SIST EN ISO 18187:2024

SLOVENSKI STANDARD
01-december-2024
Nadomešča:
SIST EN ISO 18187:2018
Kakovost tal - Kontaktni preskus za trdne vzorce z dehidrogenazno aktivnostjo
Arthrobacter globiformis (ISO 18187:2024)
Soil quality - Contact test for solid samples using the dehydrogenase activity of
Arthrobacter globiformis (ISO 18187:2024)
Bodenbeschaffenheit - Feststoffkontakttest unter Verwendung der
Dehydrogenaseaktivität von Arthrobacter globiformis (ISO 18187:2024)
Qualité du sol - Essai contact pour échantillons solides utilisant l'activité déshydrogénase
de Arthrobacter globiformis (ISO 18187:2024)
Ta slovenski standard je istoveten z: EN ISO 18187:2024
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
May 2024
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 18187:2018
English Version
Soil quality - Contact test for solid samples using the
dehydrogenase activity of Arthrobacter globiformis (ISO
18187:2024)
Qualité du sol - Essai contact pour échantillons solides Bodenbeschaffenheit - Feststoffkontakttest unter
utilisant l'activité déshydrogénase de Arthrobacter Verwendung der Dehydrogenaseaktivität von
globiformis (ISO 18187:2024) Arthrobacter globiformis (ISO 18187:2024)
This European Standard was approved by CEN on 15 May 2024.

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, Türkiye 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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18187:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 18187:2024) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with 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 2024, and conflicting national standards
shall be withdrawn at the latest by November 2024.
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.
This document supersedes EN ISO 18187:2018.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 18187:2024 has been approved by CEN as EN ISO 18187:2024 without any modification.

International
Standard
ISO 18187
Second edition
Soil quality — Contact test for solid
2024-05
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 18187:2024(en) © ISO 2024
ISO 18187:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 18187:2024(en)
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 Reagents .6
6 Apparatus . 8
7 Procedure . 9
7.1 Preparation of dilutions .9
7.2 Reference substances and positive control preparation .9
7.3 Contact test procedure .10
7.3.1 General .10
7.3.2 Aeration .10
7.3.3 Deactivation .11
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 interlaboratory test .15
Annex B (informative) Preparation of test organisms .22
Annex C (informative) Testing chemical substances .24
Bibliography .26

iii
ISO 18187:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 18187:2016), which has been technically
revised.
The main changes are as follows:
— the scope was amended to include the possibility of applying the contact assay for testing the effect of
chemicals (as detailed in Annex C);
— further details regarding other potential interfering factors (for the testing of plastic and low-density
waste materials) on the performance of the contact assay were addressed, and adequate methodological
alternatives were suggested to reduce uncertainties in the assay outcome;
— validity criteria were updated to include the range of dehydrogenase activity inhibitions expected under
three other reference substances (i.e. copper sulfate (II) pentahydrate, 3,5-dichlorophenol, zinc sulfate
heptahydrate) that are spiked into quartz sand and used as alternative positive controls in the quality
testing of waste materials;
— in Clause B.4, genome sequencing is proposed to confirm the identity of Arthrobacter globiformis, instead
of microbiological identification strips which are no longer recommended.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 18187:2024(en)
Introduction
This document 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 it uses a ubiquitous soil
[1][2]
bacterial species with high affinity to surfaces whose dehydrogenases are involved in different biological
mechanisms withstanding bacteria integrity (e.g. respiratory chains). Moreover, it has been noticed that this
[3][4][5][6][7]
parameter (dehydrogenase activity inhibition) is quite sensitive 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 [8].
This document is also based on Reference [9].
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 and in Reference [10].

v
International Standard ISO 18187:2024(en)
Soil quality — Contact test for solid samples using the
dehydrogenase activity of Arthrobacter globiformis
1 Scope
This document 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
in natural samples, such as soils and waste materials. Although not the main purpose, the contact test can
additionally be used for testing the effect of chemicals, as described in the Annex C. The test yields a result
within 6 h and can therefore be used for screening potentially contaminated test material.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of sludge and
sediment samples
ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic
conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
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.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
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 (3.13) B or
inoculum (3.12)]
.
Note 1 to entry: It is used to standardize the analysis.

ISO 18187:2024(en)
3.3
positive control
sample of a control substrate (3.6) with a mixture of known solutions [distilled water, medium (3.13) B or
inoculum (3.12)] and a reference substance
Note 1 to entry: It is used to check the sensitivity of the test organism.
3.4
blank A
blank, which sets the own fluorescence of the substrate after being deactivated
Note 1 to entry: Blank A 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 B is not added with bacteria.
3.6
control substrate
reference or standard substrate used as a control and as a dilution substrate for preparing dilution/
concentration series with test substrates (3.7), a reference substance or a test chemical
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, redox reactions, and
biosynthetic metabolic processes (e.g. the respiratory chain), which require cell integrity to be produced
[2]
Note 1 to entry: These enzymes can reduce resazurin into resorufin in the extracellular environment .
Note 2 to entry: See Reference [11].
3.10
ECx
effect concentration for x % effect
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 substrate, in milligrams per
kilogram.
ISO 18187:2024(en)
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).
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 [12]).
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: It is when preparing the test (3.7) 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
kinetics period)
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
lowest value of the dilution factor 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)]. An LID of 8 corresponds to a dilution of soil or waste of 1:8.

ISO 18187:2024(en)
4 Principle
The bacterium 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 activity,
[2]
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 the subsequent lowering
of fluorescence emission.
5 Reagents and material
5.1 Test organisms
1)
The test organism is Arthrobacter globiformis (Conn 1982) Conn and Dimmick 1947 , which is common in
soils. Arthrobacter species belong to the Microccocaceae family. They are mostly obligate aerobic organisms,
[13]
although some species can exhibit anaerobic metabolism under 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
[14]
can lead to gram variability by differential 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 bacterium strain can be achieved from commercially available freeze-dried or liquid-frozen aliquots from
culture collections, e.g. Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (strain number
2)
20124), 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 in 5.2.2 and 5.2.3 should be used 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 [15]). The reference soil
[item a)] is preferred, but if such a soil is not available, either a standard natural soil or a standard artificial
soil can 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.
TM
1) Strain number ATCC 8010 Arthrobacter globiformis (Conn) Conn and Dimmick.
2) 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 bacterium. This information is given for the convenience of users of this document and
does not constitute an endorsement by ISO of these firms.

ISO 18187:2024(en)
b) Standard natural soil with the following characteristics: C ≤ (1,7 to 2,6) %; sand (particle
org
size 0,063 mm to 2 mm) content of 50 % to 75 %; < 20 % of particles less than 0,02 mm; pH 5 to 7,5.
3)
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; pH 5,5 to 6,5).
[16]
The substrate called standard artificial soil has the following composition:
Percentage expressed on
dry mass basis
— Sphagnum peat finely ground and with no visible plant remains
5 %
(particle size ≤ 1 mm)
— Kaolinite clay containing not less than 30 % kaolinite 20 %
— Industrial quartz sand (dominant fine sand with 50 % to 75 % of
74 %
particle size 0,063 mm to 2 mm)
— Calcium carbonate 1 %
Standard 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, but other control soils (as suggested above) can be selected.
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 18400-206;
— 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, to avoid (bio)chemical changes that can
interfere with the ecotoxicity results. For long-term storage, the samples can be frozen at (−20 ± 2) °C.
NOTE For waste samples not microbially-active or not prone to (bio)chemical changes that can lead to a

variation in the ecotoxicity testing results, a storage up to two months at (4 ± 2) °C can be considered upon the
judgment of the operator (as per EN 14735).
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). Metals and organic
contaminants in the samples should be screened as this provides helpful information for data interpretation.
For the interpretation of test results, the following characteristics should be determined for each sample:
[17] [18]
a) pH in accordance with ISO 10390 for soil samples, EN 15933 for biowaste samples and
[17]
ISO 10390 for other solid wastes;
[19]
b) texture (sand, loam, silt) in accordance with ISO 11277 ;
3) 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 can be used if they can be shown to lead to the same results.

ISO 18187:2024(en)
[20] [21]
c) water content in accordance with ISO 11465 for soil samples and EN 15934 for biowaste samples;
[22]
d) water holding capacity according to ISO 11268-2 ;
[23]
e) cation exchange capacity in accordance with ISO 11260 ;
[24] [25]
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 are 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 performing the test.
When using soil or waste natural samples, dilutions can 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 to define the appropriate percentage of water content according to the level of OM or TOC in this
type of samples.
5.4 Reagents
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.

ISO 18187:2024(en)
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 D(+)-glucose, and
— 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.

ISO 18187:2024(en)
5.4.11 Benzyldimethylhexadecylammonium chloride, reference substance [also called cetalkonium
4)
chloride (C16-BAC)], CH (CH )15N(Cl)(CH )2CH C H [CAS Registry Number® (CAS RN) : 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 (between
−1 −1
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-
[26]
negative bacteria by affecting the permeability of their cytoplasmic membrane and leading to cytolysis.
5.4.12 Copper sulfate (II) pentahydrate, additional reference substance (CAS RN 7758-99-8).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume to have
−1
a final concentration of 500 mg Cu · kg soil dry mass.
5.4.13 3,5-dichlorophenol (3,5-DCP), additional reference substance (CAS RN 591-35-5).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume to have
−1
a final concentration of 100 mg 3,5-DCP · kg soil dry mass.
5.4.14 Zinc sulfate heptahydrate, additional reference substance (CAS RN 7446-20-0).
Prepare a stock solution in water (5.4.1) before spiking the quartz sand with its appropriate volume to have
−1
a final concentration of 100 mg Zn · kg 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 [27]).
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 of bacterial inoculum at 600 nm.
6.9 Fluorometer for microplates, emission at 590 nm, excitation at 535 nm.
NOTE As an example, the fluorometer 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.
4) Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society (ACS). This
information is given for the convenience of users of this document 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 18187:2024(en)
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.
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 can 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 substances and positive control preparation
A positive control should be performed every time a contact test is run.
C16-BAC (see 5.4.11) should be used as a reference substance in the testing of soil samples. In an
interlaboratory test (see Reference [6]), LUFA standard soil type 2.2 [see 5.2.2, b)] was spiked with C16-BAC
−1 [5]
at a concentration of 600 mg kg . The mean value of the calculated inhibition was 58,2 % (BAC).
For preparation of the C16-BAC positive control, spike LUFA standard soil type 2.2 [see 5.2.2, b)] with C16-
−1
BAC (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 standard
−1
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).
For the testing of waste materials (or chemicals, see Annex C), the positive control should also be prepared
in the recommended control substrate [i.e. quartz sand; see 5.2.2, c)]. Other reference substances should
be used, depending on the laboratory resources, or on the type of contaminants in the solid sample (if
known) or testing chemicals. For these situations, and in accordance with the interlaboratory test results
−1
(see Annex A), copper (II) sulfate pentahydrate (see 5.4.12; at 500 mg Cu · kg ), 3,5-DCP (see 5.4.13;
−1 −1
at 100 mg 3,5-DCP · kg ) or zinc
...