SIST EN ISO 14239:2020
(Main)Soil quality - Laboratory incubation systems for measuring the mineralization of organic chemicals in soil under aerobic conditions (ISO 14239:2017)
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
Relations
Standards Content (Sample)
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.
---------------------- Page: 1 ----------------------
SIST EN ISO 14239:2020
---------------------- Page: 2 ----------------------
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.
---------------------- Page: 3 ----------------------
SIST EN ISO 14239:2020
EN ISO 14239:2020 (E)
Contents Page
European foreword . 3
2
---------------------- Page: 4 ----------------------
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.
3
---------------------- Page: 5 ----------------------
SIST EN ISO 14239:2020
---------------------- Page: 6 ----------------------
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
---------------------- Page: 7 ----------------------
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
Ch. de Blandonnet 8 • 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
---------------------- Page: 8 ----------------------
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
14
5.2 For C-labelled test materials .20
6 Test report .20
Bibliography .21
© ISO 2017 – All rights reserved iii
---------------------- Page: 9 ----------------------
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
---------------------- Page: 10 ----------------------
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
---------------------- Page: 11 ----------------------
SIST EN ISO 14239:2020
---------------------- Page: 12 ----------------------
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
2
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
1)
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
---------------------- Page: 13 ----------------------
SIST EN ISO 14239:2020
ISO 14239:2017(E)
4.1.1 Soil collection and characterization
)
1
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
2
14
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
---------------------- Page: 14 ----------------------
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
14
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
2
14
(titration), CO (scintillation counting),
2
14
and/or C-labelled volatile products
(scintillation counting).
14
soda-lime column — free access of oxygen for long-term — CO trapped in soda lime has to be
2
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
2
14
(titration), CO (scintillation counting
2
14
or C-labelled volatile products (scintil-
lation counting).
© ISO 2017 – All rights reserved 3
---------------------- Page: 15 ----------------------
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
14
— 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-
2
phorimager or classical autoradiography.
14
miniaturized — no need for C-labeled radiolabeled — need the use of micro-GC to measure
13
respirometer compound; CO production and of GC-IRMS to esti-
2
mate its isotopic signature;
— suitable to estimate the mineraliza-
13
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
14
C-labelled test materials in soil. CO free air is drawn through the incubation vessel containing the
2
treated soil samples. The CO and organic volatiles evolved from the soil are trapped in a series of
2
absorption traps (see Figure 1).
4 © ISO 2017 – All rights reserved
---------------------- Page: 16 ----------------------
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
a
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
2
14
alkali). For studies with C-labelled compounds, CO need not be removed from the air unless there is a
2
danger of saturation of the CO traps.
2
4.3.2.2 Ethylene glycol or ethylene glycol methyl ester, for absorption of organic volatiles.
© ISO 2017 – All rights reserved 5
---------------------- Page: 17 ----------------------
SIST EN ISO 14239:2020
ISO 14239:2017(E)
3
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.
3)
14
4.3.2.6 Scintillation cocktails for determination of the CO in alkali traps .
2
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).
3
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
...
SLOVENSKI STANDARD
oSIST prEN ISO 14239:2020
01-januar-2020
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)
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: prEN ISO 14239
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 14239:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 14239:2020
---------------------- Page: 2 ----------------------
oSIST prEN 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
---------------------- Page: 3 ----------------------
oSIST prEN 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
Ch. de Blandonnet 8 • 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
---------------------- Page: 4 ----------------------
oSIST prEN 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
14
5.2 For C-labelled test materials .20
6 Test report .20
Bibliography .21
© ISO 2017 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN 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
---------------------- Page: 6 ----------------------
oSIST prEN 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
---------------------- Page: 7 ----------------------
oSIST prEN ISO 14239:2020
---------------------- Page: 8 ----------------------
oSIST prEN 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
2
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
1)
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
---------------------- Page: 9 ----------------------
oSIST prEN ISO 14239:2020
ISO 14239:2017(E)
4.1.1 Soil collection and characterization
)
1
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
2
14
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
---------------------- Page: 10 ----------------------
oSIST prEN 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
14
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
2
14
(titration), CO (scintillation counting),
2
14
and/or C-labelled volatile products
(scintillation counting).
14
soda-lime column — free access of oxygen for long-term — CO trapped in soda lime has to be
2
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
2
14
(titration), CO (scintillation counting
2
14
or C-labelled volatile products (scintil-
lation counting).
© ISO 2017 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST prEN 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
14
— 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-
2
phorimager or classical autoradiography.
14
miniaturized — no need for C-labeled radiolabeled — need the use of micro-GC to measure
13
respirometer compound; CO production and of GC-IRMS to esti-
2
mate its isotopic signature;
— suitable to estimate the mineraliza-
13
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
14
C-labelled test materials in soil. CO free air is drawn through the incubation vessel containing the
2
treated soil samples. The CO and organic volatiles evolved from the soil are trapped in a series of
2
absorption traps (see Figure 1).
4 © ISO 2017 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST prEN 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
a
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
2
14
alkali). For studies with C-labelled compounds, CO need not be removed from the air unless there is a
2
danger of saturation of the CO traps.
2
4.3.2.2 Ethylene glycol or ethylene glycol methyl ester, for absorption of organic volatiles.
© ISO 2017 – All rights reserved 5
---------------------- Page: 13 ----------------------
oSIST prEN ISO 14239:2020
ISO 14239:2017(E)
3
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.
3)
14
4.3.2.6 Scintillation cocktails for determination of the CO in alkali traps .
2
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).
3
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 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.
6 © ISO 2017 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST prEN ISO 14239:2020
ISO 14239:2017(E)
4.3.4.1 Incubation system I
Incubation of soil samples shall take place in temperature controlled incubators or rooms (4.3.3.3.). Set
up cylindrical, separately removable incubation units in the chamber (see Figure 1). The incubation
units shall contain sets of soil samples in glass dishes (4.3.3.7) (normally one incubation set consists of
6 sub-samples). Each incubation unit can be aerated separately.
In order to ensure aerobic conditions, bring a constant stream of CO -free air (4.3.2.1) through each
2
incubation unit using a membrane pump (4.3.3.4).
4.3.4.2 Incubation system II
Incubate the soil sample in a glass flask (e.g. Erlenmeyer flask) (4.3.3.8) in a temperature controlled
room or incubator (4.3.3.3). Bring a constant stream of CO -free air (4.3.2.1) through the flask.
2
4.3.4.3 Absorption of volatile products
For both systems, moisten the CO -free air passing over the soils by bubbling it through two gas wash
2
bottles (4.3.3.1) about half-filled with acidified, deionized water (approximately 1 ml of concentrated
sulfuric acid per litre of water). Distribute the water saturated air to the different incubation units via
valves (4.3.3.6).
Establish a constant flow of approximately 0,1 l/min through each incubation unit; use a flow meter
(4.3.3.5) to measure the flow rates.
For both systems, bubble the outgoing gas through an absorption system to capture volatilized parent
compound, volatile metabolite, and CO for subsequent analyses. All connections shall be made of
2
14
stainless steel or polytetrafluoroethylene (PTFE) tubing. Quantify any C-labelled compounds by
liquid scintillation counting, as appropriate.
The absorption systems consist of:
— one gas washing bottle (4.3.3.9) filled with reagent for absorption of organic volatiles (4.3.2.2 or
4.3.2.3);
— one gas washing bottle (4.3.3.9) filled with reagent for absorption of alkaline volatiles (4.3.2.4) (if
necessary);
— one gas washing bottle for absorption of CO (4.3.2.5). If high rates of CO production are expected,
2 2
a second CO trap is needed.
2
4.4 Soda-lime column system
4.4.1 Principle
14
This system allows determination of the dissipation and/or metabolism of C-labelled test materials
14
in soil. Soil treated with the C-labelled test materials is held in a flask with a ground-glass jointed
neck into which a ground-glass jointed glass column has been inserted (see Figure 2).The glass column
14 14
contains a trap for volatilized C-labelled materials and a trap for CO . Oxygen and atmospheric
2
gases other than CO move freely into and out of the flask by diffusion.
2
© ISO 2017 – All rights reserved 7
---------------------- Page: 15 ----------------------
oSIST prEN ISO 14239:2020
ISO 14239:2017(E)
Key
1 flask with 29/32 neck 6 soda lime
2 glass reflux column with 29/32 joint 7 oil- soaked glass wool plug
14
3 soda lime 8 trap for CO
2
14
4 glass wool plug 9 soil plus C-supplement
5 trap for atmospheric CO
2
Figure 2 — Incubation vessel for aerobic soil metabolism
NOTE ln addition to use with soils, the system has also been used for aerobic degradation studies with
standing or shaken sediments, pure cultures of microorganisms, algae and plant cell cultures.
4.4.2 Materials and reagents
Reagents of recognized analytical grade shall be used.
4.4.2.1 Granulated soda lime, grain size 1,5 mm to 3 mm, containing a saturation indicator.
4.4.2.2 Glass wool.
4.4.2.3 Paraffin oil solution in hexane (volume fraction of 2 %), for soaking glass wool plugs with oil.
4.4.2.4 Hydrochloric acid (HCl) (volume fraction of ~18 %), for dissolution of soda lime granules.
8 © ISO 2017 – All rights reserved
---------------------- Page: 16 ----------------------
oSIST prEN ISO 14239:2020
ISO 14239:2017(E)
4.4.2.5 CO-absorbing solution; e.g. 1 mol/l NaOH (see Figure 3) or other suitable CO trapping
2 2
4)
solutions (see Figure 4).
4.4.2.6 Scintillation cocktail, suitable for mixing with NaOH (if applicable).
4.4.3 Appa
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.