SIST EN ISO 23611-4:2012
(Main)Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and identification of soil-inhabiting nematodes (ISO 23611-4:2007)
Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and identification of soil-inhabiting nematodes (ISO 23611-4:2007)
ISO 23611-4:2007 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).
ISO 23611-4:2007 applies to all terrestrial biotopes in which nematodes occur. It is not applicable to aquatic nematodes because these nematodes do not pass through the filter.
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme, Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO 23611-4:2007)
Dieser Teil von ISO 23611 legt ein Verfahren zur Probenahme und Handhabung frei lebender Nematoden aus terrestrischen Freilandböden fest, das als eine Voraussetzung für die Verwendung dieser Tiere als Bioindikatoren (z. B. zur Beurteilung der Beschaffenheit eines Bodens als Lebensraum für Organismen) dient. Dieser Teil von ISO 23611 gilt für alle terrestrischen Biotope, in denen Nematoden vorkommen. Die allgemeine Planung der Probenahme für Freilanduntersuchungen ist in ISO 10381-1 festgelegt. Dieser Teil von ISO 23611 gilt nicht für aquatische Nematoden, da diese nicht durch das Filter hindurchgehen. Die Verfahren für einige andere Bodenorganismengruppen wie z. B. Regenwürmer, Enchytraeen oder Collembolen werden in anderen Teilen von ISO 23611 behandelt. Bei den Nematoden, die mit dem vorgeschlagenen Verfahren charakterisiert werden, handelt es sich um alle im Boden vorkommenden, frei lebenden Formen von Nematoden. Sie umfassen Nematoden, die sich nicht von Pflanzen ernähren, sowie auch ektoparasitäre pflanzenfressende Nematoden und frei lebende Stadien endoparasitärer Nematoden. Die mengenmäßige Bestimmung obligater pflanzenfressender Nematoden in Wurzeln erfordert besondere Verfahren. ANMERKUNG Grundlegende Informationen zur Ökologie von Nematoden und ihrer Verwendung als Bioindikatoren sind im Literaturverzeichnis zu finden. Dieser Teil von ISO 23611 behandelt nicht die Charakterisierung des Standortes nach bodenkundlichen Gesichtspunkten, die für die Probenahme von Wirbellosen im Boden nachdrücklich empfohlen wird, da eine enge Beziehung zwischen dem Vorkommen bestimmter Arten und abiotischen Faktoren besteht. ISO 10390, ISO 10694, ISO 11272, ISO 11274, ISO 11277, ISO 11461 und ISO 11465 sind zur Bestimmung des pH-Wertes, der Korngrößenverteilung, des C/N-Verhältnisses, des Gehaltes an organischem Kohlenstoff und des Wasserrückhaltevermögens geeigneter.
Qualité du sol - Prélèvement des invertébrés du sol - Partie 4: Prélèvement, extraction et identification des nématodes du sol (ISO 23611-4:2007)
L'ISO 23611-4:2007 sp�cifie une m�thode pour �chantillonner et manipuler les n�matodes du sol en tant que condition pr�alable pour les utiliser comme bio-indicateurs (par exemple pour �valuer la qualit� d'un sol en tant qu'habitat pour des organismes).
L'ISO 23611-4:2007 s'applique � tous les biotopes terrestres dans lesquels des n�matodes sont pr�sents. Elle n'est pas applicable pour les n�matodes aquatiques parce que ceux-ci ne traversent pas le filtre.
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 4. del: Vzorčenje, ekstrakcija in identifikacija nematod iz tal (ISO 23611-4:2007)
ISO 23611-4:2007 določa metodo za vzorčenje in delo s prostoživečimi nematodi iz poljskih tal kot nujen pogoj za uporabo teh živali kot bioindikatorjev (npr. za ocenjevanje kakovosti tal kot habitata za organizme).
ISO 23611-4:2007 velja za vse zemeljske biotope, v katerih so nematodi. Ne velja za vodne nematode, ker ti ne prehajajo skozi filter.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 23611-4:2012
01-januar-2012
.DNRYRVWWDO9]RUþHQMHQHYUHWHQþDUMHYYWOHKGHO 9]RUþHQMHHNVWUDNFLMDLQ
LGHQWLILNDFLMDQHPDWRGL]WDO ,62
Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and
identification of soil-inhabiting nematodes (ISO 23611-4:2007)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme,
Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO 23611-4:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 4: Prélèvement, extraction et
identification des nématodes du sol (ISO 23611-4:2007)
Ta slovenski standard je istoveten z: EN ISO 23611-4:2011
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 23611-4:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 23611-4:2012
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SIST EN ISO 23611-4:2012
EUROPEAN STANDARD
EN ISO 23611-4
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2011
ICS 13.080.30; 13.080.05
English Version
Soil quality - Sampling of soil invertebrates - Part 4: Sampling,
extraction and identification of soil-inhabiting nematodes (ISO
23611-4:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie Bodenbeschaffenheit - Probenahme von Wirbellosen im
4: Prélèvement, extraction et identification des nématodes Boden - Teil 4: Probenahme, Extraktion und Bestimmung
du sol (ISO 23611-4:2007) von Boden bewohnenden Nematoden (ISO 23611-4:2007)
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 23611-4:2011: E
worldwide for CEN national Members.
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SIST EN ISO 23611-4:2012
EN ISO 23611-4:2011 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 23611-4:2012
EN ISO 23611-4:2011 (E)
Foreword
The text of ISO 23611-4:2007 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 23611-4:2011 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 23611-4:2007 has been approved by CEN as a EN ISO 23611-4:2011 without any
modification.
3
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SIST EN ISO 23611-4:2012
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SIST EN ISO 23611-4:2012
INTERNATIONAL ISO
STANDARD 23611-4
First edition
2007-11-15
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 23611-4:2007(E)
©
ISO 2007
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
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ii © ISO 2007 – All rights reserved
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 2
4 Reagents. 3
5 Apparatus . 3
5.1 Sampling. 4
5.2 Extraction . 4
5.3 Counting . 4
5.4 Fixation and preparation of mass slides. 5
5.5 Identification. 5
6 Procedure . 5
6.1 General. 5
6.2 Sampling. 5
6.3 Extraction . 6
6.4 Counting . 7
6.5 Fixation and preparation of mass slides. 7
6.6 Identification. 8
7 Data assessment. 8
8 Study report. 9
Annex A (informative) Figures of equipment and methods for nematological research. 10
Annex B (informative) Information about the availability of the Oostenbrink elutriator. 13
Annex C (informative) Information about the Baermann funnel/tray extraction method . 15
Bibliography . 17
© ISO 2007 – All rights reserved iii
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(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 23611-4 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 soil-inhabiting nematodes
iv © ISO 2007 – All rights reserved
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
Introduction
This part of ISO 23611 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:
[15],[17],[28]
⎯ biological classification of soils including soil quality assessment ;
[9],[10],[13],[24]
⎯ terrestrial bio-indication and long-term monitoring ;
⎯ 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 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 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.
Nematodes occur in high numbers [(5 000 to 100 000)/kg fresh soil] and with a high (20 to 100) species
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 and
[27],[28]
omnivores . These factors make the group highly suitable as indicators for ecological soil 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 history, practical
[14] [22],[23]
advantages and disadvantages. A comprehensive overview is given by Oostenbrink and Southey .
The more recently described methods (or variants) are often developed with special interest to certain
plant-parasitic species.
[4]
Since Bongers introduced the Maturity Index, the use of nematodes in bio-indication for soil quality 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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SIST EN ISO 23611-4:2012
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SIST EN ISO 23611-4:2012
INTERNATIONAL STANDARD ISO 23611-4:2007(E)
Soil quality — Sampling of soil invertebrates —
Part 4:
Sampling, extraction and identification of soil-inhabiting
nematodes
1 Scope
This part of ISO 23611 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 part of ISO 23611 applies to all terrestrial biotopes in which nematodes occur. The sampling design of
field studies in general is specified in ISO 10381-1.
This part of ISO 23611 is not applicable to aquatic nematodes because these nematodes do not pass through
the filter. Methods for some other soil organism groups such as earthworms, enchytraeids or collembolans are
covered in other parts of ISO 23611.
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 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
nematode
small, non-segmented free-living worm (up to a few millimetres in length) belonging to the class Nematoda
NOTE Nematodes without a soil-inhabiting stage are not included in this context.
2.2
location
study area or plot that is characterized based on the composition of (among others) the nematode fauna
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
2.3
bulk-sample
composite soil sample made out of many small soil cores to get an impression of the average nematode
composition
2.4
soil sampler
tool to collect soil material in a quick and standardized way
2.5
1)
Oostenbrink elutriator
metal funnel with an upward water flow to separate nematodes from larger soil particles
See Figure A.3.
2.6
mass slide
microscopic slide on which 300 to 400 nematodes are mounted for species identification
2.7
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
2.8
colonizer – persister (cp) scale
[4],[5]
ecological classification of nematodes, proposed by Bongers
NOTE 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.
3 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, culture). 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.
[23]
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.
1) Oostenbrink elutriator is the trade name of a product supplied by firm Eijkelkamp, Giesbeek, NL
(http://www.eijkelkamp.nl). 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.
2 © ISO 2007 – All rights reserved
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
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
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.
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 from soil
[1],[14],[19],[23]
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.
[19]
Alternative extraction methods such as the Seinhorst elutriator or 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, centrifugation techniques are most suitable.
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 level, e.g. juveniles. With a new technique based on DNA analysis,
juveniles can be identified to species level. This new technique is expected to become operational within several years.
4 Reagents
4.1 Formalin [formaldehyde solution, 6 % (volume fraction)].
4.2 Paraffin, with melting point near 60 °C.
5 Apparatus
Use standard laboratory equipment and the following.
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
5.1 Sampling
5.1.1 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.
5.1.2 Plastic bucket (collection of soil samples in the field).
5.1.3 Plastic container, for mixing of the bulk-sample.
5.1.4 Sieve, with 8 mm apertures.
5.1.5 Coated bags or plastic bags or glass vessels (transport and storage).
5.1.6 Permanent marker or pre-printed labels.
5.2 Extraction
5.2.1 Beaker, of capacity 100 ml to 250 ml.
5.2.2 Balance, able to weigh 1 kg to 25 kg, for weighing the total sample mass.
1)
5.2.3 Oostenbrink elutriator (see also Annex B).
5.2.4 Three sieves, with 45 µm apertures and 30 cm diameters.
5.2.5 One sieve, with 250 µm apertures and a 10 cm diameter.
5.2.6 Plastic bowl, of capacity circa 2 l.
5.2.7 Clamping ring.
5.2.8 Extraction sieve, with 1 000 µm apertures and 16 cm diameter.
5.2.9 Milk- or cotton-wool filters.
5.2.10 Shallow trays (Petri dishes) or special extraction dishes.
5.2.11 Glass vessel, of capacity 100 ml, with a screw-cap.
5.3 Counting
5.3.1 Dissecting microscope, 10× to 50× magnification.
5.3.2 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.
5.3.3 Simple hand counting device.
5.3.4 Aquarium pump, for mixing nematode suspensions.
5.3.5 Pipette (drop glass), with adjustable volume.
5.3.6 Handling needle.
5.3.7 Bottle, of volume 100 ml.
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
5.4 Fixation and preparation of mass slides
5.4.1 Water jet pump, for concentration of suspension.
5.4.2 Glass slides, 50 mm × 76 mm.
5.4.3 Cover glasses, 45 mm × 45 mm.
5.4.4 Electric heating plate.
5.4.5 Metal stamp, 40 mm × 40 mm, for paraffin seal on glass slides.
5.5 Identification
5.5.1 Microscope, magnification 400× to 1 000×.
5.5.2 Ocular micrometer indicator.
[3]
5.5.3 Identification keys .
5.5.4 Standard form, to list the identification results.
6 Procedure
6.1 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.
6.2 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 (5.1.1) 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 (5.1.2) 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 the organic horizons from
1)
the mineral material. Small amounts of litter can also be treated in an Oostenbrink elutriator (5.2.3) to
extract the nematodes. Extraction efficiency can be enhanced by soaking and blending the organic
[16],[18]
parts .
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SIST EN ISO 23611-4:2012
ISO 23611-4:2007(E)
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 th
...
SLOVENSKI STANDARD
kSIST FprEN ISO 23611-4:2011
01-april-2011
[Not translated]
Soil quality - Sampling of soil invertebrates - Part 4: Sampling, extraction and
identification of soil-inhabiting nematodes (ISO 23611-4:2007)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 4: Probenahme,
Extraktion und Bestimmung von Boden bewohnenden Nematoden (ISO 23611-4:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 4: Prélèvement, extraction et
identification des nématodes du sol (ISO 23611-4:2007)
Ta slovenski standard je istoveten z: FprEN ISO 23611-4
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
kSIST FprEN ISO 23611-4:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN ISO 23611-4:2011
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kSIST FprEN ISO 23611-4:2011
EUROPEAN STANDARD
FINAL DRAFT
FprEN ISO 23611-4
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2010
ICS 13.080.30; 13.080.05
English Version
Soil quality - Sampling of soil invertebrates - Part 4: Sampling,
extraction and identification of soil-inhabiting nematodes (ISO
23611-4:2007)
Qualité du sol - Prélèvement des invertébrés du sol - Partie Bodenbeschaffenheit - Probenahme von Wirbellosen im
4: Prélèvement, extraction et identification des nématodes Boden - Teil 4: Probenahme, Extraktion und Bestimmung
du sol (ISO 23611-4:2007) von Boden bewohnenden Nematoden (ISO 23611-4:2007)
This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical
Committee CEN/TC 345.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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 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.
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.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprEN ISO 23611-4:2010: E
worldwide for CEN national Members.
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kSIST FprEN ISO 23611-4:2011
FprEN ISO 23611-4:2010 (E)
Contents Page
Foreword .3
2
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kSIST FprEN ISO 23611-4:2011
FprEN ISO 23611-4:2010 (E)
Foreword
The text of ISO 23611-4:2007 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as FprEN ISO 23611-4:2010 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This document is currently submitted to the Unique Acceptance Procedure.
Endorsement notice
The text of ISO 23611-4:2007 has been approved by CEN as a FprEN ISO 23611-4:2010 without any
modification.
3
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kSIST FprEN ISO 23611-4:2011
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kSIST FprEN ISO 23611-4:2011
INTERNATIONAL ISO
STANDARD 23611-4
First edition
2007-11-15
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 23611-4:2007(E)
©
ISO 2007
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
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ii © ISO 2007 – All rights reserved
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 2
4 Reagents. 3
5 Apparatus . 3
5.1 Sampling. 4
5.2 Extraction . 4
5.3 Counting . 4
5.4 Fixation and preparation of mass slides. 5
5.5 Identification. 5
6 Procedure . 5
6.1 General. 5
6.2 Sampling. 5
6.3 Extraction . 6
6.4 Counting . 7
6.5 Fixation and preparation of mass slides. 7
6.6 Identification. 8
7 Data assessment. 8
8 Study report. 9
Annex A (informative) Figures of equipment and methods for nematological research. 10
Annex B (informative) Information about the availability of the Oostenbrink elutriator. 13
Annex C (informative) Information about the Baermann funnel/tray extraction method . 15
Bibliography . 17
© ISO 2007 – All rights reserved iii
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(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 23611-4 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 soil-inhabiting nematodes
iv © ISO 2007 – All rights reserved
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
Introduction
This part of ISO 23611 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:
[15],[17],[28]
⎯ biological classification of soils including soil quality assessment ;
[9],[10],[13],[24]
⎯ terrestrial bio-indication and long-term monitoring ;
⎯ 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 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 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.
Nematodes occur in high numbers [(5 000 to 100 000)/kg fresh soil] and with a high (20 to 100) species
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 and
[27],[28]
omnivores . These factors make the group highly suitable as indicators for ecological soil 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 history, practical
[14] [22],[23]
advantages and disadvantages. A comprehensive overview is given by Oostenbrink and Southey .
The more recently described methods (or variants) are often developed with special interest to certain
plant-parasitic species.
[4]
Since Bongers introduced the Maturity Index, the use of nematodes in bio-indication for soil quality 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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kSIST FprEN ISO 23611-4:2011
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kSIST FprEN ISO 23611-4:2011
INTERNATIONAL STANDARD ISO 23611-4:2007(E)
Soil quality — Sampling of soil invertebrates —
Part 4:
Sampling, extraction and identification of soil-inhabiting
nematodes
1 Scope
This part of ISO 23611 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 part of ISO 23611 applies to all terrestrial biotopes in which nematodes occur. The sampling design of
field studies in general is specified in ISO 10381-1.
This part of ISO 23611 is not applicable to aquatic nematodes because these nematodes do not pass through
the filter. Methods for some other soil organism groups such as earthworms, enchytraeids or collembolans are
covered in other parts of ISO 23611.
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 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
nematode
small, non-segmented free-living worm (up to a few millimetres in length) belonging to the class Nematoda
NOTE Nematodes without a soil-inhabiting stage are not included in this context.
2.2
location
study area or plot that is characterized based on the composition of (among others) the nematode fauna
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
2.3
bulk-sample
composite soil sample made out of many small soil cores to get an impression of the average nematode
composition
2.4
soil sampler
tool to collect soil material in a quick and standardized way
2.5
1)
Oostenbrink elutriator
metal funnel with an upward water flow to separate nematodes from larger soil particles
See Figure A.3.
2.6
mass slide
microscopic slide on which 300 to 400 nematodes are mounted for species identification
2.7
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
2.8
colonizer – persister (cp) scale
[4],[5]
ecological classification of nematodes, proposed by Bongers
NOTE 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.
3 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, culture). 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.
[23]
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.
1) Oostenbrink elutriator is the trade name of a product supplied by firm Eijkelkamp, Giesbeek, NL
(http://www.eijkelkamp.nl). 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.
2 © ISO 2007 – All rights reserved
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
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
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.
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 from soil
[1],[14],[19],[23]
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.
[19]
Alternative extraction methods such as the Seinhorst elutriator or 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, centrifugation techniques are most suitable.
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 level, e.g. juveniles. With a new technique based on DNA analysis,
juveniles can be identified to species level. This new technique is expected to become operational within several years.
4 Reagents
4.1 Formalin [formaldehyde solution, 6 % (volume fraction)].
4.2 Paraffin, with melting point near 60 °C.
5 Apparatus
Use standard laboratory equipment and the following.
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
5.1 Sampling
5.1.1 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.
5.1.2 Plastic bucket (collection of soil samples in the field).
5.1.3 Plastic container, for mixing of the bulk-sample.
5.1.4 Sieve, with 8 mm apertures.
5.1.5 Coated bags or plastic bags or glass vessels (transport and storage).
5.1.6 Permanent marker or pre-printed labels.
5.2 Extraction
5.2.1 Beaker, of capacity 100 ml to 250 ml.
5.2.2 Balance, able to weigh 1 kg to 25 kg, for weighing the total sample mass.
1)
5.2.3 Oostenbrink elutriator (see also Annex B).
5.2.4 Three sieves, with 45 µm apertures and 30 cm diameters.
5.2.5 One sieve, with 250 µm apertures and a 10 cm diameter.
5.2.6 Plastic bowl, of capacity circa 2 l.
5.2.7 Clamping ring.
5.2.8 Extraction sieve, with 1 000 µm apertures and 16 cm diameter.
5.2.9 Milk- or cotton-wool filters.
5.2.10 Shallow trays (Petri dishes) or special extraction dishes.
5.2.11 Glass vessel, of capacity 100 ml, with a screw-cap.
5.3 Counting
5.3.1 Dissecting microscope, 10× to 50× magnification.
5.3.2 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.
5.3.3 Simple hand counting device.
5.3.4 Aquarium pump, for mixing nematode suspensions.
5.3.5 Pipette (drop glass), with adjustable volume.
5.3.6 Handling needle.
5.3.7 Bottle, of volume 100 ml.
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
5.4 Fixation and preparation of mass slides
5.4.1 Water jet pump, for concentration of suspension.
5.4.2 Glass slides, 50 mm × 76 mm.
5.4.3 Cover glasses, 45 mm × 45 mm.
5.4.4 Electric heating plate.
5.4.5 Metal stamp, 40 mm × 40 mm, for paraffin seal on glass slides.
5.5 Identification
5.5.1 Microscope, magnification 400× to 1 000×.
5.5.2 Ocular micrometer indicator.
[3]
5.5.3 Identification keys .
5.5.4 Standard form, to list the identification results.
6 Procedure
6.1 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.
6.2 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 (5.1.1) 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 (5.1.2) 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 the organic horizons from
1)
the mineral material. Small amounts of litter can also be treated in an Oostenbrink elutriator (5.2.3) to
extract the nematodes. Extraction efficiency can be enhanced by soaking and blending the organic
[16],[18]
parts .
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kSIST FprEN ISO 23611-4:2011
ISO 23611-4:2007(E)
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
2
(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 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 homogenizatio
...
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