EN ISO 16198:2015

Soil quality - Plant-based test to assess the environmental bioavailability of trace elements to plants (ISO 16198:2015)

ISO 16198:2015 specifies the plant-based test, called biotest, which enables estimation of the environmental bioavailability of trace elements to plants either basically as the concentration in shoots and roots or in a more integrative way as the net uptake flux in plants.

Bodenbeschaffenheit - Pflanzenbasierter Test zur Beurteilung der umweltrelevanten Bioverfügbarkeit von Spurenelementen für Pflanzen (ISO 16198:2015)

Qualité du sol - Test végétal pour l'évaluation de la biodisponibilité environnementale des éléments traces pour les végétaux (ISO 16198:2015)

L'ISO 16198:2015 spécifie le test végétal, dénommé «biotest» qui permet d'estimer la biodisponibilité environnementale des éléments traces pour les végétaux, soit simplement sous la forme de concentrations dans les parties aériennes et racinaires, soit de façon plus globale sous forme de flux de prélèvement net dans les végétaux.

Kakovost tal - Rastlinski biološki preskus za ocenjevanje okoljske biološke razpoložljivosti elementov v sledovih za rastline (ISO 16198:2015)

Preskus na rastlinski osnovi, v nadaljevanju biološki preskus, je preskus za ocenjevanje okoljske biološke razpoložljivosti elementov v sledovih za rastline kot vsebnost v poganjkih in koreninah ali na bolj celostni način kot neto absorpcija v rastlinah. Postopek biološkega preskusa zajema dva zaporedna koraka: (i) predhodna hidroponična rast in (ii) rast v stiku z vzorci prsti. Vsebnost elementov v sledovih v poganjkih in koreninah kot tudi njihova neto absorpcija v rastlinah se določata po koncu drugega koraka biološkega preskusa. Biološki preskus se uporablja za ocenjevanje okoljske biološke razpoložljivosti elementov v sledovih za rastline, bolj natančno poljščine, ki rastejo v prsti ali talnih materialih s prisotnostjo kisika, ob upoštevanju naslednjih alinej:
– tri rastlinske vrste (zelje, Brassica oleracea; trsikasta bilnica, Festuca arundinacea; paradižnik, Lycopersicon esculentum; glej 7.1) so predlagane v standardiziranih postopkih bioloških preskusov, uporabiti pa je mogoče tudi druge ciljne rastlinske vrste (glej dodatek A); – standardizirani postopek biološkega preskusa je potrjen za več elementov v sledovih, vključno z arzenikom (As), kadmijem (Cd), kromom (Cr), bakrom (Cu), svincem (Pb), nikljem (Ni) in cinkom (Zn), vendar so mogoči tudi drugi elementi v sledovih (glej dodatek A); – uporaba tega biološkega preskusa za prsti in talne materiale vključuje prsti, pred ali po vzorčenju polja, spremenjene s kompostom, blatom, odpadnimi vodami in drugimi (odpadnimi) materiali.

General Information

• Status: Published
• Publication Date: 13-Jan-2015
• Withdrawal Date: 30-Jul-2015
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 14-Jan-2015
• Completion Date: 14-Jan-2015
• Ref Project: SIST EN ISO 16198:2015 - Soil quality - A plant based-based biotest to assess the environmental bioavailability of trace elements to plants (ISO 16198:2015)

Overview

EN ISO 16198:2015 (ISO 16198:2015) - titled Soil quality - Plant-based test to assess the environmental bioavailability of trace elements to plants - specifies a standardized plant-based biotest for estimating the environmental bioavailability of trace elements to plants. The standard describes a laboratory procedure in which roots are grown in contact with soil (without penetrating it) so that uptake into shoots and roots - or more integratively, the net uptake flux - can be quantified. Prepared by ISO/TC 190 and adopted by CEN as EN ISO 16198:2015, it supports consistent, comparable assessments of trace element bioavailability across soils.

Key topics and requirements

The standard covers technical and procedural elements needed to run the biotest, including:

• Principle of the plant-based biotest and rationale for root–soil contact design.
• Selection of plant species and seed handling (annexes list adapted species and seed densities).
• Biotest apparatus and technical drawings for test components.
• Composition of nutrient solutions, growth-chamber climatic conditions, and pre-culture hydroponic steps.
• Soil sample pre-treatment (size reduction, sample size) and required soil analyses.
• Experimental procedure: germination, incubation with soil or soil material, plant growth period, harvests, grinding, digestion and analytical determination of trace elements.
• Validity criteria and statistical analysis requirements for biomasses and bioavailability end points.
• Reporting requirements and annexed guidance on digestion, expected biomass ranges and ring-test procedures.

Applications and users

EN ISO 16198:2015 is useful for:

• Environmental laboratories performing soil quality and contamination assessments.
• Soil scientists and ecotoxicologists quantifying bioavailability of metals and trace elements to plants.
• Regulatory bodies and consultants assessing contaminated land, risk to vegetation, and remediation effectiveness.
• Agronomists and remediation specialists comparing bioavailability across different soils or monitoring post-remediation changes.

The method provides a biologically relevant complement to chemical extraction methods by accounting for rhizosphere processes and plant uptake behavior.

Related standards

• ISO 17402 (conceptual framework for bioavailability)
• Chemical methods such as ISO 19730 (for comparison and correlation)
• Other plant-based biotests: ISO 11269‑1, ISO 11269‑2, ISO 17126, ISO 22030

Keywords: EN ISO 16198:2015, ISO 16198:2015, soil quality, plant-based biotest, bioavailability, trace elements, environmental bioavailability, soil assessment.

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2015
Kakovost tal - Rastlinski biološki preskus za ocenjevanje okoljske biološke
razpoložljivosti elementov v sledovih za rastline (ISO 16198:2015)
Soil quality - A plant based-based biotest to assess the environmental bioavailability of
trace elements to plants (ISO 16198:2015)
Bodenbeschaffenheit - Pflanzenbasierter Biotest zur Beurteilung der umweltrelevanten
Bioverfügbarkeit von Spurenelementen für Pflanzen (ISO 16198:2015)
Qualité du sol - Biotest végétal pour l'évaluation de la biodisponibilité environnementale
des éléments traces pour les végétaux (ISO 16198:2015)
Ta slovenski standard je istoveten z: EN ISO 16198:2015
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.

EUROPEAN STANDARD
EN ISO 16198
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2015
ICS 13.080.30
English Version
Soil quality - Plant-based test to assess the environmental
bioavailability of trace elements to plants (ISO 16198:2015)
Qualité du sol - Test végétal pour l'évaluation de la Bodenbeschaffenheit - Pflanzenbasierter Test zur
biodisponibilité environnementale des éléments traces pour Beurteilung der umweltrelevanten Bioverfügbarkeit von
les végétaux (ISO 16198:2015) Spurenelementen für Pflanzen (ISO 16198:2015)
This European Standard was approved by CEN on 24 October 2014.

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, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16198:2015 E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 16198:2015) has been prepared by Technical Committee ISO/TC 190 “Soil quality” in
collaboration with Technical Committee CEN/TC 345 “Characterization of soils” 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 July 2015, and conflicting national standards shall be withdrawn at the
latest by July 2015.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16198:2015 has been approved by CEN as EN ISO 16198:2015 without any modification.

INTERNATIONAL ISO
STANDARD 16198
First edition
2015-01-15
Soil quality — Plant-based test
to assess the environmental
bioavailability of trace elements to
plants
Qualité du sol — Test végétal pour l’évaluation de la biodisponibilité
environnementale des éléments traces pour les végétaux
Reference number
ISO 16198:2015(E)
©
ISO 2015
ISO 16198:2015(E)
© ISO 2015
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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2015 – All rights reserved

ISO 16198:2015(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Laboratory apparatus . 5
6 Reagents . 5
6.1 General . 5
7 Biological and growing apparatus. 6
7.1 Plant species . 6
7.2 Biotest apparatus . 6
7.3 Composition of the nutrient solutions . 9
7.4 Climatic conditions in the growth chamber.10
8 Pre-treatment and analysis of soil or soil material sample .10
8.1 Sample size and particle size reduction .10
8.2 Analyses .10
9 Experimental and analytical procedure .11
9.1 Overview of the procedure .11
9.2 Selection and preparation of seeds .11
9.3 Preculture period: Germination and pre-growth in hydroponics .11
9.4 Preparation and incubation of soil or soil material .12
9.5 Test culture period: Plant growth in contact with soil or soil material .12
9.6 Plant harvests.13
9.7 Grinding and digestion of shoots and roots .13
9.8 Analytical determination.14
10 Validity criteria .14
11 Assessment of the results .14
11.1 Determination of trace element concentrations and uptake flux in plants .14
11.2 Data presentation .16
11.3 Expression of the results .16
12 Statistical analysis .16
12.1 General .16
12.2 Plant biomasses .16
12.3 Bioavailability end points .17
13 Test report .17
Annex A (informative) Plant species adapted to the biotest procedure .19
Annex B (informative) Technical drawings of the different components of the biotest .21
Annex C (informative) Seed selection and seed density in plant pot for a range of species tested
with the standardized experimental procedure .24
Annex D (informative) Digestion and analysis of plant samples .26
Annex E (informative) Range of biomasses and trace element quantities in control plant pots .28
Annex F (informative) Ring-test .29
Bibliography .43
ISO 16198:2015(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 7, Soil and site
assessment.
iv © ISO 2015 – All rights reserved

ISO 16198:2015(E)
Introduction
One of the main objectives of ISO 17402 is to define a conceptual framework of the bioavailability of
contaminants in soils and soil materials, and to provide a guidance for the selection of methods able to
be standardized for the measurement of bioavailability. Bioavailability was thus defined according to
three successive steps:
a) “environmental availability”;
b) “environmental bioavailability”;
c) “toxicological bioavailability”.
The environmental bioavailability is consequently a prerequisite to the assessment of the toxicological
bioavailability and is directly related to the impact of pollutants on major functions of soil in the
ecosystem and more particularly to habitat and retention functions.
Environmental bioavailability can be estimated with either chemical or biological methods. In the case
of trace elements, chemical methods are usually the cheapest, easy to perform, and some of them are
already standardized at national or international level (e.g. ISO 19730). However, chemical methods
which, strictly speaking, measure the environmental availability in soils have to be correlated with
biological measurements before being used as indicators of environmental bioavailability. Whatever
chemical methods are employed, none are designed per se to address the diversity of responses observed
among different plant species or cultivars which can be attributed to a) the uptake behaviour of plants
(i.e. sensitive, tolerant, accumulator, or hyper-accumulator of trace elements) and/or b) the ability of
plants to alter the biological, physical and physical-chemical properties of their “bio-influenced zone” at
the soil-root interface, i.e. the so-called rhizosphere. It could alternatively, be suggested to apply chemical
methods directly to the rhizosphere but the sampling of the rhizosphere is definitely too tedious to be
applied routinely.
For biological methods, four standardized biotests account for rhizosphere processes as they are based
on soil-grown plants (ISO 11269-1, ISO 11269-2, ISO 17126, and ISO 22030). However, these were only
designed to predict trace element phytotoxicity, i.e. the toxicological bioavailability. In these biotests,
roots grow directly in the soil, therefore requiring a tedious washing procedure to reliably measure
trace elements accumulated in the roots. Indeed, the amount of trace elements accumulated in shoots of
non-accumulator plant species is not sufficiently sensitive to be used for assessing the environmental
bioavailability of trace elements compared to the amount accumulated in the whole plant, roots included.
Thus, there is still a need to develop biological methods accounting for rhizosphere processes and enabling
to include the root compartment in order to properly estimate the environmental bioavailability of trace
elements to plants.
Consequently, the present International Standard introduces a biotest based on the growing of roots in
contact with the soil but without penetrating it. Although this experimental design is partly artificial, it
enables a fair comparison of the bioavailability of trace elements between tested soils. In addition, the
end point measured can be more directly related to the measurement of the environmental availability
than any end point based on the measurement of toxicity.
INTERNATIONAL STANDARD ISO 16198:2015(E)
Soil quality — Plant-based test to assess the environmental
bioavailability of trace elements to plants
1 Scope
This International Standard specifies the plant-based test, hereafter called the biotest. It enables
estimation of the environmental bioavailability of trace elements to plants either basically as the
concentration in shoots and roots or in a more integrative way as the net uptake flux in plants. The
biotest procedure includes two successive steps: (i) a pre-growth of plants in hydroponics and (ii) a
growth of plants in contact with soil samples. The concentration in shoots and roots as well as the
net uptake flux of trace elements in plants are determined at the end of the second step of the biotest
procedure.
This biotest is applicable to the assessment of environmental bioavailability of trace elements to plants,
more particularly to agricultural plants, in soils or soil materials under oxic conditions, considering that
— three plant species (cabbage, Brassica oleracea; tall fescue, Festuca arundinacea; tomato, Lycopersicon
esculentum; 7.1) are suggested in the standardized biotest procedure, but additional target-plant
species can also be used (see 7.1, Annex A), and
— the standardized biotest procedure is validated for a range of trace elements including arsenic
(As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), nickel (Ni), and zinc (Zn), but
additional trace elements can also be accounted for (see Annex A).
The biotest can be applied to soils and soil materials, including soils amended before or after field
sampling with composts, sludges, wastewaters, and other (waste) materials.
NOTE 1 This biotest is not designed to assess the environmental bioavailability of trace elements that are
prone to volatilisation or resulting from uptake occurring in plant leaves following, e.g. atmospheric fallout.
NOTE 2 This biotest is not designed to assess the environmental bioavailability to plants of organic
contaminants. A similar experimental procedure could be used but the physical separation between plant roots
and soil using a polyamide mesh needs to be adapted to avoid organic contaminant sorption on the mesh.
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 3696, Water for analytical laboratory use — Specification and test methods
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
contaminated soil on the emergence and early growth of higher plants
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
ISO 16198:2015(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
contaminant
substance or agent present in the soil as a result of human activity
[SOURCE: ISO 11074:2005, 3.5.1]
Note 1 to entry: There is no assumption in this definition that harm results from the presence of the contaminant
3.2
environmental availability
fraction of contaminant physico-chemically driven by desorption processes potentially available to
organisms
[SOURCE: ISO 17402:2008, 3.4]
3.3
environmental bioavailability
fraction of the environmentally available compound which an organism takes up through physiologically
driven processes
[SOURCE: ISO 17402:2008, 3.5]
3.4
habitat function
ability of soil/soil materials to serve as a habitat for micro-organisms, plants, soil-living animals, and
their interactions (biocenosis)
[SOURCE: ISO 11074:2005, 3.4.3]
3.5
trace element
−1
chemical element in soil occurring at concentration generally less than 100 mg kg
Note 1 to entry: Given according to Reference [16].
3.6
retention function
ability of soil/soil materials to adsorb pollutants in such a way that they cannot be mobilized via the
water pathway and translocated into the terrestrial food chain
[SOURCE: ISO 11074:2005, 3.4.13]
3.7
rhizosphere
volume of soil around living roots that is influenced by root activities
Note 1 to entry: Given according to Reference [17].
3.8
soil
upper layer of the earth’s crust transformed by weathering and physical/chemical and biological
processes. It is composed of mineral particles, organic matter, water, air, and living organisms organized
in genetic soil horizons
[SOURCE: ISO 11074:2005, 2.1.8]
2 © ISO 2015 – All rights reserved

ISO 16198:2015(E)
3.9
soil material
material coming from soil and displaced and/or modified by human activity, including excavated soil,
dredged materials, manufactured soils, and treated soils and fill materials
[SOURCE: ISO 17402:2008, 3.16]
3.10
toxicological bioavailability
internal concentration of pollutant accumulated and/or related to a toxic effect
[SOURCE: ISO 17402:2008, 3.18]
4 Principle
This International Standard describes the experimental procedure of the biotest developed initially
by References [18], [19], and [20]. This biotest consists of two successive steps of plant growth (see
Figure 1). During the first step (i.e. preculture period), plant seedlings are grown in hydroponics for
14 d to achieve an adequate plant biomass and a dense, planar root mat. During the second step (i.e. test
culture period), the root mat of pre-grown plants is put in contact for 8 d with a 6 mm-thick layer of soil
sample sieved to 2 mm.
ISO 16198:2015(E)
Key
a seed germination (7 d) 8 air diffuser
b seedling pre-growth (7 d) 9 30-µm mesh
1 preculture period in hydroponics – 14 d 10 air
2 test culture period soil-plant contact – 8 d 11 root mat
3 aluminium foil 12 nutrient solution 2
4 floating platform 13 shoots
5 seeds 14 soil layer (6 mm thick)
6 plant pot 15 nutrient solution 3
7 nutrient solution 1 16 filter paper wicks
Figure 1 — The two-step procedure of the biotest
A set of control plants is harvested at the end of the preculture period in hydroponics to determine the
pools of trace elements in plant shoots and roots before exposure to soil. Whole plants (shoots and roots)
are then harvested at the end of the test culture period. Biomasses and trace element concentrations in
shoots and roots are determined. The end points of the biotest are a) the concentration of trace elements
in shoots and roots at the end of the test culture period and b) the net uptake flux of trace elements in
[21]
the whole plants during the test culture period. If these end points are usually correlated, the uptake
flux is expected to be more representative of the trace element bioavailability to plants during the test
4 © ISO 2015 – All rights reserved

ISO 16198:2015(E)
culture period (i.e. the exposure to tested soils) as, conversely to concentrations, the uptake flux does
not include the portion of trace elements taken up during the preculture period (11.1).
As plant growth during the pre-culture period is usually sufficient to prevent the occurrence of
phytotoxic symptoms induced by adverse soil chemical properties or excessive accumulation of trace
elements in plant, the biotest enables a fair comparison of trace element bioavailability over a broad
range of soils, including heavily contaminated soils.
5 Laboratory apparatus
The following equipment shall be used. All equipment that comes into contact with the sample (soils,
plants, or reagents) shall not adsorb substantially trace elements and shall not contaminate the sample.
5.1 Sieving equipment, nominal screen size 2 mm.
5.2 Crushing equipment, jaw crusher and cutting device.
5.3 Balance, with an accuracy of at least 100 mg.
5.4 Balance, with an accuracy of at least 1 mg.
5.5 Growth chamber, suitable for maintaining specific climatic conditions as specified in 7.4.
5.6 Ventilated oven, suitable for drying soil or soil material at 25 °C and shoots and roots at 50 °C.
5.7 Scissors, with blades made of zirconium oxide.
5.8 Grinder and marbles, made of zirconium oxide.
6 Reagents
6.1 General
Use reagents of analytical grade purity with a concentration of investigated trace elements (e. g. As, Cd,
-1
Co, Cr, Cu, Ni, Pb, and Zn) lower or equal to 5 mg kg . Water used shall comply with grade 3 according to
ISO 3696.
−1
6.2 Water, distilled or demineralized with a specific conductivity of at most 5 µS cm at 25 °C and a pH
within the range 5,0 to 7,5.
-1
6.3 Hydrogen peroxide (H O , 34,01 g mol ).
2 2
-1
6.4 Calcium chloride dihydrate (CaCl ·2H O, 147,07 g mol ).
2 2
-1
6.5 Boric acid (H BO , 61,83 g mol ).
3 3
-1
6.6 Calcium nitrate tetrahydrate (Ca(NO ) ·4H O, 236,15 g mol ).
3 2 2
-1
6.7 Potassium nitrate (KNO , 101,1 g mol ).
-1
6.8 Magnesium sulfate heptahydrate (MgSO ·7H O, 246,48 g mol ).
4 2
ISO 16198:2015(E)
-1
6.9 Potassium phosphate (KH PO , 136,09 g mol ).
2 4
-1
6.10 Ethylenediaminetetraacetic acid iron(III) sodium (NaFe(III)EDTA, 367,05 g mol ).
-1
6.11 Copper chloride dihydrate (CuCl ·2H O, 170,48 g mol ).
2 2
-1
6.12 Manganese chloride tetrahydrate (MnCl ·4H O, 197,91 g mol ).
2 2
-1
6.13 Zinc sulfate heptahydrate (ZnSO ·7H O, 287,54 g mol ).
4 2
-1
6.14 Sodium molybdate dihydrate (Na MoO ·2H O, 241,95 g mol ).
2 4 2
7 Biological and growing apparatus
7.1 Plant species
The three following species, i.e. cabbage (B. oleracea), tall fescue (F. arundinacea), and tomato (L.
esculentum), are used during the biotest deployment. These three plant species were selected among non-
accumulator, common agricultural species for their ability to collectively maximize the phytoavailability
of trace elements (more specifically that of As, Cd, Cu, Pb, and Zn) in soils exhibiting a broad range
[21]
of physical-chemical properties and origin of trace element. For each of the three species, the
following cultivars are recommended: castelard for B. oleracea, calina for F. arundinacea, and fline for
L. esculentum. However, in the only case where recommended cultivars are not commercially available
other cultivars may be used provided that they exhibit an adequate growth and homogeneous root mat
during the biotest procedure for the different soils tested. Specify in the test report the reasons for
selecting different cultivars than those recommended. For a given cultivar, seeds used for a single set of
experiment shall come from the same batch.
Additional species can be selected, e.g. species with specific physiological characteristics or with
ecological, agricultural or economic significance in certain regions of the world or for specific site
assessment, provided that these species exhibit an adequate growth and homogeneous root mat during
the biotest procedure for the different soils tested. A list of plant species adapted to be grown in the
biotest, but only partly validated for the standardized procedure, is given in Annex A. Specify in the test
report the reasons for selecting additional species.
NOTE 1 The biotest procedure should be used with untreated seeds (i.e. not treated with any pesticide) as
much as possible. If not feasible, specify it in the test report.
NOTE 2 If other cultivars than those recommended are used, it is to note that this can alter the biotest
measurement in a similar extent than if different species were used.
7.2 Biotest apparatus
The following apparatus shall be used. Apparatus that comes into contact with the sample (soils, plants
or reagents) shall not adsorb the component of interest and shall not contaminate the sample.
The plant-receiving pot (i.e. plant pot) is designed to contain the whole plants from the beginning of the
preculture period to the end of the test culture period. The plant pot enables plants to develop a planar
and dense root mat while maintaining a physical separation with the tested soil sample. The plant pot
consists in a cylinder fitted to an upper plate at the top and closed at the bottom with a polyamide mesh
(30 µm pore size) using an adjustable clamp (see Figure 2). The mesh shall be well tightened.
6 © ISO 2015 – All rights reserved

ISO 16198:2015(E)
Key
1 upper plate
2 cylinder
3 adjustable clamp
4 30 µm polyamide mesh
Figure 2 — Plant-receiving pot assembly
The assembly used for the first step, i.e. the pre-culture period, is designed to enable seeds to germinate
and for seedlings to develop a dense and planar mat of roots in hydroponics. This assembly enables
a close contact between seeds or seedling roots and the nutrient solution. This assembly consists in
a perforated platform floating over a tank-containing nutrient solution. Perforations passing through
the floating platform enable to lodge plant pots. The nutrient solution is continuously aerated with a
bubbling system composed of an air-pump, capillary tubes connected with derivations and diffusers
diving into the nutrient solution (see Figure 3). The floatability of the platform is critical to ensure a
homogeneous contact of all the plant pots in a tank with the aerated nutrient solution. This assembly
also limits the exposure of the nutrient solution to light radiations, thereby avoiding algal development.
ISO 16198:2015(E)
23 46
Key
1 tank
2 perforated floating platform
3 nutrient solution 1 or 2
4 air diffuser
5 air
6 tube
7 air-pump
Figure 3 — Assembly used for the preculture period
The assembly used for the second step, i.e. the test culture period, is designed to enable a close contact
between the root mat and the soil layer. It is made of two parts and three filter paper wicks sandwiched
in between: a) a contact assembly that firmly press the plant pot over the soil layer using fastenings and
b) a 0,5 dm screw-top jar filled with the nutrient solution 3 (see Figure 4). This assembly enables (i) the
root mat to be maintained in contact with the whole surface area of the soil layer, and (ii) the filter paper
wicks to remain fully moistened during the entire duration of the test culture period.
Key
1 wing nut 6 soil-receiving plate
2 plant pot 7 screw-top
3 soil layer (6 mm thick) 8 filter paper wicks
4 screw 9 screw-top jar
5 screw nut 10 nutrient solution 3
Figure 4 — Soil-plant contact assembly used for the test culture period
8 © ISO 2015 – All rights reserved

ISO 16198:2015(E)
Except for the adjustable clamp, the filter paper wicks and the polyamide mesh, all the components
of the plant-based test are reusable provided that they are subjected to a two-step washing, firstly, in
hot water to remove adhering mucilage and microbial biofilms then secondly, in a volume fraction of
10 % HNO , followed by a thorough rinsing with distilled or demineralized water. A thorough list of
the components along with specification is given in Table 1 for information and technical drawings
are given in Annex B. Home-made apparatus may be built provided that the size of the different parts
remains proportional and that component specification is similar.
Table 1 — Components of the biotest (informative)
a b
Component No. Quantity Specification
PVC, high density, high tempera-
Cylinder 1 1
ture, for food contact
Upper plate 2 1 HDPE for food contact
Plant pot
Polyamide mesh / 1 100 × 100 mm, 30 µm pore size
Adjustable clamp / 1 180 mm × 2,4 mm
12 dm , 400 mm×294 mm
c
Tank 3 1 × 165 mm, Opaque HDPE for food
contact
Perforated floating plat-
4 1 Extruded polystyrene platform
form
Preculture
3 −1
Air flow 100 - 200 dm h out-
period
Two outputs Air-pump / 1
−1
put
Capillary tube / 2 PVC, int. diam. ca. 4 mm
Derivation / 1 If necessary
Air-diffuser / 2 Ceramic diffuser, 100 × 10 mm
Opaque and white PP for food
Screwtop jar 5 1
contact
Filter paper wick / 3 Hardened ashless filter paper
HDPE for food contact, 6 mm
Test culture
Soil-receiving plate 6 1
thickness
period
d
Screw / 4 HDPE
d
Screw nut / 8 HDPE
d
Wing nut / 4 HDPE
a
Component number as referenced in the technical drawings depicted in Annex B.
b
For one item of each of the three biotest components (i.e. plant pot and apparatus for preculture and test culture
periods).
c 3
Adapted for 12 plant pots per tank filled with 6 dm of nutrient solution.
d
Only two screws can be used to reduce time-fitting, with only four screw nuts and two wing nuts.
7.3 Composition of the nutrient solutions
Three different nutrient solutions shall be prepared for the deployment of the biotest for (i) seed
germination, (ii) seedling pre-growth in hydroponics [steps (i) and (ii) are included in the preculture
period], and (iii) plant growth during the test culture period.
-3
During seed germination (preculture period), the nutrient solution 1 shall be composed of 600 µmol·dm
-3
CaCl (6.4) and 2 µmol·dm H BO (6.5).
2 3 3
During seedling growth in hydroponics (preculture period), the nutrient solution 2 shall be prepared
-3
KH PO (6.9);
by adding the nutrients in the following order and concentration: 500 µmol·dm
2 4
-3 -3 -3
2 000 µmol·dm KNO (6.7); 2 000 µmol·dm Ca(NO ) (6.6); 1 000 µmol·dm MgSO (6.8);
3 3 2 4
ISO 16198:2015(E)
-3 -3 -3 -3
0,2 µmol·dm CuCl (6.11); 10 µmol·dm H BO (6.5); 2 µmol·dm MnCl (6.12); 1 µmol·dm
2 3 3 2
-3 -3
ZnSO (6.13); 0,05 µmol·dm Na MoO (6.14); and 100 µmol·dm NaFe(III)EDTA (6.10).
4 2 4
During plant growth in the test culture period, the nutrient solution 3 shall be prepared by adding
-3 -3
the nutrients in the following order and concentration: 50 µmol·dm KH PO (6.9); 2 000 µmol·dm
2 4
-3 -3
KNO (6.7); 2 000 µmol·dm Ca(NO ) (6.6); and 1 000 µmol·dm MgSO (6.8).
3 3 2 4
Nutrient solutions shall be prepared with distilled or demineralized water (6.2).
7.4 Climatic conditions in the growth chamber
Plants can be grown in growth chamber, phytotron, or greenhouse provided that the climatic conditions
comply with those listed in Table 2. Monitor and record the temperature and the relative humidity in the
growth chamber in short intervals (<1 h). Measure and record the illumination at least at the beginning
and at the end of the biotest procedure.
Table 2 — Climatic conditions for the deployment of the biotest (normative)
Climatic conditions Day Night
Photoperiod, in h 16 8
a -2 -1 b
PAR , in µmol photons m s , at 400–700 nm 200 - 400 /
Temperature, in °C 25 ± 3 20 ± 2
Relative humidity, in % 75 ± 5 70 ± 5
a
Photosynthetically active radiation.
b
At the canopy level.
8 Pre-treatment and analysis of soil or soil material sample
8.1 Sample size and particle size reduction
The test portion shall be prepared to have a grain size less than or equal to 2 mm. The material shall
not be finely ground, which would likely alter the bioavailability. Material >2 mm of natural origin (e.g.
stones, pebbles, twigs) shall be separated and discarded from the sample. If the laboratory sample
cannot be crushed or sieved because of its water content, it is allowed, in this case only, to reduce the
water content until the laboratory sample can be sieved. The drying temperature shall not exceed 25 °C.
NOTE Drying, even below 25 °C, of samples collected from volcanic soil exhibiting allophanic properties
should be avoided. This kind of soil sample should be stored moist at 4 °C.
8.2 Analyses
Determine the following physical-chemical properties of soil or soil material:
— initial water content according to ISO 11465;
— water holding capacity according to ISO 11269-2;
— texture according to ISO 11277;
— pH in H O or CaCl according to ISO 10390;
2 2
— organic carbon concentration according to ISO 10694.
NOTE The determination of texture, pH and organic carbon concentration is necessary for the interpretation
of the results.
10 © ISO 2015 – All rights reserved

ISO 16198:2015(E)
9 Experimental and analytical procedure
9.1 Overview of the procedure
Figure 5 introduces the different steps of the procedure described hereafter.
Preculture Test culture Plant TE analysis
Seed selection
and preparation period period Harvest in plants
(9.2) (9.3) (9.5) (9.6) (9.7 & 9.8)
Soil preparation
and incubation
(9.4)
Day 0Day 14 Day 22
NOTE 1 Dark boxes stand for the two steps of the biotest procedure.
NOTE 2 TE means trace element.
Figure 5 — Sequential steps of the experimental and analytical procedures
9.2 Selection and preparation of seeds
Select the seeds to be used in the biotest (Annex C). Reject empty husks, damaged seeds, or unevenly
shaped or sized seeds.
Store selected seeds in darkness and in dry conditions, ideally at 4 °C, to preserve germination capacity.
9.3 Preculture period: Germination and pre-growth in hydroponics
Before germination, sterilize the surface of the seeds by immersion for 10 min in a volume fraction of
6 % H O , then rinse the seeds three times with distilled or demineralized water.
2 2
Then put the required number of seeds at the surface of the polyamide mesh in the plant pot, according to
the following density: 50, 80, and 40 seeds per plant pot for B. Oleracea, F. arundinacea, and L. esculentum,
respectively. Additional seed density for a range of species tested with the standardized experimental
procedure is given in Annex C.
Prepare for each soil or soil material tested a minimum of five replicates (i.e. plant pots) per plant species.
In addition, prepare a minimum of five plant pots to serve as controls of trace element concentration in
plants (particularly for the micronutrients Cu and Zn) upon completion of the preculture period. To
obtain enough plant pots with a homogeneous plant biomass to serve as controls and for the test culture
ISO 16198:2015(E)
period, multiply the number of initial plant pots to grow by a security factor of 1,2 at least. Calculate the
total number of plant pots to prepare per plant species using Formula (1):
nn=×[( nn)]+× f (1)
ps rc
where:
n is the total number of plant pots to prepare per plant species;
p
n is the number of soil or soil material tested;
s
n is the number of replicates (minimum 5);
r
n is the number of plant pots which serve as control of the preculture period (minimum 5),
c
f is the security factor (minimum 1,2).
After sowing, plant pots are passed through the floating platform (12 plant pots per floating platform)
placed at the top surface of 6 dm of the nutrient solution 1 in the tank. Germinate the seeds for two to four
days in darkness by covering the tank with an aluminium foil. When functional photosynthetic organs
(green pigmentation on cotyledons or leaves) appear, the aluminium foil can be removed. Seedlings are
then grown up to the end of the first week over the same nutrient solution. Nutrient solution 1 does not
need to be renewed.
After the first week, seedlings are grown for one additional week at the top surface of 6 dm of the
nutrient solution 2. The nutrient solution 2 shall be renewed every third day. Randomize the plant pot
position in the floating platform at each renewal. At each renewal, the preculture apparatus (tank and
floating platform) shall be replaced and thoroughly washed in hot water and then in a volume fraction
of 10 % HNO followed by a thorough rinsing with distilled or demineralized water.
During these two weeks in hydroponics, nutrient solutions shall be aerated with a bubbling system.
9.4 Preparation and incubation of soil or soil material
In parallel with the preculture period, tested samples of soil or soil material are incubated for two
weeks in darkness in similar climatic
...