Soil quality -- In situ caging of snails to assess bioaccumulation of contaminants

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.

Qualité du sol -- Encagement in situ d’escargots pour la mesure de la bioaccumulation de contaminants

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.

General Information

Status
Published
Publication Date
07-Dec-2021
Current Stage
5060 - Close of voting Proof returned by Secretariat
Start Date
05-Nov-2021
Completion Date
04-Nov-2021
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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
---------------------- Page: 1 ----------------------
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

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

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Published in Switzerland
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
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

iii
© ISO 2021 – All rights reserved
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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.
© ISO 2021 – All rights reserved
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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).

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

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.

© ISO 2021 – All rights reserved
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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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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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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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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

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.
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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 wooden storage and boxes for fasting; indelible markers, resealable bags.

6 Preparation of the organisms for the exposure

After the end of their growth (see 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 (5.2.5.). 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 Clause 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 are woken 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 necessary to wake them some hours before using in the field. They

shall be sprayed with water in the wood box. This facilitate their handling to remove them from the

wood box and placed them in the microcosm once in the field.

— if they were stored for longer periods (>1 week but < 5 months) before exposure in the field, they

should be awakened and fed with snail feed (5.2.10) for 2 days to 5 days in order they reach their

initial mass. After being awakened by spraying water in the wood box, they are placed in cages

or plastic box (see Figure C.2 in Annex C) for 2 days to 5 days and fed. Then again weighed and

distributed in homogeneous batches (see example in Annex G, Table G.1 and Figure G.1) in the wood

box in which they can be stored for a brief duration (0 to 1 week) before being again awakened and

disposed in the microcosms.

The proportion of snails not woken shall be less than 20 %. As soon as they become active (snails not

stuck to the walls of the box and starting to move), the snails shall be transferred into a box that has

been premoistened with water.

All the snails needed for the assay shall be weighed, and distributed in distinct mass classes (e.g. group

all snails from 4 g to 4,5 g, from 4,6 g to 5 g, from 5,1 g to 5,5 g, from 5,6 g to 6 g. Then, prepare group

of 15 snails each as homogeneous as possible with respect to mass (same distribution of mean group

mass, see example Annex G, Figure G.1).
NOTE Optionally, the shell diameter can be measured.

Snails for the test shall be individually weighed and placed in wooden storage boxes; 15 individuals

shall be stored per wood storage, since one microcosm shall contain 15 snails for exposure.

7 Exposure of the test organisms
7.1 General

The main steps of the bioassay are illustrated in Annex F, Figures F.5 and F.6 (an example of a table of

data is given in Annex G, Table G.1).
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ISO 24032:2021(E)
7.2 Beginning of exposure

Three microcosms shall be placed at each plot. To consider soil heterogeneity in terms of intrinsic

properties and contamination profiles, a minimum of 3 microcosms, per a certain plot area is used.

Each microcosm should contain 15 snails that are exposed to soil, humus and vegetation under natural

climatic conditions. This is the natural way of exposure of snails. Plants, humus that cover the soil (and

also soil) are a source of feeding for snails. Pieces of tiles shall be placed in the cage to provide a shelter

and a bonding surface to snails.

The snails shall be carefully removed from the wooden box, without pulling too hard to avoid braking

the shell; they shall not produce white mucus (like a white foam), which is a sign of mishandling.

NOTE 1 The number of microcosms per plot can be adapted depending on the number or mass of snail tissue

needed for analysis or in the frame of a preliminary study.

NOTE 2 If there is no shade on site, a shade mesh could be placed above the netting to reduce the heat in the

cage. Annex F, Figure F.4.

Once on the field, set up a microcosm on soil (remove stone to avoid space between microcosm and soil

to ensure that the microcosm is sufficiently buried in the soil to avoid the nails from escaping, drive the

cage in the top soil layer of 0,5 cm to 1 cm). Place the snails and the pieces of tiles used as shelters (see

Figure 1). Finally, cover the microcosms with the netting and fix the netting with the pickets. About

20 min are required for this step.
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ISO 24032:2021(E)
a) Sub-adult snail, total fresh mass 4 g to 6 g
b) Open microcosm
c) Microcosms covered by a stainless steel net-
ting (mesh size: 10 mm) securely fitted over the
top of the microcosm by 4 pickets
d) Microcosms on site

Figure 1 — In situ exposure: Active biomonitoring using microcosms where snails are exposed

7.3 End of the exposure — Starvation

All the snail from one microcosm are carefully removed and placed together, e.g. in the wood box used

to store the snails before exposure.

Back in the laboratory, snails shall be cleaned, i.e. if necessary, by removal of soil particles with a brush

and water. Then, snails shall be placed for starvation in a plastic box easy to clean (e.g. as

...

NORME ISO
INTERNATIONALE 24032
Première édition
2021-12
Qualité du sol — Encagement in situ
d’escargots pour la mesure de la
bioaccumulation de contaminants
Soil quality — In situ caging of snails to assess bioaccumulation of
contaminants
Numéro de référence
ISO 24032:2021(F)
© ISO 2021
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ISO 24032:2021(F)
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ISO 24032:2021(F)
Sommaire Page

Avant-propos .............................................................................................................................................................................................................................iv

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

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ..................................................................................................................................................................................1

3 Termes et définitions ...................................................................................................................................................................................... 1

4 Principe.......................................................................................................................................................................................................................... 2

5 Organisme et équipement d’essai ...................................................................................................................................................... 2

5.1 Matériel biologique ............................................................................................................................................................................. 2

5.2 Équipement ................................................................................................................................................................................................ 3

6 Préparation des organismes pour l’exposition ................................................................................................................... 4

7 Exposition des organismes d’essai ................................................................................................................................................... 5

7.1 Généralités ................................................................................................................................................................................................. 5

7.2 Début de l’exposition ......................................................................................................................................................................... 5

7.3 Fin de l’exposition — Jeûne ......................................................................................................................................................... 6

7.4 Prélèvement et préparation après exposition ............................................................................................................ 7

8 Calcul et expression .......................................................................................................................................................................................... 7

8.1 Généralités ................................................................................................................................................................................................. 7

8.2 Pour les métaux (métalloïdes) .................................................................................................................................................. 7

8.2.1 Valeur seuil indicative ................................................................................................................................................... 7

8.2.2 Calcul de la somme des excès de transfert des métaux (métalloïdes):

indice SET ................................................................................................................................................................................. 8

8.3 Pour les autres substances chimiques ............................................................................................................................... 9

9 Validité de l’expérience ................................................................................................................................................................................. 9

10 Rapport d’essai ...................................................................................................................................................................................................... 9

Annexe A (informative) Sources et voies d’exposition des escargots aux contaminants

sur le terrain .........................................................................................................................................................................................................10

Annexe B (informative) Principales étapes du bioessai in situ ...........................................................................................11

Annexe C (informative) Technique d’élevage des escargots ...................................................................................................15

Annexe D (informative) Exemple de composition de la nourriture pour escargots ......................................22

Annexe E (informative) Concentrations habituelles dans les viscères d’escargots subadultes

avant encagement ............................................................................................................................................................................................23

Annexe F (informative) Dispositifs d’essai recommandés pour l’exposition in situ pour

évaluer la bioaccumulation de contaminants chez les escargots .................................................................25

Annexe G (informative) Exemple de masse des escargots avant exposition .........................................................28

Annexe H (informative) Résultats de l’essai circulaire international ..........................................................................30

Annexe I (informative) Exposition ex situ pour évaluer la bioaccumulation de substances

chimiques chez les escargots ...............................................................................................................................................................48

Bibliographie ...........................................................................................................................................................................................................................56

iii
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ISO 24032:2021(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes

nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est

en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.

L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d'approbation requis pour les différents types de documents ISO. Le présent document a

été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir

www.iso.org/directives).

L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de

brevets reçues par l'ISO (voir www.iso.org/brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion

de l'ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www.iso.org/avant-propos.

Le présent document a été élaboré par le comité technique ISO/TC 190, Qualité du sol, sous-comité SC 4,

Caractérisation biologique, en collaboration avec le comité technique CEN/TC 444, Caractérisation

environnementale des matrices solides, du Comité européen de normalisation (CEN) conformément à

l’Accord de coopération technique entre l’ISO et le CEN (Accord de Vienne).

Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent

document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l’adresse www.iso.org/fr/members.html.
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ISO 24032:2021(F)
Introduction

Les escargots sont des macroinvertébrés omniprésents dans le sol, vivant à l’interface entre le sol, les

végétaux et l’air. Ces mollusques gastéropodes pulmonés sont phytophages et saprophages (niveau

trophique des consommateurs primaires et détritivores). Ils ingèrent des végétaux et du sol, et

rampent sur le sol où ils pondent leurs œufs. De ce fait, ils intègrent de nombreuses sources et voies de

contamination (voir Annexe A, Figure A.1). Les escargots participent aux échanges avec le sol et sont la

proie de divers consommateurs (invertébrés: vers luisants, larves de carabidés, ou vertébrés: oiseaux,

petits mammifères tels que les musaraignes, les hérissons et l’homme).

Parmi les espèces d’escargots, l’espèce recommandée est Cantareus aspersus O.F. Müller 1774

(synonymes: Helix aspersa aspersa, Cornu aspersum), également connue sous le nom d’escargot de jardin,

ou plus simplement «Petit-Gris» (voir Annexe A, Figure A.2). Cette espèce est un mollusque gastéropode

[9], [28]

pulmoné stylommatophore de la famille Helicidae, très répandue dans le monde . Cette espèce

paléarctique peut s’acclimater à des régions présentant des climats de types différents: méditerranéen,

océanique tempéré, continental tempéré, voire tropical. Cantareus aspersus (Müller, 1774) est d’origine

européenne et a été introduit dans le monde entier. Il est désormais présent sur tous les continents

hormis l’Antarctique. D’autre part, l’espèce est reconnue comme étant un escargot nuisible pour

l’agriculture dans certains pays et doit être traitée avec précaution.
[1]

Les escargots juvéniles font l’objet de l’ISO 15952 , qui décrit comment déterminer ex situ, c’est-à-

dire dans des conditions de laboratoire, les effets toxiques de substances chimiques ou de matrices

contaminées sur la survie et la croissance d’escargots juvéniles (1 g MF).

À l’heure actuelle, il n’existe aucun bioessai in situ normalisé permettant d’évaluer sur le terrain le

transfert de contaminants de l’environnement aux organismes de la faune du sol. En effet, bien que

[3]

l’ISO 19204 (relative à l’approche TRIADE) recommande l’application de trois éléments de preuve

combinés (chimie, écotoxicologie et écologie) et souligne l’intérêt des bio-indicateurs d’effet et

d’accumulation comme outils complémentaires pour l’évaluation des risques écologiques spécifiques à

un site, peu de bioessais sont disponibles à cette fin. Comme décrit dans l’ISO 19204:2017, Annexe A, les

analyses de la bioaccumulation dans les végétaux ou les organismes du sol sont donc utiles pour:

— évaluer la biodisponibilité réelle des contaminants du sol pour les organismes du sol;

— aborder le transfert dans la chaîne alimentaire et le risque d’empoisonnement secondaire des

consommateurs.

Dans certains cas, la bioaccumulation peut donner lieu à des effets toxiques, mais ce n’est pas toujours

[2]
le cas (voir ISO 17402 ).

L’élevage étant possible (voir l’ISO 15952:2018, Annexe B), des escargots au passé biologique connu

peuvent être utilisés sur le terrain pour analyser la biodisponibilité des contaminants présents dans

les habitats (sol, végétaux, air) en mesurant leur accumulation chez des individus encagés et exposés

pendant une période déterminée.
[10],[12],[13],[15],[19],[22],[23],[27],[29],[30]
C. aspersus peut être utilisé soit sur le terrain , soit en
[14],[18],[20],[21]

laboratoire pour évaluer le devenir et le transfert (à savoir la biodisponibilité

environnementale, ISO 17402) des substances chimiques dans les sols. Ce bio-indicateur du sol a été

appliqué sur de nombreux sites pour évaluer la fonction d’habitat et de rétention des sols. Ce bioessai

permet de déterminer la biodisponibilité des substances chimiques pour les escargots en mesurant

leur concentration dans leur masse viscérale (qui contient principalement la glande digestive et

quelques autres organes, comme décrit dans la Référence [16]). La masse viscérale est le principal site

d’accumulation des contaminants chez les escargots.
1) Disponible à l’adresse : https://inpn.mnhn.fr/espece/cd_nom/199863/tab/taxo.

2) Disponible à l’adresse : https://ecobiosoil.univ-rennes1.fr/ADEME-Bioindicateur/english/worksheet.php.

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ISO 24032:2021(F)

Le présent document décrit la méthodologie pour exposer des escargots in situ pendant 28 jours et

les préparer jusqu’à la réalisation d’analyses chimiques pour évaluer la bioaccumulation dans leurs

viscères. Ce bioessai évalue le transfert de contaminants de l’environnement aux escargots petits-gris.

Il est applicable sur le terrain (par exemple, sites pollués, sols amendés, sols après dépollution, sites

agricoles ou autres sites préoccupants et déchets) en mettant les escargots en cage pendant 28 jours

sur le site/le sol/le déchet étudié. Les escargots intègrent des substances chimiques de toutes les

sources terrestres (sol, végétaux, air). Après exposition, les concentrations en substances chimiques

sont mesurées dans la masse viscérale des escargots.

En variante, cette méthode peut être utilisée en laboratoire (ex situ) pour évaluer la bioaccumulation de

substances chimiques chez les escargots exposés uniquement au sol (voir Annexe I).

L’Annexe H présente les résultats d’un essai circulaire réalisé in situ par six laboratoires pour évaluer la

méthode d’exposition et par quatre laboratoires de l’exposition jusqu’à l’analyse chimique

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NORME INTERNATIONALE ISO 24032:2021(F)
Qualité du sol — Encagement in situ d’escargots pour la
mesure de la bioaccumulation de contaminants
1 Domaine d’application

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

[13],[19],[22]
de l’exposition .

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

[8],[10]

déchets , 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.
2 Références normatives
Le présent document ne contient aucune référence normative.
3 Termes et définitions

Pour les besoins du présent document, les termes et définitions suivants s’appliquent.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp;

— IEC Electropedia: disponible à l’adresse https:// www .electropedia .org/ .
3.1
cage

microcosme fermé permettant l’exposition des escargots par différentes voies et diverses sources

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ISO 24032:2021(F)
3.2
bioaccumulation

phénomène par lequel un produit chimique présent dans un milieu s’accumule dans un organisme

vivant

Note 1 à l'article: Ce phénomène est observé lorsque le taux d’absorption dépasse le taux d’élimination du

contaminant.
3.3
escargot inactif

escargot sans activité, généralement au sec quand il se colle (généralement par un simple disque de

mucus séché) contre les parois de la boîte dans laquelle il est placé
3.4
estivation
escargots maintenus inactifs, au sec, à une température de 15 °C à 20 °C
3.5
parcelle
zone caractéristique et représentative du site

Note 1 à l'article: Il convient de consigner les coordonnées géographiques de chaque parcelle.

3.6
site

terrain (ou entité géographique) étudié et où les microcosmes sont placés pour évaluer la biodisponibilité

des contaminants pour les escargots

Note 1 à l'article: Le site peut avoir une ou plusieurs parcelles et une ou plusieurs utilisations du sol, par exemple

un champ, un pâturage, une forêt, un site industriel ou une décharge.
4 Principe

Les escargots sont encagés dans des microcosmes sur le site étudié pendant 28 jours. Quinze escargots

petit-gris subadultes [masse corporelle de (5 ± 1) g] doivent être placés dans chaque microcosme. De la

fin de leur élevage à leur mise en place sur le sol, ils peuvent être conservés dans des boîtes en bois sèches

(boîtes rondes en bois, d’environ 12 cm de diamètre et 4 cm de haut; voir Figure 1 et Figure B.2). Ils sont

sortis d’estivation en les aspergeant d’eau quelques heures avant de les placer dans les microcosmes.

Une fois dans ceux-ci, ils sont exposés au sol, aux végétaux qui ont poussé sur site et à l’air ambiant afin

d’être soumis à des conditions d’exposition naturelles (aléas climatiques).

Après l’exposition, les escargots collectés sont ramenés au laboratoire sans nourriture pendant 48 h.

Pendant cette période de jeûne, les fèces sont retirées toutes les 24 h. Les escargots sont ensuite

congelés à –80 °C. Après décongélation, le corps mou est retiré de la coquille; la masse viscérale et le

pied (voir Annexe B, Figure B.1) sont séparés et préparés pour l’analyse chimique afin de déterminer la

concentration interne en substances chimiques. L’Annexe B présente les principales étapes.

5 Organisme et équipement d’essai
5.1 Matériel biologique

Les organismes d’essai doivent être des escargots subadultes (pour éviter les variations de masse

pendant la durée d’exposition et la dilution consécutive de la bioaccumulation par le gain de masse

pendant la croissance ou les transferts de composés aux œufs pendant les phases de la reproduction).

L’espèce recommandée est l’escargot petit-gris Cantareus aspersus (Müller, 1774) qui doit être âgé de

7 semaines à 12 semaines, avec une masse fraîche moyenne de (5 ± 1) g.

NOTE 1 Facultativement, le diamètre de la coquille peut être mesuré (moyenne ± écart-type [ET] de

25 mm ± 5 mm; min./max. de 20 mm/30 mm).
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ISO 24032:2021(F)

Les escargots doivent être sélectionnés à partir d’un élevage synchrone afin d’obtenir une population

la plus homogène possible au regard de la masse et de l’âge. Les techniques d’élevage des escargots

sont décrites à l’Annexe C. En résumé, après une période en nurserie et une période de croissance (de

3 semaines à 6 semaines, suivies de 4 semaines à 6 semaines), les escargots subadultes doivent être

utilisés directement ou après une période d’estivation. Il convient que cette période [à savoir escargot

inactif, collé sur la paroi d’une boîte sèche (boîte en plastique à éviter) dans une salle à température

contrôlée entre 15 °C et 20 °C] ne dépasse pas 5 mois. L’estivation se déroule dans des boîtes rondes en

bois (environ 12 cm de diamètre et 4 cm de haut; généralement 15 escargots par boîte, ce qui équivaut

au nombre d’escargots par microcosme).

Les escargots doivent être élevés aux fins du projet (voir Annexes C et D) ou achetés auprès d’éleveurs

d’escargots locaux.

NOTE 2 Il est possible d’utiliser un autre genre et/ou une autre espèce d’Helicidae (voir les exemples et les

conditions dans l’Annexe G de l’ISO 15952:2018).

Un contrôle de la qualité chimique des escargots subadultes sélectionnés pour l’encagement (à savoir,

des escargots non exposés) peut être effectué sur 6 escargots pour ce qui est des concentrations initiales

des substances chimiques d’intérêt (Cescargot-t0). Ces escargots témoins peuvent être sélectionnés en

même temps que les escargots utilisés pour l’encagement. L’analyse de la qualité chimique des escargots

avant l’encagement peut être effectuée en même temps que l’analyse des escargots après exposition.

Ce contrôle n’est pas obligatoire. En effet, après l’exposition, toutes les données sont comparées à la

valeur seuil indicative (VSI) (voir 8.2.1); cependant, s’il est possible d’obtenir ces données de contrôle,

cela permet d’indiquer si les escargots n’étaient pas contaminés avant l’exposition. Pour les substances

chimiques pour lesquelles aucune VSI n’est disponible, les données peuvent être comparées à différentes

valeurs (voir 8.2.2.4), parmi lesquelles les valeurs Cescargot-t0.

Les escargots subadultes utilisés doivent présenter des concentrations habituelles dans leur masse

viscérale avant l’encagement (voir Annexe E). Pour les données relatives aux HAP et aux PCB, comme

les extractions sont souvent réalisées sur des tissus frais, les données du Tableau E.1 sont exprimées

−1 −1

en µg·kg de masse fraîche de viscères (ces valeurs peuvent être converties en µg·kg MS sur la base

de ≈ 15 % de masse sèche de la masse viscérale); pour les métaux (métalloïdes), les données sont

exprimées en mg·kg de masse sèche de la masse viscérale.
5.2 Équipement

5.2.1 Microcosme, cylindres en acier inoxydable de 25 cm de diamètre et 25 cm de haut, recouverts

d’un grillage de 0,5 cm ou 1 cm.
Un exemple est présenté à la Figure 1 et à l’Annexe F, Figure F.1.

NOTE 1 D’autres dispositifs peuvent être utilisés si le matériau qui les constitue ne peut être une source

de contamination; pour certaines fins (par exemple, l’exposition des escargots à des substances chimiques

[11]

pulvérisées sur le terrain), un microcosme entièrement grillagé peut être utilisé [voir, par exemple, Référence,

qui utilise des cages en acier inoxydable de 25 cm × 25 cm × 15 cm (maillage: 1 cm) fermées par une grille en acier

inoxydable de 30 cm × 30 cm (maillage: 1 cm) et maintenues par quatre piquets (voir Annexe F, Figure F.2)].

NOTE 2 Dans certains cas, il peut être nécessaire de protéger le microcosme des prédateurs ou du bétail

(voir exemples dans l’Annexe F, Figure F.3) ou du soleil (voir Annexe F, Figure F.4).

5.2.2 Grillage, 0,5 cm ou 1 cm de grillage, également en acier inoxydable.

5.2.3 Piquets, piquets en acier inoxydable (5 mm de diamètre et de 46 cm à 72 cm de long) pour

maintenir le grillage sur la cage. La taille des piquets doit être adaptée en fonction du tassement du sol

ou de la présence de pierres.
5.2.4 Morceaux de tuiles, voir Figure 1 et Annexe F.
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ISO 24032:2021(F)

5.2.5 Boîtes de conservation en bois. Les escargots inactifs peuvent être conservés et transportés

avant exposition dans des boîtes rondes en bois (environ 12 cm de diamètre et 4 cm de haut), en plaçant

les escargots au sec à une température entre 15 °C et 20 °C (voir Figure 1, Figure B.2 et Annexe G).

5.2.6 Boîtes de jeûne et de prélèvement. Pour la préparation des escargots en laboratoire

(par exemple, pour conserver les escargots avant leur pesée individuelle), des récipients en plastique

(RP) (par exemple, en polystyrène transparent ou tout autre récipient ayant des dimensions

approximatives de 24 cm [longueur] × 10,5 cm [largeur] × 8 cm [hauteur]) peuvent être utilisés.

5.2.7 Pied à coulisse. Pied à coulisse d’une précision de 0,1 mm pour le mesurage du diamètre de la

coquille.
5.2.8 Balance. Balance analytique ayant une précision d’au moins 10 mg.
5.2.9 Eau, au minimum déionisée.

5.2.10 Nourriture, qui doit être fournie sous forme de farine avec son taux d’humidité naturel (de 5 %

à 10 %).

Afin que la croissance soit suffisante, il est recommandé d’effectuer les essais avec une nourriture

à base de farine comprenant des céréales, du fourrage, des sels minéraux et des vitamines couvrant

convenablement les besoins des escargots. Un exemple de composition de la nourriture est fourni dans

l’Annexe D.

5.2.11 Petit appareillage. Élastiques pour fermer les boîtes de conservation en bois ou les boîtes de

jeûne et de prélèvement. Ruban adhésif pour étiqueter la boîte de conservation en bois et les boîtes de

jeûne; marqueurs indélébiles; sacs refermables.
6 Préparation des organismes pour l’exposition

Après la fin de leur croissance (voir Figure C.1, croissance 1, c’est-à-dire le temps nécessaire pour

obtenir des subadultes ayant atteint la masse requise pour l’essai), les escargots doivent être conservés

inactifs dans une boîte en bois (5.2.5.). Leur masse diminue pendant cette période de conservation; c’est

pourquoi dans certains cas (tels qu’une durée de conservation supérieure à une semaine), ils doivent

être sortis d’estivation quelques jours avant le début de l’essai (voir Article 6).

En fonction de la durée de conservation entre la fin de la période de croissance (c’est-à-dire lorsque

la masse moyenne requise est atteinte; voir 5.1) et le début de l’essai sur le terrain, les escargots sont

réveillés selon les scénarios suivants:

— si les escargots sont utilisés dans la semaine qui suit leur pesée et leur répartition en lots homogènes

(15 escargots pour 1 microcosme), il est nécessaire de les réveiller quelques heures avant de

les utiliser sur le terrain. Ils doivent être aspergés d’eau dans la boîte en bois. Cela facilite leur

manipulation pour les sortir de la boîte en bois et les placer dans le microcosme une fois sur le

terrain;

— s’ils ont été conservés pendant des périodes plus longues (>1 semaine, mais < 5 mois) avant exposition

sur le terrain, il convient de les réveiller et de les nourrir avec des aliments pour escargots (5.2.10)

pendant 2 jours à 5 jours afin qu’ils atteignent leur masse initiale. Après avoir été réveillés par

aspersion d’eau dans la boîte en bois, ils sont placés dans des cages ou des boîtes en plastique (voir

Figure C.2 dans l’Annexe C) pendant 2 à 5 jours et nourris. Ils sont ensuite de nouveau pesés et

répartis en lots homogènes (voir exemple dans l’Annexe G, Tableau G.1 et Figure G.1) dans la boîte

en bois dans laquelle ils peuvent être conservés à court terme (de 0 à 1 semaine), avant d’être de

...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 24032
ISO/TC 190/SC 4
Soil quality — In situ caging of
Secretariat: AFNOR
snails to assess bioaccumulation of
Voting begins on:
2021­09­09 contaminants
Voting terminates on:
Qualité du sol — Encagement in situ d’escargots pour la mesure de la
2021­11­04
bioaccumulation de contaminants
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ISO/FDIS 24032:2021(E)
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ISO/FDIS 24032:2021(E)
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ISO/FDIS 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 ............................................................................................................................................................................ 8

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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ISO/FDIS 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 non­governmental, 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.
iv © ISO 2021 – All rights reserved
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ISO/FDIS 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:

glow­worms, 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, Cornu aspersum) also known as common garden snail, brown garden snail,

garden snail, land snail, nicked name in French “Petit­Gris” (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

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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ISO/FDIS 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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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 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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ISO/FDIS 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 sub­area 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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ISO/FDIS 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

snail­t0). 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 Csnail­t0.

The sub­adult 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

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

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.
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ISO/FDIS 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 wooden storage and boxes for fasting; indelible markers resealable bags.

6 Preparation of the organisms for the exposure

After the end of their growth (see Figure C.1, growth 1, i.e. time needed to obtain sub­adults that reached

the mass required for the test) snails shall be stored inactive in wooden box (5.2.5.). 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 Clause 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 are woken 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 necessary to wake them some hours before using in the field. They

shall be sprayed with water in the wood box. This facilitate their handling to remove them from the

wood box and placed them in the microcosm once in the field.

— if they were stored for longer periods (>1 week but < 5 months) before exposure in the field, they

should be awakened and fed with snail feed (5.2.10) for 2 days to 5 days in order they reach their

initial mass. After being awakened by spraying water in the wood box, they are placed in cages

or plastic box (see Figure C.2 in Annex C) for 2 days to 5 days and fed. Then again weighed and

distributed in homogeneous batches (see example in Annex G, Table G.1 and Figure G.1) in the wood

box in which they can be stored for a brief duration (0 to 1 week) before being again awakened and

disposed in the microcosms.

The proportion of snails not woken shall be less than 20 %. As soon as they become active (snails not

stuck to the walls of the box and starting to move), the snails shall be transferred into a box that has

been premoistened with water.

All the snails needed for the assay shall be weighed, and distributed in distinct mass classes (e.g. group

all snails from 4 g to 4,5 g, from 4,6 g to 5 g, from 5,1 g to 5,5 g, from 5,6 g to 6 g. Then, prepare group

of 15 snails each as homogeneous as possible with respect to mass (same distribution of mean group

mass, see example Annex G, Figure G.1).
NOTE Optionally, the shell diameter can be measured.

Snails for the test shall be individually weighed and placed in wooden storage boxes; 15 individuals

shall be stored per wood storage, since one microcosm shall contain 15 snails for exposure.

7 Exposure of the test organisms
7.1 General

The main steps of the bioassays are illustrated in Annex F, Figures F.5 and F.6 (an example of a table of

data is given in Annex G, Table G.1).
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ISO/FDIS 24032:2021(E)
7.2 Beginning of exposure

Three microcosms shall be placed at each plot. To consider soil heterogeneity in terms of intrinsic

properties and contamination profiles, a minimum of 3 microcosms, per a certain plot area is used.

Each microcosm should contain 15 snails that are exposed to soil, humus and vegetation under natural

climatic conditions. This is the natural way of exposure of snails. Plants, humus that cover the soil (and

also soil) are a source of feeding for snails. Pieces of tiles shall be placed in the cage to provide a shelter

and a bonding surface to snails.

The snails shall be carefully removed from the wooden box, without pulling too hard to avoid braking

the shell; they shall not produce white mucus (like a white foam), which is a sign of mishandling.

NOTE 1 The number of microcosms per plot can be adapted depending on the number or mass of snail tissue

needed for analysis or in the frame of a preliminary study.

NOTE 2 If there is no shade on site, a shade mesh could be placed above the netting to reduce the heat in the

cage. Annex F, Figure F.4.

Once on the field, set up a microcosm on soil (remove stone to avoid space between microcosm and soil

to ensure that the microcosm is sufficiently buried in the soil to avoid the nails from escaping, drive the

cage in the top soil layer of 0,5 cm to 1 cm). Place the snails and the pieces of tiles used as shelters (see

Figure 1). Finally, cover the microcosms with the netting and fix the netting with the pickets. About

20 min are required for this step.
© ISO 2021 – All rights reserved 5
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