EN ISO 23611-2:2011

SLOVENSKI STANDARD
01-januar-2012
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Soil quality - Sampling of soil invertebrates - Part 2: Sampling and extraction of micro-
arthropods (Collembola and Acarina) (ISO 23611-2:2006)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 2: Probenahme
und Extraktion von Mikroarthropoden (Collembolen und Milben) (ISO 23611-2:2006)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 2 : Prélèvement et extraction
des micro-arthropodes (Collembola et Acarina) (ISO 23611-2:2006)
Ta slovenski standard je istoveten z: EN ISO 23611-2:2011
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.

EUROPEAN STANDARD
EN ISO 23611-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2011
ICS 13.080.30; 13.080.05
English Version
Soil quality - Sampling of soil invertebrates - Part 2: Sampling
and extraction of micro-arthropods (Collembola and Acarina)
(ISO 23611-2:2006)
Qualité du sol - Prélèvement des invertébrés du sol - Partie Bodenbeschaffenheit - Probenahme von Wirbellosen im
2 : Prélèvement et extraction des micro-arthropodes Boden - Teil 2: Probenahme und Extraktion von
(Collembola et Acarina) (ISO 23611-2:2006) Mikroarthropoden (Collembolen und Milben) (ISO 23611-
2:2006)
This European Standard was approved by CEN on 17 June 2011.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-2:2011: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
The text of ISO 23611-2:2006 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 23611-2:2011 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by January 2012, and conflicting national standards shall be withdrawn at
the latest by January 2012.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
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, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 23611-2:2006 has been approved by CEN as a EN ISO 23611-2:2011 without any
modification.
INTERNATIONAL ISO
STANDARD 23611-2
First edition
2006-02-01
Soil quality — Sampling of soil
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 23611-2:2006(E)
©
ISO 2006
ISO 23611-2:2006(E)
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ii © ISO 2006 – All rights reserved

ISO 23611-2:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Terms and definitions. 1
3 Principle. 1
4 Test materials. 2
4.1 Biological material. 2
4.2 Reagents. 2
5 Apparatus. 3
6 Procedure. 4
6.1 Collecting the soil samples . 4
6.2 Extracting Collembola and Acarina from soil samples . 4
6.3 Sorting, preserving and identifying Collembola and Acarina. 5
7 Assessment of results. 6
8 Study report. 6
Annex A (informative) Species determination in collembolans and mites . 7
Annex B (informative) Alternative methods for sampling of micro-arthropods . 9
Bibliography . 10

ISO 23611-2:2006(E)
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 23611-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
ISO 23611 consists of the following parts, under the general title Soil quality — Sampling of soil invertebrates:
⎯ Part 1: Hand-sorting and formalin extraction of earthworms
⎯ Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina)
⎯ Part 3: Sampling and soil extraction of enchytraeids
⎯ Part 4: Sampling, extraction and identification of free-living stages of terrestrial nematodes
iv © ISO 2006 – All rights reserved

ISO 23611-2:2006(E)
Introduction
This part of ISO 23611 has been drawn up since there is 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 [31], [32], [35], [41],
[45], [46]);
⎯ terrestrial bioindication and long-term monitoring (e.g. References [1], [7], [17], [40], [42]).
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 decomposition
[4], [20] [9]
, whereas predacious mites may act as webmasters in soil food webs . These characteristics, allied to
a widespread taxonomic knowledge, allowed their use as study organisms in several research programmes
dealing with the impacts of forest practices (e.g. References [12], [13], [14], [15], [18], [19], [21], [22], [23], [25],
[26], [27], [28], [29], [30], [31], [33], [34], [37], [38], [39]) or crop management practices (e.g. [6], [11], [16], [24]).
These features make them suitable organisms to be used as bio-indicators of changes in soil quality,
[43]
especially due to land-use practices and pollution .

INTERNATIONAL STANDARD ISO 23611-2:2006(E)

Soil quality — Sampling of soil invertebrates —
Part 2:
Sampling and extraction of micro-arthropods (Collembola and
Acarina)
1 Scope
This part of ISO 23611 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).
Basic information on the ecology of micro-arthropods and their use can be found in the references listed in the
Bibliography.
The sampling and extraction methods of this part of ISO 23611 are applicable to almost all types of soils.
Exceptions may be soils from extreme climatic conditions (hard, frozen or flooded soils) and other matrices
than soil, e.g. tree trunks, plants or lichens. For the sampling design of field studies in general, see
ISO 10381-1.
Methods for some other soil organism groups such as earthworms are covered in other parts of ISO 23611.
This part of ISO 23611 does not cover the pedological characterization of the site which is highly
recommendable when sampling soil invertebrates. ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277,
ISO 11461 and ISO 11465 are more suitable for measuring pH, particle size distribution, C/N ratio, organic
carbon content and water-holding capacity.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
micro-arthropods
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 This group is mainly composed by mites (Acarina), springtails (Collembola), Protura, Diplura, garden
centipedes (Symphyla), Pauropoda, small centipedes and millipedes, and insects and their larvae from several orders
(Diptera, Coleoptera, etc.).
3 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 Acarida are rapidly (within a few days)
[7], [40]
extracted by behavioural methods, using a MacFadyen apparatus, and preserved for future identifications .
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).
ISO 23611-2:2006(E)
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 is advisable in the case litter is
[40]
sampled .
4 Test materials
4.1 Biological material
Collembola (springtails) are small wingless hexapods (from 150 µm up to 9 mm 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, that holds the jumping apparatus on its normal
position. This jumping appendage, the furcula (or spring), when it exists, is located in the fourth segment.
Springtails live in litter and soil, and have very distinctive life forms. They belong to the class Collembola, and
[17]
can be separated into 18 families .
Soil mites are small chelicerate arthropods related to spiders (length from 150 µm up to < 5 mm), living in soil
and litter, and also presenting very distinctive life forms. They belong to the class Arachnida, subclass Acarida,
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.
4.2 Reagents
Unless otherwise specified, use only reagents of good quality and distilled water.
4.2.1 Propan-2-ol, 80 % (volume fraction).
4.2.2 Formalin [formaldehyde solution 40 % (volume fraction)].
4.2.3 Acetic acid.
4.2.4 Phenol, C H OH, crystalline (carbolic acid).
6 5
4.2.5 Hydrogen chloride, c(HCl) from 8 mol/l to 10 mol/l.
4.2.6 2,2,2-Trichloro-1,1-ethanediol (chloral hydrate).
4.2.7 1,2,3-Trihydroxypropane (glycerine).
4.2.8 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).
4.2.9 Nesbitt clearing medium, used to clear mite specimens composed of chloral hydrate (80 g), distilled
water (50 ml) and concentrated hydrogen chloride (5 ml).
4.2.10 Lactophenol solution, used to clear mite specimens composed of lactic acid (10 ml), crystals of
phenol (3,6 g) and distilled water (5 ml).
4.2.11 2-Hydroxypropanoic acid (lactic acid), to clear and observe micro-arthropod specimens, especially
oribatid mites under the microscope.
4.2.12 Ethanol, 70 % to 75% (volume fraction), used for fixation and preservation (in this case, also in
combination with glycerine, 10:1).
2 © ISO 2006 – All rights reserved

ISO 23611-2:2006(E)
4.2.13 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).
5 Apparatus
Use standard laboratory equipment and the following.
5.1 Measuring tape.
5.2 Collecting flasks.
5.3 Wash bottle.
5.4 Forceps, pipette, fine painting brush, fine needles.
5.5 Petri dishes.
5.6 Stereomicroscope.
5.7 Microscope, with phase or interference contrast is preferable.
5.8 Microscopic slides, with excavated area in the centre, and lamellae.
5.9 Electrical heating plate.
5.10 Plastic vials.
5.11 Ceramic heating elements.
5.12 Pencil, notebook, water resistant marker, labels.
5.13 Split corer
Sampling device made of stainless steel or aluminium (40 cm long and e.g. 5,6 cm diameter may be used; the
size 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.
5.14 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.
5.15 Plastic tubes, with caps (5 cm diameter, 5 cm long), or plastic bags, for storing the soil samples.
5.16 Kempson extractor, in the case litter is sampled.
5.17 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 5.13 to 5.17, see References [7] and [40].
ISO 23611-2:2006(E)
6 Procedure
6.1 Collecting the soil samples
At each sampling point (previously defined according to sampling design rules), a soil sample is collected
using a split corer (5.13); 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 (see Clause 1), it is useful to determine the actual
moisture of the soil to be sampled.
After the sample is taken, the corer is opened 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 (5.15), but special care shall be taken during conditioning to avoid disturbing the core
structure and compaction of the soil material, that may lead to the death of animals. The time lapse between
sampling and extraction should not exceed a few days, 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 (5.17) 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 (5.15), labelled and stored.
6.2 Extracting Collembola and Acarina from soil samples
In the laboratory, animals are extracted by behavioural methods, e.g. using a MacFadyen high-gradient
extractor (5.14). 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 (5.2) with 25 ml of “von
Törne-fixative” (4.2.8).
Alternatively, a saturated solution of picric acid, a 50 % ethylene glycol solution (plus some drops of a
detergent) or even 75 % ethanol (4.2.12) may be used as fixative.
A temperature gradient is created between the upper part (where the samples are) and the lower part of the
system (where the collecting flasks are placed). Heat can be provided by ceramic heating elements (5.11),
giving approximately 10 W per sample. The collecting flasks are immersed in a cooling water bath. In some
commercial apparatus, the temperature gradient is obtained by circulating heated air in the canister area and
cooled air on the collecting area.
The temperature difference between the upper and lower parts should be around 30 °C to 35 °C, with the
upper part being heated at 45 °C to 50 °C and the lower part being cooled usually at 10 °C (maximum field
temperature). Special care shall be taken in order to avoid a fast increase in temperature in the upper part,
which may cause the rapid desiccation of the sample and the death of the animals. Therefore, it is
recommended to have a gradual increase of the temperature of the upper part, starting with approximately
5 °C above field temperature during the first three days, and intensifying the gradient for the next six to seven
days.
The extraction procedure takes nine to ten days. Afterwards, animal samples are labelled and ready to be
stored until processing (sorting and identification). Extraction should preferab
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