EN ISO 18187:2018

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

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.

Contents Page
European foreword . 3

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.

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
ISO 18187:2016(E)
© 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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CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved

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 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

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.
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.
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).
2 © ISO 2016 – All rights reserved

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
-1
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 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
1)
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.
4 © ISO 2016 – All rights reserved

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.
2)
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 sieve of 2 mm square mesh. Waste raw material (e.g.
construction waste material) should be grounded before testing (refer to EN 14735). The screening of
metals and organic contaminants in the samples is strongly advised as it provides helpful information
for data interpretation.
For interpretation of test results, the following characteristics should be determined for each sample:
a) pH in accordance with ISO 10390 for soil samples, EN 15933 for biowaste samples and ISO 10390
for other solid wastes;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465 for soil samples and EN 15934 for biowaste samples;
d) water holding capacity according to ISO 11268-2;
e) cation exchange capacity in accordance with ISO 11260;

2) LUFA Standard soil type 2.2 is the trade name of a product supplied by LUFA Speyer. This information is given
for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the
product named. Equivalent products may be used if they can be shown to lead to the same results.
ISO 18187:2016(E)
f) organic carbon in accordance with ISO 10694 for soil samples and EN 15936 for biowaste samples.
Only samples with a pH between 5 and 9 will be appropriately assessed by this contact test (see 7.4).
The water content shall be adjusted to 20 % (soil, waste material; see 5.2.2 and 5.2.3). This adjustment
shall be calculated according to the original water content of samples, which should be determined
before the preparation of the test. The moistening of samples shall be done one or two days before the
test start (see Table 1). Store the samples at (4 ± 2) °C until the test start.
When using natural samples, dilutions may also be prepared (see 7.1).
In case of testing a chemical substance in soil, a different procedure should be followed (see Annex C).
Waste materials consisting of sewage sludge with high organic matter (OM) or total organic carbon
(TOC) content should be added with more water (e.g. water content adjusted to 33 %). More tests are
being developed as to define the appropriate percentage of water content according to the level of OM
or TOC in this type of samples.
5.4 Chemicals
Unless otherwise specified, only analytical-grade reagents shall be used.
5.4.1 Water, sterilized and non-sterilized, deionized, distilled or of equivalent purity
-1
(conductivity < 10 µS·cm ).
5.4.2 Dimethyl sulfoxide solution (DMSO), C H OS, volume fraction of 4 % in distilled water.
2 6
Sterilize the solution by filtration through a polyamide membrane filter having a pore size of 0,2 μm
and using a syringe.
-1
5.4.3 Sodium hydroxide solution, NaOH of, e.g. 1 mol·l .
NOTE For the adjustment of the media pH, it can be necessary to use bases of lower or higher concentration.
-1
5.4.4 Hydrochloric acid, HCl of, e.g. 1 mol·l .
NOTE For the adjustment of the media pH, it can be necessary to use acids of lower or higher concentration.
5.4.5 Medium A, for A. globiformis stock culture (pH 7,2 to 7,4).
Dissolve
— 10 g casein peptone,
— 5 g yeast extract,
— 5 g D(+)-glucose, and
— 5 g NaCl
in water (5.4.1), make up to 1 000 ml with water and autoclave for 20 min at (121 ± 3) °C. If stored
sterilized (never opened) at (4 ± 2) °C in the dark, the solution is stable up to 12 months.
5.4.6 Medium B, for preparing the lyophilizates and the test solution.
Dilute 333,3 ml of medium A (5.4.5) in 666,6 ml of sterilized water. Or, dissolve
— 3,33 g casein peptone,
— 1,67 g yeast extract,
— 1,67 g of D(+)-glucose, and
6 © ISO 2016 – All rights reserved

ISO 18187:2016(E)
— 1,67 g NaCl
in water (5.4.1) and make the volume up to 1 000 ml with water. Autoclave the medium for 20 min at
(121 ± 3) °C. If stored sterilized (never opened) at (4 ± 2) °C in the dark, the solution is stable up to
12 months.
5.4.7 Agar (casein-peptone soymeal-peptone) slant, for a dense cell suspension of A. globiformis.
Dissolve 40 g of tryptic soy agar in water (5.4.1) and make up to 1 000 ml with water. After being
dissolved, distribute 7 ml of this medium into culture tubes (6.15). The tubes should be filled until
allowing the agar to flow just 50 mm below the neck, when the neck is laid over a horizontal 10 ml glass
pipette. Seal them with caps and then autoclave for 20 min at (121 ± 3) °C. After cooling, the tubes are
ready for use.
5.4.8 Protective medium, for freeze-drying bacteria.
Dissolve
— 20 g skim milk, and
— 5 g myo-inositol
in water (5.4.1), make up to 100 ml with water and autoclave for 10 min at (121 ± 3) °C. After autoclaving
and cooling down to (80 ± 2) °C, place the medium immediately in an ice bath to avoid caramelization. If
stored sterilized (never opened) at (4 ± 2) °C in the dark, the solution is stable up to 12 months.
5.4.9 Phosphate buffer.
Dissolve
— 8,2 g KH PO ·H O,
2 4 2
— 13,24 g K HPO ·H O,
2 4 2
— 2 g sodium acetate, C H NaO , and
2 3 2
— 2 g D(+)-glucose
in water (5.4.1) and make up to 1 000 ml with water. Adjust the pH value of the buffer to 7,0 ± 0,2 with
diluted hydrochloric acid (5.4.4) or sodium hydroxide solution (5.4.3). Autoclave the medium for 20 min
at (121 ± 3) °C. If stored at (4 ± 2) °C in the dark, the solution is stable for up to 12 months.
5.4.10 Resazurin dye solution.
Dissolve (45 ± 1) mg of resazurin in 1 000 ml of phosphate buffer (5.4.9). If stored at (4 ± 2) °C in the
dark, the blue-coloured solution is stable up to one week.
5.4.11 Benzyldimethylhexadecylammonium chloride, reference substance [also called cetalkonium
chloride (C16-BAC)], CH (CH )15N(Cl)(CH )2CH C H (CAS no.: 122-18-9).
3 2 3 2 6 5
It is a quaternary ammonium salt often used as an anti-microbial agent. C16-BAC is a homologue of
BAC (benzalkonium chloride, a C12 homologue) which is made of a mixture of alkylbenzyldimethyl-
ammonium chlorides, composed mainly of cetalkonium chloride. C16-BAC is moderately soluble in water
−1 −1
(between 2 g l and 85 g l ), and whenever needed, the solution can be sonicated to be homogenized.
If stored at (4 ± 2) °C in the dark, the solution is stable for up to one week. Its mode of action targets
mainly Gram-negative bacteria by affecting the permeability of their cytoplasmic membrane and
[19]
leading to cytolysis.
ISO 18187:2016(E)
5.4.12 Copper sulfate (II) pentahydrate, additional reference substance (CAS no. 7758-99-8).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume as
-1
to have a final concentration of 500 mg Cu kg soil dry mass.
5.4.13 3,5-dichlorophenol (3,5-DCP), additional reference substance (CAS no. 591-35-5).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume as
-1
to have a final concentration of 100 mg 3,5-DCP kg soil dry mass.
5.4.14 Zinc sulfate heptahydrate, additional reference substance (CAS no. 7446-20-0).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume as
to have a final concentration of 100 mg Zn kg-1 soil dry mass.
6 Apparatus
Use laboratory equipment and the following.
6.1 Autoclave.
6.2 Ultrasonic bath.
6.3 Sterile bench.
6.4 Freezer, -20 °C and -80 °C for storing lyophilizates and the stock cultures.
6.5 Conical flask, with cellulose stopper as a culture vessel, nominal capacity of 100 ml (e.g.
Reference [1]).
6.6 Temperature controlled incubator, capable of being maintained at (30 ± 1) °C.
6.7 Horizontal shaker.
6.8 Photometer, suitable for measuring the optical density at 600 nm.
6.9 Fluorimeter for microplates, emission at 590 nm, excitation at 535 nm.
NOTE As an example, the fluorimeter can have the following features: sensitivity around 5 pM of fluorescein
(top and bottom reading), dynamic range of five decades, filter-dependent bandpass, tungsten quartz halogen
lamp with a photomultiplier (PMT) detection system.
6.10 pH-meter.
6.11 Multipette or micropipettes, 10 000 µl, 1 000 µl, 200 µl, and 20 µl.
6.12 Tips or combitips, (suitable for 10 ml) autoclavable.
6.13 Microplates, 24 well with lid, transparent and flat bottom.
6.14 Round-bottom glass bottle and adaptable top filter, with 0,22 µm pore, if necessary for the
lyophilizer.
6.15 Glass culture tubes with caps, 16 mm × 160 mm, for agar slant.
8 © ISO 2016 – All rights reserved

ISO 18187:2016(E)
6.16 Screw-cap vials, resistant to -80 °C, nominal capacity of 1,5 ml or 2 ml.
6.17 Membrane polyamide filter, pore size 0,2 µm, diameter 25 mm, and syringe.
6.18 Water bath.
6.19 Lyophilizer.
6.20 Sieve, pore size 2 mm.
7 Procedure
7.1 Preparation of dilutions
If very toxic samples are being tested, a geometric dilution series may be prepared by mixing different
proportions of sample (soil or waste material, see 5.3) with the respective control substrate (see 5.2).
Mixture ratios of 100 %, 50 %, 25 %, 12,5 %, 6,25 % and 0 % of sample dry mass are suggested. At the
end, adjust the water content as stated in 5.2 and 5.3.
7.2 Reference substance and positive control preparation
A positive control should be performed every time a contact test is run.
C16-BAC (see 5.4.11) is recommended as a reference substance. In a round robin test (see Reference [7]),
-1
LUFA standard soil type 2.2 [see 5.2.2, b)] was spiked with C16-BAC at a concentration of 600 mg kg .
[14]
The mean value of the calculated inhibition was 58,2 % (BAC).
For preparation of the positive control, spike LUFA standard soil type 2.2 (see 5.2.2) with C16-BAC
-1
(see 5.4.11) in a concentration of 600 mg kg (soil dry mass). Prepare a stock solution by dissolving
(600 ± 2) mg of C16-BAC (see 5.4.11) in 100 ml of distilled water. Afterwards, spike 10 g of LUFA
-1
standard soil type 2.2 with 1 ml of the stock solution (6 mg ml ). Mix the soil until the substance is
homogeneously distributed. If stored at -20 °C, the prepared positive control substrate is stable for up
to six months. If using a freshly made positive control substrate, it should be prepared one or two days
before the test start for allowing soil-chemical equilibrium (see Table 1). Adjust the water content of the
substrate (see 5.2).
Besides using LUFA standard soil type 2.2, the positive control should also be prepared for the control
substrate selected according to the type of sample being tested (see 5.2).
NOTE If another reference substance is needed depending on laboratory resources or on the type of
contaminants in the solid sample (if known) or testing chemicals, and in accordance with the ring test results
-1
(see Annex A), it is suggested to use copper (II) sulfate pentahydrate (see 5.4.12; at 500 mg Cu kg ), 3,5-DCP (see
-1 -1
5.4.13; at 100 mg 3,5-DCP kg ) or zinc sulfate heptahydrate (see 5.4.14; at 100 mg Zn kg ) as additional reference
substances spiked into quartz sand. Spike this control soil one or two days before the test start (see Table 1). As
a guidance, the concentrations mentioned cause an average inhibition of A. globiformis dehydrogenase activity
between 40 % and 100 % for copper sulfate, and between 50 % and 80 % for 3,5-DCP and zinc sulfate.
7.3 Contact test procedure
7.3.1 General
One or two days (day -2 or day -1, see Table 1) before the test start, weigh (600 ± 6) mg of the pre-
moistened controls (negative and positive) (see 5.2) and test substrates (see 5.3) into the wells of a
24-well microplate. Use at least four replicates per treatment. Weigh the same amount of each substrate
ISO 18187:2016(E)
(controls and test) to prepare two (preferably three) replicates for each blank A and B (see 3.4, 3.5,
Table 1).
— Blank A — This blank sets the remaining dehydrogenase activity measured (as fluorescence) in
the different treatments after deactivation, or the own fluorescence of the substrate material (e.g.
organic matter type). The fluorescence of the blank A (controls and test samples) shall be subtracted
from the fluorescence of the respective treatments exposed to the bacteria (see 8.1.1).
— Blank B — This blank indicates the activity level of dehydrogenase in natural samples and the
efficiency of the deactivation step by comparing with blank A. Hence, blank B is a useful measurement
for data interpretation.
The blank B shall be performed in separate microplates. Store the microplates closed with the lid in the
dark at (4 ± 2) °C until further testing.
Table 1 — Overview of the test procedure
Negative Positive
Test day Step Blank B Blank A Test sample
control control
-2 or -1 Preparation of dilutions — — — — If needed
Moistening of the substrates x x x x x
Spiking of the substrates x
— — — x (if chemicals are
tested)
Uncontaminated material
(control) 600 ± 6 600 ± 6 600 ± 6 — —
(mg wet mass)
Contaminated or spiked
600 ± 6 600 ± 6 — 600 ± 6 600 ± 6
substrate (mg wet mass)
Store microplates in the dark at 4 °C until the test start (day 0)
-1 Aeration of samples — — — — if anaerobic
0 Water (ml) 0,6 0,6 0,6 0,6 0,6
a
— Pasteurization/ Deactivation of Blank A and Test plates
Inoculum (ml) — — 0,4 0,4 0,4
Medium B (ml) 0,4 0,4 — — —
Two hours of incubation at (30 ± 1) °C in the dark
Resazurin solution (ml) 0,8 0,8 0,8 0,8 0,8
Measurement of the kinetic (every 15 min) during 1 h
a
Test plates are the 24-well microplates containing the negative and positive controls, and the test samples, which are
all added with A. globiformis inoculum. The Blank A and B plates are not added with the inoculum, but only with medium B.
The blank B plates are not subjected to the pasteurization/deactivation step.
7.3.2 Aeration
In case of anaerobic soils and waste materials, aerate them one day before the test start (day -1, see
Table 1), by shaking the mixtures (600 mg ± 6 mg of sample wet mass plus 0,6 ml of sterile distilled
−1
water) up to 24 h on a horizontal shaker at a suitable frequency, e.g. 150 min , in order to achieve the
first validity criterion (see Clause 9). The water shall not be removed after aeration, if no more water is
added for the incubation (see 7.3.3).
7.3.3 Deactivation
At the test start (day 0, see Table 1) add 0,6 ml of sterilized distilled water in all replicates and proceed
with the deactivation step.
10 © ISO 2016 – All rights reserved

ISO 18187:2016(E)
The natural dehydrogenase activity of the substrates should be suppressed as far as possible by pre-
treating the samples. This is made by heating the closed microplates (except the ones for blank B
...