ISO/DTS 25008-2.2

• Abstract:

  This technical specification describes a set of physiological (Part 1) and biochemical (Part 2) parameters allowing to measure sublethal effects in higher plants exposed to soil pollutants. Part 2 is applicable to soils of unknown quality e.g. from contaminated sites, amended soils or soils after remediation either in situ or laboratory exposure assays. For in situ experiments the areas to compare shall be exposed to the same climatic conditions (humidity, temperature, sunlight). This part specifies a toolbox of methods for determining antioxidant defence enzymes activities in leaf homogenates. The method has been developed initially for lettuce but is suitable for monocotyledonous and other dicotyledonous species.

• Status: Not Published
• ICS: 13.080.05 - Examination of soils in general
• ICS: 65.020.20 - Plant growing
• Technical Committee: ISO/TC 190/SC 4 - Biological characterization
• Drafting Committee: ISO/TC 190/SC 4/WG 3 - Effects on soil flora
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 16-Sep-2026
• Completion Date: 16-Sep-2026

Overview

ISO/DTS 25008-2.2: Soil Quality - Responses of Higher Plants to Environmental Stresses - Part 2: Methods to Assess Enzyme Activities of the Antioxidant Defense System is a technical specification developed by ISO/TC 190/SC 4. This standard provides internationally harmonized laboratory and in situ methods for assessing the sublethal effects of soil pollutants and other environmental stressors on higher plants. Focusing on the biochemical parameters of plant leaves, it specifies protocols for measuring the activity of key antioxidant defense enzymes-catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD). These methods offer objective tools for soil quality assessment, particularly for contaminated, amended, or remediated soils.

Key Topics

• Antioxidant Defense Enzyme Activities: The document provides detailed methodologies to quantify the activity of critical antioxidant enzymes in plant leaf homogenates. These enzymes serve as sensitive biomarkers for oxidative stress caused by soil contaminants or adverse environmental conditions.
• Sampling Procedures: Guidance is given for selecting and collecting plant tissue samples, with special recommendations to avoid bias (e.g., harvesting only mature, healthy leaves under similar environmental conditions).
• Applicable Plant Species: Originally developed for lettuce (Lactuca sativa), the methods are broadly suitable for both monocotyledonous and dicotyledonous species, expanding their usability in various ecological and agronomic studies.
• Experimental Flexibility: The standard addresses both laboratory-controlled exposure assays and in situ experiments, making it suitable for field studies, site monitoring, and risk assessments.
• Quality Control and Statistical Analysis: It outlines the need for appropriate controls, reference soils, and statistical treatment of results to ensure reliability and interpretative value.

Applications

• Soil Pollution Assessment: The primary application is the evaluation of soil quality at contaminated, remediated, or amended sites, where direct and indirect effects on plant health need to be monitored.
• Ecotoxicological Studies: Researchers and environmental professionals can use these protocols to detect and monitor sublethal, biochemical-level impacts of pollutants-including metals, persistent organics, and agricultural residues-on plant systems.
• Agricultural and Land Management: The standard supports decision-making for land use, reclamation, and sustainable soil management by providing evidence-based measures of plant stress under varying field conditions.
• Regulatory Compliance: Laboratories and consultants conducting environmental audits and risk assessments will find standardized, reproducible methods that align with international reporting requirements.
• Research and Development: The methods can be integrated into broader phytotoxicity studies, enabling a more holistic understanding of how environmental stressors affect plant physiology in conjunction with other parameters such as germination or growth (referenced in Part 1 of ISO 25008).

Related Standards

For broader context and comprehensive soil quality assessment, the following ISO standards are referenced or recommended:

• ISO/DTS 25008-1: Physiological parameters for measuring plant responses to soil stressors.
• ISO 18763 & ISO 17126: Assessment of pollutant effects on seed germination and seedling emergence.
• ISO 11269-1 & ISO 11269-2: Methods for evaluating root growth and early plant development in response to soil contamination.
• ISO 22030: Chronic toxicity in higher plants.
• ISO 29200 & ISO 21479: Evaluation of genotoxicity and biochemical markers like leaf fatty acid profiles.
• ISO 16198, ISO 10390, ISO 10694, ISO 10930, ISO 11260, ISO 11277, ISO 22190, ISO 16965: Various methods for assessing soil chemistry, structure, and bioavailability of trace elements.

In summary, ISO/DTS 25008-2.2 supports robust, science-based approaches to soil quality monitoring by utilizing enzyme activity as sensitive indicators of plant stress, thereby helping stakeholders maintain healthy terrestrial ecosystems and meet international soil protection standards.

FINAL DRAFT
Technical
Specification
ISO/TC 190/SC 4
Soil quality — Responses of higher
Secretariat: AFNOR
plants to environmental stresses —
Voting begins on:
2026-09-16
Part 2:
Methods to assess the enzyme
Voting terminates on:
2026-11-11
activities of the antioxidant defense
system
Qualité du sol — Réponses des végétaux supérieurs aux
contraintes environnementales —
Partie 2: Méthodes d'évaluation des activités des enzymes du
système de défense antioxydant
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
Technical
Specification
ISO/TC 190/SC 4
Soil quality — Responses of higher
Secretariat: AFNOR
plants to environmental stresses —
Voting begins on:
Part 2:
Methods to assess the enzyme
Voting terminates on:
activities of the antioxidant defense
system
Qualité du sol — Réponses des végétaux supérieurs aux
contraintes environnementales —
Partie 2: Méthodes d'évaluation des activités des enzymes du
système de défense antioxydant
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 2
5.1 Chemicals and solutions .2
5.2 Equipment .3
5.3 Other materials .3
6 Plant and sampling procedures . 4
7 Measurement of enzymes activities involved in the antioxidant defence system . 4
7.1 Sample preparation .4
7.2 Catalase (CAT, EC 1.11.1.6) .5
7.3 Glutathione S-transferase (GST, EC 2.5.1.18) .5
7.4 Superoxide dismutase (SOD, EC 1.15.11) .6
8 Data analysis and interpretation . 7
Annex A (informative) Examples values for enzyme activities described in this document . 8
Bibliography . 9

iii
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization.
A list of all parts in the ISO/TS 25008 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
Introduction
Higher plants are important organisms in terrestrial ecosystems, which are often exposed to different types
of environmental stressors. These stressors induce genetic, metabolic and physiological perturbations
in plants. Within ISO/TC 190/SC4, there are currently a few standardized methods available to assess
[1]
these perturbations, mainly based on developmental traits: germination (ISO 18763 ), emergence of
[2] [3] [4]
seedlings (ISO 17126 ), plant growth (ISO 11269-1 and ISO 11269-2 , root growth and early growth
[5]
respectively), and chronic toxicity (ISO 22030 ). Only two standards address specific endpoints, i.e.
[6] [7]
genotoxicity (ISO 29200 ) or biochemical parameters for leaf fatty acid composition (ISO 21479 ).
In addition to the endpoints considered in these standards, the homeostasis of physiological processes
and oxidative biochemical responses in plants can also become impaired when exposed to stressful
environmental conditions. As a result, the resilience and plasticity of plants can become deeply constrained,
affecting their capacity to adapt, survive and thrive in terrestrial ecosystems.
[8] [9]
The ISO/TS 25008 series includes two parts, describing a set of physiological (ISO/DTS 25008-1 ) and
biochemical (this document) parameters (Figure 1), of which the combined analysis may foster a more
holistic and comprehensive evaluation of the performance of plants exposed to soil pollutants or other
environmental stresses, comparatively to the analysis of isolated parameters.
[8]
Figure 1 — Schematic representation of the parameters covered by the ISO/TS 25008 series
This document determines biochemical markers in plants exposed to environmental stresses inducing
an oxidative stress. This method allows to compare the quality of soils by measuring antioxidant defence
enzymes in leaf homogenates from plant species grown on these soils.

v
FINAL DRAFT Technical Specification ISO/DTS 25008-2.2:2026(en)
Soil quality — Responses of higher plants to environmental
stresses —
Part 2:
Methods to assess the enzyme activities of the antioxidant
defense system
1 Scope
This document describes a set of biochemical parameters allowing the measurement of sublethal effects in
higher plants exposed to soil pollutants and other stressors (e.g. drought, nutrient availability, heat stress).
This document is applicable to soils of unknown quality (e.g. from contaminated sites, amended soils or soils
after remediation), following laboratory exposure assays. This document specifies methods for measuring
antioxidant defence enzyme activities that indicate stress symptoms in the leaves of lettuce and higher
plants, either monocotyledonous or dicotyledonous species.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 11074, Soil quality — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 11074 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
This document specifies methods for the measurement of enzyme activity (catalase, CAT, EC 1.11.1.6;
glutathione-S-transferase, GST, EC 2.5.1.18 and superoxide dismutase, SOD, EC 1.15.11) in plant
homogenates. The measurement of these enzyme activities is based on the use of solutions of colorimetric or
spectrophotometric substrates, which are incubated during specific times and temperatures.
After the incubation, the reactions are stopped, and, according to the enzymatic activity considered,
the intensities of absorbance are measured with quartz cuvettes or microplate with a UV-visible
spectrophotometer.
5 Reagents and materials
5.1 Chemicals and solutions
5.1.1 Water.
Deionized water (i.e: ultra pure water, total organic carbon < 10 µg/l and conductivity < 18,2 mOhm) can be
used for solution preparation.
5.1.2 Phosphate buffer 200 mmol/l:
1)
— Na HPO (CAS Registry Number® : 7558-79-4 – 141,96 g/mol): 28,4 g per liter deionized water;
2 4
— NaH PO . H O (CAS N°: 10049-21-5 – 137,99 g/mol): 27,6 g per liter deionized water.
2 4 2
Mix both solutions according to desired pH (see 5.1.3 5.1.4).
Store the phosphate buffer for less than 1 month at 4°C.
5.1.3 Phosphate buffer 100 mmol/l, EDTA-Na 1 mmol/l, pH 7,2.
EDTA C H N O Na ∙ 2H O (CAS 6381-92-6 – 372,2 g/mol): 930,5 mg EDTA in 50 ml deionized water.
10 14 2 8 2 2
Mix 72 ml of Na HPO solution (200 mmol/l) with 28 ml NaH PO ∙ H O solution (200 mmol/l) and 4 ml
2 4 2 4 2
EDTA-Na (0,05 mol/l) solution.
Adjust final volume (200 ml) with deionized water.
Store the phosphate buffer for less than 1 month at 4°C.
5.1.4 Phosphate buffer 50 mmol/l, pH 7,0.
Mix 30,5 ml of Na HPO solution (200 mmol/l) with 19,5 ml NaH PO ∙ H O solution (200 mmol/l).
2 4 2 4 2
Adjust final volume (200 ml) with deionized water.
Prepare the phosphate buffer just before use.
5.1.5 Hydrogen peroxide (H O ) 15 mmol/l.
2 2
Mix 172 µl H O 30% (weight fraction, CAS 7722-84-1) with 100 ml phosphate buffer 50 mmol/l, pH 7,0.
2 2
Prepare the solution just before use and maintain it at 25°C .
5.1.6 Chloro-2,4-dinitrobenzene (CDNB) 100 mmol/l.
Dissolve 101,25 mg CDNB (CAS 97-00-7 – 202,5 g/mol) in 5 ml ethanol 96% (CAS 41340-36-7).
Prepare aliquots of 150 µl solutions in 1,5 ml microcentrifuge tubes and store them at 4°C.
5.1.7 Reduced glutathione (GSH) 200 mmol/l.
Dissolve 30,73 mg GSH (CAS 70-18-8 – 307,32 g/mol) in 0,5 ml phosphate buffer 100 mmol/l, EDTA-Na 1
mol/l, pH 7,2.
Keep the solution on ice before use.
1) Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
p
...

ISO/TC 190/SC 4
Secretariat: AFNOR
Date: 2026-07-1609-01
Soil quality — Responses of higher plants to environmental
stresses —
Part 2:
Methods to assess of the enzyme activities of the antioxidant defense
system
Qualité du sol — Réponses des végétaux supérieurs aux contraintes environnementales —
Partie 2: Méthodes d'évaluation des activités des enzymes du système de défense antioxydant
VVVVoVVooooottttinttiiiiing bng bng bng bng bg beeeeeeggigigigigiinnsnsnsnsnss o o o o o on:n:n:n:n:n: 202 202 202 202 202 202666666------050505050505------010101010101
VoVVoVoVoVootinttintintintining tggggg t t t t teerererererrminminminminminminatatatatatateseseseseses o o o o o onnnnnn::::::   222222000000222222666666------000000666666------262626262626

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
E-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 1
5.1 Chemicals and solutions . 1
5.2 Equipment . 3
5.3 Other materials . 3
6 Plant and sampling procedures . 4
7 Measurement of enzymes activities involved in the antioxidant defence system . 4
7.1 Sample preparation . 4
7.2 Catalase (CAT, EC 1.11.1.6) . 5
7.3 Glutathione S-transferase (GST, EC 2.5.1.18) . 5
7.4 Superoxide dismutase (SOD, EC 1.15.11) . 6
8 Data analysis and interpretation . 7
Annex A (informative) Examples values for enzyme activities described in this document. 8
Bibliography . 9

iii
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization.
A list of all parts in the ISO/TS 25008 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
Introduction
Higher plants are important organisms in terrestrial ecosystems, which are often exposed to different types
of environmental stressors. These stressors induce genetic, metabolic and physiological perturbations in
plants. Within ISO/TC 190/SC4, there are currently a few standardized methods available to assess these
[1]
perturbations, mainly based on developmental traits: germination (ISO 18763 ),), emergence of seedlings
[2] [3] [4]
(ISO 17126 ),), plant growth (ISO 11269-1 and ISO 11269-2 ,, root growth and early growth
[5]
respectively), and chronic toxicity (ISO 22030 ).). Only two standards address specific endpoints, i.e.
[6] [7]
genotoxicity (ISO 29200 )) or biochemical parameters for leaf fatty acid composition (ISO 21479 ).).
In addition to the endpoints considered in these standards, the homeostasis of physiological processes and
oxidative biochemical responses in plants can also become impaired when exposed to stressful environmental
conditions. As a result, the resilience and plasticity of plants can become deeply constrained, affecting their
capacity to adapt, survive and thrive in terrestrial ecosystems.
[8] [9]
The ISO/TS 25008 series includes two parts, describing a set of physiological (ISO/DTS 25008-1 )) and
biochemical (this document) parameters (Figure 1), of which the combined analysis may foster a more holistic
and comprehensive evaluation of the performance of plants exposed to soil pollutants or other environmental
stresses, comparatively to the analysis of isolated parameters.

[8]
Figure 1 — Schematic representation of the parameters covered by the ISO/TS 25008 series
This document determines biochemical markers in plants exposed to environmental stresses inducing an
oxidative stress. This method allows to compare the quality of soils by measuring antioxidant defence enzymes
in leaf homogenates from plant species grown on these soils.
v
Soil quality — Responses of higher plants to environmental stresses —
Part 2:
Methods to assess of the enzyme activities of the antioxidant defense
system
1 Scope
This document describes a set of biochemical parameters allowing the measurement of sublethal effects in
higher plants exposed to soil pollutants and other stressors (e.g. drought, nutrient availability, heat stress).
This document is applicable to soils of unknown quality (e.g. from contaminated sites, amended soils or soils
after remediation), following laboratory exposure assays. This document specifies methods for measuring
antioxidant defence enzyme activities that indicate stress symptoms in the leaves of lettuce and higher plants,
either monocotyledonous or dicotyledonous species.
2 Normative references
There are no normative references in this document.
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 11074, Soil quality — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 11074 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
This document specifies methods for the measurement of enzyme activity (catalase, CAT, EC 1.11.1.6;
glutathione-S-transferase, GST, EC 2.5.1.18 and superoxide dismutase, SOD, EC 1.15.11) in plant homogenates.
The measurement of these enzyme activities is based on the use of solutions of colorimetric or
spectrophotometric substrates, which are incubated during specific times and temperatures.
After the incubation, the reactions are stopped, and, according to the enzymatic activity considered, the
intensities of absorbance are measured with quartz cuvettes or microplate with a UV-visible
spectrophotometer.
5 Reagents and materials
5.1 Chemicals and solutions
5.1.1 Water.
Deionized water (i.e: ultra pure water, total organic carbon < 10 µg/l and conductivity < 18,2 mOhm) can be
used for solution preparation.
5.1.2 Phosphate buffer 200 mmol/l:
1)
— Na HPO (CAS Registry Number® : 7558-79-4 – 141,96 g/mol): 28,4 g per liter deionized water;
2 4
— NaH PO . H O (CAS N°: 10049-21-5 – 137,99 g/mol): 27,6 g per liter deionized water.
2 4 2
Mix both solutions according to desired pH (see 5.1.3 5.1.4).
Store the phosphate buffer for less than 1 month at 4°C.
5.1.3 Phosphate buffer 100 mmol/l, EDTA-Na 1 mmol/l, pH 7,2.
EDTA C H N O Na ∙ 2H O (CAS 6381-92-6 – 372,2 g/mol): 930,5 mg EDTA in 50 ml deionized water.
10 14 2 8 2 2
Mix 72 ml of Na HPO solution (200 mmol/l) with 28 ml NaH PO ∙ H O solution (200 mmol/l) and 4 ml
2 4 2 4 2
EDTA-Na (0,05 mol/l) solution.
Adjust final volume (200 ml) with deionized water.
Store the phosphate buffer for less than 1 month at 4°C.
5.1.4 Phosphate buffer 50 mmol/l, pH 7,0.
Mix 30,5 ml of Na HPO solution (200 mmol/l) with 19,5 ml NaH PO ∙ H O solution (200 mmol/l).
2 4 2 4 2
Adjust final volume (200 ml) with deionized water.
Prepare the phosphate buffer just before use.
5.1.5 Hydrogen peroxide (H O ) 15 mmol/l.
2 2
Mix 172 µl H O 30% (weight fraction, CAS 7722-84-1) with 100 ml phosphate buffer 50 mmol/l, pH 7,0.
2 2
Prepare the solution just before use and maintain it at 25°C .
5.1.6 Chloro-2,4-dinitrobenzene (CDNB) 100 mmol/l.
Dissolve 101,25 mg CDNB (CAS 97-00-7 – 202,5 g/mol) in 5 ml ethanol 96% (CAS 41340-36-7).
Prepare aliquots of 150 µl solutions in 1,5 ml microcentrifuge tubes and store them at 4°C.
5.1.7 Reduced glutathione (GSH) 200 mmol/l.
Dissolve 30,73 mg GSH (CAS 70-18-8 – 307,32 g/mol) in 0,5 ml phosphate buffer 100 mmol/l, EDTA-Na 1
mol/l, pH 7,2.
Keep the solution on ice before use.

1)
Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
product named. Equivalent products may be used if they can be shown to lead to the same results.

5.1.8 Glutathion-S-transferase (GST).
Mix 25 U GST (CAS 50812-37-8) with 1 ml deionized water.
Prepare aliquots of 10 µl solutions in 1,5 ml microcentrifuge tubes and store them at –80°C.
5.1.9 DL methionine 200 mmol/l.
Dissolve 298 mg methionine (CAS 59-51-8 – 149,21 g/mol) in 10 ml deionized water preheated at 60°C, in a
15 ml centrifuge tube.
5.1.10 (–) Riboflavin 1 mmol/l.
Dissolve 37,64 mg riboflavin (CAS 83-88-5 – 376,36 g/mol) in 100 ml deionized water in a 100 ml glass flask.
Keep the solution away from light before use.
5.1.11 Nitrotetrazolium Blue chloride (NBT) 17 mmol/l.
Dissolve 14,1 mg Nitrotetrazolium Blue chloride (CAS 298-83-9 – 817,64 g/mol) in 10 ml deionized water, in
a 15 ml centrifuge tube.
Keep the solution away from light before use.
5.1.12 Triton X-100 1 % (w/v).
Add 100 mg of Trit
...

FINAL DRAFT
Technical
Specification
ISO/DTS 25008-2
ISO/TC 190/SC 4
Soil quality — Responses of higher
Secretariat: AFNOR
plants to environmental stresses —
Voting begins on:
2026-05-01
Part 2:
Methods to assess enzyme activities
Voting terminates on:
2026-06-26
of antioxidant defence system
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/DTS 25008-2:2026(en) © ISO 2026

FINAL DRAFT
ISO/DTS 25008-2:2026(en)
Technical
Specification
ISO/DTS 25008-2
ISO/TC 190/SC 4
Soil quality — Responses of higher
Secretariat: AFNOR
plants to environmental stresses —
Voting begins on:
Part 2:
Methods to assess enzyme activities
Voting terminates on:
of antioxidant defence system
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/DTS 25008-2:2026(en) © ISO 2026

ii
ISO/DTS 25008-2:2026(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 2
5.1 Chemicals and solutions .2
5.2 Equipment .3
5.3 Other materials .3
6 Plant and sampling procedures . 4
7 Measurement of enzymes activities involved in the antioxidant defence system . 4
7.1 Sample preparation .4
7.2 Catalase (CAT, EC 1.11.1.6) .5
7.3 Glutathione S-transferase (GST, EC 2.5.1.18) .5
7.4 Superoxide dismutase (SOD, EC 1.15.11) .6
8 Data analysis and interpretation . 7
Annex A (informative) Examples values for enzyme activities described in this document . 8
Bibliography . 9

iii
ISO/DTS 25008-2:2026(en)
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization.
A list of all parts in the ISO/TS 25008 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/DTS 25008-2:2026(en)
Introduction
Higher plants are important organisms in terrestrial ecosystems, which are often exposed to different types
of environmental stressors. These stressors induce genetic, metabolic and physiological perturbations
in plants. Within ISO/TC 190/SC4, there are currently a few standardized methods available to assess
[1]
these perturbations, mainly based on developmental traits: germination (ISO 18763 ), emergence of
[2] [3] [4]
seedlings (ISO 17126 ), plant growth (ISO 11269-1 and ISO 11269-2 , root growth and early growth
[5]
respectively), and chronic toxicity (ISO 22030 ). Only two standards address specific endpoints, i.e.
[6] [7]
genotoxicity (ISO 29200 ) or biochemical parameters for leaf fatty acid composition (ISO 21479 ).
In addition to the endpoints considered in these standards, the homeostasis of physiological processes
and oxidative biochemical responses in plants can also become impaired when exposed to stressful
environmental conditions. As a result, the resilience and plasticity of plants can become deeply constrained,
affecting their capacity to adapt, survive and thrive in terrestrial ecosystems.
[8] [9]
The ISO/TS 25008 series includes two parts, describing a set of physiological (ISO/DTS 25008-1 ) and
biochemical (this document) parameters (Figure 1), of which the combined analysis may foster a more
holistic and comprehensive evaluation of the performance of plants exposed to soil pollutants or other
environmental stresses, comparatively to the analysis of isolated parameters.
[8]
Figure 1 — Schematic representation of the parameters covered by the ISO/TS 25008 series
This document determines biochemical markers in plants exposed to environmental stresses inducing
an oxidative stress. This method allows to compare the quality of soils by measuring antioxidant defence
enzymes in leaf homogenates from plant species grown on these soils.

v
FINAL DRAFT Technical Specification ISO/DTS 25008-2:2026(en)
Soil quality — Responses of higher plants to environmental
stresses —
Part 2:
Methods to assess enzyme activities of antioxidant defence
system
1 Scope
This document describes a set of biochemical parameters allowing the measurement of sublethal effects in
higher plants exposed to soil pollutants and other stressors (e.g. drought, nutrient availability, heat stress).
This document is applicable to soils of unknown quality (e.g. from contaminated sites, amended soils or soils
after remediation), following laboratory exposure assays. This document specifies methods for measuring
antioxidant defence enzyme activities that indicate stress symptoms in the leaves of lettuce and higher
plants, either monocotyledonous or dicotyledonous species.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
This document specifies methods for the measurement of enzyme activity (catalase, CAT, EC 1.11.1.6;
glutathione-S-transferase, GST, EC 2.5.1.18 and superoxide dismutase, SOD, EC 1.15.11) in plant
homogenates. The measurement of these enzyme activities is based on the use of solutions of colorimetric or
spectrophotometric substrates, which are incubated during specific times and temperatures.
After the incubation, the reactions are stopped, and, according to the enzymatic activity considered,
the intensities of absorbance are measured with quartz cuvettes or microplate with a UV-visible
spectrophotometer.
ISO/DTS 25008-2:2026(en)
5 Reagents and materials
5.1 Chemicals and solutions
5.1.1 Water.
Deionized water (i.e: ultra pure water, total organic carbon < 10 µg/l and conductivity < 18,2 mOhm) can be
used for solution preparation.
5.1.2 Phosphate buffer 200 mmol/l:
1)
— Na HPO (CAS Registry Number® : 7558-79-4 – 141,96 g/mol): 28,4 g per liter deionized water;
2 4
— NaH PO . H O (CAS N°: 10049-21-5 – 137,99 g/mol): 27,6 g per liter deionized water.
2 4 2
Mix both solutions according to desired pH (see 5.1.3 5.1.4).
Store the phosphate buffer for less than 1 month at 4°C.
5.1.3 Phosphate buffer 100 mmol/l, EDTA-Na 1 mmol/l, pH 7,2.
EDTA C H N O Na ∙ 2H O (CAS 6381-92-6 – 372,2 g/mol): 930,5 mg EDTA in 50 ml deionized water.
10 14 2 8 2 2
Mix 72 ml of Na HPO solution (200 mmol/l) with 28 ml NaH PO ∙ H O solution (200 mmol/l) and 4 ml
2 4 2 4 2
EDTA-Na (0,05 mol/l) solution.
Adjust final volume (200 ml) with deionized water.
Store the phosphate buffer for less than 1 month at 4°C.
5.1.4 Phosphate buffer 50 mmol/l, pH 7,0.
Mix 30,5 ml of Na HPO solution (200 mmol/l) with 19,5 ml NaH PO ∙ H O solution (200 mmol/l).
2 4 2 4 2
Adjust final volume (200 ml) with deionized water.
Prepare the phosphate buffer just before use.
5.1.5 Hydrogen peroxide (H O ) 15 mmol/l.
2 2
Mix 172 µl H O 30% (weight fraction, CAS 7722-84-1) with 100 ml phosphate buffer 50 mmol/l, pH 7,0.
2 2
Prepare the solution just before use and maintain it at 25°C .
5.1.6 Chloro-2,4-dinitrobenzene (CDNB) 100 mmol/l.
Dissolve 101,25 mg CDNB (CAS 97-00-7 – 202,5 g/mol) in 5 ml ethanol 96% (CAS 41340-36-7).
Prepare aliquots of 150 µl solutions in 1,5 ml microcentrifuge tubes and store them at 4°C.
5.1.7 Reduced glutathione (GSH) 200 mmol/l.
Dissolve 30,73 mg GSH (CAS 70-18-8 – 307,32 g/mol) in 0,5 ml phosphate buffer 100 mmol/l, EDTA-Na 1
mol/l, pH 7,2.
Keep the solution on ice before use.
1) Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
product named. Equivalent products may be used if they can be shown to lead to the same results.

ISO/DTS 25008-2:2026(en)
5.1.8 Glutathion-S-transferase (GST).
Mix 25 U GST (CAS 50812-37-8) with 1 ml deionized water.
Prepare aliquots of 10 µl solutions in 1,5 ml microcentr
...

ISO/DTS 25008-2
ISO/TC 190/SC 4
ISO/CD TS 25008-2(en)
Secretariat: AFNOR
Date: 2026-04-17
Soil quality — Responses of higher plants to environmental
stresses —
Part 2:
Methods to assess enzyme activities of antioxidant defence system

ISO/CD TSDTS 25008-2:2026(en)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
E-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO/CD TSDTS 25008-2:2026(en)
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents and materials . 1
5.1 Chemicals and solutions . 1
5.2 Equipment . 3
5.3 Other materials . 3
6 Plant and sampling procedures . 4
7 Measurement of enzymes activities involved in the antioxidant defence system . 4
7.1 Sample preparation . 4
7.2 Catalase (CAT, EC 1.11.1.6) . 5
7.3 Glutathione S-transferase (GST, EC 2.5.1.18) . 5
7.4 Superoxide dismutase (SOD, EC 1.15.11) . 6
8 Data analysis and interpretation . 7
Annex A (informative) Examples values for enzyme activities described in this document. 9
Bibliography . 10

iii
ISO/CD TSDTS 25008-2:2026(en)
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 drawnISO draws attention to the possibility that some of the elementsimplementation of this
document may beinvolve the subjectuse of (a) patent(s). ISO takes no position concerning the evidence,
validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this
document, ISO had not received notice of (a) patent(s) which may be required to implement this document.
However, implementers are cautioned that this may not represent the latest information, which may be
obtained from the patent database available at www.iso.org/patents. 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 ).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation onof the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO'sISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT)), see the following URL:
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil Qualityquality, Subcommittee SC 4,
Biological characterization.
A list of all parts in the ISO/TS 25008 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/CD TSDTS 25008-2:2026(en)
Introduction
Higher plants are important organisms in terrestrial ecosystems, which are often exposed to different types
of environmental stressors. These stressors induce genetic, metabolic and physiological perturbations in
plants. Within ISO TC190/TC 190/SC4, there are currently a few standardized methods available to assess
these perturbations, mainly based on developmental traits: germination (ISO 18763 ,), emergence of seedlings
(ISO 17126 ,), plant growth (ISO 11269-1 and ISO 11269-2 (, root growth and early growth respectively), and
chronic toxicity (ISO 22030 .). Only two standards address specific endpoints, i.e. genotoxicity (ISO 29200) or
biochemical parameters for leaf fatty acid composition (ISO 21479.).
In addition to the endpoints considered in these standards, the homeostasis of physiological processes and
oxidative biochemical responses in plants can also become impaired when exposed to stressful environmental
conditions also become deeply impaired. As a result, the resilience and plasticity of plants can become deeply
constrained and affect, affecting their capacity to adapt, survive and thrive in terrestrial ecosystems.
The ISO/TS 25008ISO/TS 25008 series includes two parts, describing a set of physiological (ISO/DTS 25008-
1(Part 1)) and biochemical (Part 2this document) parameters (Figure 1), of which the combined analysis may
foster a more holistic and comprehensive evaluation of the performance of plants exposed to soil pollutants
or other environmental stresses, comparatively to the analysis of isolated parameters.
v
ISO/CD TSDTS 25008-2:2026(en)
Figure 1— Schematic representation of the parameters covered by the ISO/TS 25008Part 1 and Part 2
of ISO/TS 25008 series
vi
ISO/CD TSDTS 25008-2:2026(en)
This document intends to determinedetermines biochemical markers in plants exposed to environmental
stresses inducing an oxidative stress. This method allows to compare the quality of soils by measuring
antioxidant defence enzymes in leaf homogenates from plant species grown on these soils.
vii
ISO/CD TSDTS 25008-2:2026(en)
Soil quality — Responses of higher plants to environmental stresses —
Part 2:
Methods to assess enzyme activities of antioxidant defence system
1 Scope
This document describes a set of biochemical parameters allowing the measurement of sublethal effects in
higher plants exposed to soil pollutants and other stressors (e.g. drought, nutrient availability, heat stress).
This document is applicable to soils of unknown quality (e.g. from contaminated sites, amended soils or soils
after remediation), following laboratory exposure assays. This partdocument specifies methods for measuring
antioxidant defence enzyme activities that indicatesindicate stress symptoms in the leaves of lettuce and
higher plants, either monocotyledonous or dicotyledonous species.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminologicalterminology databases for use in standardization at the following
addresses:
IEC Electropedia: available at — ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
This document specifies methods for the measurement of enzyme activity (catalase, CAT, EC 1.11.1.6;
glutathione-S-transferase, GST, EC 2.5.1.18 and superoxide dismutase, SOD, EC 1.15.11) in plant homogenates.
The measurement of these enzyme activities is based on the use of solutions of colorimetric or
spectrophotometric substrates, which are incubated during specific times and temperatures.
After the incubation, the reactions are stopped, and, according to the enzymatic activity considered, the
intensities of absorbance are measured with quartz cuvettes or microplate with a UV/-visible
spectrophotometer.
5 Reagents and materials
5.1 Chemicals and solutions
5.1.1 Water.
Deionized water (i.e: ultra pure water, total organic carbon < 10 µg/l and conductivity < 18,2 mOhm) can be
used for solution preparation.
5.1.2 Phosphate buffer 200 mmol/l:
ISO/CD TSDTS 25008-2:2026(en)
1)
— - Na HPO (CAS N°:Registry Number® : 7558-79-4 -– 141,96 g/mol): 28,4 g per liter deionized water;
2 4
— - NaH PO . H O (CAS N°: 10049-21-5 -– 137,99 g/mol): 27,6 g per liter deionized water.
2 4 2
Mix both solutions according to desiratedesired pH (see 5.1.35.1.4)).
Store the phosphate buffer for less than 1 month at 4°C.
5.1.3 Phosphate buffer 100 mmol/l, EDTA-Na 1 mmol/l, pH 7,2.
- EDTA C H N O Na .∙ 2H O (CAS N°: 6381-92-6 -– 372,2 g/mol): 930,5 mg EDTA in 50 ml deionized water.
10 14 2 8 2 2
Mix 72 ml of Na HPO solution (200 mmol/l) with 28 ml NaH PO .∙ H O solution (200 mmol/l) and 4 ml
2 4 2 4 2
EDTA-Na (0,05 mol/l) solution.
Adjust final volume (200ml200 ml) with deionized water.
Store the phosphate buffer for less than 1 month at 4°C.
5.1.4 Phosphate buffer 50 mmol/l, pH 7,0.
Mix 30,5 ml of Na HPO solution (200mmol200 mmol/l) with 19,5 ml NaH PO .∙ H O solution
2 4 2 4 2
(200mmol200 mmol/l).
Adjust final volume (200ml200 ml) with deionized water.
Prepare the phosphate buffer just before use.
5.1.5 Hydrogen peroxide (H O ) 15 mmol/l.
2 2
Mix 172 µl H O 30% (w/wweight fraction, CAS N°: 7722-84-1) with 100 ml phosphate buffer 50 mmol/l, pH
2 2
7,0.
Prepare the solution just before use and maintain it at 25°C .
5.1.6 Chloro-2,4-dinitrobenzene (CDNB) 100 mmol/l.
Dissolve 101,25 mg CDNB (CAS N°: 97-00-7 – 202,5 g/mol) in 5 ml ethanol 96% (CAS N°: 41340-36-7).
Prepare aliquots of 150 µl solutions in 1,5 ml microcentrifuge tubes and store them at 4°C.
5.1.7 Reduced glutathione (GSH) 200 mmol/l.
Dissolve 30,73 mg GSH (CAS N°: 70-18-8 – 307,32 g/mol) in 0,5 ml phosphate buffer 100 mmol/l, EDTA-Na
1 mol/l, pH 7,2.
Keep the solution on ice before use.
5.1.8 Glutathion-S-Transferasetransferase (GST)).
Mix 25 U GST (CAS N°: 50812-37-8) with 1 ml deionized water.

1)
Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the
product named. Equivalent products may be used if they can be shown to lead to the same results.

ISO/CD TSDTS 25008-2:2026(en)
Prepare aliquots of 10 µl solutions in 1,5 ml microcentrifuge tubes and store them at -–80°C.
5.1.9 DL Methioninemethionine 200 mmol/l.
Dissolve 298 mg methionine (CAS N°: 59-51-8 – 149,21 g/mol) in 10 ml deionized water preheated at 60°C, in
a 15 ml centrifuge tube.
5.1.10 (-)(–) Riboflavin 1 mmol/l.
Dissolve 37,64 mg riboflavin (CAS N°: 83-88-5 – 376,36 g/mol) in 100 ml deionized water in a 100 ml glass
flask.
Keep the solution away from light before use.
5.1.11 Nitrotetrazolium Blue chloride (NBT) 17 mmol/l.
Dissolve 14,1 mg Nitrotetrazolium Blue chloride (CAS N°: 298-83-9 – 817,64 g/mol) in 10 ml deionized water,
in a 15 ml centrifuge tube.
Keep the solution away from light before use.
5.1.12 Triton X-100 1 % (w/v)).
Add 100 mg of Triton X-100 solution (CAS N°: 9002-93-1) in 10 ml deionized water in a 15 ml centrifuge tube.
5.2 Equipment
5.2.1 Balance (± 0,001 g)).
5.2.2 Ice machine.
5.2.3 Mechanical homogenizer Ultra Turrax.
5.2.4 Thermostatic water bath at 25°C.
5.2.5 Refrigerated centrifuge at 4°C and speed at 9000 g9 000g, adaptable for microcentrifuge and
centrifuge tubes.
5.2.6 Cuvette and plate reading spectrophotometer UV-Vis for reading absorbances at (240 ± 5) nm,
(340 ± 5) nm and (560 nm ± 5) nm.
5.3 Other materials
5.3.1 Potter-Elvehjem homogenizer.
5.3.2 Bottles of 100 ml in opaque glass.
5.3.3 Microcentrifuge tubes of 1,5 ml in natural polypropylene.
5.3.4 Centrifuge tubes of 15 ml in natural polypropylene.
5.3.5 Quartz cuvettes of 1 ml.
5.3.6 96-well UV mi
...