prEN ISO 24032

Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO/DIS 24032:2020)

-

Bodenbeschaffenheit - In-situ-Käfighaltung von Schnecken zur Beurteilung der Bioakkumulation von chemischen Stoffen (ISO/DIS 24032:2020)

Dieses Dokument beschreibt ein Verfahren zur Bewertung der Bioakkumulation von Chemikalien in Schnecken, d. h. Konzentrationen von Metall(oid)en (ME) oder organischen Verbindungen (z. B. PAK und PCB), die sich in ihren Geweben angereichert haben.
In diesem Dokument wird dargelegt, wie Schnecken für die 28 tägige In situ-Käfighaltung vorbereitet werden, wie das In situ-Prüfdesign aussieht und wie die Schnecken bis zur Konservierung und weiteren Analyse gesammelt und vorbereitet werden. Falls eine kinetische Untersuchung der Akkumulation notwendig ist, ist auch eine Probenahme der Schnecken zu verschiedenen Zeitpunkten während der Exposition möglich (Gimbert et al., 2008a; Pauget et al., 2013; Mariet et al., 2017).
Diese Norm schließt analytische Verfahren aus: Vorbereitung (Extraktion und Mineralisierung) der Proben und Quantifizierung von Chemikalien. Diese fallen nicht in den Anwendungsbereich des vorliegenden Dokuments.
Das Verfahren ist auf Böden mit unterschiedlicher Nutzung (landwirtschaftliche, industrielle, Wohn  und Waldböden, vor und nach der Sanierung, auf potenziell kontaminierten Standorten usw.) sowie auf Abfallmaterialien (de Vaufleury, 2015; Bourioug et al., 2015) anwendbar und sollte vorzugsweise auf Böden mit Vegetation und/oder Humusbedeckung angewendet werden.
Das Verfahren ist vorbehaltlich bestimmter Temperaturgrenzen (frostfreier Zeitraum, d. h. hauptsächlich von April bis Oktober in gemäßigtem Klima) anwendbar.
Optional (Anhang A) kann das Verfahren im Labor verwendet werden, um die Akkumulation von Kontaminanten (und optional den SET Index (en: Sum of Excess of Transfer index) für ME, PAK, PCB) von Schnecken zu bewerten, die nur dem Boden exponiert sind.

Qualité des sols - Encagement in situ descargots pour la mesure de la bioaccumulation de contaminants (ISO/DIS 24032:2020)

Kakovost tal - Uporaba kletk s polži na terenu za oceno bioakumulacije onesnaževal (ISO/DIS 24032:2020)

General Information

• Status: Not Published
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Due Date: 27-Jul-2021
• Completion Date: 27-Jul-2021
• Ref Project: oSIST prEN ISO 24032:2021 - Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO/DIS 24032:2020)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 24032:2021
01-januar-2021
Kakovost tal - Uporaba kletk s polži na terenu za oceno bioakumulacije
onesnaževal (ISO/DIS 24032:2020)

Soil quality - In situ caging of snails to assess bioaccumulation of contaminants (ISO/DIS

24032:2020)
Bodenbeschaffenheit - In-situ-Käfighaltung von Schnecken zur Beurteilung der
Bioakkumulation von chemischen Stoffen (ISO/DIS 24032:2020)

Qualité des sols - Encagement in situ descargots pour la mesure de la bioaccumulation

de contaminants (ISO/DIS 24032:2020)
Ta slovenski standard je istoveten z: prEN ISO 24032
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 24032:2021 en,fr,de

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

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oSIST prEN ISO 24032:2021
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oSIST prEN ISO 24032:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 24032
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2020-11-18 2021-02-10
Soil quality — In situ caging of snails to assess
bioaccumulation of contaminants

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

ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 24032:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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oSIST prEN ISO 24032:2021
ISO/DIS 24032:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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

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Published in Switzerland
ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 24032:2021
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Contents Page

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

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

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

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

5 Test organism and equipment ................................................................................................................................................................ 2

5.1 Biological material ............................................................................................................................................................................... 2

5.2 Equipment ................................................................................................................................................................................................... 3

5.2.1 Microcosm ............................................................................................................................................................................. 3

5.2.2 Netting ...................................................................................................................................................................................... 3

5.2.3 Pickets ....................................................................................................................................................................................... 3

5.2.4 Pieces of tiles ....................................................................................................................................................................... 3

5.2.5 Wooden storage ................................................................................................................................................................ 4

5.2.6 Boxes for fasting, sampling ..................................................................................................................................... 4

5.2.7 Calliper rule .......................................................................................................................................................................... 4

5.2.8 Balance ..................................................................................................................................................................................... 4

5.2.9 Water .......................................................................................................................................................................................... 4

5.2.10 Feed ............................................................................................................................................................................................. 4

5.2.11 Small material .................................................................................................................................................................... 4

6 Preparation of the organisms for the exposure ................................................................................................................... 4

7 Exposure of the test organisms ............................................................................................................................................................. 5

7.1 Beginning of exposure ...................................................................................................................................................................... 5

7.2 End of the exposure — Starvation ......................................................................................................................................... 6

7.3 Sampling and preparation after exposure ....................................................................................................................... 6

8 Calculation and expression ....................................................................................................................................................................... 6

8.1 General ........................................................................................................................................................................................................... 6

8.2 For metal(loid)s...................................................................................................................................................................................... 6

8.2.1 Threshold guide value ................................................................................................................................................. 6

8.2.2 Calculation of the sum of the excess of transfer of metal(loid)s: SET index ................. 7

8.3 For other chemicals ............................................................................................................................................................................ 7

9 Validity of the experiment .......................................................................................................................................................................... 8

10 Test report ................................................................................................................................................................................................................... 8

Annex A (informative) Sources and routes of exposure of snails to contaminants in the field ...............9

Annex B (informative) Main steps of the bioassay in situ ............................................................................................................10

Annex C (informative) Breeding technique for snails......................................................................................................................12

Annex D (informative) Example of composition of snail feed .................................................................................................17

Annex E (informative) Usual concentrations in the viscera of sub-adult snails before caging .............18

Annex F (informative) Standardized forms of recommended test systems for in situ

exposure to assess bioaccumulation of contaminants in snails.......................................................................19

Annex G (informative) Example of mass of snails before exposure ..................................................................................23

Annex H (informative) Results of the international ring test ..................................................................................................25

Annex I (informative) Ex situ exposure to assess bioaccumulation of chemicals in snails .......................41

Bibliography .............................................................................................................................................................................................................................46

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Foreword

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

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

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

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

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

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

electrotechnical standardization.

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

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

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

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

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

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

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

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

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

constitute an endorsement.

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

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

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

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

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

Biological characterization.
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Introduction

Snails are ubiquitous soil macroinvertebrates living at the interface soil, plants and air. Those pulmonate

gastropod molluscs are phytophagous and saprophagous (trophic level of primary consumers and

detritivorous). They ingest vegetation and soil, crawl on the ground where they lay their eggs.

Therefore, they integrate multiple sources and routes of contamination (see Annex A – Figure A.1).

Snails participate in exchanges with soil and are preyed upon by various consumers (invertebrates:

glow-worms, ground beetle larvae, or vertebrates: birds, small mammals such as shrews, hedgehogs

and humans).

Among snail species, the recommended species is Cantareus aspersus O.F. Müller 1774 (synonyms: Helix

aspersa aspersa (O.F. Müller, 1774), Cornu aspersum; https:// inpn .mnhn .fr/ espece/ cd _nom/ 199863/

tab/ taxo) also known as common garden snail, brown garden snail, garden snail, land snail, nicked

name in French “Petit-Gris”. This species is a stylommatophoran pulmonate gastropod molluscs of the

Helicidae family, widely distributed across the world (Potts, 1975; Chevallier, 1977). This palearctic

species can be acclimated to regions with different types of climate: Mediterranean, oceanic temperate,

midcontinental temperate and even tropical. Cantareus aspersus (Müller, 1774) is of European origin

and has been introduced into all parts of the world. It is now on all continents except Antarctica. On the

other hand, the species is recognized as an agriculturally harmful snail in some countries and must be

treated carefully.

Juvenile snails are already covered in ISO 15952 (2018) that describes how to assess ex situ, i.e. in

laboratory conditions, toxic effect of chemicals or contaminated matrix on the survival and growth of

juvenile (1 g fw).

Currently there is no standardized in situ bioassay allowing the assessment in the field of the transfer

of contaminants from the environment to organisms of the soil fauna. Indeed, despite ISO 19204 (2017)

(relative to the TRIAD approach) recommends the application of three combined lines of evidence

(chemistry, ecotoxicology and ecology) and highlights the interest of bioindicators of effect and

accumulation as additional tools for site-specific ecological risk assessment, few bioassays are available

for this purpose. As described in Annex A of ISO 19204 (2017), measurements of bioaccumulation in

plants or soil organisms are thus useful to:
— assess the effective bioavailability of soil contaminants to soil organisms;

— approach the food chain transfer and the risk of secondary poisoning of consumers.

In some cases, bioaccumulation can result in toxic effects but this is not always the case

(see ISO 17402:2008).

Since farming is possible (see Annex in ISO 15952:2018), snails with a known biological past can be

used on the field to analyse bioavailability of contaminants present in the habitats (soil, plants, air) by

measuring their accumulation in individuals caged and exposed for a determined period of time.

C. aspersus can be used either in the field (Gomot-de Vaufleury and Pihan, 2000; Scheifler et al., 2006;

Regoli et al., 2006; Gimbert et al., 2008a; Pauget et al., 2013; de Vaufleury et al., 2015; Mariet et al, 2017)

or in the laboratory (Gimbert et al., 2008b; Pauget et al., 2011, 2012, 2017; Louzon et al., 2020) to

assess the fate and transfer (i.e. environmental bioavailability, ISO 17402) of chemicals in soils. This

soil bioindicator has been applied on numerous field sites (https:// ecobiosoil .univ -rennes1 .fr/ ADEME

-Bioindicateur/ english/ worksheet .php) to evaluate habitat and retention function of soils. This bioassay

allows determining the bioavailability of chemicals to snails thanks to the measurement of their

concentration in their visceral mass (which contain mainly the digestive gland and some other organs

as described in Gomot-de Vaufleury and Pihan, 2002). The visceral mass is the main site of contaminant

accumulation in snails.

This standard describes how to expose snails in situ for 28 days and how to prepare them until chemical

analysis are performed to assess bioaccumulation in their viscera. This bioassay evaluates the transfer

of contaminants from the environment to land snails.
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This test is applicable in the field (e.g. contaminated sites, amended soils, soils after remediation,

agricultural or other sites under concern and waste materials) by caging snails for 28 days on the

studied site/soil/waste. Snails integrate chemicals of all terrestrial sources (soil, plant, air). After

exposure, concentrations of chemicals are measured in the visceral mass of snails.

Optionally, the method can be used in the laboratory (ex situ) to evaluate bioaccumulation of chemicals

of snails exposed only to soil (Annex I).

The results of a ring test performed in situ by six laboratories to assess the method of exposure and by

four laboratories from exposure until to chemical analysis are shown in Annex H.
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oSIST prEN ISO 24032:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 24032:2020(E)
Soil quality — In situ caging of snails to assess
bioaccumulation of contaminants
1 Scope

This document describes a method to assess the bioaccumulation of chemicals in snails, i.e.

concentrations of metal(loid)s (ME) or organic compounds (e.g. PAHs and PCBs) accumulated in their

tissues.

This document presents how to prepare snails for caging in situ for 28 days, the in situ test design and

then how to collect and prepare the snails until conservation and further analysis. If a kinetic study of

accumulation is necessary, sampling of snails at different time-points during exposure is possible as

well (Gimbert et al., 2008a; Pauget et al., 2013; Mariet et al., 2017)

This standard excludes analytical methods: preparation (extraction and mineralization) of the samples

and quantification of chemicals. These are not in the scope of the present document.

The method is applicable for soils under different uses (agricultural, industrial, residential, forests,

before and after remediation, on potentially contaminated sites, etc.) and waste materials (de Vaufleury,

2015; Bourioug et al., 2015), and should be preferably used on soil with vegetation and/or humus cover.

The method is applicable subject to certain limits of temperature (frost-free period, i.e. mainly from

April to October in temperate region).

Optionally (Annex A) the method can be used in the laboratory to evaluate the accumulation of

contaminants (and optionally the Sum of Excess of Transfer (SET) index for ME, PAH, PCB) of snails

exposed only to soil.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 15952:2018, Soil quality — Effects of pollutants on juvenile land snails (Helicidae) — Determination of

the effects on growth by soil contamination
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

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

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
caging

closed microcosm allowing exposure of snails by various routes and several sources

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3.2
bioaccumulation

phenomenon by which a chemical present in the medium accumulates in a living organism

Note 1 to entry: This phenomenon is observed when the rate of absorption exceeds the rate of elimination of the

contaminant
3.3
inactive snails

snails without any activity, generally under dry conditions where they glue on the walls of the box in

which they are placed (generally just due to a simple dried mucus ring)
3.4
aestivation
snails kept inactive, under dry conditions, at a temperature of 15 °C to 20 °C
3.5
plot
characteristic and representative sub-area of the site
Note 1 to entry: The geographical coordinates of each plot should be recorded
3.6
site

field place (or geographical entity) under study and where the microcosms will be placed to assess the

bioavailability of contaminants to snails

Note 1 to entry: The site can present one or more plot(s) and land use, i.e., a field, a pasture, a forest, an industrial

site, a discharge
4 Principle

Snails are caged in microcosms at the study site for 28 days. Fifteen sub-adult (5 ± 1 g of the body mass)

garden snails shall be placed in each microcosm. From the end of their breeding to their placement

on the soil, they can be stored in dry wooden boxes (round wooden boxes, approximately 12 cm in

diameter and 4 cm in height, Figure 1 and B.1.),). They are awaken from aestivation by spraying them

with water a few hours before they are placed in the microcosms. Here, they will be exposed to soil as

well as plants that have grown on-site and ambient air in order to be under natural exposure conditions

(climate hazards).

After exposure, the collected snails are brought back to the laboratory and starved for 48 hours. During

the starvation, faeces are removed every 24 hours. Snails are then frozen at –80 °C. After thawing, the

soft body is removed from the shell; the visceral mass and the foot (Annex B, Figure B.1) are separated

and prepared for chemical analysis to determinate internal concentration of chemicals. Main steps are

presented in Annex B.
5 Test organism and equipment
5.1 Biological material

Test organisms shall be sub-adult snails (to avoid mass change during the exposure duration and the

consecutive dilution of the bioaccumulation per the mass gain during the growth or the transfers of

compounds to the eggs during the reproductive stages). The recommended species is the land snail

Cantareus aspersus (Müller, 1774) which shall be 7 to 12 weeks old, having a mean fresh mass of 5 ± 1 g

(with min/max of 4/6 g).

NOTE 1 Optionally the shell diameter can be measured (mean ± SD of 25 mm ± 5 mm; min/max of 20/30 mm).

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The snails shall be selected from synchronous breeding in order to form a population as homogeneous

as possible with respect to mass and age. The breeding techniques for snails are described in Annex C.

In summary, after a nursery and then a growth period (3 to 6 weeks followed by 4 to 6 weeks), the

sub-adult snails shall be used directly or after an aestivation period that should not be more than 5

months (i.e. snail inactive, fixed on the wall of a dry box (plastic box must be avoided), in a temperature-

controlled room between 15 and 20 °C). The aestivation is carried out in round wooden boxes

(approximately of 12 cm in diameter and 4 cm in height; usually 15 snails per boxes, which is equal the

number of snails per microcosm).

Snails shall be reared for the purpose of the project (see Annexes C and D) or be purchased from local

snail farmers.

NOTE 2 The use of some other genus and/or species of Helicidae is possible (see examples and conditions

in Annex G of ISO 15952:2018).

A control of the chemical quality of the subadult snails selected for the caging (i.e. unexposed snails)

can be performed on 6 snails with respect to the initial concentrations of the chemicals of interest.

These control snails can be selected at the same time as the snails used for snail caging. The analysis of

the chemical quality of snails before caging can be done at the same time as the analysis of snails after

exposure. It is not mandatory to make this control. Indeed, after exposure, all data are compared to the

threshold guide value (TGV) (see 8.2.); however, if possible to get these data, it provides an indication

that snails were uncontaminated before exposure. For chemicals for which no TGV are available, data

can be compared to various values (see 8.2.2.3) among which are Csnail-t0.

The sub-adult snails used shall present usual concentrations in the visceral mass before caging

(Annex E). For PAH and PCB data, as extraction are often made on fresh tissues, data of Table E.1. are

−1 −1

in µg.kg fresh mass of viscera (these values can be converted in µg.kg dw on the basis of ≈ 15 % dry

mass of the visceral mass); for metal(loids) in mg.kg dry mass of visceral mass..

5.2 Equipment
5.2.1 Microcosm

Stainless steel cylinders with 25 cm diameter and 25 cm height covered by a 0,5 or 1 cm mesh netting.

An example is presented in Figure 1 and in Annex F.

NOTE 1 Other device could be used if the material that constitutes them cannot be a source of contamination;

for some purpose (e.g. exposure of snails to chemicals sprayed in the field), fully screened microcosm can be used

(see for example Druart et al. (2011) that used stainless steel cages of 25 × 25 × 15 cm (mesh size of grid: 1 cm)

closed by a stainless steel grid of 30 × 30 cm (mesh size: 1 cm) held by four pickets (see Annex F, Figure F.2).

NOTE 2 In some cases, it could be necessary to protect the microcosm from predators or cattles (see examples

in Annex F Figure F.3).
5.2.2 Netting
0,5 or 1 cm mesh netting, also stainless steel.
5.2.3 Pickets

Stainless steel picket (diameter 5 mm; length 46 to 72 cm) to maintain the mesh netting on the cage.

Depending on the soil settlement or the presence of stones, the size of picket shall be adapted.

5.2.4 Pieces of tiles
See Figure 1 and Annex F.
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5.2.5 Wooden storage

Inactive snails can be stored and transported before exposure in round wooden boxes

(approximately 12 cm in diameter and 4 cm in height), with the snails under dry conditions, at a

temperature of 15 °C to 20 °C (see Figure 1, Figure F.5 and Annex G).
5.2.6 Boxes for fasting, sampling

For the preparation of snails in the laboratory (e.g. to keep the snails before individual weighing), plastic

containers (PCs) (e.g. made of transparent polystyrene or any other container having approximate

dimensions: 24 cm (length) × 10,5 cm (width) × 8 cm (height)) can be used.
5.2.7 Calliper rule

For the measurement of the shell diameter, a calliper rule having a precision of 0,1 mm.

5.2.8 Balance
One analytical balance having a precision of at least 10 mg.
5.2.9 Water
Water, of purity at least deionized.
5.2.10 Feed

The feed shall be provided in the form of flour at its natural moisture content (5 % to 10 %).

In order to obtain sufficient growth, it is recommended to carry out the tests with a flour-based feed

comprising cereals, forage, mineral salts and vitamins which properly covers the needs of the snails. An

example of feed composition is given in Annex D.
5.2.11 Small material

Elastic strips to close wooden storage or boxes for fasting, sampling. Tape to label the wooden storage

and boxes for fasting; indelible markers; Ziploc© bags.
6 Preparation of the organisms for the exposure

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

the mass required for the test) snails shall be stored inactive in wooden box (see 3.5). Their mass will

decrease during this storage period that’s why in some cases (i.e. storage for more than 1 week) they

shall be woken from aestivation few days before the start of the assay (see 6).

Depending on the duration of storage between the end of growth period (i.e. when reaching the mean

mass requested, see 5.1.) and the start of the test in the field, snails shall or not be woken by spraying

them with water into the box according to the following scenarios:

— if snails are used in the week following their weighing and distribution in homogeneous batch

(15 snails for 1 microcosm) it is just necessary to awake them some hours before using in the field.

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

...