Soil quality - Measurement of enzyme activity patterns in soil samples using colorimetric substrates in micro-well plates (ISO 20130:2018)

This document specifies a method for the measurement of several hydrolase activities (arylamidase, arylsulfatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, acid, alkaline and global phosphatases, urease) simultaneously (or not) in soil samples, using colorimetric substrates. Enzyme activities of soil vary seasonally and depend on soil chemical, physical and biological characteristics. This method can be applied either to detect harmful effects on soil enzyme activities derived from toxic substances or other anthropogenic agents in contaminated soils against a control soil, or to test chemicals.

Bodenbeschaffenheit - Messung von Enzymaktivitätsmustern in Bodenproben mit kolorimetrischen Substraten in Mikrotiterplatten (ISO 20130:2018)

Dieses Dokument legt ein Verfahren zur gemeinsamen (oder getrennten) Messung verschiedener Hydrolase-Aktivitäten (Arylamidase, Arylsulfatase, β Galactosidase, α Glucosidase, β Glucosidase, N Acetyl-Glucosa¬minidase, saure, alkalische und globale Phosphatasen, Urease) in Bodenproben unter Verwendung von kolorimetrischen Substraten fest. Die Enzymaktivitäten im Boden variieren je nach Jahreszeit und sind abhängig von chemischen, physikalischen und biologischen Eigenschaften des Bodens.
Diese Methode kann entweder zum Nachweis schädlicher Auswirkungen auf die Enzymaktivitäten im Boden durch toxische Substanzen oder andere anthropogene Stoffe in kontaminierten Böden gegenüber einem Kontrollboden oder zur Prüfung von Chemikalien angewendet werden.

Qualité du sol - Mesure de l'activité enzymatique dans des échantillons de sol en utilisant des substrats colorimétriques (ISO 20130:2018)

Le présent document spécifie une méthode de mesure simultanée (ou non) de plusieurs activités des hydrolases (arylamidase, arylsulfatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acétylglucosaminidase, phosphatases acides, alcalines et globales, uréase) dans des échantillons de sol en utilisant des substrats colorimétriques. Les activités enzymatiques du sol varient en fonction des saisons et dépendent des caractéristiques chimiques, physiques et biologiques du sol. Cette méthode peut être appliquée soit pour la détection des effets nocifs de substances toxiques ou d'autres agents anthropiques dans un sol contaminé par comparaison avec un sol de référence, soit pour la réalisation d'essais sur des produits chimiques.

Kakovost tal - Merjenje encimske aktivnosti v vzorcih tal s kolorometričnimi substrati na mikrotitrskih ploščah (ISO 20130:2018)

General Information

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

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SLOVENSKI STANDARD
SIST EN ISO 20130:2020
01-november-2020
Kakovost tal - Merjenje encimske aktivnosti v vzorcih tal s kolorometričnimi
substrati na mikrotitrskih ploščah (ISO 20130:2018)

Soil quality - Measurement of enzyme activity patterns in soil samples using colorimetric

substrates in micro-well plates (ISO 20130:2018)
Bodenbeschaffenheit - Messung von Enzymaktivitätsmustern in Bodenproben mit
kolorimetrischen Substraten in Mikrotiterplatten (ISO 20130:2018)

Qualité du sol - Mesure de l'activité enzymatique dans des échantillons de sol en utilisant

des substrats colorimétriques (ISO 20130:2018)
Ta slovenski standard je istoveten z: EN ISO 20130:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 20130:2020 en,fr,de

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

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SIST EN ISO 20130:2020
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SIST EN ISO 20130:2020
EN ISO 20130
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Measurement of enzyme activity patterns in
soil samples using colorimetric substrates in micro-well
plates (ISO 20130:2018)

Qualité du sol - Mesure de l'activité enzymatique dans Bodenbeschaffenheit - Messung von

des échantillons de sol en utilisant des substrats Enzymaktivitätsmustern in Bodenproben mit

colorimétriques (ISO 20130:2018) kolorimetrischen Substraten in Mikrotiterplatten (ISO

20130:2018)
This European Standard was approved by CEN on 13 April 2020.

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

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

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

member.

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

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

Centre has the same status as the official versions.

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

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

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

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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20130:2020 E

worldwide for CEN national Members.
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SIST EN ISO 20130:2020
EN ISO 20130:2020 (E)
Contents Page

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

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SIST EN ISO 20130:2020
EN ISO 20130:2020 (E)
European foreword

The text of ISO 20130:2018 has been prepared by Technical Committee ISO/TC 190 "Soil quality” of the

International Organization for Standardization (ISO) and has been taken over as EN ISO 20130:2020 by

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

which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by November 2020, and conflicting national standards

shall be withdrawn at the latest by November 2020.

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

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

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

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

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

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

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

United Kingdom.
Endorsement notice

The text of ISO 20130:2018 has been approved by CEN as EN ISO 20130:2020 without any modification.

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SIST EN ISO 20130:2020
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SIST EN ISO 20130:2020
INTERNATIONAL ISO
STANDARD 20130
First edition
2018-07
Soil quality — Measurement of
enzyme activity patterns in soil
samples using colorimetric substrates
in micro-well plates
Qualité du sol — Mesure de l'activité enzymatique dans des
échantillons de sol en utilisant des substrats colorimétriques
Reference number
ISO 20130:2018(E)
ISO 2018
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
Contents Page

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

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

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

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

3 Terms and definitions, symbols and abbreviated terms ............................................................................................ 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Symbols and abbreviated terms............................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reactives ....................................................................................................................................................................................................................... 2

5.1 Buffers and reageants ....................................................................................................................................................................... 2

5.2 Substrates and standards .............................................................................................................................................................. 5

5.2.1 Preparation of standard solutions .................................................................................................................... 5

5.2.2 Preparation of substrate solutions ................................................................................................................... 5

6 Apparatus and materials.............................................................................................................................................................................. 7

7 Procedure..................................................................................................................................................................................................................... 8

7.1 Establishment of calibration curves ..................................................................................................................................... 8

7.1.1 General...................................................................................................................................................................................... 8

7.1.2 Solution of PNP ...................................................................... ............................................................................................ 8

7.1.3 Solution of β-naphthylamine ................................................................................................................................. 8

7.1.4 Solution of ammonium chloride ......................................................................................................................... 8

7.2 Sampling ....................................................................................................................................................................................................... 9

7.2.1 Sample preparation ....................................................................................................................................................... 9

7.2.2 Addition of substrate .................................................................................................................................................10

7.2.3 Absorbance measurements .................................................................................................................................11

7.2.4 Measurements of urease activities ................................................................................................................12

8 Calculation of results ....................................................................................................................................................................................12

9 Expression of results .....................................................................................................................................................................................12

10 Validity criteria ...................................................................................................................................................................................................12

11 Interlaboratory validation ......................................................................................................................................................................13

12 Test report ................................................................................................................................................................................................................13

Annex A (informative) Validation of the method and intralaboratory tests for evaluating

soil enzymatic activities with colorimetric method .....................................................................................................14

Annex B (informative) International ring test for evaluating soil enzymatic activities

with colorimetric method ........................................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................28

© ISO 2018 – All rights reserved iii
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
Foreword

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

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

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

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

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

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

electrotechnical standardization.

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

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

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

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

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

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

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

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

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

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URL: www .iso .org/iso/foreword .html.

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

Biological characterization.
iv © ISO 2018 – All rights reserved
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
Introduction

Microorganisms are responsible for many key processes in the cycle of elements. Enzymes are

responsible for the degradation of organic molecules and their mineralization. The main postulate is the

microbial origin of soil enzymes, even if plant root exudates include enzymes. Extracellular enzymes in

soil play key roles in the biodegradation of organic macromolecules. The simultaneous monitoring of

several enzyme activities important in the biodegradation of organic compounds and mineralization

of carbon, nitrogen, phosphorus and sulfur in soil may reveal harmful effects caused by chemicals

and other anthropogenic impacts. However, the measurements carried out under selected laboratory

conditions using artificial substrates cannot be a substitute for the actual rate of enzymatic processes

in soil in situ.
© ISO 2018 – All rights reserved v
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SIST EN ISO 20130:2020
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SIST EN ISO 20130:2020
INTERNATIONAL STANDARD ISO 20130:2018(E)
Soil quality — Measurement of enzyme activity patterns
in soil samples using colorimetric substrates in micro-
well plates
1 Scope

This document specifies a method for the measurement of several hydrolase activities (arylamidase,

arylsulfatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, acid, alkaline

and global phosphatases, urease) simultaneously (or not) in soil samples, using colorimetric substrates.

Enzyme activities of soil vary seasonally and depend on soil chemical, physical and biological

characteristics. This method can be applied either to detect harmful effects on soil enzyme activities

derived from toxic substances or other anthropogenic agents in contaminated soils against a control

soil, or to test chemicals.
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 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic

conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

3 Terms and definitions, symbols and abbreviated terms
3.1 Terms and definitions
No terms and definitions are listed in this document.

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

— IEC Electropedia: available at http: //www .electropedia .org/
— ISO Online browsing platform: available at https: //www .iso .org/obp
3.2 Symbols and abbreviated terms
ARN Arylamidase
ARS Arylsulfatase

E.C. Enzyme code number by the Nomenclature Committee of the International Union of Bio-

chemistry and Molecular Biology (NC-IUBMB)
NAG N-acetyl-glucosaminidase
PAC acid phosphatase
PAK alkaline phosphatase
PHOS phosphatase
© ISO 2018 – All rights reserved 1
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
URE urease
β−GAL β-galactosidase
α−GLU α-glucosidase
β−GLU β-glucosidase
4 Principle

This document describes a method for the simultaneous measurement of several enzymes in soil

samples (see Table 1). It is based on the use of soil samples solutions and colorimetric substrates,

which are incubated during specific times at 25 °C ± 2 °C or 37 °C ± 2 °C in multi-well plates. After

the incubation, reactions are stopped, plates are then centrifuged and supernatants transferred into

new plates. The intensities of the coloration are measured with absorbance with a 96 wells microplate

spectrophotometer UV/visible.
Table 1 — Enzymatic activity measurements with colorimetric method
Enzyme Abbreviation N° Soil cycle Macromolecule degraded
Arylamidase ARN E.C. 3.4.11.2 Nitrogen
Arylsulfatase ARS E.C. 3.1.6.1 Sulfur Mineralization of organic sulfur
β-Galactosidase βGAL E.C. 3.2.1.22 Carbon Hemicellulose
α-Glucosidase αGLU E.C. 3.2.1.20 Carbon Starch and glycogen
β-Glucosidase βGLU E.C. 3.2.1.21 Carbon Cellulose
N-acetyl- NAG E.C. 3.2.1.52 Carbon Chitin and other β-1,4-linked
glucosaminidase glucosamine polymers
Phosphatase PHOS E.C. 3.1.4.1 Phosphorus Phosphate esters
Acid phosphatase PAC E.C. 3.1.4.1 Phosphorus Phosphate esters
Alkaline phosphatase PAK E.C. 3.1.4.1 Phosphorus Phosphate esters
Urease URE E.C. 3.5.1.5 Nitrogen Urea

An interlaboratory trial was carried out for the validation of the standard; summary of the international

ring test is given in Table 8, and the whole data of the interlaboratory validation are described in

Annex B.
5 Reactives
5.1 Buffers and reageants
5.1.1 General

The choice is made to use deionized water as medium to evaluate native soil enzyme activities at soil pH

and also to allow the analysis of multiple enzymes using the same soil suspension. The soil (in g)/water

(in ml) ratio (4:25) is optimized to maximize reaction, sensitivity and facilitate pipetting technique.

The use of the same soil solution for analysing multiple enzymes also makes data more comparable.

Arylamidase is measured with Tris buffer 50 mmol/l, pH 7,5 and acid and alkaline phosphatases are

involved with the use of Tris-HCl 50 mmol/l at pH 5,5 and Tris base 50 mmol/l at pH 11, respectively.

NOTE The volume can be adapted according to needs.
5.1.2 Tris hydrochloride 50 mmol/l pH 5,5 ± 0,1.

— Tris(hydroxymethyl)aminomethane hydrochloride (CAS N°: 1185-53-1 – Mw: 157 ,6): 7,88 g;

2 © ISO 2018 – All rights reserved
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
— deionized water: 1 000 ml;
— hydrochloric acid (HCl) (CAS N°7647-01-0) 1 mol/l.

Dissolve 7,88 g of Tris(hydroxymethyl)aminomethane hydrochloride into 800 ml deionized water and

adjust to pH 5,5 with hydrochloric acid (1 mol/l). Fill in to 1 000 ml. The storage duration shall not

exceed one month at 4 °C ± 2 °C in glass or polypropylene bottle.
5.1.3 Tris base 50 mmol/l pH 11 ± 0,1.
— Tris(hydroxymethyl)aminomethane (CAS N°: 77-86-1 - Mw: 121 ,14): 6,06 g;
— deionized water: 1 000 ml;
— sodium hydroxide (CAS N° 1310-73-2) (1 mol/l).

Dissolve 6,06 g of Tris(hydroxymethyl)aminomethane into 800 ml deionized water and adjust to pH 11

with sodium hydroxide (1 mol/l). Fill in to 1 000 ml. The storage duration shall not exceed one month

at 4 °C ± 2 °C.
5.1.4 Tris base 50 mmol/l pH 7,5 ± 0,1.
— Tris(hydroxymethyl)aminomethane (CAS N°: 77-86-1 - Mw: 121 ,14): 6,06 g;
— deionized water: 1 000 ml;
— hydrochloric acid (HCl) (CAS N°7647-01-0) 1 mol/l.

Dissolve 6,06 g of Tris(hydroxymethyl)aminomethane into 800 ml deionized water and adjust to pH 7,5

with hydrochloric acid (1 mol/l). Fill in to 1 000 ml. The storage duration shall not exceed one month at

4 °C ± 2 °C.
5.1.5 Tris base 100 mmol/l pH 12 ± 0,1.
— Tris(hydroxymethyl)aminomethane (CAS N°: 77-86-1 - Mw: 121 ,14): 12,11 g;
— deionized water: 1 000 ml;
— sodium hydroxide (CAS N° 1310-73-2) (5 mol/l).

Dissolve 12,11 g of Tris(hydroxymethyl)aminomethane into 800 ml deionized water and adjust to pH

12 with sodium hydroxide (5 M). Fill in to 1 000 ml. The storage duration shall not exceed one month at

4 °C ± 2 °C.
5.1.6 Calcium chloride dihydrate 0,5 mol/l.
— calcium chloride dihydrate (CAS N°: 10035-04-8 - Mw: 147 ,01): 14,7 g;
— Deionized water: 200 ml.

Dissolve 14,7 g of calcium chloride dihydrate in 200 ml of deionized water. The storage duration shall

not exceed one month at 4 °C ± 2 °C.
5.1.7 Salicylate reagent.
— sodium salicylate 270 mmol/l (CAS N°: 54-21-7 - Mw: 160 ,1): 865 mg;
— tri sodium citrate 145 mmol/l (CAS N°: 6132-04-3 - Mw: 294 ,1): 853 mg;
— di sodium tartrate 60 mmol/l (CAS N°: 6106-24-7 - Mw: 230 ,08): 276 mg;
— sodium nitroferricyanide 2 mmol/l (CAS N°: 13755-38-9 - Mw: 297 ,95): 12 mg;
© ISO 2018 – All rights reserved 3
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SIST EN ISO 20130:2020
ISO 20130:2018(E)
— deionized water: 20 ml.

Salicylate reagent is prepared with the 4 compounds listed above just before analysis; dissolve 865 mg

of sodium salicylate, 853 mg of tri sodium citrate, 276 mg of di sodium tartrate and 12 mg of sodium

nitroferricyanide in 20 ml of deionized water.
5.1.8 Cyanurate reagent.
— tri sodium citrate 580 mmol/l (CAS N°: 6132-04-3 - Mw: 294 ,1): 3,4 g;
— di sodium tartrate 90 mmol/l (CAS N°: 6106-24-7 - Mw: 230 ,08): 414 mg;
— lithium hydroxide 280 mmol/l (CAS N° : 1310-65-2 - Mw: 23 ,95): 134 mg;
— dichloroisocyanurate 10 mmol/l (CAS N° : 51580-86-0 - Mw: 255 ,98): 51 mg;
— deionized water: 20 ml.

Cyanurate reagent is prepared with the 4 compounds listed above just before analysis; dissolve

3,4 g of tri sodium citrate, 414 mg of di sodium tartrate, 134 mg of lithium hydroxide and 51 mg of

dichloroisocyanurate in 20 ml of deionized water.
5.1.9 Ethanol, 96 %.
— Ethanol 96 % (CAS N° 41340-36-7).
5.1.10 Acidified ethanol (0,26 mol/l HCl).
— Hydrochloric acid ACS reagent, 37 % (CAS N°7647-01-0) 4,32 ml;
— Ethanol 96 % (CAS N° 41340-36-7).

Dilute 4,32 mL of concentrated HCl into 200 ml ethanol 96 %. The storage duration shall not exceed one

month at 4 °C ± 2 °C.
5.1.11 p-dimethylaminocinnamaldehyde (DMCA) (3,5 mmol/l).
— DMCA (CAS N°: 6203-18-5 - Mw: 175 ,23): 0,12 g;
— Ethanol 96 % (CAS N° 41340-36-7).

Dissolve 0,12 g of DMCA into 200 ml ethanol 96 %. The storage duration shall not exceed one week at

−20 °C ± 2 °C.
Table 2 — Buffer utilization for enzymatic activity measurement
ARS; α-GLU;
β-GLU; β-GAL; ARN URE PAC PAK
NAG; PHOS;
Soil solution deionized water Tris base deionized water Tris HCl Trisbase
50 mmol/l, pH 7,5 50 mmol/l, 50 mmol/l, pH 11
pH 5,5
Stop/ Tris 100 mmol/l Ethanol 96 % salicylate reagent Tris 100 mmol/l pH 12
revelation pH12 Acidified ethanol
CaCl 0,5 mol/l DMCA cyanurate reagent CaCl 0,5 mol/l
2 2
4 © ISO 2018 – All rights reserved
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SIST EN ISO 20130:2020
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5.2 Substrates and standards
5.2.1 Preparation of standard solutions
5.2.1.1 Para-nitrophenol (CAS N°: 100-02-7 - PNP) at 3,6 mmol/l.
— para-nitrophenol (PNP) (Mw: 139 ,11): 10 mg;
— deionized water: 20 ml.

PNP as a powder should be stored at −20 °C ± 2 °C and protected from light. Weigh PNP carefully and

dissolve it into deionized water. Working concentration is 0,36 mM (i.e. dilution of the concentrated

solution 1/10). The storage of the concentrated and working concentrations shall not exceed two years

at −20 °C ± 2 °C. Solutions could be aliquoted for one use or maximum 3 freeze/defreeze cycles.

NOTE Paranitrophenol can cause damage to organs through prolonged or repeated exposure if swallowed

(H373) and harmful if swallowed, in contact with skin or if inhaled (H302, H312, and H332). Appropriated

ventilation and protections need to be used.
5.2.1.2 Ammonium chloride (NH Cl) at 62 mmol/l.
— ammonium chloride (CAS N°: 12125-02-9 - Mw: 53 ,49): 66,4 mg;
— deionized water: 20 ml.

Ammonium chloride as a powder can be stored at room temperature and protected from light. Weigh

ammonium chloride carefully and dissolve it into water. The concentrated solution should be stored

at −20 °C ± 2 °C for two years. The storage of the concentrated solution shall not exceed two years

at −20 °C ± 2 °C. Working concentration is 0,62 mmol/l (dilution 1/100). The storage of the working

concentration shall not exceed two years at −20 °C ± 2 °C.
5.2.1.3 β-naphthylamine, 1 mmol/l.
— β-naphthylamine (CAS N°: 91-59-8 - Mw: 143 ,19): 35,8 mg;
— ethanol 96 %: 20 ml;
— deionized water.

Dissolve 35,8 mg of β-naphthylamine into 50 ml ethanol 96 % (0,071 %). This 5 mmol/l stock solution

shall be stored at −20 °C for one year. Working concentration is 1 mmol/l (dilution 1/5 in water) and

shall be stored at −20 °C ± 2 °C for one year.

NOTE β-Naphtylamine has acute toxicity (oral, dermal, inhalation), category 4, respiratory sensitization,

category 1, and hazardous to the aquatic environment. Eyeshields , full-face particle respirator type N100

2) 3)

(US) , appropriated ventilation, specific gloves , glasses and protective screen need to be used.

5.2.2 Preparation of substrate solutions

Commercially available colorimetric substrates are delivered as powders that should be stored,

according to the specifications, frozen at −20 °C ± 2 °C or cooled at 4 °C ± 2 °C or stored at room

1) https: //www .sigmaaldrich .com/labware/labware -products .html ?TablePage = 20009868

2) http: //www .sigmaaldr ich .com/catalog/sear ch ?int erf ace = Substance & t erm =

msaadvantageseries1000fullfacerespirator1234598765+OR+msaultratwinfullfacerespirator1234598765 & focus =

product & mode = boolean

3) http: //www .sigmaaldrich .com/labware/labware -products .html ?TablePage = 9577418

© ISO 2018 – All rights reserved 5
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temperature (RT). All the substrates should be prepared in advance according to the requirements of

Table 3.

NOTE The volume needs to be sufficient for reliable weighing and measurement of substrates. It depends

also on the number of plates needed. Examples are given in Table 3.

Table 3 — Colorimetric substrates available commercially for enzymatic activity measurements

and their preparation for measures
Enzyme Substrate Molar Concentration Examples of Storage of solu-
(Storage temperature) mass preparation tions
g/mol mol/l Temperature,
duration
Arylamidase L-leucine 292,8 0,008 Dissolve −20 °C ± 2 °C
β-naphthylamide 0,2342 g in
1 year
hydrochloride 50 ml of
deionized
(−20 °C ± 2 °C)
water
CAS N°: 893–36–7
Arylsulfatase Potassium 4-nitrophenyl 257,26 0,025 Dissolve −20 °C ± 2 °C
sulfate 0,322 g in
1 year
50 ml of
(−20 °C ± 2 °C)
deionized
water
CAS N°: 6217–68–1
β-Galactosidase p-nitrophenyl β-D-ga- 301,25 0,05 Dissolve −20 °C ± 2 °C
lactopyranoside 0,753 g in
1 year
50 ml of
(−20 °C ± 2 °C)
deionized
CAS N°: 3150–24–1 water
α-Glucosidase 4-nitrophenyl α-D-glu- 301,25 0,025 Dissolve −20 °C ± 2 °C
copyranoside 0,375 g in
1 year
50 ml of
(−20 °C ± 2 °C)
deionized
water
CAS N°: 3767–28–0
β-Glucosidase 4-nitrophenyl β-D-glu- 301,25 0,05 Dissolve −20 °C ± 2 °C
copyranoside 0,753 g in
1 year
50 ml of
(−20 °C ± 2 °C)
deionized
CAS N°: 2492–87–7 water

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