Soil quality - Assessment of genotoxic effects on higher plants - Vicia faba micronucleus test (ISO 29200:2013)

The purpose of ISO 29200:2013 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.

Bodenbeschaffenheit - Beurteilung der genotoxischen Wirkungen auf höhere Pflanzen - Mikrokern-Prüfung mit Vicia faba (ISO 29200:2013)

Zweck dieser Internationalen Norm ist es, ein Verfahren zur Beurteilung der genotoxischen Wirkungen (Chromosomenbruch oder Dysfunktion der Mitosespindel) von Böden oder Bodenmaterialien auf die Seitenwurzeln einer höheren Pflanze, Vicia faba (Ackerbohne), zu beschreiben. Dieses Verfahren ermöglicht die Beurteilung der Genotoxizität (Toxizität für genetisches Material) von Boden und Boden¬material sowie Kompost, Schlamm, Abfall, Dünger, usw. Zwei Wege der Exposition können in Betracht gezogen werden: die direkte Exposition der Pflanzen mit Boden (oder Bodenmaterial), welche für das echte genotoxische Potenzial wichtig ist, und die Exposition der Pflanzen gegenüber dem Wasserextrakt des Bodens (oder Boden¬materials). Der letztere Weg der Exposition der Pflanzen gegenüber Sickerwasser oder Eluat erlaubt die Erfassung der Mutagene, die nicht an Böden adsorbieren und in die Bodenlösung gelangen können. Darüber hinaus darf die Prüfung zur Abschätzung genotoxischer Wirkungen von chemischen Substanzen und Wässern, Abwasser usw. verwendet werden.

Qualité du sol - Évaluation des effets génotoxiques sur les végétaux supérieurs - Essai des micronoyaux sur Vicia faba (ISO 29200:2013)

L'ISO 29200:2013 décrit une méthode pour évaluer les effets génotoxiques (cassure des chromosomes ou dysfonctionnement du fuseau mitotique) des sols ou des matrices solides sur une plante supérieure: Vicia faba (fève). Cette méthode permet d'évaluer la génotoxicité (toxicité vis-à-vis du matériel génétique) des sols ou des matrices solides telles que des composts, boues, déchets, matières fertilisantes, etc. Deux modes d'exposition peuvent être considérés: une exposition directe des plantes au sol (ou aux matrices solides) ce qui est représentatif du potentiel génotoxique réel et une exposition des plantes à l'extrait aqueux du sol (ou des matrices solides). Ce dernier mode d'exposition à un lixiviat ou un éluat permet de détecter les mutagènes qui ne sont pas adsorbés dans les sols et qui peuvent être transférés aux compartiments aquatiques. D'autre part, cet essai peut être utilisé pour évaluer les effets génotoxiques des substances chimiques, ainsi que des eaux, effluents, etc.

Kakovost tal - Ocenjevanje genotoksičnih učinkov na višje rastline - Mikronukleusni preskus z bobom (Vicia faba) (ISO 29200:2013)

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SLOVENSKI STANDARD
SIST EN ISO 29200:2020
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
SIST EN ISO 29200:2020 en,fr,de

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

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SIST EN ISO 29200:2020
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SIST EN ISO 29200:2020
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.
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SIST EN ISO 29200:2020
EN ISO 29200:2020 (E)
Contents Page

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

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SIST EN ISO 29200:2020
EN ISO 29200:2020 (E)
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.

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SIST EN ISO 29200:2020
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SIST EN ISO 29200:2020
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
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SIST EN ISO 29200:2020
ISO 29200:2013(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2013

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
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Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved
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SIST EN ISO 29200:2020
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 2013 – All rights reserved iii
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SIST EN ISO 29200:2020
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
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SIST EN ISO 29200:2020
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.
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SIST EN ISO 29200:2020
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SIST EN ISO 29200:2020
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
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SIST EN ISO 29200:2020
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

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
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SIST EN ISO 29200:2020
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,
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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

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 germination step is necessary to obtain secondary roots: the seeds

are cleaned with demineralised water and immersed during a period between 6 h and 24 h at ambient

temperature in demineralised water in order to hydrate them. The seed coats are then removed and

the seeds are left to germinate vertically at 24 °C ± 1 °C in continually humidified cotton (not having

undergone any chlorinated treatment) in the dark.
NOTE Other germination material may be used: vermiculite, peat, etc.

After about three days, only those seeds whose primary root length is between 3 cm and 5 cm are

selected. Their tip (around 5 mm) is then cut off in order to interrupt the growth of this main root and

to stimulate that of the secondary roots.

For solid phase exposure, the primary rooted seeds are directly placed in soils for beginning the

exposure of secondary roots.

For liquid phase exposure, the seeds are then placed, so as to immerse only the root, over a container

containing some nutritive medium (Hoagland’s medium (5.4.4)) at a temperature of 24 °C ± 1 °C in order

to induce the secondary roots sprouting. This previously oxygenated medium is renewed every 24h. The

secondary roots of the seeds reach a length of 1 cm to 2 cm after a period of four days; the seeds bearing

these secondary roots are then used for the purpose of the test.

This germination step of the Vicia faba seeds, necessary in both ways of exposure, can be started four

days and eight days respectively for solid-phase and liquid-phase exposure before beginning the test.

1) LUFA soils are an example of a suitable product available commercially. This information is given for the

convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.

4 © ISO 2013 – All rights reserved
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ISO 29200:2013(E)
6.3 Conducting of the test
6.3.1 Soils and soil materials

The dilutions of the test mixture are chosen within a geometric series with a factor not exceeding two

and shall cover a large range of concentrations (e.g. from 0,01 % to 100 % ). These mixtures are prepared

by diluting the soil with a reference soil.

Each test shall include a negative control without any test sample and a positive control (see 5.3).

The direct exposure of the plant organisms to the different concentrations of the soil is performed by

placing the germinated seeds (at least three per dilution) in a plastic pot containing 200 g of the tested

soil and/or mixtures (see Figure 1) throughout the exposure time between three and five days, according

to obtain at least ten roots of 1 cm length.
Figure 1 — Method of direct exposure of the Vicia faba seeds
6.3.2 Water extracts of soil

The concentrations of the sample under test are chosen within a geometric series with a factor not

exceeding two and shall cover a large range of concentrations (e.g. from 0,01 % to 100 % for matrices).

This range of concentrations is prepared by diluting the sample with the previously oxygenated

Hoagland’s medium (see Annex A).

Each test shall include a negative control without any test sample and a positive control (see 5.3).

At the time of the test, the different solutions to be tested are extemporaneously brought up to a

temperature of 24 °C ± 1 °C and well homogenised before exposure of plant organisms. This is carried out

by placing the germinated seeds (at least three per concentration) in a glass container having a sufficient

diameter in order to prevent, as far as possible, contact between the root tips and the container wall

throughout the exposure time. The roots are immersed in a minimum volume of 200 ml of test solution

(see Figure 2).
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SIST EN ISO 29200:2020
ISO 29200:2013(E)
Figure 2 — Method of aqueous exposure of the Vicia faba seeds

The exposure time shall be at least 30 h, which corresponds to the approximate duration of the cell cycle.

However the optimal exposure time recommended to detect genotoxic effects is 48 h to be sure that the

cell cycle is ended and for a better practicability.
6.4 Test environment

The tests are performed in a climatic chamber (with an intensity of at least 5 000 lx and 16/8 photoperiod)

at a temperature of 24 °C ± 1 °C. The liquid-phase test can also be carried out in darkness if necessary

(e.g. maleic hydrazide).
6.5 Cell preparation

At the end of the exposure period, the roots are simply removed from the water extract, or carefully

extracted from the soil. Then they are cleaned with deionised water and the last two centimeters of the

secondary roots (ten or so roots per seed, chosen at random) are sampled and placed at 4 °C for a minimum

duration of one night in Carnoy’s solution. These root tips can then be stored on a long term basis in 70 %

ethanol for a deferred observat
...

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