EN ISO 23611-4:2022

SLOVENSKI STANDARD
01-marec-2023
Nadomešča:
SIST EN ISO 23611-4:2012
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 4. del: Vzorčenje, ekstrakcija in
identifikacija nematod iz tal (ISO 23611-4:2022)
Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and
identification of soil-inhabiting nematodes (ISO 23611-4:2022)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme,
Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO 23611-4:2022)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 4 : Prélèvement, extraction
et identification des nématodes du sol (ISO 23611-4:2022)
Ta slovenski standard je istoveten z: EN ISO 23611-4:2022
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 23611-4
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2022
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-4:2011
English Version
Soil quality - Sampling of soil invertebrates - Part 4:
Sampling, extraction and identification of soil-inhabiting
nematodes (ISO 23611-4:2022)
Qualité du sol - Prélèvement des invertébrés du sol - Bodenbeschaffenheit - Probenahme von Wirbellosen
Partie 4 : Prélèvement, extraction et identification des im Boden - Teil 4: Probenahme, Extraktion und
nématodes du sol (ISO 23611-4:2022) Bestimmung von Boden bewohnenden Nematoden
(ISO 23611 4:2022)
This European Standard was approved by CEN on 16 July 2022.

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
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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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-4:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 23611-4:2022) 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 2023, and conflicting national standards
shall be withdrawn at the latest by February 2023.
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 23611-4:2011.
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 23611-4:2022 has been approved by CEN as EN ISO 23611-4:2022 without any
modification.
INTERNATIONAL ISO
STANDARD 23611-4
Second edition
2022-08
Soil quality — Sampling of soil
invertebrates —
Part 4:
Sampling, extraction and
identification of soil-inhabiting
nematodes
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 4: Prélèvement, extraction et identification des nématodes du
sol
Reference number
ISO 23611-4:2022(E)
ISO 23611-4:2022(E)
© ISO 2022
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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 23611-4:2022(E)
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
6.1 Sampling . 3
6.2 Extraction . 4
6.3 Counting . 4
6.4 Fixation and preparation of mass slides . 5
6.5 Identification . 5
7 Procedure .5
7.1 General . 5
7.2 Sampling . 5
7.3 Extraction . 6
7.4 Counting . 7
7.5 Fixation and preparation of mass slides . 8
7.6 Identification . 8
8 Data assessment .8
9 Test report . 9
Annex A (informative) Figures of equipment and methods for nematological research .11
Annex B (informative) Information about the availability of the Oostenbrink elutriator .14
Annex C (informative) Information about the Baermann funnel/tray extraction method .17
Annex D (informative) Examples of the use of soil invertebrates in soil monitoring
programmes (including presentation of their results) .19
Bibliography .24
iii
ISO 23611-4:2022(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.
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 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 23611-4:2007), which has been
technically revised. The main changes are as follows:
— examples of the use of nematodes in soil monitoring programmes have been added (including
presentation of their results) as an informative annex (see Annex D).
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 23611-4:2022(E)
Introduction
This document has been drawn up since there is 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:
[37],[42],[57]
— biological classification of soils including soil quality assessment ;
[25],[28],[31],[50]
— terrestrial bio-indication and long-term monitoring ;
[4]
— evaluation of the effects of chemicals on soil animals in the field (see ISO 11268-3 ).
Data for these purposes are gained by standardized methods since they can form the basis for far-
reaching decisions (e.g. whether a given site should be remediated or not). In fact, the lack of such
standardized methods is one of the most important reasons why bio-classification and bio-assessment
in terrestrial (i.e. soil) habitats has so far been relatively rarely used in comparison to aquatic sites.
Nematodes are an important and major part of the soil fauna. Some authors estimate that this group
[52]
is probably the most dominant one of the multicellular organisms (Metazoa) on earth . Nematodes
occur from the Antarctic to the tropics and from deep sea sediments to mountain regions. They are
active in every place with sufficient water and organic material. The species diversity and functional
[14]
variety are impressive . Nematodes are commonly known as parasites of animals and plants, but the
major part of the nematode fauna participates in decomposition processes by feeding on bacteria and
fungi.
6 -2 6 -2
Nematodes occur in high numbers (0,2 × 10 m to 9 × 10 m ) and with a high (10 to 100 species)
[12]
diversity in almost every soil sample . Moreover, there is a broad ecological spectrum of feeding types
and food web relations among the nematodes such as bacterivores, fungivores, herbivores, predators
[57],[58]
and omnivores . These factors make the group highly suitable as indicators for ecological soil
[56]
quality , but standardization of methods is urgently needed for comparison and combination of
results.
In the past 100 years, nematology has developed strongly from the viewpoint of agriculture, advisory
sampling and phytosanitary regulations because some terrestrial nematodes cause a lot of damage in
crops. With respect to methods, there are several “schools” in different parts of the world with their own
[14]
history, practical advantages and disadvantages. A comprehensive overview is given by Oostenbrink
[48],[49]
and Southey . The more recently described methods (or variants) are often developed with special
interest to certain plant parasitic species. Within the past 20 years new methods have evolved that
[21],[34],[54].
allow a DNA-based taxonomic identification of nematode species This opens the taxonomic
analysis of nematodes to a broader community of non-specialists.
[16]
Since Bongers introduced the Maturity Index, the use of nematodes in bio-indication for soil quality
[56]
has increased rapidly . Nematodes are now used for ecological soil research and monitoring in
several countries all over the world. Monitoring activities make special demands on methodology, for
instance, that a large number of soil samples is processed on a routine basis against reasonable costs.
Some of the methods originally developed for advisory sampling in agriculture are very suitable for
ecological research. They form the basis for specific variants described in this document.
The nematodes that are characterized by the proposed procedure are all the free-living forms of
nematodes found in soil. They include non-plant-feeding nematodes as well as ectoparasitic plant-
feeding nematodes and free-living stage of endoparasitic nematodes. The quantification of obligate
plant-feeding nematodes in roots requires specific methods. Basic information on the ecology of
nematodes and their use as bio-indicators can be found in the bibliography.
v
INTERNATIONAL STANDARD ISO 23611-4:2022(E)
Soil quality — Sampling of soil invertebrates —
Part 4:
Sampling, extraction and identification of soil-inhabiting
nematodes
1 Scope
This document specifies a method for sampling and handling free-living nematodes from terrestrial
field soils as a prerequisite for using them as bio-indicators (e.g. to assess the quality of a soil as a
habitat for organisms).
This document applies to all terrestrial biotopes in which nematodes occur. The sampling design of
field studies in general is specified in ISO 18400-101.
This document is not applicable to aquatic nematodes because of differences in the sample matrix
(e.g. water column). Methods for some other soil organism groups such as earthworms, collembolans
enchytraeids or macro-invertebrates are covered in ISO 23611-1, ISO 23611-2, ISO 23611-3 and
ISO 23611-5.
This document 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 include suitable procedures for measuring pH, particle size distribution, C/N ratio,
organic carbon content and water-holding capacity.
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 terminological 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
nematode
small, non-segmented free-living worm (up to a few millimetres in length) belonging to the class
Nematoda
Note 1 to entry: Nematodes without a soil-inhabiting stage are not included in this context.
3.2
location
study area or plot that is characterized based on the composition of (among others) the nematode fauna
ISO 23611-4:2022(E)
3.3
bulk-sample
composite soil sample made out of many small soil cores to get an impression of the average nematode
composition
3.4
soil sampler
tool to collect soil material in a quick and standardized way
3.5
mass slide
microscopic slide on which 300 to 400 nematodes are mounted for species identification (3.7)
3.6
identification
determination of the species, genus or family of an individual based on morphological characteristics
(mouth parts, sexual organs, body ratios) with an identification key
3.7
colonizer–persister (cp) scale
ecological classification of nematodes
[16],[17]
Note 1 to entry: Proposed by Bongers .
Note 2 to entry: The principle is analogous to the r-K life strategies during succession, distinguished in
fundamental ecology. Non-plant-feeding nematode families are classified to one of the five cp-groups. This is also
the basis for the calculation of the Maturity Index.
4 Principle
Nematodes are collected in soil samples with a small cylindrical core (diameter: approximately 2 cm;
length: 10 cm or 15 cm) or an auger (see Figure A.2). For monitoring purposes, the soil samples are
combined in a bulk-sample from a homogeneous area. The total number of samples to be taken depends
on the investigated surface area and its homogeneity (e.g. pedology, land use, crop). The individual
samples can be gathered in the field in a standard plastic bag or plastic bucket. The combined bulk-
sample is too large for direct examination and therefore it is mixed and subsampled. In the field and
during transport to the laboratory, the soil samples shall be protected against strong fluctuations in
temperature, water-loss and heavy mechanical disturbance. They can be stored for at most four weeks
at 4 °C.
[49]
NOTE 1 The sampling method described above is derived from “the Dutch Method” for determining
the infestation of a field with potato-cyst nematodes, and has been used for many years in several European
countries.
The Oostenbrink funnel method is recommended for routine extractions of soil samples, for instance
in a monitoring network. The Oostenbrink method is not the simplest one that can be used under any
circumstance. However, it has several advantages: it is highly standardized and constant in extraction
efficiency. The Oostenbrink wet funnel method combines three basic means that can be used for the
separation of nematodes from soils: washing, sieving, active movement. Therefore, it obtains better
results than any one of the basic methods individually. Further advantages are given below:
— relatively large soil samples of any soil type can be treated at once (100 g to 500 g);
— clean nematode suspensions;
— isolation of most living and active nematodes;
— there are many years of experience with enormous amounts of routine soil extractions;
— it is used in many places around the world.
ISO 23611-4:2022(E)
1)
After sampling, the nematodes are extracted from the soil using the Oostenbrink elutriator (model III)
(see Figure A.3 and Annex B). In this technique, an upward current of water separates the nematodes
[10],[33],[44],[49]
from soil particles and holds them in suspension while the heavier particles sink . This
suspension of nematodes and small particles passes through three sieves (mesh width: 45 μm). The
catch is washed from the sieves onto a cotton-wool filter (milk filter). The cotton-wool filter is mounted
on a supporting sieve and is placed in a dish with 100 ml of tap water. For three days, through their
active downwards movement, the nematodes separate themselves from the debris on the filter. Thus,
the living nematodes actively crawl through the filter in a dish with tap water.
After extraction, the nematodes are counted in 2 times 10 ml of the 100 ml suspension, then
concentrated, preserved and mounted on mass slides. Finally, at least 150 individuals or a fixed
percentage of the total number in the sample is identified under the microscope.
Mature nematodes can be identified to species level. However, populations in the soil are often
dominated by juveniles and the genera level of taxonomy is a practical (but less sensitive) way of
distinction.
[44]
Alternative extraction methods such as the Seinhorst elutriator , Baermann funnel (see Annex C)
can be useful under special circumstances, but are not recommended as general procedures because
the Oostenbrink elutriator is robust, easy to operate and usually quantitatively superior to most other
techniques. For preserved samples containing non-living organisms, the Oostenbrink elutriator method
is not suitable. Here, flotation/centrifugation methods using colloidal silica are recommended as they
[26]
allow the extraction of all the forms of nematodes .
NOTE 2 This document is not applicable for aquatic nematodes because these nematodes do not pass through
the filter. Special centrifugation techniques are available for sediment samples.
NOTE 3 Identification with a light microscope is based on morphological characteristics. In some cases, it is
not possible to recognize the specimen on species or even genus level, e.g. juveniles. New techniques, such as
[40],[54]
DNA barcoding or metabarcoding, offer a morphology-independent taxonomy, so that also juveniles can be
identified to genus or species level.
NOTE 4 The sampling of nematodes is often included in much broader monitoring programmes which try to
cover the whole soil fauna or parts of it (e.g. the mesofauna). Examples of the use of soil invertebrates are given in
Annex D. The design of such programmes is not included in this document.
5 Reagents
5.1 Formalin [formaldehyde solution, 60 ml/l].
5.2 Paraffin, with melting point near 60 °C.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Sampling
6.1.1 Soil sampler, of an open, closed or split-tube type.
EXAMPLE Grass plot sampler (diameter: 23 mm) or soil auger (see Figure A.2); commercially available.
6.1.2 Plastic bucket (collection of soil samples in the field).
1) Oostenbrink elutriator is the trade name of a product supplied e.g. by MEKU Erich Pollähne GmbH (https://
www .meku -pollaehne .de/ Nematologie/ Oostenbrink -Elutriator/ oostenbrink -elutriator .html). This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of the product
named. Equivalent products may be used if they can be shown to lead to the same results.
ISO 23611-4:2022(E)
6.1.3 Plastic container, for mixing of the bulk-sample.
6.1.4 Sieve, with 8 mm apertures.
6.1.5 Coated bags or plastic bags or glass vessels (transport and storage).
6.1.6 Permanent marker or pre-printed labels.
6.2 Extraction
6.2.1 Beaker, of capacity 100 ml to 250 ml.
6.2.2 Balance, able to weigh 1 kg to 25 kg, for weighing the total sample mass.
1)
6.2.3 Oostenbrink elutriator (see also Figure A.3, Annex B), metal funnel with an upward water
flow to separate nematodes from larger soil particles.
6.2.4 Three sieves, with 45 µm apertures and 30 cm diameters.
6.2.5 One sieve, with 250 µm apertures and a 10 cm diameter.
6.2.6 Plastic bowl, of capacity approximately 2 l.
6.2.7 Clamping ring.
6.2.8 Extraction sieve, with 1 000 µm apertures and 16 cm diameter.
6.2.9 Milk- or cotton-wool filters.
6.2.10 Shallow trays (Petri dishes) or special extraction dishes.
6.2.11 Glass vessel, of capacity 100 ml, with a screw-cap.
6.3 Counting
6.3.1 Dissecting microscope, 10× to 50× magnification.
6.3.2 Small counting dish with grid or glass slide with grid.
NOTE Counting dishes in several sizes and different grids are available from the manufacturers of laboratory
equipment. They can also be made out of small plastic Petri dishes by scratching a grid on the bottom with a
needle.
6.3.3 Simple hand counting device.
6.3.4 Aquarium pump, for mixing nematode suspensions.
6.3.5 Pipette (drop glass), with adjustable volume.
6.3.6 Handling needle.
6.3.7 Bottle, of volume 100 ml.
ISO 23611-4:2022(E)
6.4 Fixation and preparation of mass slides
6.4.1 Water jet pump, for concentration of suspension.
6.4.2 Glass slides, 50 mm × 76 mm.
6.4.3 Cover glasses, 45 mm × 45 mm.
6.4.4 Electric heating plate.
6.4.5 Metal stamp, 40 mm × 40 mm, for paraffin seal on glass slides.
6.5 Identification
6.5.1 Microscope, magnification 400× to 1 000×.
6.5.2 Ocular micrometer indicator.
[15]
6.5.3 Identification keys .
6.5.4 Standard form, to list the identification results.
7 Procedure
7.1 General
For quality assurance, each sample shall be given a unique code from the moment it is taken in the
field. This code (label) shall stay with the sample during all the processing and analysis steps. Standard
(electronic) form(s) should be used to follow the routing of the samples and collection of analysis
results. These basic data may be combined in a spreadsheet or database file for further calculations and
statistical testing.
7.2 Sampling
While the density and diversity of soil nematodes are the highest in the top 10 cm of the mineral
soil, a grass plot sampler (6.1.1) with a 10 cm or 15 cm long sampling-tube is appropriate for most
biomonitoring purposes. It is recommended to use a closed tube with a fixed length and diameter.
EXAMPLE 1 A grass plot sampler consists of a stainless-steel gouge auger (available in different dimensions)
consisting of a steel auger pipe, a collecting bucket (6.1.2) and a stick with a steel handle. Because of the conical
shape of the pipe, the sample is easily pushed toward the collecting bucket when the next sample is taken. The
sample depth is constant and soil cores can be collected easily over a large area (see Figure A.1). This device can
be used in many situations.
EXAMPLE 2 Alternatively, a soil auger can be used as a simple, cheap and quick working device. Augers are
available in different diameters. Soil samples collected with an auger are less compressed. The disadvantage is
that soil material can be lost more easily (see Figure A.2).
EXAMPLE 3 When accurate separation of soil layers is required, a split-tube sampler can be used. This
sampling device needs more handling time and is less suited for large numbers of samples and large areas (see
Figure A.2).
Samples from deeper layers can be taken with an auger to avoid excessive soil compression, or special
split-tube samplers (see Figure A.2). Organic or litter material can be included in the samples, but it
increases the numbers of nematodes found, sometimes considerably. Organic layers may be sampled
independently. In this case, a wider split-tube corer (5 cm to 10 cm) is preferred in order to separate
ISO 23611-4:2022(E)
the organic horizons from the mineral material. Small amounts of litter can also be treated in an
Oostenbrink elutriator (6.2.3) to extract the nematodes. Extraction efficiency can be enhanced by
[41],[43]
soaking and blending the organic parts .
When a representative sample is required from a specific type of ecosystem, a typical area of at least
2 2
5 000 m , and preferably 10 000 m , shall be selected. It is recommended to select an area which is
(more or less) homogeneous in terms of soil properties, vegetation and soil-use. The studied surface is
reported as part of the location information. As a rule of thumb, 100 soil cores shall be combined from
2 2
10 000 m . For smaller areas (e.g. 100 m ), approximately 25 cores are sufficient to get an impression
of the average nematode composition and to collect enough soil material. A denser sampling pattern
results in a higher accuracy in the estimation of nematode abundance and species composition. However,
there is a trade-off with the amount of subsample that is finally analysed from the homogenized
bulk soil sample. So, a very large bulk-sample does not give more information because only a small
part is analysed and homogenization cannot be completely perfect. Three hundred samples with a
grass plot sampler (diameter 23 mm) are recommended as a maximum for a composite bulk-sample.
In a biomonitoring programme, the sample density per surface area should preferably be equal in all
locations. The mass of the bulk-sample and the number of soil cores in it need to be known.
The sampling plan may be regular (grid), according to a pattern or random. For larger locations, it is
most practical to walk a zigzag pattern and take arbitrary samples along this route. In the case that a
location consists of different parcels, the number of soil samples shall be distributed over the parcels
based on their area. Samples from very atypical parts of the location such as ditches, tracks or pathways
shall be avoided.
The collected bulk-sample is homogenized in a plastic container (6.1.3). This can be done in the field
or laboratory, depending on the most practical way of working and transport. Mixing starts with
crumbling of the cores through an 8 mm aperture sieve (6.1.4). Subsequently, the soil is gently mixed
until the mass is uniform in colour and consistency. Mixing and preparation of the sample can take
more than an hour for bulk-samples from clay soils or densely rooted top soils. However, this step is
essential to all the analyses that are based on it and should be given enough attention. Coarse organic
material, roots and stones shall be removed. The final choice for details of working shall be specified in
the field sampling protocol, and again shall be uniform for the entire biomonitoring programme.
When the bulk-sample is homogenized, approximately one litre is taken out for further examination.
This can be done by several spoonful from different parts of the bulk to obtain a representative
subsample. Put the sample in a labelled plastic bag or glass vessel (6.1.5). At this point, more subsamples
can be taken from the bulk for other biotic and abiotic analyses. Soil samples for nematode analysis
shall be stored at 4 °C prior to extraction. The storage period should be kept as short as possible, four
weeks at the maximum. This temperature may not be optimal for all nematodes (e.g. Aphelenchoididae,
Anguinidae; see Reference [13]). In any case, the appropriate temperature shall be checked beforehand
when sampling outside of the holarctic region (e.g. in the tropics).
7.3 Extraction
Mix the soil (sub)sample from the field again before extraction and fill a beaker (6.2.1) with 100 ml
to 250 ml of soil. Weigh the sample in order to convert the results to a fresh-weight basis. In another
sub-part of the same soil sample, measure the soil humidity to express final density as a unit of soil dry-
weight. Store the remaining sample for abiotic analysis, unless material was collected separately in the
field from the same bulk-sample.
Prepare the Oostenbrink elutriator (6.2.3). Put the sample in the top sieve and wash the soil in the
elutriator. Specific water flow speeds depend on the type of funnel used. A detailed description of the
apparatus and the way to use it is given in References [10] and [49]. The latest version of the Oostenbrink
funnel has larger dimensions and is fully automated (see Figure A.5). It is suited for routine extraction
of large numbers of samples. The soil sample is washed into the funnel through the top sieve. When
ISO 23611-4:2022(E)
the funnel is filled by the upward stream of water, the nematodes are separated from the heavier soil
particles.
NOTE 1 If for whatever reason an Oostenbrink elutriator is not available, the Baermann funnel/tray method
can be an alternative. Details concerning its use are given in Annex C.
Unplug the funnel when the water has reached the edge and catch the water flow on a pile of three
sieves with 45 µm pore size (6.2.4). An additional top sieve (6.2.5) can be mounted on the three sieves
(6.2.4) to catch large nematodes that do not pass the final cotton-wool filter step. This sieve (6.2.5) shall
be placed directly in an extraction dish (shallow tray filled with water) (6.2.10) and stored in a cabinet
for three days. At higher ambient temperatures, the cabinet should be humidified and kept away from
heat sources. The sieves (6.2.4) are rinsed with a gentle stream of water which is caught in a plastic
bowl (6.2.6). The suspension with the nematodes can be set aside for 15 min to 30 min, during which
time the nematodes settle to the bottom. It has the advantage that the supernatant water in the plastic
bowl can be poured out more quickly. Pour the content of the bowl over a double filter [e.g. cotton-wool
(milk)] (6.2.9), which is clipped onto an extraction sieve (6.2.8) with a clamping ring (6.2.7). Place the
extraction sieve (6.2.8) with debris in an extraction dish (6.2.10). The active nematodes in the debris
crawl through the filter (6.2.9) in the extraction dish (6.2.10) with 100 ml of tap water during a period
of three days. It is important that the filter (6.2.9) stays in contact with the water in the extraction dish
(6.2.10). If necessary, add tap water during the three-day period. After three days, both the 250 µm
pore size sieve (6.2.5) and the sieve with cotton-wool filters (6.2.9) are removed and the content of the
extraction dishes (6.2.10) can be poured into a glass vessel of 100 ml (6.2.11). Label the vessels with the
correct sample codes. Subsequently, the nematodes can be counted.
NOTE 2 Other filter material than a cotton-wool filter is acceptable as long as it has been shown that its
properties equal those of cotton wool with respect to nematode extraction (i.e. no change in pore size after
absorption of water).
NOTE 3 This method allows the user to extract only mobile forms. Therefore, the count does not take into
account the motionless forms (eggs, dead organisms in the soil). This type of extraction does not give the exact
value of the number of nematodes (at any stage of development) at the time of the extraction. For preserved
samples containing non-living organisms, this method is not suitable (see Clause 4).
NOTE 4 The last extraction phase lasts for a period of three days. In this step, the cotton-wool filters (in a
supporting sieve) are placed in a shallow dish with 100 ml tap water. Experimental results show that 59 % of
[53]
the nematodes crawl out during the first 24 h, 73 % after two days, and 82 % are caught after three days .
These numbers are based on a comparison with the nematodes found after seven days. For practical reasons, a
standard extraction period of three days is chosen. A longer extraction time has the disadvantages that the water
in the dishes evaporates, nematodes can die, and eggs present in the debris possibly hatch.
7.4 Counting
Before counting, combine the contents of the two vessels (6.2.11) containing the nematodes from one
soil sample. Wait at least 2 h to be sure that the nematodes have sunk to the bottom. Concentrate the
suspensions (decanting or suction) so that suspensions fit in one 100-ml bottle (6.3.7 or 6.2.11). If
necessary, add tap water to the vessel until it contains exactly 100 ml. Mix the suspension with air
(6.3.4) thoroughly (3 min to 5 min). Immediately afterwards, take out 10 ml mixed suspension with
a pipette (6.3.5) and put it in a counting dish (6.3.2). Do this in duplicate. Count the number (6.3.3)
of nematodes under a stereomicroscope (6.3.1) while moving the counting dish slowly around. The
result of the two counts shall be within 10 % difference. Otherwise, the suspension was probably not
homogenous enough and the counting procedure shall be repeated. Note the number of nematodes in
each counting dish on a standard form. The average number of the duplicate count, scaled up to 100 ml
suspension, is the estimation of the nematode abundance in the extracted amount (volume or weight)
of soil. Put the counted subsample back in the 100-ml bottle (6.3.7). Next, the nematodes shall be heat
killed and fixed with formalin (5.1).
The volume of the suspension in the vessels and the volume of the mixed suspension taken out with
the pipette for counting depend on the density of the nematodes in the vessels. Although a suspension
volume of 100 ml and the amount taken out for counting (10 ml) work well in most cases, this can be
changed. Almost all nematodes from the soil sample should be counted if the nematode density is very
ISO 23611-4:2022(E)
low, while only 1 % of suspension may include already too many nematode individuals if the original
density in the soil is very high.
WARNING — Appropriate precautions should be taken when dealing with formalin to avoid
danger from inhalation or skin exposure (such as wearing gloves and handling the samples
in a fume cupboard). According to the “Material Safety Data Sheet” for formaldehyde 370 ml/l
solution as published by producing companies, the compound is a skin sensitizer and is
considered to be a carcinogen (humans: limited evidence; animals: sufficient evidence). It is
notified in industrialized countries and is allowed for scientific use.
7.5 Fixation and preparation of mass slides
If recently counted, let the nematodes sink to the bottom of the vessel for several hours. Meanwhile, the
suspension may be kept in a refrigerator at 4 °C. Concentrate the suspension until about 5 mm of water
is left in the vessel. This can be done with a small pipette (6.3.5) connected to a (water flow) vacuum
pump (6.4.1). A piece of plankton gauze, (10 µm pore size) stretched over the opening of the pipette,
prevents the loss of nematodes. Add approximately 10 ml of boiling water to the concentrated sample
to kill and stretch the nematodes. Add approximately 30 ml formalin (5.1) to preserve the sample.
The sample can then be kept for a reasonably long period (i.e. several months) and mass slides can be
prepared for species identification.
Concentrate the sample again before the preparation of mass slides. Prepare the slides (6.4.2) by adding
a small paraffin square (ridge) (5.2) with a heated stamp (6.4.5). Press the stamp briefly on the paraffin
so that it can melt and stick to the stamp. Then put the stamp on the glass slide. Make two slides for
every sample. Add two drops of (shaken) suspension to each slide. Put a cover glass (6.4.3) over the
paraffin square of approximately 40 mm by 40 mm and the nematodes in the drops of fixative. Heat the
slide on a hot plate (6.4.4) to 65 °C so that the paraffin melts again. Remove the slides directly otherwise
the nematodes can be damaged. The paraffin becomes solid and the nematodes are mounted in the
slides. Mark the slides with the number of the sample and A or B. The slides can be stored for several
months.
NOTE The selection of the fixation method depends on the objective of the study. For a taxonomic
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