Soil quality - Determination of potential nitrification and inhibition of nitrification - Rapid test by ammonium oxidation (ISO 15685:2012)

This International Standard specifies a rapid method for the determination of the potential rate of ammonium
oxidation and inhibition of nitrification in soils. This method is suitable for all soils containing a population of
nitrifying microorganisms. It can be used as a rapid screening test for monitoring soil quality and quality of
wastes, and is suitable for testing the effects of cultivation methods, chemical substances [except volatiles,
i.e. H > 1 (Henry’s constant)], extracts of biosolids and pollution in soils.

Bodenbeschaffenheit - Bestimmung der potentiellen Nitrifizierung und Hemmung der Nitrifizierung - Schnellverfahren mittels Ammoniumoxidation (ISO 15685:2012)

Diese Internationale Norm legt ein Schnellverfahren zur Bestimmung der potentiellen Ammoniumoxidation und der Hemmung der Nitrifizierung in Böden fest. Dieses Verfahren ist geeignet für die Anwendung bei allen Böden, die eine Population an nitrifizierenden Mikroorganismen enthalten. Es kann als schnelles Übersichtsverfahren zur Überwachung der Bodenbeschaffenheit und der Beschaffenheit von Abfällen verwendet werden und ist für die Prüfung der Einflüsse von Kultivierungsverfahren, zusätzlichen Chemikalien [außer flüchtige Substanzen, d. h. H > 1 (Henry-Konstante)], Eluaten von Biofeststoffen und Verunreinigungen von Böden geeignet.

Qualité du sol - Détermination de la nitrification potentielle et inhibition de la nitrification - Essai rapide par oxydation de l'ammonium (ISO 15685:2012)

Kakovost tal - Določevanje potencialne nitrifikacije in zaviranja nitrifikacije - Hitri preskus z oksidacijo amonija (ISO 15685:2012)

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
25-May-2020
Due Date
30-Jul-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 15685:2020
01-november-2020

Kakovost tal - Določevanje potencialne nitrifikacije in zaviranja nitrifikacije - Hitri

preskus z oksidacijo amonija (ISO 15685:2012)

Soil quality - Determination of potential nitrification and inhibition of nitrification - Rapid

test by ammonium oxidation (ISO 15685:2012)
Bodenbeschaffenheit - Bestimmung der potentiellen Nitrifizierung und Hemmung der
Nitrifizierung - Schnellverfahren mittels Ammoniumoxidation (ISO 15685:2012)

Qualité du sol - Détermination de la nitrification potentielle et inhibition de la nitrification -

Essai rapide par oxydation de l'ammonium (ISO 15685:2012)
Ta slovenski standard je istoveten z: EN ISO 15685:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 15685:2020 en,fr,de

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

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SIST EN ISO 15685:2020
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SIST EN ISO 15685:2020
EN ISO 15685
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Determination of potential nitrification and
inhibition of nitrification - Rapid test by ammonium
oxidation (ISO 15685:2012)

Qualité du sol - Détermination de la nitrification Bodenbeschaffenheit - Bestimmung der potentiellen

potentielle et inhibition de la nitrification - Essai rapide Nitrifizierung und Hemmung der Nitrifizierung -

par oxydation de l'ammonium (ISO 15685:2012) Schnellverfahren mittels Ammoniumoxidation (ISO

15685:2012)
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 15685:2020 E

worldwide for CEN national Members.
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SIST EN ISO 15685:2020
EN ISO 15685:2020 (E)
Contents Page

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

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SIST EN ISO 15685:2020
EN ISO 15685:2020 (E)
European foreword

The text of ISO 15685:2012 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 15685:2020 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 15685:2012 has been approved by CEN as EN ISO 15685:2020 without any modification.

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SIST EN ISO 15685:2020
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SIST EN ISO 15685:2020
INTERNATIONAL ISO
STANDARD 15685
Second edition
2012-07-15
Soil quality — Determination of potential
nitrification and inhibition of nitrification —
Rapid test by ammonium oxidation
Qualité du sol — Détermination de la nitrification potentielle et inhibition
de la nitrification — Essai rapide par oxydation de l’ammonium
Reference number
ISO 15685:2012(E)
ISO 2012
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SIST EN ISO 15685:2020
ISO 15685:2012(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 15685:2020
ISO 15685:2012(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 .............................................................................................................................................................. 3

7 Sampling, storage and characterization of samples ................................................................................. 3

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

8.1 Testing soils .......................................................................................................................................................... 4

8.2 Testing the effect of chemicals ........................................................................................................................ 4

8.3 Testing polluted soils ......................................................................................................................................... 4

8.4 Testing water extracts of biosolids ................................................................................................................ 5

9 Calculations .......................................................................................................................................................... 5

10 Test report ............................................................................................................................................................. 5

Annex A (informative) Results of a laboratory ring test ........................................................................................... 7

Annex B (informative) Micro-scale measurement of potential nitrification rates.............................................. 9

Bibliography ..................................................................................................................................................................... 11

© ISO 2012 – All rights reserved iii
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SIST EN ISO 15685:2020
ISO 15685:2012(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 15685 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 15685:2004), which has been technically revised.

iv © ISO 2012 – All rights reserved
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SIST EN ISO 15685:2020
INTERNATIONAL STANDARD ISO 15685:2012(E)
Soil quality — Determination of potential nitrification and
inhibition of nitrification — Rapid test by ammonium oxidation
1 Scope

This International Standard specifies a rapid method for the determination of the potential rate of ammonium

oxidation and inhibition of nitrification in soils. This method is suitable for all soils containing a population of

nitrifying microorganisms. It can be used as a rapid screening test for monitoring soil quality and quality of

wastes, and is suitable for testing the effects of cultivation methods, chemical substances [except volatiles,

i.e. H > 1 (Henry’s constant)], extracts of biosolids and pollution in soils.
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 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using

barium chloride solution

ISO 11261, Soil quality — Determination of total nitrogen — Modified Kjeldahl method

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

ISO 14256-2, Soil quality — Determination of nitrate, nitrite and ammonium in field-moist soils by extraction

with potassium chloride solution — Part 2: Automated method with segmented flow analysis

EN 14735, Characterization of waste — Preparation of waste samples for ecotoxicity tests

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
inhibitory dose

amount of a chemical added to soil that effectively inhibits biological activity by a stated percentage after a

given time, in comparison with an untreated control

NOTE It is expressed as a percentage. For example, ID25 and ID50 indicate a 25 % and 50 % inhibition of biological

activity, respectively.
© ISO 2012 – All rights reserved 1
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SIST EN ISO 15685:2020
ISO 15685:2012(E)
3.2
biosolids

organic products used in agriculture, which include sewage sludge, compost, manure and some industrial wastes

4 Principle

Ammonium oxidation, the first step in autotrophic nitrification in soil, is used to assess the potential activity of

microbial nitrifying populations. Autotrophic ammonium-oxidizing bacteria are exposed to ammonium sulfate

in a soil slurry buffered at pH 7,2. Oxidation of the nitrite performed by nitrite-oxidizing bacteria in the slurry

is inhibited by the addition of sodium chlorate. The subsequent accumulation of nitrite is measured over a 6 h

incubation period, and is taken as an estimate of the potential activity of ammonium-oxidizing bacteria. As the

generation time of ammonia-oxidizing bacteria is long (> 10 h), the method provides a measure of the potential

activity of the nitrifying population at the time of sampling. It does not measure growth of the nitrifying population.

Chemical test substances and extracts of biosolids can be tested by adding them to the slurry at various

concentrations. The test can be used for comparison of different soils (e.g. polluted and reference soils).

Effects of polluted soils, waste samples and other solid materials on nitrification can be evaluated by measuring

the method on mixtures of these samples with a reference soil.

Substances known to be active at pH < 7,2 should be added in due time before starting the test to allow

substances to exhibit their effects on nitrifying bacteria.
5 Reagents
5.1 Distilled water.
5.2 Potassium dihydrogenphosphate, c(KH PO = 0,2 mol/l).
2 4
5.3 Dipotassium hydrogenphosphate, c(K HPO = 0,2 mol/l).
2 4
5.4 Sodium chlorate, c(NaClO = 0,5 mol/l).
5.5 Diammonium sulfate, (NH ) SO .
4 2 4
5.6 Sodium hydrogencarbonate, c(NaHCO = 5 mmol/l).
5.7 Potassium chloride, c(KCl = 4 mol/l).
5.8 Stock solution A.
Prepare stock solution A as follows.
Potassium dihydrogenphosphate, KH PO (5.2) 28 ml
2 4
Dipotassium hydrogenphosphate, K HPO (5.3) 72 ml
2 4
Distilled water (5.1) 100 ml
5.9 Test medium.
Prepare the test medium as follows.
Stock solution A (5.8) 10 ml
Sodium chlorate, NaClO (5.4) 10 ml to 30 ml
Diammonium sulfate, (NH ) SO (5.5) 0,198 g
4 2 4
2 © ISO 2012 – All rights reserved
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SIST EN ISO 15685:2020
ISO 15685:2012(E)
Dilute to 1 000 ml with distilled water (5.1).

The test medium contains 1 mmol/l of potassium phosphate buffer, 5 mmol/l to 15 mmol/l of sodium chlorate

and 1,5 mmol/l of diammonium sulfate, and has a pH of approximately 7,2.

The sodium chlorate concentration selected within the range given should be sufficient for effective inhibition

of biological nitrate formation, while not having negative effects on ammonium oxidation. If necessary, test the

influence of the sodium chlorate concentration.

Test chemicals to be added to the test medium shall be dissolved in the phosphate buffer described and added

before diluting to 1 l. For test chemicals with low water solubility, prepare solutions by mechanical dispersion

or by using vehicles such as organic solvents, emulsifiers or dispergents of low toxicity to ammonium-oxidizing

bacteria. If any vehicle is used to add a test chemical to the slurry or a soil, additional control shall be performed

to assess possible effects of this vehicle on ammonium oxidation.

Add extracts of biosolids, when tested, separately to the test medium before dilution to 1 l.

NOTE When a carbon source is needed, e.g. when testing biosolids with high nitrate levels, add 10 ml of

NaHCO (5.6) to the test medium before diluting to 1 l.
6 Apparatus
6.1 Ordinary laboratory equipment.
6.2 Orbital shaking incubator, thermostatically controlled.
7 Sampling, storage and characterization of samples

Guidance on sampling, sample processing and storage is given in ISO 10381-6. For the testing of chemicals

or biosolids, or for mixing tests with polluted soils, experimental soil with an ammonium-oxidizing activity of

between 200 ng N/g and 800
...

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