EN ISO 29200:2020

SLOVENSKI STANDARD
01-november-2020
Kakovost tal - Ocenjevanje genotoksičnih učinkov na višje rastline -
Mikronukleusni preskus z bobom (Vicia faba) (ISO 29200:2013)
Soil quality - Assessment of genotoxic effects on higher plants - Vicia faba micronucleus
test (ISO 29200:2013)
Bodenbeschaffenheit - Beurteilung der genotoxischen Wirkungen auf höhere Pflanzen -
Mikrokern-Prüfung mit Vicia faba (ISO 29200:2013)
Qualité du sol - Évaluation des effets génotoxiques sur les végétaux supérieurs - Essai
des micronoyaux sur Vicia faba (ISO 29200:2013)
Ta slovenski standard je istoveten z: EN ISO 29200:2020
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 29200
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Assessment of genotoxic effects on higher
plants - Vicia faba micronucleus test (ISO 29200:2013)
Qualité du sol - Évaluation des effets génotoxiques sur Bodenbeschaffenheit - Beurteilung der genotoxischen
les végétaux supérieurs - Essai des micronoyaux sur Wirkungen auf höhere Pflanzen - Mikrokern-Prüfung
Vicia faba (ISO 29200:2013) mit Vicia faba (ISO 29200:2013)
This European Standard was approved by CEN on 13 April 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 29200:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 29200:2013 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 29200:2020 by
Technical Committee CEN/TC 444 “Environmental characterization of solid matrices” the secretariat of
which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by November 2020, and conflicting national standards
shall be withdrawn at the latest by November 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 29200:2013 has been approved by CEN as EN ISO 29200:2020 without any modification.

INTERNATIONAL ISO
STANDARD 29200
First edition
2013-09-01
Soil quality — Assessment of
genotoxic effects on higher plants —
Vicia faba micronucleus test
Qualité du sol — Évaluation des effets génotoxiques sur les végétaux
supérieurs — Essai des micronoyaux sur Vicia faba
Reference number
ISO 29200:2013(E)
©
ISO 2013
ISO 29200:2013(E)
© ISO 2013
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Published in Switzerland
ii © ISO 2013 – All rights reserved

ISO 29200:2013(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative reference . 1
3 Terms and definitions . 1
4 Principle . 2
5 Plants, test equipment and reagents . 2
5.1 Equipment . 2
5.2 Test organism . 2
5.3 Reference substance . 2
5.4 Reagents. 3
6 Protocols . 3
6.1 Preparation of the soil to be tested . 3
6.2 Preparation of the seeds . 4
6.3 Conducting of the test . 5
6.4 Test environment . 6
6.5 Cell preparation . 6
7 Assessment of the results . 8
7.1 Presentation of the data . 8
7.2 Statistical analysis . 8
7.3 Interpretation of the results . 8
8 Validity criteria . 8
9 Test report . 8
Annex A (informative) Composition of Hoagland’s medium .10
Annex B (informative) Testing chemicals added to soils .11
Annex C (informative) Results of the interlaboratory test conducted within the framework of
NF T 90-327
.............................................................................................................................................................................................................12
Annex D (informative) Results of the interlaboratory test conducted on the reference substance
and an industrial contaminated soil .14
Bibliography .18
ISO 29200:2013(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. 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. 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.
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
iv © ISO 2013 – All rights reserved

ISO 29200:2013(E)
Introduction
In the field of assessment of the quality of soils and soil materials, it appears necessary to determine
in vivo their genotoxic potential which may be induced by pollution or by a decontamination process.
Indeed, genotoxic agents have the ability to damage the genome of living organisms or to interfere with
its functioning, but they are not always detected by chemical analysis or classical ecotoxicological tests.
Actually, genotoxic effects are often observed at sublethal concentrations, where no toxic effect (e.g.
survival or growth) can be observed in the short term but some long term effects may be feared in living
organisms. Moreover, higher plants, like Vicia faba (broad bean) are ecologically relevant to assess soils
and soil materials quality.
INTERNATIONAL STANDARD ISO 29200:2013(E)
Soil quality — Assessment of genotoxic effects on higher
plants — Vicia faba micronucleus test
1 Scope
The purpose of this International Standard is to describe a method for assessing genotoxic effects
(chromosome breakage or dysfunction of the mitotic spindle) of soils or soil materials on the secondary
roots of a higher plant: Vicia faba (broad bean). This method allows the assessment of genotoxicity
(toxicity for genetic material) of soils and soil materials like compost, sludge, waste, fertilizing matters,
etc. Two ways of exposure can be considered: a direct exposure of plants to the soil (or soil material)
which is relevant for the real genotoxic potential and an exposure of plants to the water extract of the
soil (or soil material). This last way of exposure to a leachate or an eluate allows the detection of the
mutagens which are not adsorbed to soils and which may be transferred to aquatic compartments.
Moreover, this test may be used to evaluate genotoxic effects of chemical substances and to waters,
effluents, etc.
2 Normative reference
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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level
using barium chloride solution
ISO 11269-2:2012, 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 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method
ISO/TS 21268-1, Soil quality — Leaching procedures for subsequent chemical and ecotoxicological testing
of soil and soil materials — Part 1: Batch test using a liquid to solid ratio of 2 l/kg dry matter
ISO/TS 21268-2, Soil quality — Leaching procedures for subsequent chemical and ecotoxicological testing
of soil and soil materials — Part 2: Batch test using a liquid to solid ratio of 10 l/kg dry matter
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
control soil
uncontaminated substrate used as control and dilution medium for preparing dilution series with test
soils or test materials
EXAMPLE compost, sludge, waste, chemicals
ISO 29200:2013(E)
3.2
mitotic index
number of cells in division per 1 000 cells observed when all of the stages of the mitosis are taken into
account, from the prophase (when the chromosomes begin to condense) up to the telophase (when the
chromatin of the two nuclei formed at each pole of the cell finishes decondensing).
3.3
test mixture
mixture of test material (soil, compost, sludge, waste or chemical) with control soil
4 Principle
This genotoxicity test is based on the detection of micronuclei in the cells of the secondary root tips of
Vicia faba (broad bean). The micronuclei, visible in the cytoplasm of the cells, result from a chromosome
break (effect of clastogenic substances) or from a dysfunction of the mitotic spindle (effect of aneugens).
In both cases, the fragments of chromosomes or the entire chromosomes cannot migrate to one of the
poles of the spindle at the time of the anaphase of the mitotic division and therefore form one (or more)
micronucleus.
The micronucleus frequency is determined in the control root cells and in those which have been exposed
to the soil (or soil material) or the water extract of the soil being tested. A statistical test then enables to
determine the significativeness of data.
5 Plants, test equipment and reagents
5.1 Equipment
The exposure of the plants to the soils and soil materials under test is performed in plastic pots (diameter:
9 cm, height: 10 cm).
Exposure to water extract of soils is carried out in glass containers (e.g. glass beaker of capacity 200 ml).
A microscope equipped with an objective with x 400 magnification is required for studying the
microscopic effects of the cells.
5.2 Test organism
The plant selected for its high sensitivity to micropollutants and for its ease of obtention is Vicia faba
(broad bean), Aguadulce, with a very long pod. This higher plant forms part of the family of pulses and
of the dicotyledoneae class.
Seeds coated with insecticides and/or fungicides should not be used.
5.3 Reference substance
Maleic hydrazide is recommended as a reference substance. The positive control is carried out at
-5
the concentration of 10 M, 1,12 mg/kg and 1,12 mg/l for solid-phase and liquid-phase exposures
respectively.
The preparation of this photodegradable substance as well as the exposure of the plant organisms to the
solution shall be carried out in the dark.
2 © ISO 2013 – All rights reserved

ISO 29200:2013(E)
5.4 Reagents
5.4.1 Carnoy’s solution
Carnoy’s solution is composed of glacial acetic acid and of ethanol in respective volume proportions of
25 % and 75 % and shall be prepared extemporaneously.
5.4.2 Hydrolysis solution
A solution of HCl with concentration 1 mol/l enables to conduct the hydrolysis of the roots.
5.4.3 Staining solution
The staining solution used for specifically highlighting the DNA is 1 % orcein diluted in 45 % acetic acid.
This mixture is brought to the boil during 10 min, then filtered after cooling down. When it is used, it is
important to filter the staining solution after each use in order to prevent the forming of orcein crystals
which could be confused with micronuclei during the microscopic examination of the cells.
NOTE Other specific DNA staining solution can be used.
5.4.4 Hoagland’s medium
Nutritive medium of which the composition is given in Annex A.
5.4.5 Intermediate solvent
Dimethyl sulfoxide (DMSO), at a maximal concentration of 1 %.
NOTE Any other appropriate water-miscible solvent whose genotoxic innocuousness has been previously
established may be used.
6 Protocols
6.1 Preparation of the soil to be tested
6.1.1 Chemical substances
Chemical substances may be tested: their preparation is explained in Annex B.
6.1.2 Soils and soil materials
Whatever the soil to be tested (sampled from a contaminated site or from a remediated soil, or other
soil materials like compost, sludge, waste, fertilizing matters, etc.), it should have pH values after
sieving within a range that is not toxic to Vicia faba. Soils under test should be sieved by 4 mm mesh and
thoroughly mixed and should be stored as shortly as possible, in the dark at 4 °C ± 2 °C in accordance with
ISO 10381-6 using containers that minimise losses of soil contaminants by volatilisation and sorption to
the container walls. Soil pH should not be corrected.
For each soil to be tested, the following characteristics should be determined:
— Soil texture classification,
— pH in accordance with ISO 10390,
— Water content in accordance with ISO 11465,
— Water holding capacity according to Annex B of ISO 11269-2:2012,
ISO 29200:2013(E)
— Cationic exchange capacity in accordance with ISO 11260,
— Organic matter content in accordance with ISO 10694.
The soil mixtures are placed in plastic pots with a moisture content of 70 % of water-holding capacity.
6.1.3 Control soil
1)
Either reference or standard natural soils can be used as control soil, e.g. LUFA soils previously air
dried at room temperature, sieved between 2 mm and 5 mm, with clay (< 2µm) content < 25 % , silt (2 µm
-50 µm) content < 45 % , soil organic matter content between 1,5 % and 5 % , pH between 5 and 8 .
water
When comparing soils of known and unknown quality, the control soil and soil under test should be of
the same textural class, and be as similar as practicable in all respects other than the presence of the
chemical or contaminant being investigated. Indeed, significant differences in soil characteristics other
than the presence of contaminants may lead to differences in plant cell division, so in micronucleus
frequency and may induce false positive test results.
NOTE Although mitotic index is not modified by pH between 4 and 9, it is recommended to use a control soil
with a pH between 5 and 8 for a better genotoxicity assessment of chemicals.
water
6.1.4 Water extracts of soil
Water extracts of soils or soil materials are prepared, as rapidly as possible after receipt of the sample
at the laboratory, with a leaching test according to one of the protocols described in ISO/TS 21268-1 or
ISO/TS 21268-2 or EN 14735. However, the eluates obtained shall not be filtered but can be decanted
during 2 h. In this case, the supernatant phase is sampled and stored in the dark at a temperature of 4 °C
± 3 °C up until the test is carried out which shall take place at the maximum 24 h after the leaching stage.
The Hoagland’s medium is used for the negative control and to prepare the dilutions of the water extract.
6.2 Preparation of the seeds
Seeds (approximately three times higher than the required number) are selected from the stock of seeds
stored at 4 °C in the dark. Then a
...