SIST EN ISO 14852:2018
(Main)Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium - Method by analysis of evolved carbon dioxide (ISO 14852:2018)
Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium - Method by analysis of evolved carbon dioxide (ISO 14852:2018)
This document specifies a method, by measuring the amount of carbon dioxide evolved, for the
determination of the degree of aerobic biodegradability of plastic materials, including those containing
formulation additives. The test material is exposed in a synthetic medium under standardized
laboratory conditions to an inoculum from activated sludge, mature compost or soil under aerobic,
mesophilic conditions.
If an unadapted activated sludge is used as the inoculum, the test result can be used to assess the aerobic
biodegradation processes which occur in a waste water treatment plant environment. If a mixed or preexposed
inoculum is used, the method can be used to investigate the potential biodegradability of a test
material.
The conditions used in this document do not necessarily correspond to the optimum conditions allowing
maximum biodegradation to occur, but this test method is designed to measure the biodegradation of
plastic materials and give an indication of their potential biodegradability.
The method enables the assessment of the biodegradation to be improved by calculating a carbon
balance (optional, see Annex C).
The method applies to the following materials:
— natural and/or synthetic polymers, copolymers or mixtures thereof;
— plastic materials which contain additives such as plasticizers, colorants or other compounds;
— water-soluble polymers;
— materials which, under the test conditions, do not inhibit the microorganisms present in the
inoculum. Inhibitory effects can be determined using an inhibition control or by another appropriate
method (see, for example, ISO 8192[1]). If the test material is inhibitory to the inoculum, a lower test
concentration, another inoculum or a pre-exposed inoculum can be used.
Bestimmung der vollständigen aeroben Bioabbaubarkeit von Kunststoff-Materialien in einem wässrigen Medium - Verfahren mittels Analyse des freigesetzten Kohlenstoffdioxides (ISO 14852:2018)
Évaluation de la biodégradabilité aérobie ultime des matériaux plastiques en milieu aqueux - Méthode par analyse du dioxyde de carbone libéré (ISO 14852:2018)
Le présent document spécifie une méthode d'évaluation du taux de biodégradabilité aérobie des matériaux plastiques, y compris ceux contenant des additifs, par la détermination de la quantité de dioxyde de carbone libéré. Le matériau d'essai en milieu synthétique est exposé dans des conditions de laboratoire normalisées à un inoculum provenant de boues activées, de compost mature ou de sol dans des conditions aérobies et mésophiles.
En cas d'utilisation comme inoculum de boues activées non adaptées, le résultat d'essai peut être utilisé pour évaluer les processus de biodégradation aérobie qui se produisent dans une installation de traitement des eaux résiduaires. Si on utilise un inoculum mélangé ou pré-exposé, la méthode permet d'étudier la biodégradabilité potentielle du matériau d'essai.
Les conditions utilisées dans le présent document ne correspondent pas nécessairement aux conditions optimales permettant d'obtenir le taux maximal de biodégradation; cependant, cette méthode d'essai est conçue pour mesurer la biodégradation des matériaux plastiques et pour donner une indication de leur biodégradabilité potentielle.
La méthode permet d'affiner l'évaluation de la biodégradation par le calcul d'un bilan carbone (facultatif, voir l'Annexe C).
La présente méthode s'applique aux matériaux suivants:
— polymères naturels et/ou synthétiques, copolymères ou mélanges de ceux-ci;
— matériaux plastiques contenant des additifs tels que plastifiants, colorants ou tout autre composé;
— polymères hydrosolubles;
— matériaux n'ayant pas d'effet inhibiteur dans les conditions d'essai sur les micro-organismes présents dans l'inoculum. Les effets inhibiteurs peuvent être déterminés en utilisant un dispositif de contrôle de l'inhibition ou par toute autre méthode appropriée (voir, par exemple, l'ISO 8192[1]). Si le matériau d'essai a un effet inhibiteur vis-à-vis de l'inoculum, il est possible d'utiliser une plus faible concentration, un autre inoculum ou un inoculum pré-exposé.
Določanje končne aerobne biorazgradljivosti polimernih materialov v vodnem mediju - Metoda z analizo sproščenega ogljikovega dioksida (ISO 14852:2018)
Ta dokument določa metodo za določanje stopnje aerobne biorazgradljivosti polimernih materialov, vključno s takšnimi, ki vsebujejo formulacijske dodatke, z merjenjem količine sproščenega ogljikovega dioksida. Preskusni material je izpostavljen inokulumu iz aktivnega blata, zrelega komposta ali zemljine pod aerobnimi mezofilnimi pogoji v sintetičnem mediju pod standardiziranimi laboratorijskimi pogoji.
Če je kot inokulum uporabljeno neprilagojeno aktivno blato, je mogoče rezultate preskusa uporabiti za dostop do procesov aerobne biorazgraditve, do katerih prihaja v okolju čistilne naprave za odpadno vodo. Če je uporabljen mešan ali znova izpostavljen inokulum, je mogoče metodo uporabiti za ugotavljanje potencialne biorazgradljivosti preskusnega
materiala.
Pogoji, ki so uporabljeni v tem dokumentu, niso nujno enaki idealnim pogojem, pri katerih pride do največje stopnje biorazgradljivosti, vendar je ta metoda zasnovana za merjenje biorazgraditve polimernih materialov in podaja oceno njihove potencialne biorazgradljivosti.
Metoda omogoča izboljšanje ocene biorazgradljivosti z izračunom ravnovesja
ogljika (izbirno, glej dodatek C).
Ta metoda se uporablja za naslednje materiale:
– naravne in/ali sintetične polimere, kopolimere ali njihove mešanice;
– polimerne materiale, ki vsebujejo dodatke, kot so mehčala, barvila ali druge spojine;
– polimere, topne v vodi;
– materiale, ki v preskusnih pogojih ne zavirajo delovanja mikroorganizmov, prisotnih v inokulumu. Zaviralne učinke je mogoče določiti na podlagi spremljanja zaviranja ali z drugo ustrezno metodo (glej npr. standard ISO 8192[1]). Če preskusni material zavira inokulum, se lahko uporabi manjša preskusna koncentracija, drug inokulum ali predhodno izpostavljeni inokulum.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 14852:2018
01-december-2018
1DGRPHãþD
SIST EN ISO 14852:2004
'RORþDQMHNRQþQHDHUREQHELRUD]JUDGOMLYRVWLSROLPHUQLKPDWHULDORYYYRGQHP
PHGLMX0HWRGD]DQDOL]RVSURãþHQHJDRJOMLNRYHJDGLRNVLGD ,62
Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous
medium - Method by analysis of evolved carbon dioxide (ISO 14852:2018)
Bestimmung der vollständigen aeroben Bioabbaubarkeit von Kunststoff-Materialien in
einem wässrigen Medium - Verfahren mittels Analyse des freigesetzten
Kohlenstoffdioxides (ISO 14852:2018)
Évaluation de la biodégradabilité aérobie ultime des matériaux plastiques en milieu
aqueux - Méthode par analyse du dioxyde de carbone libéré (ISO 14852:2018)
Ta slovenski standard je istoveten z: EN ISO 14852:2018
ICS:
83.080.01 Polimerni materiali na Plastics in general
splošno
SIST EN ISO 14852:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 14852:2018
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SIST EN ISO 14852:2018
EN ISO 14852
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2018
EUROPÄISCHE NORM
ICS 83.080.01 Supersedes EN ISO 14852:2004
English Version
Determination of the ultimate aerobic biodegradability of
plastic materials in an aqueous medium - Method by
analysis of evolved carbon dioxide (ISO 14852:2018)
Évaluation de la biodégradabilité aérobie ultime des Bestimmung der vollständigen aeroben
matériaux plastiques en milieu aqueux - Méthode par Bioabbaubarkeit von Kunststoff-Materialien in einem
analyse du dioxyde de carbone libéré (ISO wässrigen Medium - Verfahren mittels Analyse des
14852:2018) freigesetzten Kohlenstoffdioxides (ISO 14852:2018)
This European Standard was approved by CEN on 26 August 2018.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14852:2018 E
worldwide for CEN national Members.
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SIST EN ISO 14852:2018
EN ISO 14852:2018 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 14852:2018
EN ISO 14852:2018 (E)
European foreword
This document (EN ISO 14852:2018) has been prepared by Technical Committee ISO/TC 61 "Plastics"
in collaboration with Technical Committee CEN/TC 249 “Plastics” the secretariat of which is held by
NBN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by April 2019, and conflicting national standards shall be
withdrawn at the latest by April 2019.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 14852:2004.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 14852:2018 has been approved by CEN as EN ISO 14852:2018 without any modification.
3
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SIST EN ISO 14852:2018
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SIST EN ISO 14852:2018
INTERNATIONAL ISO
STANDARD 14852
Second edition
2018-09
Determination of the ultimate aerobic
biodegradability of plastic materials
in an aqueous medium — Method by
analysis of evolved carbon dioxide
Évaluation de la biodégradabilité aérobie ultime des matériaux
plastiques en milieu aqueux — Méthode par analyse du dioxyde de
carbone libéré
Reference number
ISO 14852:2018(E)
©
ISO 2018
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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 2018 – All rights reserved
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Test environment . 4
6 Reagents . 4
7 Apparatus . 6
8 Procedure. 7
8.1 Test material . 7
8.2 Reference material . 7
8.3 Preparation of the inoculum . 7
8.3.1 General. 7
8.3.2 Inoculum from wastewater-treatment plants . 8
8.3.3 Inoculum from soil and/or compost. 8
8.4 Test . 8
9 Calculation and expression of results .10
9.1 Calculation .10
9.1.1 Theoretical amount of carbon dioxide evolved by the test material.10
9.1.2 Percentage biodegradation from CO evolution .10
2
9.2 Expression and interpretation of results .10
10 Validity of results .11
11 Test report .11
Annex A (informative) Principle of a system for measuring evolved carbon dioxide (example) .13
Annex B (informative) Examples of methods for the determination of evolved carbon dioxide .14
Annex C (informative) Example of the determination of a carbon balance .16
Annex D (informative) Example of a determination of the amount of water insoluble
polymer remaining at the end of a biodegradation test and the molecular mass of
the polymer .18
Bibliography .19
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 14, Plastics
and environment.
This second edition cancels and replaces the first edition (ISO 14852:1999), which has been technically
revised. It also incorporates the Technical Corrigendum ISO 14852:1999/Cor.1:2005 and contains the
following changes:
— the validity criteria has been revised to comply with ISO 14855;
— in the introduction, an obsolete, potentially misleading paragraph has been deleted;
— the normative reference clause has been updated;
— the “Terms and definitions” clause has been revised and updated;
— the test methods have been updated for better comprehension.
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 2018 – All rights reserved
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
Introduction
With the increasing use of plastics, their recovery and disposal have become a major issue. As a first
priority, recovery should be promoted. Biodegradable plastics are now emerging as one of the options
available to solve such environmental problems. Plastic materials, such as products or packaging, which
are sent to composting facilities should be potentially biodegradable. Therefore, it is very important to
determine the potential biodegradability of such materials and to obtain an indication of their potential
biodegradability.
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SIST EN ISO 14852:2018
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SIST EN ISO 14852:2018
INTERNATIONAL STANDARD ISO 14852:2018(E)
Determination of the ultimate aerobic biodegradability
of plastic materials in an aqueous medium — Method by
analysis of evolved carbon dioxide
WARNING — Sewage, activated sludge, soil and compost may contain potentially pathogenic
organisms. Therefore, appropriate precautions should be taken when handling them. Toxic test
compounds and those whose properties are unknown should be handled with care.
1 Scope
This document specifies a method, by measuring the amount of carbon dioxide evolved, for the
determination of the degree of aerobic biodegradability of plastic materials, including those containing
formulation additives. The test material is exposed in a synthetic medium under standardized
laboratory conditions to an inoculum from activated sludge, mature compost or soil under aerobic,
mesophilic conditions.
If an unadapted activated sludge is used as the inoculum, the test result can be used to assess the aerobic
biodegradation processes which occur in a waste water treatment plant environment. If a mixed or pre-
exposed inoculum is used, the method can be used to investigate the potential biodegradability of a test
material.
The conditions used in this document do not necessarily correspond to the optimum conditions allowing
maximum biodegradation to occur, but this test method is designed to measure the biodegradation of
plastic materials and give an indication of their potential biodegradability.
The method enables the assessment of the biodegradation to be improved by calculating a carbon
balance (optional, see Annex C).
The method applies to the following materials:
— natural and/or synthetic polymers, copolymers or mixtures thereof;
— plastic materials which contain additives such as plasticizers, colorants or other compounds;
— water-soluble polymers;
— materials which, under the test conditions, do not inhibit the microorganisms present in the
inoculum. Inhibitory effects can be determined using an inhibition control or by another appropriate
[1]
method (see, for example, ISO 8192 ). If the test material is inhibitory to the inoculum, a lower test
concentration, another inoculum or a pre-exposed inoculum can be used.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved
organic carbon (DOC)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
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 https: //www .electropedia .org/
3.1
ultimate aerobic biodegradation
breakdown of an organic compound by microorganisms in the presence of oxygen into carbon dioxide,
water and mineral salts of any other elements present (mineralization) plus new biomass
3.2
activated sludge
mixture of organic materials and biomass produced in the aerobic treatment of waste water by the
growth of bacteria and other microorganisms in the presence of dissolved oxygen
3.3
concentration of suspended solids in an activated sludge
amount of solids obtained by filtration or centrifugation of a known volume of activated sludge and
drying at about 105 °C to constant mass
3.4
dissolved inorganic carbon
DIC
part of the inorganic carbon in water which cannot be removed by specified phase separation
−2
Note 1 to entry: Phase separation can be achieved for example by centrifugation at 40 000 m⋅s for 15 min or by
membrane filtration using membranes with pores of 0,2 µm to 0,45 µm diameter.
3.5
theoretical amount of evolved carbon dioxide
ThCO
2
maximum theoretical amount of carbon dioxide evolved after completely oxidizing a chemical
compound, calculated from the molecular formula
Note 1 to entry: It is expressed as milligrams of carbon dioxide evolved per milligram or gram of test compound.
3.6
total organic carbon
TOC
amount of carbon bound in an organic compound
Note 1 to entry: It is expressed as milligrams of carbon per 100 mg of the compound.
[SOURCE: ISO 17556:2012, 3.14]
3.7
dissolved organic carbon
DOC
part of the organic carbon in water which cannot be removed by specified phase separation
−2
Note 1 to entry: Phase separation can be achieved for example by centrifugation at 40 000 m⋅s for 15 min or by
membrane filtration using membranes with pores of 0,2 µm to 0,45 µm diameter.
3.8
lag phase
time from the start of a test until adaptation and/or selection of the degrading microorganisms is
achieved and the degree of biodegradation of a chemical compound or organic matter has increased to
about 10 % of the maximum level of biodegradation
Note 1 to entry: It is measured in days.
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
3.9
maximum level of biodegradation
degree of biodegradation of a chemical compound or organic matter in a test, above which no further
biodegradation takes place during the test
Note 1 to entry: It is measured in per cent.
3.10
biodegradation phase
time from the end of the lag phase of a test until the plateau phase has been reached
Note 1 to entry: It is measured in days.
3.11
plateau phase
time from the end of the biodegradation phase until the end of a test
Note 1 to entry: It is measured in days.
3.12
pre-exposure
pre-incubation of an inoculum in the presence of the chemical compound or organic matter under test,
with the aim of enhancing the ability of the inoculum to biodegrade the test material by adaptation
and/or selection of the microorganisms
3.13
pre-conditioning
pre-incubation of an inoculum under the conditions of the subsequent test in the absence of the chemical
compound or organic matter under test, with the aim of improving the test by acclimatization of the
microorganisms to the test conditions
3.14
inoculum
microorganisms or other material used in an inoculation
Note 1 to entry: Also called inoculant.
3.15
inoculation
introduction of microorganisms into a culture medium in order to start a biological process
4 Principle
The biodegradability of a plastic material is determined using aerobic, mesophilic microorganisms in
an aqueous system. The test mixture contains an inorganic medium, the organic test material (the sole
source of carbon and energy) with a concentration between 100 mg/l and 2 000 mg/l of organic carbon,
and activated sludge or a suspension of active soil or compost as the inoculum. If higher concentrations
of test material are used then an optimised test medium should be applied.
NOTE Lower concentrations such as between 20 mg/l and 40 mg/l of organic carbon have been tested and
found suitable.
The mixture is agitated in test flasks and aerated with carbon-dioxide-free air over a period of time
depending on the biodegradation kinetics, but not exceeding 6 months. The carbon dioxide evolved
during the microbial degradation is determined by a suitable analytical method, examples of which are
given in Annexes A and B.
The level of biodegradation is determined by comparing the amount of carbon dioxide evolved with
the theoretical amount (ThCO ) and expressed in per cent. The test result is the maximum level of
2
biodegradation, determined from the plateau phase of the biodegradation curve. Optionally, a carbon
balance may be calculated to give additional information on the biodegradation (see Annex C).
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
Unlike ISO 9439, which is used for a variety of organic compounds, this document is specially designed
for the determination of the biodegradation of plastic materials. The special requirements necessary
affect the choice of the inoculum and the test medium, and there is the possibility of improving the
evaluation of the biodegradability by calculating a carbon balance.
5 Test environment
Incubation shall take place in the dark or in diffuse light in an enclosure which is free from vapours
inhibitory to microorganisms and which is maintained at a constant temperature, preferably between
20 °C and 25 °C, to an accuracy of ±1 °C.
6 Reagents
Use only reagents of recognized analytical grade.
6.1 Distilled or deionized water, free of toxic substances (copper in particular) and containing less
than 2 mg/l of DOC.
6.2 Test medium.
Depending on the purpose of the test, different test media may be used. For example, if a test material
is used at higher concentrations, use the optimized test medium (6.2.2) with higher buffering capacity
and nutrient concentrations.
6.2.1 Standard test medium.
6.2.1.1 Solution A.
Dissolve the following in water (6.1) and make up to 1 000 ml.
anhydrous potassium dihydrogen phosphate (KH PO ) 8,5 g
2 4
anhydrous dipotassium hydrogen phosphate (K HPO ) 21,75 g
2 4
disodium hydrogen phosphate dihydrate (Na HPO ⋅ 2H O) 33,4 g
2 4 2
ammonium chloride (NH Cl) 0,5 g
4
The correct composition of the solution can be checked by measuring the pH, which should be 7,4.
6.2.1.2 Solution B.
Dissolve 22,5 g of magnesium sulfate heptahydrate (MgSO ⋅ 7H O) in water (6.1) and make up to
4 2
1 000 ml.
6.2.1.3 Solution C.
Dissolve 36,4 g of calcium chloride dihydrate (CaCl ⋅ 2H O) in water (6.1) and make up to 1 000 ml.
2 2
6.2.1.4 Solution D.
Dissolve 0,25 g of iron(III) chloride hexahydrate (FeCl ⋅ 6H O) in water (6.1) and make up to 1 000 ml.
3 2
Prepare this solution freshly before use to avoid precipitation, or add a drop of concentrated
hydrochloric acid (HCl) or a drop of 0,4 g/l aqueous solution of ethylenediaminetetraacetic acid (EDTA).
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
6.2.1.5 Preparation.
To prepare 1 l of test medium, add the following, to about 500 ml of water (6.1):
— 10 ml of solution A (6.2.1.1);
— 1 ml of each of solutions B (6.2.1.2), C (6.2.1.3), D (6.2.1.4).
Make up to 1 000 ml with water (6.1).
Prepare the test medium freshly before use. The solutions A up to C may be stored up to 6 months in the
dark at room temperature. The same applies for solution D in case HCl or EDTA has been added.
6.2.2 Optimized test medium.
This optimized medium is highly buffered and contains more inorganic nutrients. This is necessary to
keep the pH constant in the system during the test, even at high concentrations of the test material. The
medium contains about 2 400 mg/l of phosphorus and 50 mg/l of nitrogen and is therefore suitable
for concentrations in the test material of up to 2 000 mg/l of organic carbon. If higher or lower test-
material concentrations are used, increase or decrease respectively the nitrogen content to keep the
C:N ratio at about 40:1.
6.2.2.1 Solution E.
Dissolve the following in water (6.1) and make up to 1 000 ml.
anhydrous potassium dihydrogen phosphate (KH PO ) 37,5 g
2 4
disodium hydrogen phosphate dihydrate (Na HPO ⋅ 2H O) 87,3 g
2 4 2
ammonium chloride (NH Cl) 2,0 g
4
6.2.2.2 Solution F (trace-element solution, optional).
Dissolve in 10 ml of aqueous HCl solution (25 %, 7,7 mol/l), in the following sequence:
a) 70 mg of ZnCl ;
2
b) 100 mg of MnCl ⋅ 4H O;
2 2
c) 6 mg of H BO ;
3 3
d) 190 mg of CoCl ⋅ 6H O;
2 2
e) 3 mg of CuCl ⋅ 2H O;
2 2
f) 240 mg of NiCl ⋅ 6H O;
2 2
g) 36 mg of Na MoO ⋅ 2H O;
2 4 2
h) 33 mg of Na WO ⋅ 2H O;
2 4 2
i) 26 mg of Na SeO ⋅ 5H O.
2 3 2
Make up to 1 000 ml with water (6.1).
6.2.2.3 Solution G (vitamin solution, optional).
Dissolve in 100 ml of water (6.1) 0,6 mg of biotine, 2,0 mg of niacinamide, 2,0 mg of p-aminobenzoate,
1,0 mg of panthotenic acid, 10,0 mg of pyridoxal hydrochloride, 5,0 mg of cyanocobalamine, 2,0 mg of
folic acid, 5,0 mg of riboflavin, 5,0 mg of DL-thioctic acid and 1,0 mg of thiamine dichloride or use a
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SIST EN ISO 14852:2018
ISO 14852:2018(E)
solution of 15 mg of yeast extract in 100 ml of water (6.1). Filter the solution for sterilization using
membrane filters (see 7.6).
Solutions E and F are optional and are not required if a sufficient concentration of the inoculum is
used, e.g. activated sludge, soil or compost. It is recommended that 1 ml portions be prepared and kept
refrigerated until use.
6.2.2.4 Preparation.
To prepare 1 litre of test medium, add, to about 800 ml of water (6.1),
— 100 ml of solution E (6.2.2.1), and
— 1 ml of each of solutions B (6.2.1.2), C (6.2.1.3), D (6.2.1.4) and, optionally, F (6.2.2.2) and G (6.2.2.3).
Make up to 1 000 ml with water (6.1) and measure the pH.
The correct composition of the test medium can be checked by measuring the pH, which should be
7,0 ± 0,2.
6.3 Pyrophosphate solution.
Dissolve 2,66 g of sodium pyrophosphate (Na P O ) in water (6.1) and make up to 1 000 ml.
4 2 7
7 Apparatus
7.1 General. Ensure that all glassware is thoroughly cleaned and, in particular, free from organic or
toxic matter.
Required is usual laboratory equipment, plus the following.
7.2 Test flasks: glass vessels (e.g. bottles or conical flasks) designed to allow gas purging and shaking
or stirring, and fitted with tubing impermeable to CO . The vessels shall be located in a c
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