SIST EN ISO 23611-2:2024

SLOVENSKI STANDARD
01-december-2024
Nadomešča:
SIST EN ISO 23611-2:2012
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 2. del: Vzorčenje in ekstrakcija
mikročlenonožcev: skakači (Collembola) in pršice (Acarina) (ISO 23611-2:2024)
Soil quality - Sampling of soil invertebrates - Part 2: Sampling and extraction of micro-
arthropods (Collembola and Acarina) (ISO 23611-2:2024)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 2: Probenahme
und Extraktion von Mikroarthropoden (Collembolen und Milben) (ISO 23611-2:2024)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 2: Prélèvement et extraction
des micro-arthropodes (Collembola et Acarina) (ISO 23611-2:2024)
Ta slovenski standard je istoveten z: EN ISO 23611-2:2024
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 23611-2
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2024
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-2:2011
English Version
Soil quality - Sampling of soil invertebrates - Part 2:
Sampling and extraction of micro-arthropods (Collembola
and Acarina) (ISO 23611-2:2024)
Qualité du sol - Prélèvement des invertébrés du sol - Bodenbeschaffenheit - Probenahme von Wirbellosen
Partie 2: Prélèvement et extraction des micro- im Boden - Teil 2: Probenahme und Extraktion von
arthropodes (Collembola et Acarina) (ISO 23611- Mikroarthropoden (Collembolen und Milben) (ISO
2:2024) 23611-2:2024)
This European Standard was approved by CEN on 2 May 2024.

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.

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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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-2:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23611-2:2024) 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 November 2024, and conflicting national standards
shall be withdrawn at the latest by November 2024.
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 23611-2:2011.
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 23611-2:2024 has been approved by CEN as EN ISO 23611-2:2024 without any
modification.
International
Standard
ISO 23611-2
Second edition
Soil quality — Sampling of soil
2024-04
invertebrates —
Part 2:
Sampling and extraction of micro-
arthropods (Collembola and
Acarina)
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 2: Prélèvement et extraction des micro-arthropodes
(Collembola et Acarina)
Reference number
ISO 23611-2:2024(en) © ISO 2024

ISO 23611-2:2024(en)
© ISO 2024
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
ii
ISO 23611-2:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Test materials . 2
5.1 Biological material .2
5.2 Reagents .2
6 Apparatus . 3
7 Procedure . 4
7.1 Collecting the soil samples . .4
7.2 Extracting Collembola and Acarina from soil samples .4
7.3 Sorting, preserving and identifying Collembola and Acarina .5
7.3.1 Sorting and preserving .5
7.3.2 Identification .5
8 A ssessment of results . 6
9 Study report . 6
Annex A (informative) Species determination in collembolans and mites . 8
Annex B (informative) Alternative method for sampling of micro-arthropods . 9
Bibliography .10

iii
ISO 23611-2:2024(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
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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 document 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)
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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 second edition cancels and replaces the first edition (ISO 23611-2:2006), which has been technically
revised.
The main changes are as follows:
— an additional Note was added in 7.3.2.1 with the description of an alternative method to the classic pre-
heating techniques for specimen preparation for Collembola taxonomic identification;
— the bibliographic references list was revised and updated.
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 23611-2:2024(en)
Introduction
This document was prepared in response to a growing need for the standardization of sampling and
extraction methods of soil micro-arthropods. These methods are needed for the following purposes:
— biological classification of soils including soil quality assessment (e.g. References [19], [24], [27], [30],
[36], [40], [41]);
— terrestrial bioindication and long-term monitoring (e.g. References [3], [12], [14], [19], [31], [34], [37]).
Data collected by standardized methods can be more accurately evaluated, allowing more reliable
comparisons between sites (e.g. polluted versus non-polluted sites, changes in land-use practices).
From the several micro-arthropod groups, Collembola and Acarina are the most studied in soil ecology.
Their relevance for the soil system comes from their high abundance and diversity, and also from their
role in key biological processes. Collembola and Oribatid mites act mainly as catalysts in organic matter
[6],[21] [21],[26]
decomposition, whereas predacious mites can act as webmasters in soil food webs. These
characteristics, allied to a widespread taxonomic knowledge, allow their use as study organisms in several
research programmes dealing with the impacts of forest practices (e.g. References [8], [16], [17], [18], [22],
[23], [24], [28], [29], [32], [33], [35], [42]) or crop management practices (e.g [2], [7], [10], [13], [20], [25],
[43], [44].). These features make them suitable organisms to be used as bio-indicators of changes in soil
[38]
quality, especially due to land-use practices and pollution .
[45]
For the sampling design of field studies in general, see ISO 18400-104 for general guidance on the
development of site investigation strategies and detailed guidance on the development of sampling
strategies.
Methods for other soil organism groups, such as earthworms, enchytraeids, nematodes and macro-
[52] [53] [54] [55]
invertebrates are covered in ISO 23611-1 , ISO 23611-3 , ISO 23611-4 and ISO 23611-5 ,
respectively.
v
International Standard ISO 23611-2:2024(en)
Soil quality — Sampling of soil invertebrates —
Part 2:
Sampling and extraction of micro-arthropods (Collembola
and Acarina)
1 Scope
This document specifies a method for sampling, extracting and preserving collembolans and mites from
field soils as a prerequisite for using these animals as bio-indicators (e.g. to assess the quality of a soil as a
habitat for organisms).
The sampling and extraction methods of this document are applicable to almost all types of soils. Exceptions
can be soils from extreme climatic conditions (hard, frozen or flooded soils) and other matrices than soil,
e.g. tree trunks, plants or lichens.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain 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
micro-arthropod
group which is defined by its small size (range size from 100 µm to a few millimetres) making up a significant
part of the below-ground food web in many terrestrial ecosystems
Note 1 to entry: This group is mainly composed by mites (Acarina), springtails (Collembola), Protura, Diplura, garden
centipedes (Symphyla), Pauropoda, small centipedes and millipedes, small arachnids (spiders and pseudoscorpions),
and insects and their larvae from several orders (Hymenoptera, Diptera, Coleoptera, etc.).
4 Principle
Soil samples are collected in the field using a split corer. Soil cores are placed in plastic tubes (or plastic
bags) and transported to the laboratory. Afterwards, Collembola and Acarina are rapidly (within a few days)
extracted by behavioural methods, using a MacFadyen apparatus, and preserved for future identifications.
[12],[34]
In addition, preparation techniques are also described. Finally, abundance values can be recalculated
related to area (usually 1 m ), volume or weight (usually 1 kg).
NOTE Alternative methods for extraction can be used under special circumstances. Flotation methods (e.g. the
heptane flotation method) can be used in clay or loamy soils and a Kempson extractor (6.18) can be used in the case
[34]
litter is sampled .
ISO 23611-2:2024(en)
5 Test materials
5.1 Biological material
Collembola (springtails) are small wingless hexapods (from 150 µm up to 9 mm in length), having a
distinctive head with a pair of antennae, without true compound eyes, with six abdominal segments and
three pre-genital appendages in the abdomen. In the first segment, there is the ventral tube (or collophore)
that is used for adhering to smooth surfaces. The name Collembola comes from this structure (from Greek
colla = glue and embolon = bar). In the third segment, there is the tenaculum, which holds the jumping
apparatus on its normal position. This jumping appendage, the furcula (or spring), is located in the fourth
segment, when present. Springtails live in litter and soil, and have very distinctive life forms. They belong to
[4]
the class Collembola and can be separated into 33 families .
Soil mites are small chelicerate arthropods related to spiders (length from 150 µm up to < 5 mm), living
in soil and litter and presenting very distinctive life forms. They belong to the class Arachnida, subclass
Acarina, and can be separated into four groups: Cryptostigmata (Oribatida), Mesostigmata (Gamasida),
Prostigmata (Trombidiformes) and Astigmata.
NOTE Some hints for the taxonomy of springtails and mites are given in Annex A.
5.2 Reagents
Unless otherwise specified, use only reagents of good quality and distilled water.
5.2.1 Propan-2-ol, 80 % (volume fraction).
5.2.2 Formalin [formaldehyde solution 40 % (volume fraction)].
5.2.3 Acetic acid.
5.2.4 Phenol, C H OH, crystalline (carbolic acid).
6 5
5.2.5 Hydrogen chloride, c(HCl) from 8 mol/l to 10 mol/l.
5.2.6 2,2,2-Trichloro-1,1-ethanediol (chloral hydrate).
5.2.7 1,2,3-Trihydroxypropane (glycerine).
5.2.8 von Törne fixative, used to preserve the extracted animals and composed by Propan-2-ol (80 %),
formalin (40 %) and glacial acetic acid (a volume fraction 10:0,3:0,03).
5.2.9 Nesbitt clearing medium, used to clear mite specimens composed of chloral hydrate (80 g), distilled
water (50 ml) and concentrated hydrogen chloride (5 ml).
5.2.10 Lactophenol solution, used to clear mite specimens composed of lactic acid (10 ml), crystals of
phenol (3,6 g) and distilled water (5 ml).
5.2.11 2-Hydroxypropanoic acid (lactic acid), to clear and observe micro-arthropod specimens, especially
oribatid mites under the microscope.
5.2.12 Ethanol, 70 % to 75 % (volume fraction), used for fixation and preservation (in this case, also in
combination with glycerine, 10:1).
5.2.13 Hoyer’s medium, used to mount Collembola specimens composed of distilled water (50 ml), gum-
arabic (30 g), chloral hydrate (200 g) and glycerine (20 ml).

ISO 23611-2:2024(en)
5.2.14 DNA extraction buffer (SNET buffer solution), used to clear collembolans.
5.2.15 Protease K solution, used to clear collembolans.
5.2.16 Ethanol 35 % (volume fraction), used for preservation of the specimens.
5.2.17 Formol 3 %, used for preservation of the specimens.
5.2.18 Marc André 2 medium, to clear and provide the best optical properties to the specimens for
identification.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Measuring tape.
6.2 Collecting flasks.
6.3 Wash bottle.
6.4 Forceps, pipette, fine painting brush, fine needles.
6.5 Petri dishes.
6.6 Stereomicroscope.
6.7 Microscope, with phase or interference contrast is preferable.
6.8 Microscopic slides, with excavated area in the centre, and lamellae.
6.9 Electrical heating plate.
6.10 Plastic vials.
6.11 Ceramic heating elements.
6.12 Pencil, notebook, water resistant marker, labels.
6.13 Split corer
Sampling device made of stainless steel or aluminium (40 cm long and e.g. 5,6 cm diameter may be used; the
length and diameter should not differ considerably from these numbers, in order to maintain comparable
conditions), used to collect soil cores (samples). It can be composed of two independent parts that fit together
along the corer main axis or it can consist of one tube. On the top, it has a handle and, on the bottom, a
cutting edge.
6.14 Glass vials.
6.15 Drying oven.
ISO 23611-2:2024(en)
6.16 MacFadyen apparatus
High-gradient (multiple) device used to extract micro-arthropods from soil samples. The principle is to create
an artificial temperature gradient between the canister where the sample is placed (hot) and the collecting
device below (cold), inducing a negative thermotactic (at the same time, a positive hygrotactic, negative
phototactic, and positive skototactic) behaviour on the animals that, by this way, leave the soil sample.
6.17 Plastic tubes, with caps (5 cm diameter, 5 cm long), or plastic bags, for storing the soil samples.
6.18 Kempson extractor, in the case litter is sampled.
6.19 Sample frame, 25 cm × 25 cm × 15 cm, made of stainless steel and with sharpened edges, to sample
animals from the litter layer.
NOTE For details concerning the equipment in 6.13, and 6.16 to 6.19, see References [12] and [34].
7 Procedure
7.1 Collecting the soil samples
At each sampling point (previously defined according to sampling design rules), a soil sample is collected
using a split corer (6.13); for flooded soils the same corer may be employed, but an auger tip should be
present to retain the soil after extraction.
NOTE In addition to the general characterization of the site, it is useful to determine the actual moisture of the
soil to be sampled.
After the sample is taken, the corer is opened (a picture of the soil core profile can complement the site
characterization) and the soil core is separated into litter layer (including the humus horizon) and the
upper 10 cm of the mineral soil. Generally, 5 cm layers are used for the upper part of the mineral horizon, but
if a finer analysis is required, thinner layers can be defined. The depth of the litter layer should be registered.
After this procedure, each layer is conditioned in plastic tubes; these are sealed with caps, labelled, and
stored for transportation to the laboratory. Plastic bags can be used as substitutes of the plastic tubes (6.17),
but special care shall be taken during conditioning to avoid disturbing the core structure and compaction
of the soil material, that can lead to the death of animals. The time lapse between sampling and extraction
should be recorded and should not exceed 5 days (if the samples remain at 20 °C ± 2 °C and the soil is kept
moist), in order to avoid undesirable side effects due to confinement and shifts in micro populations.
If sampling of animals is restricted to the litter layer, a sample frame (6.19) is used instead. The frame is
pressed into the litter by hand. Directly afterwards, the litter inside the frame is collected and the litter
samples are placed in plastic bags (6.17), labelled, and stored.
When sampling in soil, the site should be physico-chemically characterized. In particular, pH, particle
size distribution, C/N ratio, organic carbon content and water-holding capacity should be measured using
[46] [47] [48] [49] [50] [51]
ISO 10390 , ISO 10694 , ISO 11274 , ISO 11277 , ISO 11461 , ISO 11465 .
7.2 Extracting Collembola and Acarina from soil samples
In the laboratory, animals are extracted by behavioural methods, e.g. using a MacFadyen high-gradient
extractor (6.16). Each sample core is placed inverted into the canister having a plastic or metal net (2 mm
mesh size) on the bottom. This is connected to a funnel attached to a collecting flask (6.2) with 25 ml of “von
Törne-fixative” (5.2.8).
Alternatively, a saturated solution of picric acid, a 50 % ethylene glycol solution (plus some drops of a
detergent) or even 75 % ethanol (5.2.12) may be used as fixative.
A temperature gradient is created between the upper part (where the samples are placed) and the lower
part of the system (where the collecting flasks are placed). Heat can be provided by ceramic heating
elements (6.11), giving approximately 10 W per sample. The collecting flasks are immersed in a cooling

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