SIST EN ISO 15952:2018

SLOVENSKI STANDARD
oSIST prEN ISO 15952:2017
01-junij-2017
Kakovost tal - Vpliv onesnaževal na juvenilne (mladostniške) stadije kopenskih
polžev (Helicidae) - Ugotavljanje vplivov na rast zaradi onesnaženja tal (ISO/DIS
15952:2017)
Soil quality - Effects of pollutants on juvenile land snails (Helicidae) - Determination of
the effects on growth by soil contamination (ISO/DIS 15952:2017)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Jungtiere von Landschnecken -
Bestimmung der Wirkungen auf das Wachstum durch Bodenverunreinigung (ISO/DIS
15952:2017)
Qualité du sol - Effets des polluants vis-à-vis des escargots juvéniles (Helicidae) -
Détermination des effets sur la croissance par contamination du sol (ISO/DIS
15952:2017)
Ta slovenski standard je istoveten z: prEN ISO 15952
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 15952:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 15952:2017

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oSIST prEN ISO 15952:2017
DRAFT INTERNATIONAL STANDARD
ISO/DIS 15952
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2017-04-10 2017-07-02
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
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 15952:2017(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 2017

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oSIST prEN ISO 15952:2017
ISO/DIS 15952:2017(E) ISO/DIS 15952

Contents Page
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Test environment . 3
6 Reagents . 3
7 Apparatus . 5
8 Storage and preparation of the samples . 5
9 Procedure . 6
10 Reference substance . 9
11 Calculations and expression of results . 10
12 Validity of test . 12
13 Test report . 13
Annex A (normative) Static method . 14
Annex B (informative) Breeding technique for snails . 15
Annex C (informative) Example of composition of snail feed . 20
Annex D (informative) Example of table of data . 21
Annex E (informative) Example of results with Helix aspersa aspersa. 22
Annex F (informative) Determination of the effects on growth by food contamination . 25
Annex G (informative) Test performance with other snail species . 29


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iii
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ii © ISO 2017 – All rights reserved

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oSIST prEN ISO 15952:2017
ISO/DIS 15952
Contents Page
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Test environment . 3
6 Reagents . 3
7 Apparatus . 5
8 Storage and preparation of the samples . 5
9 Procedure . 6
10 Reference substance . 9
11 Calculations and expression of results . 10
12 Validity of test . 12
13 Test report . 13
Annex A (normative) Static method . 14
Annex B (informative) Breeding technique for snails . 15
Annex C (informative) Example of composition of snail feed . 20
Annex D (informative) Example of table of data . 21
Annex E (informative) Example of results with Helix aspersa aspersa. 22
Annex F (informative) Determination of the effects on growth by food contamination . 25
Annex G (informative) Test performance with other snail species . 29
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oSIST prEN ISO 15952:2017
ISO/DIS 15952
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 15952 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
This second/third/. edition cancels and replaces the first/second/. edition (ISO 15952:2006), [clause(s) /
subclause(s) / table(s) / figure(s) / annex(es)] of which [has / have] been technically revised.
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oSIST prEN ISO 15952:2017
ISO/DIS 15952
Introduction
Because of the limited amount of data available concerning toxicity of contaminants on soil organisms, the
problems of assessing 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 standards assess
[5]
acute toxicity [earthworms (ISO 11268-1) and coleoptera larvae ] and three other standards assess sublethal
[2] [1] [3]
effects of soil contaminants on reproduction (earthworms , Collembola , Enchytraeidae ). In the biological
cycles of organisms, it appears that growth is, like reproduction, a fundamental ecophysiological parameter to be
[37]
taken into consideration for the sustainability of species and ecosystems .
[15, 17 to 19, 31, 32, 39 to 41]
Snails are relevant ecological indicators for assessing the quality of soils , as they are
characteristic of the soil surface layer (saprophagous and phytophagous) of which a large part of the biological
[6], [17], [28]
cycle takes place in the soil (egg-laying, hatching, 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
[6], [17], [30]
and finally shell and organic 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
[8, 10 to 14, 16, 17,
indicator: easy to sample and identify, they are widely distributed; they accumulate contaminants
21, 23, 28, 29, 32 to 47] [6], [9], [31]
; their ecological and physiological characteristics are well-known ; and they are now
[21], [25, [31]
easy to breed under controlled conditions . Their susceptibility to common contaminants of their
[10 to 15, 18 to 27, 29, 32 to 37, 36 to 47]
environment has been demonstrated .
This International Standard describes a method for determining the effects on survival and growth of young snails
of substances, preparations, 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
[55]
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 on all continents except
[9]
Antarctica .
Indeed, in their natural environment, snails integrate the contaminants by contact (with various substrates such
[6], [28]
as soil, soil leachates, plant litter), by ingestion (of plants and soil), as well as through the respiratory tract .
So, for specific testing purposes (evaluation of the toxicity of a pesticide, for example), another test design, which
is focussed on exposure via food uptake, is optionally available (Annex F and Reference [5]).
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oSIST prEN ISO 15952:2017

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oSIST prEN ISO 15952:2017
DRAFT INTERNATIONAL STANDARD ISO/DIS 15952
Soil quality — Effects of pollutants on juvenile land snails
(Helicidae) — Determination of the effects on growth by soil
contamination
1 Scope
This International Standard 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 referenced documents are indispensable for the application 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 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to Eisenia
fetida/Eisenia andrei
ISO 11269-2, 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
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oSIST prEN ISO 15952:2017
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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
3.4
growth
increase in the biomass, i.e. in the total fresh mass (body and shell) of the organisms and increase in the maximum
shell diameter, between the start and completion of the test
Note 1 to entry: It is expressed in the form of a growth coefficient.
3.5
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 EC50 means the concentration estimated to reduce growth at the end of the test to 50 % compared to the
control.
3.6
median lethal concentration
LC
50
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
3.7
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 2 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.
3.8
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 3 to entry: The NOEC is the concentration just below the LOEC.
Note 4 to entry: For 3.5, 3.6, 3.7 and 3.8, results are given:
 in dry mass of test substance or preparation per dry mass of the test substrate;
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 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 days to test mixtures
containing the test substance, preparation or matrix at different concentrations. The test mixtures are freshly
prepared and renewed every 7 days.
According to the objectives, the test mixtures may be prepared with artificial soil (6.3.2) or with a suitable natural
soil (6.3.3).
The snails are fed during the test with uncontaminated food.
The effects on growth (fresh mass and shell diameter) and on survival are measured after 28 days of exposure
(optionally, effects could be measured every 7 days during 28 days).
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 days with
respect to the fresh mass [EC (28 days)] and to the shell diameter [EC (28 days)] or other values of EC .
50,m 50,d 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 days)].
50
For particular applications, various parameters (EC , NOEC, LOEC, LC ) can be assessed (optional) after exposure
x 50
periods lower than 28 days (7 days, 14 days or 21 days).
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 lux.
6 Reagents
6.1 Water, of purity at least deionized
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
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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 box (7.1) that has been moistened with water (6.1). The bottom of this box either can be 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 to 3 days), 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 (6.3.2) or a suitable natural soil (6.3.3) is used as
test substrate.
NOTE 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 assess 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 by ISO 11268-1).
Table 1 — Composition of artificial soil
Composition Percentage expressed in dry mass
Sphagnum peat air-dried and finely ground (2  1) mm without any
10 %
visible plant remains.
Kaolinite clay, preferably containing not less than 30 % kaolinite. 20 %
Air-dried industrial quartz sand (predominantly fine sand with Approximately 69 % (depending on the amount of
more than 50 % by mass of particle size 0,05 mm to 0,2 mm). CaCO needed).
3
Calcium carbonate (CaCO , pulverised, analytical grade) to bring Approximately 0,3 % to 1,0 %
3
the pH of the wetted artificial soil to 6,0  0,5.
The artificial soil shall be prepared, at least two days 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 two days to equilibrate acidity. To
determine pH and the maximum water-holding capacity, the dry artificial soil shall be pre moistened one or two
days 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 according to 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-
3
holding capacity of the artificial soil shall be determined according to ISO 11274 or to Annex C of ISO 11269-
2:2012.
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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 Annex C of ISO 11269-2:2012;
 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.
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 (6.3.3) that can be used as control or dilution substrate,
shall be determined.
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8.2 Waste material
The samples of waste material received at the laboratory shall be stored according to 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
...