EN ISO 24032:2021

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

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.

Contents Page
European foreword . 3

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.

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 24032:2021(E)
© 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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 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.
iv
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.
v
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.
vi
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.
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
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.
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).
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.
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 in Figure C.2).
During starvation, snails shall be starved for two days (until they produce no more faeces). During this
starvation period, the faeces shall be removed every 12 h to avoid that snails re-eat the faeces. It is
recommended to weigh the snails at the end of exposure and after starvation before freezing.
NOTE As the mass is influenced by the weather in the field, weighing the snails after starvation and a
homogeneous hydration facilitates the comparisons between snails exposed under quite different meteorological
conditions, or between experiments performed at different years). Optionally, the shell diameter can be
measured.
ISO 24032:2021(E)
Snails are then frozen at –80 °C. They can be frozen in resealable bags or any other container that can
be effectively closed.
Optionally a –20 °C freezer can be used if no –80 °C freezer is available. A -80 °C freezer allows to kill the
snails by freeze drying more rapidly. It is also required for appropriate conservation before additional
biomarkers analysis.
7.4 Sampling and preparation after exposure
For preparation of the visceral mass, the snails shall be thawed. Depending of the temperature of the
room, wait until the soft body is completely soft (without presence of ice in the body). After thawing,
the snails shall be weighed, the soft body (i.e. foot + visceral mass) shall be removed from the shell and
the visceral mass separated from the foot for analysis of chemicals (see step 3 in Figure B.3).
The removal of the visceral mass requires about 10 min for unskilled operator and 2 min for skilled.
Two snails per microcosm shall be randomly sampled after 28 days of exposure. The total number of
snails that shall be sampled for metal(loid)s analysis is two per microcosm, resulting in a total of six
individuals per plot: three microcosms x two snail/microcosm). The remaining snails [13 snails (if no
mortality occurred during exposure)] can be stored frozen for further analysis. It provides a safety
margin in case of mortality, and also allows to obtain enough biological material if analysis of other
pollutants [polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans (PCDD/Fs), rare
earths, polybrominated compounds, etc.] or biomarkers are needed.
NOTE 1 For the analysis of organic compounds, if the mass of the viscera is not sufficient for individual
analysis, the visceral masses of two or more snails can be pooled to reach the required mass of sample for
analysis.
NOTE 2 If only one microcosm is used on one plot (e.g. in a preliminary study), six snails are sampled in the
microcosm.
8 Calculation and expression
8.1 General
Two ways are currently possible: one for metal(loid)s for which guide values are available, and other
chemicals for which no guide value is available at the time of publication.
8.2 For metal(loid)s
8.2.1 Threshold guide value
For 14 metal(loid)s threshold guide value [(TGV) previously named internal concentrations of
[22]
reference (CIRef) ] have been determined in snails using the metal concentrations in snails exposed
on unpolluted sites (n = 150) (see Table 1, Figure 2).
They allow to calculate the SET index (sum of the excess of transfer) to provide an evaluation of the
abnormal transfer of metal(loid)s to snails. Briefly, the ME concentration in snails after 28 days
exposure on the studied site are divided by the TGV for each ME to calculate the accumulation quotient
[18],[23],[24],[25],[26]
(AQ); then the AQ-1 for each ME are added to provide the SET index .
ISO 24032:2021(E)
Table 1 — Threshold guide value (TGV) of metal(loid)s in the viscera of snails after 1 month
[23]
exposure on uncontaminated sites
ME As Cd Co Cu Cr Hg Mo Ni Pb Sb Sn Sr Tl Zn
TGV-in situ
0,307 2,27 6,676 184,7 2,01 0,198 4,428 5,249 12,9 0,076 0,058 125,7 0,259 1 490
−1
(mg kg )
NOTE  TGV are median value (see Figure 2).
Key
X uncontaminated plots
-1
Y C (mg.kg )
snail
-1
A TGV Cd 2,27 mg.kg
Figure 2 — Example of calculation of the TGV for cadmium
8.2.2 Calculation of the sum of the excess of transfer of metal(loid)s: SET index
8.2.2.1 General
To identify the metal transfer from the environment to snails, the median of the snail’s viscera
concentration is compared to the TGV. If the median concentration in the snail exposed to the plot
under investigation is higher than the TGV, then the soil presents an abnormal metal transfer to snail.
8.2.2.2 Calculation of the accumulation quotient (AQ)
AQ = [Csnail-28d]/TGV for each metal(loid)s
With [Csnail-28d] = median concentration of the metal(loid) in the viscera of the 6 snails exposed on
the studied plot.
An AQ > 1 identifies an excess of transfer.
8.2.2.3 Calculation of the sum of the excess of transfer of metal(loid)s: SET plot and SET site
SETplot = Σ(AQ-1) and
SETsite = Σ(AQ-1) / nplot
8.2.2.4 If the TGV is not available for a studied metal(loid)s
C snail-28d can be compared either to:
a) the Csnail-28d of snails caged on a control site (i.e. uncontaminated site);
b) or to the Csnail-28d of snails reared in the laboratory during the exposure of snails on site (e.g. if it
is not possible to find a plot on an uncontaminated site to serve as control);
c) or at least to the initial concentration of snails (i.e. before exposure): Csnail-t0.
ISO 24032:2021(E)
8.3 For other chemicals
For PAH, PCB, pesticides or any other chemicals for which no in situ TGV are available, Csnail-28d shall
be compared to guide value as described in 8.2.2.4.
9 Validity of the experiment
The results are considered to be valid if the following conditions are met:
— the percentage of the mortality observed in the control containers (see 8.2.2.4) is less than or equal
to 30 % at the end of the test.
10 Test report
The test report shall refer to this document and shall include the following information:
a) a reference to this document, i.e. ISO 24032:2021;
b) the description of the site and plot(s) of the site under study;
c) pictures of the studied site/plots;
d) environmental information (rainfall, min and max temperature measured or on the basis of a
meteorological station near the studied zone) during exposure;
e) data available on the soil, the site (physico-chemical data);
f) geographical location of the microcosms (postal code, municipality, exact GPS coordinates (decimal
degree or DMS: degree minute second and/or WGS world geodetic system);
g) description of the vegetal and the humus cover;
h) mass of the snails (total fresh mass) at the start of the test (when placed in the wooden box for
transport) and after exposure (as stated in Clause 7);
i) percentage of the survival in each microcosm, and the mean (±standard deviation) for
the 3 microcosms per studied zone on the site;
j) the description of the obvious or pathological symptoms (e.g. snails producing a liquid, or showing
a swelling shiny foot), or of the noticeable modifications in behaviour (e.g. sign of lethargy not
withdrawing in the snail when handled), observed on the testing organisms;
k) any other manipulation not specified in this document and any events likely to have influenced the
results.
ISO 24032:2021(E)
Annex A
(informative)
Sources and routes of exposure of snails to contaminants in the
field
Key
S soil solution
T total quantity of pollutants in the soil
L pollutants poorly linked to the solid phase
H pollutants strongly linked to the solid phase
Exposure route Exposure source

digestive plant
solid phase of the
dermal
soil
liquid phase of the
respiratory
soil
air
NOTE Adapted from Reference [31].
Figure A.1 — Sources and routes of exposure of snails to contaminants in the field
Figure A.2 — Landsnail Cantareus aspersus — Sub-adult
ISO 24032:2021(E)
Annex B
(informative)
Main steps of the bioassay in situ
ISO 24032:2021(E)
Figure B.1 — Main steps of the bioassay
ISO 24032:2021(E)
NOTE Transport to the site and back to the laboratory.
Figure B.2 — Wood bow for snail storage and transport
a) Removal of the soft body from the shell (pull the white muscle at the base of the columella)
b) Separation of the organs constituting the viscera from the “foot”
ISO 24032:2021(E)
c)
Key
1 foot c kidney
2 viscera d part 2 lung
a part 1 lung e hepatopancreas (large and small lobes) = liver
b mantle edge f anterior part of the digestive tract
Figure B.3 — Preparation of the viscera after thawing
ISO 24032:2021(E)
Annex C
(informative)
Breeding technique for snails
C.1 General
The young snails used for the toxicity tests are obtained through the so-called “out-of-ground” breeding
technique, because it takes place in a building within a controlled environment,
...