prEN ISO 23611-4

Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and identification of soil-inhabiting nematodes (ISO/DIS 23611-4:2021)

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.

Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme, Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO/DIS 23611 4:2021)

Qualité du sol - Prélèvement des invertébrés du sol - Partie 4 : Prélèvement, extraction et identification des nématodes du sol (ISO/DIS 23611-4:2021)

Le présent document spécifie une méthode pour échantillonner et manipuler les nématodes du sol en tant que condition préalable pour les utiliser comme bio-indicateurs (par exemple pour évaluer la qualité d’un sol en tant qu’habitat pour des organismes).
Le présent document s’applique à tous les biotopes terrestres dans lesquels des nématodes sont présents. Le mode opératoire d’échantillonnage pour les études de terrain est spécifié de façon générale dans l’ISO 18400-101.
Le présent document n’est pas applicable aux nématodes aquatiques en raison des différences dans la matrice d’échantillonnage (par exemple, la colonne d’eau). Des méthodes destinées à certains autres groupes d’organismes tels que les vers de terre, les collemboles, les enchytréides ou les macro-invertébrés, sont décrites dans l’ISO 23611-1, l’ISO 23611-2, l’ISO 23611-3 et l’ISO 23611-5.
Le présent document ne couvre pas la caractérisation pédologique du site qui est particulièrement recommandée lors du prélèvement d’invertébrés du sol. L’ISO 10390, l’ISO 10694, l’ISO 11272, l’ISO 11274, l’ISO 11277, l’ISO 11461 et l’ISO 11465 décrivent des modes opératoires appropriés pour mesurer le pH, la répartition granulométrique, le rapport C/N, la teneur en carbone organique et la capacité de rétention en eau.

Kakovost tal - Vzorčenje nevretenčarjev v tleh - 4. del: Vzorčenje, ekstrakcija in identifikacija nematod iz tal (ISO/DIS 23611-4:2021)

General Information

• Status: Not Published
• ICS: 13.080.05 - Examination of soils in general
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6055 - CEN Ratification completed (DOR) - Publishing
• Due Date: 02-Sep-2021
• Ref Project: oSIST prEN ISO 23611-4:2021 - Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and identification of soil-inhabiting nematodes (ISO/DIS 23611-4:2021)

RELATIONS

• Revised: EN ISO 23611-4:2011 - Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and identification of soil-inhabiting nematodes (ISO 23611-4:2007)
• Effective Date: 13-Feb-2019

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 23611-4:2021
01-november-2021

Kakovost tal - Vzorčenje nevretenčarjev v tleh - 4. del: Vzorčenje, ekstrakcija in

identifikacija nematod iz tal (ISO/DIS 23611-4:2021)
Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and
identification of soil-inhabiting nematodes (ISO/DIS 23611-4:2021)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme,
Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO/DIS 23611
4:2021)

Qualité du sol - Prélèvement des invertébrés du sol - Partie 4 : Prélèvement, extraction

et identification des nématodes du sol (ISO/DIS 23611-4:2021)
Ta slovenski standard je istoveten z: prEN ISO 23611-4
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 23611-4:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 23611-4:2021
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oSIST prEN ISO 23611-4:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23611-4
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-10-04 2021-12-27
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
ICS: 13.080.05; 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 23611-4:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2021
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oSIST prEN ISO 23611-4:2021
ISO/DIS 23611-4:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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

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ISO copyright office
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Published in Switzerland
ii © ISO 2021 – All rights reserved
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oSIST prEN ISO 23611-4:2021
ISO/DIS 23611-4:2021(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 ....................................................................................................................................................................................................... 4

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.................................................................................................................................................................................................... 9

9 Test report ................................................................................................................................................................................................................10

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 ......................16

Annex D (informative) Examples of the use of soil invertebrates in soil monitoring

programmes (including presentation of their results) .............................................................................................18

Bibliography .............................................................................................................................................................................................................................23

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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.

This second edition cancels and replaces the first edition (ISO 23611-4:2007), which has been

technically revised. The main change to the previous edition is as follows:

— addition of examples of nematods monitoring programmes (including presentation of their results)

as an informative annex.
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 2021 – All rights reserved
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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:
[36],[41],[56]
— biological classification of soils including soil quality assessment ;
[24],[27],[30],[49]
— terrestrial bio-indication and long-term monitoring ;
[33]
— evaluation of the effects of chemicals on soil animals (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

[51]

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

Nematodes occur in high numbers (0,2 – 9 × 10 m ) and with a high (10 to 100) species diversity

[12]

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

[56],[57]

and omnivores . These factors make the group highly suitable as indicators for ecological soil

[55]

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

[47],[48]

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

[20],[33],[53]

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

[55]

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.

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oSIST prEN ISO 23611-4:2021
DRAFT INTERNATIONAL STANDARD ISO/DIS 23611-4:2021(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.

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.

NOTE Basic information on the ecology of nematodes and their use as bio-indicators can be found in the

bibliography.

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.

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3.2
location

study area or plot that is characterized based on the composition of (among others) the nematode fauna

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
Oostenbrink elutriator

metal funnel with an upward water flow to separate nematodes from larger soil particles

See Figure A.3.
3.6
mass slide

microscopic slide on which 300 to 400 nematodes are mounted for species identification

3.7
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
3.8
colonizer – persister (cp) scale
[16],[17]
ecological classification of nematodes, proposed by Bongers

Note 1 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: circa 2 cm; length:

10 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, and 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.

[48]

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 most simple 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

1) Oostenbrink elutriator is the trade name of a product supplied e.g. by MEKU Erich Pollähne GmbH (http://

www .meku -pollaehne .de/ Nematologie/ Oostenbrink -Elutriator/ oostenbrink -elutriator .html). This information is

given for the convenience of users of this International Standard 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.

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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.

After sampling, the nematodes are extracted from the soil using the Oostenbrink elutriator 1) (model

III) (see Figure A.3 and Annex B). In this technique, an upward current of water separates the nematodes

[10],[32],[43],[48]

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 × 10 % 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.
[43]

Alternative extraction methods such as the Seinhorst elutriator , Baermann funnel (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. As an alternative, flotiation extraction using colloidal silica are recommended for preserved

[25]
samples containing non-living organisms

NOTE 2 This part of ISO 23611 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 Determination 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

[39],[53]

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 nematods 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, 6 % (volume fraction)].
5.2 Paraffin, with melting point near 60 °C.
6 Apparatus
Use standard laboratory equipment and the following.
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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 (Figures A.2 and A.3); commercially available.

6.1.2 Plastic bucket (collection of soil samples in the field).
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.
6.2.3 Oostenbrink elutriator (see also Annex B).
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 circa 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.
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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.
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.
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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

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

[40] [42]
soaking and blending the organic parts , .

When a representative sample is required from a specific type of ecosystem, a typical area of at least

0,5 ha, and preferably 1 ha, 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 1 ha. For

smaller areas (e.g. 100 m ), circa 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
...