Soil quality - Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions (ISO 14239:2017)

ISO 14239:2017 specifies six suitable incubation systems for measuring the rates and extent of mineralization of organic compounds in soil by measurement of carbon dioxide (CO2) evolution. All incubation systems are applicable to soluble or insoluble compounds but choice of system depends on the overall purposes of the study.
ISO 14239:2017 does not apply to the use of such systems for material balance studies, which are often test-substance specific.

Bodenbeschaffenheit - Laboratoriumsinkubationssysteme zur Bestimmung der Mineralisierung von organischen Chemikalien im Boden unter aeroben Bedingungen (ISO 14239:2017)

Dieses Dokument legt sechs geeignete Inkubationssysteme für die Bestimmung der Rate und des Umfangs der Mineralisierung organischer Verbindungen im Boden durch Messung der Kohlenstoffdioxidentwicklung (CO2) fest. Alle Inkubationssysteme sind für lösliche oder unlösliche Verbindungen verwendbar, jedoch hängt die Wahl des Systems vom Zweck der Untersuchung ab.
Dieses Dokument gilt nicht für die Anwendung solcher Systeme für Materialbilanzuntersuchungen, die oft prüfsubstanzspezifisch sind.

Qualité du sol - Systèmes d'incubation de laboratoire destinés à la mesure de la minéralisation de produits chimiques organiques dans le sol en conditions aérobies (ISO 14239:2017)

ISO 14239:2017 définit six systèmes d'incubation appropriés permettant de mesurer les vitesses et l'étendue de la minéralisation de composés organiques dans le sol par mesurage du dégagement de dioxyde de carbone (CO2). Tous les systèmes d'incubation peuvent être utilisés avec des composés solubles ou insolubles, mais le choix du système dépend des objectifs globaux de l'étude.
ISO 14239:2017 ne s'applique pas à l'utilisation desdits systèmes pour les bilans de masse, qui sont souvent spécifiques de la substance d'essai.

Kakovost tal - Laboratorijski inkubacijski sistemi za merjenje mineralizacije organskih spojin v tleh pri aerobnih pogojih (ISO 14239:2017)

General Information

Status
Published
Public Enquiry End Date
02-Feb-2020
Publication Date
23-Sep-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Aug-2020
Due Date
22-Oct-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 14239:2020
01-november-2020
Nadomešča:
SIST ISO 14239:2001
Kakovost tal - Laboratorijski inkubacijski sistemi za merjenje mineralizacije
organskih spojin v tleh pri aerobnih pogojih (ISO 14239:2017)

Soil quality - Laboratory incubation systems for measuring the mineralization of organic

chemicals in soil under aerobic conditions (ISO 14239:2017)
Bodenbeschaffenheit - Laboratoriumsinkubationssysteme zur Bestimmung der
Mineralisierung von organischen Chemikalien im Boden unter aeroben Bedingungen
(ISO 14239:2017)
Qualité du sol - Systèmes d'incubation de laboratoire destinés à la mesure de la

minéralisation de produits chimiques organiques dans le sol en conditions aérobies (ISO

14239:2017)
Ta slovenski standard je istoveten z: EN ISO 14239:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 14239:2020 en,fr,de

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

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SIST EN ISO 14239:2020
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SIST EN ISO 14239:2020
EN ISO 14239
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Laboratory incubation systems for measuring
the mineralization of organic chemicals in soil under
aerobic conditions (ISO 14239:2017)
Qualité du sol - Systèmes d'incubation de laboratoire Bodenbeschaffenheit -

destinés à la mesure de la minéralisation de produits Laboratoriumsinkubationssysteme zur Bestimmung

chimiques organiques dans le sol en conditions der Mineralisierung von organischen Chemikalien im

aérobies (ISO 14239:2017) Boden unter aeroben Bedingungen (ISO 14239:2017)
This European Standard was approved by CEN on 13 April 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14239:2020 E

worldwide for CEN national Members.
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SIST EN ISO 14239:2020
EN ISO 14239:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 14239:2020
EN ISO 14239:2020 (E)
European foreword

The text of ISO 14239:2017 has been prepared by TTechnical Committee ISO/TC 190 "Soil quality” of

the International Organization for Standardization (ISO) and has been taken over as EN ISO 14239:2020

by Technical Committee CEN/TC 444 “Environmental characterization of solid matrices” the secretariat

of which is held by NEN.

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 October 2020, and conflicting national standards shall

be withdrawn at the latest by October 2020.

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.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 14239:2017 has been approved by CEN as EN ISO 14239:2020 without any modification.

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SIST EN ISO 14239:2020
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SIST EN ISO 14239:2020
INTERNATIONAL ISO
STANDARD 14239
Second edition
2017-07
Soil quality — Laboratory incubation
systems for measuring the
mineralization of organic chemicals in
soil under aerobic conditions
Qualité du sol — Systèmes d’incubation de laboratoire destinés à la
mesure de la minéralisation de produits chimiques organiques dans le
sol en conditions aérobies
Reference number
ISO 14239:2017(E)
ISO 2017
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

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
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Methods ......................................................................................................................................................................................................................... 1

4.1 General requirements ....................................................................................................................................................................... 1

4.1.1 Soil collection and characterization ................................................................................................................ 2

4.1.2 Test material ........................................................................................................................................................................ 2

4.1.3 Incubation conditions ........................................................................................................................................... ....... 2

4.2 Choice of incubation systems ..................................................................................................................................................... 2

4.3 Flow-through system ......................................................................................................................................................................... 4

4.3.1 Principle .................................................................................................................................................................................. 4

4.3.2 Materials and reagents ............................................................................................................................................... 5

4.3.3 Apparatus and glassware ......................................................................................................................................... 6

4.3.4 Procedure ............................................................................................................................................................................... 6

4.4 Soda-lime column system .............................................................................................................................................................. 7

4.4.1 Principle .................................................................................................................................................................................. 7

4.4.2 Materials and reagents ............................................................................................................................................... 8

4.4.3 Apparatus and glassware ......................................................................................................................................... 9

4.4.4 Procedure ............................................................................................................................................................................... 9

4.5 Biometer system .................................................................................................................................................................................12

4.5.1 Principle ...............................................................................................................................................................................12

4.5.2 Materials and reagents ............................................................................................................................................13

4.5.3 Apparatus and glassware ......................................................................................................................................14

4.5.4 Procedure ............................................................................................................................................................................14

4.6 Radiorespirometer ...........................................................................................................................................................................14

4.6.1 Principle ...............................................................................................................................................................................14

4.6.2 Materials and reagents ............................................................................................................................................15

4.6.3 Apparatus, glass- and plastic-ware ...............................................................................................................15

4.6.4 Procedure ............................................................................................................................................................................15

4.7 Microradiorespirometer ..............................................................................................................................................................16

4.7.1 Principle ...............................................................................................................................................................................16

4.7.2 Materials and reagents ............................................................................................................................................16

4.7.3 Apparatus, and plastic-ware. ..............................................................................................................................16

4.7.4 Procedure ............................................................................................................................................................................17

4.8 Miniaturized respirometer ........................................................................................................................................................17

4.8.1 Principle ...............................................................................................................................................................................17

4.8.2 Materials and reagents ............................................................................................................................................18

4.8.3 Apparatus, and plastic-ware ...............................................................................................................................18

4.8.4 Procedure ............................................................................................................................................................................19

5 Calculation and expression of results ..........................................................................................................................................19

5.1 For unlabelled test materials ...................................................................................................................................................19

5.2 For C-labelled test materials ...............................................................................................................................................20

6 Test report ................................................................................................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................21

© ISO 2017 – All rights reserved iii
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the voluntary nature of standards, on the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the WTO

principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary

information

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological methods.

This second edition cancels and replaces the first edition (ISO 14239:1997), which has been technically

revised. The main changes are the inclusion of two additional incubation systems.

iv © ISO 2017 – All rights reserved
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Introduction

This document describes incubation systems for determining the mineralization of organic compounds

in soil under aerobic conditions.

Mineralization is only one of the parameters which can be used to assess the biodegradation of organic

compounds in soil. If mineralization is not extensive, this does not necessarily mean that the test

material is not biodegradable. Material balance studies to assess the production of metabolites, in

addition to mineralization studies, provide a comprehensive assessment of biodegradation.

It is essential that this document be used in conjunction with ISO 11266, which gives general guidance

on the information needed to assess the potential of an organic compound to be degraded in soil.

Depending on the aim of the study, it is feasible to use a range of incubation conditions, described below,

and different methods of analysis.
© ISO 2017 – All rights reserved v
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SIST EN ISO 14239:2020
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SIST EN ISO 14239:2020
INTERNATIONAL STANDARD ISO 14239:2017(E)
Soil quality — Laboratory incubation systems for
measuring the mineralization of organic chemicals in soil
under aerobic conditions

WARNING — The methods in this document use several materials of a hazardous nature. Due

care is necessary in their handling and disposal. In particular, all pertinent national regulations

should be complied with.
1 Scope

This document specifies six suitable incubation systems for measuring the rates and extent of

mineralization of organic compounds in soil by measurement of carbon dioxide (CO ) evolution. All

incubation systems are applicable to soluble or insoluble compounds but choice of system depends on

the overall purposes of the study.

This document does not apply to the use of such systems for material balance studies, which are often

test-substance specific.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 11266, Soil quality — Guidance on laboratory testing for biodegradation of organic chemicals in soil

under aerobic conditions

ISO 11269-2:2012, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects

of contaminated soil on the emergence and early growth of higher plants

ISO 11274, Soil quality — Determination of the water-retention characteristic — Laboratory methods

ISO 18400-206, Soil quality — Sampling — Part 206: Guidance on the collection, handling and storage of

soil for the assessment of biological functional and structural endpoints in the laboratory

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at http:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 Methods
4.1 General requirements

The following procedures shall be followed, whichever incubation system is selected.

1) Under preparation. Stage at the time of publication: ISO/DIS 18400-206:2017.
© ISO 2017 – All rights reserved 1
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
4.1.1 Soil collection and characterization

Soil shall be collected and handled in accordance with ISO 18400-206 . The soil shall be characterized

in accordance with ISO 11266.
4.1.2 Test material
The test material shall be characterized in accordance with ISO 11266.
4.1.3 Incubation conditions

The following conditions shall be used unless there is a specific reason for using different conditions:—

Temperature: (20 ± 2) °C

— Pore water pressure of soil: −0,01 MPa to −0,03 MPa (measured to ±5 %) as determined in

with ISO 11274 (or between 40 % and 60 % max. water holding capacity (WHC measured to ±5 %)

accordance
in accordance with ISO 11269-2:2012, Annex A)
— Incubation: in the dark

The incubation conditions should be reported in the test report. If they differ from those above, the

reasons for changing them should also be reported in the test report.

A temperature of (20 ± 2) °C has been chosen as a standard for comparative purposes and because it

gives relatively rapid results. Temperatures outside this range can be used if they are more appropriate

(for example, because of local conditions, lack of cooling equipment).
4.2 Choice of incubation systems
One of the six systems described in this document shall be used:
— the flow-through system (4.3);
— the soda-lime column system (4.4);
— the biometer system (4.5);
— the radiorespirometer (4.6);
— the microradiorespirometer (4.7);
— the miniaturized respirometer (4.8).

Data on the mineralization of organic chemicals can most reliably be obtained from experiments with

radiolabelled compounds.

Recoveries of CO in the six systems can be measured using known quantities of unlabelled or

C-labelled calcium carbonate and adding sufficient hydrochloric acid to dissolve fully the calcium

carbonate.

The main advantages and disadvantages of the systems are described in Table 1 below.

2 © ISO 2017 – All rights reserved
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Table 1 — Advantages and disadvantages of the incubation systems
Device Advantages Disadvantages

flow-through system — sufficient oxygen for long-term, aero- — difficulties with complete recoveries

bic degradation studies; when volatile C-compounds are under
investigation;
— uses standard laboratory glassware;
— sensitivity to leaks in the system.
— allows measurement of unlabelled CO
(titration), CO (scintillation counting),
and/or C-labelled volatile products
(scintillation counting).

soda-lime column — free access of oxygen for long-term — CO trapped in soda lime has to be

system degradation studies; released and re-adsorbed in liquid for
scintillation counting;
— uses standard laboratory glassware;
— water content of soils has to be adjust-
requires little space;
ed at least once per month.
— adaptable without changes for use
with standing or shaken aerobic sedi-
ments, pure cultures of microorganisms,
algae or plant cell cultures;
— problem-free incubation under vari-
ous environmental conditions;
— full recoveries of applied radioactivity
in short- or long-term material balance
studies.
biometer system — requires little space; — not ideal for long-term incubations
due to lack of free access of air and re-
— adaptable without changes for use with
duction of partial pressure of oxygen in
standing cultures of aerobic sediments;
chamber during incubation;
— pure cultures of microorganisms or
— requires special glassware.
algae;
— problem-free incubation under var-
ious environmental conditions; ease of
measurement of non-radioactive CO
(titration), CO (scintillation counting
or C-labelled volatile products (scintil-
lation counting).
© ISO 2017 – All rights reserved 3
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Table 1 (continued)
Device Advantages Disadvantages

radiorespirometer — use of standard laboratory glassware; — NaOH traps have to be regularly

replaced by new ones (to avoid their
— easy to set up;
saturation);
— requires little space;
— water content of soil has to be adjust-
— adaptable to standing or shaken ed at least once every two weeks.
aerobic sediments or pure cultures of
microorganisms;
— good recovery of applied radioactivity
for mass balance.

microradiorespirom- — use of 24-wells microplate; — not ideal for long term incubation;

eter
— easy to set up; — not enough soils C mass balance;
— requires very little space; — need to have from five to ten biological
repeats to take into account the variabil-
— relatively high throughput analysis.
ity of the measure due to the relatively
small amount of soil analyzed;
14
— difficult CO counting using phos-
phorimager or classical autoradiography.

miniaturized — no need for C-labeled radiolabeled — need the use of micro-GC to measure

respirometer compound; CO production and of GC-IRMS to esti-
mate its isotopic signature;
— suitable to estimate the mineraliza-
tion of different kinds of C-labelled — not ideal for long term incubation
substrates in small soil samples; because of the lack of oxygen due to the
incubation of soil in an air-tight device
— allows analysis of functional and
molecular characteristics on the same
micro-samples.
4.3 Flow-through system
4.3.1 Principle

This method allows determination of the dissipation and/or metabolism of non-radioactive or

C-labelled test materials in soil. CO free air is drawn through the incubation vessel containing the

treated soil samples. The CO and organic volatiles evolved from the soil are trapped in a series of

absorption traps (see Figure 1).
4 © ISO 2017 – All rights reserved
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SIST EN ISO 14239:2020
ISO 14239:2017(E)
Key
1 flow-through monitor 8 sample
2 valve for maintaining a slight pressure 9 incubation chamber
3 reservoir 10 pump
4 wash bottle 11 collector
5 incubation unit 12 valve for flow-through regulation
6 valve 13 absorption traps
7 distribution board Gas supply.
Figure 1 — Example of flow-through incubation system
4.3.2 Materials and reagents
Reagents of recognized analytical grade shall be used.

4.3.2.1 Source of CO-free air (e.g. obtained by passing air through an aqueous solution of strong

alkali). For studies with C-labelled compounds, CO need not be removed from the air unless there is a

danger of saturation of the CO traps.

4.3.2.2 Ethylene glycol or ethylene glycol methyl ester, for absorption of organic volatiles.

© ISO 2017 – All rights reserved 5
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SIST EN ISO 14239:2020
ISO 14239:2017(E)

4.3.2.3 Polyurethane foam trap, density 16 kg/m for absorption of organic volatiles.

4.3.2.4 Sulfuric acid, c(H SO ) = 0,5 mol/l, for absorption of alkaline volatiles.

2 4

4.3.2.5 Sodium or potassium hydroxide solution, c(KOH) [or (NaOH)] = 0,1 mol/l to 0,5 mol/l for

14 2)
absorption of nonradioactive CO ; or scintillation cocktail for absorption of CO
2 2 .

WARNING — If the scintillation cocktail is used as a trap, volatile organic amines and solvents

can accumulate in toxic concentrations and there is danger of explosion. Therefore it is essential

that the work area is well ventilated.
4.3.2.6 Scintillation cocktails for determination of the CO in alkali traps .
4.3.3 Apparatus and glassware
4.3.3.1 Liquid scintillation counter.
4.3.3.2 Scintillation vials.
4.3.3.3 Temperature-controlled incubator or room (±2° C).
4.3.3.4 Membrane pump (capacity, approximately 2,8 m /h).
4.3.3.5 Flow meter.
4.3.3.6 Flow-restrictor valves.
4.3.3.7 Glass dishes for system I, e.g. moist soil (equivalent to 50 g dry mass)
— diameter 5 cm, height 5 cm – for samples equivalent to 50 g air-dried soil
— diameter 9,5 cm, height 5 cm – for samples equivalent to 300 g air-dried soil
4.3.3.8 Erlenmeyer flask (250 ml) for system II.
4.3.3.9 Gas washing bottles (100 ml) for absorption traps.
4.3.3.10 Gas washing bottles (200 ml to 500 ml) for moistening the air.
4.3.4 Procedure

Choose incubation system I or II described in 4.3.4.1 or 4.3.4.2. System I is more applicable when many

samples shall be incubated in limited space, system II requires more space but is applicable for small-

scale experiments.

2) Carbosorb (Canberra Packard) and Oxisolve (Zinsser) are examples of suitable products available

commercially. This information is given for the convenience of users of this International Standard and does not

constitute an endorsement by ISO of these products.

3) Hionic fluor and Optifluor (Canberra Packard) are examples of suitable products availab

...

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