ISO/FDIS 23611-1

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

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.

Qualité du sol — Prélèvement des invertébrés du sol — Partie 1: Tri manuel et extraction des vers de terre

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.

General Information

• Status: Not Published
• ICS: 13.080.05 - Examination of soils in general
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: ISO/TC 190/SC 4 - Biological characterization
• Drafting Committee: ISO/TC 190/SC 4/WG 2 - Effects on soil fauna
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 30-Mar-2026
• Completion Date: 30-Mar-2026

Relations

• Consolidates: FprEN ISO 23611-1 - Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO/FDIS 23611-1:2026)
• Effective Date: 12-Feb-2026
• Revises: ISO 23611-1:2018 - Soil quality — Sampling of soil invertebrates — Part 1: Hand-sorting and extraction of earthworms
• Effective Date: 28-Oct-2023

Overview

ISO/FDIS 23611-1:2026 specifies standardized methods for sampling and handling earthworms in field soils, supporting their use as bioindicators in soil quality assessment. Developed by ISO Technical Committee 190, Subcommittee 4, this international standard provides clear procedures for collecting, extracting, preserving, and analyzing earthworms, which play a critical role in terrestrial ecosystems. The standard is applicable to all terrestrial habitats where earthworms are present, ensuring a consistent approach to soil invertebrate sampling for environmental monitoring and research.

Key Topics

• Standardized Sampling Methods: Detailed procedures for both hand-sorting and chemical extraction (using AITC) of earthworms, ensuring reliable and repeatable field studies.
• Bioindicator Value: Earthworms are recognized as key bioindicators due to their sensitivity to soil conditions and pollutants. This standard supports their use in soil quality monitoring and environmental impact assessments.
• Sample Handling and Preservation: Guidance on effective preservation-including ethanol and formalin methods-ensures samples remain suitable for various analyses, including morphological examination and genetic studies.
• Field Equipment and Safety: Recommendations for equipment (forceps, spades, containers, etc.) and important safety considerations when handling chemical extractants like allyl isothiocyanate (AITC) and formalin.
• Identification and Biomass Determination: Procedures for earthworm identification (live or preserved) and accurate biomass measurements, supporting ecological and taxonomic studies.
• Ecological Categories: Overview of earthworm ecological types (endogeic, epigeic, and anecic) and guidance for working with different species in varying climates and habitats.

Applications

ISO/FDIS 23611-1 is used by:

• Environmental Monitoring Agencies: To assess soil quality, track remediation efforts, and monitor the effects of land use or pollutants.
• Soil Scientists and Ecologists: For ecological research, biodiversity studies, and long-term monitoring programs involving earthworm communities.
• Agriculture and Land Management: Informing sustainable practices and evaluating soil health as it relates to crop productivity and ecosystem services.
• Environmental Consultancies: Conducting impact assessments and compliance studies for land development, waste management, and contamination remediation projects.
• Academic Researchers: Supporting standard methods in soil zoology, taxonomy, and biomonitoring, and facilitating comparative studies across different regions.

Consistent methods ensure that results are comparable across projects and geographic areas, aiding in data sharing and international research collaborations.

Related Standards

For a comprehensive approach to soil quality assessment and soil invertebrate sampling, consider:

• ISO 18400-101: Provides principles for sampling strategies in field studies, including design and implementation.
• ISO 11268-3: Guidance for assessing the impact of pollutants on earthworm populations in the field.
• ISO 23611-5: Describes methods for sampling macrofauna (including earthworms) more broadly.
• Other ISO 23611 Parts: Cover protocols for other soil organism groups such as collembolans and enchytraeids.

These related standards, referenced within ISO/FDIS 23611-1, ensure harmonized methodologies within the context of broader soil quality and biodiversity monitoring programs.

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Keywords: earthworm sampling, soil invertebrates, soil quality, bioindicator, environmental monitoring, ISO standard, hand-sorting, AITC extraction, soil fauna, international standards, environmental impact assessment, earthworm preservation.

Standards Content (Sample)

FINAL DRAFT
International
Standard
ISO/TC 190/SC 4
Soil quality — Sampling of soil
Secretariat: AFNOR
invertebrates —
Voting begins on:
2026-03-30
Part 1:
Hand-sorting and extraction of
Voting terminates on:
2026-05-25
earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 190/SC 4
Soil quality — Sampling of soil
Secretariat: AFNOR
invertebrates —
Voting begins on:
Part 1:
Hand-sorting and extraction of
Voting terminates on:
earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
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Published in Switzerland Reference number
ii
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
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
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
organo-mineral casts (often with higher organic matter contents than the ingested soil) on the surface of
the soil at the entrance of its 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
FINAL DRAFT International Standard ISO/FDIS 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.

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 (occurring in many regions world-wide,
mainly the Northern hemisphere) earthworms, mainly belonging to the family Lumbricidae, are given in Annex B. In
the Southern hemisphere the earthworm species of the Lumbricidae family are less common, except for e.g. Australia
[29] [29]
and New Zealand. In the latter, they dominate the earthworm fauna, 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).

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

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

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 th
...

ISO/DISFDIS 23611-1:2026(en)
ISO/TC 190/SC 4
Secretariat: AFNOR
Date: 2026-02-1803-16
Soil quality — Sampling of soil invertebrates — —
Part 1: Handsorting
Hand-sorting and extraction of earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
FDIS stage
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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
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Phone: + 41 22 749 01 11
EmailE-mail: [e-mail]
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Published in Switzerland
ii
ISO/DISFDIS 23611-1:2026(en)
Contents
Foreword . iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 3
6 Apparatus . 3
7 Procedure . 3
7.1 Sampling of the earthworms . 3
7.2 Preservation . 7
7.3 Determination of biomass . 7
7.4 Preparation of a composite sample for community DNA metabarcoding . 8
8 Data assessment . 8
9 Test report . 8
Annex A (informative) Other methods for sampling . 10
Annex B (informative) Species identification in earthworms . 11
Annex C (normative) Determination of maximum water-holding capacity . 12
Bibliography . 13

iii
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'sISO’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.
iv
ISO/DISFDIS 23611-1:2026(en)
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.
v
Introduction
This document has beenwas originally drawn up since there was abecause 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][9][24][36][39][51] ][[51]
— — biological classification of soils including soil quality assessment; ;
[2]][[16]][[18]][[45][2][16][18][45][50] ][[50]
— — terrestrial bio-indication and long-term monitoring; ;
[35] [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 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, 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] [27][29][41]][[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] [28]
tropical region. . Since Darwin (1881) (see Reference [11][11]),), their influence on soil structure (e.g.
aeration, water holding capacity) and on soil functions like(e.g. litter decomposition and nutrient cycling) is
[14] [14]
well-known. . 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 carcinogenic and
[13] [13]
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][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][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 onof the
[12] [12]
hazard classification &and labelling and of properties of concern. . Hence, although not currently labelled
as carcinogenic like formalin, AITC shallmust 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][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 of the two
[38] [38]
methods. .
vi
ISO/DISFDIS 23611-1:2026(en)
[6] [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 organo-mineral casts (often
with higher organic matter contents than the ingested soil) on the surface of the soil at the entrance of its
galleries. These categories are evident in temperate regions. But, especially in tropical and subtropical regions,
[4] [4]
there are blends of categories, likesuch as “epi-endogeic” ” , and the anecic category is not as common as in
15 [15]
the temperate region, with a predominance of endogeics especially in 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][20] [23][23]
The sampling design of field studies in general is given in ISO 18400-101 and ISO 23611-6 ,, and
guidance on the determination of effects of pollutants on earthworms in field situations is given in ISO 11268-
1 1)
3 . . These aspects can vary according to the national requirements or the climatic/ and regional conditions
of the site to be sampled.
This Standard 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’).
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’).

vii
DRAFT International Standard ISO/DIS 23611-1:2025(en)

Soil quality — Sampling of soil invertebrates — —
Part 1: Handsorting
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,
etc.)) 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, Soil qualitySludge and solid environmental matrices — Determination of dry matter andresidue or
water content and calculation of the dry matter fraction 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 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 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.
EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae and Rhinodrilidae (Latin America),
Eudrilidae (Africa) or Megascolecidae [Asia, North America (Pacific Coast)].
3.2 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 likesuch 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][7] [29] [31] [47].
3.3 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 aboutapproximately 100 years while the second method using the new
[10]][[33] [10][33][52]][[52]
extraction 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 aliveon 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] [22]
ISO 23611-5 . The design of such programmes is not included in this document.
NOTE 3 Some hintssuggestions for the taxonomytaxonomic identification of peregrine (occurring in many regions
world-wide, mainly the Northern hemisphere) earthworms, mainly belonging to the family Lumbricidae, are given in
Annex B. In the Southern hemisphere the earthworm species of the Lumbricidae family are less common, except for e.g.
[29] [29]
Australia and New Zealand . In the latter, they dominate the earthworm fauna, with 15 lumbricid species
[29] [29]
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][3] and[8] [8]. P.
corethrurus usually can be identified live, but depending on the location of sampling (e.g.,. Central America or Northern
South America), it couldcan be confused with other Pontoscolex species.
© ISO #### 2026 – All rights reserved
ISO/DISFDIS 23611-1:20252026(en)
5 Reagents
5.1 5.1 Allyl-isothiocyanate (AITC), synthetic grade (, approximately 94 % to 97 % (volume
fraction)).).
5.2 5.2 Isopropanol, 100 % (volume fraction).
5.3 5.3 Ethanol, 70 % to 80 % (volume fraction).
5.4 5.4 Formalin, formaldehyde solution 4 % (volume fraction), for storage purposes only.
5.5 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 6.1 Plastic vessels, capacities 250 ml and 500 ml, for storing the earthworms.
6.2 6.2 Rubber gloves.
6.3 6.3 Forceps.
2 2
6.4 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 6.5 Spade or shovel.
6.6 6.6 Stereomicroscope, with low magnification (×10 to ×40).
6.7 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 6.8 Water-can, preferably 20 l, with water (5 l to 10 l per sampling plot).
6.9 6.9 Watering can, preferably 10 l.
6.10 6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 6.11 Thermometer, for measuring air temperature.
6.12 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, independentregardless of the type of chemical
expellant (e.g. References [10][10] and [25] [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, midsummer
[29] [29]
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 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 least
[37] [37]
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] 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) a) pH in accordance with ISO 10390;
b) b) texture (sand, loam, silt) in accordance with ISO 11277;
c) c) water content in accordance with ISO 11465;
d) d) water holding capacity as specified in Annex C;
e) e) cationic exchange capacity in accordance with ISO 11260;
f) 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 regions), a smaller
2 [40] [40] 2[53] [53]
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(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(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(6.2)) and small ones by using
forceps (6.3(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
© ISO #### 2026 – All rights reserved
ISO/DISFDIS 23611-1:20252026(en)
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(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 fraction)
[21][21]
and storage of individuals in a freezer at ≤–20 °C (see ISO 21286) ). The vessels shall be labelled and
observations (e.g. whether earthworms have been in a quiescence stage) should be recorded in the notebook
(6.10(6.10).).
An immediate fixation in 4 % formalin (volume fraction) (5.4(5.4)) is possible, but not recommended due to
the fact that, 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 useapplication of molecular
methods would not be possible anymore since formalin degrades DNA.
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 (aboutapproximately 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 for, allowing a more exact determination of the biomass, but in contrast to
keeping them, 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(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, an amount of 1 g
of AITC (5.1(5.1)) is dissolved in 50 ml of isopropanol (5.2(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 another 15 min.
Large earthworms should be collected by hand using rubber gloves (6.2(6.2)) and small earthworms by
forceps (6.3(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 th
...