ISO/TS 22939:2019

TECHNICAL ISO/TS
SPECIFICATION 22939
Second edition
2019-08
Soil quality — Measurement of
enzyme activity patterns in soil
samples using fluorogenic substrates
in micro-well plates
Qualité du sol — Mesure en microplaques de l'activité enzymatique
dans des échantillons de sol en utilisant des substrats fluorogènes
Reference number
©
ISO 2019
© ISO 2019
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ii © ISO 2019 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 1
5 Principle . 1
6 Reagents . 2
6.1 Buffers . 2
6.2 Substrates and standards . 3
6.2.1 Preparation of standard solutions . 3
6.2.2 Preparation of substrate solutions . 3
6.2.3 Preparation of multi-well plates . 3
6.2.4 Preparation of standard plates . 3
6.2.5 Preparation of substrate plates . 4
6.2.6 Fluorogenic substrates . 4
7 Apparatus and materials. 5
8 Procedure. 6
8.1 Sampling . 6
8.2 Sample preparation . 6
8.2.1 Homogenization . 6
8.2.2 Preparation of dilutions . 6
8.2.3 Sample distribution . 7
8.3 Incubation . 7
8.4 Fluorescence measurements . 7
9 Calculation of results . 7
10 Expression of results . 7
11 Test report . 8
Annex A (informative) Guidance on the use of freshly prepared substrates .9
Annex B (informative) Example of a graph for calculation .11
Bibliography .13
Foreword
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This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
This second edition cancels and replaces the first edition (ISO/TS 22939:2010), which has been
technically revised. The main changes compared to the previous edition are as follows:
— Clause 3 “Terms and definitions” added;
— 6.2.4: unit corrected in (40 ml to 40 µl);
— 6.2.6, Table 1 (Chitinase change E.C. 3.2.1.30 to E.C.3.2.1.52 and Alanin-aminopeptidase E.C.
3.4.11.12 to E.C. 3.4.11.2).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved

Introduction
Micro-organisms are responsible for many key processes in the cycle of elements. Enzymes play
key roles in the degradation and mineralization of organic macromolecules. The main postulate is
the microbial origin of soil enzymes, even if plant root exudates include enzymes. The simultaneous
monitoring of several enzyme activities important in the biodegradation of organic compounds
and mineralization of C, N, P and S in soil may reveal harmful effects caused by chemicals and other
anthropogenic impacts (e.g. acidification, compaction). 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.
TECHNICAL SPECIFICATION ISO/TS 22939:2019(E)
Soil quality — Measurement of enzyme activity patterns
in soil samples using fluorogenic substrates in micro-
well plates
1 Scope
This document specifies a method for the measurement of several enzyme activities (arylsulfatase,
α −glucosidase, β -glucosidase, Cellubisidase, β -Xylosidase, phosphodiesterase (PDE), chitinase,
phosphomonoesterase (PME), leucine-aminopeptidase, Alanine-aminopeptidase) simultaneously (or
not) using fluorigenic substrates in soil samples. Enzyme activities of soil vary seasonally and depend on
the chemical, physical and biological characteristics of soil. Its application for the detection of harmful
effects of toxic chemicals or other anthropogenic impacts depends on the simultaneous comparison
of enzyme activities in a control soil similar to the test soil, or on exposure tests with chemicals or
treatments.
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
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
3 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:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
4 Abbreviated terms
E.C. Enzyme code number defined by the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB)
SOM Soil organic matter content
MUB Modified universal buffer
5 Principle
This document describes a method for the simultaneous measurements of several enzymes in soil
samples. It is based on the use of soil samples diluted in buffer containing fluorogenic substrates, which
are incubated for 3 h at (30 ± 2) °C in multi-well plates. After the incubation the enzyme activities are
[1][2]
measured as fluorescence with a plate-reading fluorometer . The method described is based on
dried standard and substrate plates enabling storage and limiting bias due to differences between
reagent batches, and also enabling comparison between reagent batches. Annex A describes a method
utilizing freshly prepared reagents, which has a clearly defined and exact incubation period. The
advantage of the use of freshly prepared substrates is that an instrument for lyophilization is not
required.
6 Reagents
6.1 Buffers
6.1.1 General
The selection of the buffer depends on the soil sample because the pH strongly affects enzyme
activities. Sodium acetate buffer, 0,5 mol/l, at pH 5,5 has been used for acid soils with a high organic
matter content. The use of the modified universal buffer (MUB) at the pH of the soil sample gives the
flexibility necessary for coverage of a broad spectrum of different soils. Adequate stability of substrates
at different buffers needs to be ensured. Good stability has been observed in 0,5 mol/l sodium acetate
[3]
buffer at pH 5,5 .
6.1.2 Sodium acetate buffer, 0,5 mol/l, pH 5,5.
— sodium acetate trihydrate (CAS N°: 6131-90-4 – 136,08 g/mol): 68,04 g;
— deionized water 1 000 ml;
— acetic acid (CAS N°: 64-19-7 – 60,05 g/mol): >99,8 %.
Dissolve sodium acetate trihydrate in water (e.g. 800 ml)
...