SIST EN ISO 23611-1:2026

• Abstract:

  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 is applicable to all terrestrial biotopes in which earthworms occur. This document does not apply to semi-terrestrial soils (i.e. soils that are partly aquatic, such as bogs, beaches, marshes, stream margins) and it can be difficult to use under extreme climatic or geographical conditions (e.g. in high mountains). Methods for other soil organism groups, such as micro-arthropods and enchytraeids (mesofauna), are covered in other parts of the ISO 23611 series.

• Status: Published
• Public Enquiry End Date: 14-Sep-2025
• Publication Date: 17-Aug-2026
• ICS: 13.080.05 - Examination of soils in general
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 22-Jul-2026
• Due Date: 26-Sep-2026
• Completion Date: 18-Aug-2026
• Ref Project: EN ISO 23611-1:2026 - Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2026)

• Replaces: SIST EN ISO 23611-1:2018 - Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2018)
• Effective Date: 01-Sep-2026

Overview

SIST EN ISO 23611-1:2026 – Soil quality – Sampling of soil invertebrates – Part 1: Hand-sorting and extraction of earthworms establishes standardized procedures for sampling and handling earthworms from field soils. Earthworms serve as vital bioindicators for assessing soil quality, ecosystem health, and the impact of pollutants and land management practices. This standard provides guidelines specific to terrestrial environments (excluding semi-terrestrial soils such as marshes or stream margins) and is intended for use in soil monitoring, ecological research, and environmental assessments. Developed by the Slovenian Institute for Standardization (SIST) as part of the international ISO 23611 series, this standard harmonizes field techniques to ensure consistent, reliable data for environmental professionals and researchers.

Key Topics

• Sampling Methods: Describes the combination of physical (hand-sorting) and chemical (allyl isothiocyanate, AITC) extraction techniques for earthworm collection.
• Field Procedure: Outlines steps for selecting sampling sites, determining plot size, soil excavation, and worm collection, tailored to soil type and expected earthworm density.
• Preservation and Storage: Details protocols for fixing and preserving collected specimens in ethanol or formalin, with notes for DNA analysis and taxonomic identification.
• Biomass Calculation: Includes guidance for measuring both fresh and dry biomass of earthworm samples, supporting soil biodiversity and ecological studies.
• Data Assessment: Recommends parameters such as soil pH, organic carbon content, texture, and cation exchange capacity to contextualize biological findings.
• Safety and Environmental Care: Emphasizes the use of personal protective equipment (PPE) when handling chemicals such as AITC and formalin, and outlines environmentally conscious procedures.

Applications

• Soil Quality Monitoring: Enables land managers and researchers to use earthworm abundance and diversity as sensitive indicators of environmental changes, pollution, and land management impacts.
• Ecological Research: Supports studies on earthworm community structure, habitat preferences, species identification, and biodiversity, facilitating comparability across regions and research projects.
• Environmental Impact Assessment: Provides a standardized framework to assess the effects of chemicals, remediation efforts, or agricultural practices on soil fauna, informing policy and regulatory decisions.
• Bioindicator Programs: Forms the biological foundation for long-term soil monitoring programs at local, national, and international scales, supporting sustainable land use and conservation planning.
• Taxonomic and Genetic Studies: Offers guidance for preserving earthworms for both morphological identification and modern genetic analysis, including DNA metabarcoding approaches.

Related Standards

For comprehensive soil quality assessment and sampling of other soil invertebrate groups, the following standards are relevant:

• ISO 23611 Series: Includes additional parts detailing sampling methods for micro-arthropods and enchytraeids (mesofauna).
• ISO 18400-101: General principles for soil sampling in field studies.
• ISO 11268-3: Guidance on assessing the effects of pollutants on earthworms in field scenarios.
• ISO 10390: Determination of pH in soil.
• ISO 10694: Determination of organic and total carbon in soil.
• ISO 11260: Measurement of cation exchange capacity and base saturation.
• ISO 11277: Soil particle size distribution analysis.
• ISO 11465: Procedures for determining water content and dry matter in soil.
• ISO 21286: Identification of test species by DNA barcoding.

Practical Value

Implementing SIST EN ISO 23611-1:2026 improves the reliability and comparability of earthworm sampling results across diverse soil environments. This aids environmental scientists, regulatory bodies, and land managers in making informed decisions based on high-quality, standardized biological data. By supporting soil health assessments, pollution studies, and ecotoxicological research, the standard contributes to better soil management and conservation of terrestrial ecosystems.

SLOVENSKI STANDARD
01-september-2026
Nadomešča:
SIST EN ISO 23611-1:2018
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 1. del: Ročno razvrščanje
deževnikov in njihova ekstrakcija (ISO 23611-1:2026)
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of
earthworms (ISO 23611-1:2026)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese
und Extraktion von Regenwürmern (ISO 23611-1:2026)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction
des vers de terre (ISO 23611-1:2026)
Ta slovenski standard je istoveten z: EN ISO 23611-1:2026
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 23611-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2026
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-1:2018
English Version
Soil quality - Sampling of soil invertebrates - Part 1: Hand-
sorting and extraction of earthworms (ISO 23611-1:2026)
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
23611-1:2026) Regenwürmern (ISO 23611-1:2026)
This European Standard was approved by CEN on 26 June 2026.

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, Türkiye 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
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-1:2026 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23611-1:2026) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “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 2027, and conflicting national standards shall
be withdrawn at the latest by January 2027.
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 23611-1:2018.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 23611-1:2026 has been approved by CEN as EN ISO 23611-1:2026 without any
modification.
International
Standard
ISO 23611-1
Third edition
Soil quality — Sampling of soil
2026-06
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 23611-1:2026(en) © ISO 2026

ISO 23611-1:2026(en)
© ISO 2026
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]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO 23611-1:2026(en)
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 .7
7.4 Preparation of a composite sample for community DNA metabarcoding .7
8 Data assessment . 8
9 Test report . 8
Annex A (informative) Other methods for sampling . 9
Annex B (informative) Species identification in earthworms .10
Annex C (normative) Determination of maximum water-holding capacity .11
Bibliography .12

iii
ISO 23611-1:2026(en)
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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 23611-1:2018), which has been technically
revised.
The main changes are:
— addition of a description of ecological categories of earthworms;
— addition of information related to live identification of earthworms;
— revised description for determination of biomass;
— addition of a section on the preparation of composite samples for community DNA metabarcoding;
— revision of Annex A on other methods of earthworm sampling;
— removal of the former informative Annex C on the modified TSBF method;
— removal of the outdated informative Annex E with examples of earthworm monitoring programmes
(including presentation of their results).
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 23611-1:2026(en)
Introduction
This document was originally drawn up because of the 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:
[9][24][36][39][51]
— biological classification of soils including soil quality assessment;
[2][16][18][45][50]
— terrestrial bio-indication and long-term monitoring;
[35]
— evaluation of the effects of chemicals on soil animals in the field.
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 bio-classification and bio-assessment in terrestrial (i.e.
soil) habitats has so far been used relatively rarely 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, the sampling of earthworms is described.
Originally, the methods described in this document were developed for taxonomical and ecological studies,
investigating the role of earthworms (macrofauna) in various soil ecosystems. These animals are without
[27][29][41]
doubt the most important soil invertebrates in temperate regions. This does not necessarily imply
that earthworms are less important in tropical soils. In fact, there is wide evidence that earthworms can
be as important as or even more important than the abundant termites and ants in many locations of the
[28]
tropical region. Since Darwin (1881) (see Reference [11]), their influence on soil structure (e.g. aeration,
water holding capacity) and on soil functions (e.g. litter decomposition and nutrient cycling) is well-known.
[14]
Due to their often very high biomass they are also important in many terrestrial food-webs.
In earlier versions of this document the chemical formalin was recommended as extraction fluid. Since
then, evidence has increased that formalin has properties of concern, mainly in terms of human toxicity.
According to the European Chemicals Agency (ECHA) chemical database, this chemical is labelled as
[13]
carcinogenic and suspected to be mutagenic (Categories Carc. 1B and Muta. 2). In addition, negative
effects on non-target organisms (including soil microorganisms, mesofauna and plants) have been reported
(e.g. see Reference [10]). Therefore, this substance has been replaced.
Due to the growing reservations against the use of formalin, several alternatives have been studied. In
Reference [52] 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. The ECHA chemical database provides an overview
[12]
of the hazard classification and labelling and of properties of concern. Hence, although not currently
labelled as carcinogenic like formalin, AITC must only be used with appropriate safety precautions.
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 [33] no differences were found in numbers or biomass
of earthworms extracted at crop sites when using either formalin or AITC as extractant. 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
earthworm 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 and exchangeability
[38]
of the two methods.
[6]
Earthworms can be divided into three main ecological categories: endogeic, epigeic and anecic. Endogeic
species vary greatly in size, with little or no pigmentation, are geophagous, live in the soil and rarely rise
to the surface, creating galleries with little connectivity to the soil surface. Epigeics tend to have smaller
sizes, are dorsally or completely pigmented, live on or near the soil surface or in places with accumulation
of organic matter (composters, bromeliads, rotten trunks, epiphytic soils), ingest little soil and produce
basically organic casts. Anecics often move to the soil surface, have dorsal pigmentation especially in the
anterior part of the body, are usually large in size and produce vertical galleries and accumulations of

v
ISO 23611-1:2026(en)
organo-mineral casts (often with higher organic matter contents than the ingested soil) on the surface of
the soil at the entrance of their galleries. These categories are evident in temperate regions. But, especially
[4]
in tropical and subtropical regions, there are blends of categories, such as “epi-endogeic” , and the anecic
category is not as common as in the temperate region, with a predominance of endogeics especially in
[15]
agroecosystems .
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.
[20] [23]
The sampling design of field studies in general is given in ISO 18400-101 and ISO 23611-6 , and guidance
1)
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 and regional conditions of the
site to be sampled.
1) This document will be replaced by an Organisation for Economic Co-operation and Development (OECD) document
(either a Test Guideline or Guidance Document) currently under development (OECD project no. 2.47 ‘New Test Guideline
on Determination of Effects on Earthworms in Field Studies’).

vi
International Standard ISO 23611-1:2026(en)
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 is applicable to all terrestrial biotopes in which earthworms occur. This document
does not apply to semi-terrestrial soils (i.e. soils that are partly aquatic, such as bogs, beaches, marshes,
stream margins) and it can be difficult to use under extreme climatic or geographical conditions (e.g. in
high mountains). Methods for other soil organism groups, such as micro-arthropods and enchytraeids
(mesofauna), are covered in other parts of the ISO 23611 series.
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, treated biowaste and sludge — 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, Sludge and solid environmental matrices — Determination of dry residue or water content and
calculation of the dry matter fraction on a mass basis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
earthworm
megadrile soil-inhabiting invertebrate belonging to the order Crassiclitellata (class Clitellata, phylum
Annelida) and classified as soil macrofauna
Note 1 to entry: The length of adult individuals can vary from a few centimetres to more than 1 m.

ISO 23611-1:2026(en)
EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae and Rhinodrilidae (Latin America),
Eudrilidae (Africa) or Megascolecidae [Asia, North America (Pacific Coast)].
3.2
peregrine species
earthworm species occurring in many regions world-wide today, usually introduced by man
Note 1 to entry: The pan-tropical species Pontoscolex corethrurus (probably coming from Northern Brazil and/or
the Guyanas) is the most common species in the world. Other well-known examples of peregrine species are several
lumbricid species such as Aporrectodea caliginosa (originally coming from Eurasia, but now living also in the Americas
and Oceania).
Note 2 to entry: See References [7],[29],[31],[47].
3.3
clitellum
ring-shaped or saddle-shaped epidermal thickening found near the anterior portion of mature earthworms,
that 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 the earthworms from the soil by applying AITC.
The first method is known for approximately 100 years while the second method using the new extraction
[10][33][52]
fluid was first proposed in 2003. After extraction, the earthworms are fixed and taken to the
laboratory. There they are preserved in a way that they can be stored indefinitely in a collection (e.g. for
taxonomical purposes). In addition, the determination of the biomass of earthworms is described. In addition
to identification of fixed earthworms, some species (especially Lumbricidae, and often P. corethrurus)
can also be identified alive (see Annex B). Thus, after extraction, taxonomic identification and biomass
determination are carried out on the living animals rather than after fixation. An advantage of performing
the taxonomic identification on living animals is that, in many cases, juvenile animals can also be identified
to species level. Finally, abundance and biomass values can be recalculated to area (usually 1 m ) or, more
rarely, volume parameters.
NOTE 1 Alternative methods and combinations thereof can be useful under special circumstances (e.g. electrical
extraction) but are beyond the scope of this document (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 method for sampling edaphic macrofauna is described in
[22]
ISO 23611-5. The design of such programmes is not included in this document.
NOTE 3 Some suggestions for the taxonomic identification of peregrine species of earthworms, mainly belonging to
the family Lumbricidae, are given in Annex B. In the Southern hemisphere the earthworm species of the Lumbricidae
[29]
family are less common, except for e.g. Australia and New Zealand. In the latter, they dominate the earthworm fauna,
[29]
with 15 lumbricid species occurring. The most common peregrine species in the world is Pontoscolex corethrurus,
belonging to Family Rhinodrilidae, but species of the Family Megascolecidae, Acanthodrilidae and Benhamiidae
can also be common in some regions. The identification of these species can be performed using keys available in
References [3] and [8]. P. corethrurus usually can be identified live, but depending on the location of sampling (e.g.
Central America or Northern South America), it can be confused with other Pontoscolex species.
5 Reagents
5.1 Allyl-isothiocyanate (AITC), synthetic grade, approximately 94 % to 97 % (volume fraction).
5.2 Isopropanol, 100 % (volume fraction).

ISO 23611-1:2026(en)
5.3 Ethanol, 70 % to 80 % (volume fraction).
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 earthworms.
6.2 Rubber gloves.
6.3 Forceps.
2 2
6.4 Folding table, piece of thick plastic sheeting, 1 m to 2 m or large bucket, e.g. 100 l, to collect the
soil for hand-sorting.
6.5 Spade or shovel.
6.6 Stereomicroscope, with low magnification (×10 to ×40).
6.7 Balance, weight range from 0,01 g to 200 g; If weighing is carried out at the species level, a scale with
an accuracy of 0,001 g or 0,000 1 g can be necessary.
6.8 Water-can, preferably 20 l, with water (5 l to 10 l per sampling plot).
6.9 Watering can, preferably 10 l.
6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 Thermometer, for measuring air temperature.
6.12 Drying oven, 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, regardless of the type of chemical
expellant (e.g. References [10] and [25]).
Sampling should be done at times of the year when the animals are not forced by the environmental
conditions (i.e. low soil moisture or high temperatures, or both) into diapause (i.e. when they are not
reacting to AITC). In temperate regions, such unfavourable sampling times occur in winter and, in particular,
[29]
midsummer periods. Earthworms sampled from the same plot but sampled under the two different
methods should be stored in separate individual plastic vessels. After the end of the sampling process, the
excavated and examined soil is returned to the original sampling plot. When no deep-burrowing animals

ISO 23611-1:2026(en)
are occurring at a given site, AITC extraction is not necessary. At sites where giant earthworms are living
(parts of South America, Southeast Asia and Australia), hand-sorting can be inappropriate and should be at
[37]
least complemented by chemical extraction, in order to collect the large species.
NOTE Usually, earthworms are identified after preservation. However, the most common peregrine lumbricid
[26]
species can also be identified alive. This offers the advantage that many juvenile stages of these species can also be
identified to species level based on their characteristic colouring and behaviour (see 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 earthworms in a small portion of soil
from the sampling site is recommended. In the case of AITC extraction, the earthworms should be rinsed in
tap water 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 C;
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
earthworms. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms
have approximate lengths between 1 cm and 20 cm. At places with a low density of earthworms [e.g. soils
with low pH (< 4,5) or which are anthropogenically used such as crop sites], larger plots (i.e. 1 m ) can be
necessary. On the other hand, at sites with a high earthworm density (e.g. many meadows in temperate
2 [40] 2 [53]
regions), a smaller plot of 1/8 m is sufficient. Even smaller sample sizes (e.g. 1/16 m ) can lead to
very low, and thus variable, individual earthworm numbers per sample, which in turn leads to an increase in
sample numbers (e.g. 16 replicates).
The soil is removed by means of a spade or shovel (6.5) up to a depth of 20 cm to 30 cm from this plot
(20 cm are suitable for many temperate sites, but the depth also depends on site properties such as the
depth of the plough layer). The excavated soil is spread out, e.g. on a folding table or a piece of plastic or
collected in a large bucket (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. The soil is searched cautiously
for earthworms. Large earthworms are collected by hand using rubber gloves (6.2) and small ones by
using forceps (6.3). To avoid autotomy and further damage to the earthworms, the animals should only be
touched at the anterior part of the body. If earthworms 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. Pieces of earthworms that cannot be identified as either the head
or tail should not be quantified for abundance unless there is only one. Sometimes the pieces can be united
back as an incomplete earthworm and be accounted as a juvenile. The pieces shall be weighed together with
all individuals and fragments to determine the total biomass of the sample.
NOTE 1 With the naked eye, the front end of adult earthworms 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 (volume fraction) (5.3). It is
recommended to exchange the ethanol solution after 24 h. The volume of liquid in the vessel should be at
least 3 times the volume occupied by the earthworms. If the use of molecular methods for the identification
of earthworms is planned, preference should be given to ethanol at a concentration of ≥95 % (volume
[21]
fraction) and storage of individuals in a freezer at ≤–20 °C (see ISO 21286) . The vessels shall be labelled

ISO 23611-1:2026(en)
and observations (e.g. whether earthworms have been in a quiescence stage) should be recorded in the
notebook (6.10).
An immediate fixation in 4 % formalin (volume fraction) (5.4) is possible, but not recommended, because the
handling of this compound should be minimised as much as possible (in particular under field conditions)
and because formalin degrades DNA, thus preventing the application of molecular methods.
NOTE 2 In order to avoid morphological changes (e.g. an inversion of the prostomium) due to immediate fixation in
ethanol, the individual earthworms can be put briefly (approximately one minute) into warm (e.g. 30 °C to 40 °C) tap
water. Alternatively, they can be relaxed in sparkling water or 5 % diluted ethanol (volume fraction) for up to 3 min
before fixation. After that they can be transferred to 70 % ethanol solution (volume fraction).
Large earthworms or a high number of earthworms with high body water content can dilute 70 % ethanol
(volume fraction) used as fixative solution, causing them to become soggy and lose important morphological
features necessary for taxonomic identification. In such cases, it is recommended to use a more concentrated
ethanol solution., e.g. 80 % (volume fraction).
For live identification, the earthworms are transferred to suitable containers (e.g. plastic jars with a volume
of at least 0,5 l). The earthworms are kept either on moist blotting paper or in moist soil. The earthworms
will excrete on moist blotting paper, allowing a more exact determination of the biomass, but, unlike when
they are kept in soil, they only have a limited shelf life and shall therefore be identified within at most 3 days.
Damaged specimens need to be separated and immediately fixed since a dead earthworm in a sampling
container leads to the death of the entire batch in a short time.
The earthworms are transported to the laboratory in cooling boxes and stored in the refrigerator at 4 °C to
8 °C.
7.1.3 AITC extraction
The same sampling point from which the topsoil 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 point) beforehand to
the sampling points using large water-cans (6.8). AITC readily degrades in water, and less rapidly also in
isopropanol. Degradation is enhanced by sunlight and warmth. To prepare the AITC solution, 1 g of AITC
(5.1) is dissolved in 50 ml of isopropanol (5.2) under a fume hood in the laboratory up to one week prior to
use, and the resulting stock solution is topped up with water to a total volume of 10 l immediately before
use in the field. The density of AITC is 1,013 kg/l; i.e. using a volume fraction, the concentration would be
given as 98,7 ml/l. The diluted AITC solution is carefully and evenly applied into the sampling point from
which the topsoil has been removed for hand-sorting. The solution should be applied in several portions
(usually 2 to 3) according to the seepage capacity of the soil until 5 l to 10 l of AITC-solution have been
added, depending on the soil properties (in cases where the sampling plot is larger than 0,25 m , the amount
of AITC-solution shall be increased accordingly). During the application, the plot shall be observed in order
to collect all earthworms appearing on the soil surface of the sampling plot. The sampling is finished 30 min
after the application of the last watering can. If earthworms continue to emerge, this duration should be
prolonged by 15 min.
Large earthworms should be collected by hand using rubber gloves (6.2) and small earthworms by forceps
(6.3). The repellent AITC produces rapid earthworm emergence from the soil. Earthworms should only be
collected when the largest (preferably whole) portion of the body becomes visible, otherwise damage or
retraction back into the soil occurs. To avoid autotomy and damage of the earthworms, the animals should
only be touched at the anterior part of the body, usually in front of the clitellum. The collected earthworms
should immediately be fixed in 70 % ethanol (volume fraction) (5.3) using 250 ml or 500 ml plastic vessels
(6.1) for at least 30 min and should be replaced within 24 h for a new 70 % ethanol solution (volume fraction)
in order to guarantee adequate fixation and preservation of the collected individuals over the long-term.
The vessels shall be labelled, and any observations should be recorded in the notebook (6.10).
At sites where giant earthworms occur (South America, Southeast Asia, and Australia), and where hand-
sorting is not appropriate to get them, AITC-solution should be applied on an area of 4 m (its amount shall
[37]
be increased accordingly). Before doing so, herbs and litter shall be removed from the soil surface. In all
other respects, the sampling process is the same.

ISO 23611-1:2026(en)
It is not necessary to perform the AITC extraction if no vertical burrowers (especially Lumbricus terrestris
or Aporrectodea longa) are living at a given site (e.g. in very acid soils). The occurrence of these large
earthworms is indicated by surface casts and/or collected litter at their burrow openings (diameter ≥
0,5 cm) which are detectable before excavation of the soil used for hand-sorting. These species do not occur
[44]
in some places (e.g. Brazil), and anecic species of other families produce vertical galleries. Some of them
produce casts on the surface, but the presence of these does not necessarily indicate the presence of anecic
earthworms. Therefore, it is interesting to prospect before sampling using AITC to specify its use or not in a
given location.
NOTE 1 In soils with high clay content, the digging of the sampling pit can close tightly many earthworm burrows
at the bottom of the pit, thus preventing the infiltration of AITC and/or the emergence of deep burrowing earthworms.
In such conditions, the smeared burrow openings can be opened before the AITC application (e.g. using a knife).
Large earthworms or a high number of earthworms containing much water kept in 70 % ethanol (volume
fraction) can dilute the ethanol, become soggy and lose their morphological features necessary for
taxonomic identification. In such cases, it is recommended to use a more concentrated ethanol solution., e.g.
80 % (volume fraction).
WARNING — AITC is a hazardous material. The ECHA chemical database provides an overview of
[12]
the hazard classification and labelling and of properties of concern. This document does not
claim to address all related safety issues. It is the responsibility of the user of this document to take
appropriate measures for the safety and health protection of personnel before applying the methods
described in this document.
For live identification, the earthworms are transferred to suitable containers (e.g. plastic jars with a volume
of at least 0,5 l). The earthworms are kept either on moist blotting paper or in moist soil. The earthworms
will excrete on moist blotting paper, allowing a more exact determination of the biomass, but, unlike when
they are kept in soil, they only have a limited shelf life and shall therefore be identified within at most 3 days.
Damaged specimens need to be separated and immediately fixed since a dead earthworm in a sampling
container leads to the death of the entire batch in a short time.
The earthworms are transported to the laboratory in cooling boxes and stored in the refrigerator at 4 °C to
8 °C.
7.2 Preservation
The following methods are possible.
a) After fixation, the animals can be kept in 4 % formalin (volume fraction) (5.4) for four days at minimum,
but preferably one or two weeks. Afterwards, the earthworms can be stored for an unlimited period in
70 % to 80 % ethanol (volume fraction) (5.3).
b) An alternative method is to fix the earthworms immediately after collection in a mixture of 70 % to
80 % ethanol (volume fraction) (5.3) and 4 % formalin (volume fraction) (5.4) in a ratio of 98 % to
2 % (volume fraction). This preservation liquid is replaced by fresh preservation liquid on the day after
sampling at the latest.
c) The use of concentrated ethanol (95 % volume fraction or higher) as a fixative and preservative solution
does not adequately preserve some internal structures (e.g. calciferous glands) necessary for the
identification and description of some species (especially some native tropical species). As there is no
better alternative for earthworms, whenever possible, use formaldehyde between 4 % and 8 % (volume
fraction) to preserve earthworms. For long-term conservation, earthworms can be kept in formaldehyde
solution for 30 days and then can be transferred to 70 % to 80 % ethanol (volume fraction).
WARNING — Formaldehyde is a hazardous material. The ECHA chemical database provides an
[13]
overview of the hazard classification and labelling and of properties of concern. This document
does not claim to address all related safety issues. It is the responsibility of the user of this document
to take appropriate measures for the safety and health protection of personnel before applying the
methods described in this document.

ISO 23611-1:2026(en)
Preservation in pure ethanol should be avoided since the outer surface of the earthworms sometimes
becomes very hard, so that important features are not visible anymore.
In case the earthworm tissue shall be preserved for biochemical or genetical studies (see 7.4), preservation
[25]
in formalin is not recommended. Instead, the earthworms shall be fixed and stored in ethanol (≥95 %
volume fraction).
7.3 Determination of biomass
Determination of biomass is performed using the fixed or the preserved material. The animals are rinsed in
water, quickly dried on a piece of paper and subsequently the mass is determined using a suitable balance
(6.7). Afterwards the earthworms are stored again in 70 % to 80 % ethanol (volume fraction) (5.3) or in the
ethanol/formalin mixture. If the earthworms have been identified alive, they can be weighed directly or
after allowing them to void their guts (e.g. overnight in moist paper towels). Earthworms are weighed either
individually or in groups, according to species and age stage.
Biomass of earthworms collected in the field is highly variable due to variations in the state of hydratio
...