1、 ISO 2012 Soil quality Effects of pollutants on earthworms Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei Qualit du sol Effets des polluants vis-vis des vers de terre Partie 2: Dtermination des effets sur la reproduction de Eisenia fetida/ Eisenia andrei INTERNATIO
2、NAL STANDARD ISO 11268-2 Second edition 2012-11-01 Reference number ISO 11268-2:2012(E) ISO 11268-2:2012(E) ii ISO 2012 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2012 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or
3、by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-
4、mail copyrightiso.org Web www.iso.org Published in Switzerland ISO 11268-2:2012(E) ISO 2012 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 T erms and definitions . 2 4 Principle . 3 5 Reagents and material . 4 6 Apparatus 5 7 Procedure 6 7.1 E
5、xperimental design . 6 7.2 Preparation of test mixture . 7 7.3 Addition of the earthworms 8 7.4 Test conditions and measurements . 8 7.5 Reference substance 9 8 Calculation and expression of results . 9 8.1 Calculation 9 8.2 Expression of results . 9 9 Validity of the test 9 10 Statistical analysis
6、9 10.1 General . 9 10.2 Single-concentration tests 10 10.3 Multi-concentration tests 10 11 Test report . 11 Annex A (informative) Determination of the chronic toxicity of chemicals on earthworms under tropical conditions 13 Annex B (informative) Culturing of Eisenia fetida and Eisenia andrei .15 Ann
7、ex C (informative) Determination of water holding capacity of artificial soil 16 Annex D (informative) Techniques for counting juvenile worms hatched from cocoons.17 Annex E (informative) Performance of the method .18 Bibliography .20 ISO 11268-2:2012(E) Foreword ISO (the International Organization
8、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 t
9、he 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. Interna
10、tional 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. Publicatio
11、n 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
12、. 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 1 1268 consists of the following parts, under the general title So
13、il 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 effects in field situations iv ISO 2012 All rights reser
14、ved ISO 11268-2:2012(E) Introduction Ecotoxicological 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 ISO 17616 35 ). ISO 15799 includes a list and short characterization of re
15、commended 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 potentially reaching the groundwater by the water path (retention function of
16、 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 chronic test for reproduction are available. This part of ISO 1 1268 des
17、cribes 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 testing chemicals added to standard soils (e.g. artificial soil) for t
18、heir 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 representatives of soil fauna and earthworms in particular. Background infor
19、mation 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 other species have not been proven to be more sensitive in general, an
20、d 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 European Union 11 . ISO 2012 All rights reserved v Soil quality Effect
21、s of pollutants on earthworms Part 2: Determination of effects on reproduction of Eisenia fetida/ Eisenia andrei WARNING Contaminated soils may contain unknown mixtures of toxic, mutagenic, or otherwise harmful chemicals or infectious microorganisms. Occupational health risks may arise from dust or
22、evaporated chemicals during handling and incubation. Precautions should be taken to avoid skin contact. 1 Scope This part of ISO 11268 specifies one of the methods for evaluating the habitat function of soils and determining the effects of soil contaminants and chemicals on the reproduction of Eisen
23、ia 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 remediation, agricultural or other sites concerned, and waste materials. Effects of substances are assess
24、ed 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 the study, the control and dilution substrate (dilution series of contaminated soil) should be either an
25、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 under temperate as well as under tropical conditions. The method is not applicable to volatile substances
26、, 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 account the persistence of the substance during the test. 2 Normative references The following referen
27、ced 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,
28、 handling and storage of soil under aerobic conditions for the assessment of microbiological processes, biomass and diversity 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
29、 11260, Soil quality Determination of effective cation exchange capacity and base saturation level using barium chloride solution ISO 11268-1, Soil quality Effects of pollutants on earthworms Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei ISO 11277, Soil quality Determinati
30、on of particle size distribution in mineral soil material Method by sieving and sedimentation ISO 11465, Soil quality Determination of dry matter and water content on a mass basis Gravimetric method INTERNATIONAL STANDARD ISO 11268-2:2012(E) ISO 2012 All rights reserved 1 ISO 11268-2:2012(E) 3 T erm
31、s and definitio ns For the purposes of this document, the following terms and definitions apply. 3.1 contaminant substance or agent present in the soil as a result of human activity ISO 15176:2002 3.2 growth increase in biomass (i.e. the fresh mass of organisms) NOTE It is expressed as a percentage
32、of the fresh mass of organisms at the start of the test. 3.3 reproduction mean number of offspring per test vessel after eight weeks incubation under the specified test conditions 3.4 ER x (effective rate) or EC x(effective concentration) x % effect rate or concentration of the test sample or test s
33、ubstance at which reproduction is reduced by x % compared to the control 3.5 limit test single concentration test consisting of at least four replicates each, the test sample without any dilution or the highest concentration of test substance mixed into the control soil and the control 3.6 lowest ob
34、served 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 observed NOTE The LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All
35、 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 been selected. 3.7 NOER (no observed effective rate) or NOEC (no observed effect concentr
36、ation) 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 reduced reproduction or mass alteration (error probability: p 0,05) NOTE The NOEC is e
37、xpressed as a percentage of test-soil dry mass per test-mixture dry mass. 3.8 reference soil uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar properties (nutrient concentrations, pH, organic carbon content and texture) to the test soil 3.9 standa
38、rd soil field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are within a known range EXAMPLE Euro-Soils 11 , artificial soil 27 , LUFA standard soil 23 . NOTE The properties of standard soils can differ from those of the test soil. 2 ISO 2012 All
39、rights reserved ISO 11268-2:2012(E) 3.10 control soil reference or standard soil used as a control and as a medium for preparing dilution series with test samples or a reference substance, which fulfils the validity criteria NOTE In the case of natural soil, it is advisable to demonstrate its suitab
40、ility for a test and for achieving the test validity criteria before using the soil in a definitive test. 3.11 test mixture mixture of contaminated soil or the test substance with a control soil (3.10) NOTE Test mixtures are given in percent of contaminated soil based on soil dry mass. 3.12 test mix
41、ture ratio ratio of test soil to control soil in a test mixture NOTE Different ratios may be applied in a dilution series to establish a dose-response relationship. 4 Principle The effects on reproduction of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed to the test soil are co
42、mpared to those observed for samples exposed to a control soil. If appropriate, effects based on exposure to a dilution range of contaminated soil or range of concentrations of a test substance are determined. In addition, observations on growth and survival of adult earthworms are recorded. Test mi
43、xtures are prepared at the start of the test and are not renewed within the test period. After four weeks, adult worms are removed from the test containers and effects on mortality and biomass are measured by counting and weighing. The effect on reproduction as the definitive end point is measured b
44、y counting the number of offspring hatched from the cocoons after an additional period of four weeks. The results obtained from the tests are compared with a control soil or, if appropriate, are used to determine the dilutions or concentrations which cause no effects on biomass, mortality and reprod
45、uction (NOER/NOEC) and the dilution (concentration) resulting in x % reduction of juveniles hatched from cocoons compared to the control (ER x /EC x , 56 d), respectively. If testing a dilution or concentration series, all test dilutions/concentrations above the LOER/LOEC shall have a harmful effect
46、 equal to, or greater than, that observed at the LOER/LOEC. Where there is no prior knowledge of the dilution/concentration of the test soil/test substance likely to have an effect, then it is useful to conduct the test in two steps: a preliminary test carried out, in accordance with ISO 11268-1, to
47、 give an indication of the effect dilution/concentration and of the dilution/concentration giving no mortality (NOER/NOEC). Dilutions/concentrations to be used in the definitive test can then be selected. the definitive test to determine sublethal effects of (dilutions of) contaminated soil or the c
48、oncentration of a chemical which, when evenly mixed into the standard soil, causes no significant effects on numbers of offspring hatched from cocoons compared with the control (NOER/NOEC), and the lowest concentration causing effects (LOER/LOEC). NOTE The use of a reference soil is an essential req
49、uirement to demonstrate the present status of the test population, and to avoid misinterpretation of results. ISO 2012 All rights reserved 3 ISO 11268-2:2012(E) 5 Reagents and material 5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia andrei 15, 19, 20,which are between two months and one year old, with a clitellum, and a wet mass between 300 mg and 600 mg (E. fetida) and between 250 mg and 600 mg (E. andrei). Select worms used for
