EN ISO 23753-1:2019

Soil quality - Determination of dehydrogenases activity in soils - Part 1: Method using triphenyltetrazolium chloride (TTC) (ISO 23753-1:2019)

This document specifies a method for determining the activity of dehydrogenases enzymes in soil using 2,3,5-triphenyltetrazolium chloride (TTC).

Bodenbeschaffenheit - Bestimmung der Dehydrogenaseaktivität in Böden - Teil 1: Verfahren mit Triphenyltetrazoliumchlorid (TTC) (ISO 23753-1:2019)

Dieses Dokument legt ein Verfahren zur Bestimmung der Aktivität von Dehydrogenaseenzymen im Boden mit 2,3,5 Triphenyltetrazoliumchlorid (TTC) fest.

Qualité du sol - Détermination de l'activité des déshydrogénases dans les sols - Partie 1: Méthode au chlorure de triphényltétrazolium (CTT) (ISO 23753-1:2019)

Le présent document spécifie une méthode de détermination de l'activité des enzymes déshydrogénases dans le sol à l'aide de chlorure de 2,3,5-triphényltétrazolium (CTT).

Kakovost tal - Določevanje aktivnosti dehidrogenaze v tleh - 1. del: Metoda s trifeniltetrazolijevim kloridom (TTC) (ISO 23753-1:2019)

Ta dokument določa metodo za določevanje aktivnosti dehidrogenaze encimov v tleh z uporabo
2,3,5-trifeniltetrazolijevega klorida (TTC).

General Information

• Status: Published
• Publication Date: 19-Mar-2019
• Withdrawal Date: 29-Sep-2019
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 20-Mar-2019
• Completion Date: 20-Mar-2019
• Ref Project: SIST EN ISO 23753-1:2019 - Soil quality - Determination of dehydrogenases activity in soils - Part 1: Method using triphenyltetrazolium chloride (TTC) (ISO 23753-1:2019)

Relations

• Replaces: EN ISO 23753-1:2011 - Soil Quality - Determination of dehydrogenase activity in soil - Part 1: Method using triphenyltetrazolium chloride (TTC) (ISO 23753-1:2005)
• Effective Date: 27-Mar-2019
• Amended By: EN ISO 23753-1:2019/A1:2020 - Soil quality - Determination of dehydrogenases activity in soils - Part 1: Method using triphenyltetrazolium chloride (TTC) - Amendment 1 (ISO 23753-1:2019/Amd 1:2020)
• Effective Date: 13-Nov-2019

Overview

EN ISO 23753-1:2019 (CEN adoption of ISO 23753-1:2019) specifies a standardized laboratory method for measuring dehydrogenase activity in soils using 2,3,5‑triphenyltetrazolium chloride (TTC). Dehydrogenases are intracellular microbial enzymes linked to respiratory activity; their activity is used as an indicator of viable, physiologically active soil microbial communities and overall soil quality.

Key topics and technical requirements

• Principle: TTC is added to a soil sample; microbially mediated reduction produces triphenylformazan (TPF), which is extracted with ethanol and quantified by spectrophotometry at 485 nm.
• Core conditions:
  • Incubation at 25 °C ± 1 °C for 6 h.
  • Substrate (TTC) prepared in Tris buffer (100 mmol/l, pH 7.6); recommended TTC concentration in the procedure is noted (e.g., 300 mmol/l preparation, 10%).
  • Extraction solvent: analytical grade ethanol (96 %) is specified; other solvents (e.g., acetone) are noted as alternatives.
• Reagents and calibration:
  • Preparation of Tris buffer (100 mmol/l, pH 7.6), TTC substrate solution, and TPF stock/working solutions for standard curves.
  • Establishment of a TPF standard curve for quantification and validity checks.
• Sampling and handling:
  • Follow ISO 18400‑206 for soil collection and handling.
  • If sieved fresh samples cannot be analysed immediately, storage up to 7 days at 4 °C ± 2 °C is acceptable.
  • Dry matter determination per ISO 11465.
• Limitations and validity:
  • Abiotic reducers (e.g., Fe(II), sulfides), coal or carbonaceous materials, or very organic-rich substrates can bias results; mitigation checks (spiking with TPF and recovery) are recommended.
  • Storage time affects enzyme activity - samples with different storage times should not be compared.

Applications and users

This TTC-based soil enzyme assay is used for:

• Environmental and agricultural soil quality monitoring
• Research on soil microbial activity, biogeochemical cycles and soil management impacts
• Assessing effects of contamination, remediation and land-use changes Primary users include environmental laboratories, academic researchers, agronomists, soil ecologists, and regulatory agencies involved in soil health and ecosystem assessments.

Related standards

• ISO 11465 - Determination of dry matter and water content (gravimetric)
• ISO 18400‑206 - Sampling: collection, handling and storage for microbiological assessment
• EN ISO 23753‑1:2019 supersedes the previous edition (EN ISO 23753‑1:2011)

Keywords: soil quality, dehydrogenase activity, TTC method, triphenyltetrazolium chloride, soil enzyme assay, ISO 23753-1, microbial activity assay.

Standards Content (Sample)

SLOVENSKI STANDARD
01-julij-2019
Nadomešča:
SIST EN ISO 23753-1:2011
Kakovost tal - Določevanje aktivnosti dehidrogenaze v tleh - 1. del: Metoda s
trifeniltetrazolijevim kloridom (TTC) (ISO 23753-1:2019)
Soil quality - Determination of dehydrogenases activity in soils - Part 1: Method using
triphenyltetrazolium chloride (TTC) (ISO 23753-1:2019)
Bodenbeschaffenheit - Bestimmung der Dehydrogenaseaktivität in Böden - Teil 1:
Verfahren mit Triphenyltetrazoliumchlorid (TTC) (ISO 23753-1:2019)
Qualité du sol - Détermination de l'activité des déshydrogénases dans les sols - Partie 1:
Méthode au chlorure de triphényltétrazolium (CTT) (ISO 23753-1:2019)
Ta slovenski standard je istoveten z: EN ISO 23753-1:2019
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 23753-1
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2019
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 23753-1:2011
English Version
Soil quality - Determination of dehydrogenases activity in
soils - Part 1: Method using triphenyltetrazolium chloride
(TTC) (ISO 23753-1:2019)
Qualité du sol - Détermination de l'activité des Bodenbeschaffenheit - Bestimmung der
déshydrogénases dans les sols - Partie 1: Méthode au Dehydrogenaseaktivität in Böden - Teil 1: Verfahren
chlorure de triphényltétrazolium (CTT) (ISO 23753- mit Triphenyltetrazoliumchlorid (TTC) (ISO 23753-
1:2019) 1:2019)
This European Standard was approved by CEN on 4 February 2019.

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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23753-1:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23753-1:2019) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “Test methods for 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 September 2019, and conflicting national standards
shall be withdrawn at the latest by September 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 23753-1:2011.
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 23753-1:2019 has been approved by CEN as EN ISO 23753-1:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 23753-1
Second edition
2019-02
Soil quality — Determination of
dehydrogenases activity in soils —
Part 1:
Method using triphenyltetrazolium
chloride (TTC)
Qualité du sol — Détermination de l'activité des déshydrogénases
dans les sols —
Partie 1: Méthode au chlorure de triphényltétrazolium (CTT)
Reference number
ISO 23753-1:2019(E)
©
ISO 2019
ISO 23753-1:2019(E)
© ISO 2019
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
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Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 23753-1:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Limitations . 1
6 Reagents and materials . 2
7 Apparatus . 3
8 Procedure. 3
8.1 Establishment of standard curve . 3
8.2 Sampling . 3
9 Calculation . 4
10 Validity criteria . 4
10.1 Standard curve . 4
10.2 Samples . 5
11 Test report . 5
Annex A (informative) Results of modified parameters . 6
Bibliography . 9
ISO 23753-1:2019(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
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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).
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expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
This second edition cancels and replaces the first edition (ISO 23753-1:2005), which has been
technically revised. The main changes compared to the previous edition are as follows:
— a new Clause 5 “Limitations” has been added;
— in Clause 6, reagents and their preparation have been updated to new results [e.g. use of less toxic
solvent (ethanol), substrate concentration of 60 mmol/l of TTC, concentration of Tris buffer of
100 mmol/l at pH 7,6, incubation time of 6 h];
— Tables 1 and 2 have been added;
— Clause 10 "Validity criteria" has been added;
— a new Annex A “Results of modified parameters” has been added;
— Clause 2 “Normative references” and the Bibliography have been updated.
A list of all the parts in the ISO 23753 series can be found on the ISO website.
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 2019 – All rights reserved

ISO 23753-1:2019(E)
Introduction
The soil microflora is responsible for the decomposition and conversion of organic substances, carbon,
nitrogen, sulfur and phosphorus cycles, soil aggregates stability and as a food source for microbivores.
Dehydrogenases, as intracellular enzymes and respiratory chain components of the microbial cells, play
a major role in the production of energy by organisms. They oxidize organic compounds by transferring
...