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1、 Reference number ISO 16387:2004(E) ISO 2004 INTERNATIONAL STANDARD ISO 16387 First edition 2004-02-01 Soil quality Effects of pollutants on Enchytraeidae (Enchytraeus sp.) Determination of effects on reproduction and survival Qualit du sol Effets des polluants sur les Enchytraeidae (Enchytraeus sp.
2、) Dtermination des effets sur la reproduction et la survie Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license fro
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6、he address given below. ISO 2004 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below
7、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 copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2004 All rights reserved Copyright International Organization for Standardi
8、zation Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 16387:2004(E) ISO 2004 All rights reserved iii Contents Page Forewordiv Introduction v 1 Scop
9、e1 2 Normative references .1 3 Terms and definitions.1 4 Principle.2 5 Materials.3 6 Apparatus.4 7 Test environment5 8 Procedure.5 8.1 Preparation of the test substrates.5 8.2 Preparation of test vessel contents 6 8.3 Range-finding test.6 8.4 Definitive test.6 8.5 Reference substance7 8.6 Summary an
10、d timetable of the test .8 9 Calculation and expression of results9 9.1 General.9 9.2 Range-finding test.9 9.3 Definitive test.9 10 Validity of the test .10 11 Test report10 Annex A (informative) Detailed description of extraction techniques11 Annex B (normative) Determination of the effects of cont
11、aminated soil on Enchytraeidae reproduction12 Annex C (informative) Determination of the pH value 17 Annex D (informative) Determination of maximum water-holding capacity.18 Annex E (informative) Conditions for culture of Enchytraeus sp19 Annex F (informative) Test procedure using other Enchytraeus
12、species21 Annex G (informative) Overview of the statistical assessment of data (NOEC determination) .23 Bibliography .24 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53
13、MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 16387:2004(E) iv ISO 2004 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 Internationa
14、l 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,
15、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 commi
16、ttees 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 po
17、ssibility 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 16387 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. Copyright Internat
18、ional Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 16387:2004(E) ISO 2004 All rights reserved v Introduction Thi
19、s International Standard has been drawn up taking into consideration test procedures recommended by the International Organization for Standardization (ISO) and the Organization for Economic Cooperation and Development (OECD) (see Clause 2 and Bibliography). The method described was developed for te
20、sting the effects of chemicals added to an artificial soil. An adaptation for testing or comparing soils to assess, for example, the effects of remediation treatments is given in Annex B. It can also be adapted for assessing sublethal effects and determining no-effect levels for pesticides. Soil-dwe
21、lling annelids of the genus Enchytraeus are ecologically relevant, i.e. they are abundant in many soils where earthworms are scarce, but can also reach high population densities in soils well inhabited by earthworms. Enchytraeidae can be used in laboratory tests as well as in semi-field and field st
22、udies. From a practical point of view, many Enchytraeus species are easy to handle and breed, and their generation time is significantly shorter than that of earthworms the test duration for a reproduction test with Enchytraeidae is 4 weeks to 6 weeks, compared to 12 weeks (including synchronization
23、) with earthworms. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Orga
24、nization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 16387:2004(E) ISO 2004 All rights reserved 1 Soi
25、l quality Effects of pollutants on Enchytraeidae (Enchytraeus sp.) Determination of effects on reproduction and survival 1 Scope This International Standard describes a method for determining the effects of substances or contaminated soils on reproduction and on survival of the worm Enchytraeus albi
26、dus (Enchytraeidae). The animals are exposed to the substances by dermal and alimentary uptake using a defined artificial soil substrate to which specified amounts of that substance are added, or by using a soil substrate of unknown quality. This International Standard is applicable to test substanc
27、es that are either insoluble or soluble in water, although the method of application differs. The method is not applicable to volatile test 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 0,0
28、13 3 Pa at 25 C. The water solubility and the vapour pressure of the test substance should be known. Additionally, information on the persistence of the test substance in soil is desirable. NOTE 1 Basic information on the ecology and ecotoxicology of Enchytraeidae in the terrestrial environment can
29、be found in the bibliographic references. NOTE 2 The stability of the test substance cannot be ensured over the test period. No provision is made in the test method for monitoring the persistence of the test substance. NOTE 3 Recommendations for adapting the method to comparing or monitoring soil qu
30、ality are given in Annex B. 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
31、. ISO 11268-2:1998, Soil quality Effects of pollutants on earthworms (Eisenia fetida) Part 2: Determination of effects on reproduction OECD Guideline No. 207, 1984, Earthworm Acute Toxicity Tests, Organization for Economic Cooperation and Development, Paris 3 Terms and definitions For the purposes o
32、f this document, the following terms and definitions apply. 3.1 reproduction increase in the mean number of juveniles per test vessel after 6 weeks 3.2 concentration lethal to 50 % of the test organisms LC50 that concentration of the test substance which kills 50 % of the test animals within the per
33、iod of the range- finding test or the definitive test Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS
34、 -,-,- ISO 16387:2004(E) 2 ISO 2004 All rights reserved NOTE 1 The LC50 is expressed as mass of test substance per dry mass of the test substrate. NOTE 2 It is the median lethal concentration. 3.3 lowest observed effect concentration LOEC lowest tested concentration at which the test substance is ob
35、served to have a statistically significant effect on reproduction (probability p 0,05) within a given exposure time NOTE In this test, the effect on reproduction (number of juveniles) is used as test parameter. 3.5 effect concentration ECx concentration at which a specific effect is detected where x
36、 is the percentage (10, 25, 50) of this effect, e.g. on reproduction, in relation to a control NOTE For example, EC50 means the concentration estimated to reduce the reproduction rate at the end of the test to 50 % compared to the control. All effect concentrations are expressed as mass of test subs
37、tance per dry mass of the test substrate. 4 Principle Adult Enchytraeidae worms are exposed to a range of concentrations of the test substance mixed in artificial soil. The test can be divided into two distinct steps: a short (2 weeks) range-finding test in which the range of toxic effects (mainly m
38、ortality) is determined, and a long-term (6 weeks) definitive test in which the survival of parental worms and the fecundity (number of juveniles) are measured. Therefore, the test is usually conducted as follows. a) For test substances of unknown toxicity exposure, it is recommended to conduct a ra
39、nge-finding test for a period of 14 d, indicating the concentrations for total mortality and for the absence of mortality. The resulting dose-response relationship is important for the proper design of the definitive test. b) The definitive test is designed to determine the concentration of a test s
40、ubstance mixed into the artificial soil that causes a defined significant or specified effect on reproduction. This test design includes the investigation of lethal effects on the parental Enchytraeidae. The total duration of the definitive test is 6 weeks (if another Enchytraeus species than E. alb
41、idus is used, it can be shorter). After the first 3 weeks, the adult worms are removed, the number of living worms and morphological changes (e.g. body lesions or fragmentation of the worm) are recorded. After another 3 weeks, the number of offspring hatched from the cocoons is counted. The NOEC and
42、 the ECx for reproduction are determined. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/12/2007 20:58:53 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 1
43、6387:2004(E) ISO 2004 All rights reserved 3 5 Materials The test substrate and the test substance are set up with standard laboratory equipment and kept in glass vessels. 5.1 Biological material. The recommended test species is Enchytraeus albidus Hence 1837 (white potworm; Enchytraeidae, Oligochaet
44、a, Annelida). E. albidus is one of the largest enchytraeid species, measuring 15 mm to 40 mm, and has a world-wide distribution (see Bibliography). It can easily be recognised by two characteristics: four setae per bundle ventrally, and the very long seminal duct in the clitellum region as well as s
45、ome segments behind it. The species can be found in marine, limnic and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and only rarely in meadows. This broad ecological tolerance and some morphological variations indicate that the species may consist of several races (or e
46、cotypes). E. albidus can be obtained commercially, since it is sold as food for fish. It should be verified whether such a culture is contaminated by other, usually smaller species (see Bibliography). If contamination occurs, all worms are washed in water in a Petri dish. With the help of a stereomi
47、croscope, large adult specimens of E. albidus are selected to start a new culture. All other worms of the original culture are discarded. E. albidus can be bred easily in a wide range of organic waste materials (see Annex E) and has a short life cycle, reaching maturity between 33 d (at 18 C) and 74
48、 d (at 12 C). Only cultures which have been kept in the laboratory for at least 5 weeks (one generation cycle) without problems can be used for testing purposes. Other species of the genus Enchytraeus, e.g. the true soil-inhabiting but smaller species E. buchholzi Vejdovsky 1879 or E. crypticus Westheide and Graefe 1992, are also suitable as test organisms (see Annex F). If other species of Enchytraeus are used, they shall be clearly identified and the rationale for the selectio