SIST EN ISO 24032:2022
(Main)Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO 24032:2021)
Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO 24032:2021)
This document describes a method to assess the bioaccumulation of chemicals in snails, i.e.
concentrations of metal(loid)s (ME) or organic compounds [e.g. polycyclic aromatic hydrocarbons
(PAHs) and polychlorinated biphenyls (PCBs)] accumulated in their tissues.
This document presents how to prepare snails for caging in situ for 28 days, the in situ test design and
then how to collect and prepare the snails until conservation and further analysis. If a kinetic study of
accumulation is necessary, sampling of snails at different time-points during exposure is possible as
well [13],[19],[22].
This document excludes analytical methods. Preparation (extraction and mineralization) of the samples
and quantification of chemicals are not in the scope of the present document.
The method is applicable for soils under different uses (agricultural, industrial, residential, forests,
before and after remediation, on potentially contaminated sites, etc.) and waste materials [8],[10],
preferably with vegetation and/or humus cover.
The method is applicable subject to certain limits of temperature (frost-free period, i.e. mainly from
April to October in temperate region).
Optionally (see Annex I), the method can be used in the laboratory to evaluate the accumulation of
contaminants [and optionally, the sum of excess of transfer (SET) index for ME, PAH, PCB] of snails
exposed only to soil
Bodenbeschaffenheit - In-situ-Käfighaltung von Schnecken zur Beurteilung der Bioakkumulation von chemischen Stoffen (ISO 24032:2021)
Dieses Dokument beschreibt ein Verfahren zur Bewertung der Bioakkumulation von Chemikalien in Schnecken, d. h. Konzentrationen von Metall(oid)en (ME) oder organischen Verbindungen, z. B. polyzyklische aromatische Kohlenwasserstoffe (PAK) und polychlorierte Biphenyle (PCB), die sich in ihren Geweben angereichert haben.
In diesem Dokument wird dargelegt, wie Schnecken für die 28 tägige In situ-Käfighaltung vorbereitet werden, wie das In situ-Prüfdesign aussieht und wie die Schnecken bis zur Konservierung und weiteren Analyse gesammelt und vorbereitet werden. Falls eine kinetische Untersuchung der Akkumulation notwendig ist, ist auch eine Probenahme der Schnecken zu verschiedenen Zeitpunkten während der Exposition möglich [13] [19] [22].
Dieses Dokument schließt analytische Verfahren aus. Die Vorbereitung (Extraktion und Mineralisierung) der Proben und Quantifizierung von Chemikalien fallen nicht in den Anwendungsbereich des vorliegenden Dokuments.
Das Verfahren ist auf Böden mit unterschiedlicher Nutzung (landwirtschaftliche, industrielle, Wohn und Waldböden, vor und nach der Sanierung, auf potenziell kontaminierten Standorten usw.) sowie auf Abfallmaterialien [8] [10] anwendbar, vorzugsweise auf Böden mit Vegetation und/oder Humusbedeckung.
Das Verfahren ist vorbehaltlich bestimmter Temperaturgrenzen (frostfreier Zeitraum, d. h. hauptsächlich von April bis Oktober in gemäßigtem Klima) anwendbar.
Optional (siehe Anhang I) kann das Verfahren im Labor verwendet werden, um die Akkumulation von Kontaminanten (und optional die Summe des Übertragungsüberschusses (en: Sum of Excess of Transfer) (SET) index für ME, PAK, PCB) von Schnecken zu bewerten, die nur dem Boden exponiert sind.
Qualité du sol - Encagement in situ d’escargots pour la mesure de la bioaccumulation de contaminants (ISO 24032:2021)
Le présent document décrit une méthode permettant d’évaluer la bioaccumulation de substances chimiques chez les escargots, c’est-à-dire les concentrations de métaux (métalloïdes) (ME) ou de composés organiques [tels que les hydrocarbures aromatiques polycycliques (HAP) et les polychlorobiphényles (PCB)] accumulés dans leurs tissus.
Le présent document présente la méthodologie pour préparer les escargots pour un encagement in situ pendant 28 jours, la description de l’essai in situ, puis la méthodologie de collecte et de préparation des escargots jusqu’à leur conservation pour analyse ultérieure. Si une étude cinétique de l’accumulation est nécessaire, il est également possible de prélever des échantillons d’escargots à différents moments de l’exposition[13],[19],[22].
Le présent document exclut les méthodes analytiques. La préparation (extraction et minéralisation) des échantillons et la quantification des substances chimiques ne font pas partie du domaine d’application du présent document.
La méthode est applicable à des sols destinés à différentes utilisations (agricole, industrielle, résidentielle, forestière, avant et après dépollution, sur des sites potentiellement pollués, etc.) et aux déchets[8],[10], de préférence sur des sols recouverts d’une couverture végétale et/ou d’une couche d’humus.
La méthode est applicable sous réserve de certaines limites de température (période sans gel, c’est-à-dire généralement d’avril à octobre en région tempérée).
En option (voir Annexe I), la méthode peut être utilisée en laboratoire pour évaluer l’accumulation de contaminants [et, facultativement, l’indice Somme des excès de transfert (SET) pour les ME, HAP et PCB] des escargots exposés uniquement au sol.
Kakovost tal - Uporaba kletk s polži na terenu za oceno bioakumulacije onesnaževal (ISO 24032:2021)
Ta dokument opisuje metodo za ocenjevanje bioakumulacije kemikalij v polžih, tj. koncentracij kovin in metaloidov (ME) oziroma organskih spojin [npr. policikličnih aromatskih ogljikovodikov (PAH) in polikloriranih bifenilov (PCB)], nakopičenih v njihovih tkivih. V tem dokumentu je predstavljeno, kako pripraviti polže za 28-dnevno nastanitev v kletke na kraju samem, vključno z načrtom preskusa na kraju samem ter nadalje s postopkom zbiranja in priprave polžev do skladiščenja oziroma nadaljnje analize. Če je potrebna kinetična študija akumulacije, je mogoče izvesti tudi vzorčenje polžev ob različnem času med izpostavljenostjo [13], [19], [22]. Ta dokument vključuje analitične metode. Priprava (ekstrakcija in mineralizacija) vzorcev in količinska opredelitev kemikalij ne spadata na področje uporabe tega dokumenta. Metoda se uporablja za tla v okviru različnih vrst uporabe (kmetijska, industrijska ali stanovanjska uporaba, gozdovi, pred sanacijo in po njej, na potencialno onesnaženih območjih itd.) in odpadne materiale [8], [10], po možnosti z rastlinjem in/ali humusom. Metoda se uporablja ob upoštevanju določenih omejitev glede temperature (obdobje brez zmrzali, tj. od aprila do oktobra v območjih z zmernim podnebjem). Metodo je mogoče izbirno (glej dodatek I) uporabiti v laboratoriju za vrednotenje akumulacije onesnaževal [in izbirno indeksa vsote presežnega prenosa (SET) za kovine in metaloide, policiklične aromatske ogljikovodike ter poliklorirane bifenile] v polžih, izpostavljenih izključno tlom.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 24032:2022
01-marec-2022
Kakovost tal - Uporaba kletk s polži na terenu za oceno bioakumulacije
onesnaževal (ISO 24032:2021)
Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO
24032:2021)
Bodenbeschaffenheit - In-situ-Käfighaltung von Schnecken zur Beurteilung der
Bioakkumulation von chemischen Stoffen (ISO 24032:2021)
Qualité du sol - Encagement in situ d’escargots pour la mesure de la bioaccumulation de
contaminants (ISO 24032:2021)
Ta slovenski standard je istoveten z: EN ISO 24032:2021
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 24032:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 24032:2022
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SIST EN ISO 24032:2022
EN ISO 24032
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - In situ caging of snails to assess
bioaccumulation of contaminants (ISO 24032:2021)
Qualité du sol - Encagement in situ d'escargots pour la Bodenbeschaffenheit - In-situ-Käfighaltung von
mesure de la bioaccumulation de contaminants (ISO Schnecken zur Beurteilung der Bioakkumulation von
24032:2021) chemischen Stoffen (ISO 24032:2021)
This European Standard was approved by CEN on 5 December 2021.
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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 24032:2021 E
worldwide for CEN national Members.
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SIST EN ISO 24032:2022
EN ISO 24032:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 24032:2022
EN ISO 24032:2021 (E)
European foreword
This document (EN ISO 24032:2021) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with 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 June 2022, and conflicting national standards shall be
withdrawn at the latest by June 2022.
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.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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 24032:2021 has been approved by CEN as EN ISO 24032:2021 without any modification.
3
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SIST EN ISO 24032:2022
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SIST EN ISO 24032:2022
INTERNATIONAL ISO
STANDARD 24032
First edition
2021-12
Soil quality — In situ caging of
snails to assess bioaccumulation of
contaminants
Qualité du sol — Encagement in situ d’escargots pour la mesure de la
bioaccumulation de contaminants
Reference number
ISO 24032:2021(E)
© ISO 2021
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test organism and equipment . 2
5.1 Biological material . 2
5.2 Equipment . 3
6 Preparation of the organisms for the exposure . 4
7 Exposure of the test organisms . 4
7.1 General . 4
7.2 Beginning of exposure . 5
7.3 End of the exposure — Starvation . 6
7.4 Sampling and preparation after exposure . 7
8 Calculation and expression .7
8.1 General . 7
8.2 For metal(loid)s . 7
8.2.1 Threshold guide value . 7
8.2.2 Calculation of the sum of the excess of transfer of metal(loid)s: SET index . 8
8.3 For other chemicals . 9
9 Validity of the experiment . 9
10 Test report . 9
Annex A (informative) Sources and routes of exposure of snails to contaminants in the field .10
Annex B (informative) Main steps of the bioassay in situ .11
Annex C (informative) Breeding technique for snails .15
Annex D (informative) Example of composition of snail feed .22
Annex E (informative) Usual concentrations in the viscera of sub-adult snails before caging.23
Annex F (informative) Recommended test systems for in situ exposure to assess
bioaccumulation of contaminants in snails .25
Annex G (informative) Example of mass of snails before exposure .28
Annex H (informative) Results of the international ring test .30
Annex I (informative) Ex situ exposure to assess bioaccumulation of chemicals in snails .48
Bibliography .56
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SIST EN ISO 24032:2022
ISO 24032:2021(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 of 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
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with
the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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.
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
Introduction
Snails are ubiquitous soil macroinvertebrates living at the interface soil, plants and air. Those pulmonate
gastropod molluscs are phytophagous and saprophagous (trophic level of primary consumers and
detritivorous). They ingest vegetation and soil, and crawl on the ground where they lay their eggs.
Therefore, snails integrate multiple sources and routes of contamination (see Annex A, Figure A.1).
Snails participate in exchanges with soil and are preyed upon by various consumers (invertebrates:
glow-worms, ground beetle larvae, or vertebrates: birds, small mammals such as shrews, hedgehogs
and humans).
1)
Among snail species, the recommended species is Cantareus aspersus O.F. Müller 1774 (synonyms:
Helix aspersa aspersa, Cornu aspersum) also known as common garden snail, brown garden snail,
garden snail, land snail, nicked name in French “Petit-Gris” (see Annex A, Figure A.2). This species is
a stylommatophoran pulmonate gastropod molluscs of the Helicidae family, widely distributed across
[9],[28]
the world . This palearctic species can be acclimated to regions with different types of climate:
Mediterranean, oceanic temperate, midcontinental temperate and even tropical. Cantareus aspersus
(Müller, 1774) is of European origin and has been introduced into all parts of the world. It is now on all
continents except Antarctica. On the other hand, the species is recognized as an agriculturally harmful
snail in some countries and must be treated carefully.
[1]
Juvenile snails are already covered in ISO 15952 that describes how to assess ex situ, i.e. in laboratory
conditions, toxic effect of chemicals or contaminated matrix on the survival and growth of juvenile
(1 g fw).
Currently there is no standardized in situ bioassay allowing the assessment in the field of the transfer
[3]
of contaminants from the environment to organisms of the soil fauna. Indeed, despite ISO 19204
(relative to the TRIAD approach) which recommends the application of three combined lines of
evidence (chemistry, ecotoxicology and ecology) and highlights the interest of bioindicators of effect
and accumulation as additional tools for site-specific ecological risk assessment, few bioassays are
available for this purpose. As described in ISO 19204:2017, Annex A, measurements of bioaccumulation
in plants or soil organisms are thus useful to:
— assess the effective bioavailability of soil contaminants to soil organisms;
— approach the food chain transfer and the risk of secondary poisoning of consumers.
In some cases, bioaccumulation can result in toxic effects but this is not always the case (see
[2]
ISO 17402 ).
Since farming is possible (see ISO 15952:2018, Annex B), snails with a known biological past can be
used on the field to analyse bioavailability of contaminants present in the habitats (soil, plants, air) by
measuring their accumulation in individuals caged and exposed for a determined period of time.
[10],[12],[13],[15],[19],[22],[23],[27],[29],[30]
C. aspersus can be used either in the field or in the laboratory
[14],[18],[20],[21]
to assess the fate and transfer (i.e. environmental bioavailability, ISO 17402) of chemicals
2)
in soils. This soil bioindicator has been applied on numerous field sites to evaluate habitat and
retention function of soils. This bioassay allows determining the bioavailability of chemicals to snails
thanks to the measurement of their concentration in their visceral mass (which contain mainly the
digestive gland and some other organs as described in Reference [16]). The visceral mass is the main
site of contaminant accumulation in snails.
This document describes how to expose snails in situ for 28 days and how to prepare them until
chemical analysis are performed to assess bioaccumulation in their viscera. This bioassay evaluates the
transfer of contaminants from the environment to land snails.
1) Available from: https://inpn.mnhn.fr/espece/cd_nom/199863/tab/taxo.
2) Available from: https://ecobiosoil.univ-rennes1.fr/ADEME-Bioindicateur/english/worksheet.php.
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
This test is applicable in the field (e.g. contaminated sites, amended soils, soils after remediation,
agricultural or other sites under concern and waste materials) by caging snails for 28 days on the
studied site/soil/waste. Snails integrate chemicals of all terrestrial sources (soil, plant, air). After
exposure, concentrations of chemicals are measured in the visceral mass of snails.
Optionally, the method can be used in the laboratory (ex situ) to evaluate bioaccumulation of chemicals
of snails exposed only to soil (see Annex I).
The results of a ring test performed in situ by six laboratories to assess the method of exposure and by
four laboratories from exposure until to chemical analysis are shown in Annex H.
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SIST EN ISO 24032:2022
INTERNATIONAL STANDARD ISO 24032:2021(E)
Soil quality — In situ caging of snails to assess
bioaccumulation of contaminants
1 Scope
This document describes a method to assess the bioaccumulation of chemicals in snails, i.e.
concentrations of metal(loid)s (ME) or organic compounds [e.g. polycyclic aromatic hydrocarbons
(PAHs) and polychlorinated biphenyls (PCBs)] accumulated in their tissues.
This document presents how to prepare snails for caging in situ for 28 days, the in situ test design and
then how to collect and prepare the snails until conservation and further analysis. If a kinetic study of
accumulation is necessary, sampling of snails at different time-points during exposure is possible as
[13],[19],[22]
well .
This document excludes analytical methods. Preparation (extraction and mineralization) of the samples
and quantification of chemicals are not in the scope of the present document.
The method is applicable for soils under different uses (agricultural, industrial, residential, forests,
[8],[10]
before and after remediation, on potentially contaminated sites, etc.) and waste materials ,
preferably with vegetation and/or humus cover.
The method is applicable subject to certain limits of temperature (frost-free period, i.e. mainly from
April to October in temperate region).
Optionally (see Annex I), the method can be used in the laboratory to evaluate the accumulation of
contaminants [and optionally, the sum of excess of transfer (SET) index for ME, PAH, PCB] of snails
exposed only to soil.
2 Normative references
There are no normative references in this document.
3 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 https:// www .electropedia .org/
3.1
caging
closed microcosm allowing exposure of snails by various routes and several sources
3.2
bioaccumulation
phenomenon by which a chemical present in the medium accumulates in a living organism
Note 1 to entry: This phenomenon is observed when the rate of absorption exceeds the rate of elimination of the
contaminant.
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
3.3
inactive snail
snail without any activity, generally under dry conditions where they glue on the walls of the box in
which they are placed (generally just due to a simple dried mucus ring)
3.4
aestivation
snails kept inactive, under dry conditions, at a temperature of 15 °C to 20 °C
3.5
plot
characteristic and representative sub-area of the site
Note 1 to entry: The geographical coordinates of each plot should be recorded.
3.6
site
field place (or geographical entity) under study and where the microcosms are placed to assess the
bioavailability of contaminants to snails
Note 1 to entry: The site can present one or more plot(s) and land use, i.e. a field, a pasture, a forest, an industrial
site, a discharge.
4 Principle
Snails are caged in microcosms at the study site for 28 days. Fifteen sub-adult [(5 ± 1) g of the body mass]
garden snails shall be placed in each microcosm. From the end of their breeding to their placement on
the soil, they can be stored inactive in dry wooden boxes (round wooden boxes, approximately 12 cm in
diameter and 4 cm in height; see Figure 1 and Figure B.2). They are woken from aestivation by spraying
them with water a few hours before they are placed in the microcosms. Here, they are exposed to soil as
well as plants that have grown on-site and ambient air in order to be under natural exposure conditions
(climate hazards).
After exposure, the collected snails are brought back to the laboratory and starved for 48 h. During
the starvation, faeces are removed every 24 h. Snails are then frozen at –80 °C. After thawing, the soft
body is removed from the shell; the visceral mass and the foot (see Annex B, Figure B.1) are separated
and prepared for chemical analysis to determinate internal concentration of chemicals. Main steps are
presented in Annex B.
5 Test organism and equipment
5.1 Biological material
Test organisms shall be sub-adult snails (to avoid mass change during the exposure duration and the
consecutive dilution of the bioaccumulation per the mass gain during the growth or the transfers of
compounds to the eggs during the reproductive stages). The recommended species is the land snail
Cantareus aspersus (Müller, 1774) which shall be 7 weeks to 12 weeks old, having a mean fresh mass
of (5 ± 1) g.
NOTE 1 Optionally, the shell diameter can be measured (mean ± SD of 25 mm ± 5 mm; min/max of 20
mm/30 mm).
The snails shall be selected from synchronous breeding in order to form a population as homogeneous
as possible with respect to mass and age. The breeding techniques for snails are described in Annex C.
In summary, after a nursery and a growth period (3 weeks to 6 weeks followed by 4 weeks to 6 weeks),
the sub-adult snails shall be used directly or after an aestivation period that should not be more than 5
months [i.e. snail inactive, fixed on the wall of a dry box (plastic box shall be avoided), in a temperature-
controlled room between 15 °C and 20 °C]. The aestivation is carried out in round wooden boxes
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
(approximately of 12 cm in diameter and 4 cm in height; usually 15 snails per boxes, which is equal the
number of snails per microcosm).
Snails shall be reared for the purpose of the project (see Annexes C and D) or be purchased from local
snail farmers.
NOTE 2 The use of some other genus and/or species of Helicidae is possible (see examples and conditions
in ISO 15952:2018, Annex G).
A control of the chemical quality of the subadult snails selected for the caging (i.e. unexposed snails)
can be performed on 6 snails with respect to the initial concentrations of the chemicals of interest (C
snail-t0). These control snails can be selected at the same time as the snails used for snail caging. The
analysis of the chemical quality of snails before caging can be done at the same time as the analysis
of snails after exposure. It is not mandatory to make this control. Indeed, after exposure, all data
are compared to the threshold guide value (TGV) (see 8.2.1); however, if possible to get these data, it
provides an indication that snails were uncontaminated before exposure. For chemicals for which no
TGV are available, data can be compared to various values (see 8.2.2.4) among which are Csnail-t0.
The sub-adult snails used shall present usual concentrations in the visceral mass before caging (see
Annex E). For PAH and PCB data, as extraction are often made on fresh tissues, the data of Table E.1 are
−1 −1
in µg.kg fresh mass of viscera (these values can be converted in µg.kg dw on the basis of ≈ 15 % dry
−1
mass of the visceral mass); for metal(loids), the data are in mg.kg dry mass of visceral mass.
5.2 Equipment
5.2.1 Microcosm, stainless steel cylinders with 25 cm diameter and 25 cm height covered by a 0,5 cm
or 1 cm mesh netting.
An example is presented in Figure 1 and in Annex F, Figure F.1.
NOTE 1 Other devices can be used if the material that constitutes them cannot be a source of contamination;
for some purpose (e.g. exposure of snails to chemicals sprayed in the field), fully screened microcosm can be used
[see for example Reference [11] that used stainless steel cages of 25 cm × 25 cm × 15 cm (mesh size of grid: 1 cm)
closed by a stainless steel grid of 30 cm × 30 cm (mesh size: 1 cm) held by four pickets (see Annex F, Figure F.2)].
NOTE 2 In some cases, it can be necessary to protect the microcosm from predators or cattle (see examples in
Annex F, Figure F.3) or from the sun (see Annex F, Figure F.4).
5.2.2 Netting, 0,5 cm or 1 cm mesh netting, also stainless steel.
5.2.3 Pickets, stainless steel picket (diameter 5 mm; length 46 cm to 72 cm) to maintain the mesh
netting on the cage. Depending on the soil settlement or the presence of stones, the size of picket shall
be adapted.
5.2.4 Pieces of tiles, see Figure 1 and Annex F.
5.2.5 Wooden storage. Inactive snails can be stored and transported before exposure in round
wooden boxes (approximately 12 cm in diameter and 4 cm in height), with the snails under dry
conditions, at a temperature of 15 °C to 20 °C (see Figure 1, Figure B.2 and Annex G).
5.2.6 Boxes for fasting, sampling. For the preparation of snails in the laboratory [e.g. to keep the
snails before individual weighing), plastic containers (PCs) (e.g. made of transparent polystyrene or
any other container having approximate dimensions: 24 cm (length) × 10,5 cm (width) × 8 cm (height)]
can be used.
5.2.7 Calliper rule. For the measurement of the shell diameter, a calliper rule having a precision
of 0,1 mm.
3
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SIST EN ISO 24032:2022
ISO 24032:2021(E)
5.2.8 Balance. One analytical balance having a precision of at least 10 mg.
5.2.9 Water, of purity at least deionized.
5.2.10 Feed, which shall be provided in the form of flour at its natural moisture content (5 % to 10 %).
In order to obtain sufficient growth, it is recommended to carry out the tests with a flour-based feed
comprising cereals, forage, mineral salts and vitamins which properly covers the needs of the snails. An
example of feed composition is given in Annex D.
5.2.11 Small material. Elastic strips to close wooden storage or boxes for fasting, sampling. Tape to
label the wood
...
SLOVENSKI STANDARD
oSIST prEN ISO 24032:2021
01-januar-2021
Kakovost tal - Uporaba kletk s polži na terenu za oceno bioakumulacije
onesnaževal (ISO/DIS 24032:2020)
Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO/DIS
24032:2020)
Bodenbeschaffenheit - In-situ-Käfighaltung von Schnecken zur Beurteilung der
Bioakkumulation von chemischen Stoffen (ISO/DIS 24032:2020)
Qualité des sols - Encagement in situ descargots pour la mesure de la bioaccumulation
de contaminants (ISO/DIS 24032:2020)
Ta slovenski standard je istoveten z: prEN ISO 24032
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 24032:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 24032:2021
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oSIST prEN ISO 24032:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 24032
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2020-11-18 2021-02-10
Soil quality — In situ caging of snails to assess
bioaccumulation of contaminants
Qualité du sol — Encagement in situ d’escargots pour la mesure de la bioaccumulation de contaminants
ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 24032:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
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ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test organism and equipment . 2
5.1 Biological material . 2
5.2 Equipment . 3
5.2.1 Microcosm . 3
5.2.2 Netting . 3
5.2.3 Pickets . 3
5.2.4 Pieces of tiles . 3
5.2.5 Wooden storage . 4
5.2.6 Boxes for fasting, sampling . 4
5.2.7 Calliper rule . 4
5.2.8 Balance . 4
5.2.9 Water . 4
5.2.10 Feed . 4
5.2.11 Small material . 4
6 Preparation of the organisms for the exposure . 4
7 Exposure of the test organisms . 5
7.1 Beginning of exposure . 5
7.2 End of the exposure — Starvation . 6
7.3 Sampling and preparation after exposure . 6
8 Calculation and expression . 6
8.1 General . 6
8.2 For metal(loid)s. 6
8.2.1 Threshold guide value . 6
8.2.2 Calculation of the sum of the excess of transfer of metal(loid)s: SET index . 7
8.3 For other chemicals . 7
9 Validity of the experiment . 8
10 Test report . 8
Annex A (informative) Sources and routes of exposure of snails to contaminants in the field .9
Annex B (informative) Main steps of the bioassay in situ .10
Annex C (informative) Breeding technique for snails.12
Annex D (informative) Example of composition of snail feed .17
Annex E (informative) Usual concentrations in the viscera of sub-adult snails before caging .18
Annex F (informative) Standardized forms of recommended test systems for in situ
exposure to assess bioaccumulation of contaminants in snails.19
Annex G (informative) Example of mass of snails before exposure .23
Annex H (informative) Results of the international ring test .25
Annex I (informative) Ex situ exposure to assess bioaccumulation of chemicals in snails .41
Bibliography .46
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(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.
iv © ISO 2020 – All rights reserved
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(E)
Introduction
Snails are ubiquitous soil macroinvertebrates living at the interface soil, plants and air. Those pulmonate
gastropod molluscs are phytophagous and saprophagous (trophic level of primary consumers and
detritivorous). They ingest vegetation and soil, crawl on the ground where they lay their eggs.
Therefore, they integrate multiple sources and routes of contamination (see Annex A – Figure A.1).
Snails participate in exchanges with soil and are preyed upon by various consumers (invertebrates:
glow-worms, ground beetle larvae, or vertebrates: birds, small mammals such as shrews, hedgehogs
and humans).
Among snail species, the recommended species is Cantareus aspersus O.F. Müller 1774 (synonyms: Helix
aspersa aspersa (O.F. Müller, 1774), Cornu aspersum; https:// inpn .mnhn .fr/ espece/ cd _nom/ 199863/
tab/ taxo) also known as common garden snail, brown garden snail, garden snail, land snail, nicked
name in French “Petit-Gris”. This species is a stylommatophoran pulmonate gastropod molluscs of the
Helicidae family, widely distributed across the world (Potts, 1975; Chevallier, 1977). This palearctic
species can be acclimated to regions with different types of climate: Mediterranean, oceanic temperate,
midcontinental temperate and even tropical. Cantareus aspersus (Müller, 1774) is of European origin
and has been introduced into all parts of the world. It is now on all continents except Antarctica. On the
other hand, the species is recognized as an agriculturally harmful snail in some countries and must be
treated carefully.
Juvenile snails are already covered in ISO 15952 (2018) that describes how to assess ex situ, i.e. in
laboratory conditions, toxic effect of chemicals or contaminated matrix on the survival and growth of
juvenile (1 g fw).
Currently there is no standardized in situ bioassay allowing the assessment in the field of the transfer
of contaminants from the environment to organisms of the soil fauna. Indeed, despite ISO 19204 (2017)
(relative to the TRIAD approach) recommends the application of three combined lines of evidence
(chemistry, ecotoxicology and ecology) and highlights the interest of bioindicators of effect and
accumulation as additional tools for site-specific ecological risk assessment, few bioassays are available
for this purpose. As described in Annex A of ISO 19204 (2017), measurements of bioaccumulation in
plants or soil organisms are thus useful to:
— assess the effective bioavailability of soil contaminants to soil organisms;
— approach the food chain transfer and the risk of secondary poisoning of consumers.
In some cases, bioaccumulation can result in toxic effects but this is not always the case
(see ISO 17402:2008).
Since farming is possible (see Annex in ISO 15952:2018), snails with a known biological past can be
used on the field to analyse bioavailability of contaminants present in the habitats (soil, plants, air) by
measuring their accumulation in individuals caged and exposed for a determined period of time.
C. aspersus can be used either in the field (Gomot-de Vaufleury and Pihan, 2000; Scheifler et al., 2006;
Regoli et al., 2006; Gimbert et al., 2008a; Pauget et al., 2013; de Vaufleury et al., 2015; Mariet et al, 2017)
or in the laboratory (Gimbert et al., 2008b; Pauget et al., 2011, 2012, 2017; Louzon et al., 2020) to
assess the fate and transfer (i.e. environmental bioavailability, ISO 17402) of chemicals in soils. This
soil bioindicator has been applied on numerous field sites (https:// ecobiosoil .univ -rennes1 .fr/ ADEME
-Bioindicateur/ english/ worksheet .php) to evaluate habitat and retention function of soils. This bioassay
allows determining the bioavailability of chemicals to snails thanks to the measurement of their
concentration in their visceral mass (which contain mainly the digestive gland and some other organs
as described in Gomot-de Vaufleury and Pihan, 2002). The visceral mass is the main site of contaminant
accumulation in snails.
This standard describes how to expose snails in situ for 28 days and how to prepare them until chemical
analysis are performed to assess bioaccumulation in their viscera. This bioassay evaluates the transfer
of contaminants from the environment to land snails.
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(E)
This test is applicable in the field (e.g. contaminated sites, amended soils, soils after remediation,
agricultural or other sites under concern and waste materials) by caging snails for 28 days on the
studied site/soil/waste. Snails integrate chemicals of all terrestrial sources (soil, plant, air). After
exposure, concentrations of chemicals are measured in the visceral mass of snails.
Optionally, the method can be used in the laboratory (ex situ) to evaluate bioaccumulation of chemicals
of snails exposed only to soil (Annex I).
The results of a ring test performed in situ by six laboratories to assess the method of exposure and by
four laboratories from exposure until to chemical analysis are shown in Annex H.
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oSIST prEN ISO 24032:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 24032:2020(E)
Soil quality — In situ caging of snails to assess
bioaccumulation of contaminants
1 Scope
This document describes a method to assess the bioaccumulation of chemicals in snails, i.e.
concentrations of metal(loid)s (ME) or organic compounds (e.g. PAHs and PCBs) accumulated in their
tissues.
This document presents how to prepare snails for caging in situ for 28 days, the in situ test design and
then how to collect and prepare the snails until conservation and further analysis. If a kinetic study of
accumulation is necessary, sampling of snails at different time-points during exposure is possible as
well (Gimbert et al., 2008a; Pauget et al., 2013; Mariet et al., 2017)
This standard excludes analytical methods: preparation (extraction and mineralization) of the samples
and quantification of chemicals. These are not in the scope of the present document.
The method is applicable for soils under different uses (agricultural, industrial, residential, forests,
before and after remediation, on potentially contaminated sites, etc.) and waste materials (de Vaufleury,
2015; Bourioug et al., 2015), and should be preferably used on soil with vegetation and/or humus cover.
The method is applicable subject to certain limits of temperature (frost-free period, i.e. mainly from
April to October in temperate region).
Optionally (Annex A) the method can be used in the laboratory to evaluate the accumulation of
contaminants (and optionally the Sum of Excess of Transfer (SET) index for ME, PAH, PCB) of snails
exposed only to soil.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 15952:2018, Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of
the effects on growth by soil contamination
3 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
caging
closed microcosm allowing exposure of snails by various routes and several sources
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ISO/DIS 24032:2020(E)
3.2
bioaccumulation
phenomenon by which a chemical present in the medium accumulates in a living organism
Note 1 to entry: This phenomenon is observed when the rate of absorption exceeds the rate of elimination of the
contaminant
3.3
inactive snails
snails without any activity, generally under dry conditions where they glue on the walls of the box in
which they are placed (generally just due to a simple dried mucus ring)
3.4
aestivation
snails kept inactive, under dry conditions, at a temperature of 15 °C to 20 °C
3.5
plot
characteristic and representative sub-area of the site
Note 1 to entry: The geographical coordinates of each plot should be recorded
3.6
site
field place (or geographical entity) under study and where the microcosms will be placed to assess the
bioavailability of contaminants to snails
Note 1 to entry: The site can present one or more plot(s) and land use, i.e., a field, a pasture, a forest, an industrial
site, a discharge
4 Principle
Snails are caged in microcosms at the study site for 28 days. Fifteen sub-adult (5 ± 1 g of the body mass)
garden snails shall be placed in each microcosm. From the end of their breeding to their placement
on the soil, they can be stored in dry wooden boxes (round wooden boxes, approximately 12 cm in
diameter and 4 cm in height, Figure 1 and B.1.),). They are awaken from aestivation by spraying them
with water a few hours before they are placed in the microcosms. Here, they will be exposed to soil as
well as plants that have grown on-site and ambient air in order to be under natural exposure conditions
(climate hazards).
After exposure, the collected snails are brought back to the laboratory and starved for 48 hours. During
the starvation, faeces are removed every 24 hours. Snails are then frozen at –80 °C. After thawing, the
soft body is removed from the shell; the visceral mass and the foot (Annex B, Figure B.1) are separated
and prepared for chemical analysis to determinate internal concentration of chemicals. Main steps are
presented in Annex B.
5 Test organism and equipment
5.1 Biological material
Test organisms shall be sub-adult snails (to avoid mass change during the exposure duration and the
consecutive dilution of the bioaccumulation per the mass gain during the growth or the transfers of
compounds to the eggs during the reproductive stages). The recommended species is the land snail
Cantareus aspersus (Müller, 1774) which shall be 7 to 12 weeks old, having a mean fresh mass of 5 ± 1 g
(with min/max of 4/6 g).
NOTE 1 Optionally the shell diameter can be measured (mean ± SD of 25 mm ± 5 mm; min/max of 20/30 mm).
2 © ISO 2020 – All rights reserved
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The snails shall be selected from synchronous breeding in order to form a population as homogeneous
as possible with respect to mass and age. The breeding techniques for snails are described in Annex C.
In summary, after a nursery and then a growth period (3 to 6 weeks followed by 4 to 6 weeks), the
sub-adult snails shall be used directly or after an aestivation period that should not be more than 5
months (i.e. snail inactive, fixed on the wall of a dry box (plastic box must be avoided), in a temperature-
controlled room between 15 and 20 °C). The aestivation is carried out in round wooden boxes
(approximately of 12 cm in diameter and 4 cm in height; usually 15 snails per boxes, which is equal the
number of snails per microcosm).
Snails shall be reared for the purpose of the project (see Annexes C and D) or be purchased from local
snail farmers.
NOTE 2 The use of some other genus and/or species of Helicidae is possible (see examples and conditions
in Annex G of ISO 15952:2018).
A control of the chemical quality of the subadult snails selected for the caging (i.e. unexposed snails)
can be performed on 6 snails with respect to the initial concentrations of the chemicals of interest.
These control snails can be selected at the same time as the snails used for snail caging. The analysis of
the chemical quality of snails before caging can be done at the same time as the analysis of snails after
exposure. It is not mandatory to make this control. Indeed, after exposure, all data are compared to the
threshold guide value (TGV) (see 8.2.); however, if possible to get these data, it provides an indication
that snails were uncontaminated before exposure. For chemicals for which no TGV are available, data
can be compared to various values (see 8.2.2.3) among which are Csnail-t0.
The sub-adult snails used shall present usual concentrations in the visceral mass before caging
(Annex E). For PAH and PCB data, as extraction are often made on fresh tissues, data of Table E.1. are
−1 −1
in µg.kg fresh mass of viscera (these values can be converted in µg.kg dw on the basis of ≈ 15 % dry
−1
mass of the visceral mass); for metal(loids) in mg.kg dry mass of visceral mass.
5.2 Equipment
5.2.1 Microcosm
Stainless steel cylinders with 25 cm diameter and 25 cm height covered by a 0,5 or 1 cm mesh netting.
An example is presented in Figure 1 and in Annex F.
NOTE 1 Other device could be used if the material that constitutes them cannot be a source of contamination;
for some purpose (e.g. exposure of snails to chemicals sprayed in the field), fully screened microcosm can be used
(see for example Druart et al. (2011) that used stainless steel cages of 25 × 25 × 15 cm (mesh size of grid: 1 cm)
closed by a stainless steel grid of 30 × 30 cm (mesh size: 1 cm) held by four pickets (see Annex F, Figure F.2).
NOTE 2 In some cases, it could be necessary to protect the microcosm from predators or cattles (see examples
in Annex F Figure F.3).
5.2.2 Netting
0,5 or 1 cm mesh netting, also stainless steel.
5.2.3 Pickets
Stainless steel picket (diameter 5 mm; length 46 to 72 cm) to maintain the mesh netting on the cage.
Depending on the soil settlement or the presence of stones, the size of picket shall be adapted.
5.2.4 Pieces of tiles
See Figure 1 and Annex F.
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5.2.5 Wooden storage
Inactive snails can be stored and transported before exposure in round wooden boxes
(approximately 12 cm in diameter and 4 cm in height), with the snails under dry conditions, at a
temperature of 15 °C to 20 °C (see Figure 1, Figure F.5 and Annex G).
5.2.6 Boxes for fasting, sampling
For the preparation of snails in the laboratory (e.g. to keep the snails before individual weighing), plastic
containers (PCs) (e.g. made of transparent polystyrene or any other container having approximate
dimensions: 24 cm (length) × 10,5 cm (width) × 8 cm (height)) can be used.
5.2.7 Calliper rule
For the measurement of the shell diameter, a calliper rule having a precision of 0,1 mm.
5.2.8 Balance
One analytical balance having a precision of at least 10 mg.
5.2.9 Water
Water, of purity at least deionized.
5.2.10 Feed
The feed shall be provided in the form of flour at its natural moisture content (5 % to 10 %).
In order to obtain sufficient growth, it is recommended to carry out the tests with a flour-based feed
comprising cereals, forage, mineral salts and vitamins which properly covers the needs of the snails. An
example of feed composition is given in Annex D.
5.2.11 Small material
Elastic strips to close wooden storage or boxes for fasting, sampling. Tape to label the wooden storage
and boxes for fasting; indelible markers; Ziploc© bags.
6 Preparation of the organisms for the exposure
After the end of their growth (Figure C.1, growth 1, i.e. time needed to obtain sub-adults that reached
the mass required for the test) snails shall be stored inactive in wooden box (see 3.5). Their mass will
decrease during this storage period that’s why in some cases (i.e. storage for more than 1 week) they
shall be woken from aestivation few days before the start of the assay (see 6).
Depending on the duration of storage between the end of growth period (i.e. when reaching the mean
mass requested, see 5.1.) and the start of the test in the field, snails shall or not be woken by spraying
them with water into the box according to the following scenarios:
— if snails are used in the week following their weighing and distribution in homogeneous batch
(15 snails for 1 microcosm) it is just necessary to awake them some hours before using in the field.
They shall be sprayed with water. This facilitate their handling to remove them from the wood b
...
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