1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 20963:2011Soil quality Effects ofpollutants on insect larvae(Oxythyrea funesta) Determination of acute toxicity(ISO 20963:2005)BS EN ISO 20963:2011 BRITISH STANDARDNati
2、onal forewordThis British Standard is the UK implementation of EN ISO20963:2011. It is identical to ISO 20963:2005.The UK participation in its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can beobtained on request to its sec
3、retary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2011ISBN 978 0 580 73158 7ICS 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was publis
4、hed under the authority of theStandards Policy and Strategy Committee on 31 July 2011.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 20963 June 2011 ICS 13.080.30 English Version Soil quality - Effects of pollutants on insect larvae (Oxy
5、thyrea funesta) - Determination of acute toxicity (ISO 20963:2005) Qualit du sol - Effets des polluants vis-vis des larves dinsectes (Oxythyrea funesta) - Dtermination de la toxicit aigu (ISO 20963:2005) Bodenbeschaffenheit - Auswirkungen von Schadstoffen auf Insektenlarven (Oxythyrea funesta) - Bes
6、timmung der akuten Toxizitt (ISO 20963:2005) 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
7、. 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 t
8、ranslation 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, Fi
9、nland, 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 COMIT EUROPEN DE NORMALISATION EUROPISCHES
10、 KOMITEE FR 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 20963:2011: EBS EN ISO 20963:2011EN ISO 20963:2011 (E) 3 Foreword The text of ISO 20963:2005 has been
11、 prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 20963:2011 by Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN. This European Standard shall be g
12、iven 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
13、 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: Aust
14、ria, 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.
15、Endorsement notice The text of ISO 20963:2005 has been approved by CEN as a EN ISO 20963:2011 without any modification. BS EN ISO 20963:2011ISO 20963:2005(E) ISO 2005 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Pri
16、nciple . 2 5 Test environment 3 6 Reagents 3 7 Apparatus. 4 8 Procedure. 4 8.1 Test design 4 8.2 Preparation of test mixture 5 8.3 Preparation of control container . 6 8.4 Food addition 6 8.5 Introduction of the biological material 6 8.6 Test conditions and measurements 6 8.7 Reference substance 6 9
17、 Expression of results 7 9.1 Calculation. 7 9.2 Expression of results 8 10 Validity of the test . 8 11 Test report 8 Annex A (informative) Example of breeding technique for Oxythyrea funesta . 9 Annex B (informative) Results of the collaborative trial carried out in France 11 Bibliography . 12 BS EN
18、 ISO 20963:2011ISO 20963:2005(E) iv ISO 2005 All rights reservedForeword 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 co
19、mmittees. 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 th
20、e 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 Internati
21、onal 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
22、the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 20963 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. BS EN ISO 20963:2011ISO 20963:2005(E) ISO 2005 All rights reserved vIntroducti
23、on This International Standard describes a method for the determination of the acute toxicity of contaminated soils and chemicals to the larvae of Oxythyrea funesta, a phytophagous coleopteran (Scarabaeidae, Cetoniinae) with wide geographic distribution (Europe, North Africa and the Middle East). Ox
24、ythyrea funesta has many characteristics which make it suitable for soil quality monitoring or testing effects of chemicals: ecological relevance: this type of organism contributes in many ways to soil structure by stimulating soil aeration and drainage; the first stages of development, i.e. incubat
25、ion of eggs, larval cycle and pupation, are underground; the larvae of Oxythyrea funesta are tolerant to modifications of the test substrate granulometry; this species can be bred under controlled conditions. BS EN ISO 20963:2011BS EN ISO 20963:2011INTERNATIONAL STANDARD ISO 20963:2005(E) ISO 2005 A
26、ll rights reserved 1Soil quality Effects of pollutants on insect larvae (Oxythyrea funesta) Determination of acute toxicity 1 Scope This International Standard describes a method for the determination of the effects of contaminated soils and substances on the survival of the larvae of Oxythyrea fune
27、sta. The larvae are exposed to the pollutants by cuticular and alimentary uptake. For contaminated soils, the effects on the survival are determined in the test soil and in a control soil. Depending on the objectives of the study, the control and dilution substrates (dilution series of contaminated
28、soil) are either uncontaminated soil comparable to the soil sample to be tested or artificial soil substrate. Effects of substances are assessed using a defined artificial soil substrate. This International Standard is not applicable to volatile substances, i.e. substances for which Henrys constant
29、or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,001 33 Pa at 25 C. NOTE This method does not take into account the possible degradation of the substances or pollutants during the test. 2 Normative references The following referenced documents are
30、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 stor
31、age of soil for the assessment of aerobic microbial processes in the laboratory ISO 10390, Soil quality Determination of pH ISO 11268-1, Soil quality Effects of pollutants on earthworms (Eisenia fetida) Part 1: Determination of acute toxicity using artificial soil substrate ISO 11269-2:1), Soil qual
32、ity Determination of the effects of pollutants on soil flora Part 2: Effects of chemicals on the emergence and growth of higher plants 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 1) To be published. (Revision of ISO 11269-2:1995) BS EN ISO 20
33、963:2011ISO 20963:2005(E) 2 ISO 2005 All rights reserved3.1 lethal concentration 50 LC 50 median lethal concentration of test substance or percent dilution of contaminated soil, which kills 50 % of the test organism within the test period 3.2 lethal concentration x LCx concentration of the test subs
34、tance or percent dilution of contaminated soil which kills x % of the test organism within the test period NOTE x is the percentage (10, 20, 25) of this effect. 3.3 Lowest Observed Effect Concentration LOEC lowest tested concentration of test substance or contaminated soil at which a statistically s
35、ignificant effect is observed compared with the control NOTE All test concentrations above the LOEC have a harmful effect equal or greater than those observed at the LOEC. 3.4 No Observed Effect Concentration NOEC highest tested concentration of test substance or contaminated soil at which no statis
36、tically significant effect is observed compared with the control NOTE The NOEC is the test concentration immediately below the LOEC. 3.5 test mixture mixture of the test substance with the test substrate, mixture of contaminated soil with the test substrate or mixture of contaminated soil with an un
37、contaminated soil comparable to the soil sample to be tested 3.6 diapause interruption of metabolism during egg, larva, pupa or imago development 4 Principle Larvae of Cetoniinae (species Oxythyrea funesta) are exposed to a dilution range of contaminated soil or to a range of concentrations of test
38、substance. The mortality of larvae is determined after 10 days. Test mixtures are prepared at the start of the test and are not renewed within the test period. The results obtained from the test are compared with a control and are used to determine the concentration which causes mortality of 50 % of
39、 the larvae (LC 5010days). The test is conducted in two steps: a preliminary test to determine appropriate dilution/concentration range in the final test; the definitive test to determine the dilutions/concentrations causing between 10 % and 90 % mortality, which yields the test result. BS EN ISO 20
40、963:2011ISO 20963:2005(E) ISO 2005 All rights reserved 3It may also be possible to determine the effects of contaminated soils or substances on the growth of larvae (optional). The increase in mass within the test period allows this criterion to be considered as complementary to mortality, in order
41、to assess the effects of contaminated soils or substances. 5 Test environment Tests shall be performed at a temperature of (26 1) C in complete darkness. 6 Reagents 6.1 Biological material The species used in the test is Oxythyrea funesta (Scarabaeidae, Cetoniinae). Third-instar larvae with a fresh
42、mass within the range 100 mg to 200 mg are required to perform the test. The larvae shall be healthy, without any bites or other visible injuries. NOTE Depending on the breeding conditions, described in Annex A, larvae approximately two weeks old are suitable for the test. Larvae of similar size sha
43、ll be selected. The difference in mass between the smallest and the largest larva within a single test container shall not exceed 50 mg. Eliminate the particles of breeding substrate stuck to the integument using, for example, a soft brush before weighing the larvae. It is also possible to leave the
44、 animals to move along on slightly moist paper in order to eliminate the breeding substrate stuck to the integument. Synchronisation of breeding is necessary. An example of breeding technique for Oxythyrea funesta is given in Annex A. 6.2 Test substrate The mass of substrate used per glass container
45、 (7.1) shall be equivalent to 300 g (dry mass). The substrate, called artificial soil, shall have the following composition (in accordance with ISO 11268-1): sphagnum peat, air-dried, finely ground and with no visible plant remains: 10 % (expressed on a dry mass basis); kaolinite clay containing not
46、 less than 30 % kaolinite: 20 % (expressed on a dry mass basis); industrial quartz sand (dominant fine sand with more than 50 % particle size between 0,05 mm and 0,20 mm): 70 % (expressed on a dry mass basis). Add pulverised calcium carbonate (CaCO3), of recognised analytical grade, as necessary to
47、bring the pH of the wetted substrate to 6,0 0,5 (commonly between 0,5 % and 1 % of the mass of the dry ingredients). Prepare the artificial soil by mixing the dry constituents listed above thoroughly in a large-scale laboratory mixer. The amount of calcium carbonate required can vary, depending on p
48、roperties of the individual batch of sphagnum peat, and should be determined by weighing subsamples immediately before the test. Store the mixed artificial soil at room temperature. To determine pH and the maximum water-holding capacity, pre-moisten the dry artificial soil at least two days before s
49、tarting the test by adding deionized water to obtain half of the required final water content of 50 % of the maximum water-holding capacity. BS EN ISO 20963:2011ISO 20963:2005(E) 4 ISO 2005 All rights reservedDetermine the water-holding capacity in accordance with ISO 11269-2:, Annex A, and pH in accordance with ISO 10390. If the measured pH is not within the required range, add a sufficient amount of CaCO3or prepare a new batch of artificial soil. 6.3 Larvae food, i.e. dried and finely ground cow-du
