Soil quality - Sampling of soil invertebrates - Part 3: Sampling and extraction of enchytraeids (ISO 23611-3:2019)

This document 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 is included in the Bibliography.
This document applies to all terrestrial biotopes in which enchytraeids occur. The sampling design of field studies in general is given in ISO 18400-101. 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 arthropods are given in ISO 23611-1, ISO 23611-2, ISO 23611-4 and ISO 23611-5.
This document is not applicable for very wet or flooded 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 soil properties, climate and land use). However, such a characterization is not covered by this document. 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.

Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme und Extraktion von Enchytraeen (ISO 23611-3:2019)

Dieses Dokument 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.
Dieses Dokument gilt für alle terrestrischen Biotope, in denen Enchytraeen leben. Der für Freiland¬unter-suchungen im Allgemeinen gültige Probenahmeplan ist bereits in ISO 18400 101 festgelegt. Diese Einzel-heiten können in Abhängigkeit von den klimatischen/regionalen Bedingungen des zu beprobenden Stand-orts variieren. Verweisung [6] vermittelt einen Überblick zur Bestimmung der Wirkungen von Verun-reinigungen auf Enchytraeen unter Freilandbedingungen.
Die Verfahren für einige andere Bodenorganismengruppen, wie Regenwürmer und Arthropoden, sind in ISO 23611 1, ISO 23611 2, ISO 23611 4 und ISO 23611 5 festgelegt.
Dieses Dokument gilt nicht für nasse oder überflutete Böden und kann unter extremen klimatischen oder geographischen Bedingungen (z. B. im Hochgebirge) schwierig anzu¬wenden sein.
Für die Probenahme von Wirbellosen in Böden wird nachdrücklich empfohlen, den Standort zu charakterisieren (z. B. Berücksichtigung der Bodeneigenschaften, des Klimas und der Bodennutzung). Eine derartige Charakterisierung wird jedoch in diesem Dokument 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 23611-3:2019)

Le présent document spécifie une méthode pour le prélèvement, la manipulation et l'extraction des enchytréides du sol prélevés 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).
Les informations de base concernant l'écologie des enchytréides et leur utilisation en tant que bio-indicateurs dans l'environnement terrestre sont disponibles dans la Bibliographie.
Le présent document s'applique à tous les biotopes terrestres dans lesquels des enchytréides sont présents. La conception de l'échantillonnage pour les études sur le terrain est, de manière générale, décrite dans l'ISO 18400‑101. Ces détails peuvent varier en fonction des conditions climatiques/régionales du site sur lequel l'échantillon doit être prélevé et un aperçu de la détermination des effets sur site des polluants vis-à-vis des enchytréides est fourni dans la Référence [6].
Des méthodes applicables à d'autres groupes d'organismes du sol, comme les vers de terre ou les arthropodes, sont données dans les normes ISO 23611‑1, ISO 23611‑2, ISO 23611‑4 et ISO 23611‑5.
Le présent document n'est pas applicable aux sols très humides ou inondés et peut s'avérer difficile à mettre en œuvre dans des conditions climatiques ou géographiques extrêmes (en haute montagne, par exemple).
Lors du prélèvement d'invertébrés du sol, il est vivement recommandé de caractériser le site (par exemple, du point de vue des propriétés du sol, du climat et de l'utilisation du terrain). Cependant, cette caractérisation n'est pas traitée dans le présent document. Les normes ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 et ISO 11465 sont plus appropriées pour le mesurage du pH, de la répartition granulométrique, du rapport C/N, de la teneur en carbone organique et de la capacité de rétention d'eau.

Kakovost tal - Vzorčenje nevretenčarjev v tleh - 3. del: Vzorčenje in ekstrakcija enhitrej iz tal (ISO 23611-3:2019)

Ta standard določa metodo za vzorčenje enhitrejev iz tal na kopenskem terenu ter rokovanje z njimi kot predpogoj za uporabo teh živali kot bioloških indikatorjev (npr. za oceno kakovosti tal kot habitata za organizme). Osnovne informacije o ekologiji enhitrejev in njihovi uporabi kot bioloških indikatorjev v kopenskem okolju so vključene v bibliografiji. Ta dokument se uporablja za vse kopenske biotope, v katerih se pojavljajo enhitreji. Načrtovanje vzorčenja za terenske raziskave na splošno je podano v standardu ISO 18400-101. Te podrobnosti se lahko razlikujejo glede na podnebne/regionalne razmere območja, ki ga je treba vzorčiti, pregled nad ugotavljanjem učinkov onesnaževal na enhitreje v terenskih razmerah pa je naveden v referencah [6]. Metode za nekatere druge skupine talnih organizmov, kot so deževniki ali členonožci, so podane v standardih ISO 23611-1, ISO 23611-2, ISO 23611-4 in ISO 23611-5. Ta dokument se ne uporablja za zelo vlažna ali poplavljena tla, uporaba za ekstremne podnebne ali geografske razmere (npr. v visokogorju) pa je lahko otežena. Pri vzorčenju nevretenčarjev iz tal je zelo priporočljivo opisati območje (npr. lastnosti tal, podnebje in raba tal). Vendar tak opis v tem dokumentu ni zajet. Za merjenje vrednosti pH, porazdelitve velikosti delcev, razmerja C/N, vsebnosti organskega ogljika in zmogljivosti zadrževanja vode so primernejši standardi ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 in ISO 11465.

General Information

Status
Published
Publication Date
01-Oct-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
02-Oct-2019
Completion Date
02-Oct-2019

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SLOVENSKI STANDARD
SIST EN ISO 23611-3:2020
01-januar-2020
Nadomešča:
SIST EN ISO 23611-3:2012

Kakovost tal - Vzorčenje nevretenčarjev v tleh - 3. del: Vzorčenje in ekstrakcija

enhitrej iz tal (ISO 23611-3:2019)

Soil quality - Sampling of soil invertebrates - Part 3: Sampling and extraction of

enchytraeids (ISO 23611-3:2019)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 3: Probenahme
und Bodenextraktion von Enchytraeen (ISO 23611-3:2019)

Qualité du sol - Prélèvement des invertébrés du sol - Partie 3: Prélèvement et extraction

des enchytréides (ISO 23611-3:2019)
Ta slovenski standard je istoveten z: EN ISO 23611-3:2019
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 23611-3:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 23611-3:2020
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SIST EN ISO 23611-3:2020
EN ISO 23611-3
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 13.080.05; 13.080.30 Supersedes EN ISO 23611-3:2011
English Version
Soil quality - Sampling of soil invertebrates - Part 3:
Sampling and extraction of enchytraeids (ISO 23611-
3:2019)

Qualité du sol - Prélèvement des invertébrés du sol - Bodenbeschaffenheit - Probenahme von Wirbellosen

Partie 3: Prélèvement et extraction des enchytréides im Boden - Teil 3: Probenahme und Bodenextraktion

(ISO 23611-3:2019) von Enchytraeen (ISO 23611-3:2019)
This European Standard was approved by CEN on 12 August 2019.

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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-3:2019 E

worldwide for CEN national Members.
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SIST EN ISO 23611-3:2020
EN ISO 23611-3:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 23611-3:2020
EN ISO 23611-3:2019 (E)
European foreword

This document (EN ISO 23611-3:2019) has been prepared by Technical Committee ISO/TC 190 "Soil

quality" in collaboration with Technical Committee CEN/TC 444 “Test methods for environmental

characterization of solid matrices” 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 April 2020, and conflicting national standards shall be

withdrawn at the latest by April 2020.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 23611-3:2011.

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, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 23611-3:2019 has been approved by CEN as EN ISO 23611-3:2019 without any

modification.
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SIST EN ISO 23611-3:2020
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SIST EN ISO 23611-3:2020
INTERNATIONAL ISO
STANDARD 23611-3
Second edition
2019-08
Soil quality — Sampling of soil
invertebrates —
Part 3:
Sampling and extraction of
enchytraeids
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 3: Prélèvement et extraction des enchytréides
Reference number
ISO 23611-3:2019(E)
ISO 2019
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 2

7 Procedure..................................................................................................................................................................................................................... 3

7.1 Soil sampling ............................................................................................................................................................................................. 3

7.2 Extraction of the enchytraeids .................................................................................................................................................. 3

7.3 Microscopic identification ............................................................................................................................................................. 4

7.4 Preservation of Enchytraeidae .................................................................................................................................................. 5

7.5 Validity of the extraction process............................................................................................................................................ 5

7.6 Determination of biomass ............................................................................................................................................................. 5

8 Data assessment.................................................................................................................................................................................................... 5

9 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Quick extraction of enchytraeids ................................................................................................................ 7

Annex B (informative) Examples of the use of soil invertebrates in soil monitoring

programmes (including presentation of their results) ................................................................................................ 9

Bibliography .............................................................................................................................................................................................................................11

© ISO 2019 – All rights reserved iii
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological characterization.

This second edition cancels and replaces the first edition (ISO 23611-3:2007), which has been technically

revised. The main changes to the previous edition are as follows:

— addition of examples of enchytraeid monitoring programmes (including presentation of their

results) as an informative annex.
A list of all parts in the ISO 23611 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)
Introduction

This document has been developed to address 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 [4], [25], [27],

[31], [36]);

— terrestrial bioindication and long-term monitoring (e.g. References [4], [30]);

— evaluation of the effects of chemicals on soil animals (References [18], [26], [28]).

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 with 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

[7]

to the most important soil invertebrates in temperate regions (mainly in acidic soils ). Their influence

[17][23]

on soil functions like litter decomposition and nutrient cycling is well known . Due to their often

very high numbers, and their population biomass, they are also important in many terrestrial food-

[6]

webs . Some species have unintentionally been distributed by humans 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 standards

(e.g. ISO 14240-1, ISO 14240-2, ISO 17601, ISO/TS 29843-1 and ISO/TS 29843-2.
© ISO 2019 – All rights reserved v
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SIST EN ISO 23611-3:2020
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SIST EN ISO 23611-3:2020
INTERNATIONAL STANDARD ISO 23611-3:2019(E)
Soil quality — Sampling of soil invertebrates —
Part 3:
Sampling and extraction of enchytraeids
1 Scope

This document 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 is included in the Bibliography.

This document applies to all terrestrial biotopes in which enchytraeids occur. The sampling design of

field studies in general is given in ISO 18400-101. 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 arthropods are given in

ISO 23611-1, ISO 23611-2, ISO 23611-4 and ISO 23611-5.

This document is not applicable for very wet or flooded 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

soil properties, climate and land use). However, such a characterization is not covered by this document.

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 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp/
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
enchytraeid

small soil-inhabiting worm (a few millimetres to several centimetres in length) belonging to the family

Enchytraeidae, class Oligochaeta, superclass Clitellata, phylum Annelida
[35]
Note 1 to entry: The common name for enchytraeid is potworm .
EXAMPLE Species of the genera Enchytraeus, Fridericia or Cognettia.
© ISO 2019 – All rights reserved 1
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)
4 Principle

Enchytraeids at a certain site are sampled from the soil by using a split soil corer (diameter usually

3 cm to 6 cm) (6.1). 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.

[12][20][24]

(This approach has been well 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).

The determination of the biomass of enchytraeids is also described in this document. 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). Examples of the use of soil invertebrates are given in

Annex B. The design of such programmes is not included in this document (but see e.g. Reference [3]).

NOTE 2 Some hints for the taxonomy of enchytraeids are given in the Bibliography.

5 Reagents
5.1 Tap water (without toxic properties, e.g. due to copper contamination).
5.2 Ethanol, 70 % (volume fraction).

5.3 Bengalred, 4,5,6,7-Tetrachloro-2',4',5',7'-tetraiodofluorescein formulated as a staining agent.

5.4 Bouin’s fixative, buffered solution of formaldehyde, acetic acid and picric acid.

5.5 Paracarmin, staining agent, prepared as a mixture of carmine acid, aluminium chloride and

calcium chloride solved in ethanol.

5.6 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

20 38 2 19 30 2
fraction) Canadoesen (C H O).
21 40
6 Apparatus

6.1 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) and a plastic or wooden impact-absorbing hammer.

6.2 Plastic bags (e.g. 1-l freezer bags); general store.
6.3 Temperature recorder or a minimum/maximum-thermometer.

6.4 Plastic bowls, diameter approximately 20 cm, height approximately 10 cm; general store.

6.5 Plastic sieves, diameter approximately 15 cm, mesh width approximately 1,0 mm; general store.

6.6 60 W bulbs as a heating device; general store.

6.7 Glassware, for example petri dishes (square format) with a size of 8 cm × 8 cm or small glass

vessels (e.g. 50 ml).
2 © ISO 2019 – All rights reserved
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)
6.8 Large, sharp knife.
6.9 Refrigerator.
6.10 Dissecting microscope with low magnification (10 times to 40 times).

6.11 Microscope with high magnification (60 times to 400 times) and equipped with an interference

lighting device.
6.12 Spring steel pincers (flat).
6.13 Transfer tool, pasteur pipette, soft steel forceps or a hooked needle.
7 Procedure
7.1 Soil sampling

The soil samples to be used for the investigation of the enchytraeid community are taken destructively

by means of a split soil corer (6.1). The corer is carefully pressed into the soil. The depth depends on

the land use and soil type, but usually varies from 10 cm (e.g. forests) up to 30 cm (e.g. crop sites), i.e.

those layers in which the bulk of the enchytraeids are living. In rare cases, for example if thick roots are

present, a plastic or wooden impact-absorbing 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 in height) with a knife (6.8). These soil cylinders may be stored in

small plastic bags (6.2) in a refrigerator (6.9) at approximately 4 °C to 6 °C for a period of preferably not

[9]

longer than one week to two weeks (storage should not exceed one month in any case ). The soil corer

is cleaned with water afterwards.
7.2 Extraction of the enchytraeids

In principle, the extraction of the worms from the soil is caused by their active movement through the

water-saturated sample into the water-filled bowl (6.4).

The extraction should commence as soon as possible after the sampling (see 7.1). The bowls (6.4) are

carefully filled up with tap water (5.1). 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 samples

in the sieve shall be completely submerged and the bottoms 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 2 d to 7 d and extraction 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 according to organizational requirements and the number of individuals in a sample.

However, the worms quickly die if an oxygen deficiency occurs. In order to avoid this problem, the

water should be changed after 18 h to 24 h, and again after 48 h (if extraction period exceeds 2 d). For

that purpose the sieve with the sample should be carefully transferred to a bowl with fresh water. An

acceleration of the extraction using a heat source [e.g. a 60 W bulb (6.6)] placed above the sample can be

helpful, but should be carefully used (i.e. slow temperature increase over at least 3 h), since otherwise

— species-specifically — many animals, especially juveniles and fragmentation stages, remain in the

soil (see Annex A).

NOTE 1 In order to reduce the amount of debris at the bottom of the extraction bowls, a fine wiping cloth

(mesh size 1 mm) can be put in the sieve before the soil sample is put in.

At the end of the extraction procedure, the sieves are removed. The requirements for the disposal of the

soil are given in the appropriate national regulations. The water is slowly and carefully decanted from

the bowl. The finest fraction of soil at the bottom of the bowls should not be disturbed (see Figure 2). A

small amount of water (up to a height of 5 mm to 10 mm) shall remain in the bowls. Subsequently, the

© ISO 2019 – All rights reserved 3
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)

finest fraction of soil is suspended in the overlying water, placed in a petri dish (6.7) and briefly stored

until soil particles have settled and the water becomes clear. Since the whitish worms are heavier than

water, but are rarely able to hide themselves in the narrow soil layer, they can easily be collected out

of the petri dish under a dissecting microscope (6.10). For this transfer, a soft steel forceps, a Pasteur

pipette or a hooked needle (6.13) can be used, but in any case, damaging of the worms shall be avoided.

The most convenient way of counting the total number is to divide the surface of the petri dish into

parallel rows which are checked one after another. Due to their white colour, the worms are clearly

visible against the usually brownish soil particles. The animals are transferred to small plastic or glass

vessels (e.g. 20 ml).

The number of samples which can be extracted simultaneously is theoretically unlimited. However, due

to the size of the water bowls, space limitations can occur. Since they (i.e. at least the water) shall be

cooled, usually only up to 40 to 50 samples can be processed at one time. These limitations can be

overcome by carrying out the procedure in a cool room, for example a cellar.

NOTE 2 In rare cases, the enchytraeids can be confused with Diptera larvae (which very often possess

brownish or black head capsules) or nematodes (non-peristaltic movement; usually smaller and faster moving

than oligochaetes). Additionally, fungal hyphae or fine root material can be mistaken for enchytraeids, since they

can possess the same length and colour. However, they always lack the segmentation of oligochaete worms.

Figure 1 — Extraction bowl with soil sample
Figure 2 — Sediment layer (including enchytraeids)
7.3 Microscopic identification

The microscopic identification should be done as soon as possible, because the animals die in water

after some days, even if stored in a refrigerator (6.9). A soft steel forceps, a Pasteur pipette or a hooked

needle (6.13) can be used carefully to transfer the animals with a drop of water to a slide. If the worms

are moving too fast on the slide, they can be anaesthetised with CO (e.g. by using a drop of mineral

water with gas, but it should be used with care, otherwise the worms are killed).

NOTE Identification of the enchytraeids is difficult. Therefore, in many cases only the number of animals

[20] [32]

is determined. Otherwise, keys for European enchytraeid species and for species of the genus Fridericia ,

as well as other publications, are available. A compromise can be the use of a site-specific key, since usually

only three to 25 species occur at any given site (in this case, often worms fixed in ethanol can be identified to

the species level). An overview of the information (e.g. parameters, drawings) needed for the identification of a

certain species is given in Reference [11].
4 © ISO 2019 – All rights reserved
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SIST EN ISO 23611-3:2020
ISO 23611-3:2019(E)

In the case of high individual numbers (>100) a sample partitioning can be carried out. Partitioning

shall guarantee that all species (whether big or small) are parted to a known degree. Therefore, this

process shall be described in detail.
7.4 Preservation of Enchytraeidae

Enchytraeidae can be preserved for further investigations (e.g. species descriptions) in 70 % (volume

[33]

fraction) ethanol (5.2) . However, the preservation is accompanied by a loss of visible morphological

details. Animals difficult to identify or those selected as reference specimen may also be identified

after a fixing in Bouin (5.4), respective colouring in Paracarmin (5.5) and storing in Canada-balm (5.6)

(which can be relatively elaborate). For species identification, the use of a microscope (6.11) equipped

with differential interference contrast (DIC) is strongly recommended.
7.5 Validity of the extraction process

Extraction efficiency can be checked by fixing soil samples with ethanol (96 %), which are taken in

parallel to other field samples. The soil is spread in a thin layer on the bottom of a flat plastic vessel

(e.g. Bellaplast 16 cm × 11 cm) and then the ethanol is added. Afterwards, some drops of Bengalred

(5.3) are applied to the ethanol. After one day, the bright-red coloured worms can easily be counted.

However, this procedure is only necessary when using samples from an unknown site for the first time.

Additionally, this check shall be done with six to eight replicates since the variability of enchytraeid

numbers can be quite high.
7.6 Determination of biomass

For an estimation of the ecological role (e.g. in the soil food-web) of enchytraeids at a certain site, the

determination of their biomass is necessary. Since direct weighing is difficult due to the low individual

mass of most species, potential corruption from changing gut content and, in particular, quick

desiccation, the biomass may also be indirectly estimated by the following methods:

[1][2]

— creation of specific species calibration curves to the ratio of length and mass then measurement

of the length of the animals in a sample;

— automatic calculation of the mass by computer-assisted measuring of the length-mass-ratio

of embedded individuals and following computation with the largely constant density of

[23]
enchytraeids .
8 Data assessment
The following measurement en
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