SIST EN ISO 15952:2012
(Main)Soil quality - Effects of pollutants on juvenile land snails (Helicidae) - Determination of the effects on growth by soil contamination (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)
ISO 15952:2005 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; and soils (contaminated or of unknown quality) or waste materials. A static method is also described.
ISO 15952:2005 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 possible changes 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.
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Jungtiere von Landschnecken - Bestimmung der Wirkungen auf das Wachstum durch Bodenverunreinigung (ISO 15952:2006)
Diese Internationale Norm legt ein semistatisches (halbkontinuierliches) Verfahren zur Bestimmung der Wirkungen von Kontaminanten auf das Wachstum und Überleben von jungen Schnecken, üblicherweise Helix aspersa aspersa Müller (Cornu aspersum), fest. Die Tiere werden unter Verwendung eines Prüfsubstrats (künstlicher oder natürlicher Boden in Abhängigkeit von der Zielsetzung der Studie), dem festgelegte Mengen von Folgendem zugesetzt werden, über den Haut und Verdauungspfad exponiert:
Substanzen oder Zubereitungen;
Böden (verunreinigte (kontaminierte) oder von unbekannter Beschaffenheit) oder Abfälle.
Zusätzlich zu dem semistatischen Verfahren darf ein statisches Verfahren angewendet werden (optional). Dieses Verfahren ist in Anhang A beschrieben.
Diese Internationale Norm ist nicht auf flüchtige Substanzen anwendbar, d. h. Substanzen, bei denen die Henry Konstante H oder der Luft/Wasser Verteilungskoeffizient größer als 1 ist, oder bei denen der Dampfdruck bei 25 °C 0,013 3 Pa überschreitet.
Diese Prüfung berücksichtigt mögliche Veränderungen der Prüfsubstanz, der Zubereitung, des Bodens oder Abfalls, weil im Verlauf der 28 tägigen Prüfdauer die Prüfmischung alle 7 Tage hergestellt und ausgewechselt wird.
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)
L'ISO 15952:2005 s'applique à une méthode semi-statique pour la détermination des effets de contaminants sur la croissance et la survie d'escargots juvéniles, généralement Helix aspersa aspersa Müller. Les animaux sont exposés par les voies cutanée et digestive à un substrat d'essai (sol artificiel ou naturel selon l'objectif de l'étude) auquel sont ajoutées des quantités définies de substances ou de préparations, de sols (contaminés ou de qualité inconnue) ou de déchets. Une méthode statique est aussi décrite.
L'ISO 15952:2005 ne s'applique pas aux substances volatiles, c'est-à-dire aux substances dont la constante de Henry, H, ou le coefficient de partage air/eau est supérieur à 1, ou pour lesquelles la pression de vapeur est supérieure à 0,013 3 Pa à 25 °C.
Cet essai prend en considération le changement éventuel de la substance d'essai, de la préparation, du sol ou des déchets, étant donné que le mélange d'essai est préparé et renouvelé tous les 7 jours pendant la période d'essai de 28 jours.
Kakovost tal - Vpliv onesnaževal na juvenilne stadije kopenskih polžev (Helicidae) - Ugotavljanje vplivov na rast zaradi onesnaženja tal (ISO 15952:2006)
ISO 15952:2005 določa semistatično metodo ugotavljanja vpliva onesnaževal na rast in preživetje polžev, po navadi Helix aspersa aspersa Müller, v juvenilnih stadijih. Živali so prek kože in prebavnega trakta izpostavljene preskusnemu substratu (umetnim ali naravnim tlem, glede na namen raziskave), ki se mu dodajo opredeljene količine naslednjega: snovi ali pripravki ter tla (onesnažena ali neznane količine) ali odpadni materiali. Opisana je tudi statična metoda.
ISO 15952:2005 ne velja za hlapne snovi, tj. snovi, pri katerih je Henryjeva konstanta, H, ali porazdelitveni koeficient zrak/voda večji od 1 ali pri katerih parni tlak presega 0,013 3 Pa pri 25 °C.
Ta preskus upošteva morebitne spremembe v preskusni snovi, pripravku, tleh ali odpadnem materialu, ker se preskusna zmes pripravlja in obnavlja vsakih sedem dni med 28-dnevnim preskusnim obdobjem.
General Information
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Standards Content (Sample)
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
SIST EN ISO 15952:2012
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
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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
EN ISO 15952:2011 (E) 2 Contents Page Foreword .3 SIST EN ISO 15952:2012
EN ISO 15952:2011 (E) 3 Foreword The text of ISO 15952:2006 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 15952:2011 by Technical Committee CEN/TC 345 “Characterization of soils” 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 December 2011, and conflicting national standards shall be withdrawn at the latest by December 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 15952:2006 has been approved by CEN as a EN ISO 15952:2011 without any modification.
SIST EN ISO 15952:2012
SIST 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
SIST EN ISO 15952:2012
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SIST EN ISO 15952:2012
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
SIST EN ISO 15952:2012
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]).
SIST EN ISO 15952:2012
SIST EN ISO 15952:2012
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 s
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.[Not translated]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:FprEN ISO 15952kSIST FprEN ISO 15952:2011en,fr,de01-april-2011kSIST FprEN ISO 15952:2011SLOVENSKI
STANDARD
kSIST FprEN ISO 15952:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
FINAL DRAFT
FprEN ISO 15952
December 2010 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 draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical Committee CEN/TC 345.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
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.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and shall not be referred to as a European Standard.
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B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. FprEN ISO 15952:2010: EkSIST FprEN ISO 15952:2011
FprEN ISO 15952:2010 (E) 2 Contents Page Foreword .3 kSIST FprEN ISO 15952:2011
FprEN ISO 15952:2010 (E) 3 Foreword The text of ISO 15952:2006 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as FprEN ISO 15952:2010 by Technical Committee CEN/TC 345 “Characterization of soils”, the secretariat of which is held by NEN. This document is currently submitted to the Unique Acceptance Procedure. Endorsement notice The text of ISO 15952:2006 has been approved by CEN as an FprEN ISO 15952:2010 without any modification.
kSIST FprEN ISO 15952:2011
kSIST FprEN ISO 15952:2011
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
kSIST FprEN ISO 15952:2011
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kSIST FprEN ISO 15952:2011
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
kSIST FprEN ISO 15952:2011
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. kSIST FprEN ISO 15952:2011
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]).
kSIST FprEN ISO 15952:2011
kSIST FprEN ISO 15952:2011
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 kSIST FprEN ISO 15952:2011
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). kSIST FprEN ISO 15952:2011
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. kSIST FprEN ISO 15952:2011
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. kSIST FprEN ISO 15952:2011
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. kSIST FprEN ISO 15952:2011
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 t
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