Soil quality - Determination of abundance and activity of soil microflora using respiration curves (ISO 17155:2012)

This International Standard specifies a test method for determining the activity of active aerobic, eterotrophic
microbial biomass in soils. This method is applicable to the monitoring of soil quality and to the evaluation of
the ecotoxic potential of soils and soil materials. It is also applicable for soils sampled along contamination
gradients in the field and to soils that are contaminated experimentally in the field or in the laboratory.

Bodenbeschaffenheit - Bestimmung der Abundanz und Aktivität der Bodenmikroflora mit Hilfe von Atmungskurven (ISO 17155:2012)

Diese Internationale Norm legt ein Untersuchungsverfahren zur Bestimmung der Aktivität der aktiven aeroben heterotrophen mikrobiellen Biomasse in Böden fest. Das Verfahren gilt für die Bewertung der Bodenqualität und die Einschätzung des ökotoxischen Potenzials von Böden und Bodenmaterialien. Es gilt auch für Böden, die entlang von Verunreinigungsgradienten im Feld entnommen wurden und für Böden, die im Feld oder im Laboratorium experimentell verunreinigt wurden.

Qualité du sol - Détermination de l'abondance et de l'activité de la microflore du sol à l'aide de courbes de respiration (ISO 17155:2012)

1 Domaine d'application
La présente Norme internationale spécifie une méthode d'essai pour déterminer l'activité de la biomasse microbienne hétérotrophe aérobie active dans les sols. Cette méthode s'applique à la surveillance de la qualité du sol et à l'évaluation du potentiel écotoxique des sols et des matériaux de type sol. Elle s'applique également aux sols prélevés le long de gradients de contamination sur le terrain et aux sols qui sont délibérément contaminés sur le terrain ou en laboratoire à des fins d'analyse.

Kakovost tal - Določevanje številčnosti in aktivnosti mikroflore tal z dihalnimi krivuljami (ISO 17155:2012)

General Information

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

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SLOVENSKI STANDARD
SIST EN ISO 17155:2020
01-november-2020
Kakovost tal - Določevanje številčnosti in aktivnosti mikroflore tal z dihalnimi
krivuljami (ISO 17155:2012)

Soil quality - Determination of abundance and activity of soil microflora using respiration

curves (ISO 17155:2012)

Bodenbeschaffenheit - Bestimmung der Abundanz und Aktivität der Bodenmikroflora mit

Hilfe von Atmungskurven (ISO 17155:2012)

Qualité du sol - Détermination de l'abondance et de l'activité de la microflore du sol à

l'aide de courbes de respiration (ISO 17155:2012)
Ta slovenski standard je istoveten z: EN ISO 17155:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 17155:2020 en,fr,de

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

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SIST EN ISO 17155:2020
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SIST EN ISO 17155:2020
EN ISO 17155
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Determination of abundance and activity of
soil microflora using respiration curves (ISO 17155:2012)

Qualité du sol - Détermination de l'abondance et de Bodenbeschaffenheit - Bestimmung der Abundanz und

l'activité de la microflore du sol à l'aide de courbes de Aktivität der Bodenmikroflora mit Hilfe von

respiration (ISO 17155:2012) Atmungskurven (ISO 17155:2012)
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 17155:2020 E

worldwide for CEN national Members.
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SIST EN ISO 17155:2020
EN ISO 17155:2020 (E)
Contents Page

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

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SIST EN ISO 17155:2020
EN ISO 17155:2020 (E)
European foreword

The text of ISO 17155:2012 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 17155:2020 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 17155:2012 has been approved by CEN as EN ISO 17155:2020 without any modification.

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SIST EN ISO 17155:2020
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SIST EN ISO 17155:2020
INTERNATIONAL ISO
STANDARD 17155
Second edition
2012-10-01
Soil quality — Determination of
abundance and activity of soil microflora
using respiration curves
Qualité du sol — Détermination de l’abondance et de l’activité de la
microflore du sol à l’aide de courbes de respiration
Reference number
ISO 17155:2012(E)
ISO 2012
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SIST EN ISO 17155:2020
ISO 17155:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s

member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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SIST EN ISO 17155:2020
ISO 17155:2012(E)
Contents Page

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

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

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

3 Terms and definitions ......................................................................................................................................... 1

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

5 Reagents ................................................................................................................................................................ 2

6 Apparatus .............................................................................................................................................................. 2

7 Sampling ................................................................................................................................................................ 3

7.1 Sample quantities ................................................................................................................................................ 3

7.2 Sampling and storage ........................................................................................................................................ 3

7.3 Soil sample characteristics .............................................................................................................................. 4

8 Procedure .............................................................................................................................................................. 4

8.1 Test .......................................................................................................................................................................... 4

8.2 Toxicity testing ..................................................................................................................................................... 4

9 Calculation ............................................................................................................................................................ 5

9.1 Microbial parameters .......................................................................................................................................... 5

9.2 Interpretation of data .......................................................................................................................................... 6

10 Test report ............................................................................................................................................................. 8

Annex A (informative) Results of a laboratory ring test carried out in Germany .............................................. 9

Annex B (informative) Comparison of microbial biomass determination by respiration curve

[1]

measurement (this International Standard) and substrate-induced respiration (ISO 14240-1 ) 11

Bibliography .....................................................................................................................................................................13

© ISO 2012 – All rights reserved iii
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SIST EN ISO 17155:2020
ISO 17155:2012(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 17155 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.

This second edition cancels and replaces the first edition (ISO 17155:2002), which has been technically revised.

iv © ISO 2012 – All rights reserved
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SIST EN ISO 17155:2020
INTERNATIONAL STANDARD ISO 17155:2012(E)
Soil quality — Determination of abundance and activity of soil
microflora using respiration curves
1 Scope

This International Standard specifies a test method for determining the activity of active aerobic, heterotrophic

microbial biomass in soils. This method is applicable to the monitoring of soil quality and to the evaluation of

the ecotoxic potential of soils and soil materials. It is also applicable for soils sampled along contamination

gradients in the field and to soils that are contaminated experimentally in the field or in the laboratory.

2 Normative references

The following referenced documents are indispensable for the application 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the 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)

ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by

sieving and sedimentation

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method

ISO 14238, Soil quality — Biological methods — Determination of nitrogen mineralization and nitrification in

soils and the influence of chemicals on these processes
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
basal respiration rate

constant mass of CO released or mass of O consumed per unit mass of soil per unit time without substrate addition

2 2
NOTE See Figure 1 for a typical basal respiration curve.
3.2
substrate-induced respiration rate

constant mass of CO released or mass of O consumed per unit mass of soil per unit time shortly after

2 2
addition of a carbon substrate
NOTE 1 See Figure 1 for a typical substrate-induced respiration curve.

NOTE 2 If glucose is used as a carbon substrate, microbial biomass can be determined from the substrate-induced

[1]
respiration rate (see ISO 14240-1 ).
© ISO 2012 – All rights reserved 1
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SIST EN ISO 17155:2020
ISO 17155:2012(E)
3.3
respiratory activation quotient
basal respiration rate divided by substrate-induced respiration rate
Q = (1)
3.4
specific growth rate

exponent representing respiration rate per unit of time during the exponential phase of growth

NOTE See Equation (3).
3.5
time to the peak maximum
peakmax
time from addition of substrate to the maximum respiration rate
NOTE 1 See Figure 1.

NOTE 2 The time to the peak maximum also reflects the viability of the growing organisms.

3.6
cumulative CO evolution or O consumption
2 2

total area bounded by the line of the soil respiration rate curve to the time axis from time of the addition of

substrate to the time of peak maximum (t )
peakmax
NOTE See Figure 1.
3.7
soil material

material composed of excavated soil, dredged materials, manufactured soils, treated soils or fill materials

4 Principle

The CO production or O consumption (respiration rate) from unamended soils as well as the decomposition of

2 2

an easily degraded substrate (glucose + ammonium + phosphate) is monitored regularly (e.g. every hour). From

the CO production or O consumption data, the different microbial parameters (basal respiration, substrate-

2 2
induced respiration, respiratory activation quotient, t , C ) can be calculated.
peakmax R
5 Reagents
5.1 Glucose, C H O .
6 12 6
5.2 Potassium dihydrogenphosphate, KH PO .
2 4
5.3 Diammonium sulfate, (NH ) SO .
4 2 4

5.4 Substrate, consisting of a mixture of 80 g of glucose (5.1), 13 g of diammonium sulfate (5.3), and 2 g of

KH PO (5.2), which is thoroughly ground and mixed in a mortar.
2 4
6 Apparatus
Ordinary laboratory equipment and 6.1.
2 © ISO 2012 – All rights reserved
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SIST EN ISO 17155:2020
ISO 17155:2012(E)

6.1 Respirometer for continuous measurement of CO evolution or O consumption, maintained at a

2 2
[2]

constant temperature (preferably 20 °C). Suitable examples of equipment are given in ISO 16072.

Key
X t h time
−1 −1
Y µg g h CO or O respiration rates
dm 2 2
C cumulative CO evolution or O consumption
R 2 2
dp/dt rate of product formation after substrate addition
respiration rate of K-strategist at the time of substrate addition
r respiration rate of r-strategist at the time of substrate addition
t time to the peak maximum
peakmax
specific growth rate
1 R substrate-induced respiration R = K + r (at t = 0)
S S
2 R basal respiration
3 substrate addition

Figure 1 — Soil respiration rate before and after addition of an easily degraded substrate

7 Sampling
7.1 Sample quantities

Choose the size of the soil samples taking into account the apparatus (6.1) used, the organic matter content of

the samples (7.3) and the soil needed for sample characterization (7.3). It is recommended that at least three

replicates per sample be measured.
7.2 Sampling and storage

The recommendations in ISO 10381-6 for collection, handling and storage of soil samples shall be followed.

© ISO 2012 – All rights reserved 3
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SIST EN ISO 17155:2020
ISO 17155:2012(E)
7.3 Soil sample characteristics

Soil samples generating soil respiration curves can be obtained from mineral, organic, polluted, and unpolluted

soils. Determine the following characteristics for each soil sample:
— particle size distribution in accordance with ISO 11277;
— water content in accordance with ISO 11465;
— water-holding capacity in accordance with Annex A of ISO 14238:2012;
— pH in accordance with ISO 10390;
— organic matter content in accordance with ISO 10694.
8 Procedure
8.1 Test

Pre-incubate moist soil samples (preferably 40 % to 60 % of maximum water holding capacity or 0,01 MPa to

0,03 MPa suction pressure) at 20 °C for 3 d to 4 d before the beginning of the measurement. Measure the basal

respiration of the sub-samples first. Measure the respiration rates until constant rates are obtained.

After measuring the basal respiration, add 10 mg of the substrate (5.4) per gram soil (dry mass) and mix

homogeneously with a spatula into the soil samples. If the organic matter content is >5 %, add 0,2 g of the

substrate per gram humus (see References [4][5]).
8.2 Toxicity testing

In principle, testing the influence of chemicals should also be possible with the method. Up to the time of

publication, there is only scarce experience available in the literature.

To determine the influence of chemicals on the abundance and activity of soil microorganisms, a soil with low

content of organic carbon (mass fraction between 0,5 % and 1,5 %). Particles of size <20 µm should not exceed

20 % mass fraction in order to provide a high degree of bioavailablity.

The effect of chemicals on the soil microbial activity can be determined as follows. Using a range-finding test,

determine the concentration rang
...

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