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

ISO/DTS 25008-2.2 is an ISO technical specification for soil quality testing based on higher-plant responses to environmental stress. It specifies laboratory and in situ methods for measuring antioxidant defence enzyme activities in leaf homogenates, mainly catalase (CAT), glutathione S-transferase (GST) and superoxide dismutase (SOD), so users can compare soils of unknown quality and look for sublethal plant effects.

What does ISO/DTS 25008-2.2 specify?

ISO/DTS 25008-2.2 covers biochemical indicators for plants exposed to soil pollutants and other stressors such as drought, nutrient availability and heat stress. It is intended for soils of unknown quality, including contaminated sites, amended soils and remediated soils.

The document is organized into scope, references, terms and definitions, principle, reagents and materials, plant and sampling procedures, measurement of enzyme activities, and data analysis and interpretation. Annex A provides example enzyme activity values.

For in situ work, the areas being compared are to be exposed to the same climatic conditions, which keeps humidity, temperature and sunlight from distorting the comparison. The methods were developed for lettuce but are stated to be suitable for monocotyledonous and other dicotyledonous species.

What are the key requirements of ISO/DTS 25008-2.2?

Sampling and sample handling

Clause 6 places strong emphasis on comparable leaf sampling. Leaves should be fresh, green and of similar size, and the same leaf part should be taken across individuals when only part of a leaf is sampled.

For controlled-condition studies, the document uses repeated plant replicates and fresh processing at each sampling time. For field and in situ studies, reference and affected areas are compared, and multiple individuals per species are sampled so the plant response reflects the site rather than one unusual plant.

Preparation and quality control

Clause 7.1 requires fresh leaf tissue to be homogenized in chilled phosphate buffer with EDTA, centrifuged, and analyzed from the supernatant without delay. The work is carried out in triplicate, and result reliability is checked with the relative standard deviation (RSD).

That matters in practice because these enzyme activities can change quickly after harvest. Cold handling, immediate analysis and replicate measurements reduce avoidable variation and make results easier to defend in reporting.

Enzyme-specific methods

Clause 7.2 measures CAT by the fall in absorbance after reaction with hydrogen peroxide in quartz cuvettes. Clause 7.3 measures GST by the rise in absorbance in a UV microplate assay using glutathione and CDNB, with GST used as a positive control.

Clause 7.4 measures SOD by inhibition of photochemical NBT reduction under light in the presence of riboflavin. In practice, the three assays give different views of oxidative stress, so a laboratory can choose one marker or combine several markers depending on the study goal.

Expression and interpretation

Clause 8 requires enzyme activity to be expressed per gram of fresh sample using the prescribed corrections for blank, dilution and sample mass. It also recommends statistical comparison of treatments with a t test for two groups or one-way ANOVA for several groups.

The document also advises using reference soils or reference areas and negative controls. That matters because enzyme activity varies with plant species, genotype, leaf position, developmental stage and stress duration, so results should be interpreted against a local control.

What terms does ISO/DTS 25008-2.2 define?

• Catalase (CAT) - an antioxidant enzyme measured here by how fast it reduces hydrogen peroxide in the assay.
• Glutathione S-transferase (GST) - an enzyme measured with GSH and CDNB to track biochemical stress responses.
• Superoxide dismutase (SOD) - an antioxidant enzyme measured by how strongly it suppresses photochemical NBT reduction.
• Relative standard deviation (RSD) - a repeatability check used on triplicate results to judge analytical consistency.
• UV-visible spectrophotometer (UV-Vis) - the instrument used to read absorbance changes in cuvettes or microplates.
• Leaf homogenate - the buffered, centrifuged leaf extract that supplies the enzyme activity for measurement.

Who uses ISO/DTS 25008-2.2?

ISO/DTS 25008-2.2 is used by soil and environmental laboratories, plant physiology and ecotoxicology teams, remediation specialists, and risk assessors. It is relevant where users need to compare contaminated, amended or remediated soils, or track plant stress in laboratory or field studies.

It is also useful for agricultural and site-specific assessments where one crop species or a representative set of plants is sampled. In practice, users apply it to sample planning, enzyme testing, data comparison and interpretation of plant stress responses.

Which standards are used with ISO/DTS 25008-2.2?

• ISO 11074 - provides the soil quality vocabulary used for the terms and definitions section.
• ISO/DTS 25008-1 - the companion part in the same series, covering physiological parameters that can be combined with the biochemical markers in this document.
• EN ISO 16198 - supports plant-based assessment of the bioavailability of trace elements.
• ISO 10390, ISO 10694, ISO 10930, ISO 11260, ISO 11277, ISO 22190 and ISO 16965 - provide supporting soil and element-characterization methods that help relate enzyme responses to soil chemistry and contamination.

What does the ISO/DTS 25008-2.2 document contain?

The document contains reagent recipes, equipment lists and practical sampling guidance for leaf-based enzyme testing. It then gives separate assay set-ups for CAT, GST and SOD, including control/sample arrangements and calculation formulas.

It also contains the data-analysis section that points users toward statistical comparison and combined interpretation with other measurements. Annex A supplies non-normative example activity values from literature for lettuce grown on different uncontaminated soils.

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
...