oSIST prEN ISO 23611-1:2025

SLOVENSKI STANDARD
01-september-2025
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 1. del: Ročno razvrščanje
deževnikov in njihova ekstrakcija (ISO/DIS 23611-1:2025)
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of
earthworms (ISO/DIS 23611-1:2025)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese
und Extraktion von Regenwürmern (ISO/DIS 23611-1:2025)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction
des vers de terre (ISO/DIS 23611-1:2025)
Ta slovenski standard je istoveten z: prEN ISO 23611-1
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.

DRAFT
International
Standard
ISO/DIS 23611-1
ISO/TC 190/SC 4
Soil quality — Sampling of soil
Secretariat: AFNOR
invertebrates —
Voting begins on:
Part 1: 2025-06-20
Hand-sorting and extraction of
Voting terminates on:
2025-09-12
earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
ICS: 13.080.05; 13.080.30
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Reference number
ISO/DIS 23611-1:2025(en)
DRAFT
ISO/DIS 23611-1:2025(en)
International
Standard
ISO/DIS 23611-1
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
ICS: 13.080.05; 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2025
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland Reference number
ISO/DIS 23611-1:2025(en)
ii
ISO/DIS 23611-1:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 3
6 Apparatus . 3
7 Procedure . 4
7.1 Sampling of the earthworms.4
7.1.1 General .4
7.1.2 Hand-sorting .4
7.1.3 AITC extraction .5
7.2 Preservation .6
7.3 Determination of biomass .6
7.4 Preparation of a composite sample for community DNA metabarcoding .7
8 Data assessment . 7
9 Test report . 7
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/DIS 23611-1:2025(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,
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with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types
of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent
rights identified during the development of the document will be in the Introduction and/or on the ISO list of
patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization.
This third edition cancels and replaces the second edition (ISO 23611-1:2018), which has been technically
revised. The main changes are:
— addition of a definition for ecological categories of earthworms;
— addition of a section on the preparation of composite samples for community DNA metabarcoding;
— removal of the 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/DIS 23611-1:2025(en)
Introduction
This Standard has been originally drawn up since there was a growing need for the standardization of
terrestrial zoological field methods. Such methods, mainly covering the sampling, extraction and handling
of soil invertebrates, are necessary for the following purposes:
[9][19][30][33][45]
— biological classification of soils including soil quality assessment ;
[2][15][17][39][44]
— terrestrial bio-indication and long-term monitoring ;
[29]
— 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 relatively rarely been used in comparison to aquatic sites.
Since it is neither possible nor useful to standardize methods for all soil organisms, the most important ones
have been selected. In this document sampling of earthworms is described.
Originally, the methods described in this document were developed for taxonomical and ecological studies,
investigating the role of earthworms (macrofauna) in various soil ecosystems. These animals are without
[21][23][35]
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 may
be as important as or even more important than the abundant termites and ants in many locations of the
[22]
tropical region. Since Darwin (1881) (see Reference [11]), their influence on soil structure (e.g. aeration,
[14]
water holding capacity) and soil functions like litter decomposition and nutrient cycling is 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 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
[13]
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 [46] 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. In the ECHA chemical database, properties of concern
[12]
‘skin sensitising’ and ‘respiratory sensitising’ are listed. It is also toxic and hazardous to the aquatic
environment. Hence, although less harmful than formalin, AITC shall 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 [27] 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
worm numbers extracted with AITC versus formalin in a Bland-Altman graph (a common way to compare
a gold-standard method to an alternative method in the medical sciences), no significant bias of the AITC
method as compared to the formalin method was found, indicating the similarity/exchangeability of the two
[32]
methods .
Basic information on the ecology of earthworms and their use as bioindicators in the terrestrial environment
can be found in the references listed in the Bibliography.

v
DRAFT International Standard ISO/DIS 23611-1:2025(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 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
1)
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.
This document is not applicable for semi-terrestrial soils (i.e. soils that are partly aquatic like 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 ISO 23611.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 10390, Soil, 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 quality — Determination of dry matter and water content on a mass basis — Gravimetric method
ISO 21286, Soil quality — Identification of ecotoxicological test species by DNA barcoding
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/
1) 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’).

ISO/DIS 23611-1:2025(en)
3.1
earthworms
megadrile soil-inhabiting earthworms 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
earthworms 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 example of peregrine species are several
lumbricid species like Aporrectodea caliginosa (originally coming from Eurasia, but now living also in the Americas
and Australia).
Note 2 to entry: See References [7] [23] [25] [41].
3.3
clitellum
ring or saddle shaped epidermal thickening only in mature worms which is near the anterior portion and
eventually forms the cocoon
3.4
clitellum
[6]
Earthworms can be divided into three main ecological categories: endogeic, epigeic and anecic. Endogeic
species vary greatly in size, are generally pigmented, 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 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 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
[4]
regions, there are blends of categories, like “epi-endogeic” and the anecics category are not found until the
current days.
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 about 100 years while the second method using the new extraction fluid
[10][27][46]
was first proposed in 2003. After extraction, the earthworms are fixed and transported to the
laboratory. There they are preserved in a way that they can be stored in a collection indefinitely (e.g. for
taxonomical purposes). In addition, the determination of the biomass of earthworms is described. Finally,
abundance and biomass values can be recalculated to area (usually 1 m ) or, more rarely, volume parameters.
NOTE 1 Alternative methods can be useful under special circumstances (e.g. electrical extraction), but cannot be
recommended as a general quantitative procedure (see Annex A).
NOTE 2 The sampling of earthworms is often included in much broader monitoring programs, trying to cover
the whole soil fauna or parts of it (e.g. the macrofauna). The standard for sampling edaphic macrofauna is found in
ISO 23611-6. The design of such programmes is not included in this document.

ISO/DIS 23611-1:2025(en)
NOTE 3 Some hints for the taxonomy 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 rare (except for the species Eisenia andrei, the
compost earthworm, found mainly in composters). The most common peregrine species in the world is Pontoscolex
corethrurus, belonging to Family Rhinidrilidae, but species of the Family Megascolecidae, Acanthodrilidae and
Benhamiidae may also be common in some regions. The identification of these species can be performed using keys
[3] [8]
available in, and .
5 Reagents
5.1 Allyl-isothiocyanate (AITC), synthetic grade (about 94 % to 97 % (volume fraction)).
5.2 Isopropanol, 100 % (volume fraction).
5.3 Ethanol, 70-80 % (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 worms.
6.2 Rubber gloves.
6.3 Forceps.
2 2
6.4 Piece of thick plastic sheeting, 1 m to 2 m .
6.5 Spade or shovel.
6.6 Stereomicroscope, with low magnification (×10 to ×40).
6.7 Balance, weigh 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 may be necessary.
6.8 Water-can, preferably 20 l, with water (20 l per sampling plot).
6.9 Watering can.
6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 Thermometer, e.g. for measuring air temperature.
6.12 Drying cabinet, for soil moisture determination.

ISO/DIS 23611-1:2025(en)
7 Procedure
7.1 Sampling of the earthworms
7.1.1 General
Sampling of earthworms is done by a combination of two different methods: hand-sorting and AITC
extraction. Based on several comparative studies, the combination of a physical and a chemical method is
clearly recommended in the various reviews on earthworm ecology, independent from the type of chemical
expellant (e.g. References [10] and[20]).
Sampling should be done at times of the year when the animals are not forced by the environmental
conditions (i.e. low soil moisture and/or high temperatures) into diapause (i.e. are not reacting to AITC). In
[23]
temperate regions, such unfavourable sampling times are winter and, in particular, 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 may not be appropriate but at least needs to be complemented by chemical
[31]
extraction, in order to collect the large species .
NOTE Usually the earthworms are determined after preservation, but if the species spectrum of a sampling site is
[43]
well known, worms can also be determined alive (see also Annex B).
In case the collected earthworms are to be used for further analysis or testing, e.g. for biomarker
measurements or for use in bioassays, storage or incubation of the worms in a small portion of soil from
the sampling site is recommended. In the case of AITC extraction, rinsing the worms in tap water is needed
before incubation in soil.
For the interpretation of test results, the following characteristics shall be determined for the field site to be
studied:
a) pH in accordance with ISO 10390;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465;
d) water holding capacity as specified in Annex 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
worms. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms have
approximately a length between 1 cm and 20 cm. At places with a low density of earthworms [e.g. soils with
low pH (< 4,5) or which are anthropogenically used like crop sites], larger plots (i.e. 1 m ) may be necessary.
On the other hand, at sites with a high earthworm density (e.g. many meadows in temperate regions), a
2 [34] 2[47]
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 worm 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 sole). The excavated soil is spread out, e.g. on a piece of plastic (6.4). This can be done in the field
but, especially in periods of bad weather, the whole procedure can also be performed in the laboratory or
greenhouse. Afterwards, the soil is searched cautiously for earthworms. 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

ISO/DIS 23611-1:2025(en)
to the worms, the animals should only be touched at the anterior part of the body. If worms are cut by the
spade used to dig out the soil, both parts are collected in order to measure the correct biomass, whereas
only front parts are counted when determining the number of individuals. Pieces of earthworms that cannot
be identified as eith
...