of a thermostated tank. Tank water is inside a recirculation system that in

turn is connected to a cryothermostat that allows water recirculation at a temperature of 23 °C ± 2 °C.

In order to achieve constant agitation of the microbial suspension and samples, the equipment is

provided with a system comprising an array of 12 magnets coupled to 12 motors placed underneath

the tank base, in such a way that each magnet corresponds to one reaction flask. Agitation of the

microbial suspension and samples is achieved by putting a magnet inside each flask. Agitation speed (in

revolutions per minute, rpm) is set using specific hardware.
Important quantification parameters are referred to in Table B.1, Annex B.

The CO value of the samples placed in the flasks is sequentially measured by a multiplexer system. This

system comprises a rotating drum with 12 inlet channels and one outlet, which is directly connected to an

airtight tank where the CO sensor is located. Every air outlet of the reaction flasks is connected to an air

inlet of the multiplexer system. By a single rotation, the drum selects just one of the inlets, establishing a

direct connection between the selected inlet of one of the reaction flasks and the tank where the detector

is located, and blocking the rest of the inlets. A stepper motor makes the multiplexer system rotate to

select the relevant inlet, and specifically designed hardware and the corresponding firmware control

which of the 12 inlets is selected at all times. This way, it is possible to save the information relative to

the CO evolved (ppm) and the respective air flow (l/h) of a given flask at a given time.

The minimum test medium and the inoculum are added to the Erlenmeyer flasks. The volume of the

inoculum in each flask varies according to the degree of activity, ranging between 10 % and 20 % of the

total volume (inoculum + minimum test medium), which amounts to 1 l. If the inoculum is from urban

wastewater the total volume (inoculum + minimum medium) can increase up to 40 % of the total volume.

Then the inlet and outlet connectors of the CO detector are installed. The agitation and the temperature

are switched on and the test is started on the computer, keeping it in operation for a period of 16 h

(overnight) in order to properly condition the microorganisms present in the medium. Afterwards,

collagen (in the positive controls) and leather (in the samples) are added.
The biodegradation equipment featu
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 20136
IULTCS/IUC 37
IULTCS
Leather — Determination of
Voting begins on:
2020-03-16
degradability by micro-organisms
Voting terminates on:
Cuir — Détermination de la dégradabilité par les micro-organismes
2020-05-11
ISO/CEN PARALLEL PROCESSING
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 numbers
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 20136:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
IULTCS/IUC 37:2020(E)
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 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
---------------------- Page: 2 ----------------------
ISO/FDIS 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

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ISO/FDIS 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.
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ISO/FDIS 20136:2020(E)
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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.
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ISO/FDIS 20136:2020(E)
FINAL DRAFT 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.

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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.

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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.

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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.

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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 General

The equipment works continuously in an open system in which the air free of CO (7.6) circulates

throughout the system impelled by a pump (see Figures B.1 to B.5, Annex B). To increase the amount of

oxygen dissolved in the liquid phase, the intake of air into the Erlenmeyer flask is made through the use

of an air diffuser incorporated into the distilling head that is in contact with the liquid medium.

The air flow that goes into each Erlenmeyer flask is controlled by a system of individual pressure

gauges. The system also features a digital air flow quantification system. Digital data for each

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measurement and each Erlenmeyer flask are saved to a file and are subsequently converted into l/h

based on a calibration curve.

The equipment is provided with a thermostatic system capable of regulating the temperature of the

Erlenmeyer flasks by means of a thermostated tank. Tank water is inside a recirculation system that in

turn is connected to a cryothermostat that allows water recirculation at a temperature of 23 °C ± 2 °C.

In order to achieve constant agitation of the microbial suspension and samples, the equipment is

provided with a system comprising an array of 12 magnets coupled to 12 motors placed underneath

the tank base, in such a way that each magnet corresponds to one reaction flask. Agitation of the

microbial suspension and samples is achieved by putting a magnet inside each flask. Agitation speed (in

revolutions per minute, rpm) is set using specific hardware.
Important quantification parameters are referred to in Table B.1, Annex B.

The CO value of the samples placed in the flasks is sequentially measured by a multiplexer system. This

system comprises a rotating drum with 12 inlet channels and one outlet, which is directly connected to an

airtight tank where the CO sensor is located. Every air outlet of the reaction flasks is connected to an air

inlet of the multiplexer system. By a single rotation, the drum selects just one of the inlets, establishing a

direct connection between the selected inlet of one of the reaction flasks and the tank where the detector

is located, and blocking the rest of the inlets. A stepper motor makes the multiplexer system rotate to

select the relevant inlet, and specifically designed hardware and the corresponding firmware control

which of the 12 inlets is selected at all times. This way, it is possible to save the information relative to

the CO evolved (ppm) and the respective air flow (l/h) of a given flask at a given time.

The minimum test medium and the inoculum are added to the Erlenmeyer flasks. The volume of the

inoculum in each flask varies according to the degree of activity, ranging between 10

...

NORME ISO
INTERNATIONALE 20136
IULTCS/IUC 37
Deuxième édition
2020-06
Cuir — Détermination de la
dégradabilité par les micro-
organismes
Leather — Determination of degradability by micro-organisms
Numéros de référence
ISO 20136:2020(F)
IULTCS/IUC 37:2020(F)
ISO 2020
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ISO 20136:2020(F)
IULTCS/IUC 37:2020(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.

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Publié en Suisse
ii © ISO 2020 – Tous droits réservés
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ISO 20136:2020(F)
IULTCS/IUC 37:2020(F)
Sommaire Page

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

Introduction ................................................................................................................................................................................................................................vi

1 Domaine d'application ................................................................................................................................................................................... 1

2 Référence normatives ..................................................................................................................................................................................... 1

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

4 Symboles et termes abrégés ..................................................................................................................................................................... 2

5 Principe .......................................................................................................................................................................................................................... 2

5.1 Généralités .................................................................................................................................................................................................. 2

5.2 Évaluation de la biodégradation par titrage manuel: méthode A ............................................................... 3

5.3 Évaluation de la biodégradation par détection infrarouge (IR): méthode B ..................................... 3

6 Substances chimiques ..................................................................................................................................................................................... 3

7 Appareillage et matériel ............................................................................................................................................................................... 4

8 Mode opératoire.................................................................................................................................................................................................... 7

8.1 Collecte et préparation de l'inoculum ................................................................................................................................. 7

8.2 Préparation du matériau d’essai et du matériau de référence ...................................................................... 7

8.3 Conditions d'essai et période d'incubation .................................................................................................................... 8

8.4 Fin de l'essai .............................................................................................................................................................................................. 8

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

9.1 Évaluation de la biodégradation par titrage manuel (méthode A) ............................................................ 8

9.1.1 Détermination de la teneur en carbone organique ............................................................................ 8

9.1.2 Détermination de la quantité de dioxyde de carbone (CO ) produite ............................... 9

9.1.3 Correction tenant compte de la normalité de HCl ............................................................................... 9

9.1.4 Pourcentage de biodégradation à partir du dioxyde de carbone (CO ) dégagé ........ 9

9.2 Évaluation de la biodégradation par détection infrarouge (IR) (méthode B) ...............................10

9.2.1 Détermination de la teneur en carbone organique .........................................................................10

9.2.2 Détermination de la quantité de dioxyde de carbone (CO ) produite ............................10

9.2.3 Pourcentage de biodégradation à partir des données de CO .................................................

2 10

10 Expression des résultats............................................................................................................................................................................15

11 Validité des résultats .....................................................................................................................................................................................15

12 Rapport d'essai ...................................................................................................................................................................................................15

Annexe A (informative) Détermination du degré et de la vitesse de dégradation du matériau ..........16

Annexe B (informative) Détermination quantitative de la biodégradation du cuir .........................................19

Annexe C (informative) Données comparatives de biodégradabilité utilisant différentes

eaux résiduaires de tannerie ou eaux usées ........................................................................................................................23

Bibliographie ...........................................................................................................................................................................................................................24

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ISO 20136:2020(F)
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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 la Commission des essais chimiques de l'Union internationale des

sociétés de techniciens et chimistes du cuir (commission IUC, IULTCS), en collaboration avec le comité

technique du Comité européen de normalisation (CEN) CEN/TC 289, Cuir, dont le secrétariat est tenu

par l'UNI, conformément à l'Accord de coopération technique entre l'ISO et le CEN (Accord de Vienne).

L'IULTCS est une organisation mondiale de sociétés professionnelles des industries du cuir fondée

en 1897 ayant pour mission de favoriser l'avancement des sciences et technologies du cuir. L'IULTCS

a trois commissions, qui sont responsables de l'établissement des méthodes internationales

d'échantillonnage et d'essai des cuirs. L'ISO reconnaît l'IULTCS en tant qu'organisme international à

activités normatives pour l'élaboration de méthodes d'essai relatives au cuir.

Cette deuxième édition annule et remplace la première édition (ISO 20136:2017), qui a fait l'objet d'une

révision technique. Les principales modifications par rapport à l'édition précédente sont les suivantes:

— dans la première édition, la méthode B décrivait un système à circuit d'oxygène (O ) fermé.

L'inconvénient de ce système résidait dans la diminution de la concentration en oxygène avec le

temps, qui se traduisait par la diminution de l'activité des micro-organismes. Un système à circuit

d'oxygène ouvert a désormais été mis au point; dans ce système où l'oxygène n'est pas limité,

l'activité des micro-organismes est toujours optimale;

— une explication concernant la méthode de calcul menant aux résultats complète la méthode B. Le

dioxyde de carbone (CO ) accumulé durant l'essai (zone située sous la courbe illustrant les moles

de CO en fonction du temps) est calculé;

— la possibilité d'utiliser des eaux usées municipales comme inoculum à la place des eaux résiduaires

de tannerie a été incluse;
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— une nouvelle annexe, l’Annexe C, qui compare la biodégradabilité en présence d’inoculums de

différentes origines, a été ajoutée.

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.
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Introduction

L'un des problèmes importants auxquels est confrontée l'industrie de la chaussure est le traitement

des déchets. Ces déchets, et notamment le cuir, bien que considérés comme non dangereux par la

réglementation actuelle, sont produits en grandes quantités et finissent pour la plupart en décharge, où

leur temps de dégradation naturelle dépasse de loin la durée de vie utile des produits.

Face à ce problème, la recherche s’oriente de plus en plus vers des agents de tannage alternatifs qui

confèrent au cuir les mêmes propriétés que celles obtenues au moyen des agents actuellement employés,

en réduisant toutefois le temps de biodégradation dans la nature.

Le présent document permet le mesurage de la biodégradabilité du cuir dans un système liquide en

utilisant des micro-organismes comme inoculum. L’essai est considéré comme valide lorsque le

pourcentage de dégradation du collagène (témoin positif) est d’au moins 70 % sur une période de

cinquante jours (50 d) au maximum. Le degré de biodégradabilité d’un échantillon de cuir (matériau

d’essai) est déterminé par comparaison de sa valeur de dégradabilité, en pourcentage, avec la valeur

de dégradabilité, en pourcentage, obtenue avec du collagène soumis au même essai pendant la même

durée. Plus les pourcentages de dégradabilité sont proches, plus le temps de biodégradation dans la

nature est court. Par conséquent, les matériaux d’essai dont les pourcentages de dégradabilité sont très

inférieurs à celui du collagène mettront plus de temps à se biodégrader dans la nature.

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ISO 20136:2020(F)
NORME INTERNATIONALE
IULTCS/IUC 37:2020(F)
Cuir — Détermination de la dégradabilité par les micro-
organismes
1 Domaine d'application

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 CO 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.
2 Référence normatives
Le présent document ne contient aucune référence normative.
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 http:// www .electropedia .org/
3.1
filtre poreux n° 1
diffuseur à taille de pores comprise entre 100 μm et 160 μm
Note 1 à l'article: Il s’agit du mesurage normal.
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3.2
inoculum
boues activées d'eaux résiduaires de tannage

Note 1 à l'article: En l'absence de tannerie à proximité, des eaux usées urbaines peuvent servir d'inoculum.

4 Symboles et termes abrégés
AMP adsorption modulée en pression
atm atmosphère normale, unité de pression définie comme égale à 101 325 Pa
[Ba(OH) ] hydroxyde de baryum
C carbone
CO dioxyde de carbone

GL18 pas de vis utilisé avec des fioles Erlenmeyer H-SA V40/45 (capacité 5 000 ml)

GL14 pas de vis utilisé avec des fioles Erlenmeyer H-SA V29/32 (capacité 2 000 ml)

H-SA V 29/32 dimensions intérieure et extérieure, en millimètres, de l'embouchure

des fioles Erlenmeyer

H-SA V H40/45 dimensions intérieure et extérieure, en millimètres, de l'embouchure

des fioles Erlenmeyer
IR infrarouge
−6 −1
ppm parties par million (10 ), par exemple 1 mg par kilogramme (mg.kg )
Q débit d'air, en moles, passant à travers le système en une heure (mol.h )
nar
Q débit de CO , en moles, passant à travers le système en une heure (mol.h )
nCO2 2
5 Principe
5.1 Généralités

La méthode consiste à quantifier le CO dégagé au cours de la dégradation du collagène, polymère

constitué d'acides aminés polymérisés, sous l'action de micro-organismes présents dans les boues des

cuves de traitement biologique des tanneries. Le CO est dégagé dans des proportions stœchiométriques

par rapport à la quantité de carbone (C) présente dans ledit polymère. Le pourcentage initial de carbone

présent dans chacun des échantillons soumis à essai est déterminé par une analyse élémentaire.

Le CO accumulé durant l'essai est converti en pourcentage de biodégradation à l'aide d'équations

mathématiques. Les essais doivent être réalisés en double en présence d'un témoin positif, constitué

d'un milieu d'essai minimum (6.3), d'un inoculum (boues activées d'eaux résiduaires de tannage) et de

collagène, et d'un témoin négatif, constitué seulement d'un milieu d'essai minimum et d'un inoculum.

L'essai doit être considéré comme valide si le degré de biodégradation du témoin positif (collagène pur)

est égal ou supérieur à 70 %.

Les essais doivent être effectués en utilisant un équipement capable de fournir les conditions

nécessaires à la réalisation de l'essai. Il convient que l'agitation, la température et le débit d'air exempt

de CO soient contrôlés.

Le pourcentage initial de carbone (C) présent dans le collagène étudié est déterminé par l'analyse

élémentaire de l'éprouvette. Le pourcentage de biodégradation n'inclut pas la quantité de carbone

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convertie en une nouvelle biomasse cellulaire qui n'a pas été métabolisée en dioxyde de carbone au

cours de l'essai.
5.2 Évaluation de la biodégradation par titrage manuel: méthode A

Cette méthode d'essai détermine le pourcentage de biodégradation de peaux, tannées ou non tannées,

par le mesurage indirect du CO dégagé au cours de la dégradation du collagène, le principal constituant

de la peau, sous l'action des micro-organismes présents dans les eaux résiduaires de tannage.

Le CO dégagé durant l'essai est déterminé de façon indirecte par le biais de la réaction de l'hydroxyde

de baryum [Ba(OH) ] avec le CO , qui donne un précipité, le carbonate de baryum (BaCO ). La quantité

2 2 3

de CO dégagée est déterminée par titrage du [Ba(OH) ] restant, non précipité, avec une solution d'acide

2 2

chlorhydrique à 0,05 mol.l . Ces mesurages sont effectués quotidiennement tout au long de l'essai.

Pour évaluer la biodégradabilité, on procède au mesurage indirect du CO dégagé en fonction du temps

et on calcule le degré de biodégradation par la différence entre le pourcentage initial de carbone présent

dans le collagène et la teneur résiduelle en carbone organique soluble n'ayant pas été transformé en CO

au cours du processus (voir Annexe A, Figures A.1 à A.3).
5.3 Évaluation de la biodégradation par détection infrarouge (IR): méthode B

Cette méthode consiste à déterminer la biodégradation en quantifiant le CO dégagé tout au long de la

dégradation du collagène, au moyen de la détection infrarouge directe et de la surveillance continue

de la concentration de CO , en utilisant un équipement pouvant évaluer simultanément douze fioles

Erlenmeyer
(voir Annexe B, Figures B.1 à B.5).

L'équipement (voir Annexe B, Figure B.1) est apte à mesurer la valeur de CO de plusieurs échantillons

contenus dans différentes fioles Erlenmeyer. La quantité de CO dégagée au cours de la dégradation de

l'échantillon sous l'action des micro-organismes est mesurée par un détecteur infrarouge. Ce détecteur

est géré par un système de multiplexage comprenant un tambour rotatif comportant douze canaux

d'entrée, de sorte que chaque sortie d'air des fioles Erlenmeyer est reliée à une entrée d'air du système

de multiplexage. Le tambour est pourvu d'une sortie directement reliée à un débitmètre d'air qui

mesure le débit (l.h ) et, ensuite, à une cuve étanche à l'air qui contient le capteur de CO . L'Annexe B

(voir Tableau B.1) résume les paramètres, les unités de mesure et la plage des valeurs de détection. Les

valeurs du débit d'air et de la concentration de CO sont sauvegardées dans un système d'acquisition de

données relié à un ordinateur.
6 Substances chimiques
®1)

6.1 Eau déionisée ou ultrapure (Milli Q ), exempte de matières toxiques, d'une résistivité

supérieure à 18 MΩ/cm.

6.2 Solutions mères, utiliser uniquement des réactifs de qualité analytique. Les solutions mères

employées dans les essais sont les mêmes pour les deux méthodes utilisées dans le présent document.

Préparer des solutions mères synthétiques par dissolution dans de l'eau distillée (6.1), au volume de 1 l

et dans des fioles séparées, des substances suivantes:
6.2.1 Chlorure ferrique (FeCl ·6H O), 1,00 g.
3 2
6.2.2 Sulfate de magnésium (MgSO ·7H O), 22,50 g.
4 2
6.2.3 Chlorure de calcium (CaCl ·2H O), 36,43 g.
2 2

1) Milli Q est un exemple de produit approprié disponible dans le commerce. Cette information est donnée

à l'intention des utilisateurs du présent document et ne signifie nullement que l’ISO approuve ou recommande

l'emploi exclusif du produit ainsi désigné.
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ISO 20136:2020(F)
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6.2.4 Tampon phosphate:
— dihydrogénophosphate de potassium (KH PO ), 8,50 g;
2 4
— potassium phosphate dibasique trihydraté (K HPO ·3H O), 28,50 g;
2 4 2
— di-sodium hydrogénophosphate (Na HPO ), 17,68 g;
2 4
— chlorure d’ammonium (NH Cl), 1,70 g.
6.2.5 Sulfate d’ammonium [(NH ) SO ], 40,00 g.
4 2 4
6.3 Milieu d’essai minimum

Le milieu d’essai minimum doit contenir les solutions mères suivantes, diluées à 1 l avec de l’eau

déionisée:
6.3.1 Solution mère de chlorure ferrique (6.2.1), 2 ml.
6.3.2 Solution mère de sulfate de magnésium (6.2.2), 2 ml.
6.3.3 Solution mère de chlorure de calcium (6.2.3), 2 ml.
6.3.4 Solution mère de tampon phosphate (6.2.4), 4 ml.
6.3.5 Solution mère de sulfate d'ammonium (6.2.5), 2 ml.
®2)

6.4 Éprouvettes: utiliser du collagène de type I (Sigma ou similaire) comme témoin positif. Les

éprouvettes doivent être constituées, pour l'essentiel, de polymères naturels ou de cuir de tannerie

utilisés pour la fabrication de vêtements en cuir.

6.5 Pour la méthode A seulement: préparer une solution d'hydroxyde de baryum [Ba(OH) ]

à 0,025 mol.l par dissolution de 4,0 g de [Ba(OH) ] par litre d'eau distillée. Filtrer pour éliminer la

matière solide, vérifier la molarité par titrage avec un acide standard. Stocker la solution claire obtenue

dans un récipient scellé pour éviter l'absorption du CO de l'air. Il est conseillé de préparer 5 l de solution

en une fois lors de la réalisation d'une série d'essais.
6.6 Acide chlorhydrique, à 0,05 mol.l .
7 Appareillage et matériel
Équipement courant de laboratoire et, notamment:
7.1 Balance analytique, permettant une lecture à 0,000 1 g près.
7.2 Pipettes, d'une capacité de 5 ml à 25 ml.
7.3 Micropipettes, d'une capacité de 500 μl et 1 000 μl.

7.4 Fioles de pré-traitement et fioles (pour la méthode A seulement), de différentes capacités.

2) Sigma® est un exemple de produit approprié disponible dans le commerce. Cette information est donnée

à l'intention des utilisateurs du présent document et ne signifie nullement que l’ISO approuve ou recommande

l'emploi exclusif du produit ainsi désigné.
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7.5 Burettes, d'une capacité de 100 ml.

7.6 Source d'air exempt de CO autonome, constituée d'un compresseur silencieux relié à un

système d'adsorption modulée en pression (AMP) muni d'un tamis moléculaire.

7.7 Sépiolite pour filtrer les impuretés et l'humidité du système de ventilation.

7.8 Bouchons, tuyau en plastique souple, imperméable au CO .
7.9 Récipients d’essai

7.9.1 Méthode A: huit fioles Erlenmeyer de 5 l (fioles de réaction) pour chaque essai (deux témoins

et deux éprouvettes par essai). Des fioles Erlenmeyer H-SA V H40/45 d'une capacité de 5 000 ml doivent

être utilisées, ainsi que des têtes de distillation V2 à pas de vis GL18 et un filtre poreux n° 1 (diffuseur).

Le volume total de liquide (milieu de culture + inoculum) doit être de 2,5 l.

7.9.2 Méthode B: douze fioles d'un volume d'essai de 1 l (fioles de réaction) pourvues d'une tête de

distillation et d'un diffuseur d'air, utilisées pour effectuer les essais (deux témoins et quatre échantillons,

en double). Les fioles Erlenmeyer doivent avoir une capacité de 2 000 ml et 3 traits. Il doit s'agir de

modèles du type H-SA V 29/32 (SQ13). Elles doivent être pourvues de têtes de distillation V2 à pas de

vis GL14 (entrée d'air de 6 mm et sortie d'air de 8 mm) et d'un filtre poreux n° 1 (diffuseur). Le volume

total de liquide (milieu de culture + inoculum) doit être de 1 l.
7.10 Équipement d'essai
7.10.1 Évaluation de la biodégradation par titrage manuel (équipement A)

L'équipement A fonctionne de façon à faire buller l'air exempt de CO à travers une série de sept

fioles Erlenmeyer (fioles de pré-traitement) qui piègent le dioxyde de carbone résiduel présent dans

l'écoulement d'air provenant du dispositif AMP (7.6). Le système se divise ensuite en huit lignes

contrôlées par huit robinets qui permettent un contrôle indépendant de l'écoulement; ces huit lignes

alimentent à leur tour huit fioles Erlenmeyer (fioles de réaction) situées à l'intérieur de la cuve. La sortie

de chacune des huit fioles Erlenmeyer est directement reliée à une série de trois fioles Erlenmeyer en

verre (fioles d'analyse) reliées entre elles, chacune contenant 100 ml de [Ba(OH) ] à 0,025 mol.l , d'où

seront tirés les résultats (voir Annexe A, Figures A.2 et A.3).

L'équipement comprend également un thermostat qui permet de réguler la température des fioles de

réaction grâce à un système de recirculation d'eau en circuit fermé. L'essai est réalisé à (23 ± 1) °C. Les

fioles de réaction sont constamment agitées à 24 r.min (mouvement de va-et-vient) pendant toute la

durée de l'essai.

Le volume d'inoculum de chaque fiole varie en fonction de son degré d'activité, représentant entre 10 %

et 20 % du volume total (inoculum + milieu d’essai minimum), qui est de 2,5 l. Si l'inoculum provient

d'eaux usées urbaines, le volume total (inoculum + milieu minimum) peut augmenter de jusqu'à 40 %.

L'air quittant le générateur doit passer par le système AMP, lequel devra avoir fonctionné pendant 16 h

(toute la nuit) avant le début de l'essai afin de garantir l'obtention d'une concentration de CO stable,

inférieure à 1 ppm, dans l'écoulement d'air.

Pendant l'essai, un écoulement d'air exempt de CO de débit constant, égal à 150 ml.min , alimente

chaque fiole de réaction. Le débit d'air est régulièrement vérifié au niveau de chaque sortie au moyen de

débitmètres gradués afin de s'assurer que le système ne présente aucune fuite.

Le CO dégagé par la digestion aérobie des éprouvettes par les micro-organismes est quantifié en

mesurant le niveau de carbonatation de l'hydroxyde de baryum [Ba(OH )] à 0,025 mol.l contenu dans

les trois fioles d'analyse reliées à chaque fiole de réaction. Les fioles d'analyse sont remplacées toutes

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les 24 h par d'autres fioles contenant la même quantité initiale d'hydroxyde de baryum [Ba(OH )]

à 0,025 mol.l .

Les valeurs quotidiennes de quantification de la carbonatation du [Ba(OH )] sont saisies dans un tableur

qui les convertit en pourcentages de biodégradation (Article 10).

7.10.2 Évaluation de la biodégradation par détection infrarouge (IR) (équipement B)

7.10.2.1 Généralités

L'équipement fonctionne en continu en circuit ouvert, l'air exempt de CO (7.6) circulant à travers tout

le système sous l'action d'une pompe (voir Annexe B, Figures B.1 à B.5). Pour augmenter la quantité

d'oxygène dissoute dans la phase liquide, l'air entrant dans la fiole Erlenmeyer passe à travers un

diffuseur d'air intégré à la tête de distillation, qui est en contact avec le milieu liquide.

Le débit d'air entrant dans chaque fiole Erlenmeyer est contrôlé par un système de manomètres

individuels. Le système comprend également un système de quantification du débit numérique. Les

données numériques correspondant à chaque mesurage et à chaque fiole Erlenmeyer sont sauvegardées

dans un fichier; elles sont ensuite converties en l.h à l'aide d'une courbe d'étalonnage.

L'équipement est pourvu d'un système thermostati
...

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