EN ISO 15952:2011

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kakovost tal - Vpliv onesnaževal na juvenilne stadije kopenskih polžev (Helicidae) - Ugotavljanje vplivov na rast zaradi onesnaženja tal
(ISO 15952:2006)Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Jungtiere von Landschnecken - Bestimmung der Wirkungen auf das Wachstum durch Bodenverunreinigung (ISO 15952:2006)Qualité du sol - Effets des polluants vis-à-vis des escargots juvéniles (Helicidae) - Détermination des effets sur la croissance par contamination du sol (ISO 15952:2006)Soil quality - Effects of pollutants on juvenile land snails (Helicidae) - Determination of the effects on growth by soil contamination (ISO 15952:2006)13.080.30Biološke lastnosti talBiological properties of soilsICS:Ta slovenski standard je istoveten z:EN ISO 15952:2011SIST EN ISO 15952:2012en,fr,de01-januar-2012SIST EN ISO 15952:2012SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN ISO 15952
June 2011 ICS 13.080.30 English Version
Soil quality - Effects of pollutants on juvenile land snails (Helicidae) - Determination of the effects on growth by soil contamination (ISO 15952:2006)
Qualité du sol - Effets des polluants vis-à-vis des escargots juvéniles (Helicidae) - Détermination des effets sur la croissance par contamination du sol (ISO 15952:2006)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Jungtiere von Landschnecken - Bestimmung der Wirkungen auf das Wachstum durch Bodenverunreinigung (ISO 15952:2006) This European Standard was approved by CEN on 10 June 2011.
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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 15952:2011: ESIST EN ISO 15952:2012

Reference numberISO 15952:2006(E)© ISO 2006
INTERNATIONAL STANDARD ISO15952First edition2006-02-15Soil 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
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ii © ISO 2006 – All rights reserved
ISO 15952:2006(E) © ISO 2006 – All rights reserved iiiContents Page Foreword.iv Introduction.v 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.6 8.1 Soil to be tested.6 8.2 Waste material.6 9 Procedure.6 9.1 Preparation of the test.6 9.2 Distribution of the 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 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 for Helix aspersa aspersa.13 13 Test report.13 Annex A (normative)
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.26 Annex G (informative)
Test performance with other snail species.30 Bibliography.31
ISO 15952:2006(E) iv © ISO 2006 – All rights reserved 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. SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved vIntroduction 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 acute toxicity [earthworms (ISO 11268-1) and coleoptera larvae [5]] and three other standards assess sublethal effects of soil contaminants on reproduction (earthworms [2], Collembola [1], Enchytraeidae [3]). 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 and ecosystems [33]. Snails are pertinent ecological indicators for assessing the quality of soils [15], 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, initial stages of development, hibernation, etc.) [6], [17], [26]. 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 matter when they die) [6], [17], [28]. 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 [8, 10 to 14, 16, 17, 19, 21, 26, 27, 35 to 43]; their ecological and physiological characteristics are well-known [6], [9], [29]; and they are now easy to breed under controlled conditions [19], [23, [29]. Their susceptibility to common contaminants of their environment has been demonstrated [10 to 15, 18 to 27, 32, 33, 36 to 42]. 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 snail, land snail, “Petit-Gris”). 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 Antarctica [9]. 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 respiratory tract [6], [26]. 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 [4]).
INTERNATIONAL STANDARD ISO 15952:2006(E) © ISO 2006 – All rights reserved 1Soil 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 the determination of 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 or preparations; ⎯ soils (contaminated or of unknown quality) or waste materials. 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 volatile substances, i.e. substances for which the Henry constant, H, or the air/water partition coefficient is over 1, or for which the vapour pressure is over 0,013 3 Pa at 25 °C. This test takes into account the possible change in the test substance, preparation, soil or waste material because the test mixture is prepared and renewed every 7 days during the 28-day test period. 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory ISO 10390, Soil quality — Determination of pH 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 (Eisenia fetida) — Part 1: Determination of acute toxicity using artificial soil substrate ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of chemicals on the emergence and 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 SIST EN ISO 15952:2012

ISO 15952:2006(E) 2 © ISO 2006 – All rights reserved 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 It is expressed in the form of a growth coefficient. 3.5 effect concentration ECx 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 LC50 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 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 1 The NOEC is the concentration just below the LOEC. NOTE 2 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; ⎯ in mass percentage of the tested matrix in the test mixture (expressed in dry mass). SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved 34 Principle Juvenile land snails (usually Helix aspersa aspersa Müller) are exposed during a period of 28 days to a test mixture containing the test substance, preparation or matrix at different concentrations. The test mixture is freshly prepared and renewed every 7 days. According to the objectives, the test mixture 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 [EC50,m (28 days)] and to the shell diameter [EC50,d (28 days)] or other values of ECx. 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 [LC50(28 days)]. For particular applications, various parameters (ECx, NOEC, LOEC, LC50) can be assessed (optional) after exposure 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 lux 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 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. SIST EN ISO 15952:2012

ISO 15952:2006(E) 4 © ISO 2006 – All rights reserved 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 by 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 visible plant remains. 10 % Kaolinite clay, preferably containing not less than 30 % kaolinite. 20 % Air-dried industrial quartz sand (predominantly fine sand with more than 50 % by mass of particle size 0,05 mm to 0,2 mm). Approximately 69 % (depending on the amount of CaCO3 needed). Calcium carbonate (CaCO3, pulverised, analytical grade) to bring the pH of the wetted artificial soil to 6,0 ± 0,5. Approximately 0,3 % to 1,0 %
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 CaCO3 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 according to ISO 11274 or to Annex A of ISO 11269-2. SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved 56.3.3 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 A of ISO 11269-2; ⎯ 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 snails1). 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 polystyrene2) 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 Containers for food 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.
1) The snail feed “Helixal” manufactured and distributed by Établissements Chays Frères, 6, rue du Collège, BP 21, 25800 Valdahon, France, or the INRA formulation snail feed manufactured and distributed by Établissements Berton SARL, Lieu-dit Berton / Départementale 23, 85510 Le Boupère, France, or the snail feed manufactured and distributed by UCAAB, rue de l'Église, BP 19, 02400 Château-Thierry Cedex, France, are examples of suitable products available on the market. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of these products. 2) The disposable transparent polystyrene mouse boxes referenced E1DBBAC001 distributed by Charles River Laboratories France, BP 0109, 69592 L'Arbresle Cedex, France, are examples of suitable products available on the market. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of these products. SIST EN ISO 15952:2012

ISO 15952:2006(E) 6 © ISO 2006 – All rights reserved 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 10381-6. 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. 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 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 Final 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 provoked effect is between 10 % and 90 %. For the definitive test, three replicates are carried out per concentration. SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved 79.1.2 Preparation of the test mixtures 9.1.2.1 General The test mixture (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 (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 Annex A of ISO 11269-2). 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 Annex A of ISO 11269-2), 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 (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. Pour the thus 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 Annex A of ISO 11269-2), 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. SIST EN ISO 15952:2012

ISO 15952:2006(E) 8 © ISO 2006 – All rights reserved 9.1.2.5 Solid matrixes 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 (6.3.2) or natural soil (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 to Annex A of ISO 11269-2), 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 solid 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 products. Measure the pH for each test concentration according to ISO 10390. Continue the test as specified in 9.2. 9.2 Distribution of the test mixture In preparation for the test, add sufficient test mixture (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 (5). Observe them regularly and note any anomaly that could interfere with the conducting of the test. SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved 99.5.2 Three times a week 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 (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). NOTE 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 Every 7 days If effects need to be assessed 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 days). Every 7 days: ⎯ 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 (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 [11] or [41]), or any noticeable modifications in behaviour (e.g. lethargy on the test mixture, lack of feeding), observed on the snails. SIST EN ISO 15952:2012

ISO 15952:2006(E) 10 © ISO 2006 – All rights reserved At the end of the test, measure the pH of a control container and of a container 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. Optional: at the end of the test (28 days), after final weighing and measurement of the shell diameter, let the snails of each container fasted during 48 h in moist boxes without test substrate (until they no longer excrete), then deep-freeze them in view of possibly analysing the concentration of contaminants present in their tissues or organs. 10 Reference substance A control test is regularly conducted with a reference substance to check the occurrence of changes in the sensitivity of the test organisms. NOTE Experience has shown that cadmium chloride is a suitable reference substance. On a regular basis, determine the values of EC50,m (28 days) and EC50,d (28 days) of the cadmium chloride by applying the protocol described in this International Standard. WARNING — Appropriate precautions should be taken when dealing with cadmium chloride which is a harmful substance. The EC50,m (28 days) of the CdCl2 shall be between 350 mg and 650 mg of Cd per kg of dry test substrate. The EC50,d (28 days) of the CdCl2 shall be between 500 mg and 800 mg of Cd per kg of dry test substrate. Mention the obtained values and their obtaining dates in the test report. Four laboratories participated in 2000 in an interlaboratory test concerning cadmium chloride. The results are given in Table 2. Table 2 — Results of the interlaboratory test with Helix aspersa aspersa (substrate contamination; semi-static method) Substance EC50,m (28 days) mg Cd/kg EC50,d (28 days) mg Cd/kg
Mean Range Mean Range CdCl2 500 398 to 622 600 507 to 781 11 Calculations and expression of results 11.1 Calculations For each concentration, determine the percentage of mortality, if any, at the time of the final test. At the end of the test (28 days) (optional: week by week), calculate the mean masses and the mean shell diameters, and the associated standard deviations, of the snails for each container and for each concentration. An example of a data table is given in Annex D. For each container, calculate the biomass growth coefficient (designated kGC,m) and the shell diameter growth coefficient (designated kGC,d) according respectively to Equations (1) and (2). ()0GC,0100tntmtmmkm−=× (1) SIST EN ISO 15952:2012

ISO 15952:2006(E) © ISO 2006 – All rights reserved 11where kGC,m is the biomass growth coefficient; tnm is the mean mass of the snails per replicate at time tn, in grams (g); 0tm is the mean mass of the snails per replicate at time t0, in grams (g). ()0GC,0100tntdtddkd−=× (2) where kGC,d is the shell diameter growth coefficient; dtn is the mean shell diameter of the snails per replicate at time tn, in millimetres (mm); dt0 is the mean shell diameter of the snails per replicate at time t0, in millimetres (mm). For each concentration, calculate the mean percentage of biomass growth inhibition (designated I,mp) and of shell diameter growth inhibition (designated I,dp) according respectively to Equations (3) and (4). ()()()0000I,000''100tnttntmtntmmmmpmm−−−=×− (3) where I,mp is the mean percentage of biomass growth inhibition; 0tnm is the mean mass of the snails at time tn in the control, in grams (g); 00tm is the mean mass of the snails at time t0 in the control, in grams (g); 'tnm is the mean mass of the snail per concentration at time tn, in grams (g); 0'tm is the mean mass of the snail per concentration at time t0, in grams (g). ()()()0000I,000''100tnttntdtntddddpdd−−−=×− (4) where I,dp is the mean percentage of shell diameter growth inhibition; 0tnd is the mean shell diameter at time tn in the control, in millimetres (mm); 00td is the mean shell diameter at time t0 in the control, in millimetres (mm); 'tnd is the mean shell diameter per concentration at time tn, in millimetres (mm); 0'td is the mean shell diameter per concentration at time t0, in millimetres (mm). SIST EN ISO 15952:2012

ISO 15952:2006(E) 12 © ISO 2006 – All rights reserved 11.2 Expression of results For each concentration and at various times of measurement, present the percentage of mortality for each replicate, and for the parameters masses and diameter of shell: ⎯ the calculated mean values and the standard deviation per replicate; ⎯ the growth coefficient per replicate (see examples in E.1 and E.2); ⎯ the mean percentage of growth inhibition (see examples in E.3 and E.4); ⎯ a graphic presentation of the results of the test, giving a clear image of the dose-response relationship for the effects on growth. It is possible (optional) to determine the LOE
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