SIST EN ISO 23611-1:2011
(Main)Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)
ISO 23611-1:2006 specifies a method for sampling and handling earthworms 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).
ISO 23611-1:2006 applies to all terrestrial biotopes in which earthworms occur. It is not applicable for semi-terrestrial soils and it can be difficult to use under extreme climatic or geographical conditions (e.g. in high mountains).
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Formalinextraktion von Regenwürmern (ISO 23611-1:2006)
Dieser Teil von ISO 23611 legt ein Verfahren zur Probenahme und Behandlung von Regenwürmern aus Freilandböden fest, mit dessen Hilfe die Voraussetzungen für die Verwendung dieser Tiere als Bioindikatoren (z. B. zur Beurteilung der Beschaffenheit eines Bodens als Lebensraum für Organismen) geschaffen werden. Grundlegende Informationen zur Ökologie von Regenwürmern und ihrer Verwendung als Bioindikatoren in der terrestrischen Umwelt lassen sich den im Literaturverzeichnis aufgeführten Quellen entnehmen. Dieser Teil von ISO 23611 gilt für alle terrestrischen Biotope, in denen Regenwürmer leben. Der für Freilanduntersuchungen im Allgemeinen gültige Probenahmeplan ist bereits in ISO 10381-1 (siehe auch [38]) festgelegt, und eine Anleitung zur Bestimmung der Wirkungen von Schadstoffen auf Regenwürmer unter Freilandbedingungen wird in ISO 11268-3 gegeben. Diese Einzelheiten können den nationalen Anforderun-gen entsprechend oder in Abhängigkeit von den klimatischen/regionalen Bedingungen des zu beprobenden Standorts variieren (siehe auch Anhang C). Dieser Teil von ISO 23611 gilt nicht für semiterrestrische Böden. Die Anwendung unter extremen klimatischen oder geographischen Bedingungen (z. B. im Hochgebirge) kann schwierig sein. Die Verfahren für einige andere Bodenorganismengruppen wie z. B. Collembolen werden in anderen Teilen von ISO 23611 behandelt. Dieser Teil von ISO 23611 behandelt nicht die Charakterisierung des Standortes nach bodenkundlichen Gesichtspunkten, wie sie für die Probenahme von Wirbellosen im Boden nachdrücklich empfohlen wird. Vor allem sollten der pH-Wert, die Korngrößenverteilung, das C/N-Verhältnis, der Gehalt an organischem Kohlenstoff und das Wasserrückhaltevermögen nach ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 und ISO 11465 bestimmt werden.
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1 : Tri manuel et extraction au formol des vers de terre (ISO 23611-1:2006)
L'ISO 23611-1:2006 spécifie une méthode pour le prélèvement et la manipulation de vers de terre sur le terrain, comme prérequis à l'utilisation de ces animaux en tant que bio-indicateurs, par exemple pour évaluer la qualité d'un sol en tant qu'habitat pour des organismes.
L'ISO 23611-1:2006 est utilisée pour tous les biotopes terrestres dans lesquels il est possible de trouver des vers de terre. Elle ne s'applique pas aux sols semi-terrestres et il peut être difficile de l'appliquer dans des conditions climatiques ou géographiques extrêmes (en haute montagne par exemple).
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 1. del: Ročno razvrščanje deževnikov in njihova ekstrakcija s formalinom (ISO 23611-1:2006)
ISO 23611-1:2006 določa metodo za vzorčenje in delo z deževniki iz poljskih tal kot nujen pogoj za uporabo teh živali kot bioindikatorjev (npr. za ocenjevanje kakovosti tal kot habitata za organizme).
ISO 23611-1:2006 velja za vse zemeljske biotope, v katerih so deževniki. Ne velja za polzemeljska tla in se lahko s težavo uporablja v ekstremnih klimatskih ali geografskih razmerah (npr. v visokih gorah.
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.]YUãþDQMHBodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Formalinextraktion von Regenwürmern (ISO 23611-1:2006)Qualité du sol - Prélèvement des invertébrés du sol - Partie 1 : Tri manuel et extraction au formol des vers de terre (ISO 23611-1:2006)Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)13.080.30Biološke lastnosti talBiological properties of soilsICS:Ta slovenski standard je istoveten z:EN ISO 23611-1:2011SIST EN ISO 23611-1:2011en,fr,de01-december-2011SIST EN ISO 23611-1:2011SLOVENSKI
STANDARD
SIST EN ISO 23611-1:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN ISO 23611-1
July 2011 ICS 13.080.30; 13.080.05 English Version
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)
Qualité du sol - Prélèvement des invertébrés du sol -Partie 1 : Tri manuel et extraction au formol des vers de terre (ISO 23611-1:2006)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Formalinextraktion von Regenwürmern (ISO 23611-1:2006) 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-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 translation 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, Finland, 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
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR 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 23611-1:2011: ESIST EN ISO 23611-1:2011
EN ISO 23611-1:2011 (E) 2 Contents Page Foreword .3 SIST EN ISO 23611-1:2011
EN ISO 23611-1:2011 (E) 3 Foreword The text of ISO 23611-1:2006 has 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 23611-1:2011 by Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN. This European Standard shall 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 document 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: 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 Kingdom. Endorsement notice The text of ISO 23611-1:2006 has been approved by CEN as a EN ISO 23611-1:2011 without any modification.
SIST EN ISO 23611-1:2011
SIST EN ISO 23611-1:2011
Reference numberISO 23611-1:2006(E)© ISO 2006
INTERNATIONAL STANDARD ISO23611-1First edition2006-02-01Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and formalin extraction of earthworms Qualité du sol — Prélèvement des invertébrés du sol — Partie 1: Tri manuel et extraction au formol des vers de terre
SIST EN ISO 23611-1:2011
ISO 23611-1:2006(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the 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 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 or ISO's 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
copyright@iso.org Web
www.iso.org Published in Switzerland
ii © ISO 2006 – All rights reserved
SIST EN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved iiiContents Page Foreword.iv Introduction.v 1 Scope.1 2 Terms and definitions.1 3 Principle.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 Annex C (informative)
The modified TSBF Method.9 Bibliography.10
SIST EN ISO 23611-1:2011
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 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, 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 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 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 23611-1 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 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) ⎯ Part 3: Sampling and soil extraction of enchytraeids ⎯ Part 4: Sampling, extraction and identification of free-living stages of terrestrial nematodes SIST EN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved vIntroduction 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 following 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 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 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 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 food-webs. Since it is neither possible nor useful to standardize methods for all soil organisms, the most important ones have been selected. SIST EN ISO 23611-1:2011
SIST EN ISO 23611-1:2011
INTERNATIONAL STANDARD ISO 23611-1:2006(E) © ISO 2006 – All rights reserved 1Soil 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 earthworms 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 in 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 is 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 geographical 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. ISO 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 apply. 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) 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, 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]. SIST EN ISO 23611-1:2011
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 eventually 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 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 described. 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 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 parts 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 [formaldehyde 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 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). SIST EN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved 35.9 Watering can. 5.10 Pencil, 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 methods: 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 conditions (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, should 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, formalin 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). NOTE 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 recommended. In the case of formalin extraction, rinsing the worms in tap water is needed before incubation in soil. 6.1.2 Hand-sorting Due to the individual size of the worms, a large plot shall be identified. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms have approximately a length between 1 cm and 20 cm. However, at places with a low density of earthworms [e.g. soils with low pH (< 4,5) or which are anthropogenically used like crop sites], larger plots (i.e. 1 m2) are recommended (ISO 11268-3). On the other hand, at sites with a high earthworm density (e.g. many meadows in temperate regions), a smaller plot of 1/8 m2 is sufficient [24]. Even smaller sample sizes (e.g. 1/16 m2 ; [36]) can lead to very low, and thus variable, individual worm numbers per sample, which in turn leads to an increase in sample numbers (i.e. 16 replicates). In any case, the soil is removed by means of a spade or shovel (5.5) up to a depth of 20 cm from this plot (20 cm are suitable for many temperate sites, but the depth also depends on the site properties). The excavated soil is spread out on a piece of plastic (5.4); this can be done in the field but, especially in periods of bad weather, the whole procedure can also be performed in the laboratory or greenhouse. Afterwards, the soil is searched cautiously for earthworms. Big earthworms are collected by hand using plastic hand gloves (5.2) and small ones by using forceps (5.3). To avoid autotomy and further damage of the worms, the animals should only be touched at the anterior part of the body. If worms are cut by the spade used to dig out the soil, both parts are collected in order to measure the correct biomass, whereas only front parts are counted when determining the number of individuals. SIST EN ISO 23611-1:2011
ISO 23611-1:2006(E) 4 © ISO 2006 – All rights reserved NOTE 1 With a naked eye, the front end of adult worms can be identified by the position of the clitellum: it is always located closer to the head than to the tail. The collected earthworms should immediately be fixed in 70 % ethanol
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.[Not translated]Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Formalinextraktion von Regenwürmern (ISO 23611-1:2006)Qualité du sol - Prélèvement des invertébrés du sol - Partie 1 : Tri manuel et extraction au formol des vers de terre (ISO 23611-1:2006)Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)13.080.30Biološke lastnosti talBiological properties of soilsICS:Ta slovenski standard je istoveten z:FprEN ISO 23611-1kSIST FprEN ISO 23611-1:2011en,fr,de01-april-2011kSIST FprEN ISO 23611-1:2011SLOVENSKI
STANDARD
kSIST FprEN ISO 23611-1:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
FINAL DRAFT
FprEN ISO 23611-1
December 2010 ICS 13.080.30 English Version
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)
Qualité du sol - Prélèvement des invertébrés du sol -Partie 1 : Tri manuel et extraction au formol des vers de terre (ISO 23611-1:2006)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Formalinextraktion von Regenwürmern (ISO 23611-1:2006) This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical Committee CEN/TC 345.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language made by translation 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, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. FprEN ISO 23611-1:2010: EkSIST FprEN ISO 23611-1:2011
FprEN ISO 23611-1:2010 (E) 2 Contents Page Foreword .3 kSIST FprEN ISO 23611-1:2011
FprEN ISO 23611-1:2010 (E) 3 Foreword The text of ISO 23611-1:2006 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the International Organization for Standardization (ISO) and has been taken over as FprEN ISO 23611-1:2010 by Technical Committee CEN/TC 345 “Characterization of soils”, the secretariat of which is held by NEN. This document is currently submitted to the Unique Acceptance Procedure. Endorsement notice The text of ISO 23611-1:2006 has been approved by CEN as an FprEN ISO 23611-1:2010 without any modification.
kSIST FprEN ISO 23611-1:2011
kSIST FprEN ISO 23611-1:2011
Reference numberISO 23611-1:2006(E)© ISO 2006
INTERNATIONAL STANDARD ISO23611-1First edition2006-02-01Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and formalin extraction of earthworms Qualité du sol — Prélèvement des invertébrés du sol — Partie 1: Tri manuel et extraction au formol des vers de terre
kSIST FprEN ISO 23611-1:2011
ISO 23611-1:2006(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the 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 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 or ISO's 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
copyright@iso.org Web
www.iso.org Published in Switzerland
ii © ISO 2006 – All rights reserved
kSIST FprEN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved iiiContents Page Foreword.iv Introduction.v 1 Scope.1 2 Terms and definitions.1 3 Principle.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 Annex C (informative)
The modified TSBF Method.9 Bibliography.10
kSIST FprEN ISO 23611-1:2011
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 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, 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 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 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 23611-1 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 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) ⎯ Part 3: Sampling and soil extraction of enchytraeids ⎯ Part 4: Sampling, extraction and identification of free-living stages of terrestrial nematodes kSIST FprEN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved vIntroduction 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 following 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 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 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 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 food-webs. Since it is neither possible nor useful to standardize methods for all soil organisms, the most important ones have been selected. kSIST FprEN ISO 23611-1:2011
kSIST FprEN ISO 23611-1:2011
INTERNATIONAL STANDARD ISO 23611-1:2006(E) © ISO 2006 – All rights reserved 1Soil 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 earthworms 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 in 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 is 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 geographical 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. ISO 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 apply. 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) 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, 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]. kSIST FprEN ISO 23611-1:2011
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 eventually 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 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 described. 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 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 parts 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 [formaldehyde 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 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). kSIST FprEN ISO 23611-1:2011
ISO 23611-1:2006(E) © ISO 2006 – All rights reserved 35.9 Watering can. 5.10 Pencil, 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 methods: 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 conditions (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, should 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, formalin 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). NOTE 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 recommended. In the case of formalin extraction, rinsing the worms in tap water is needed before incubation in soil. 6.1.2 Hand-sorting Due to the individual size of the worms, a large plot shall be identified. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms have approximately a length between 1 cm and 20 cm. However, at places with a low density of earthworms [e.g. soils with low pH (< 4,5) or which are anthropogenically used like crop sites], larger plots (i.e. 1 m2) are recommended (ISO 11268-3). On the other hand, at sites with a high earthworm density (e.g. many meadows in temperate regions), a smaller plot of 1/8 m2 is sufficient [24]. Even smaller sample sizes (e.g. 1/16 m2 ; [36]) can lead to very low, and thus variable, individual worm numbers per sample, which in turn leads to an increase in sample numbers (i.e. 16 replicates). In any case, the soil is removed by means of a spade or shovel (5.5) up to a depth of 20 cm from this plot (20 cm are suitable for many temperate sites, but the depth also depends on the site properties). The excavated soil is spread out on a piece of plastic (5.4); this can be done in the field but, especially in periods of bad weather, the whole procedure can also be performed in the laboratory or greenhouse. Afterwards, the soil is searched cautiously for earthworms. Big earthworms are collected by hand using plastic hand gloves (5.2) and small ones by using forceps (5.3). To avoid autotomy and further damage of the worms, the animals should only be touched at the anterior part of the body. If worms are cut by the spade used to dig out the soil, both parts are collected in order to measure the correct biomass, whereas only front parts are counted when determining the number of individuals. kSIST FprEN ISO 23611-1:2011
ISO 23611-1:2006(E) 4 © ISO 2006 – All rights reserved NOTE 1 With a naked eye, the front end of adult worms can be identified by the position of the clitellum: it is always located closer to the head than to the tail. The collected earthworms should immediately be fixed in 70 % ethanol (4.3) using the 250 ml or 500 ml plastic vessels (5.1) for at least half an hour, but not longer than 24 h. The vessels shall be labelled and observa
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