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1、 Reference number ISO 23611-1:2006(E) ISO 2006 INTERNATIONAL STANDARD ISO 23611-1 First edition 2006-02-01 Soil quality Sampling of soil invertebrates Part 1: Hand-sorting and formalin extraction of earthworms Qualit du sol Prlvement des invertbrs du sol Partie 1: Tri manuel et extraction au formol
2、des vers de terre Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 23611-1:2006(E) PDF discl
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5、e file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2006 All ri
6、ghts 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 or ISOs member body in the country of t
7、he 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 2006 All rights reserved Copyright International Organization for Standardization Provided by IHS under license wi
8、th ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 23611-1:2006(E) ISO 2006 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope . 1 2 Terms and definitions. 1 3 P
9、rinciple. 2 4 Reagents 2 5 Apparatus 2 6 Procedure 3 6.1 Sampling of the earthworms . 3 6.2 Preservation 5 6.3 Determination of biomass 5 7 Data assessment. 5 8 Study report. 6 Annex A (informative) Other methods for sampling. 7 Annex B (informative) Species determination in peregrine earthworms . 8
10、 Annex C (informative) The modified TSBF Method 9 Bibliography. 10 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without lic
11、ense from IHS -,-,- ISO 23611-1:2006(E) iv ISO 2006 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 International Standards is normally carried out through ISO tech
12、nical 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, also take part in the work. ISO collaborates closely
13、 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 committees is to prepare International Standards. Draft I
14、nternational 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
15、 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 23611-1 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. ISO 23611 consists of the following parts, under the general title S
16、oil quality Sampling of soil invertebrates: Part 1: Hand-sorting and formalin extraction of earthworms Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina) Part 3: Sampling and soil extraction of enchytraeids Part 4: Sampling, extraction and identification of free-living stag
17、es of terrestrial nematodes Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 23611-1:2006(E)
18、 ISO 2006 All rights reserved v Introduction This part of ISO 23611 has been drawn up since there is a growing need for the standardisation of terrestrial zoological field methods. Such methods, mainly covering the sampling, extraction and handling of soil invertebrates, are necessary for the follow
19、ing purposes: biological classification of soils including soil quality assessment 21, 26, 34; terrestrial bioindication and long-term monitoring 9, 12, 28; evaluation of the effects of chemicals on soil animals (ISO 11268-3). Data for these purposes are gained by standardized methods since they can
20、 form the basis for far-reaching decisions (e.g. whether a given site should be remediated or not). In fact, the lack of such standardised methods is one of the most important reasons why bio-classification and bio-assessment in terrestrial (i.e. soil) habitats has so far relatively rarely been used
21、 in comparison to aquatic sites. Originally, the methods described here were developed for taxonomical and ecological studies, investigating the role of earthworms in various soil ecosystems. These animals are without doubt the most important soil invertebrates in temperate regions and, to a lesser
22、extent, in tropical soils 25, 13, 15. Since Darwin (1881), their influence on soil structure (e.g. aeration, water holding capacity) and soil functions like litter decomposition and nutrient cycling is well-known 8. Due to their often very high biomass they are also important in many terrestrial foo
23、d-webs. Since it is neither possible nor useful to standardize methods for all soil organisms, the most important ones have been selected. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04
24、/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or network
25、ing permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 23611-1:2006(E) ISO 2006 All rights reserved 1 Soil quality Sampling of soil invertebrates Part 1: Hand-sorting and formalin extraction of earthworms 1 Scope This part of ISO 23611 specifies a method for sampling and handling ea
26、rthworms from field soils as a prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for organisms). Basic information on the ecology of earthworms and their use as bioindicators in the terrestrial environment can be found in the references listed i
27、n the bibliography. This part of ISO 23611 applies to all terrestrial biotopes in which earthworms occur. The sampling design of field studies in general is specified in ISO 10381-1 (see also Reference 38) and guidance on the determination of effects of pollutants on earthworms in field situations i
28、s given in ISO 11268-3. These details can vary according to the national requirements or the climatic/regional conditions of the site to be sampled (see also Annex C). This part of ISO 23611 is not applicable for semi-terrestrial soils and it can be difficult to use under extreme climatic or geograp
29、hical conditions (e.g. in high mountains). Methods for some other soil organism groups, such as collembolans, are covered in other parts of ISO 23611. This part of ISO 23611 does not cover the pedological characterization of the site which is highly recommendable when sampling soil invertebrates. IS
30、O 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 and ISO 11465 are more suitable for measuring pH, particle size distribution, C/N ratio, organic carbon content and water-holding capacity. 2 Terms and definitions For the purposes of this document, the following terms and definitions ap
31、ply. 2.1 earthworm megadrile soil-inhabiting earthworms (length of adult individuals: few centimetres to more than 1 m) belonging to the order Oligochaeta (class Clitellata, phylum Annelida) EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae (Latin America), Eudrilidae (Africa
32、) or Megascolecidae Asia, North America (Pacific Coast). 2.2 peregrine species earthworms occurring in many regions world-wide today, usually introduced by man NOTE 1 Well-known examples of peregrine species are several lumbricid species like Aporrectodea caliginosa (originally coming from Eurasia,
33、but now living also in the Americas and Australia) or the pan-tropical species Pontoscolex corethrurus (probably coming from Northern Brazil and/or the Guyanas). NOTE 2 See Reference 15. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Tec
34、hnical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 23611-1:2006(E) 2 ISO 2006 All rights reserved 2.3 clitellum ring or saddle of epidermal thickening only in mature worms which is near the anterior and even
35、tually forms the cocoon 3 Principle Earthworms at a certain site are sampled from the soil by using a combination of two different methods: hand-sorting animals from a certain area (e.g. 0,25 m2) or volume (e.g. 50 cm 50 cm 20 cm); extraction of worms from the soil by applying formalin. Both methods
36、 are well-known for a long time 5,20. After extraction, the earthworms are fixed and transported to the laboratory. There they are preserved in a way that they can be stored in a collection indefinitely (e.g. for taxonomical purposes). In addition, the determination of the biomass of earthworms is d
37、escribed. Finally, abundance and biomass values can be recalculated to area (usually 1 m2) or, more rarely, volume parameters. NOTE 1 Alternative methods can be useful under special circumstances (e.g. electrical extraction or the use of mustard as a chemical expellent), but cannot be recommended as
38、 a general procedure (see Annex A). In addition, the use of other extraction chemicals than formalin seems to have no advantages concerning extraction efficiency 11; 33. NOTE 2 The sampling of earthworms is often included in much broader monitoring programs, trying to cover the whole soil fauna or p
39、arts of it (e.g. the macrofauna). The design of such programs is not included in this part of ISO 23611 1. NOTE 3 Some hints for the taxonomy of peregrine (occurring in many regions world-wide) earthworms, mainly belonging to the family Lumbricidae, are given in Annex B. 4 Reagents 4.1 Formalin form
40、aldehyde solution 4 % (volume fraction). 4.2 Formalin formaldehyde solution 37 % (volume fraction). 4.3 Ethanol, 70 % (volume fraction). 5 Apparatus Use standard laboratory equipment and the following. 5.1 Plastic vessels, capacities 250 ml and 500 ml, for storing the worms. 5.2 Plastic gloves. 5.3
41、Forceps. 5.4 Piece of thick plastic sheeting, 1 m2 to 2 m2. 5.5 Spade or shovel. 5.6 Dissecting microscope, with low magnification (10 to 40 times). 5.7 Balance, able to weigh from 0,01 g to 200 g. 5.8 Water-can, preferably 20 l, with water (20 l per sampling plot). Copyright International Organizat
42、ion for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 19:36:41 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 23611-1:2006(E) ISO 2006 All rights reserved 3 5.9 Watering can. 5.10 Pencil
43、, notebook, water resistant marker, labels that go in the vessel. 5.11 Thermometer, e.g. for measuring air temperature. 5.12 Drying cabinet, for soil moisture determination. 6 Procedure 6.1 Sampling of the earthworms 6.1.1 General Sampling of earthworms is done by a combination of two different meth
44、ods: hand-sorting and formalin extraction. Based on several comparative studies, this combination is clearly recommended in the various reviews on earthworm ecology (e.g. References 7, 8, 15). Sampling should be done at times of the year where the animals are not forced by the environmental conditio
45、ns (i.e. low soil moisture and/or high temperatures) into diapause (i.e. are not reacting to formalin). In temperate regions, such unfavourable sampling times are winter and, in particular, midsummer periods 15. Earthworms sampled from the same plot, but sampled under the two different methods, shou
46、ld be stored in individual plastic vessels. After the end of the sampling process, the excavated and examined soil is returned to the original sampling plot. In some cases, it is appropriate to use only one of the two methods; e.g. when no deep-burrowing animals are occurring at a given site, formal
47、in extraction is not necessary. On the other hand, at sites where giant earthworms are living (parts of South America, South East Asia and Australia), hand- sorting is not useful 22. A very similar method, known as modified TSBF method, is particularly suitable for tropical regions (see Annex C). NO
48、TE Usually the earthworms are determined after preservation, but if the species spectrum of a sampling site is well known, worms can also be determined alive 30; see also Annex B. In the case collected earthworms are to be used for further analysis or testing, e.g. for biomarker measurements or for use in bioassays, storage or incubation of the worms in a small portion of soil from the sampling site is recommende