Soil quality - Contact test for solid samples using the dehydrogenase activity of Arthrobacter globiformis (ISO 18187:2016)

ISO 18187:2016 specifies a rapid method for assessing solid samples in an aerobic suspension, by determining the inhibition of dehydrogenase activity of Arthrobacter globiformis using the redox dye resazurin.
It is applicable for assessing the effect of water-soluble and solid matter bounded non-volatile contaminants of natural samples, such as soils and waste materials. The test yields a result within 6 h and can therefore be used for screening potentially contaminated material.

Bodenbeschaffenheit - Feststoffkontakttest unter Verwendung der Dehydrogenaseaktivität von Arthrobacter globiformis (ISO 18187:2016)

Diese Internationale Norm legt ein Schnellverfahren zur Beurteilung von Feststoffproben in einer aeroben Suspension durch Bestimmung der Hemmung der Dehydrogenaseaktivität von Arthrobacter globiformis unter Verwendung des Redox-Farbstoffes Resazurin fest.
Sie gilt für die Beurteilung der Auswirkung von wasserlöslichen und feststoffgebundenen nicht flüchtigen Verunreinigungen in natürlichen Proben, wie Böden und Abfällen. Die Prüfung führt innerhalb von 6 h zu einem Ergebnis und kann deshalb zum Screening potentiell verunreinigten Materials eingesetzt werden.

Qualité du sol - Essai contact pour échantillons solides utilisant l'activité déshydrogénase de Arthrobacter globiformis (ISO 18187:2016)

ISO 18187:2016 spécifie une méthode rapide d'évaluation d'échantillons solides en suspension aérobie, en déterminant l'inhibition de l'activité déshydrogénase de Arthrobacter globiformis à l'aide d'un indicateur redox coloré, la résazurine.
Cette méthode est applicable à l'évaluation de l'effet de contaminants non volatils, liés aux matières solides ou solubles dans l'eau, présents dans des échantillons naturels tels que des sols et des déchets. L'essai délivre un résultat en 6 h et peut donc être utilisé pour le criblage de matériaux potentiellement contaminés.

Kakovost tal - Kontaktni preskus za trdne vzorce z dehidrogenazno aktivnostjo Arthrobacter globiformis (ISO 18187:2016)

Standard ISO 18187:2016 določa hitro metodo za ocenjevanje trdnih vzorcev v aerobni suspenziji z ugotavljanjem zaviranja dehidrogenazne aktivnosti Arthrobacter globiformis z uporabo redoks barvila resazurin.
Uporablja se za ocenjevanje učinka vodotopnih in trdnih nehlapnih onesnaževal v naravnih vzorcih, kot so prsti in odpadni materiali. Test zagotovi rezultate v roku 6 ur, zato se lahko uporablja za preverjanje potencialno kontaminiranega materiala.

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Published
Publication Date
20-Feb-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
21-Feb-2018
Completion Date
21-Feb-2018

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SLOVENSKI STANDARD
SIST EN ISO 18187:2018
01-julij-2018
Kakovost tal - Kontaktni preskus za trdne vzorce z dehidrogenazno aktivnostjo
Arthrobacter globiformis (ISO 18187:2016)

Soil quality - Contact test for solid samples using the dehydrogenase activity of

Arthrobacter globiformis (ISO 18187:2016)
Bodenbeschaffenheit - Feststoffkontakttest unter Verwendung der
Dehydrogenaseaktivität von Arthrobacter globiformis (ISO 18187:2016)

Qualité du sol - Essai contact pour échantillons solides utilisant l'activité déshydrogénase

de Arthrobacter globiformis (ISO 18187:2016)
Ta slovenski standard je istoveten z: EN ISO 18187:2018
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 18187:2018 en,fr,de

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

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SIST EN ISO 18187:2018
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SIST EN ISO 18187:2018
EN ISO 18187
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2018
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Contact test for solid samples using the
dehydrogenase activity of Arthrobacter globiformis (ISO
18187:2016)

Qualité du sol - Essai contact pour échantillons solides Bodenbeschaffenheit - Feststoffkontakttest unter

utilisant l'activité déshydrogénase de Arthrobacter Verwendung der Dehydrogenaseaktivität von

globiformis (ISO 18187:2016) Arthrobacter globiformis (ISO 18187:2016)
This European Standard was approved by CEN on 14 February 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18187:2018 E

worldwide for CEN national Members.
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SIST EN ISO 18187:2018
EN ISO 18187:2018 (E)
Contents Page

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

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SIST EN ISO 18187:2018
EN ISO 18187:2018 (E)
European foreword

The text of ISO 18187:2016 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 18187:2018 by

Technical Committee CEN/TC 444 “Test methods for 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 August 2018, and conflicting national standards shall

be withdrawn at the latest by August 2018.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 18187:2016 has been approved by CEN as EN ISO 18187:2018 without any modification.

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SIST EN ISO 18187:2018
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SIST EN ISO 18187:2018
INTERNATIONAL ISO
STANDARD 18187
First edition
2016-05-01
Soil quality — Contact test for solid
samples using the dehydrogenase
activity of Arthrobacter globiformis
Qualité du sol — Essai contact pour échantillons solides
utilisant l’activité déshydrogénase de Arthrobacter globiformis
Reference number
ISO 18187:2016(E)
ISO 2016
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SIST EN ISO 18187:2018
ISO 18187:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, 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.
ISO copyright office
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 18187:2018
ISO 18187:2016(E)
Contents Page

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

Introduction ..................................................................................................................................................................................................................................v

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

5 Reagents and material .................................................................................................................................................................................... 4

5.1 Test organisms ........................................................................................................................................................................................ 4

5.2 Control substrates ................................................................................................................................................................................ 4

5.2.1 General...................................................................................................................................................................................... 4

5.2.2 Control for soils ................................................................................................................................................................. 4

5.2.3 Control for waste material ....................................................................................................................................... 5

5.3 Test substrates ........................................................................................................................................................................................ 5

5.4 Chemicals ..................................................................................................................................................................................................... 6

6 Apparatus ..................................................................................................................................................................................................................... 8

7 Procedure..................................................................................................................................................................................................................... 9

7.1 Preparation of dilutions .................................................................................................................................................................. 9

7.2 Reference substance and positive control preparation ....................................................................................... 9

7.3 Contact test procedure ..................................................................................................................................................................... 9

7.3.1 General...................................................................................................................................................................................... 9

7.3.2 Aeration ................................................................................................................................................................................10

7.3.3 Deactivation ......................................................................................................................................................................10

7.3.4 Preparation of the inoculum ...............................................................................................................................11

7.3.5 Incubation and fluorescence measurement ..........................................................................................11

7.4 Interferences ..........................................................................................................................................................................................11

8 Calculation and expression of results ..........................................................................................................................................12

8.1 Calculation ...............................................................................................................................................................................................12

8.1.1 Relative fluorescence ................................................................................................................................................12

8.1.2 Determining the percentage of inhibition ..............................................................................................12

8.2 Expression of results .......................................................................................................................................................................12

9 Validity of the test .............................................................................................................................................................................................13

10 Statistical analysis ............................................................................................................................................................................................13

11 Test report ................................................................................................................................................................................................................14

Annex A (informative) Results on the ring test .......................................................................................................................................15

Annex B (informative) Preparation of test organisms ....................................................................................................................21

Annex C (informative) Testing chemical substances .........................................................................................................................23

Bibliography .............................................................................................................................................................................................................................25

© ISO 2016 – All rights reserved iii
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SIST EN ISO 18187:2018
ISO 18187:2016(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.

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 meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological

methods.
iv © ISO 2016 – All rights reserved
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SIST EN ISO 18187:2018
ISO 18187:2016(E)
Introduction

This International Standard describes the miniaturized solid contact assay with Arthrobacter globiformis

that allows the preliminary assessment of solid material (i.e. soil and waste materials) within 6 h. The

principle of the assay relies on dehydrogenase activity inhibition of an added test organism, caused by

bioavailable toxic substances in soil and waste samples. This is an ecologically relevant assay as far as

[16][6]

it uses a ubiquitous soil bacteria species with high affinity to surfaces which dehydrogenases are

involved in different biological mechanisms withstanding bacteria integrity (e.g. respiratory chains).

Moreover, it has been noticed that this parameter (dehydrogenase activity inhibition) is quite sensitive

[19][10][14][15]
to different toxic substances.

Overall, this assay is non-labour-intensive, rapid, cost-effective and sensitive, providing results that

improve the physical and chemical assessment of natural samples while allowing a quick indication of

their biological effects.

The miniaturized solid contact assay is based on the solid contact assay established by Reference [7].

This International Standard is also based on Reference [23].

The results of an interlaboratory trial towards the evaluation of test variability to assess different

waste and soil samples, as well as chemicals, are presented in Annex A.
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SIST EN ISO 18187:2018
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SIST EN ISO 18187:2018
INTERNATIONAL STANDARD ISO 18187:2016(E)
Soil quality — Contact test for solid samples using the
dehydrogenase activity of Arthrobacter globiformis
1 Scope

This International Standard specifies a rapid method for assessing solid samples in an aerobic

suspension, by determining the inhibition of dehydrogenase activity of Arthrobacter globiformis using

the redox dye resazurin.

It is applicable for assessing the effect of water-soluble and solid matter bounded non-volatile

contaminants of natural samples, such as soils and waste materials. The test yields a result within 6 h

and can therefore be used for screening potentially contaminated material.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of sludge

and sediment samples

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

CEN/TR 15310-1, Characterization of waste — Sampling of waste materials — Part 1: Guidance on

selection and application of criteria for sampling under various conditions

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
contact time
exposure period of the bacteria to a suspension of solid matter
3.2
negative control

sample of a control substrate (3.6) with a mixture of known solutions [distilled water, medium B or

inoculum (3.12)].
Note 1 to entry: It is used to standardize the analysis.
3.3
positive control

sample of a control substrate (3.6) with a mixture of known solutions [distilled water, medium B or

inoculum (3.12)] and a reference substance
Note 1 to entry: It is used to check the sensitivity of the test organism.
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SIST EN ISO 18187:2018
ISO 18187:2016(E)
3.4
blank A
blank, which sets the own fluorescence of the substrate after being deactivated
Note 1 to entry: Blank is not added with bacteria.
3.5
blank B

blank, which sets the natural fluorescence of the substrate without being deactivated

Note 1 to entry: Blank is not added with bacteria.
3.6
control substrate

reference or standard substrate used as a control and as medium (3.13) for preparing

dilution/concentration series with test substrates (3.7) or a reference substance

EXAMPLE Quartz sand or LUFA standard soil type 2.2.
3.7
test substrate

natural or artificial substrate that is naturally contaminated or spiked with a test chemical

Note 1 to entry: The test substrate is the test material (3.8) after being prepared for testing (e.g. sieved) and/or

diluted with a control substrate (3.6).
3.8
test material
original sample of soil or waste material without any changes (e.g. sieving)
3.9
dehydrogenase activity

activity of hydrogen-abstracting enzymes which are involved in many energy and biosynthesis

metabolic processes (e.g. the respiratory chain) and which require cell integrity to be produced

[6]

Note 1 to entry: These enzymes can reduce resazurin into resorufin in the extracellular environment.

Note 2 to entry: See Reference [21].
3.10
effect concentration for x % effect
ECx

concentration (mass fraction) of a test substance or sample that causes x % of an effect on a given

endpoint within a given exposure period when compared with a control

EXAMPLE An EC50 is a concentration estimated to cause an effect on a test end point in 50 % of an exposed

population over a defined exposure period.

Note 1 to entry: The ECx is expressed as a percentage of soil or waste tested per dry mass of soil mixture. When

chemicals are tested, the ECx is expressed as mass of the test substance per dry mass of soil, in milligrams per

kilogram.
3.11
freeze-dried bacteria

bacterial culture preserved through the water removing of a frozen cell suspension by sublimation

under reduced vacuum pressure

Note 1 to entry: The preserved cultures can be stored at (-20 ± 2) °C. The bacteria are active after being

reconstituted with sterilized distilled water [20 min to 30 min at (6 ± 2) °C] and ready to be used in the test, see

7.3.4 b).
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SIST EN ISO 18187:2018
ISO 18187:2016(E)
3.12
inoculum
suspension of bacteria used to inoculate a nutrient solution
3.13
medium
aqueous nutritive solution required for bacterial growth
3.14
optical density of bacterial inoculum

measurement of the attenuation of a light beam passing through a bacterial suspension at 600 nm (used

to determine the cell count indirectly)

Note 1 to entry: In a bacterial test, the absorbance is usually measured as FAU (formazine attenuation units) at

600 nm (see Reference [3]).
3.15
test start

moment when the substrates, reagents and the bacterial inoculum (3.12) are prepared immediately

before the incubation and reaction period

Note 1 to entry: Here is when preparing the test and control substrates (3.6) for incubation (i.e. Table 1, day 0).

3.16
reaction time

time it takes for the enzyme to react (from the addition of the resazurin solution until the end of the

reaction)
3.17
slope

quotient of the relative fluorescence (3.18) variation along the reaction time (3.16) between 15 min

and 45 min

Note 1 to entry: The slope (expressed as min ) results from fitting a linear regression model to the fluorescence

readings over time.
3.18
relative fluorescence

fluorescence measured for each treatment (control and test) after subtracting the fluorescence of the

respective blank A (3.4)
3.19
stock culture

bacterial culture obtained from a pure strain culture acquired from a certified laboratory

Note 1 to entry: This stock culture provides an inoculum (3.12) for the pre-culture in the test procedure.

3.20
lowest ineffective dilution
LID-value

lowest value of the dilution factor (LID) for which the test does not give an ecotoxicological relevant

reduction
Note 1 to entry: The LID is expressed as the reciprocal value of dilution.

EXAMPLE An often used dilution series is 1/2/4/8/16 [= 100 %/50 %/25 %/12,5 %/6,25 % test substrate

(3.7) to control substrate (3.6)]. A LID 8 corresponds to a dilution of soil or waste of 1 : 8.

© ISO 2016 – All rights reserved 3
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ISO 18187:2016(E)
4 Principle

The bacteria Arthrobacter globiformis is added to the solid material and incubated at (30 ± 1) °C for

2 h. After this contact time, the non-toxic redox dye resazurin is added. Due to the dehydrogenase

[6]

activity, resazurin is transformed into resorufin, in the extracellular environment. Resorufin can be

detected fluorometrically (excitation at 535 nm, emission at 590 nm) in the presence of solid matter.

The increase of resorufin is determined by measuring the fluorescence every 15 min for a period of

1 h. In order to determine the inhibition of the dehydrogenase activity, the rate of resorufin increase

in the sample is compared with the rate of resorufin increase in the control. In the presence of toxic

substances, an inhibition of dehydrogenase activity is expected. This is reflected by the reduction of

resorufin production and subsequent lowering of fluorescence emission.
5 Reagents and material
5.1 Test organisms

The test organism is Arthrobacter globiformis (Conn 1982) Conn and Dimmick 1947 (strain number

ATCC 8010), which is common in soils. Arthrobacter species belong to the Microccocaceae family. They

are mostly obligate aerobic organisms, although some species may exhibit anaerobic metabolism under

[9]

limiting oxygen conditions. Arthrobacter spp. are chemoheterotrophic, and present pleomorphic

characteristics, since they show a rod-to-coccus morphology change as they enter in the stationary

phase. Although Arthrobacter is gram-positive, it can stain gram-negative during the log-phase.

Variations in the cell wall thickness along the bacteria growth can lead to gram variability by differential

[22]

staining of the granules. However, this characteristic does not induce a differential sensitivity

between assays, as far as an inoculum in exponential growth phase is used during the reaction time.

Arthrobacter globiformis is classified in the risk group I — non-pathogenic organism.

The bacteria strain can be achieved from commercially available freeze-dried or liquid-dried reagents,

or from culture collections, e.g. Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, or

ARS Culture collection NCAUR. The bacterial suspensions used for toxicity measurements shall be

freshly prepared from stock cultures or directly used from a ready-to-use freeze-dried batch. The stock

culturing and freeze drying process of the bacteria is described in Annex B.
5.2 Control substrates
5.2.1 General

The control substrates selected from the options presented below are to prepare both the negative

(addition of distilled water, see 5.2.2, 5.2.3) and positive (addition of the reference substance, see 7.2)

controls. The moistening of the control substrates (soil or waste material) shall be made one or two

days before the test start (see Table 1). Store the substrate(s) at (4 ± 2) °C until the test start.

5.2.2 Control for soils

There are three possibilities for the choice of the control soil (see also Reference [4]). The reference soil

a) is preferred, but if such a soil is not available, either a standard natural soil or a standard artificial

soil may be used. In any case, the water content of the control soil should be adjusted to 20 %.

a) If reference soils from uncontaminated areas near a contaminated site are available, they should

be treated and characterized like the soils to be tested. If a toxic contamination or unusual soil

properties cannot be ruled out, standard control soils b) or c) should be preferred.

1) Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) GmbH, Mascheroder Weg 10, D-38124

Braunschweig, Germany; or ARS (Agricultural Research Service) Culture collection (also known as NRRL) belonging

to the National Center for Agricultural Utilization Research (NCAUR), 1815 N, University Street, Peoria, Illinois

61604, USA are examples of firms that sell this bacteria. This information is given for the convenience of users of

this document and does not constitute an endorsement by ISO of these firms.
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ISO 18187:2016(E)

b) Standard natural soil with the following characteristics: C ≤ (1,7 to 2,6) %; sand (particle size

org

0,063 mm to 2 mm) content of 50 % to 75 %; <20 % of particles less than 0,02 mm; pH 5 to 7,5.

EXAMPLE LUFA standard soil type 2.2.

c) Standard artificial soil or quartz sand (with 50 % to 75 % of sand with particle size between

0,063 mm and 2 mm).
[17]
The substrate called artificial soil has the following composition:
Percentage expressed on
dry mass basis
— Sphagnum peat finely ground and with no visible plant remains
(particle size ≤1 mm) 5 %
— Kaolinite clay containing not less than 30 % kaolinite 20 %
— Industrial quartz sand (dominant fine sand with 50 % to 75 % of par-
ticle size 0,063 mm to 2 mm) 74 %
— Calcium carbonate 1 %

Artificial soil prepared with modified peat and quartz sand particle size should be analysed more in

detail. The presence of low density particles (e.g. peat) in this artificial substrate that are likely to float

can influence the fluorescence readings.
5.2.3 Control for waste material
Quartz sand, see 5.2.2 c). The quartz sand should have a water content of 20 %.
5.3 Test substrates

The samples (soil or waste material) should be tested as soon as possible after sampling. Collect samples

as specified
— for soil in ISO 10381-6, or
— for waste materials in ISO 5667-15, EN 14735 and CEN/TR 15310-1.

Store them in the dark at (4 ± 2) °C for not more than two weeks. For long-term storage, the samples

may be frozen at (-20 ± 2) °C.
Soil and waste samples shall be passed through a siev
...

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