EN ISO 23611-5:2024

SLOVENSKI STANDARD
01-marec-2025
Nadomešča:
SIST EN ISO 23611-5:2013
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 5. del: Vzorčenje in ekstrakcija
velikih nevretenčarjev v tleh (ISO 23611-5:2024)
Soil quality - Sampling of soil invertebrates - Part 5: Sampling and extraction of soil
macro-invertebrates (ISO 23611-5:2024)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 5: Probenahme
und Extraktion von Makroinvertebraten (Großwirbellosen) im Boden (ISO 23611-5:2024)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 5: Prélèvement et extraction
des macro-invertébrés du sol (ISO 23611-5:2024)
Ta slovenski standard je istoveten z: EN ISO 23611-5:2024
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 23611-5
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2024
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-5:2013
English Version
Soil quality - Sampling of soil invertebrates - Part 5:
Sampling and extraction of soil macro-invertebrates (ISO
23611-5:2024)
Qualité du sol - Prélèvement des invertébrés du sol - Bodenbeschaffenheit - Probenahme von Wirbellosen
Partie 5: Prélèvement et extraction des macro- im Boden - Teil 5: Probenahme und Extraktion von
invertébrés du sol (ISO 23611-5:2024) Makroinvertebraten (Großwirbellosen) im Boden (ISO
23611-5:2024)
This European Standard was approved by CEN on 26 August 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.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-5:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23611-5:2024) 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 February 2025, and conflicting national standards
shall be withdrawn at the latest by February 2025.
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 23611-5:2013.
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 23611-5:2024 has been approved by CEN as EN ISO 23611-5:2024 without any
modification.
International
Standard
ISO 23611-5
Second edition
Soil quality — Sampling of soil
2024-08
invertebrates —
Part 5:
Sampling and extraction of soil
macro-invertebrates
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 5: Prélèvement et extraction des macro-invertébrés du sol
Reference number
ISO 23611-5:2024(en) © ISO 2024

ISO 23611-5:2024(en)
© ISO 2024
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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 23611-5:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 2
7 Field procedure . 3
7.1 General .3
7.2 Collecting macro-invertebrates from the litter zone .3
7.3 Collecting macro-invertebrates from soil .3
7.3.1 General .3
7.3.2 Temperate regions .3
7.3.3 Tropical regions.4
8 Laboratory procedure . 4
8.1 Treatment of collected samples .4
8.2 Preservation of specimens .5
8.3 Biomass determination .5
9 A ssessment of results . 6
10 Test report . 6
Annex A (informative) Background information . 7
Annex B (informative) Sampling soil macro-fauna using pitfall traps . 8
[68]
Annex C (informative) Monitoring example with pitfall traps . 9
Bibliography . 14

iii
ISO 23611-5:2024(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
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 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)
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 second edition cancels and replaces the first edition (ISO 23611-5:2011), which has been technically
revised.
The main changes are as follows:
— Two informative Annexes were added at the end of the document. Annex B describes the procedures to
be adopted when sampling macro-fauna using pitfall traps and Annex C presents a monitoring example
with pitfall traps.
— The bibliographic references list was revised and updated in the entire document.
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 23611-5:2024(en)
Introduction
This document was prepared in response to a need to standardize sampling and extraction methods for soil
macro-invertebrates globally. These methods are needed for the following purposes:
— biological classification of soils, including soil quality assessment (e.g. References [14], [28] and [37]);
— terrestrial bio-indication and long-term monitoring (e.g. References [65], [74], [75] and [76]).
Data collected using standardized methods can be evaluated more accurately as they allow more reliable
comparison between sites (e.g. polluted vs non-polluted sites, changes in land-use practices).
Soils of the world host an abundance of highly diverse macro-invertebrate communities. Their biology
and ecology have been widely studied. Soil invertebrates are irreplaceable actors of soil formation and
conservation in natural ecosystems. Their relevance to the soil system comes from their abundance and
diversity, and also from their role in key biological processes. They are sensitive indicators of soil quality
and recognized actors of its fertility (e.g. References [58] and [52]). Among the wide diversity of species,
adaptive strategies and size ranges represented, one specific group, also called “soil ecosystem engineers”,
includes large invertebrates that determine the activities of other smaller organisms through the mechanical
activities they produce in soil (e.g. References [18] and [46]).
Soil macro-invertebrates span a wide range of ecological functions in soil: decomposition of organic matter,
through their own activity and by stimulating the soil's microbiological activity (e.g. References [2], [3] and
[36]), predation that plays an important part in food webs (e.g. References [9], [51], [56], [59] and [63]),
soil aggregation by the production of organo-mineral structures (e.g. nests, galleries, casts) that can last
for days, months or years, soil bioturbation (e.g. Reference [28]), etc. These characteristics, coupled with
in-depth taxonomic knowledge, have enabled their use as study organisms in several research programmes
dealing with the impacts of forest practices (e.g. References [11], [36], [47], [57], [60] and [70]) or crop
management practices (e.g. References [8], [19], [27], [29], [30], [33], [38], [55] and [62]). These features
make them suitable organisms for use as bio-indicators of changes in soil quality, especially with respect to
land-use practices and pollution (e.g. References [21], [35], [45], [48], [49], [54], [60] and [74]).
The method proposed in this document covers the sampling of all soil macro-invertebrates. However, the
sampling of earthworms is already covered in ISO 23611-1. This alternative sampling method for earthworms
is described in ISO 23611-1:2018, Annex C.
The method proposed in this document is a prerequisite for using macro-invertebrates as bio-indicators
(e.g. to assess the quality of a soil as a habitat for organisms). The main premise of this method is rapid
assessment (completing the sampling of a plot in one or two days with only basic equipment and a small
number of field assistants) in order to be able to address all the taxonomic groups of soil macro-invertebrates
at the same time and in the same place. The Tropical Soil Biology and Fertility (TSBF) method has evolved
and some modifications have been introduced in order to use it in temperate regions.
A sampling design is specified in ISO 23611-6.
NOTE The method specified in this document is based on guidelines developed under the Tropical Soil Biology
[1]
and Fertility Program (TSBF method).

v
International Standard ISO 23611-5:2024(en)
Soil quality — Sampling of soil invertebrates —
Part 5:
Sampling and extraction of soil macro-invertebrates
1 Scope
This document specifies a method for sampling, extracting and preserving macro-invertebrates from soils,
including the litter zone.
The sampling and extraction methods in this document are applicable to almost all types of soil, with the
exception of soils in extreme climatic conditions (hard, frozen or flooded soils) and matrices other 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
macro-invertebrate
soil organism whose longest dimension is greater than 10 mm
EXAMPLE These include especially the following groups: Oligochaeta, Gastropoda, Chilopoda, Diplopoda, Isopoda,
Arachnida, plus various insects: Coleoptera, Orthoptera, Hymenoptera, Hemiptera, Dermaptera, Lepidoptera (larvae)
and Diptera (larvae).
Note 1 to entry: See Annex A for further details.
3.2
blotted mass
mass of individuals after preservation in formalin or ethanol (when the substance used for preservation has
been absorbed by the tissues)
4 Principle
Soil macro-invertebrates are collected in the field using a metallic frame to delimit the soil surface of the
sampling point. Macro-invertebrates present in litter and soil are picked up separately. In temperate regions,
a reagent is used to extract macro-invertebrates from soil. The sampling is completed by hand-sorting.
Animals are preserved and transported to the laboratory for further identification (e.g. References [4], [5],
[6], [7], [10], [12], [13], [16], [17], [22], [24], [25], [26], [31], [32], [34], [42], [43], [44], [50], [53], [64], [66],
[67], [71], [72], [73] and [77]). Abundance values are usually recalculated relative to area (1 m ).

ISO 23611-5:2024(en)
5 Reagents
5.1 Ethanol, (70 % volume fraction).
5.2 Formalin (formaldehyde solution), 4 % (volume fraction).
Both 70 % ethanol and 4 % formalin should be available for the preservation of specimens (4 % formalin is
more suitable for taxa with soft body parts, which can be transferred to ethanol after about 4 d fixation).
5.3 Formalin, 0,2 % (volume fraction), prepared by diluting 25 ml of formalin (39 %) in 5 l of water, for
soil macro-invertebrate extraction.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Petri dishes.
6.2 Stereo-microscope.
6.3 Plastic vials.
6.4 Entomological forceps.
6.5 Pencil, notebook, water-resistant marker, labels.
6.6 Tape measures.
6.7 Knife (cut glass).
6.8 Spade.
6.9 Plastic-weave produce sacks, for spreading on the ground.
6.10 Precision balance.
6.11 Large flat plastic trays (500 mm × 400 mm × 100 mm), for sorting the soil and litter.
6.12 Trowel.
6.13 Small plastic trays.
6.14 Fine forceps (or entomological forceps), pipette, fine paint brushes.
6.15 Sample vials, in various sizes with secure alcohol-tight caps (plastic throw away or plastic/glass
reusable vials).
6.16 Indian-ink pen (waterproof).
6.17 Stiff card for labels, ranging compass.
6.18 Large strong plastic bags (sealable).

ISO 23611-5:2024(en)
6.19 Table and plastic chairs, for sorting.
6.20 Cover, for protection from heavy rain.
6.21 Chemical protection gloves, suitable for working with formalin.
6.22 Metallic frame, preferably 250 mm × 250 mm.
Sample frame (250 mm × 250 mm × 50 mm) made of stainless steel and with sharpened edges to delimit the
sampling point where animals are sampled from the litter layer and soil.
6.23 Watering can.
6.24 Pair of scissors, to cut vegetation inside the frame.
6.25 Field balances.
7 Field procedure
7.1 General
Sampling should take place when accessible biodiversity is thought to be largest. In temperate regions, it
corresponds to spring or autumn; and in the tropics, it should take place towards the end of the rainy season.
When sampling soil invertebrates, 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 ISO 10390, ISO 10694, ISO 11274, ISO 11277, ISO 11461, ISO 11465. Natural minerals present in the site
soil should also be described.
7.2 Collecting macro-invertebrates from the litter zone
At each sampling point (= monolith) (previously defined according to sampling design rules), a litter sample
is collected using a metallic frame (6.22). The metallic frame is pressed into the litter by hand. The litter
inside the frame is removed and checked manually in the field using a large tray (6.11). Litter invertebrates
are preserved in 4 % formalin (5.2).
7.3 Collecting macro-invertebrates from soil
7.3.1 General
In temperate countries, the extraction of soil macro-invertebrates is carried out in two steps (see 7.3.2.1
and 7.3.2.2), while in tropical countries only the second step shall be performed (see 7.3.3). In both cases,
extraction of macro-invertebrates may be complemented by the use of pitfall traps (see Annexes B and C for
further details).
7.3.2 Temperate regions
7.3.2.1 Formalin extraction
The soil surface delimited by the metallic frame (6.22) is sprayed with 0,2 % formalin (5.3) using a watering
can (6.23). Two applications of 1,5 l of formalin are performed at intervals of about 10 min. Soil invertebrates
coming up to the surface are collected and preserved in vials (6.3) containing formalin (5.2).

ISO 23611-5:2024(en)
7.3.2.2 Hand-sorting of “passive” macro-invertebrates
At the end of the formalin extraction, the metallic frame (6.22) is removed and the upper 150 mm of soil is
excavated within the frame area (250 mm × 250 mm). The excavated soil is placed in a plastic bag (6.18) that
can be closed with a cover to prevent animals from escaping from the soil sample.
Appropriate sub-samples of soil are taken from the container and spread on a large tray (6.11). Macro-
invertebrates are collected and preserved in vials (6.3) with formalin (5.2). When hand-sorting is finished,
the excavated soil is replaced to avoid creating holes on the sampling site.
7.3.3 Tropical regions
In tropical countries, soil macro-invertebrates are sampled using a 250 mm × 250 mm × 300 mm deep soil
monolith. The monolith is isolated by cutting with a spade (6.8) a few centimetres outside the quadrate
(metallic frame) and then digging a 20 mm wide by 300 mm deep trench around it. This facilitates cutting of
the sample into horizontal strata and collecting animals escaping from the block.
The delimited block is divided into three layers, 0 mm to 100 mm, 100 mm to 200 mm and 200 mm
to 300 mm; and the soil and litter material is hand-sorted in trays (6.11). Since formalin is not applied in
tropical regions, the sampling depth should be doubled in order to be sure to collect endogeic and anecic
species of earthworms.
For social insects, special measures should be considered that take account of their high abundance and
marked patchiness; a nest can contain millions of individuals, of which none are sampled by a short transect,
and the contribution of the species concerned to a macrofaunal assemblage can thus be completely missed.
On the other hand, a highly populated nest sampled directly by a monolith can lead to a large overestimation
of the overall numerical or biomass density. In general, the TSBF transect should be placed to avoid direct
contact with termite and ant nests. For discussions, see References [35] and [36]. The protocol for a
100 m × 2 m transect designed to assess termite biodiversity (and feeding group representation) is given
in Reference [48]. In suitable circumstances, this protocol can also be deployed in parallel with the TSBF
transect.
NOTE Besides the general characterization of the site, it is useful to determine the actual moisture of the soil to be
sampled.
8 Laboratory procedure
8.1 Treatment of collected samples
In the laboratory, samples are cleaned with either distilled or tap water in a Petri dish with the help of a
brush or placing the organisms on a 0,5 mm to 1 mm sieve under the tap. Afterwards, the animals are placed
in new vials (6.15) with ethanol (70 % volume fraction) (5.1). Organisms with soft body parts are kept in
formalin for at least 4 d, or forever if possible.
For taxonomic identification, specimens are placed on petri dishes (6.1) and observed under the stereo-
microscope (6.2). A practical way to identify macro-invertebrates is to group them into orders first. Each
order is then identified into families and each family into species using taxonomy keys (examples of
taxonomy keys are the References [4], [5], [6], [7], [10], [12], [13], [17], [22], [24], [25], [26], [31], [32], [34],
[42], [43], [44], [53], [66], [67], [72], [73] and [77]).
Ideally, taxonomic determination should be based on the species level. If identification of species levels
fails due to time constraints, taxonomic expertise or missing taxonomic keys, e.g. mainly in tropical
regions, sorting to genus (and some higher taxonomic units) represents a good compromise between the
morphospecies and ordinal level approaches, especially as this allows most specimens to be assigned to a
functional group.
WARNING — Appropriate precautions (i.e. gloves, mask) should be taken when dealing with formalin
to avoid danger from inhalation or skin exposure. According to the Material Safety Data Sheet for
formaldehyde 37 % solution published by producing companies, the compound is a skin sensitizer

ISO 23611-5:2024(en)
and is considered to be carcinogenic (humans: limited evidence; animals: sufficient evidence). It is
legally notified in industrialized countries for scientific use.
8.2 Preservation of specimens
From any mixed soil sample of macrofauna, the following steps should be followed in order to obtain
standardized preserved specimens.
a) If the animal has no soft body parts, the organisms should be preserved in 70 % ethanol (commercial
ethanol should be diluted).
b) If the animal has soft body parts, the organism should be fixed in 4 % formalin and should, if possible, be
preserved in the same solution. Alternatively, 80 % ethanol may be used (if the organism has been fixed
during at least 4 d with 4 % formalin).
c) In all cases, samples should be stored separately in different vials, according to the smallest unit of
analysis (i.e. a monolith if the data are compared at that level).
d) Every vial should be labelled without using code numbers and should at least be written using
permanent ink, like Indian or Chinese ink, and using sturdy paper like goatskin parchment. Every label
should contain the following information:
— country;
— region;
— locality;
— collector’s name;
— date of collection.
e) For storing specimens:
— use vials (or glass tubes) that are not degraded by the ethanol or formalin, with screw caps;
— monitor levels of ethanol and formalin in order to keep them constant;
— store vials away from direct sunlight;
— change the preserving solution of each vial once every five years.
8.3 Biomass determination
Determination of biomass is performed using the preserved material. The animal's surface should be gently
dried with filter paper, then weighed using a precision balance (0,001 g).
It is virtually impossible to keep invertebrates alive after their capture in order to measure fresh masses.
In most cases, invertebrates are conserved in 70 % (volume fraction) ethanol or 4 % (volume fraction)
formalin. The latter is recommended for earthworms that should at least be fixed in formalin before being
kept in 70 % ethanol. Preservation always involves a decrease in mass, as body water is extracted by osmotic
forces. The amount lost can vary between 15 % and 40 %, depending on the water content of the animal and
its physiological state. Since most studies only aim to compare different sites and/or situations, mass loss is
not likely to distort the result. If accurate fresh mass data are necessary, it is easy to keep an aliquot of each
group and compare the mass, alive and fixed, a few days after fixation.

ISO 23611-5:2024(en)
9 Assessment of r esults
The following measurement end points can be used for the bioclassification of a soil, including bio-indication
or biomonitoring (e.g. anthropogenic stress-like chemicals or land-use changes):
— abundance (number of individuals per area);
— biomass;
— number of species or other taxonomically or ecologically defined groups;
— diversity indices (alpha, beta and gamma diversity).
Firstly, the number of individuals (total number by species or group) is counted and expressed as individuals
per sample. Secondly, the total abundance of individuals is multiplied by a factor (16) to obtain the number
of individuals per square metre.
Fresh mass measured in the field is the ideal way to calculate biomass. Failing this, the use of blotted mass,
after preservation, is acceptable. Other methods are reported in the literature, for example fresh mass
after blotting, dry mass at 60 °C overnight, drying to constant mass at higher temperatures, degutted fresh
mass, degutted dry mass, fresh mass multiplied by a constant (for assumed water content) and head width
(referenced to a calibration curve). However, these have less biological meaning than fresh mass.
10 Test report
The test report shall include at least the following information:
a) a reference to this document, i.e. ISO 23611-5:2024;
b) a full description of the study design and procedures;
c) characterization of the study site (especially soil properties);
d) sampling method;
e) description of the sampling conditions, including date and duration and time of the day of sampling in
the field and weather parameters like air temperature and humidity, rain or snow, etc.;
f) details of the extraction procedure of the biological material;
g) values recalculated to 1 m or another standard size, if necessary;
h) a summary of the results obtained;
i) a discussion of the results;
j) all information, including all measured raw data and all problems which have occurred or developed
during all phases of the study.

ISO 23611-5:2024(en)
Annex A
(informative)
Background information
Soil macro-invertebrates can also be defined as organisms belonging to taxa of which over 90 % of
specimens are visible to the naked eye. Soil macro-invertebrates comprise the following groups: Oligochaeta
(Annelida), Gastropoda (snails and slugs), Coleoptera (larvae and adults), Isoptera, Diplopoda, Chilopoda,
Hymenoptera, Arachnida, Dyctioptera, Orthoptera, Hemiptera, Dermaptera, Isopoda, Lepidoptera (larvae)
and Diptera (larvae). It specifically excludes groups with a relatively small number of specimens visible
to the naked eye such as Nematoda (e.g. Mermithidae), Enchytraeidae, Collembola, Acarina, Symphyla,
Pauropoda and Diplura. Core taxonomic units should be adopted as standard units for macrofaunal sampling.
The choice of 17 main taxa was made during the IBOY Workshop using the MDB (Macrofauna Data Base)
containing information about 32 countries and almost 1 000 sampled sites. The 17 taxa, Oligochaeta (order
Opisthopora), Coleoptera (larvae and adults), Isoptera, Diplopoda, Chilopoda, Formicidae, Gastropoda,
Aranaea, Blattoidea, Orthoptera, Dermaptera, Isopoda, Hemiptera, Lepidoptera larvae, Diptera (larvae and
adults) and residues (insects and non-insects), correspond to the most important soil macro-invertebrates
in terms of abundance and biomass.
The choice of a 250 mm × 250 mm × 300 mm monolith size is based on extensive, although largely empirical,
experience. First used by Zajonc (1956), it has been proposed as a standard for the Tropical Soil Biology and
[1][54]
Fertility Program. This monolith size is the same for both tropical and temperate soils. The aim was to
propose a method that was not excessively time-consuming, but which can provide an accurate assessment
of the composition and structure of soil macro-invertebrate communities. The method has been extremely
successful and has become a standard used in several hundred sites. Although studies aimed at specific
groups, especially termites, earthworms or ants, prefer different sample sizes, the size proposed represents
a very good compromise that allows a reasonable number of replicates to be made and the representation of
most orders in one single sample. Larger samples are excessively time-consuming and do not allow enough
replicates to be made. In most cases, a group of four well-trained persons can sort out 10 samples a day.
Unpublished field studies have shown that 15 to 20 samples are necessary in a single site to reduce variance
to a reasonably low proportion of mean (< 20 %). However, a comparison of sites with different plant cover
or soils and/or which have been subjected to different management options exposes significant differences
using as little as five samples, provided adapted statistical treatment (often multivariate analyses) is used.

ISO 23611-5:2024(en)
Annex B
(informative)
Sampling soil macro-fauna using pitfall traps
Assessing the diversity and activity of surface-dwelling fauna can be of paramount importance when
interpreting TSBF (Tropical Soil Biology and Fertility) collected data in the scope of soil ecosystem
functions and services, when trying to unearth patterns and possible interactions at a larger spatial scale
in the complex soil-based networks on terrestrial ecosystems or when aiming at assessing the status of soil
biodiversity. A common practice that may complement TSBF method, and that allows estimating soil epigeic
macro-fauna diversity and activity in ecological studies, resorts to the use of pitfall traps. This method can
be used to obtain quantitative estimates of epigeic fauna relative abundances. The principle behind these
traps is that organisms living in soil or having a preference for soil habitat are likely to accidentally fall in
open traps set at ground-level while moving on the soil surface. Once set on field, these traps can capture any
organisms passing by, at a rate that should be proportional to their activity, thus allowing for an estimate of
this parameter as well.
Depending on the average size of targeted organisms, different sizes of pitfall traps can be used; for a survey
on soil epigeic macro-fauna in general, plastic cups, with an opening of 8 cm to 10 cm in diameter and 9 cm
to 10 cm in height can be used. The pitfall should have a nested-cups design, comprising one cup to be
inserted on a pre-dug hole in soil, so that their top (opening) is lined with the bottom of the leaf litter or the
soil level, and an inner cup to collect the organisms. The nested-cup design makes the installation of the
pitfalls and its collection from the field easiest and reduces the amount of soil and debris that can fall into
[20,40]
the pitfall. Although bait can be used to amplify the capture rate or to target specific organisms, pitfall
traps in ecological studies, aimed at addressing the overall diversity, should be passive traps, so the cups
set in a field are usually only filled to half capacity with a preserving solution. A good preserving solution
should kill quickly (to prevent escape of organisms and predation), preserve the internal and external
organs of trapped organisms and minimize evaporation. The type and dilution of the preserving solution
depends on the objectives of the study; but when DNA isolation for further analysis is planned, non-diluted
propylene glycol or 96 % ethanol should be used. Propylene glycol is colourless, hygroscopic, resistant to
evaporation and non-toxic, and has a low surface tension, when compared to ethylene glycol, which is also
[40,41]
widely used. Ethanol can be an alternative when trapping time and weather conditions are not prone
[61]
to high evaporation rates. As an alternative, ethylene glycol (car anti freezing agent) diluted at 50 % with
water can also be used. Although some studies have reported ethylene glycol at 50 % as a suitable sampling
[39]
solution for DNA isolation, the former solutions are usually adopted when a DNA analysis is intended.
To prevent pitfall traps from flooding or being filled with falling litter and also to prevent larger non-target
organisms (e.g. small mammals and reptiles) from entering the trap, the inner cup surface may be covered
with a stone or other natural materials, always leaving enough space between each element to allow easy
movement of the epigeic fauna. A roof of plastic with the same diameter of the inner cup can also be used and
[20]
placed at a height of approximately 3 cm from the opening of the trap. Studies proved that pitfall roofs or
[20,23]
rain guards have no negative effects on pitfall catches; however, depending on the area the roof, it can
catch the attention of free-roaming animals, leading to the destruction of the pitfall. The number of traps to
be set in a field and the frequency of sample collection depend on the focus of the study, this being a versatile
method that can be applied to several sampling design methods. However, when considering each pitfall
trap as an independent experimental unit, only one pitfall should be placed per square meter. For a general
survey in temperate climate, pitfall traps should be left for a period of 7 to 14 days in the field (different
time intervals may be considered according to different climate zones and when using baited pitfall traps).
Sample collection can be done by either removing the trap from the soil and using a lid on the cup to keep
its content secured or by percolating the trap content over a disposable absorbent cloth (with the help of a
sieve) or a 50 µm net, that should then be tied and placed in a labelled cup with 96 % ethanol for storage to
[41]
ensure good DNA preservation.
Small parts of the organism’s body can be preserved separately for DNA extraction (e.g. legs), leaving the
rest of the material available for morphological analysis. The latter should be preserved in 70 % ethanol for
long-term storage. Processing of collected samples in laboratory may be similar to that of the TSBF method.

ISO 23611-5:2024(en)
Annex C
(informative)
[68]
Monitoring example with pitfall traps
C.1 Study aim
This study was conducted to evaluate the spatial distances between local communities and the intensity of
habitat heterogeneity at a given spatial scale. This evaluation aimed to assess, at different scales, the relative
importance of spatial and environmental factors that shape edaphic (Collembola) and epigeous (Carabidae)
communities.
Although pitfall trap method has been proposed as a sampling method for macro-invertebrates, this method
was selected for the study as it also covers specific epigeic mesofauna groups (such as Collembola).
C.2 Study area
Sampling was carried out in a typical agro-forest mosaic - a Mediterranean cork-oak field (Quercus suber
L.) - located in the consolidated alluvial plain of the Tagus River, in “Companhia das Lezírias” (Alcochete,
approximately 20 km east of Lisbon), Portugal (approximately 388 530 N, 088 520 W). The past land-use
history and management shaped the Cork-oak agro-forest systems at the landscape scale. Sampling sites
comprised four non-identical landscape windows (LW, 1 km each), chosen along a land-use management
gradient, from unmanaged woodland (LW1) to areas subjected to traditional management practices
such as forestry (LW2, LW3 and LW4) and pastures (LW3 and LW4). Thereby, while LW1 and LW2 were
dominated by closed cork-oak woodlands (less managed forests), in LW3 and LW4 open woodlands and
pasture lands predominated (highly managed forests) (Table C.1, see Reference [69] for more details).
The characterization of the landscape windows (LW1, LW2, LW3 and LW4) chosen for this assessment is
presented in Table C.1. This table also shows the mean values of environmental variables (and SD when
applicable) related to the microhabitat of the plot, the management of the patch and the landscape structure
within each LW. The indicators used in forestry, pastoral and agricultural variables served as the basis for
measuring management intensity, while landscape variables were recorded in agreement with FRAGSTATs’
Class metrics for each mainland-use type (Forest – F, Pastures/Grassland – G).
...