1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 23611-3:2011Soil quality Sampling of soilinvertebratesPart 3: Sampling and soil extraction ofenchytraeids (ISO 23611-3:2007)BS EN ISO 23611-3:2011 BRITISH STANDARDNatio
2、nal forewordThis British Standard is the UK implementation of EN ISO23611-3:2011. It is identical to ISO 23611-3:2007.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
3、secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2011ISBN 978 0 580 73164 8ICS 13.080.05; 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Stand
4、ard was published under the authority of theStandards Policy and Strategy Committee on 31 July 2011.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 23611-3 July 2011 ICS 13.080.30; 13.080.05 English Version Soil quality - Sampling of soil
5、 invertebrates - Part 3: Sampling and soil extraction of enchytraeids (ISO 23611-3:2007) Qualit du sol - Prlvement des invertbrs du sol - Partie 3: Prlvement et extraction des enchytrides (ISO 23611-3:2007) Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme und Bodenextra
6、ktion von Enchytraeen (ISO 23611-3:2007) This European Standard was approved by CEN on 17 June 2011. CEN members are bound to 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
7、-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 trans
8、lation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finlan
9、d, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOM
10、ITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 23611-3:2011: EBS EN ISO 23611-3:2011EN ISO 23611-3:2011 (E) 3 Foreword The text of ISO 23611-3:2007 has
11、been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 23611-3:2011 by Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN. This European Standard shal
12、l be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 2012, and conflicting national standards shall be withdrawn at the latest by January 2012. Attention is drawn to the possibility that some of the elements of this docu
13、ment may be the subject of patent rights. CEN and/or CENELEC 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:
14、Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingd
15、om. Endorsement notice The text of ISO 23611-3:2007 has been approved by CEN as a EN ISO 23611-3:2011 without any modification. BS EN ISO 23611-3:2011ISO 23611-3:2007(E) ISO 2007 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 4 R
16、eagents 2 5 Apparatus 2 6 Procedure 3 6.1 Soil sampling. 3 6.2 Extraction of the enchytraeids 3 6.3 Microscopic identification . 4 6.4 Preservation of Enchytraeidae 4 6.5 Validity of the extraction process . 4 6.6 Determination of biomass 5 7 Data assessment. 5 8 Test report . 5 Annex A (informative
17、) Species identification in enchytraeids. 7 Annex B (informative) Quick extraction of enchytraeids . 8 Bibliography . 10 BS EN ISO 23611-3:2011ISO 23611-3:2007(E) iv ISO 2007 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national st
18、andards 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 represented on that committee. International o
19、rganizations, 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 drafted in accordance with the rules giv
20、en 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 Standard requires approval by at least
21、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 23611-3 was prepared by Technical Committee ISO/TC 190,
22、 Soil quality, Subcommittee SC 4, Biological methods. ISO 23611 consists of the following parts, under the general title Soil 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
23、) Part 3: Sampling and soil extraction of enchytraeids Part 4: Sampling, extraction and identification of soil-inhabiting nematodes BS EN ISO 23611-3:2011ISO 23611-3:2007(E) ISO 2007 All rights reserved vIntroduction This part of ISO 23611 has been drawn up since there is a growing need for the stan
24、dardization of terrestrial zoological field methods. Such methods, mainly covering the sampling, extraction and handling of soil invertebrates, are needed for the following purposes: biological classification of soils including soil quality assessment (e.g. References 21, 25, 27); terrestrial bioind
25、ication and long-term monitoring (e.g. References 13, 26); evaluation of the effects of chemicals on soil animals (References 15, 22). Data for these purposes are gained by standardized methods since they can form the basis for far-reaching decisions (e.g. whether a given site should be remediated o
26、r not). In fact, the lack of such standardized methods is one of the most important reasons why biological classification concepts in terrestrial (i.e. soil) habitats have so far been relatively rarely used in comparison to aquatic sites. Originally, the methods described here were developed for tax
27、onomical and ecological studies, investigating the role of enchytraeids in various soil ecosystems. These animals without doubt belong to the most important soil invertebrates in temperate regions (mainly in acidic soils 5). Their influence on soil functions like litter decomposition and nutrient cy
28、cling is well-known 14, 19. Due to their number which is often very high (and to their population biomass), they are also important in many terrestrial food-webs 4. Some species have unintentionally been distributed by man in many soils of the world. Since it is neither possible nor useful to standa
29、rdize methods for all soil organisms, the most important ones have been selected. Microbiological parameters are already covered by existing ISO guidelines (e.g. ISO 10381-6 29, ISO 14240-1 37and ISO 14240-2 38). BS EN ISO 23611-3:2011BS EN ISO 23611-3:2011INTERNATIONAL STANDARD ISO 23611-3:2007(E)
30、ISO 2007 All rights reserved 1Soil quality Sampling of soil invertebrates Part 3: Sampling and soil extraction of enchytraeids 1 Scope This part of ISO 23611 specifies a method for sampling, handling and extracting enchytraeids from terrestrial field soils as a prerequisite for using these animals a
31、s bioindicators (e.g. to assess the quality of a soil as a habitat for organisms). Basic information on the ecology of enchytraeids and their use as bioindicators in the terrestrial environment are included in the Bibliography. This part of ISO 23611 applies to all terrestrial biotopes in which ench
32、ytraeids occur. The sampling design of field studies in general is specified in ISO 10381-1. These details can vary according to the climatic/regional conditions of the site to be sampled and an overview on the determination of effects of pollutants on enchytraeids in field situations is given in Re
33、ference 6. Methods for some other soil organism groups such as earthworms or micro-arthropods are specified in ISO 23611-1 and ISO 23611-2. This part of ISO 23611 is not applicable for semi-terrestrial (i.e. living in or close to the pure water) soils and might be difficult to use under extreme clim
34、atic or geographical conditions (e.g. in high mountains). When sampling soil invertebrates, it is highly recommendable to characterize the site (e.g. concerning climate and land use). However, such a characterization is not covered by this part of ISO 23611. ISO 10390, ISO 10694, ISO 11272, ISO 1127
35、4, 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 apply. 2.1 enchytraeids small soil-inhabi
36、ting worms (a few millimetres to several centimetres in length) belonging to the family Enchytraeidae, order Oligochaeta (class Clitellata, phylum Annelida) EXAMPLE Species of the genera Enchytraeus, Fridericia or Cognettia. 3 Principle Enchytraeids at a certain site are sampled from the soil by usi
37、ng a split corer (diameter usually 3 cm to 6 cm). After sampling, the soil samples containing the enchytraeids are transported to the laboratory. Then the enchytraeids are extracted from soil by means of a wet extraction method. (This approach has been well-known for a long time 11, 17, 20.) After e
38、xtraction, the enchytraeids are identified alive and, if required, preserved in such a way that they can be stored in a collection indefinitely (e.g. for taxonomical purposes). BS EN ISO 23611-3:2011ISO 23611-3:2007(E) 2 ISO 2007 All rights reservedThe determination of the biomass of enchytraeids is
39、 also described in this part of ISO 23611. The abundance and biomass values can be recalculated to the area of the soil corer or, more rarely, volume parameters. NOTE 1 The sampling of enchytraeids is often included in much broader monitoring programmes which try to cover the whole soil fauna or par
40、ts of it (e.g. the mesofauna). The design of such programmes is not included in this part of ISO 23611 (but see e.g. Reference 3). NOTE 2 Some hints for the taxonomy of enchytraeids are given in Annex A. 4 Reagents 4.1 Tap water (without toxic properties, e.g. due to copper contamination). 4.2 Ethan
41、ol, 70 % (volume fraction). 4.3 Bengalred, 4,5,6,7-Tetrachloro-2,4,5,7-tetraiodofluorescein formulated as a staining agent. 4.4 Bouins fixative, buffered solution of formaldehyde, acetic acid and picric acid. 4.5 Paracarmin, staining agent, prepared as a mixture of carmine acid, aluminium chloride a
42、nd calcium chloride solved in ethanol. 4.6 Canada-balm, natural yellowish viscous fluid containing 13 % to 14 % (volume fraction) Canadin acid (C20H38O2), 48 % to 50 % (volume fraction) - and -Canadinol acid (C19H30O2) and 5 % (volume fraction) Canadoesen (C21H40O). 5 Apparatus 5.1 Split soil corer
43、(e.g. diameter 3 cm to 6 cm; extracted core length 10 cm to 30 cm); length in total variable (depending whether or not a handle is used). 5.2 Plastic bags (e.g. 1-l freezer bags); general store. 5.3 Temperature recorder or a minimum/maximum-thermometer. 5.4 Plastic bowls, diameter approximately 20 c
44、m, height approximately 10 cm; general store. 5.5 Plastic sieves, diameter approximately 15 cm, mesh width approximately 0,5 mm; general store. 5.6 60-W bulbs as a heating device; general store. 5.7 Glassware, e.g. petri dishes (square format) with a size of 8 cm 8 cm or small glass vessels (e.g. 50
45、 ml). 5.8 Sharp, large knife. 5.9 Refrigerator. 5.10 Dissecting microscope with low magnification (10 to 40 times). 5.11 Microscope with high magnification (60 to 400 times). 5.12 Spring steel pincers (flat). 5.13 Eppendorf pipette, a soft steel forceps or a hooked needle. BS EN ISO 23611-3:2011ISO
46、23611-3:2007(E) ISO 2007 All rights reserved 36 Procedure 6.1 Soil sampling The soil samples to be used for the investigation of the enchytraeid community are taken destructively by means of a soil corer (5.1). The corer is carefully pressed into the soil. The depth depends on the soil type, but usu
47、ally varies between 10 cm (e.g. forests) and up to 30 cm (e.g. crop sites), i.e. those layers in which the bulk of the enchytraeids are living. In rare cases, e.g. if thick roots are present, a plastic or wooden hammer can be used to take the samples. After removing the soil corer, its valve is open
48、ed and the soil core is carefully taken out by hand. The core is divided into cylinders (e.g. 3 cm to 4 cm height) with a knife (5.8). These soil cylinders may be stored in small plastic bags (5.2) in a refrigerator (5.9) at approximately 4 C to 6 C for a period of preferably not longer than one to
49、two weeks (storage should not exceed one month in any case 7). The soil corer is cleaned with water afterwards. 6.2 Extraction of the enchytraeids In principle, the extraction of the worms from the soil is caused by their active movement in the water-saturated sample. The extraction should commence as soon as possible after the sampling (see 6.1). The bowls (5.4) are carefully filled up with tap water (4.1) until the empty sieves (5.5) are completely covered by the water. The s
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