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)

General Information

Status
Published
Publication Date
05-May-2020
Withdrawal Date
29-Nov-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
06-May-2020
Due Date
07-Mar-2022
Completion Date
06-May-2020

Buy Standard

Standard
EN ISO 29200:2020 - BARVE
English language
26 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day
Draft
prEN ISO 29200:2020 - BARVE
English language
23 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (Sample)

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.

---------------------- Page: 1 ----------------------
SIST EN ISO 29200:2020

---------------------- Page: 2 ----------------------
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.

---------------------- Page: 3 ----------------------
SIST EN ISO 29200:2020
EN ISO 29200:2020 (E)
Contents Page
European foreword . 3

2

---------------------- Page: 4 ----------------------
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.

3

---------------------- Page: 5 ----------------------
SIST EN ISO 29200:2020

---------------------- Page: 6 ----------------------
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

---------------------- Page: 7 ----------------------
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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

---------------------- Page: 8 ----------------------
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

---------------------- Page: 9 ----------------------
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

---------------------- Page: 10 ----------------------
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.
© ISO 2013 – All rights reserved v

---------------------- Page: 11 ----------------------
SIST EN ISO 29200:2020

---------------------- Page: 12 ----------------------
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
© ISO 2013 – All rights reserved 1

---------------------- Page: 13 ----------------------
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
-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

---------------------- Page: 14 ----------------------
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,
© ISO 2013 – All rights reserved 3

---------------------- Page: 15 ----------------------
SIST EN ISO 29200:2020
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 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

---------------------- Page: 16 ----------------------
SIST EN ISO 29200:2020
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).
© ISO 2013 – All rights reserved 5

---------------------- Page: 17 ----------------------
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
...

SLOVENSKI STANDARD
oSIST prEN ISO 29200:2020
01-januar-2020
[Not translated]
Soil quality - Assessment of genotoxic effects on higher plants - Vicia faba micronucleus
test (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: prEN ISO 29200
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 29200:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 29200:2020

---------------------- Page: 2 ----------------------
oSIST prEN 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

---------------------- Page: 3 ----------------------
oSIST prEN 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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

---------------------- Page: 4 ----------------------
oSIST prEN 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

---------------------- Page: 5 ----------------------
oSIST prEN 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

---------------------- Page: 6 ----------------------
oSIST prEN 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.
© ISO 2013 – All rights reserved v

---------------------- Page: 7 ----------------------
oSIST prEN ISO 29200:2020

---------------------- Page: 8 ----------------------
oSIST prEN 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
© ISO 2013 – All rights reserved 1

---------------------- Page: 9 ----------------------
oSIST prEN 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
-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

---------------------- Page: 10 ----------------------
oSIST prEN 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,
© ISO 2013 – All rights reserved 3

---------------------- Page: 11 ----------------------
oSIST prEN ISO 29200:2020
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 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

---------------------- Page: 12 ----------------------
oSIST prEN ISO 29200:2020
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).
© ISO 2013 – All rights reserved 5

---------------------- Page: 13 ----------------------
oSIST prEN 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 observation or else can be hydrolysed in the case of an immediate observation.
The root tips are then placed in distilled water for 10 min, hydrolysed in the hydrolysis solution at 60 °C
for 6 min and retransferred into distilled water for a few minutes.
For root cell observation, place a root tip on a slide after wiping it cautiously. Remove the first millimeter
corresponding to the root cap and the meristematic region and, with the help of a scalpel, retain only
the second millimeter which is made up of the subsequent generation of cells obtained after mitosis.
Micronuclei scoring shall be done in this particular cells region (see Figure 3). All these steps may be
done on a black background to see the different cell regions.
The staining of the DNA is carried out by crushing the root tips after addition of a drop of orcein; the
coverslip can then be placed in position and squeezed to obtain a single layer of cells.
6 © ISO 2013 – All rights reserved

---------------------- Page: 14 ----------------------
oSIST prEN ISO 29200:2020
ISO 29200:2013(E)

It is recommended to carry out at least two cell spreadings (obtained with two different roots) per seed.
1 mm 1 mm
Figure 3 — Longitudinal section of Vicia faba root showing the root cap, the meristem and cells
region to be selected for micronuclei scoring
It is preferable to perform the scoring under blindfold conditions prior to their examination so that
the person conducting the test is not influenced when counting the micronuclei (see Figure 4). The
micronuclei observed in cells in division shall not be taken into account when determining micronucleus
frequency. The results are expressed in number of micronuclei per 1 000 cells in interphase.
Figure 4 — Micronuclei in the cells of the root tips of Vicia faba (x 400)
During the micronuclei scoring, it is highly recommended to verify the proper progress of the cell division
(an essential condition for the micronuclei formation) by determining the proportion of cells in mitotic
division (commonly called mitotic index). A genotoxic effect can be masked by a cytotoxic one (toxic with
respect to the cell functioning) which would induce an underestimation of the genotoxic potential of the
sample under test. The results are expressed in number of cells in division per 1 000 cells observed.
All 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).
The microscopic examination of the slides is carried out under a magnification of 400. A minimum
number of two slides is prepared for each of the three replicates for each concentration. Considering
that 1 000 cells per slide are observed, the averages and standard deviations are therefore calculated on
a minimum of 6 000 cells for each concentration.
© ISO 2013 – All rights reserved 7
Root cap
Meristem
Cells for
micronuclei
scoring

---------------------- Page: 15 ----------------------
oSIST prEN ISO 29200:2020
ISO 29200:2013(E)

7 Assessment of the results
7.1 Presentation of the data
The results of the negative and positive controls and of the concentrations under test are expressed in
terms of average number of micronuclei per 1 000 cells observed.
7.2 Statistical analysis
The use of a non-parametric method (e.g. the Kruskal-Wallis test followed by Dunn’s multiple comparison
test) is recommended in order to highlight the significant differences between the control and test
concentrations.
7.3 Interpretation of the results
7.3.1 Positive test
The test is considered as positive if a statistically significant result with respect to the negative control
is detected for at least one of the test concentration.
7.3.2 Negative test
The test is considered as negative if, with respect to the negative control, no statistically significant
positive response is observed for the tested concentrations. It is noteworthy that the absence of
micronuclei in root cells may be due to a dysfunction of mitosis: in that case, DNA breakages are not
excluded from the nucleus and do not form any micronucleus. Consequently,
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.