SIST EN ISO 23611-3:2012
(Main)Soil quality - Sampling of soil invertebrates - Part 3: Sampling and soil extraction of enchytraeids (ISO 23611-3:2007)
Soil quality - Sampling of soil invertebrates - Part 3: Sampling and soil extraction of enchytraeids (ISO 23611-3:2007)
ISO 23611-3:2007 specifies a method for sampling, handling and extracting enchytraeids from terrestrial 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). It applies to all terrestrial biotopes in which enchytraeids occur.
ISO 23611-3:2007 is not applicable for semi-terrestrial soils and might be difficult to use under extreme climatic or geographical conditions.
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme und Bodenextraktion von Enchytraeen (ISO 23611-3:2007)
Dieser Teil von ISO 23611 legt ein Verfahren zur Probenahme, Handhabung und Extraktion von Enchytraeen aus terrestrischen 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 Enchytraeen 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 Enchytraeen leben. Der für Freilanduntersuchungen im Allgemeinen gültige Probenahmeplan ist bereits in ISO 10381-1 festgelegt. Diese Einzelheiten können in Abhängigkeit von den klimatischen/regionalen Bedingungen des zu beprobenden Standorts variieren. Referenz [6] vermittelt einen Überblick zur Bestimmung der Wirkungen von Verunreinigungen auf Enchytraeen unter Freilandbedingungen. Die Verfahren für einige andere Bodenorganismengruppen, wie Regenwürmer und Mikroarthropoden, sind in ISO 23611-1 und ISO 23611-2 festgelegt. Dieser Teil von ISO 23611 gilt nicht für semi-terrestrische Böden (d. h. in oder nahe reinem Wasser lebend) und kann unter extremen klimatischen oder geographischen Bedingungen (z. B. im Hochgebirge) schwierig anzuwenden sein. Für die Probenahme von Wirbellosen in Böden wird nachdrücklich empfohlen, den Standort zu charakterisieren (z. B. Berücksichtigung des Klimas und der Bodennutzung). Eine derartige Charakterisierung wird jedoch in diesem Teil von ISO 23611 nicht behandelt. ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 und ISO 11465 sind zur Bestimmung des pH-Wertes, der Korngrößenverteilung, des C/N-Verhältnisses, des organischen Kohlenstoffgehalts und des Wasserrückhaltevermögens geeigneter.
Qualité du sol - Prélèvement des invertébrés du sol - Partie 3: Prélèvement et extraction des enchytréides (ISO 23611-3:2007)
L'ISO 23611-3:2007 sp�cifie une m�thode pour le pr�l�vement, la manipulation et l'extraction des enchytra�ides du sol pr�lev�s sur le terrain comme pr�-requis � l'utilisation de ces animaux en tant que bio-indicateurs (par exemple, pour �valuer la qualit� d'un sol en tant qu'habitat pour ces organismes). La pr�sente partie de l'ISO 23611 s'applique � tous les biotopes terrestres dans lesquels il est possible de trouver des enchytra�ides.
L'ISO 23611-3:2007 n'est pas applicable � des sols semi-terrestres et peut �tre d'utilisation difficile dans des conditions climatiques ou g�ographiques extr�mes.
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 3. del: Vzorčenje in ekstrakcija enhitrej iz tal (ISO 23611-3:2007)
ISO 23611-3:2007 določa metodo za vzorčenje, delo in ekstrakcijo enhitrej iz poljskih tal kot nujen pogoj za uporabo teh živali kot bioindikatorjev (npr. za ocenjevanje kakovosti tal kot habitata za organizme). Velja za vse zemeljske biotope, v katerih so enhitreje.
ISO 23611-3:2007 ne velja za polzemeljska tla in je morda težje uporaben v ekstremnih klimatskih ali geografskih razmerah.
General Information
Relations
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme und Bodenextraktion von Enchytraeen (ISO 23611-3:2007)Qualité du sol - Prélèvement des invertébrés du sol - Partie 3: Prélèvement et extraction des enchytréides (ISO 23611-3:2007)Soil quality - Sampling of soil invertebrates - Part 3: Sampling and soil extraction of enchytraeids (ISO 23611-3:2007)13.080.30Biološke lastnosti talBiological properties of soilsICS:Ta slovenski standard je istoveten z:EN ISO 23611-3:2011SIST EN ISO 23611-3:2012en,fr,de01-januar-2012SIST EN ISO 23611-3:2012SLOVENSKI
STANDARD
SIST EN ISO 23611-3:2012
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN ISO 23611-3
July 2011 ICS 13.080.30; 13.080.05 English Version
Soil quality - Sampling of soil invertebrates - Part 3: Sampling and soil extraction of enchytraeids (ISO 23611-3:2007)
Qualité du sol - Prélèvement des invertébrés du sol -Partie 3: Prélèvement et extraction des enchytréides (ISO 23611-3:2007)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme und Bodenextraktion 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-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 23611-3:2011: ESIST EN ISO 23611-3:2012
EN ISO 23611-3:2011 (E) 2 Contents Page Foreword .3 SIST EN ISO 23611-3:2012
EN ISO 23611-3:2011 (E) 3 Foreword The text of ISO 23611-3:2007 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 23611-3:2011 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 23611-3:2007 has been approved by CEN as a EN ISO 23611-3:2011 without any modification.
SIST EN ISO 23611-3:2012
SIST EN ISO 23611-3:2012
Reference numberISO 23611-3:2007(E)© ISO 2007
INTERNATIONAL STANDARD ISO23611-3First edition2007-05-01Soil quality — Sampling of soil invertebrates — Part 3: Sampling and soil extraction of enchytraeids Qualité du sol — Prélèvement des invertébrés du sol — Partie 3: Prélèvement et extraction des enchytréides
SIST EN ISO 23611-3:2012
ISO 23611-3:2007(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.
COPYRIGHT PROTECTED DOCUMENT
©
ISO 2007 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 2007 – All rights reserved
SIST EN ISO 23611-3:2012
ISO 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 Reagents.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)
Species identification in enchytraeids.7 Annex B (informative)
Quick extraction of enchytraeids.8 Bibliography.10
SIST EN ISO 23611-3:2012
ISO 23611-3:2007(E) iv © ISO 2007 – 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 23611-3 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 soil-inhabiting nematodes SIST EN ISO 23611-3:2012
ISO 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 standardization 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 bioindication 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 or 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 taxonomical 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 cycling 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 standardize 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 [37] and ISO 14240-2 [38])]. SIST EN ISO 23611-3:2012
SIST EN ISO 23611-3:2012
INTERNATIONAL STANDARD ISO 23611-3:2007(E) © 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 as 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 enchytraeids 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 Reference [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 climatic 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 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 enchytraeids small soil-inhabiting 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 using 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 extraction, 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). SIST EN ISO 23611-3:2012
ISO 23611-3:2007(E) 2 © ISO 2007 – All rights reserved The determination of the biomass of enchytraeids is 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 parts 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 Ethanol, 70 % (volume fraction). 4.3 Bengalred, 4,5,6,7-Tetrachloro-2',4',5',7'-tetraiodofluorescein formulated as a staining agent. 4.4 Bouin’s fixative, buffered solution of formaldehyde, acetic acid and picric acid. 4.5 Paracarmin, staining agent, prepared as a mixture of carmine acid, aluminium chloride and 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) α- and β-Canadinol acid (C19H30O2) and 5 % (volume fraction) Canadoesen (C21H40O). 5 Apparatus 5.1 Split soil corer (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 cm, 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 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. SIST EN ISO 23611-3:2012
ISO 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 usually 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 opened 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 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 samples (i.e. soil cylinders) are put in the sieves, and are, if necessary (e.g. in cases of heavy loam soils), carefully broken apart by hand (see Figure 1). The bottom of the sieves should not reach the bottom of the bowls. To ensure an extraction efficiency of Enchytraeidae from the samples of more than 90 %, the extraction of soil should last for 4 d to 7 d and of litter for 0,5 d to 2 d at (12 ± 2) °C (water temperature). The dur
...
SLOVENSKI STANDARD
kSIST FprEN ISO 23611-3:2011
01-april-2011
[Not translated]
Soil quality - Sampling of soil invertebrates - Part 3: Sampling and soil extraction of
enchytraeids (ISO 23611-3:2007)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme
und Bodenextraktion von Enchytraeen (ISO 23611-3:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 3: Prélèvement et extraction
des enchytréides (ISO 23611-3:2007)
Ta slovenski standard je istoveten z: FprEN ISO 23611-3
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
kSIST FprEN ISO 23611-3:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN ISO 23611-3:2011
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kSIST FprEN ISO 23611-3:2011
EUROPEAN STANDARD
FINAL DRAFT
FprEN ISO 23611-3
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2010
ICS 13.080.30; 13.080.05
English Version
Soil quality - Sampling of soil invertebrates - Part 3: Sampling
and soil extraction of enchytraeids (ISO 23611-3:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie Bodenbeschaffenheit - Probenahme von Wirbellosen im
3: Prélèvement et extraction des enchytréides (ISO 23611- Boden - Teil 3: Probenahme und Bodenextraktion von
3:2007) Enchytraeen (ISO 23611-3:2007)
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 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 Ref. No. FprEN ISO 23611-3:2010: E
worldwide for CEN national Members.
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kSIST FprEN ISO 23611-3:2011
FprEN ISO 23611-3:2010 (E)
Contents Page
Foreword .3
2
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kSIST FprEN ISO 23611-3:2011
FprEN ISO 23611-3:2010 (E)
Foreword
The text of ISO 23611-3:2007 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 23611-3:2010 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 23611-3:2007 has been approved by CEN as a FprEN ISO 23611-3:2010 without any
modification.
3
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kSIST FprEN ISO 23611-3:2011
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kSIST FprEN ISO 23611-3:2011
INTERNATIONAL ISO
STANDARD 23611-3
First edition
2007-05-01
Soil quality — Sampling of soil
invertebrates —
Part 3:
Sampling and soil extraction of
enchytraeids
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 3: Prélèvement et extraction des enchytréides
Reference number
ISO 23611-3:2007(E)
©
ISO 2007
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kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(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.
COPYRIGHT PROTECTED DOCUMENT
© ISO 2007
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 2007 – All rights reserved
---------------------- Page: 8 ----------------------
kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 1
4 Reagents. 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) Species identification in enchytraeids. 7
Annex B (informative) Quick extraction of enchytraeids . 8
Bibliography . 10
© ISO 2007 – All rights reserved iii
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kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(E)
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 23611-3 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 soil-inhabiting nematodes
iv © ISO 2007 – All rights reserved
---------------------- Page: 10 ----------------------
kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(E)
Introduction
This part of ISO 23611 has been drawn up since there is a growing need for the standardization 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 bioindication 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 or 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 taxonomical and ecological studies, investigating
the role of enchytraeids in various soil ecosystems. These animals without doubt belong to the most important
[5]
soil invertebrates in temperate regions (mainly in acidic soils ). Their influence on soil functions like litter
[14], [19]
decomposition and nutrient cycling is well-known . Due to their number which is often very high (and to
[4]
their population biomass), they are also important in many terrestrial food-webs . Some species have
unintentionally been distributed by man in many soils of the world.
Since it is neither possible nor useful to standardize methods for all soil organisms, the most important
ones have been selected. [Microbiological parameters are already covered by existing ISO guidelines
[29] [37] [38]
(e.g. ISO 10381-6 , ISO 14240-1 and ISO 14240-2 )].
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kSIST FprEN ISO 23611-3:2011
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kSIST FprEN ISO 23611-3:2011
INTERNATIONAL STANDARD ISO 23611-3:2007(E)
Soil 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 as 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 enchytraeids 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 Reference [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 climatic 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 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
enchytraeids
small soil-inhabiting 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 using 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-
[11], [17], [20]
known for a long time .) After extraction, 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).
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kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(E)
The determination of the biomass of enchytraeids is 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 parts 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 Ethanol, 70 % (volume fraction).
4.3 Bengalred, 4,5,6,7-Tetrachloro-2',4',5',7'-tetraiodofluorescein formulated as a staining agent.
4.4 Bouin’s fixative, buffered solution of formaldehyde, acetic acid and picric acid.
4.5 Paracarmin, staining agent, prepared as a mixture of carmine acid, aluminium chloride and calcium
chloride solved in ethanol.
4.6 Canada-balm, natural yellowish viscous fluid containing 13 % to 14 % (volume fraction) Canadin acid
(C H O ), 48 % to 50 % (volume fraction) α- and β-Canadinol acid (C H O ) and 5 % (volume fraction)
20 38 2 19 30 2
Canadoesen (C H O).
21 40
5 Apparatus
5.1 Split soil corer (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 cm, 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 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.
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kSIST FprEN ISO 23611-3:2011
ISO 23611-3:2007(E)
6 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
usually 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 opened 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 two weeks (storage should not exceed one month in any
[7]
case ). 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
samples (i.e. soil cylinders) are put in the sieves, and are, if necessary (e.g. in cases of heavy loam soils),
carefully broken apart by hand (see Figure 1). The bottom of the sieves should not reach the bottom of the
bowls. To ensure an extraction efficiency of Enchytraeidae from the samples of more than 90 %, the
extraction of soil should last for 4 d to 7 d and of litter for 0,5 d to 2 d at (12 ± 2) °C (water temperature). The
duration depends mainly on the organic content of the sample. These times can be modified accordi
...
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