1、 ISO 2012 Soil quality Effects of pollutants on earthworms Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei Qualit du sol Effets des polluants vis-vis des vers de terre Partie 1: Dtermination de la toxicit aigu vis-vis de Eisenia fetida/ Eisenia andrei INTERNATIONAL STANDARD
2、ISO 11268-1 Second edition 2012-11-01 Reference number ISO 11268-1:2012(E) ISO 11268-1:2012(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 by any means,
3、 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-mail copyrigh
4、tiso.org Web www.iso.org Published in Switzerland ISO 11268-1:2012(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 . 3 6 Apparatus 5 7 Procedure 5 7.1 Experimental d
5、esign . 5 7.2 Preparation of test mixture . 6 7.3 Addition of the earthworms 8 7.4 Test conditions and measurements . 8 7.5 Reference substance 8 8 Calculation and expression of results . 8 8.1 Calculation 8 8.2 Expression of results . 8 9 Validity of the test 9 10 Statistical analysis 9 10.1 Genera
6、l . 9 10.2 Single-concentration tests 9 10.3 Multi-concentration tests 10 11 Test report . 11 Annex A (informative) Determination of the acute toxicity of chemicals (in particular pesticides) on earthworms under tropical test conditions 12 Annex B (informative) Culturing of Eisenia fetida and Eiseni
7、a andrei .14 Annex C (informative) Determination of water holding capacity of artificial soil 15 Annex D (informative) Background information on the acute effect of boric acid on earthworms .16 Bibliography .17 ISO 11268-1:2012(E) Foreword ISO (the International Organization for Standardization) is
8、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 represent
9、ed 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 dra
10、fted 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 St
11、andard 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 11268-1 was prepar
12、ed by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. This second edition cancels and replaces the first edition (ISO 11268-1:1993), which has been technically revised. ISO 1 1268 consists of the following parts, under the general title Soil quality Effects of po
13、llutants 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 reserved ISO 11268-1:2012(E)
14、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 1579933 and ISO 1761634 ). ISO 15799 includes a list and short characterization of recommended and standardize
15、d 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 soils), whereas terrest
16、rial 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 11268 describes a method that is b
17、ased on the determination of the acute toxicity of contaminated soils to 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 a standard soil (e.g. artificial soil) for their acute toxic potent
18、ial 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 information on the ecology of ear
19、thworms 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, and the database and experienc
20、e in testing soils is small 151623 . This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the Organization for Economic Cooperation and Development 2627and by the European Union 9 . ISO 2012 All rights reserved v Soil quality Effects of pollutants on earthwor
21、ms Part 1: Determination of acute toxicity to 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 evaporated chemicals during handling
22、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 acute toxicity of soil contaminants and chemicals to Eisenia fetida/Eisenia andrei by dermal and alimentary
23、uptake. It 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 assessed using a standard soil, preferably a defined artificial soil su
24、bstrate. For contaminated soils, the effects on survival 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 uncontaminated soil comparable to the soil sample to
25、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, i.e. substances for which H (Henrys constant) or th
26、e 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 possible degradation of the substances or contaminants during the test. 2 Normative references The following referenced documents are indispens
27、able 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 so
28、il 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 11260, Soil quality Determ
29、ination of effective cation exchange capacity and base saturation level using barium chloride solution ISO 11277, Soil quality Determination of particle size distribution in mineral soil material Method by sieving and sedimentation ISO 11465, Soil quality Determination of dry matter and water conten
30、t on a mass basis Gravimetric method INTERNATIONAL STANDARD ISO 11268-1:2012(E) ISO 2012 All rights reserved 1 ISO 11268-1:2012(E) 3 T erms 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 resul
31、t of human activity ISO 15176:2002 3.2 survival percentage of living worms at the end of the test period 3.3 mortality percentage of dead or missing worms at the end of the test period 3.4 LC 50 lethal concentration median lethal percentage of a test sample in a reference or a standard control soil,
32、 or concentration of a substance in the test sample, which kills 50 % of the test animals within the test period NOTE The LC 50is expressed as a percentage of test-soil dry mass per test-mixture dry mass. 3.5 limit test single concentration test consisting of at least four replicates each, the test
33、sample without any dilution or the highest concentration of test substance mixed into the control soil and the control 3.6 lowest 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 statisti
34、cally significant effect is observed NOTE The 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
35、given for how the LOEC and NOEC (3.7) have been selected. 3.7 NOEC no observed effect concentration test soil percentage immediately below the LOEC or, highest tested concentration of a test substance which, when compared to the control, has no statistically significant lethal or other effect such a
36、s mass alteration (error probability: p 0,05) NOTE The NOEC is expressed 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,
37、 organic carbon content and texture) to the test soil 3.9 standard 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 21 , artificial soil 26LUFA Standard Soil 24 . 2 ISO 2012 All rights reserved I
38、SO 11268-1:2012(E) NOTE The properties of standard soils can differ from those of the test soil. 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 cas
39、e of natural soil, it is advisable to demonstrate its suitability 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 percen
40、tage of contaminated soil based on soil dry mass. 3.12 test mixture 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 percent mortality of adult earthworms (species: Eisenia
41、fetida or Eisenia andrei) exposed to the test soil are compared 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 after seven days and 14 days. Test
42、 mixtures are prepared at the start of the test and are not renewed within the test period. The results obtained from the tests are compared with a control and are used to determine the dilutions or concentrations which cause no effects on biomass and survival (NOEC) and the mortality of 50 % of ear
43、thworms (LC 50 , 14 days). The test is conducted in two steps: a preliminary test, which gives an approximate indication of the dilutions (concentrations) responsible for total mortality and for the absence of mortality, which serves to determine the range of concentrations for the definitive test;
44、the definitive test to determine the dilutions (concentrations) causing between 10 % and 90 % mortality, which yields the test result. If the preliminary test shows no mortality, a limit test (see 7.1.3) may be performed as the definitive test. NOTE The use of a reference soil is an essential requir
45、ement to demonstrate the present status of the test population, and to avoid misinterpretation of results. 5 Reagents and material 5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia andrei 12 1719at least three months old, with a clitellum and a wet mass b
46、etween 300 mg and 600 mg (E. fetida) and between 250 mg and 600 mg (E. andrei). Select worms used for the test to form, as far as is practicable, a homogeneous population from the standpoint of size and mass. Worms should preferably be selected from a synchronized culture with a relatively homogeneo
47、us age structure. Before the test, wash them with potable water. NOTE An example of culturing Eisenia fetida/Eisenia andrei is given in Annex B. ISO 2012 All rights reserved 3 ISO 11268-1:2012(E) 5.2 Test sample, which may consist of field-collected soil or control soil amended by the test substance
48、. 5.2.1 Field-collected soils, soil or waste materials The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or manure) under consideration
49、 for possible land disposal. Test samples shall be sieved by 4 mm mesh and thoroughly mixed. If necessary, soil may be air-dried without heating before sieving. Storage of test samples should be as short as possible. Store the soil in accordance with ISO 10381-6 using containers that minimize losses of soil contaminants by volatilization and sorption to the container walls. Soil pH should not be corrected as it can influence bioavailability of soil contaminants. For interpret
