SIST EN ISO 20130:2020

SLOVENSKI STANDARD
oSIST prEN ISO 20130:2020
01-januar-2020
Kakovost tal - Merjenje encimske aktivnosti v vzorcih tal s kolorometričnimi
substrati (ISO 20130:2018)
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 des échantillons de sol en utilisant
des substrats colorimétriques (ISO 20130:2018)
Ta slovenski standard je istoveten z: prEN ISO 20130
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 20130:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 20130:2020

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oSIST prEN 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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oSIST prEN 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
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Published in Switzerland
ii © ISO 2018 – All rights reserved

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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
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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
constitute an endorsement.
For an explanation on 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 the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
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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.
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oSIST prEN 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
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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;
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— 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;
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— 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
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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.
4
— 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
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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
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.
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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).
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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
4
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
[
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