SIST EN ISO 15952:2018

SLOVENSKI STANDARD
01-december-2018
1DGRPHãþD
SIST EN ISO 15952:2012
Kakovost tal - Vpliv onesnaževal na juvenilne (mladostniške) stadije kopenskih
polžev (Helicidae) - Ugotavljanje vplivov na rast zaradi onesnaženja tal (ISO
15952:2018)
Soil quality - Effects of pollutants on juvenile land snails (Helicidae) - Determination of
the effects on growth by soil contamination (ISO 15952:2018)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Jungtiere von Landschnecken -
Bestimmung der Wirkungen auf das Wachstum durch Bodenverunreinigung (ISO
15952:2018)
Qualité du sol - Effets des polluants vis-à-vis des escargots juvéniles (Helicidae) -
Détermination des effets sur la croissance par contamination du sol (ISO 15952:2018)
Ta slovenski standard je istoveten z: EN ISO 15952:2018
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 15952
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2018
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 15952:2011
English Version
Soil quality - Effects of pollutants on juvenile land snails
(Helicidae) - Determination of the effects on growth by soil
contamination (ISO 15952:2018)
Qualité du sol - Effets des polluants vis-à-vis des Bodenbeschaffenheit - Wirkungen von Schadstoffen
escargots juvéniles (Helicidae) - Détermination des auf Jungtiere von Landschnecken - Bestimmung der
effets sur la croissance par contamination du sol (ISO Wirkungen auf das Wachstum durch
15952:2018) Bodenverunreinigung (ISO 15952:2018)
This European Standard was approved by CEN on 15 June 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15952:2018 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 15952:2018) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “Test methods for 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 January 2019, and conflicting national standards shall
be withdrawn at the latest by January 2019.
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.
This document supersedes EN ISO 15952:2011.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 15952:2018 has been approved by CEN as EN ISO 15952:2018 without any modification.

INTERNATIONAL ISO
STANDARD 15952
Second edition
2018-06
Soil quality — Effects of pollutants
on juvenile land snails (Helicidae) —
Determination of the effects on growth
by soil contamination
Qualité du sol — Effets des polluants vis-à-vis des escargots juvéniles
(Helicidae) — Détermination des effets sur la croissance par
contamination du sol
Reference number
ISO 15952:2018(E)
©
ISO 2018
ISO 15952:2018(E)
© ISO 2018
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

ISO 15952:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Test environment . 3
6 Reagents . 3
7 Apparatus . 5
8 Storage and preparation of the samples . 6
8.1 Soil to be tested . 6
8.2 Waste material . 6
9 Procedure. 6
9.1 Preparation of the test . 6
9.1.1 Selection of the concentrations to be tested . 6
9.1.2 Preparation of test mixtures . 7
9.2 Distribution of test mixture . 8
9.3 Introduction of the feed. 8
9.4 Introduction of the biological reagent . 8
9.5 Handling during the tests . 8
9.5.1 General. 8
9.5.2 Routine care . 9
9.5.3 Weekly task . 9
10 Reference substance .10
11 Calculations and expression of results .10
11.1 Calculations .10
11.2 Expression of results .12
12 Validity of test .13
13 Test report .13
Annex A (informative) Static method .15
Annex B (informative) Breeding technique for snails .16
Annex C (informative) Example of composition of snail feed .21
Annex D (informative) Example of table of data .22
Annex E (informative) Example of results with Helix aspersa aspersa .23
Annex F (informative) Determination of the effects on growth by food contamination .27
Annex G (informative) Suitable other snail species .30
Bibliography .31
ISO 15952:2018(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
This second edition cancels and replaces the first edition (ISO 15952:2006), which has been technically
revised.
iv © ISO 2018 – All rights reserved

ISO 15952:2018(E)
Introduction
Because of the limited amount of data available concerning toxicity of contaminants on soil organisms,
the ecotoxicity of soils and waste are cause for serious concern at both national and international
levels. Currently available tests use soil-fauna organisms restricted to annelid (earthworms and
Enchytraeidae) and arthropod phyla (insects: Collembola and Coleoptera). Among the latter, two
[6]
standards assess acute toxicity [earthworms (ISO 11268-1) and coleoptera larvae and three other
[3] [2]
standards address sublethal effects of soil contaminants on reproduction (earthworms , Collembola ,
[4]
Enchytraeidae ). In the biological cycles of organisms, it appears that growth is, like reproduction, a
fundamental ecophysiological parameter to be taken into consideration for the sustainability of species
[38]
and ecosystems .
Snails are relevant ecological indicators for assessing the quality of soils(See References [16][18]
to [20][32][33][40] to [42]), as they are characteristic of the soil surface layer (saprophagous and
phytophagous) of which a large part of the biological cycle takes place in the soil (egg-laying, hatching,
[7][18][29]
initial stages of development, hibernation, etc.) . During the other phases of their cycle, they eat
soil and are in contact with the soil via their moist pedal sole (foot) covered with mucus and participate
in the permanent exchanges with the soil (water, mineral salts, excrement and finally shell and organic
[7][18][31]
matter when they die) . In addition, they constitute an important link between plants, fauna
and soil microorganisms. They correspond fully to the criteria for a good biological indicator: easy to
sample and identify, they are widely distributed; they accumulate contaminants (See References[9],[11]
to [15],[17][18][22][24][29][30],[33] to [48]); their ecological and physiological characteristics are
[7][10][32] [22][26][32]
well-known ; and they are now easy to breed under controlled conditions . Their
susceptibility to common contaminants of their environment has been demonstrated (See References
[11] to [16],[19] to [28],[30],[33] to [38],[37] to [48]).
This International Standard describes a method for determining the effects on survival and growth of
young snails of substances, preparations (i.e. a mixture or solution composed of two or more substances),
soils or waste materials added to an artificial or a natural soil. The described method is thus applicable
to test contaminated soils or to compare different uncontaminated soils. The recommended species is
Helix aspersa aspersa Müller (also commonly called: common garden snail, brown garden snail, garden
[56]
snail, land snail, “Petit-Gris”; synonyms: Cantareus aspersus, Cornu aspersum ). Among land snails
(stylommatophoran pulmonate gastropod molluscs of the Helicidae family), Helix aspersa aspersa Müller
is the most ubiquitous. This palearctic species can be acclimated to regions with different types of
climate: Mediterranean, oceanic temperate, midcontinental temperate and even tropical. Helix aspersa
aspersa Müller is of European origin and has been introduced into all parts of the world. They are now
[10]
on all continents except Antarctica .
Indeed, in their natural environment, snails integrate the contaminants by contact (with various
substrates such as soil, soil leachates, plant litter), by ingestion (of plants and soil), as well as through the
[7][29]
respiratory tract . So, for specific testing purposes (evaluation of pesticide toxicity, for example),
another test design, which is focussed on exposure via food uptake, is optionally available (Annex F and
Reference [6]).
INTERNATIONAL STANDARD ISO 15952:2018(E)
Soil quality — Effects of pollutants on juvenile land snails
(Helicidae) — Determination of the effects on growth by
soil contamination
1 Scope
This document specifies a semi-static method for determining the effects of contaminants on growth
and survival of young snails, usually Helix aspersa aspersa Müller. The animals are exposed via the
cutaneous and digestive route using a test substrate (artificial or natural soil according to the objective
of the study) to which defined amounts of the following are added:
— substances, mixtures or preparations;
— soils (contaminated or of unknown quality) or waste materials.
This test takes into account the possible changes in the test substance, preparation, soil or waste
material because the test mixtures are prepared and renewed every week during the 28-day test period.
A static method may be implemented in addition to the semi-static method (optional). This method is
described in Annex A.
This method does not apply to substances for which the air/soil partition coefficient is greater than one,
or to substances with vapour pressure exceeding 300 Pa, at 25 °C.
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 10390, Soil quality — Determination of pH
ISO 18400-206, Soil quality — Sampling — Part 206: Guidance on the collection, handling and storage of
soil for the assessment of biological functional and structural endpoints in the laboratory
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity
to Eisenia fetida/Eisenia andrei
ISO 11269-2:2012, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects
of contaminated soil on the emergence and early growth of higher plants
ISO 11274, Soil quality — Determination of the water-retention characteristic — Laboratory methods
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —
Gravimetric method
EN 14735, Characterization of waste — Preparation of waste samples for ecotoxicity tests
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO 15952:2018(E)
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http: //www .electropedia .org/
— ISO Online browsing platform: available at https: //www .iso .org/obp
3.1
test substrate
artificial soil or natural soil used as control and dilution substrate
3.2
matrix
soil or waste material under test
3.3
test mixture
mixture of the test substance, preparation or matrix with the test substrate (3.1)
3.4
growth
increase in the biomass, i.e. in the total fresh mass (body with shell) of the organisms
between the start and completion of the test
Note 1 to entry: It is expressed as a biomass growth coefficient k .
GC,m
3.5
growth
increase in the maximum shell diameter, between the start and completion of the test
Note 1 to entry: It is expressed as a shell diameter growth coefficient k .
GC,d
3.6
effect concentration
EC
x
concentration at which a specific effect is detected; x is the percentage (10, 25, 50) of this effect, e.g.
growth inhibition
EXAMPLE EC means the concentration estimated to reduce growth at the end of the test to 50 % compared
to the control (EC and EC for biomass growth and shell growth respectively).
50,m 50,d
3.7
median lethal concentration
LC
concentration of the substance, of the test preparation initially present, or the concentration of the
matrix causing the death of 50 % of the snails submitted to testing
3.8
lowest observed effect concentration
LOEC
lowest tested concentration at which the test substance is observed to have a statistically significant
effect (p < 0,05) when compared with the control
Note 1 to entry: All test concentrations above the LOEC have a harmful effect equal to or greater than those
observed at the LOEC. When these two conditions cannot be satisfied, a full explanation should be given for how
the LOEC (and hence the NOEC) has been selected.
2 © ISO 2018 – All rights reserved

ISO 15952:2018(E)
3.9
no observed effect concentration
NOEC
test concentration immediately below the LOEC, which, when compared with the control, has no
statistically significant effect (p > 0,05) within a given exposure time
Note 1 to entry: The NOEC is the concentration just below the LOEC.
Note 2 to entry: For 3.6, 3.7, 3.8 and 3.9, results are given:
—  in dry mass of test substance or preparation per dry mass of the test substrate (3.1);
—  in mass percentage of the tested matrix in the test mixture (expressed in dry mass).
4 Principle
Juvenile land snails (usually Helix aspersa aspersa Müller) are exposed during a period of 28 d to test
mixtures containing the test substance, preparation or matrix at different concentrations. The test
mixtures are freshly prepared and renewed every 7 d.
According to the objectives of the study, the test mixtures may be prepared with artificial soil (see
6.3.2) or with a suitable natural soil (see 6.3.3).
The snails are fed during the test with uncontaminated food.
The effects on growth (biomass and shell diameter) and on survival are measured after 28 d of exposure
(optionally, effects could be measured every 7 days during 28 d).
The results obtained during testing are compared with those of a control to determine the NOEC or
LOEC and to allow the estimation of the concentration which reduces the growth of the snails by 50 %
within 28 d with respect to the fresh mass [EC (28 d)] and to the shell diameter [EC (28 days)]
50,m 50,d
or other values of EC .
x
If the concentrations selected result in lethal effects, the results obtained during testing are compared
with those of a control and used for estimating the concentration which causes the death of 50 % of the
snails [LC (28 d)].
For particular applications, various parameters (EC , NOEC, LOEC, LC ) can be determined (optional)
x 50
after exposure periods lower than 28 d (7 d, 14 d or 21 d).
The test is conducted in two stages:
— a preliminary test intended to indicate both the non-observed effect concentration, NOEC, and the
complete growth inhibition. The resulting dose-response relationship is important for the proper
design of the definitive test;
— a definitive test specifying the concentrations which cause between 10 % and 90 % of growth
inhibition. It is not necessary to perform a final test where the preliminary test has not revealed
any inhibitory effects at the maximum concentration tested.
5 Test environment
The test shall be carried out at a temperature of (20 ± 2) °C under a day-night photoperiod of 18 h to
6 h. The illumination intensity (artificial light of daylight type), without any natural light in the test
containers shall be 50 to 100 lx.
6 Reagents
6.1 Water, of purity at least deionized.
ISO 15952:2018(E)
6.2 Biological material.
Test organisms shall be juvenile snails. The recommended species is Helix aspersa aspersa Müller (also
known as Cantareus aspersus and Cornu aspersum) which shall be 3 to 5 weeks old, having a mean fresh
mass of (1 ± 0,3) g and a shell diameter of (15,5 ± 1) mm.
NOTE The use of some other genus and/or species of Helicidae is possible (see examples and conditions in
Annex G).
The snails shall be selected from synchronous breeding in order to form a population as homogeneous
as possible with respect to size, mass and age. The breeding techniques for snails are described in
Annex B. After a nursery period (3 to 5 weeks, see Annex B), the young snails shall be used after at
least 1 week of aestivation and no more than 5 months. The aestivation is carried out in round wooden
boxes (approximately 12 cm in diameter and 4 cm in height), with the snails under dry conditions, at a
temperature of 17 °C to 20 °C.
Two to three days before starting the test, snails shall be woken by spraying water (6.1) into the boxes
used for aestivation. The proportion of snails not woken shall be less than 10 %. As soon as they have
resumed activity (snails not stuck to the walls of the box and which are beginning to move about),
the snails shall be transferred to a test container (7.1) that has been moistened with water (6.1). The
bottom of this box either is covered with absorbent paper that has also been moistened, or can contain
some test substrate (6.3) moistened to 50 % to 60 % of its water-holding capacity. Between waking and
the start of the test (2 d to 3 d), the snails shall be fed (6.4).
6.3 Test substrate.
6.3.1 General
According to the objectives of the study, either an artificial soil (see 6.3.2) or a suitable natural soil (see
6.3.3) is used as test substrate. The test substrate can be used dry or raw (i.e. without dehydration
prior to use for natural soil).
Artificial soil may be used as a control and dilution substrate to assess the effect of a substance or of a
preparation, or to compare different soils or waste, or to determine the effects of a contaminated soil.
Natural soil (field soil) may be used as a control and dilution substrate in order to assess, for example,
the effect of the incorporation of wastewater treatment plant sludge into the field soil or to test the
effect of a contaminated soil (in this case an uncontaminated soil comparable to the soil sample to be
tested ought to be used).
6.3.2 Artificial soil
The artificial soil shall have the following composition (as defined in ISO 11268-1).
See Table 1.
Table 1 — Composition of artificial soil
Composition Percentage expressed in dry mass
Sphagnum peat air-dried and finely ground (2 ± 1) mm
without any visible plant remains.
Kaolinite clay, preferably containing not less than 30 %
kaolinite.
Air-dried industrial quartz sand (predominantly fine sand Approximately 69 (depending on the amount of
with more than 50 % by mass of particle size 0,05 mm to CaCO needed).
0,2 mm).
Calcium carbonate (CaCO , pulverised, analytical grade) to Approximately 0,3 to 1,0
bring the pH of the wetted artificial soil to 6,0 ± 0,5.
4 © ISO 2018 – All rights reserved

ISO 15952:2018(E)
The artificial soil shall be prepared, at least 2 d prior to starting the test, by mixing the dry constituents
listed above thoroughly in a large-scale laboratory mixer. The amount of calcium carbonate required
might vary, depending on the properties of the individual batch (mainly the peat) and should be
determined by measuring subsamples immediately before the test.
The mixed artificial soil shall be stored at room temperature for at least 2 d to equilibrate acidity. To
determine pH and the maximum water-holding capacity, the dry artificial soil is pre moistened one or
2 d before starting the test by adding deionized water to obtain half of the required final water content
of 50 % to 60 % of the maximum water-holding capacity.
The pH value shall be measured in accordance with ISO 10390. If the measured pH is not within the
required range, a sufficient amount of CaCO shall be added or a new batch of artificial soil shall be
prepared. The maximum water-holding capacity of the artificial soil shall be determined in accordance
with ISO 11274 or ISO 11269-2:2012, Annex C.
6.3.3 Natural soil
Determine the following parameters on the selected natural soil which shall be sieved through a 4-mm
square mesh sieve to remove large fragments:
— pH, according to ISO 10390;
— water-holding capacity, according to ISO 11274 or ISO 11269-2:2012, Annex C;
— water content, according to ISO 11465;
— content of organic matter, according to ISO 10694.
It is also recommended to determine the cation exchange capacity, according to ISO 11260.
6.4 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 C.
7 Apparatus
Use ordinary laboratory apparatus and the following.
7.1 Test containers.
)
Disposable mouse boxes made of transparent polystyrene or any other container having a volume of
approximately 1,6 l [advised approximate dimensions: 24 cm (length) × 10,5 cm (width) × 8 cm (height)].
7.2 Feed containers.
Petri dishes, approximately 5,5 cm in diameter and approximately 1 cm in height or any other containers
of equivalent dimensions.
7.3 Calliper rule, having a precision of 0,1 mm.
7.4 Balances.
One analytical balance having a precision of at least 1 mg. Two other balances, one having a precision of
0,1 g, another having a precision of 1 g.
ISO 15952:2018(E)
8 Storage and preparation of the samples
8.1 Soil to be tested
The soil samples received at the laboratory shall be stored in accordance with ISO 18400-206.
The soil sample submitted for testing shall be sieved through a 4-mm square mesh sieve to remove
coarse fragments.
For each soil, the same characteristics than for natural soil (see 6.3.3) that can be used as control or
dilution substrate, shall be determined.
8.2 Waste material
The samples of waste material received at the laboratory shall be stored in accordance with EN 14735
[less than 2 months at (4 ± 3) °C].
For conducting the tests, the grading of the waste shall be less than 4 mm. Where this condition is not
fulfilled, the particle size of the waste material shall be reduced so that all of the particles pass through
a 4-mm square mesh sieve.
9 Procedure
9.1 Preparation of the test
9.1.1 Selection of the concentrations to be tested
9.1.1.1 Preliminary test
This test is performed within a wide range of concentrations.
— Four concentrations of the substance or preparation and one control (e.g. 0 mg/kg; 50 mg/kg;
100 mg/kg; 500 mg/kg and 1 000 mg/kg of test substrate) with five snails per concentration and
per container. The preliminary test may be conducted without replication.
— Four percentages of the matrix under examination and one control (e.g. 0 %; 12,5 %; 25 %; 50 %;
100 %) with five snails per percentage and per container. The preliminary test may be conducted
without replication.
9.1.1.2 Definitive test
Select a range of at least five concentrations of the test substance, preparation or matrix according
to a geometric progression, so as to cover and extend beyond the range of those concentrations or
percentages which in the preliminary test did not have any effect on the growth or which inhibited it
completely. The ratio of this geometric progression shall preferably not exceed 2.
If the ratio exceeds 2, it is necessary to have available two concentrations for which the observed effect
is between 10 % and 90 %.
For the definitive test, three replicates are carried out per concentration.
6 © ISO 2018 – All rights reserved

ISO 15952:2018(E)
9.1.2 Preparation of test mixtures
9.1.2.1 General
The test mixture (see 3.3) is made up of test substrate and of test substance, preparation or matrix.
Prepare enough test mixture in order to cover the bottom of the test container with a layer of the test
mixture of at least 1 cm.
If the test substance is used in the raw state (i.e. without dehydration prior to use), take into account
its moisture rate so as to express the concentrations in milligrams of substance or of preparation per
kilogram of dry test substrate and, for the matrixes, in mass percentage of matrix (expressed in dry
mass) in the test mixture (expressed in dry mass).
9.1.2.2 Water-soluble or emulsifiable substances and preparations
For each examined concentration, dissolve the appropriate quantity of test substance or preparation
required for obtaining the desired concentration in the same water (6.1) used for moistening the test
substrate. Spray the solution over the dry or raw test substrate (6.3), then mix carefully.
The final test mixture shall have a moisture content corresponding to 50 % to 60 % of its total water-
holding capacity (determined according to ISO 11274 or according to ISO 11269-2:2012, Annex C).
Measure the pH for each test concentration according to ISO 10390.
Proceed likewise for the control treatment apart from the addition of test substance or preparation.
Continue the test as specified in 9.2.
9.1.2.3 Water-insoluble substances and preparations, but soluble in organic solvents
Dissolve the quantity of test substance or preparation required for obtaining the desired concentration
into a volatile solvent (e.g. methanol or acetone). Spray the obtained solution over the dry or raw
test substrate (6.3). Carefully mix the totality and let the organic solvent to evaporate under a fume
cupboard for 24 h.
Moisten the mixture with water (6.1) up to 50 % to 60 % of its total water-holding capacity (determined
according to ISO 11274 or according to ISO 11269-2:2012, Annex C), then mix carefully.
Measure the pH for each test concentration according to ISO 10390.
Proceed likewise for the control treatment apart from the addition of test substance or preparation.
Continue the test as specified in 9.2.
9.1.2.4 Substances and preparations insoluble in both water and organic solvents
For a substance or preparation that is insoluble in a volatile solvent, prepare a mixture of 10 g of
industrial quartz sand (see 6.3.2) (previously sampled from the quantity of sand required for the
preparation of the test substrate) and of the quantity of test substance or preparation required in order
to obtain the desired concentration. Tranfer the obtained mixture into a container containing the dry
or raw test substrate (6.3) (except the 10 g used for the contamination). Mix carefully.
Moisten the mixture with water (6.1) up to 50 % to 60 % of its total water-holding capacity (determined
according to ISO 11274 or according to ISO 11269-2:2012, Annex C), then mix carefully.
Measure the pH for each test concentration according to ISO 10390.
Proceed likewise for the control treatment apart from the addition of test substance or preparation.
Continue the test as specified in 9.2.
ISO 15952:2018(E)
9.1.2.5 Matrix
Increasing proportions of test matrix are mixed to the dry or raw test substrate (6.3) (e.g. 0 %; 12,5 %;
25 %; 50 %; 100 %).
The control treatment corresponds to 0 % of test matrix, i.e. 100 % of artificial soil (see 6.3.2) or natural
soil (see 6.3.3).
Moisten the mixture with water (6.1) up to 50 % to 60 % of its total water-holding capacity (determined
according to ISO 11274 or according toISO 11269-2:2012, Annex C), then mix carefully. (The added
water corresponds to the volume of water required in order to rehydrate the quantity of test substrate
of the mixture and to the volume of water required in order to rehydrate the quantity of matrix of the
mixture.)
If it is necessary to reduce the humidity of the matrixes, do it by dehydration outdoors or in a drying
oven at a temperature not exceeding 30 °C, in order to limit the loss of volatile substances.
Measure the pH for each test concentration according to ISO 10390.
Continue the test as specified in 9.2.
9.2 Distribution of test mixture
In preparation for the test, add sufficient test mixture (see 9.1.2) into the test containers (7.1) to fill the
bottoms of the test containers to a depth of at least 1 cm.
NOTE If the test substrate (6.3) is artificial soil, the quantity of test mixture is about 140 g (dry mass) for
each test container.
Smooth the surface of the test mixture and compact the soil slightly.
9.3 Introduction of the feed
Place the container (7.2) containing the feed (6.4) on the bottom of the test container (7.1). The feed
shall be provided ad libitum.
9.4 Introduction of the biological reagent
Select five snails (6.2) randomly for each per test container (7.1).
9.5 Handling during the tests
9.5.1 General
Cover the containers with a transparent perforated sheet [e.g. in polyalkylmethacrylate (Plexiglas) of
approximate dimensions 26,5 cm × 13,5 cm] held in place by any appropriate device during the first
two weeks of the test. During the following two weeks, use, to form the lid, a second container (7.1)
turned upside down. This arrangement doubles the volume of the test chamber thus avoiding a negative
group effect on the growth of the snails (see Figure B.2).
NOTE The plate and the container used to cover the test containers can be perforated by 3 to 4 holes with a
diameter smaller than 2 mm.
Place the test containers with the snails in conditions of the test (see Clause 5). Observe them regularly
and note any anomaly that could interfere with the conducting of the test.
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ISO 15952:2018(E)
9.5.2 Routine care
Three times a week (for example Monday, Wednesday and Friday) perform the following operations for
each test container.
— Using a spatula, regularly remove the excrement on the test mixture in order to avoid its accumulation
and the development of mould.
— Clean the side walls of the container with absorbent paper moistened with water (6.1) and wash the
lid with tap water, then dry it and remoisten it with water (6.1).
— Moisten the test mixture (see 9.1.2) by spraying it with water (6.1) so that it is at 50 % to 60 % of its
water-holding capacity. To ensure that the moisture content of the test mixture remains at 50 % to
60 % throughout the test duration, it is possible to prepare a container without snails which will be
weighed regularly in order to estimate the quantity of water to be sprayed into the test containers.
— Renew the feed (6.4).
It is advisable to carry out the operations described above at regular times, if possible (morning or
afternoon).
Note the mortality, if any.
9.5.3 Weekly task
If effects need to be determined weekly (optional, see Clause 4), snails are weighed and measured every
week. If not, snails are weighed and measured only at the end of the test (28 d).
Every 7 d:
— prior to changing the feed and cleaning the side walls and the lid, weigh the snails individually (with
a precision of 0,1 g) and measure the shell diameter (with a precision of 0,1 mm); see Figure 1;
— renew the test mixture (see 9.1.2) by a freshly-prepared one.
Figure 1 — Measure of the shell diameter, i.e. the longest size that can be measured (white
arrow on the photo)
The mass of the snails may be uncertain if they have test substrate on their shell or their foot. Before
each weighing, using a spatula, remove the substrate from the shell or the foot. It is possible to leave
the snails to move about on the clean container lid or on slightly moist paper so that they get rid of the
substrate adhering to their foot.
Note the obvious or pathological symptoms (e.g. excessive production of mucus, extended oedematous
body, drooping eyestalk like those described in References [12] or [42]), or any noticeable modifications
in behaviour (e.g. lethargy on the test mixture, lack of feeding), observed on the snails.
At the end of the test, the pH of a control container and of a container shall be measured for each
concentration in accordance with ISO 10390. It is recommended to measure the pH of any containers in
which the mortality rate or growth rate is unusual.
ISO 15952:2018(E)
Optional: at the end of the test (28 days), after final weighing and
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