SIST EN ISO 23611-1:2018
(Main)Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2018)
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of earthworms (ISO 23611-1:2018)
This document specifies a method for sampling and handling earthworms from field soils as a
prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for
organisms).
This document applies to all terrestrial biotopes in which earthworms occur. The sampling design
of field studies in general is given in ISO 18400-101 and guidance on the determination of effects of
pollutants on earthworms in field situations is given in ISO 11268-3. These aspects can vary according
to the national requirements or the climatic/regional conditions of the site to be sampled (see also
Annex C).
This document is not applicable for semi-terrestrial soils and it can be difficult to use under extreme
climatic or geographical conditions (e.g. in high mountains). Methods for some other soil organism
groups, such as collembolans, are covered in other parts of ISO 23611.
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese und Extraktion von Regenwürmern (ISO 23611-1:2018)
Dieser Teil von ISO 23611 legt ein Verfahren zur Probenahme und Behandlung von Regenwürmern aus Freilandböden fest, mit dessen Hilfe die Voraussetzungen für die Verwendung dieser Tiere als Bioindikatoren (z. B. zur Beurteilung der Beschaffenheit eines Bodens als Lebensraum für Organismen) geschaffen werden.
Grundlegende Informationen zur Ökologie von Regenwürmern und ihrer Verwendung als Bioindikatoren in der terrestrischen Umwelt können den im Literaturverzeichnis aufgeführten Quellen entnommen werden.
Dieser Teil von ISO 23611 gilt für alle terrestrischen Biotope, in denen Regenwürmer leben. Der für Freilanduntersuchungen im Allgemeinen gültige Probenahmeplan ist bereits in ISO 10381-1 festgelegt, und eine Anleitung zur Bestimmung der Wirkungen von Schadstoffen auf Regenwürmer unter Freilandbedingungen wird in ISO 11268-3 gegeben. Diese Einzelheiten können den nationalen Anforderun¬gen entsprechend oder in Abhängigkeit von den klimatischen/regionalen Bedingungen des zu beprobenden Standorts variieren (siehe auch Anhang C).
Dieser Teil von ISO 23611 gilt nicht für semiterrestrische Böden. Die Anwendung unter extremen klimatischen oder geographischen Bedingungen (z. B. im Hochgebirge) kann schwierig sein. Die Verfahren für einige andere Bodenorganismengruppen wie z. B. Collembolen werden in anderen Teilen von ISO 23611 behandelt.
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction des vers de terre (ISO 23611-1:2018)
Le présent document spécifie une méthode pour le prélèvement et la manipulation de vers de terre sur le terrain, comme prérequis à l'utilisation de ces animaux en tant que bio-indicateurs (par exemple, pour évaluer la qualité d'un sol en tant qu'habitat pour des organismes).
Le présent document est utilisé pour tous les biotopes terrestres abritant des vers de terre. La conception de l'échantillonnage pour les études sur le terrain est, de manière générale, décrite dans l'ISO 18400‑101 et les lignes directrices relatives à la détermination des effets sur site des polluants vis-à-vis des vers de terre sont données dans l'ISO 11268‑3. Ces aspects peuvent varier en fonction des exigences nationales ou des conditions climatiques/régionales du site sur lequel l'échantillon doit être prélevé (voir également l'Annexe C).
Le présent document ne s'applique pas aux sols semi-terrestres et il peut s'avérer difficile de l'appliquer dans des conditions climatiques ou géographiques extrêmes (en haute montagne, par exemple). Les méthodes pour quelques autres groupes d'organismes du sol, tels que les Collemboles, sont traitées dans d'autres parties de l'ISO 23611.
Kakovost tal - Vzorčenje nevretenčarjev v tleh - 1. del: Ročno razvrščanje deževnikov in njihova ekstrakcija (ISO 23611-1:2018)
Ta dokument določa metodo za vzorčenje deževnikov iz tal na terenu ter rokovanje z njimi kot predpogoj za uporabo teh živali kot bioloških indikatorjev (npr. za oceno kakovosti tal kot habitata za organizme).
Ta dokument se uporablja za vse kopenske biotope, v katerih se pojavljajo deževniki. Načrtovanje vzorčenja pri terenskih raziskavah je na splošno določeno v standardu ISO 18400-101, smernice za določevanje učinkov onesnaževal na deževnike v okoliščinah na terenu pa so določene v standardu ISO 11268-3. Ti vidiki se lahko razlikujejo glede na nacionalne zahteve ali podnebne razmere/regionalne pogoje lokacije, kjer bo potekalo vzorčenje (glej tudi dodatek C).
Ta dokument se ne uporablja za polkopenska tla, uporaba za ekstremne podnebne ali geografske razmere (npr. v visokogorju) pa je lahko otežena. Metode za nekatere druge skupine organizmov v tleh, kot so skakači, so obravnavane v drugih delih standarda ISO 23611.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 23611-1:2018
01-december-2018
1DGRPHãþD
SIST EN ISO 23611-1:2011
.DNRYRVWWDO9]RUþHQMHQHYUHWHQþDUMHYYWOHKGHO 5RþQRUD]YUãþDQMH
GHåHYQLNRYLQQMLKRYDHNVWUDNFLMD ,62
Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of
earthworms (ISO 23611-1:2018)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese
und Extraktion von Regenwürmern (ISO 23611-1:2018)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction
des vers de terre (ISO 23611-1:2018)
Ta slovenski standard je istoveten z: EN ISO 23611-1:2018
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 23611-1:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
EN ISO 23611-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2018
EUROPÄISCHE NORM
ICS 13.080.30; 13.080.05 Supersedes EN ISO 23611-1:2011
English Version
Soil quality - Sampling of soil invertebrates - Part 1: Hand-
sorting and extraction of earthworms (ISO 23611-1:2018)
Qualité du sol - Prélèvement des invertébrés du sol - Bodenbeschaffenheit - Probenahme von Wirbellosen
Partie 1: Tri manuel et extraction des vers de terre (ISO im Boden - Teil 1: Handauslese und Extraktion von
23611-1:2018) Regenwürmern (ISO 23611-1:2018)
This European Standard was approved by CEN on 3 May 2018.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23611-1:2018 E
worldwide for CEN national Members.
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SIST EN ISO 23611-1:2018
EN ISO 23611-1:2018 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 23611-1:2018
EN ISO 23611-1:2018 (E)
European foreword
This document (EN ISO 23611-1:2018) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “Test methods for 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 January 2019, and conflicting national standards shall
be withdrawn at the latest by January 2019.
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 23611-1:2011.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 23611-1:2018 has been approved by CEN as EN ISO 23611-1:2018 without any
modification.
3
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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
INTERNATIONAL ISO
STANDARD 23611-1
Second edition
2018-05
Soil quality — Sampling of soil
invertebrates —
Part 1:
Hand-sorting and extraction of
earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
Reference number
ISO 23611-1:2018(E)
©
ISO 2018
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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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Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 3
7 Procedure. 3
7.1 Sampling of the earthworms . 3
7.1.1 General. 3
7.1.2 Hand-sorting . 4
7.1.3 AITC extraction . 5
7.2 Preservation . 6
7.3 Determination of biomass . 6
8 Data assessment. 6
9 Test report . 7
Annex A (informative) Other methods for sampling . 8
Annex B (informative) Species determination in earthworms . 9
Annex C (informative) The modified TSBF method .10
Annex D (normative) Determination of maximum water-holding capacity .11
Annex E (informative) Examples of earthworm monitoring programmes (including
presentation of their results) .12
Bibliography .16
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(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 on 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 the following URL: 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 23611-1:2006), which has been technically
revised. The main changes are:
— the use of a new chemical extraction compound, AITC (allyl-isothiocyanate), instead of formalin;
— the addition of examples of earthworm monitoring programmes (including presentation of their
results) as an informative Annex E.
A list of all parts in the ISO 23611 series can be found on the ISO website.
iv © ISO 2018 – All rights reserved
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(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:
[25][31][39]
— biological classification of soils including soil quality assessment ;
[11][14][33]
— 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
standardised methods is one of the most important reasons why bio-classification and bio-assessment
in terrestrial (i.e. soil) habitats has so far relatively rarely been used in comparison to aquatic sites.
Since it is neither possible nor useful to standardize methods for all soil organisms, the most important
ones have been selected. In this document sampling of earthworms is described.
Originally, the methods described in this document were developed for taxonomical and ecological
studies, investigating the role of earthworms in various soil ecosystems. These animals are without
doubt the most important soil invertebrates in temperate regions and, to a lesser extent, in boreal and
[30][16][18]
tropical soils . Since Darwin (1881) (see Reference [8]), their influence on soil structure (e.g.
aeration, water holding capacity) and soil functions like litter decomposition and nutrient cycling is well-
[10]
known . Due to their often very high biomass they are also important in many terrestrial food-webs.
In the previous version of this document the chemical formalin was recommended as extraction fluid.
However, within the last years evidence increased that formalin does have critical properties, mainly
in terms of human toxicity. In December 2012, the Risk Assessment Committee (RAC) of the European
Chemicals Agency (ECHA) stated that there is sufficient scientific evidence to classify this chemical as
“probably carcinogenic for humans (Category Ib). In addition, negative effects on non-target organisms
(including soil microorganisms, mesofauna and plants) have been reported (e.g. see Reference [7]).
Therefore, this substance has been replaced.
Due to the growing reservations against the use of formalin, several alternatives have been studied. In
Reference [40] allyl isothiocyanate (AITC) was tested for its effectiveness as a chemical expellant for
sampling earthworms. AITC is a natural breakdown product of glucosinolates in many Cruciferae, i.e.
it is the component imparting the sharp taste of mustard. According to the European Chemical Agency
(ECHA), there is no concern regarding its use under outdoor conditions.
Over the last years, some studies have been performed in which the extraction efficiency of formalin
and AITC were compared at the same sites and dates. According to Reference [22] no differences
were found in numbers or biomass of earthworms extracted at crop sites when using either formalin
or AITC as extractant. In a recent unpublished review (see Reference [28]) no significant differences
were reported in earthworm numbers/biomass when comparing the efficiency of the two extraction
chemicals. Also, no interaction was found on the sampling sites between the extractant and the site,
indicating that no site-specific differences were observed in extraction efficiency of the extractants.
When plotting the correlation between worm numbers extracted with AITC versus formalin in a
Bland-Altman graph (a common way to compare a gold-standard method to an alternative method in
the medical sciences), no significant bias of the AITC method as compared to the formalin method was
found, indicating the similarity / exchangeability of the two methods.
Basic information on the ecology of earthworms and their use as bioindicators in the terrestrial
environment can be found in the references listed in the Bibliography.
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SIST EN ISO 23611-1:2018
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SIST EN ISO 23611-1:2018
INTERNATIONAL STANDARD ISO 23611-1:2018(E)
Soil quality — Sampling of soil invertebrates —
Part 1:
Hand-sorting and extraction of earthworms
1 Scope
This document specifies a method for sampling and handling earthworms from field soils as a
prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for
organisms).
This document applies to all terrestrial biotopes in which earthworms occur. The sampling design
of field studies in general is given in ISO 18400-101 and guidance on the determination of effects of
pollutants on earthworms in field situations is given in ISO 11268-3. These aspects can vary according
to the national requirements or the climatic/regional conditions of the site to be sampled (see also
Annex C).
This document is not applicable for semi-terrestrial soils and it can be difficult to use under extreme
climatic or geographical conditions (e.g. in high mountains). Methods for some other soil organism
groups, such as collembolans, are covered in other parts of ISO 23611.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level
using barium chloride solution
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —
Gravimetric method
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 http: //www .electropedia .org/
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
3.1
earthworms
megadrile soil-inhabiting earthworms belonging to the order Oligochaeta (class Clitellata, phylum
Annelida)
Note 1 to entry: The length of adult individuals can vary from a few centimetres to more than 1 m.
EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae (Latin America), Eudrilidae
(Africa) or Megascolecidae [Asia, North America (Pacific Coast)].
3.2
peregrine species
earthworms occurring in many regions world-wide today, usually introduced by man
Note 1 to entry: Well-known examples of peregrine species are several lumbricid species like Aporrectodea
caliginosa (originally coming from Eurasia, but now living also in the Americas and Australia) or the pan-tropical
species Pontoscolex corethrurus (probably coming from Northern Brazil and/or the Guyanas).
Note 2 to entry: See Reference [18].
3.3
clitellum
ring or saddle shaped epidermal thickening only in mature worms which is near the anterior and
eventually forms the cocoon
4 Principle
Earthworms at a certain site are sampled from the soil by using a combination of two different methods:
2
— hand-sorting animals from a certain area (e.g. 0,25 m ) of varying depth, depending on land use (e.g.
at crop sites: 20 cm), soil properties and the scope of the sampling;
— extraction of worms from the soil by applying AITC.
The first method is known for about 100 years while the second method using the new extraction fluid
[7][22][40]
was proposed about 15 years ago . After extraction, the earthworms are fixed and transported
to the laboratory. There they are preserved in a way that they can be stored in a collection indefinitely
(e.g. for taxonomical purposes). In addition, the determination of the biomass of earthworms is
2
described. Finally, abundance and biomass values can be recalculated to area (usually 1 m ) or, more
rarely, volume parameters.
NOTE 1 Alternative methods can be useful under special circumstances (e.g. electrical extraction), but cannot
be recommended as a general procedure (see Annex A).
NOTE 2 The sampling of earthworms is often included in much broader monitoring programs, trying to cover
the whole soil fauna or parts of it (e.g. the macrofauna). The design of such programmes is not included in this
document.
NOTE 3 Some hints for the taxonomy of peregrine (occurring in many regions world-wide) earthworms,
mainly belonging to the family Lumbricidae, are given in Annex B.
5 Reagents
5.1 Allyl-isothiocyanate (AITC), synthetic grade (about 94 % to 97 % (volume fraction).
5.2 Isopropanol, 100 % (volume fraction).
5.3 Ethanol, 70 % (volume fraction).
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
5.4 Formalin, formaldehyde solution 4 % (volume fraction), for storage purposes only.
5.5 Ethanol, 95 % (volume fraction), for storage purposes when using genetic methods such as
barcoding.
6 Apparatus
Use standard laboratory equipment and the following.
6.1 Plastic vessels, capacities 250 ml and 500 ml, for storing the worms.
6.2 Rubber gloves.
6.3 Forceps.
2 2
6.4 Piece of thick plastic sheeting, 1 m to 2 m .
6.5 Spade or shovel.
6.6 Dissecting microscope, with low magnification (×10 to ×40).
6.7 Balance, weigh range from 0,01 g to 200 g.
6.8 Water-can, preferably 20 l, with water (20 l per sampling plot).
6.9 Watering can.
6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 Thermometer, e.g. for measuring air temperature.
6.12 Drying cabinet, for soil moisture determination.
7 Procedure
7.1 Sampling of the earthworms
7.1.1 General
Sampling of earthworms is done by a combination of two different methods: hand-sorting and AITC
extraction. Based on several comparative studies, the combination of a physical and a chemical method
is clearly recommended in the various reviews on earthworm ecology, independent from the type of
chemical expellant (e.g. References [9], [10] and [18]).
Sampling should be done at times of the year when the animals are not forced by the environmental
conditions (i.e. low soil moisture and/or high temperatures) into diapause (i.e. are not reacting to AITC).
In temperate regions, such unfavourable sampling times are winter and, in particular, midsummer
[18]
periods . Earthworms sampled from the same plot, but sampled under the two different methods,
should be stored in individual plastic vessels. After the end of the sampling process, the excavated and
examined soil is returned to the original sampling plot. In some cases, it is appropriate to use only one
of the two methods; e.g. when no deep-burrowing animals are occurring at a given site, AITC extraction
is not necessary. On the other hand, at sites where giant earthworms are living (parts of South America,
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
[26]
South East Asia and Australia), hand-sorting is not useful . A very similar method, known as modified
TSBF method, is particularly suitable for tropical regions (see Annex C).
NOTE Usually the earthworms are determined after preservation, but if the species spectrum of a sampling
[35]
site is well known, worms can also be determined alive (see also Annex B).
In case the collected earthworms are to be used for further analysis or testing, e.g. for biomarker
measurements or for use in bioassays, storage or incubation of the worms in a small portion of soil from
the sampling site is recommended. In the case of AITC extraction, rinsing the worms in tap water is
needed before incubation in soil.
For the interpretation of test results, the following characteristics shall be determined for the field site
to be studied:
a) pH in accordance with ISO 10390;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465;
d) water holding capacity as specified in Annex D;
e) cationic exchange capacity in accordance with ISO 11260;
f) organic carbon in accordance with ISO 10694.
7.1.2 Hand-sorting
The size of the sample plot should be chosen according to the expected mean size and density of the
worms. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms have
approximately a length between 1 cm and 20 cm. However, at places with a low density of earthworms
2
[e.g. soils with low pH (< 4,5) or which are anthropogenically used like crop sites], larger plots (i.e. 1 m )
are recommended (see ISO 11268-3). On the other hand, at sites with a high earthworm density (e.g.
2 [29]
many meadows in temperate regions), a smaller plot of 1/8 m is sufficient . Even smaller sample
2[41]
sizes (e.g. 1/16 m ) can lead to very low, and thus variable, individual worm numbers per sample,
which in turn leads to an increase in sample numbers (e.g. 16 replicates).
In any case, the soil is removed by means of a spade or shovel (6.5) up to a depth of 20 cm from this
plot (20 cm are suitable for many temperate sites, but the depth also depends on the site properties).
The excavated soil is spread out on a piece of plastic (6.4). This can be done in the field but, especially
in periods of bad weather, the whole procedure can also be performed in the laboratory or greenhouse.
Afterwards, the soil is searched cautiously for earthworms. Big earthworms are collected by hand
using rubber gloves (6.2) and small ones by using forceps (6.3). To avoid autotomy and further damage
of the worms, the animals should only be touched at the anterior part of the body. If worms are cut by
the spade used to dig out the soil, both parts are collected in order to measure the correct biomass,
whereas only front parts are counted when determining the number of individuals.
NOTE 1 With a naked eye, the front end of adult worms can be identified by the position of the clitellum: it is
always located closer to the head than to the tail.
The collected earthworms should immediately be fixed in 70 % ethanol (5.3) using the 250 ml or 500 ml
plastic vessels (6.1) for at least 0,5 h, but not longer than 24 h. In case the ethanol solution is diluted by
body fluids and/or contains soil particles, the ethanol solution shall be exchanged after 24 h to 48 h.
The vessels shall be labelled and observations (e.g. whether worms have been in a quiescence stage)
should be recorded in the notebook (6.10).
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SIST EN ISO 23611-1:2018
ISO 23611-1:2018(E)
An immediate fixation in 4 % formalin (volume fraction) (5.4) is possible, but not recommended due
to the fact that the handling of this compound should be minimised as much as possible (in particular
under field conditions).
NOTE 2 In order to avoid morphological changes (e.g. an inversion of the prostomium) due to immediate
fixation in ethanol, the individual worms can be put briefly (about one minute) into warm (e.g. 30 °C to 40 °C) tap
water. The earthworms relax in the water, and after that they can be transferred to ethanol.
7.1.3 AITC extraction
The same plot, from which the top soil has been removed for hand-sorting, is used for AITC extraction.
A sufficient amount of water shall be transported (5 l to 10 l per sampling plot) beforehand to the plots
using large water-cans (6.8). To p
...
SLOVENSKI STANDARD
oSIST prEN ISO 23611-1:2017
01-januar-2017
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Soil quality - Sampling of soil invertebrates - Part 1: Hand-sorting and extraction of
earthworms (ISO/DIS 23611-1:2016)
Bodenbeschaffenheit - Probenahme von Wirbellosen im Boden - Teil 1: Handauslese
und Extraktion von Regenwürmern (ISO/DIS 23611-1:2016)
Qualité du sol - Prélèvement des invertébrés du sol - Partie 1: Tri manuel et extraction
des vers de terre (ISO/DIS 23611-1:2016)
Ta slovenski standard je istoveten z: prEN ISO 23611-1
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 23611-1:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 23611-1:2017
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oSIST prEN ISO 23611-1:2017
DRAFT INTERNATIONAL STANDARD
ISO/DIS 23611-1
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2016-11-16 2017-02-07
Soil quality — Sampling of soil invertebrates —
Part 1:
Hand-sorting and extraction of earthworms
Qualité du sol — Prélèvement des invertébrés du sol —
Partie 1: Tri manuel et extraction des vers de terre
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,
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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 23611-1:2016(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 2016
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oSIST prEN ISO 23611-1:2017
ISO/DIS 23611-1:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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ii © ISO 2016 – All rights reserved
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oSIST prEN ISO 23611-1:2017
ISO/DIS 23611-1:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 2
7 Procedure. 3
7.1 Sampling of the earthworms . 3
7.1.1 General. 3
7.1.2 Hand-sorting . 4
7.1.3 AITC extraction . 4
7.2 Preservation . 5
7.3 Determination of biomass . 5
8 Data assessment. 6
9 Test report . 6
Annex A (informative) Other methods for sampling . 8
Annex B (informative) Species determination in earthworms . 9
Annex C (informative) The modified TSBF Method .10
Annex D (informative) Determination of maximum water-holding capacity .11
Bibliography .12
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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.
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 23611-1 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
This second edition cancels and replaces the first edition (ISO 23611-1:2006), which has been technically
revised.
ISO 23611 consists of the following parts, under the general title Soil quality — Sampling of soil
invertebrates:
— Part 1: Hand-sorting and extraction of earthworms
— Part 2: Sampling and extraction of micro-arthropods (Collembola and Acarina)
— Part 3: Sampling and soil extraction of enchytraeids
— Part 4: Sampling, extraction and identification of free-living stages of terrestrial nematodes
— Part 5: Sampling and extraction of soil macro-invertebrates
— Part 6: Guidance for the design of sampling programmes with soil invertebrates
iv © ISO 2016 – All rights reserved
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Introduction
This part of ISO 23611 has been drawn up since there is a growing need for the standardisation of
terrestrial zoological field methods. Such methods, mainly covering the sampling, extraction and
handling of soil invertebrates, are necessary for the following purposes:
[25] [31] [39]
— biological classification of soils including soil quality assessment , , ;
[11] [14] [33]
— 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
standardised methods is one of the most important reasons why bio-classification and bio-assessment
in terrestrial (i.e. soil) habitats has so far relatively rarely been used in comparison to aquatic sites.
Since it is neither possible nor useful to standardize methods for all soil organisms, the most important
ones have been selected. In this part of ISO 23611 sampling of earthworms is described.
Originally, the methods described in this International Standard were developed for taxonomical and
ecological studies, investigating the role of earthworms in various soil ecosystems. These animals are
without doubt the most important soil invertebrates in temperate regions and, to a lesser extent, in
[30] [16] [18]
tropical soils , , . Since Darwin (1881) [see 8], their influence on soil structure (e.g. aeration,
water holding capacity) and soil functions like litter decomposition and nutrient cycling is well-
[10]
known . Due to their often very high biomass they are also important in many terrestrial food-webs.
In the previous version of this International Standard the chemical formalin was recommended as
extraction fluid. However, within the last years evidence increased that formalin does have critical
properties, mainly in terms of human toxicity. In December 2012, the Risk Assessment Committee
(RAC) of the European Chemicals Agency (ECHA) stated that there is sufficient scientific evidence to
classify this chemical as “probably carcinogenic for humans (Category Ib). In addition, negative effects
on non-target organisms (including soil microorganisms, mesofauna and plants) have been reported
(e.g. [7]). Therefore, this substance had been replaced.
Due to the growing reservations against the use of formalin several alternatives have been studied.
In [40] there was tested allyl isothiocyanate (AITC) for its effectiveness as a chemical expellant for
sampling earthworms. AITC is a natural breakdown product of glucosinolates in many Cruciferae, i.e.
it is the component imparting the sharp taste of mustard. According to the European Chemical Agency
(ECHA), there is no regulatory action in the context of human or environmental risk assessment
considered, i.e. there is no concern regarding its use.
Within the last years, some studies have been performed in which the extraction efficiency of formalin
and AITC were compared at the same sites and dates. According to [22] there were not found differences
in numbers or biomass of earthworms extracted at crop sites when using either formalin or AITC as
extractant. In a recent unpublished review [see 28] there were not found significant differences in
earthworm numbers/biomass when comparing the efficiency of the two extraction chemicals. Also no
effect was found for the interactions extractant site, indicating that no site-specific differences were
observed in extraction efficiency of the extractants. When plotting the correlation between worm
numbers extracted with AITC vs. formol in a Bland-Altman graph (a common way to compare a gold-
standard method to an alternative method in the medical sciences), no significant bias of the AITC
method as compared to the formalin method was found, indicating the similarity / exchangeability of
the two methods.
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oSIST prEN ISO 23611-1:2017
DRAFT INTERNATIONAL STANDARD ISO/DIS 23611-1:2016(E)
Soil quality — Sampling of soil invertebrates —
Part 1:
Hand-sorting and extraction of earthworms
1 Scope
This part of ISO 23611 specifies a method for sampling and handling earthworms from field soils as a
prerequisite for using these animals as bioindicators (e.g. to assess the quality of a soil as a habitat for
organisms).
Basic information on the ecology of earthworms and their use as bioindicators in the terrestrial
environment can be found in the references listed in the bibliography.
This part of ISO 23611 applies to all terrestrial biotopes in which earthworms occur. The sampling
design of field studies in general is specified in ISO 10381-1 and guidance on the determination of
effects of pollutants on earthworms in field situations is given in ISO 11268-3. These details can vary
according to the national requirements or the climatic/regional conditions of the site to be sampled
(see also Annex C).
This part of ISO 23611 is not applicable for semi-terrestrial soils and it can be difficult to use under
extreme climatic or geographical conditions (e.g. in high mountains). Methods for some other soil
organism groups, such as collembolans, are covered in other parts of ISO 23611.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil
under aerobic conditions for the assessment of microbiological processes, biomass and diversity in the
laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level
using barium chloride solution
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —
Gravimetric method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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3.1
earthworm
megadrile soil-inhabiting earthworms (length of adult individuals: few centimetres to more than 1 m)
belonging to the order Oligochaeta (class Clitellata, phylum Annelida)
EXAMPLE Species of the families Lumbricidae (Holarctic), Glossoscolecidae (Latin America), Eudrilidae
(Africa) or Megascolecidae [Asia, North America (Pacific Coast)].
3.2
peregrine species
earthworms occurring in many regions world-wide today, usually introduced by man
Note 1 to entry: Well-known examples of peregrine species are several lumbricid species like Aporrectodea
caliginosa (originally coming from Eurasia, but now living also in the Americas and Australia) or the pan-tropical
species Pontoscolex corethrurus (probably coming from Northern Brazil and/or the Guyanas).
Note 2 to entry: See Reference [18 ].
3.3
clitellum
ring or saddle of epidermal thickening only in mature worms which is near the anterior and eventually
forms the cocoon
4 Principle
Earthworms at a certain site are sampled from the soil by using a combination of two different methods:
2
— hand-sorting animals from a certain area (e.g. 0,25 m ) or volume (e.g. 50 cm × 50 cm × 20 cm);
— extraction of worms from the soil by applying AITC.
The former method is known for about 100 years while the new extraction fluid was proposed about
[6] [40]
15 years ago , . After extraction, the earthworms are fixed and transported to the laboratory. There
they are preserved in a way that they can be stored in a collection indefinitely (e.g. for taxonomical
purposes). In addition, the determination of the biomass of earthworms is described. Finally, abundance
2
and biomass values can be recalculated to area (usually 1 m ) or, more rarely, volume parameters.
NOTE 1 Alternative methods can be useful under special circumstances (e.g. electrical extraction), but cannot
be recommended as a general procedure (see Annex A)
NOTE 2 The sampling of earthworms is often included in much broader monitoring programs, trying to cover
the whole soil fauna or parts of it (e.g. the macrofauna). The design of such programs is not included in this part of
ISO 23611.
NOTE 3 Some hints for the taxonomy of peregrine (occurring in many regions world-wide) earthworms,
mainly belonging to the family Lumbricidae, are given in Annex B.
5 Reagents
5.1 Formalin [formaldehyde solution 4 % (volume fraction)].
5.2 Allyl-isothiocyanate (AITC) [synthetic grade (about 94 % to 97 % (volume fraction))].
5.3 Ethanol, [70 % (volume fraction)].
6 Apparatus
Use standard laboratory equipment and the following.
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6.1 Plastic vessels, capacities 250 ml and 500 ml, for storing the worms.
6.2 Rubber gloves.
6.3 Forceps.
2 2
6.4 Piece of thick plastic sheeting, 1 m to 2 m .
6.5 Spade or shovel.
6.6 Dissecting microscope, with low magnification (10 to 40 times).
6.7 Balance, weigh range from 0,01 g to 200 g.
6.8 Water-can, preferably 20 l, with water (20 l per sampling plot).
6.9 Watering can.
6.10 Pencil, notebook, water resistant marker, labels that go in the vessel.
6.11 Thermometer, e.g. for measuring air temperature.
6.12 Drying cabinet, for soil moisture determination.
7 Procedure
7.1 Sampling of the earthworms
7.1.1 General
Sampling of earthworms is done by a combination of two different methods: hand-sorting and AITC
extraction. Based on several comparative studies, the combination of a physical and a chemical method
is clearly recommended in the various reviews on earthworm ecology, independent from the type of
chemical expellent (e.g. references [9], [10], [18]).
Sampling should be done at times of the year when the animals are not forced by the environmental
conditions (i.e. low soil moisture and/or high temperatures) into diapause (i.e. are not reacting to AITC).
In temperate regions, such unfavourable sampling times are winter and, in particular, midsummer
[18]
periods . Earthworms sampled from the same plot, but sampled under the two different methods,
should be stored in individual plastic vessels. After the end of the sampling process, the excavated and
examined soil is returned to the original sampling plot. In some cases, it is appropriate to use only
one of the two methods; e. g. when no deep-burrowing animals are occurring at a given site, AITC
extraction is not necessary. On the other hand, at sites where giant earthworms are living (parts of
[26]
South America, South East Asia and Australia), hand-sorting is not useful . A very similar method,
known as modified TSBF method, is particularly suitable for tropical regions (see Annex C).
NOTE Usually the earthworms are determined after preservation, but if the species spectrum of a sampling
[35]
site is well known, worms can also be determined alive (see also Annex B).
In case the collected earthworms are to be used for further analysis or testing, e. g. for biomarker
measurements or for use in bioassays, storage or incubation of the worms in a small portion of soil from
the sampling site is recommended. In the case of AITC extraction, rinsing the worms in tap water is
needed before incubation in soil.
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For the interpretation of test results, the following characteristics shall be determined for the soil
sampled from the field site to be studied:
a) pH in accordance with ISO 10390,
b) texture (sand, loam, silt) in accordance with ISO 11277,
c) water content in accordance with ISO 11465,
d) water holding capacity according to Annex D,
e) cationic exchange capacity in accordance with ISO 11260,
f) organic carbon in accordance with ISO 10694.
7.1.2 Hand-sorting
The size of the sample plot should be chosen according to the expected mean size and density of the
worms. A square of 50 cm × 50 cm is often sufficient in the Holarctic where most adult earthworms have
approximately a length between 1 cm and 20 cm. However, at places with a low density of earthworms
2
[e.g. soils with low pH (< 4,5) or which are anthropogenically used like crop sites], larger plots (i.e. 1 m )
are recommended (see ISO 11268-3). On the other hand, at sites with a high earthworm density (e.g.
2 [29]
many meadows in temperate regions), a smaller plot of 1/8 m is sufficient . Even smaller sample
2 [41]
sizes (e.g. 1/16 m ; ) can lead to very low, and thus variable, individual worm numbers per sample,
which in turn leads to an increase in sample numbers (e.g. 16 replicates).
In any case, the soil is removed by means of a spade or shovel (6.5) up to a depth of 20 cm from this
plot (20 cm are suitable for many temperate sites, but the depth also depends on the site properties).
The excavated soil is spread out on a piece of plastic (6.4). This can be done in the field but, especially
in periods of bad weather, the whole procedure can also be performed in the laboratory or greenhouse.
Afterwards, the soil is searched cautiously for earthworms. Big earthworms are collected by hand
using rubber gloves (6.2) and small ones by using forceps (6.3). To avoid autotomy and further damage
of the worms, the animals should only be touched at the anterior part of the body. If worms are cut by
the spade used to dig out the soil, both parts are collected in order to measure the correct biomass,
whereas only front parts are counted when determining the number of individuals.
NOTE 1 With a naked eye, the front end of adult worms can be identified by the position of the clitellum: it is
always located closer to the head than to the tail.
The collected earthworms should immediately be fixed in 70 % ethanol (5.3) using the 250 ml or
500 ml plastic vessels (6.1) for at least half an hour, but not longer than 24 h. The vessels shall be
labelled and observations (e.g. whether worms have been in a quiescence stage) should be recorded in
the notebook (6.10).
An immediate fixation in 4 % formalin (volume fraction) (5.1) is possible, but not recommended due
to the fact that the handling of this compound should be minimised as much as possible (in particular
under field conditions).
NOTE 2 In order to avoid morphological changes (e.g. an inversion of the prostomium) due to immediate
fixation in ethanol, the individual worms can be put briefly (about one minute) into warm tap water. The
earthworms relax in the water, and after that they can be transferred to ethanol.
7.1.3 AITC extraction
The same plot, from which the top soil has been removed for hand-sorting, is used for AITC extraction.
A sufficient amount of water shall be transported (10 l per sampling plot) beforehand to the plots using
large water-cans (6.8). To prepare the AITC solution, an amount of 1 g of AITC is dissolved in 50 ml of
isopropanol, and the resulting solution is made up with water to a total volume of 10 l. The diluted AITC
solution is carefully and evenly applied into the plot from which the top soil has been removed for hand-
sorting. The solution should be applied in several portions (usually 2 to 3) according to the seepage
capacity of the soil until 5 l to10 l of AITC-solution, depending on the soil properties, are added. During
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the application, the plot shall be observed in order to collect all earthworms appearing on the soil surface
of the sampling plot. The sampling is finished 30 min after the application of the last watering can.
AITC readily decays in water, and also in the stock solution in isopropanol. Decay is enhanced by
sunlight and warmth. Thus the final solution shall be prepared immediately before use.
Large earthworms should be collected by hand using rubber gloves (6.2) and small earthworms by
forceps (6.3). The repellent AITC produces rapid earthworm emergence from the soil. Worms should
only be collected when the largest (preferably whole) portion of the body becomes visible, otherwise
damage or retraction back into the soil occurs. To avoid autotomy and damage of the worms, the animals
should only be touched at the anterior part of the body, usually in front of the clitellum. The collected
earthworms should immediately be fixed in 70 % ethanol (volume fraction) (5.3) using the 250 ml or
500 ml plastic vessels (6.1) for at least 30 min but not longer than 24 h. The vessels shall be labelled and
any observations should be recorded in the notebook (6.10).
At sites where giant earthworms are occurring (South America, South East Asia, and Australia), and
2
where hand-sorting is not appropriate to get them, AITC should be applied on an area of 4 m . Before
doing so, herbs and litter shall be removed from the soil surface. In all other respects, the sampling
process is the same.
It is not necessary to perform the AITC extraction if no vertical burrowers (especially Lumbricus
terrestris or Aporrectodea longa) are living at a given site (e.g. in very acid soils). The occurrence of
these large worms is indicated by surface casts and collected litter at their burrow openings (diameter
approximately 0,5 cm) which are easily detectable before excavation of the soil used for hand-sorting.
Therefore, any decision about the use of AITC shall be taken on a case-by-case basis.
NOTE In soils with high clay content, the digging of the sampling pit can close tightly many earthworm burrows
at the bottom of the pit, thus preventing the infiltration of AITC and/or the emergence of deep burrowing worms. In
such conditions, the smeared burrow openings can be opened before the AITC application (e.g. using a knife).
WARNING — Appropriate precautions (i.e. gloves and protective clothing) shall be taken when
dealing with AITC to avoid danger from inhalation or skin exposure. According to the “Material
Safety Data Sheet” for AITC as published by producing companies, the compound is irritating
to the skin and to the eyes and toxic to aquatic organisms. It is legally notified in industrialised
countries for commercial (e.g. as a biofumigant) and scientific use.
7.2 Preservation
Two methods are possible:
a) after fixation, the animals can be kept in 4 % formalin (volume fraction) (5.1) for four days at
minimum, but preferably one or two weeks. Afterwards, the worms can be stored for an unlimited
period in 70 % ethanol (volume fraction) (5.3);
b) an alternative method is to fix the worms immediately after collection in a mixture of 70 % ethanol
(volume fraction) (5.3) and 4 % formalin (volume fraction) (5.1) in a ratio of 98 % to 2 %. This
preservation liquid is replaced by fresh preservation liquid on the day after sampling at the latest.
Preservation in pure ethanol should be avoided since the outer surface of the worms sometimes
becomes very soft, so that important features are not visible any more.
In the case that earthworm tissue shall be preserved for biochemical or genetical studies, preservation
[15]
in formalin is not recommended .
7.3 Determination of biomass
Determination of biomass is performe
...
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