SIST EN ISO 20130:2020
(Main)Soil quality - Measurement of enzyme activity patterns in soil samples using colorimetric substrates in micro-well plates (ISO 20130:2018)
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
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20130:2020 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
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.
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
ISO 20130:2018(E)
© 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.
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Published in Switzerland
ii © ISO 2018 – All rights reserved
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 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
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
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For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
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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
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.
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 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
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 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
ISO 20130:2018(E)
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,
1)
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 20130:2018(E)
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
a
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
a
Sigma-Aldrich is an example of producer of colorimetric substrates. This information is given for the convenience of
users of this international standard and does not constitute an endorsement by ISO of these products. Equivalent products
may be used if they can be shown to lead to the same results.
NOTE All Substrates need to be manipulated with appropriated ventilation, specific gloves, and protective screen.
6 © ISO 2018 – All rights reserved
ISO 20130:2018(E)
Table 3 (continued)
a
Enzyme Substrate Molar Concentration Examples of Storage of solu-
(Storage temperature) mass preparation tions
g/mol mol/l Temperature,
duration
N-acetyl- para-nitrophenyl N-acethyl 342,31 0,01 Dissolve −20 °C ± 2 °C
glucosaminidase β-D glucopyranoside 0,171 g in
1 year
50 ml of
(−20 °C ± 2 °C)
deionized
CAS N°: 3459–18–5 water
Phosphatase 4-nitro-phenylphosphate 371,12 0,05 Dissolve −20 °C ± 2 °C
disodium salt 0,928 g in
Acid phosphatase 1 year
hexahydrate 50 ml of
and deionized
(+4 °C ± 2 °C)
water
Alkaline
CAS N°: 333338–18–4
phosphatase
Urease Urea > 98 % 60,06 0,4 Dissolve +4 °C
0,480 5 g in
(RT) 1 week
20 ml of
deionized
CAS N°: 57–13–6
water
a
Sigma-Aldrich is an example of producer of colorimetric substrates. This information is given for the convenience of
users of this international standard and does not constitute an endorsement by ISO of these products. Equivalent products
may be used if they can be shown to lead to the same results.
NOTE All Substrates need to be manipulated with appropriated ventilation, specific gloves, and protective screen.
6 Apparatus and materials
In addition to usual laboratory equipment the following materials are required:
6.1 Sieves, with grid size 2 mm, up to 5 mm according to soil texture and humidity.
NOTE Other grid sizes can be used depending on the soil texture.
6.2 Balance (±0,001 g).
6.3 Orbital shaker.
6.4 Multi-well plates, 96 wells, polystyrene, with flat bottoms but without treatment, with lids.
6.5 Automatic dispenser for water (optional).
NOTE Compared with manual pipetting, an automatic dispenser decreases the uncertainty of volumes
dispensed significantly.
6.6 Multichannel micropipettes (50 µL and 200 µL).
6.7 Incubators, set at 25 °C ± 2 °C and 37 °C ± 2 °C.
6.8 Plate centrifuge, with temperature regulation at 20 °C and acceleration of 1 500 g.
6.9 Plate reading spectrophotometer, with reading at 405 nm ± 5 nm, 540 nm ± 5 nm and
650 nm ± 5 nm (monochromator or BP ± 10 nm).
ISO 20130:2018(E)
7 Procedure
7.1 Establishment of calibration curves
7.1.1 General
Calibration curves require several concentrations of para-nitro phenol (PNP), ammonium chloride
(NH Cl) or β-naphthylamine, at least in duplicates, preferably in triplicates; all the volumes are given
per well. Homogenizations are realized with a micropipette and 2 or 3 aspirations/ejections. Exposure
to light shall be limited during preparation of standard curves. Examples of calibration curves are given
in Annex B.
7.1.2 Solution of PNP
The stock solution of PNP with a concentration of 0,36 mmol/l is used to establish the calibration curve.
Distribute the volumes needed into multi-well plate wells for concentrations 0 nmol/ml, 14 nmol/
ml, 29 nmol/ml, 72 nmol/ml, 140 nmol/ml, 220 nmol/ml, 290 nmol/ml and 360 nmol/ml in replicate
(Table 4).
Table 4 — Preparation of calibration curve of para-nitrophenol in 96 wells microplate
[PNP]
0 14 29 72 140 220 290 360
nmol/ml
PNP (µl) 0 5 10 25 50 75 100 125
Water (µl) 125 120 115 100 75 50 25 0
To reveal yellow coloration of PNP, 25 µl of water, 25 µl of calcium chloride and 100 µl of basic Tris base
100 mmol/l pH 12 are added. After homogenization, 200 µl are transferred in new plates and reading of
absorbance is realized with a microplate spectrophotometer at 405 nm.
7.1.3 Solution of β-naphthylamine
The working solution of β-naphthylamine with a concentration of 1 mmol/l, is used to produce the
standard curve. Distribute the volumes needed into multi-well plate wells for concentrations 0 nmol/
ml, 10 nmol/ml, 20 nmol/ml, 50 nmol/ml, 100 nmol/ml, 200 nmol/ml (Table 5).
Table 5 — Preparation of calibration curve of β-naphthylamine in 96 wells microplate
[β-Naphthylamine]
0 10 20 50 100 200
nmol/ml
β-Naphthylamine (µl) 0 1 2 5 10 20
Tris buffer 50 mmol/l pH 7,5 (µl) 50 49 48 45 40 30
Ethanol 96 % (µl) 50 50 50 50 50 50
To reveal coloration of β-naphthylamine, 100 µl acidified ethanol and 100 µl of
p-dimethylaminocinnamaldehyde (DMCA) are added and mixed. After 20 min incubation in the dark at
room temperature, the absorbance is read with a microplates spectrophotometer UV/visible at 540 nm.
7.1.4 Solution of ammonium chloride
The stock solution of ammonium chloride with a concentration of 62 mmol/l is diluted 1/100 and
working solution with 0,62 mmol/L (620 nmol/ml) is used to produce the standard curve. Distribute
the volumes needed into multi-well plate wells for concentrations 0 nmol/ml, 6 nmol/ml, 16 nmol/ml,
31 nmol/ml, 62 nmol/ml, 155 nmol/ml, 233 nmol/ml and 310 nmol/ml in replicate (Table 6).
8 © ISO 2018 – All rights reserved
ISO 20130:2018(E)
Table 6 — Preparation of calibration curve of ammonium chloride in 96 wells microplate
[NH Cl]
0 6 16 31 62 155 233 310
nmol/ml
NH Cl (µl) 0 2 5 10 20 50 75 100
Water (µl) 200 198 195 190 180 150 125 100
To quantify NH Cl concentration, 40 µl of the salicylate reagent are added to each well. After a 3 min
reaction period, 40 µl of cyanurate reagent are dispensed into each well, and each well homogenized
then incubated in dark for 30 min (absorbance is stable for two hours). After incubation, plates are
homogenized with a micropipette (2 or 3 aspirations/ejections) and 200 µl are transferred in new plates
and the reading of absorbances is realized with a microplates spectrophotometer UV/visible at 650 nm.
7.2 Sampling
7.2.1 Sample preparation
7.2.1.1 Homogenization
Take and handle soil samples shall be as specified in ISO 18400-206. A composite sample taken from the
field, homogenized and sieved through 2 mm mesh (up to 5 mm according to soil texture and humidity),
has been observed to yield reasonably low uncertainty of measurement for soil samples.
To improve the link between in situ activities, samples should be stored at 15 °C ± 2 °C until four days.
But, if it is not possible, weakest modifications will occur with storage at −80 °C ± 5 °C. The storage at
−20 °C ± 2 °C before or after sieving is not suitable.
The sieved soil is homogenized, and triplicate of exactly 4 g are weighed and deposited into flat bottom
flask (30 to 60 ml). Twenty-five ml of deionized water and cross shaped stirring bars are added.
−1
Containers are closed and homogenized for 10 min on orbital agitator (250 min ).
7.2.1.2 Preparation of sample plates
Cross shaped stir bars cross are added in flat bottom flask and soil suspension shall stay under stirring
during pipetting. Use multichannel micropipettes (one to three channels) to distribute each soil
suspension in microplates, in four replicate wells, with a specific volume according to Table 7 (part A),
and place the lid on each plate. Three wells are analytical points and one well is the blank (to reveal
chemical interactions with soil compounds). Prepare a same number of plates as enzyme tested (one
plate per one enzyme).
NOTE 1 An example of plate schedule is described in Figure 1.
NOTE 2 There is a possibility that humic soils necessitate the use of enlarged cones for the pipetting.
ISO 20130:2018(E)
Key
S1 sample number
a, b, c triplicate of sample
T control well for each soil
Figure 1 — Example of sample organization in plates for each enzyme
7.2.2 Addition of substrate
Simple organization would occur if one plate is used for one enzyme. When all samples are distributed
in plates, use multichannel micropipettes to distribute each substrate, in three replicates and
homogenized with 2 or 3 aspirations/ejections, and incubate the multi-well plates (Table 7 part A).
After incubation, reactions are stopped, plates are centrifuged 5 min at 1 500 g and the supernatants
are transferred in new plates. Then, absorbance is measured on a spectrophotometer for microplates.
Table 7 part B summarizes protocols for each enzyme measurement.
Table 7 — Specific experimental details for enzyme activity measurements
PHOS
A
ARN ARS β-GAL α-GLU β-GLU NAG PAC URE
INCUBATION
PAK
Soil solution (µl) 125 50
Substrate (µl) 25 40
Deionized water
0 (+40 µl in
(µl)
control wells)
Incubation time 2H 4H 2H 1H 1H 1H 0H30 3H
Incubation
37 °C 25 °C
temperature
B
STOP REACTION
10 © ISO 2018 – All rights reserved
ISO 20130:2018(E)
Table 7 (continued)
PHOS
A
ARN ARS β-GAL α-GLU β-GLU NAG PAC URE
INCUBATION
PAK
In each well:
In each well:
In each well:
40 µl salicylate
25 µl CaCl reagent
150 µl Ethanol
Stop reaction
96 %
100 µl Trizma pH 12 40 µl cyanurate
reagent
homogenization
homogenization
homogenization
25 µl in
Substrate 25 µl in control wells
control wells
30 min, RT,
Incubation
in dark
Centrifugation 5 min, 1 500g
Transfer 100 µl 200 µl
100 µl acidified
ethanol
Revelation 100 µl DMCA
20 min, RT,
in dark
Reading λ = 540 nm λ = 405 nm λ = 650 nm
NOTE 1 Incubation temperature affects reaction rates and optimum depends on the enzymes. For specific
purposes, a different temperature as described in this document can be used, e.g. in situ temperature.
NOTE 2 For urease activity, it is necessary to quantify free ammonium brought by urea solution and subtract
it from ammonium quantified after soil incubation to obtain specific ammonium released by urease. Urea control
is prepared with 40 µl urea, 200 µl of deionized water, 40 µl salicylate reagent and 40 µl cyanurate reagent. After
30 min of incubation, 200 µl are transferred into new wells and absorbances are reading at 650 nm.
After incubation, the reactions are stopped according to enzyme specific protocols (Table 7).
7.2.3 Absorbance measurements
7.2.3.1 Measurements of enzyme activities with PNP as reaction product (ARS, β-GAL, α-GLU,
β-GLU, NAG, PHOS, PAC, PAK)
The addition of 25 µl of calcium chloride and 100 µl of basic Tris buffer pH 12 stops the reaction, and
it is the key action to eliminate colloids which introduce turbidity in solutions and increase pH for
PNP quantification. Add 25 µl of substrate in control wells and homogenise with 2 or 3 aspirations/
rejections. Furthermore, plates are centrifuged five minutes at 1 500 g and 20 °C, and 200 µl of the
supernatant transferred in a new plate. The reading of absorbance is realized with a microplates
spectrophotometer UV/visible at λ = 405 nm.
7.2.3.2 Measurements of arylamidase activities (ARN)
After incubation, add 150 µl ethanol 96 % in each well (including controls) and 25 µl substrate solution
in control wells. The plate is centrifuged 5 min at 1500 g and 100 µl of supernatant are transferred into
a new plate. To reveal the quantity of naphthylamine produced, 100 µl of acidified ethanol and 100 µl
of DMCA were added in all wells and homogenized with a micropipette (2 aspirations/ejections). After
20 min, reading of absorbance is realized with a microplates spectrophotometer UV/visible at 540 nm.
ISO 20130:2018(E)
7.2.4 Measurements of urease activities
After incubation of 3 h, 40 µl of the salicylate reagent are added to each well, including controls. After
a 3 min reaction period, 40 µl of cyanurate reagent is dispensed into each well including controls.
Colorimetric reaction is achieved in 30 min and stable for two hours. Furthermore, plates are centrifuged
five minutes at 1 500 g and 20 °C, and 200 µl of the supernatant are transferred in a new plate. The
reading of absorbance is performed with a microplate spectrophotometer UV/visible at λ = 650 nm.
8 Calculation of results
The standard curve is plotted for PNP or β-naphthylamine and ammonium chloride nanomolar
concentration (nmol/ml) versus absorbance. The PNP or β-naphthylamine or ammonium chloride
concentration of blank (C ) and sample (C ) are read from the standard curve.
b s
Examples of calibration curves are given in Annex B.
The result is calculated by subtracting measurement of blank from triplicate of sample and multiplying
the difference with dilution factor (D) and soil volume (V ), and dividing with reaction time and dry
ss
mass of sample (W ).
ds
()CC−×DV×
sb ss
A= (1)
RT×W
ds
where
A is the enzymatic activity in mU/g of dry sample (nmol/min/g of dry sample);
C is the concentration of product formed in sample (nmol/ml);
s
C is the concentration of product formed in blank (nmol/ml);
b
D is the dilution of sample in microplate;
V is the volume of sample solution (ml);
ss
RT is the reaction time (min);
W is the mass of dry sample (g).
ds
9 Expression of results
Results are expressed as milliunit for one gram of dry soil corresponding to nmole of PNP, β-naphthylamine
or ammonium chloride released per minute and g soil dry mass of sample. The measurements necessary
for each expression, percentage of dry matter determination shall be carried out.
−1
NOTE The more practical and commonly unit used for enzymatic activity is Units (U) = 1 μmol min . For
soil, activity expressed as milliunit for one gram of dry soil (mU/g DS).
Soil characteristics vary widely due to geography, climate and land use. The interpretation of results
cannot, presently, be based on set limit values for each enzyme activity. The experimental design shall
facilitate comparisons with a control soil or between samples from relevant sites.
10 Validity criteria
a) The standard curve is valid if the following criteria are met (according to the results of the ring test
summarized in Annex B):
— r ≥ 0,990;
12 © ISO 2018 – All rights reserved
ISO 20130:2018(E)
— the absorbance for PNP standard curve range is within [0,04 - 1,8] ± 10 %;
— the absorbance for β-naphtylamine standard curve range is within [0,06 - 2,3] ± 10 %;
— the absorbance for ammonium standard curve range is within [0,05 - 1,6] ± 10 %.
b) For the samples:
— Triplicates % CV shall be ≤ 15 %;
— Urea blank absorbance shall be ≤ 0,2;
— For urease measurements, sample blank shall be ≤ 1;
— Differences of absorbance for sample assay and blank shall be ≥ 0,02 to 0,05 according to
spectrophotometer sensibility and repeatability.
11 Interlaboratory validation
Summary of the international ring test is given in Table 8, and the whole data of the interlaboratory
validation are described in Annex B.
Table 8 — synthetic data of interlaboratory study
α-GLU β-GL
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