EN ISO 23611-1:2018

Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2018)

This document 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).
This document applies to all terrestrial biotopes in which earthworms occur. The sampling design of field studies in general is given in ISO 18400‑101 and guidance on the determination of effects of pollutants on earthworms in field situations is given in ISO 11268‑3. These aspects can vary according to the national requirements or the climatic/regional conditions of the site to be sampled (see also Annex C).
This document 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.

Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Extraktion von Regenwürmern (ISO 23611-1:2018)

Dieses Dokument 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.
Dieses Dokument gilt für alle terrestrischen Biotope, in denen Regenwürmer leben. Der für Freilanduntersuchungen im Allgemeinen gültige Probenahmeplan ist bereits in ISO 18400-101 angegeben, und eine Anleitung zur Bestimmung der Wirkungen von Schadstoffen auf Regenwürmer unter Freilandbedingungen wird in ISO 11268-3 gegeben. Diese Aspekte können den nationalen Anforderungen entsprechend oder in Abhängigkeit von den klimatischen/regionalen Bedingungen des zu beprobenden Standorts variieren (siehe auch Anhang C).
Dieses Dokument 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.

Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction des vers de terre (ISO 23611-1:2018)

Le présent document 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).
Le présent document est utilisé pour tous les biotopes terrestres abritant des vers de terre. La conception de l'échantillonnage pour les études sur le terrain est, de manière générale, décrite dans l'ISO 18400‑101 et les lignes directrices relatives à la détermination des effets sur site des polluants vis-à-vis des vers de terre sont données dans l'ISO 11268‑3. Ces aspects peuvent varier en fonction des exigences nationales ou des conditions climatiques/régionales du site sur lequel l'échantillon doit être prélevé (voir également l'Annexe C).
Le présent document ne s'applique pas aux sols semi-terrestres et il peut s'avérer difficile de l'appliquer dans des conditions climatiques ou géographiques extrêmes (en haute montagne, par exemple). Les méthodes pour quelques autres groupes d'organismes du sol, tels que les Collemboles, sont traitées dans d'autres parties de l'ISO 23611.

Kakovost tal - Vzorčenje nevretenčarjev v tleh - 1. del: Ročno razvrščanje deževnikov in njihova ekstrakcija (ISO 23611-1:2018)

Ta dokument določa metodo za vzorčenje deževnikov iz tal na terenu ter rokovanje z njimi kot predpogoj za uporabo teh živali kot bioloških indikatorjev (npr. za oceno kakovosti tal kot habitata za organizme).
Ta dokument se uporablja za vse kopenske biotope, v katerih se pojavljajo deževniki. Načrtovanje vzorčenja pri terenskih raziskavah je na splošno določeno v standardu ISO 18400-101, smernice za določevanje učinkov onesnaževal na deževnike v okoliščinah na terenu pa so določene v standardu ISO 11268-3. Ti vidiki se lahko razlikujejo glede na nacionalne zahteve ali podnebne razmere/regionalne pogoje lokacije, kjer bo potekalo vzorčenje (glej tudi dodatek C).
Ta dokument se ne uporablja za polkopenska tla, uporaba za ekstremne podnebne ali geografske razmere (npr. v visokogorju) pa je lahko otežena. Metode za nekatere druge skupine organizmov v tleh, kot so skakači, so obravnavane v drugih delih standarda ISO 23611.

General Information

• Status: Published
• Publication Date: 30-Oct-2018
• Withdrawal Date: 30-Jan-2019
• ICS: 13.080.05 - Examination of soils in general
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 11-Jul-2018
• Completion Date: 11-Jul-2018
• Ref Project: SIST EN ISO 23611-1:2018 - Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2018)

Relations

• Replaces: EN ISO 23611-1:2011 - Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and formalin extraction of earthworms (ISO 23611-1:2006)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 23611-1:2018
01-december-2018
1DGRPHãþD
SIST EN ISO 23611-1:2011
.DNRYRVWWDO9]RUþHQMHQHYUHWHQþDUMHYYWOHKGHO5RþQRUD]YUãþDQMH
GHåHYQLNRYLQQMLKRYDHNVWUDNFLMD,62

Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of

earthworms (ISO 23611-1:2018)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese
und Extraktion von Regenwürmern (ISO 23611-1:2018)

Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction

des vers de terre (ISO 23611-1:2018)
Ta slovenski standard je istoveten z: EN ISO 23611-1:2018
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 23611-1:2018 en,fr,de

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

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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
EN ISO 23611-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2018
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-1:2011
English Version
Soil quality - Sampling of soil invertebrates - Part 1: Hand-
sorting and extraction of earthworms (ISO 23611-1:2018)

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

Partie 1: Tri manuel et extraction des vers de terre (ISO im Boden - Teil 1: Handauslese und Extraktion von

23611-1:2018) Regenwürmern (ISO 23611-1:2018)
This European Standard was approved by CEN on 3 May 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-1:2018 E

worldwide for CEN national Members.
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SIST EN ISO 23611-1:2018
EN ISO 23611-1:2018 (E)
Contents Page

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

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SIST EN ISO 23611-1:2018
EN ISO 23611-1:2018 (E)
European foreword

This document (EN ISO 23611-1:2018) 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 January 2019, and conflicting national standards shall

be withdrawn at the latest by January 2019.

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 23611-1:2011.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 23611-1:2018 has been approved by CEN as EN ISO 23611-1:2018 without any

modification.
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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
INTERNATIONAL ISO
STANDARD 23611-1
Second edition
2018-05
Soil quality — Sampling of soil
invertebrates —
Part 1:
Hand-sorting and extraction of
earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
Reference number
ISO 23611-1:2018(E)
ISO 2018
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(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 ..................................................................................................................................................................................................................... 3

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

7.1 Sampling of the earthworms ....................................................................................................................................................... 3

7.1.1 General...................................................................................................................................................................................... 3

7.1.2 Hand-sorting ....................................................................................................................................................................... 4

7.1.3 AITC extraction .................................................................................................................................................................. 5

7.2 Preservation .............................................................................................................................................................................................. 6

7.3 Determination of biomass ............................................................................................................................................................. 6

8 Data assessment.................................................................................................................................................................................................... 6

9 Test report ................................................................................................................................................................................................................... 7

Annex A (informative) Other methods for sampling ........................................................................................................................... 8

Annex B (informative) Species determination in earthworms ................................................................................................. 9

Annex C (informative) The modified TSBF method ............................................................................................................................10

Annex D (normative) Determination of maximum water-holding capacity ............................................................11

Annex E (informative) Examples of earthworm monitoring programmes (including

presentation of their results) ...............................................................................................................................................................12

Bibliography .............................................................................................................................................................................................................................16

© ISO 2018 – All rights reserved iii
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(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 on 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 the following URL: 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 23611-1:2006), which has been technically

revised. The main changes are:

— the use of a new chemical extraction compound, AITC (allyl-isothiocyanate), instead of formalin;

— the addition of examples of earthworm monitoring programmes (including presentation of their

results) as an informative Annex E.
A list of all parts in the ISO 23611 series can be found on the ISO website.
iv © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
Introduction

This document 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 necessary for the following purposes:
[25][31][39]
— biological classification of soils including soil quality assessment ;
[11][14][33]
— terrestrial bio-indication and long-term monitoring ;
— 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.

Since it is neither possible nor useful to standardize methods for all soil organisms, the most important

ones have been selected. In this document sampling of earthworms is described.

Originally, the methods described in this document 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 boreal and

[30][16][18]

tropical soils . Since Darwin (1881) (see Reference [8]), their influence on soil structure (e.g.

aeration, water holding capacity) and soil functions like litter decomposition and nutrient cycling is well-

[10]

known . Due to their often very high biomass they are also important in many terrestrial food-webs.

In the previous version of this document the chemical formalin was recommended as extraction fluid.

However, within the last years evidence increased that formalin does have critical properties, mainly

in terms of human toxicity. In December 2012, the Risk Assessment Committee (RAC) of the European

Chemicals Agency (ECHA) stated that there is sufficient scientific evidence to classify this chemical as

“probably carcinogenic for humans (Category Ib). In addition, negative effects on non-target organisms

(including soil microorganisms, mesofauna and plants) have been reported (e.g. see Reference [7]).

Therefore, this substance has been replaced.

Due to the growing reservations against the use of formalin, several alternatives have been studied. In

Reference [40] allyl isothiocyanate (AITC) was tested for its effectiveness as a chemical expellant for

sampling earthworms. AITC is a natural breakdown product of glucosinolates in many Cruciferae, i.e.

it is the component imparting the sharp taste of mustard. According to the European Chemical Agency

(ECHA), there is no concern regarding its use under outdoor conditions.

Over the last years, some studies have been performed in which the extraction efficiency of formalin

and AITC were compared at the same sites and dates. According to Reference [22] no differences

were found in numbers or biomass of earthworms extracted at crop sites when using either formalin

or AITC as extractant. In a recent unpublished review (see Reference [28]) no significant differences

were reported in earthworm numbers/biomass when comparing the efficiency of the two extraction

chemicals. Also, no interaction was found on the sampling sites between the extractant and the site,

indicating that no site-specific differences were observed in extraction efficiency of the extractants.

When plotting the correlation between worm numbers extracted with AITC versus formalin in a

Bland-Altman graph (a common way to compare a gold-standard method to an alternative method in

the medical sciences), no significant bias of the AITC method as compared to the formalin method was

found, indicating the similarity / exchangeability of the two methods.

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.
© ISO 2018 – All rights reserved v
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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
INTERNATIONAL STANDARD ISO 23611-1:2018(E)
Soil quality — Sampling of soil invertebrates —
Part 1:
Hand-sorting and extraction of earthworms
1 Scope

This document 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).

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

of field studies in general is given in ISO 18400-101 and guidance on the determination of effects of

pollutants on earthworms in field situations is given in ISO 11268-3. These aspects can vary according

to the national requirements or the climatic/regional conditions of the site to be sampled (see also

Annex C).

This document 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.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10390, Soil quality — Determination of pH

ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary

analysis)

ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level

using barium chloride solution

ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by

sieving and sedimentation

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —

Gravimetric method
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/
© ISO 2018 – All rights reserved 1
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
3.1
earthworms

megadrile soil-inhabiting earthworms belonging to the order Oligochaeta (class Clitellata, phylum

Annelida)

Note 1 to entry: The length of adult individuals can vary from a few centimetres to more than 1 m.

EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae (Latin America), Eudrilidae

(Africa) or Megascolecidae [Asia, North America (Pacific Coast)].
3.2
peregrine species
earthworms occurring in many regions world-wide today, usually introduced by man

Note 1 to entry: 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 to entry: See Reference [18].
3.3
clitellum

ring or saddle shaped epidermal thickening only in mature worms which is near the anterior and

eventually forms the cocoon
4 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 m ) of varying depth, depending on land use (e.g.

at crop sites: 20 cm), soil properties and the scope of the sampling;
— extraction of worms from the soil by applying AITC.

The first method is known for about 100 years while the second method using the new extraction fluid

[7][22][40]

was proposed about 15 years ago . 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 m ) or, more

rarely, volume parameters.

NOTE 1 Alternative methods can be useful under special circumstances (e.g. electrical extraction), but cannot

be recommended as a general procedure (see Annex A).

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 programmes is not included in this

document.

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.
5 Reagents

5.1 Allyl-isothiocyanate (AITC), synthetic grade (about 94 % to 97 % (volume fraction).

5.2 Isopropanol, 100 % (volume fraction).
5.3 Ethanol, 70 % (volume fraction).
2 © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)

5.4 Formalin, formaldehyde solution 4 % (volume fraction), for storage purposes only.

5.5 Ethanol, 95 % (volume fraction), for storage purposes when using genetic methods such as

barcoding.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Plastic vessels, capacities 250 ml and 500 ml, for storing the worms.
6.2 Rubber gloves.
6.3 Forceps.
2 2
6.4 Piece of thick plastic sheeting, 1 m to 2 m .
6.5 Spade or shovel.
6.6 Dissecting microscope, with low magnification (×10 to ×40).
6.7 Balance, weigh range from 0,01 g to 200 g.
6.8 Water-can, preferably 20 l, with water (20 l per sampling plot).
6.9 Watering can.
6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 Thermometer, e.g. for measuring air temperature.
6.12 Drying cabinet, for soil moisture determination.
7 Procedure
7.1 Sampling of the earthworms
7.1.1 General

Sampling of earthworms is done by a combination of two different methods: hand-sorting and AITC

extraction. Based on several comparative studies, the combination of a physical and a chemical method

is clearly recommended in the various reviews on earthworm ecology, independent from the type of

chemical expellant (e.g. References [9], [10] and [18]).

Sampling should be done at times of the year when 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 AITC).

In temperate regions, such unfavourable sampling times are winter and, in particular, midsummer

[18]

periods . 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, AITC extraction

is not necessary. On the other hand, at sites where giant earthworms are living (parts of South America,

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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
[26]

South East Asia and Australia), hand-sorting is not useful . 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

[35]
site is well known, worms can also be determined alive (see also Annex B).

In case the 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 AITC extraction, rinsing the worms in tap water is

needed before incubation in soil.

For the interpretation of test results, the following characteristics shall be determined for the field site

to be studied:
a) pH in accordance with ISO 10390;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465;
d) water holding capacity as specified in Annex D;
e) cationic exchange capacity in accordance with ISO 11260;
f) organic carbon in accordance with ISO 10694.
7.1.2 Hand-sorting

The size of the sample plot should be chosen according to the expected mean size and density of the

worms. 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 m )

are recommended (see ISO 11268-3). On the other hand, at sites with a high earthworm density (e.g.

2 [29]

many meadows in temperate regions), a smaller plot of 1/8 m is sufficient . Even smaller sample

2[41]

sizes (e.g. 1/16 m ) can lead to very low, and thus variable, individual worm numbers per sample,

which in turn leads to an increase in sample numbers (e.g. 16 replicates).

In any case, the soil is removed by means of a spade or shovel (6.5) 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 (6.4). 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 rubber gloves (6.2) and small ones by using forceps (6.3). 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.

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 (5.3) using the 250 ml or 500 ml

plastic vessels (6.1) for at least 0,5 h, but not longer than 24 h. In case the ethanol solution is diluted by

body fluids and/or contains soil particles, the ethanol solution shall be exchanged after 24 h to 48 h.

The vessels shall be labelled and observations (e.g. whether worms have been in a quiescence stage)

should be recorded in the notebook (6.10).
4 © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)

An immediate fixation in 4 % formalin (volume fraction) (5.4) is possible, but not recommended due

to the fact that the handling of this compound should be minimised as much as possible (in particular

under field conditions).

NOTE 2 In order to avoid morphological changes (e.g. an inversion of the prostomium) due to immediate

fixation in ethanol, the individual worms can be put briefly (about one minute) into warm (e.g. 30 °C to 40 °C) tap

water. The earthworms relax in the water, and after that they can be transferred to ethanol.

7.1.3 AITC extraction

The same plot, from which the top soil has been removed for hand-sorting, is used for AITC extraction.

A sufficient amount of water shall be transported (5 l to 10 l per sampling plot) beforehand to the plots

using large water-cans (6.8). To p
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