1、BSI Standards PublicationBS EN ISO 11268-2:2015Soil quality Effects ofpollutants on earthwormsPart 2: Determination of effects onreproduction of Eisenia fetida/Eisenia andrei(ISO 11268-2:2012)BS EN ISO 11268-2:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN
2、 ISO11268-2:2015. It is identical to ISO 11268-2:2012. It supersedes BS7755-4.2.2:1998 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can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 secretary.Th
3、is publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 89037 6ICS 13.080.30Compliance with a British Standard cannot confer im
4、munity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 August 2015.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11268-2 August 2015 ICS 13.080.30 English
5、 Version Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012) Qualit du sol - Effets des polluants vis-vis des vers de terre - Partie 2: Dtermination des effets sur la reproduction de Eisenia fetida/
6、Eisenia andrei (ISO 11268-2:2012) Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwrmer - Teil 2: Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia andrei (ISO 11268-2:2012) This European Standard was approved by CEN on 6 August 2015. CEN members are bound to
7、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-CEN
8、ELEC 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 th
9、e same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxemb
10、ourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussel
11、s 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11268-2:2015 EBS EN ISO 11268-2:2015EN ISO 11268-2:2015 (E) 3 European foreword The text of ISO 11268-2:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quali
12、ty” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 11268-2:2015 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 pub
13、lication of an identical text or by endorsement, at the latest by February 2016, and conflicting national standards shall be withdrawn at the latest by February 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENE
14、LEC 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 Republ
15、ic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endors
16、ement notice The text of ISO 11268-2:2012 has been approved by CEN as EN ISO 11268-2:2015 without any modification. BS EN ISO 11268-2:2015ISO 11268-2:2012(E) ISO 2012 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope 12 Normative references . 13 Terms and definitions . 24 Principl
17、e . 35 Reagents and material . 46 Apparatus 57 Procedure 67.1 Experimental design . 67.2 Preparation of test mixture . 77.3 Addition of the earthworms 87.4 Test conditions and measurements . 87.5 Reference substance 98 Calculation and expression of results . 98.1 Calculation 98.2 Expression of resul
18、ts . 99 Validity of the test 910 Statistical analysis 910.1 General . 910.2 Single-concentration tests 1010.3 Multi-concentration tests 1011 Test report . 11Annex A (informative) Determination of the chronic toxicity of chemicals on earthworms under tropical conditions 13Annex B (informative) Cultur
19、ing of Eisenia fetida and Eisenia andrei .15Annex C (informative) Determination of water holding capacity of artificial soil 16Annex D (informative) Techniques for counting juvenile worms hatched from cocoons.17Annex E (informative) Performance of the method .18Bibliography .20BS EN ISO 11268-2:2015
20、ISO 11268-2:2012(E)ForewordISO (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 sub
21、ject 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 (I
22、EC) 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 committe
23、es 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 he
24、ld responsible for identifying any or all such patent rights.ISO 11268-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.This second edition cancels and replaces the first edition (ISO 11268-2:1998), which has been technically revised.ISO 11268 con
25、sists of the following parts, under the general title Soil quality Effects of pollutants on earthworms: Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei Part 3: Guidance on the determination of
26、effects in field situationsiv ISO 2012 All rights reservedBS EN ISO 11268-2:2015ISO 11268-2:2012(E)IntroductionEcotoxicological test systems are applied to obtain information about the effects of contaminants in soil and are proposed to complement conventional chemical analysis (see ISO 15799 34and
27、ISO 17616 35). ISO 15799 includes a list and short characterization of recommended and standardized test systems and ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with soil eluate are applied to obtain information about the fraction of contaminants pote
28、ntially reaching the groundwater by the water path (retention function of soils), whereas terrestrial test systems are used to assess the habitat function of soils. As standardized test systems using earthworms as indicator organisms for the habitat function of soil, an acute test for survival and a
29、 chronic test for reproduction are available.This part of ISO 11268 describes a method that is based on the determination of sublethal effects of contaminated soils on adult earthworms of the species Eisenia fetida (Savigny 1826) and Eisenia andrei (Andr 1963). Optionally, the method can be used for
30、 testing chemicals added to standard soils (e.g. artificial soil) for their sublethal hazard potential to earthworms. Finally, information is provided on how to use this method for testing chemicals under tropical conditions (see Annex A).Eisenia fetida and Eisenia andrei are considered to be repres
31、entatives of soil fauna and earthworms in particular. Background information on the ecology of earthworms and their use in ecotoxicological testing is available. Other species, e.g. Aporrectodea caliginosa, Lumbricus rubellus and Lumbricus terrestris, have also been used as test organisms. These or
32、other species have not been proven to be more sensitive in general, and the database and experience in testing soils is small 1617.This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the Organization for Economic Cooperation and Development 2728and by the Eu
33、ropean Union 11. ISO 2012 All rights reserved vBS EN ISO 11268-2:2015BS EN ISO 11268-2:2015Soil quality Effects of pollutants on earthworms Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andreiWARNING Contaminated soils may contain unknown mixtures of toxic, mutagenic, or
34、 otherwise harmful chemicals or infectious microorganisms. Occupational health risks may arise from dust or evaporated chemicals during handling and incubation. Precautions should be taken to avoid skin contact.1 ScopeThis part of ISO 11268 specifies one of the methods for evaluating the habitat fun
35、ction of soils and determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after
36、 remediation, agricultural or other sites concerned, and waste materials.Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For contaminated soils, the effects are determined in the test soil and in a control soil. According to the objective of
37、the study, the control and dilution substrate (dilution series of contaminated soil) should be either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard soil (e.g. artificial soil).Information is provided on how to use this method for testing chemicals u
38、nder temperate as well as under tropical conditions.The method is not applicable to volatile substances, i.e. substances for which H (Henrys constant) or the air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 C.This method does not take into
39、account the persistence of the substance during the test.2 Normative referencesThe 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 (inclu
40、ding any amendments) applies.ISO 10381-6, Soil quality Sampling Part 6: Guidance on the collection, handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratoryISO 10390, Soil quality Determination of pHISO 10694, Soil
41、 quality Determination of organic and total carbon after dry combustion (elementary analysis)ISO 11260, Soil quality Determination of effective cation exchange capacity and base saturation level using barium chloride solutionISO 11268-1, Soil quality Effects of pollutants on earthworms Part 1: Deter
42、mination of acute toxicity to Eisenia fetida/Eisenia andreiISO 11277, Soil quality Determination of particle size distribution in mineral soil material Method by sieving and sedimentationISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric methodINTERNATI
43、ONAL STANDARD ISO 11268-2:2012(E) ISO 2012 All rights reserved 1BS EN ISO 11268-2:2015ISO 11268-2:2012(E)3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1contaminantsubstance or agent present in the soil as a result of human activityISO 15176:200
44、23.2growthincrease in biomass (i.e. the fresh mass of organisms)NOTE It is expressed as a percentage of the fresh mass of organisms at the start of the test.3.3reproductionmean number of offspring per test vessel after eight weeks incubation under the specified test conditions3.4ERx (effective rate)
45、 or ECx(effective concentration)x % effect rate or concentration of the test sample or test substance at which reproduction is reduced by x % compared to the control3.5limit testsingle concentration test consisting of at least four replicates each, the test sample without any dilution or the highest
46、 concentration of test substance mixed into the control soil and the control3.6lowest observed effect rate (LOER) or effect concentration (LOEC)lowest tested percentage of a test sample in a control soil or concentration of a substance at which a statistically significant effect is observedNOTE The
47、LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test mixtures above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If this condition cannot be satisfied, an explanation should be given for how the LOEC and NOEC (3.7) have bee
48、n selected.3.7NOER (no observed effective rate) or NOEC (no observed effect concentration)test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test substance which, when compared to the control, has no statistically significant lethal or other effect such as red
49、uced reproduction or mass alteration (error probability: p 2, where Kowis the octanol/water coefficient) or ionizing substances into account, 5 % of peat have proven to be sufficient for maintaining the desired structure of the artificial soil.NOTE 2 It has been demonstrated that Eisenia fetida can comply with the validity criteria, even as regards reproduction, when tested in field soils with lower organic carbon content (e.g. 2,7 %) 18, and experience shows that this can be achieved in artificial soil with 5
