Soil quality - Determination of the effects of pollutants on soil flora - Leaf fatty acid composition of plants to assess soil quality (ISO 21479:2019)

This document describes a method to compare the quality of soils by determining the fatty acid composition of the leaves of plant species grown in these soils.
This method does not make it possible to determine an optimal value of the Omega-3 index and, therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as homogeneous). The method can only be used to compare the quality of soils between various areas.
This method is applicable to:
— soils from contaminated sites;
— amended soils;
— soils after remediation;
? soil with waste products (e.g. slurry, manure, sludge or composts).
Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing these values to those obtained from control soils (see Annex B).

Bodenbeschaffenheit - Bestimmung der Wirkungen von Schadstoffen auf die Bodenflora - Fettsäurezusammensetzung in Blättern zur Beurteilung der Bodenbeschaffenheit (ISO 21479:2019)

Gegenstand dieses Dokuments ist ein Verfahren zum Vergleichen der Beschaffenheit von Böden durch Bestimmen der Fettsäurezusammensetzung in den Blättern von Pflanzen, die auf diesen Böden wachsen.
Dieses Verfahren ermöglicht keine Bestimmung des optimalen Werts des Omega 3 Index. Daher kann es nicht dazu verwendet werden, die intrinsische Qualität des Bodens auf einer bestimmten (als homogen angesehenen) Fläche zu bestimmen. Das Verfahren kann nur dazu verwendet werden, die Beschaffenheit von Böden auf verschiedenen Flächen miteinander zu vergleichen.
Das Verfahren ist anwendbar auf:
   Böden von kontaminierten Standorten;
   verbesserte Böden;
   Böden nach Altlastensanierungen;
   Böden, auf die Abfallprodukte (z. B. Flüssigmist, Gülle, Schlamm oder Kompost) aufgebracht wurden.
Alternativ kann die Beschaffenheit von Böden beurteilt werden, indem der Omega 3 Index von Lactuca sativa Keimlingen bestimmt wird, die in diesen Böden unter kontrollierten Bedingungen (d. h. in einer Wachstumskammer) gezogen werden, und diese Werte mit den bei Kontrollböden erhaltenen Werten verglichen werden (siehe Anhang B).

Qualité du sol - Détermination des effets des polluants sur la flore du sol - Composition en acides gras foliaires des plantes utilisées pour évaluer la qualité du sol (ISO 21479:2019)

Le présent document décrit une méthode visant à comparer la qualité des sols en déterminant la composition en acides gras des feuilles d'espèces végétales poussant sur ces sols.
Cette méthode ne permet pas de déterminer une valeur optimale de l'indice Oméga-3 et ne peut donc pas être utilisée pour déterminer la qualité intrinsèque d'un sol d'une zone spécifique (considérée homogène). La méthode peut être utilisée uniquement pour comparer la qualité des sols entre plusieurs zones.
Cette méthode est applicable à:
— des sols provenant de sites contaminés;
— des sols amendés;
— des sols après remédiation;
— des sols contenant des produits résiduaires (par exemple lisier, fumier, boues ou composts).
La qualité des sols peut aussi être évaluée en déterminant l'indice Oméga-3 de plantules de Lactuca sativa poussant dans ces sols dans des conditions contrôlées (c'est-à-dire, enceinte phytotronique) et en comparant ces valeurs avec celles obtenues à partir de sols témoins (voir l'Annexe B).

Kakovost tal - Določanje učinkov onesnaževal na floro tal - Sestava maščobnih kislin v listih rastlin za oceno kakovosti tal (ISO 21479:2019)

General Information

Status
Published
Public Enquiry End Date
02-Feb-2020
Publication Date
23-Sep-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Aug-2020
Due Date
22-Oct-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 21479:2020
01-november-2020
Kakovost tal - Določanje učinkov onesnaževal na floro tal - Sestava maščobnih
kislin v listih rastlin za oceno kakovosti tal (ISO 21479:2019)

Soil quality - Determination of the effects of pollutants on soil flora - Leaf fatty acid

composition of plants to assess soil quality (ISO 21479:2019)

Bodenbeschaffenheit - Bestimmung der Wirkungen von Schadstoffen auf die Bodenflora

- Fettsäurezusammensetzung in Blättern zur Beurteilung der Bodenbeschaffenheit (ISO

21479:2019)

Qualité du sol - Détermination des effets des polluants sur la flore du sol - Composition

en acides gras foliaires des plantes utilisées pour évaluer la qualité du sol (ISO

21479:2019)
Ta slovenski standard je istoveten z: EN ISO 21479:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 21479:2020 en,fr,de

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

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SIST EN ISO 21479:2020
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SIST EN ISO 21479:2020
EN ISO 21479
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Determination of the effects of pollutants on
soil flora - Leaf fatty acid composition of plants to assess
soil quality (ISO 21479:2019)

Qualité du sol - Détermination des effets des polluants Bodenbeschaffenheit - Bestimmung der Wirkungen

sur la flore du sol - Composition en acides gras foliaires von Schadstoffen auf die Bodenflora -

des plantes utilisées pour évaluer la qualité du sol (ISO Zusammensetzung von Fettsäuren in Blättern zur

21479:2019) Beurteilung der Bodenbeschaffenheit (ISO
21479:2019)
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 21479:2020 E

worldwide for CEN national Members.
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SIST EN ISO 21479:2020
EN ISO 21479:2020 (E)
Contents Page

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

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SIST EN ISO 21479:2020
EN ISO 21479:2020 (E)
European foreword

The text of ISO 21479:2019 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 21479:2020 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 21479:2019 has been approved by CEN as EN ISO 21479:2020 without any modification.

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SIST EN ISO 21479:2020
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SIST EN ISO 21479:2020
INTERNATIONAL ISO
STANDARD 21479
First edition
2019-06
Soil quality — Determination of the
effects of pollutants on soil flora —
Leaf fatty acid composition of plants
used to assess soil quality
Qualité du sol — Détermination des effets des polluants sur la flore du
sol — Composition en acides gras foliaires des plantes utilisées pour
évaluer la qualité du sol
Reference number
ISO 21479:2019(E)
ISO 2019
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms, definitions and abbreviated terms ................................................................................................................................ 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 Abbreviated terms ............................................................................................................................................................................... 2

4 Principle ........................................................................................................................................................................................................................ 2

5 Apparatus and reagents ................................................................................................................................................................................ 2

5.1 Apparatus .................................................................................................................................................................................................... 2

5.2 Reagents........................................................................................................................................................................................................ 3

6 Sampling strategies ........................................................................................................................................................................................... 3

7 Sampling of leaf tissues ................................................................................................................................................................................. 3

8 Obtaining, extraction and analyses of FAMES ......................................................................................................................... 4

8.1 Contamination control ..................................................................................................................................................................... 4

8.2 Obtaining and extraction of FAMES from plant leaves ......................................................................................... 4

8.3 Analysis of FAMES ................................................................................................................................................................................ 4

9 Test report ................................................................................................................................................................................................................... 6

9.1 A reference to this document, i.e. ISO 21479 ................................................................................................................ 6

9.2 Description of the site and areas analysed ..................................................................................................................... 6

9.3 Leaf sampling ........................................................................................................................................................................................... 6

9.4 Fatty acid composition ..................................................................................................................................................................... 6

9.5 Conclusion ................................................................................................................................................................................................... 6

Annex A (informative) Results of the ring test ........................................................................................................................................... 7

Annex B (informative) Assessment of soil quality by determining the Omega-3 index of

Lactuca sativa seedlings grown ex situ under controlled conditions .........................................................14

Annex C (informative) Plant species previously successfully used to assess soils of

contaminated sites (organic and/or metals) .......................................................................................................................16

Annex D (informative) Variation of the Omega-3 index as function of harvest time, plant

size and leaf development .......................................................................................................................................................................17

Annex E (informative) Effect of the quantity of foliar tissues on the FAMES composition ........................19

Annex F (informative) Example of chromatogram obtained after the FAMES analysis of

foliar tissues ...........................................................................................................................................................................................................20

Annex G (informative) Recommended mathematical method to rate soils of areas when

some sampled plant species are not found in all areas ............................................................................................21

Bibliography .............................................................................................................................................................................................................................23

© ISO 2019 – All rights reserved iii
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following

URL: www .iso .org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological characterization.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
Introduction

Among the more than 150 ISO standards on soil quality that have been developed, less than 40 address

living organisms, and among them only five address higher plants. This is despite the importance of

monitoring the adverse effects of soil quality on living organisms.
[1]

One of these five standards addresses genotoxicity , and four of them address emergence and/or

[2-5]

growth inhibition . It therefore appears that these International Standards are focused either on a

very specific effect (genotoxicity), or on effects great enough to induce developmental (and, therefore,

visible) phenotypes (emergence or growth inhibition of young seedlings) in soils sampled in the field.

Hence, more sensitive/earlier bio-indicators of the adverse effects of pollutants on plants, such as the

“Omega-3 index”, are needed.

The assessment of soil contaminant effects by the Omega-3 index is based on the leaf fatty acid

composition of angiosperm species grown in sites of concern. The use of the Omega-3 index has proven

to be appropriate for highlighting the presence of metallic and organic contaminants (herbicides, etc.)

in the soils. With this aim, physical and chemical properties (pH, N/P/K content) of soils should also be

[12]

determined because plant fatty acid composition may vary as a function of nutrient content and pH

may influence chemical compound bioavailability. It should be noted that this bio-indicator has proved

to be more sensitive (i.e. responding to lower doses of contaminants) than the biometric parameters of

[6][14]

rate of germination and biomass . Hence, this makes it possible to gain evidence of adverse effects

of soils on plants that could not be highlighted by the rate of germination or biomass. Additionally, for in

situ assessment purposes, it can be difficult to observe evident effects on the rate of germination and/

or biomass of plants.

It should be noted that from a practical point of view, especially with plant species harvested in the

field, and in comparison with other bio-indicators, the Omega-3 index presents several advantages.

— For fatty acid analysis, only 20 mg to 50 mg of fresh leaf tissues per sample are needed. Hence, this

is not destructive for plants, and there is not a problem with getting enough tissues of one species

from a given area.

— Samples of plant tissues can be stored in methanol for several days at room temperature prior to

analyses.

— It is not necessary to find a particular species at a site, and that a priori any species (often chosen

among the most representative) can be sampled (Clause 6).

The results of a ring test performed by six individual laboratories to assess the reproducibility and the

repeatability of the method are shown in Annex A. The results obtained by the same investigator with

the same sample and the same measuring instrument over a short period of time are shown in Annex B.

© ISO 2019 – All rights reserved v
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SIST EN ISO 21479:2020
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SIST EN ISO 21479:2020
INTERNATIONAL STANDARD ISO 21479:2019(E)
Soil quality — Determination of the effects of pollutants on
soil flora — Leaf fatty acid composition of plants used to
assess soil quality

WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic, or

otherwise harmful chemicals or infectious micro-organisms. Occupational health risks can

arise from dust or evaporated chemicals. Furthermore, plants might take up chemicals from the

soil and safety measures should also be considered when handling the test plants.

1 Scope

This document describes a method to compare the quality of soils by determining the fatty acid

composition of the leaves of plant species grown in these soils.

This method does not make it possible to determine an optimal value of the Omega-3 index and,

therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as

homogeneous). The method can only be used to compare the quality of soils between various areas.

This method is applicable to:
— soils from contaminated sites;
— amended soils;
— soils after remediation;
— soil with waste products (e.g. slurry, manure, sludge or composts).

Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa

seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing

these values to those obtained from control soils (see Annex B).
2 Normative references
There are no normative references in this document.
3 Terms, definitions and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1.1
Omega-3 index
% C18:3/(%C18:0 + % C18:1 + % C18:2)
Note 1 to entry: The Omega-3 index has no unit.
© ISO 2019 – All rights reserved 1
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
3.2 Abbreviated terms
For the purposes of this document, the following abbreviated term applies.

FAME(s) Fatty Acid Methyl Ester(s); C16:0: palmitic acid methyl ester; C16:1: palmitoleic methyl

ester; C18:0: stearic acid methyl ester; C18:1: oleic acid methyl ester; C18:2: linoleic acid

methyl ester; C18:3: linolenic acid methyl ester.
4 Principle

The method is used to assess the quality of soils by determining the fatty acid composition of the leaves

of angiosperm species (see Annex C and [9-14][18]) grown on these soils. After sampling leaf tissues,

their fatty acid composition is determined. For this, transesterification is carried out on the foliar

tissues and the fatty acid methyl esters obtained are analysed by gas chromatography. After analysis,

the % C18:3 / (% C18:0 + % C18:1 + % C18:2) ratio is calculated. The lower this ratio, the higher the

adverse effects on plants induced by soils is [6][9-14][18].
5 Apparatus and reagents
5.1 Apparatus

In addition to the standard laboratory equipment, the following apparatus are required.

5.1.1 Scissors to cut leaves.

5.1.2 Graduated glass pipette, to add sulfuric acid (H SO ) to methanol, pipettes to dispense the

2 4

mixture into glass culture tubes (1 ml/tube), and Pasteur pipettes for recovering hexane after extraction

of FAMEs.

5.1.3 Glass culture tubes (e.g. 1,3 × 10 cm) with polytetrafluoroethylene seal screw caps. These culture

tubes were numbered on adhesive tape (and not directly on the glass, to prevent any risk of erasing).

Tubes were checked to ensure they were not chipped (in order to guarantee their leak-tight seal).

5.1.4 System (e.g. heating block) for heating the tubes to 80 °C.

5.1.5 Benchtop centrifuge for centrifuging the tubes to 200 g to 300 g and separating the aqueous

phase from hexane.

5.1.6 Gas chromatograph vials with inserts and screws caps with a polytetrafluoroethylene septum.

5.1.7 Gas chromatograph equipped with a Flame Ionisation Detector (FID) and a capillary column for

separating and quantifying methyl esters of fatty acids with 12 carbon atoms to 22 carbon atoms, and for

each aliphatic chain length to separate the saturated, mono-, di- and tri-unsaturated esters.

Note 1 Most of the time, the studies that led to the preparation of this document were carried out using a gas

chromatograph (Hewlett Packard 5890 series II or Hewlett Packard 7890A) on a Carbowax 1,2 micron, 0,53 mm

diameter, 15 m long capillary column (Altech, Deerfield, IL., USA) or on a DB-WAX 1 micron, 0,53 mm diameter,

15 m long capillary column (Agilent, Santa Clara, CA., USA), helium being the carrier gas .

1) This information is given for the convenience of users of this standard and does not constitute an endorsement

by ISO of these products.
2 © ISO 2019 – All rights reserved
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SIST EN ISO 21479:2020
ISO 21479:2019(E)
5.2 Reagents

5.2.1 Methanol (99 %) and sulfuric acid (H SO ), components of the transesterification solution.

2 4
5.2.2 Distilled water and hexane (99 %) for extracting the FAMEs.
6 Sampling strategies

Because plant fatty acid composition can vary as a function of climatic conditions, the compared areas

should share the same climatic conditions (humidity, temperature, sunlight). In addition, because the

Omega-3 index is an early indicator, its measurement is not relevant when a strong visual phenotype

(highly reduced biomass, high leaf chlorosis, etc.) is detected for plants having grown in one area, and

not detected in another area.

Depending on the aim of the study, one or several angiosperm species can be sampled from each area

of interest. For most of the studies, even if only one species can be used for the assessment of a given

site (a metallurgic landfill soil for example), it is recommended to use several species (if possible three

to eight). By using only one species, it is possible to serendipitously sample a highly resistant (or

sensitive) species. In addition, the larger the number of species sampled, the more representative the

results will be of a “soil quality” for the overall phytocoenosis. Hence, in this case, the various areas

of the site are first prospected, and species to sample are chosen among the most representative

examples, common to all areas to the extent possible. One leaf (or a piece of a leaf when whole leaves are

too large to be entirely immerged in 1 ml of methanol/H SO , see 8.2) from four to eight individuals per

2 4

species should be sampled per area. Some plant species previously successfully used to assess the soils

of contaminated sites (by organic compounds and/or metals) are indicated in Annex C.

When it is not possible to sample the same species in all the areas, it remains possible to determine

the Omega-3 index but, in this case: (i) all the species sampled in a given area should be present and

sampled on at least one other area and (ii) all pairs of areas should share at least one species to be

sampled.

Note that for the assessment of agricultural practices, the only plant species to sample is usually the

only one of interest, namely the cultivated crop. When only one species is sampled, the leaf (or a piece of

leaf) of 6 to 12 individuals per area is harvested.
7 Sampling of leaf tissues

The following recommendations should be followed to sample leaf tissues suitable for subsequent

analysis:

— as the transesterification response involves obtaining fatty acid methyl esters from biological

samples, and the presence of water leads to hydrolysis of the esters formed, the presence of external

water on the biological samples must be avoided. Hence, if leaves are wet, before sampling, it is

necessary to remove water from their surface by the use of an absorbent paper;

— do not sample leaves under hydric (drought) or biotic (pathogens) stress. Only green leaves should

be harvested;

— harvest leaves on plants of similar size. Consequently, harvest of leaves from small plants in one

area and leaves from tall plants in another should not be undertaken to measure the Omega-3 index

(see Annex D and [12]);

— as a precautionary measure, we recommend harvesting only mature leaves and to disregard

developing ones (see Annex D);

— when only a part of the leaves from a given species is sampled, harvest the same part of the leaves

(the distal part for example) for all individuals;
© ISO 2019 – All rights reserved 3
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SIST EN ISO 21479:2020
ISO 21479:2019(E)

— as a precautionary measure, it is recommended to harvest all the plants within 2 h to 3 h (see Annex D).

8 Obtaining, extraction and analyses of FAMES
8.1 Contamination control

To prevent contamination, it is necessary to avoid any contact between the solutions with plastic, parafilm

or glue, etc. To ensure the absence of contaminations (e.g. protocol errors, contaminated solutions, etc.),

a test should be performed before each series of analyses by following the same protocol described in 8.2

and 8.3, but without biological tissues in the culture tubes. After GC analyses, with the exception of the

peak corresponding to hexane, the profile of the gas chromatogram should not display peaks.

Avoid any contact of the solutions with plastic: this recommendation does not apply to the pipette tips

used for collecting the solution of methanol/H SO (40/1) or hexane.
2 4
8.2 Obtaining and extraction of FAMES from plant leaves

Introduce the foliar tissues (approximately 1 cm × 1 cm) into the culture tubes (see 5.1.3) containing

1 ml of a solution of methanol/H SO (40/1). Seal the tubes using a screw cap equipped with a

2 4

polytetrafluoroethylene seal. Heat them for 1 h at 80 °C. With the methanol boiling at 72 °C at a pressure

of 1 atmosphere, it is mandatory to avoid any evaporation so as to cause saturation vapour pressure in

the tubes. It is, therefore, important that they are perfectly plugged. It is also necessary to visually

check (every 5 min for 20 min, then every 10 min) that the solution of methanol/H SO does not boil

2 4

for the duration of the heating. If during the heating the contents of the tube boil, lower the tube into

the ice to cool it then completely unscrew and retighten the cap. Readjust the volume, if necessary, to

1 ml by adding methanol. If the contents are still boiling afterwards, take another tube and another cap

and transfer into it the contents of the defective tube. The fatty acid composition of tissues in the tubes

where the solution of methanol/H SO has (almost) totally evaporated during the heating should not be

2 4
analysed.

After 1 h of heating at 80 °C, cool the tubes (e.g. put them on ice). First add 750 µl of 99 % hexane,

then 1,5 ml of H O. Shake vigorously by hand for 20 sec. The use of a vortex should be avoided.

Centrifuge the tubes at 200 g to 300 g for 5 min to 10 min to obtain two phases. Using a Pasteur pipette,

transfer 200 µl to 400 µl of hexane (upper phase) into a CG vial equipped with an insert. Close the vial

using a screw-opening cap equipped with a silicone septum. Colle
...

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