INTERNATIONAL STANDARD ISO 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) COPYRIGHT PROTECTED DOCUMENT © ISO 2017, Published in Switzerland All rights reserved Unless otherwise specified, no part o f 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 o f the requester ISO copyright o ffice Ch de Blandonnet • CP 401 CH-1214 Vernier, Geneva, Switzerland Tel +41 22 749 01 11 Fax +41 22 749 09 47 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 Scope Normative references Terms and definitions Symbols and abbreviated terms Principle Chemicals 5.1 5.2 Apparatus and materials Procedure 8.5 Collection and preparation of the inoculum Preparation of the test material and reference material Test conditions and incubation period Test equipment 8.4.1 Equipment for the assessment o f biodegradation by manual titration (equipment A) 8.4.2 Equipment for the assessment o f biodegradation by IR detection (equipment B) End of the test 9.1 Equipment for the assessment o f biodegradation by manual titration (equipmen t A) 8.1 8.2 8.3 8.4 Quantification 9.2 10 11 12 Method A: assessment o f biodegradation by manual titration Method B: assessment o f biodegradation by infrared detection 9.1.1 Determination of the organic carbon content 9.1.2 Determination of the amount of carbon dioxide produced (Method A) 9.1.3 Correcting for normality o f HCl 9.1.4 Percentage of biodegradation from carbon dioxide evolved Equipment for the assessment o f biodegradation by IR detection (Method B) 9.2.1 Determination of the organic carbon content 9.2.2 Determination of the amount of carbon dioxide (CO produced) 9.2.3 Percentage of biodegradation from CO data Expression of results 10 Validity of results 10 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 2017 – All rights reserved iii 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 o f 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 o f electrotechnical standardization The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part In particular the different approval criteria needed for the di fferent types o f ISO documents should be noted This document was dra fted in accordance with the editorial rules of the ISO/IEC Directives, Part (see www.iso org/directives) Attention is drawn to the possibility that some o f the elements o f this document may be the subject o f patent rights ISO shall not be held responsible for identi fying any or all such patent rights Details o f any patent rights identified during the development o f 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 in formation given for the convenience o f users and does not constitute an endorsement For an explanation on the voluntary nature o f standards, the meaning o f ISO specific terms and expressions related to formity assessment, as well as in formation about ISO’s adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso org/iso/foreword html This document was prepared by the Chemical Tests Commission o f the International Union o f 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 o f pro fessional leather societies to urther the advancement o f leather science and technology IULTCS has three Commissions, which are f responsible for establishing international method for the sampling and testing of leather ISO recognizes IULTCS as an international standardizing body for the preparation o f test methods for leather iv © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Introduction One o f the big problems faced by the footwear industry is waste treatment Although this waste, especially in the case o f 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 o f 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 bu ffing 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 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 than the use ful li fe o f the final products There fore, the use o f additives that are less harm ful to the Within this sector, the development o f 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 o f this form o f analysis in a test time o f no more than 35 days © ISO 2017 – All rights reserved v ISO 20136:2017(E) IULTCS/IUC 37:2017(E) INTERNATIONAL STANDARD Leather — Determination of degradability by microorganisms Scope T h i s c u ment s p e c i fie s a te s t me tho d to de term i ne the degre e a nd rate o f aerobic bio de gradation o f h ide s a nd ski n s o f d i fferent an i ma l origi n, whe ther they a re ta n ne d or no t, th rough the i nd i re c t determination of CO2 T he te s t m ater i a l aqueous medium is pro duce d by the degradation o f col lagen exposed to an i no c u lu m (ac ti vate d s lud ge fro m ta n ner y wa s te water) in an T he cond ition s e s tabli s he d i n th i s c u ment corre s p ond to op ti mu m l ab orator y cond ition s to ach ieve the ma xi mu m level o f bio degradation However, they may no t ne ce s s a ri ly corre s p ond to the op ti mu m conditions or maximum level of biodegradation in the natural medium In general, the experimental procedure covers the determination of the degradation degree and f f dioxide produced throughout the test For this purpose, the testing equipment complies with strict rate o the materi a l u nder control le d cond ition s , wh ich a l lows the ana lys i s o the evolve d c arb on re qu i rements with regard to flow, temp eratu re a nd agitation control This method applies to the following materials: — natu l p olymers o f an i ma l s troma (a ni ma l ti s s ue/s ki n s) , — animal hides and skins tanned (leather) using organic or inorganic tanning agents, — le athers that, u nder te s ti ng cond ition s , no t i n h ibit the ac tivity o f m icro orga n i s m s pre s ent i n the inoculum Normative references There are no normative references in this document Terms and definitions For the pu r p o s e s o f th i s c u ment, the fol lowi ng term s and defi n ition s 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 diffuser with pore size from 100 microns to 160 microns N o te to entr y: T h i s me a s u rement i s s ta nd a rd 3.2 inoculum ac ti vate d sludge from ta nner y wa s tewater © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Symbols and abbreviated terms [Ba(OH) ] C CO2 GL18 GL14 H-SA V 29/32 barium hydroxide H-SA V H40/45 inner and outer measures in millimetres o f the orifice o f the mouth o f the Erlenmeyer® flasks IR PSA infrared pressure swing adsorption carbon carbon dioxide threads are used with H-SA V40/45 Erlenmeyer® flasks (5 000 ml volume) threads are used with H-SA V29/32 Erlenmeyer® flasks (2 000 ml volume) inner and outer measures in millimetres o f the orifice o f the mouth o f the Erlenmeyer® flasks 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 CO2 evolved during the degradation of collagen, which is the major constituent o f the skin, by the action o f the microorganisms present in tannery wastewater The CO2 evolved during the test is indirectly determined through the reaction of [Ba(OH) ] with CO2 , which is precipitated as barium carbonate (BaCO3 ) The amount of CO2 evolved is determined 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 di fference between the initial carbon percentage present in collagen and the remaining soluble organic carbon content that has not been transformed into CO2 in the course of the process The initial carbon percentage (C) present in the collagen under study is determined by the elemental analysis o f the test specimen The biodegradation percentage does not include the amount o f 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 CO2 -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 o f 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 o f the CO evolved throughout the degradation o f collagen, by means o f the direct IR detection and continuous monitoring of the CO2 concentration The equipment comprises a reaction unit made up of a closed set of © 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 o f carbon (C) present in the collagen under study is determined through the elemental analysis o f each sample The percentage o f biodegradation does not include the quantity o f 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 CO2 -free air inflow should be controlled The tests are conducted in duplicate in the presence o f a positive control, composed o f a synthetic medium, microorganisms and collagen, and a negative control, composed only o f a synthetic medium and an inoculum, allowing the assessment o f five di fferent leather samples that can be evaluated in duplicate 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 o f the reaction flasks specific o f each methodology (method A: a final liquid volume o f 2,68 l; method B: a final liquid volume o f l) 6.1 Deionised or ultrapure (Milli Q® 1) ) 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 o f the following in distilled water to l: 6.2.1.1 Ferric chloride (FeCl3 •6H O), 1,00 g 6.2.1.2 Magnesium sulfate (MgSO 4•7H O), 22,5 g 6.2.1.3 Calcium chloride (CaCl2 •2H O), 36,43 g Phosphate buffer KH HPO 8,5 g, K2 HPO 4•3H O 28,5 g, Na2 HPO 17,68 g, and NH 4Cl 1,7 g, for a total of 56,38 g 6.2.1.4 6.2.1.5 6.2.2 Ammonium sulfate [(NH 4) SO 4], 40 g The test medium shall contain the following reagents diluted to l with high-quality distilled water: 6.2.2.1 Magnesium sulfate solution, ml 6.2.2.2 Calcium chloride solution, ml 6.2.2.3 Phosphate buffer solution, ml 6.2.2.4 Ferric chloride solution, ml 1) Milli Q® is an example o f a suitable product available commercially This information is given for the convenience o f users o f this document and does not constitute an endorsement by ISO o f this product © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Ammonium sulfate solution, ml 6.2.2.5 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 o f leather clothing 6.3 Only for Method A: Barium hydroxide solution, 0,025 mol/l, is prepared dissolving 4,0 g 6.4 [Ba(OH) ] per litre o f 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 CO2 from the air It is recommended that l be prepared at a time when running a series of tests Apparatus and materials The usual laboratory equipment and, in particular, the following: 7.1 Analytical balance , capable of reading to 0,000 g 7.2 Pipettes , ml to 25 ml capacity 7.3 Micro-pipettes , 500 μl and 000 μl V o l u m e t r i c f l a s , l k 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 the equipment specially conceived for this test (see Annex A and Figure A.1) This equipment allows four test specimens to be analyzed in duplicate (two test specimens and two controls) It also allows agitation, experiment temperature and CO2 -free air inflow to be controlled 7.5.2 Autonomous CO -free air source , consisting of a noiseless compressor connected to a PSA 7.5.3 Sepiolite to filter impurities and humidity from the ventilation system (pressure swing adsorption) system provided with a molecular sieve, from Peak Scientific, model PG14L T e s t f l a s k s 7.5.4.1 Eight l Erlenmeyer® 2) flasks (reaction flasks) for each test (two controls and two test specimens per test) 000 ml H-SA V H40/45 Erlenmeyer® flasks shall be used, as well as V2 distilling heads with GL18 threads and a filter pore no di ffuser Connect the PSA equipment (7.5.2 ) to four glass flasks and two plastic flasks connected in series using silicone tubing The first three flasks shall contain 700 ml o f 10 mol/l sodium hydroxide (NaOH) The fourth flask shall be empty The fi fth flask shall contain 700 ml o f 0,025 mol/l [Ba(OH) ] The sixth flask shall be empty 7.5.4.2 2) Erlenmeyer® is an example o f a suitable product available commercially This information is given for the convenience o f users o f this document and does not constitute an endorsement by ISO o f this product © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Q u a n t i f i c a t i o n 9.1 Equipment for the assessment of biodegradation by manual titration (equipment A) 9.1.1 Determination of the organic carbon content D e term i ne by elementa l ana lys i s the to ta l organ ic c arb on content o f the te s t materi a l T h i s a l lows the the ore tic a l ma xi mu m quantity o f c arb on d ioxide evolution to b e c a lc u l ate d T he materi a l h as w % ( p ercentage b y weight) o f c arb on; w/10 • g o f materi a l charge s = Y g c a rb on ch arge d to E rlen meyer® fl as k a) C + O2 → CO 2 b) g C yield 4 g C O c) Y mg C yield 4/1 • Y mg C O 9.1.2 Determination of the amount of carbon dioxide produced (Method A) Correct for the amount of carbon dioxide produced with the negative control (culture medium + i no c u lu m) b y s ub trac ti ng negative control titration from te s t materi a l titration with , mol/ l H C l a) [Ba(OH) ] + CO2 + H2O b) [Ba(OH) 2 + H2O c) m moles of CO2 = (m moles HCl)/2 → B aC O ] + HC l → B aC l 9.1.3 Correcting for normality of HCl a) m moles of CO2 = [(0,05 mol/l) • ml HCl]/2 b) mg of CO2 = [(0,05 mol/l) • ml HCl • 44]/2 = 1,1 • ml HCl Hence, the amou nt o f c arb on d ioxide evolve d i n mg i s ob tai ne d b y mu ltiplyi ng the H C l titration b y ,1 9.1.4 Percentage of biodegradation from carbon dioxide evolved The percentage of biodegradation from carbon dioxide evolved is calculated as shown below: a) (g CO2 produced/g CO2 theoretical) • 100 b) [(1 ,1 • m l HC l • ) /(4 • y) ] • 10 9.2 Equipment for the assessment of biodegradation by IR detection (Method B) 9.2.1 Determination of the organic carbon content The total organic carbon content of the material being tested is determined through an elemental ana lys i s This a l lows the ma xi mum amou nt o f C O2 th at c an be generate d in e ach te s t ru n to be the ore tic a l ly c a lc u l ate d T he materi a l h as w % (weight p ercentage) o f c arb on; w/10 • g o f s a mp le = Y g o f c arb on put i nto the fl as k a) C + O2 b) → CO 2 g C yield 4 g C O © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) c) Y g C yield 4/1 • Y g C O 9.2.2 CO2 Determination of the amount of carbon dioxide (CO produced) pro duce d du ri ng the degradation o f le ather i s acc u mu late d i n the clo s e d s ys tem and me as u re d u s i ng the re frac tive i ndex (RI ) pre s ent i n the qua nti fic ation e qu ipment For th i s pu r p o s e, the s en s ors are previou s ly c a l ibrate d b e twe en % a nd % with m i xtu re s o f c a l ibration gas e s T he re s u lts ob tai ne d are s tore d i n tx t format i n a table that c a n b e tran s forme d i nto an E xcel® s pre ad she e t conversion of % CO2 to % of biodegradation 9.2.3 for the l ater Percentage of biodegradation from CO data The percentage of biodegradation from carbon dioxide produced is calculated as shown below: The % of CO2 produced is converted into a % of biodegradation of the samples from the general equation of the gases The % CO2 convert them into biodegradation percentages through the general formula of gases (PV = nRT), where: va lue s ob tai ne d i n a l l me a s u ri ng e quipment a re mathematic a l ly pro ce s s e d to P i s the ambient atmo s pheric pre s s u re on the s a mple that dep end s on a ltitude; V i s the to ta l volu me (fl as ks + tub e s + I R de te c tor) ; N i s the nu mb er o f mole s o f the ga s m i xtu re; R i s the ga s s ta nt; T is the temperature of the gaseous phase in degrees Kelvin (K) This formula allows the determination of the number of moles of the gas mixture as a reference for the transformation of CO2 f the following mathematical steps: CO2 = n • % CO (CO CO2 • MCO (g of CO 2 /MCO T) • 100] (% biodegradation), where nCO2 is the number of CO2 moles in the gaseous phase a) %CO2 = CO2 b) MCO2 = CO2 mass determined for a given % of CO2 c) MCO2 T = Theoretical CO2 mass obtained from the % of organic carbon in the sample (see 9.2.1) p ercentage s i nto % o mole s → nC O mas s → nC O bio degradation For s uch m, it i s ne ce s s ar y to c arr y out mole s) ; ); % B io degradation → [(MC O p ercentage qua nti fie d b y the I R e quipment at ever y i n s ta nt qua nti fie d © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) 10 Expression of results The percentage of degradation of each test material is presented as a relative percentage, calculated on the basis of the absolute value of collagen degradation converted to 100 %, according to Formula (1): X = ( B • 00 ) / A (1) where A B X i s the % ab s olute bio degradation o f col lagen (70 % or more) ; i s the % ab s olute bio degradation o f e ach s a mp le; i s the % rel ati ve bio de gradabi l ity o f e ach s ample 11 Validity of results The test shall be considered as valid if the degree of biodegradation of the reference material (collagen) Clause 8, term A) i n the re ac tion fl as ks i s e qua l to or h igher than 70 % i n ab s olute term s (s e e 12 Test report The test report shall include at least the following: a) a re ference to th i s c u ment, i e I S O 01 6; b) information on the inoculum including source, date of collection, storage, handling and potential accl i mation to te s t materi a l; c) c arb on (C ) content o f the te s t materi a l, b o th the col lagen ( p o s itive control) and the le ather s a mp le s; d) percent of theoretical aerobic biodegradation for each leather tested and the standard control materi a l (col lagen) ; e) accumulative average carbon dioxide evolution over time until plateau should be reported and d i s playe d graph ic a l ly a s lag-pha s e and s lop e (rate) ; f) % relative of biodegradation of each sample 10 © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Annex A (informative) Determination of the degree and rate of degradation of the material A.1 Principle T he bio de gradabi l ity as s e s s ment e quipment i s a comp ac t u n it th at s b e en s p e c i fic a l ly conceive d for te s ti ng bio degrad ation o f le ather H owever, its appl ic abi l ity c a n b e ex trap olate d to the s tudy o f the biolo gic a l degradation o f any tex ti le (e g fabrics , we ave s , e tc ) or p olymeric materi a l ( pl as tics) , a s long as the relevant adj u s tments are made to the me tho dolo g y, e s p e ci a l ly with regard s to the i no c u lu m used The test method therefore covers the determination of the degree and rate of degradation of the throughout f free materi a l u nder control le d lab orator y cond ition s b as e d on the ana lys i s o f the evolution o f C O the te s t For th i s pu rp o s e, the u n it compl ie s with s tric t re qu i rements re erri ng to flow control (C O r) , therma l control and agitation control (to - and- fro mo tion) T h i s u n it was develop e d to s i mpl i fy the exp eri menta l pro ce s s , a l lowi ng a l l the op erationa l control s to b e acce s s e d from one e a s i ly acce s s ible and understandable control panel situated on the top of the equipment (Figures A.1 and A.2) © ISO 2017 – All rights reserved 11 ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Figure A.1 — View of the unit for biodegradability assessment 12 © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Figure A.2 — Volumetric view of the unit for biodegradability assessment © ISO 2017 – All rights reserved 13 ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Key air pump PSA system pre-treatment flasks flow meter reaction flasks analysis flasks Figure A.3 — Diagram of the unit for biodegradability assessment 14 © ISO 2017 – All rights reserved ISO 20136:2017(E) IULTCS/IUC 37:2017(E) Figure A.3 shows a diagram of the unit for biodegradability assessment from the point o f view of the basic experimental procedure As can be seen, the unit is provided with an autonomous clean CO2 -free air generation system consisting o f a noiseless compressor (specially conceived for a non-industrial use in research laboratories, with a noise level