SIST EN ISO 11268-2:2023
(Main)Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei and other earthworm species (ISO 11268-2:2023)
Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei and other earthworm species (ISO 11268-2:2023)
This document specifies one of the methods for evaluating the habitat function of soils and determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites concerned, and waste materials.
This method is designed mainly for determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method also includes technical information on how to use it with other environmentally relevant earthworm species: e.g., Aporrectodea caliginosa and Dendrodrilus rubidus (see Annexes B and C).
This method does not apply to substances for which the air/soil partition coefficient is greater than one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C. This method does not take into account the persistence of the substance during the test.
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2: Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia andrei und andere Regenwurmarten (ISO 11268-2:2023)
Dieses Dokument legt ein Verfahren zur Bewertung der Lebensraumfunktion von Böden und zur Bestimmung der akuten Toxizität von Bodenverunreinigungen und Chemikalien gegenüber Eisenia fetida/Eisenia andrei bei Aufnahme über die Haut und durch die Nahrung fest. Das Verfahren ist anwendbar auf Böden und Bodenmaterialien von unbekannter Qualität, die z. B. von verunreinigten, landwirtschaftlich beeinflussten oder anderen betroffenen Standorten stammen, oder bei denen es sich um veränderte Böden, Böden nach der Sanierung, oder um Abfallmaterialien handelt.
Die Wirkungen von Substanzen werden in einem Standardboden, vorzugsweise in einer definierten Kunsterde bewertet. Bei verunreinigten Böden werden die Wirkungen auf das Überleben im zu prüfenden Boden und in einem Kontrollboden bestimmt. Entsprechend der Zielsetzung der Studie sollten die Kontrolle und das für die Herstellung der Verdünnungsreihen des verunreinigten Bodens verwendete Substrat entweder ein mit der zu prüfenden Bodenprobe vergleichbarer, nicht verunreinigter Boden (Referenzboden) oder ein Standardboden (z. B. Kunsterde) sein.
Diese Methode ist hauptsächlich für die Bestimmung der Auswirkungen von Bodenverunreinigungen und Chemikalien auf die Vermehrung von Eisenia fetida/Eisenia andrei bestimmt. Außerdem werden technische Informationen darüber gegeben, wie Eisenia fetida/andrei für die Prüfung von Chemikalien unter tropischen Bedingungen eingesetzt werden können (siehe Anhang A). Schließlich enthält diese Methode auch technische Informationen darüber, wie sie mit anderen umweltrelevanten Regenwurmarten verwendet werden kann: z. B. Aporrectodea caliginosa und Dendrodrilus rubidus (siehe Anhang B und Anhang C).
Diese Methode ist nicht anzuwenden für Stoffe, deren Verteilungskoeffizient Luft/Boden größer als 1 ist, oder für Stoffe mit einem Dampfdruck von mehr als 300 Pa bei 25 °C. Bei dieser Methode wird die Persistenz der Substanz während der Prüfung nicht berücksichtigt. Dieses Verfahren berücksichtigt nicht den mö-glichen Abbau der Substanz während der Prüfung.
Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 2: Détermination des effets sur la reproduction de Eisenia fetida/Eisenia andrei et d’autres espèces de vers de terre (ISO 11268-2:2023)
Le présent document spécifie l’une des méthodes permettant d’évaluer la fonction d’habitat des sols et de déterminer les effets de polluants et de produits chimiques présents dans le sol sur la reproduction d’Eisenia fetida/Eisenia andrei par absorption cutanée et ingestion. Cet essai chronique s’applique aux sols et aux matériaux de type sol de qualité inconnue, provenant par exemple de sites pollués, de sols amendés, de sols après dépollution, de sols agricoles ou autres sites d’intérêt et de déchets.
Cette méthode est principalement destinée à déterminer les effets de polluants et de produits chimiques présents dans le sol sur la reproduction d’Eisenia fetida/Eisenia andrei. Des informations techniques sont également fournies sur la manière d’utiliser Eisenia fetida/andrei pour évaluer des produits chimiques dans des conditions tropicales (voir l’Annexe A). Enfin, cette méthode intègre également des informations techniques sur la manière de l’utiliser avec d’autres espèces de vers de terre pertinentes sur le plan environnemental: par exemple, Dendrodrilus rubidus et Aporrectodea caliginosa (voir les Annexes B et C).
Cette méthode ne s’applique ni aux substances pour lesquelles le coefficient de partage air/sol est supérieur à un, ni aux substances pour lesquelles la pression de vapeur à 25 °C excède 300 Pa. Cette méthode ne tient pas compte de la rémanence de la substance pendant l’essai.
Kakovost tal - Učinki onesnaževal na deževnike - 2. del: Določanje učinkov na reprodukcijo Eisenia fetida/Eisenia andrei in drugih vrst deževnikov (ISO 11268-2:2023)
Ta dokument določa eno od metod za ocenjevanje funkcije tal habitata ter določevanje učinkov onesnaževal tal in kemikalij na reprodukcijo Eisenia fetida/Eisenia andrei z dermalnim ali prehranskim vnosom. Ta kronični preskus se uporablja za tla in talne materiale neznane kakovosti, na primer tla iz onesnaženih območij, obogatena tla, tla po sanaciji, kmetijska in druga problematična mesta ter odpadne materiale.
Ta metoda je predvsem zasnovana za določevanje učinkov onesnaževal tal in kemikalij na reprodukcijo Eisenia fetida/Eisenia andrei. Tehnične informacije so zagotovljene tudi za način uporabe Eisenia fetida/andrei za preskušanje kemikalij v tropskih pogojih (glej dodatek A). Ta metoda nenazadnje vključuje tehnične informacije, kako jo uporabljati pri drugih okoljsko pomembnih vrstah deževnikov: npr. Aporrectodea caliginosa in Dendrodrilus rubidus (glej dodatka B in C).
Ta metoda se uporablja za snovi, pri katerih je koeficient deleža zraka/vode večji od ena, ali za snovi, pri katerih parni tlak pri 25 °C presega 300 Pa. Pri tej metodi se ne upošteva obstojnost snovi med preskusom.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11268-2:2023
01-junij-2023
Nadomešča:
SIST EN ISO 11268-2:2015
Kakovost tal - Učinki onesnaževal na deževnike - 2. del: Določanje učinkov na
reprodukcijo Eisenia fetida/Eisenia andrei in drugih vrst deževnikov (ISO 11268-
2:2023)
Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei and other earthworm species (ISO 11268-
2:2023)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2:
Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia
andrei und andere Regenwurmarten (ISO 11268-2:2023)
Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 2: Détermination
des effets sur la reproduction de Eisenia fetida/Eisenia andrei et d’autres espèces de
vers de terre (ISO 11268-2:2023)
Ta slovenski standard je istoveten z: EN ISO 11268-2:2023
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 11268-2:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11268-2:2023
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SIST EN ISO 11268-2:2023
EN ISO 11268-2
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 11268-2:2015
English Version
Soil quality - Effects of pollutants on earthworms - Part 2:
Determination of effects on reproduction of Eisenia
fetida/Eisenia andrei and other earthworm species (ISO
11268-2:2023)
Qualité du sol - Effets des polluants vis-à-vis des vers Bodenbeschaffenheit - Wirkungen von Schadstoffen
de terre - Partie 2: Détermination des effets sur la auf Regenwürmer - Teil 2: Bestimmung der Wirkung
reproduction de Eisenia fetida/Eisenia andrei et auf die Reproduktionsleistung von Eisenia
d'autres espèces de vers de terre (ISO 11268-2:2023) fetida/Eisenia andrei und andere Regenwurmarten
(ISO 11268-2:2023)
This European Standard was approved by CEN on 6 January 2023.
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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye 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
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11268-2:2023 E
worldwide for CEN national Members.
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SIST EN ISO 11268-2:2023
EN ISO 11268-2:2023 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 11268-2:2023
EN ISO 11268-2:2023 (E)
European foreword
This document (EN ISO 11268-2:2023) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “Environmental characterization of
solid matrices” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by August 2023, and conflicting national standards shall
be withdrawn at the latest by August 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 11268-2:2015.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 11268-2:2023 has been approved by CEN as EN ISO 11268-2:2023 without any
modification.
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SIST EN ISO 11268-2:2023
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SIST EN ISO 11268-2:2023
INTERNATIONAL ISO
STANDARD 11268-2
Third edition
2023-01
Soil quality — Effects of pollutants on
earthworms —
Part 2:
Determination of effects on
reproduction of Eisenia fetida/Eisenia
andrei and other earthworm species
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 2: Détermination des effets sur la reproduction de Eisenia
fetida/Eisenia andrei et d’autres espèces de vers de terre
Reference number
ISO 11268-2:2023(E)
© ISO 2023
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents and material . 4
6 Apparatus . 6
7 Procedure .6
7.1 Experimental design . 6
7.1.1 General . 6
7.1.2 Preliminary test . 7
7.1.3 Definitive test . 7
7.2 Preparation of test mixtures . 7
7.2.1 Testing of contaminated soil . 7
7.2.2 Testing substances added to the control soil . 8
7.2.3 Preparation of control container . 8
7.3 Addition of the earthworms . 9
7.4 Test conditions and measurements . 9
7.5 Reference substance . 9
8 Calculation and expression of results .10
8.1 Calculation . 10
8.2 Expression of results . . 10
9 Validity of the test .10
10 Statistical analysis .10
10.1 General . 10
10.2 Single-concentration tests . 10
10.3 Multi-concentration tests . 11
10.3.1 Preliminary test . 11
10.3.2 Definitive test . 11
11 Test report .12
Annex A (informative) Determination of the chronic toxicity of chemicals on Eisenia fetida/
Eisenia andrei under tropical conditions .13
Annex B (informative) Culturing and testing procedures for assessing the effects
of contaminants on juvenile production using the earthworm, Dendrodilus rubidus .15
Annex C (informative) Culturing and testing procedures for assessing the effects
of contaminants on adult survival and cocoon production and hatchability using
the earthworm, Aporrectodea caliginosa s.s. .19
Annex D (informative) Culturing of Eisenia fetida and Eisenia andrei .23
Annex E (normative) Determination of water holding capacity .24
Annex F (informative) Techniques for counting juvenile worms hatched from cocoons.25
Annex G (informative) Experience with performance criteria of the test .26
Bibliography .33
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 444, Environmental characterization, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 11268-2:2012), which has been
technically revised.
The main changes are as follows:
— modification of the concentration for the reference substance (boric acid);
— inclusion of alternative species of earthworms – Dendrodrilus rubidus, Aporrectodea caliginosa – in
informative annexes; information on their taxonomy and ecology as well as their specific testing
requirements have also been added.
A list of all the parts in the ISO 11268 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in
soil and are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616).
ISO 15799 includes a list and short characterization of recommended and standardized test systems
and ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with
soil eluate are applied to obtain information about the fraction of contaminants potentially reaching
the groundwater by the water path (retention function of soils), whereas terrestrial test systems are
used to assess the habitat function of soils.
This document describes a method that is based on the determination of sublethal effects of
contaminated soils on adult earthworms of the species Eisenia fetida (Savigny 1826) and Eisenia andrei
(André 1963). Optionally, the method can be used for testing chemicals added to standard soils (e.g.
artificial soil) for their sublethal hazard potential to earthworms. Finally, information is provided on
how to use this method for testing chemicals or test soil under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular in temperate regions. Background information on their earthworm ecology and their use in
ecotoxicological testing is available. However, these species do not occur regularly in agricultural lands
(crop sites and grasslands) or forests in these regions. In addition, they are not representative of boreal
or tropical regions. Therefore, other species such as Dendrodilus rubidus (an epigeic litter inhabitant in
boreal regions) and Aporrectodea caliginosa (an endogeic mineral dweller in temperate regions) have
been added as potential alternative test species (see Annexes B and C). These alternative earthworm
species have been used as ecotoxicological test species for some time, however, testing experience has
been limited to specific countries.
This document has been drawn up taking into consideration test procedures adopted by the
[45][46] [29]
Organization for Economic Cooperation and Development and by the European Union .
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SIST EN ISO 11268-2:2023
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SIST EN ISO 11268-2:2023
INTERNATIONAL STANDARD ISO 11268-2:2023(E)
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei and other earthworm species
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or
otherwise harmful chemicals or infectious microorganisms. Occupational health risks may
arise from dust or evaporated chemicals during handling and incubation. Precautions should be
taken to avoid skin contact.
1 Scope
This document specifies one of the methods for evaluating the habitat function of soils and determining
the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by
dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown
quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites
concerned, and waste materials.
This method is designed mainly for determining the effects of soil contaminants and chemicals on
the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use
Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method
also includes technical information on how to use it with other environmentally relevant earthworm
species: e.g. Dendrodrilus rubidus and Aporrectodea caliginosa (see Annexes B and C).
This method does not apply to substances for which the air/soil partition coefficient is greater than
one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C. This method does not take into
account the persistence of the substance during the test.
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, treated biowaste and sludge – 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
ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic
conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
contaminant
substance or agent present in the soil as a result of human activity
[SOURCE: ISO 15176:2019, 3.2.6]
3.2
growth
increase in biomass (i.e. the fresh mass of organisms)
Note 1 to entry: It is expressed as a percentage of the fresh mass of organisms at the start of the test.
3.3
reproduction
mean number of offspring per test container after eight weeks’ incubation under the specified test
conditions
3.4
ER
x
EC
x
effective rate
effective concentration
x % effect rate or concentration of the test sample or test substance at which reproduction (3.3) is
reduced by x % compared to the control
3.5
limit test
single concentration test consisting of at least four replicates each, the test sample without any dilution
or the highest concentration of test substance mixed into the control soil (3.10) and the control
3.6
LOER
LOEC
lowest observed effect rate
lowest observed effect concentration
lowest tested percentage of a test sample in a control soil (3.10) or concentration of a substance at which
a statistically significant effect is observed
Note 1 to entry: The LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test
mixtures (3.11) above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If
this condition cannot be satisfied, an explanation should be given for how the LOEC and NOEC (3.7) have been
selected.
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
3.7
NOER
NOEC
no observed effective rate
no observed effect concentration
test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test
substance which, when compared to the control, has no statistically significant lethal or other effect
such as reduced reproduction (3.3) or mass alteration (error probability: p < 0,05)
Note 1 to entry: The NOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass.
3.8
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar
properties (nutrient concentrations, pH, organic carbon content and texture) to the test soil
3.9
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are
within a known range
[29] [46] [40]
EXAMPLE Euro-Soils, artificial soil, LUFA standard soil .
Note 1 to entry: The properties of standard soils can differ from those of the test soil.
3.10
control soil
reference (3.8) or standard soil (3.9) used as a control and as a medium for preparing dilution series
with test samples or a reference substance, which fulfils the validity criteria
Note 1 to entry: In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving
the test validity criteria before using the soil in a definitive test.
3.11
test mixture
mixture of contaminated soil or the test substance with a control soil (3.10)
Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.
4 Principle
The effects on reproduction of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed
to the test soil are compared to those observed for samples exposed to a control soil. If appropriate,
effects based on exposure to a dilution range of contaminated soil or range of concentrations of a test
substance are determined. In addition, observations on growth and survival of adult earthworms are
recorded. Test mixtures are prepared at the start of the test and are not renewed within the test period.
After four weeks, adult worms are removed from the test containers and effects on mortality and
biomass are measured by counting and weighing. The effect on reproduction as the definitive end point
is measured by counting the number of offspring hatched from the cocoons after an additional period
of four weeks. The results obtained from the tests are compared with a control soil or, if appropriate,
are used to determine the dilutions or concentrations which cause no effects on biomass, mortality
and reproduction (NOER/NOEC) and the dilution (concentration) resulting in x % reduction of juveniles
hatched from cocoons compared to the control (ER /EC , 56 d), respectively.
x x
If testing a dilution or concentration series, all test dilutions/concentrations above the LOER/LOEC
shall have a harmful effect equal to, or greater than that observed at the LOER/LOEC. Where there is
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SIST EN ISO 11268-2:2023
ISO 11268-2:2023(E)
no prior knowledge of the dilution/concentration of the test soil/test substance likely to have an effect,
then it is recommended to conduct the test in two steps:
— a preliminary test carried out, in accordance with ISO 11268-1, to give an indication of the effect
dilution/concentration and of the dilution/concentration giving no mortality (NOER/NOEC);
dilutions/concentrations to be used in the definitive test can then be selected;
— a definitive test to determine sublethal effects of (dilutions of) contaminated soil or the concentration
of a chemical which, when evenly mixed into the standard soil, causes no significant effects on
numbers of offspring hatched from cocoons compared with the control (NOER/NOEC), and the
lowest concentration causing effects (LOER/LOEC).
NOTE A reference soil is used to demonstrate the appropriate status of the test population, and to avoid
misinterpretation of results.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate.
For contaminated soils, the effects are determined in the test soil and in a control soil. According to the
objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be
either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard
soil (e.g. artificial soil).
Alternative species of earthworms and their respectiv
...
SLOVENSKI STANDARD
oSIST prEN ISO 11268-2:2022
01-januar-2022
Kakovost tal - Učinki onesnaževal na deževnike - 2. del: Določanje učinkov na
reprodukcijo Eisenia fetida/Eisenia andrei (ISO 11268-2:2021)
Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei (ISO/DIS 11268-2:2021)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2:
Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia
andrei (ISO/DIS 11268-2:2021)
Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 2: Détermination
des effets sur la reproduction de Eisenia fetida/Eisenia andrei (ISO/DIS 11268-2:2021)
Ta slovenski standard je istoveten z: prEN ISO 11268-2
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 11268-2:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 11268-2:2022
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oSIST prEN ISO 11268-2:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11268-2
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-11-16 2022-02-08
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 2: Détermination des effets sur la reproduction de Eisenia fetida/Eisenia andrei
ICS: 13.080.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
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STANDARDS MAY ON OCCASION HAVE TO
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WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 11268-2:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2021
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oSIST prEN ISO 11268-2:2022
ISO/DIS 11268-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
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ISO/DIS 11268-2:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents and material . 4
6 Apparatus . 6
7 Procedure .6
7.1 Experimental design . 6
7.1.1 General . 6
7.1.2 Preliminary test . 7
7.1.3 Definitive test . 7
7.2 Preparation of test mixtures . 7
7.2.1 Testing of contaminated soil . 7
7.2.2 Testing substances added to the control soil . 8
7.2.3 Preparation of control container . 8
7.3 Addition of the earthworms . 8
7.4 Test conditions and measurements . 9
7.5 Reference substance . 9
8 Calculation and expression of results . 9
8.1 Calculation . 9
8.2 Expression of results . . 9
9 Validity of the test .10
10 Statistical analysis .10
10.1 General . 10
10.2 Single-concentration tests . 10
10.3 Multi-concentration tests . 11
10.3.1 Preliminary test . 11
10.3.2 Definitive test . 11
11 Test report .12
Annex A (informative) Determination of the chronic toxicity of chemicals on Eisenia fetida/
Eisenia andrei under tropical conditions .13
Annex B (informative) Culturing and testing procedures for assessing the effects
of contaminants on juvenile production using the earthworm, Dendrodilus rubidus,
representative of boreal and taiga eco-zones in northern temperate regions .15
Annex C (informative) Culturing and testing procedures for assessing the effects
of contaminants on adult survival and cocoon production and hatchability using
the earthworm, Aporrectodea caliginosa s.s., representative of temperate regions
and agricultural soils .19
Annex D (informative) Culturing of Eisenia fetida and Eisenia andrei .23
Annex E (informative) Determination of water holding capacity .24
Annex F (informative) Techniques for counting juvenile worms hatched from cocoons.25
Annex G (informative) Experience with performance criteria of the test .26
Bibliography .33
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
This third edition cancels and replaces the second edition (ISO 11268-2:2012), which has been
technically revised. The main changes compared to the previous edition are as follows:
— Modification of the concentration for the reference substance (boric acid);
— Inclusion of alternative species of earthworms – Dendrodrilus rubidus, Aporrectodea caliginosa – In
informative annexes. Information on their taxonomy and ecology as well as their specific testing
requirements have also been added.
A list of all the parts in the ISO 11268 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
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Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil
[2] [3]
and are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616 ).
ISO 15799 includes a list and short characterization of recommended and standardized test systems
and ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with
soil eluate are applied to obtain information about the fraction of contaminants potentially reaching
the groundwater by the water path (retention function of soils), whereas terrestrial test systems are
used to assess the habitat function of soils. As standardized test systems using earthworms as indicator
organisms for the habitat function of soil, an acute test for survival and a chronic test for reproduction
are available.
This document describes a method that is based on the determination of sublethal effects of
contaminated soils on adult earthworms of the species Eisenia fetida (Savigny 1826) and Eisenia andrei
(André 1963). Optionally, the method can be used for testing chemicals added to standard soils (e.g.
artificial soil) for their sublethal hazard potential to earthworms. Finally, information is provided on
how to use this method for testing chemicals or test soil under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular in temperate regions. Background information on their earthworm ecology and their use in
ecotoxicological testing is available. However, these species do not occur regularly in agricultural lands
(crop sites and grasslands) or forests in these regions. In addition, they are not representative of boreal
or tropical regions. Therefore, other species such as Aporrectodea caliginosa (an endogeic mineral
dweller in temperate regions) and Dendrodilus rubidus (an epigeic litter inhabitant in boreal regions)
have been added as potential alternative test species (Annexes B, C). These alternative earthworm
species have been used as ecotoxicological test species for some time, but testing experience has been
limited to specific countries.
This document has been drawn up taking into consideration test procedures adopted by the
[43][44] [27]
Organization for Economic Cooperation and Development and by the European Union .
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oSIST prEN ISO 11268-2:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 11268-2:2021(E)
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or
otherwise harmful chemicals or infectious microorganisms. Occupational health risks may
arise from dust or evaporated chemicals during handling and incubation. Precautions should be
taken to avoid skin contact.
1 Scope
This document specifies one of the methods for evaluating the habitat function of soils and determining
the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by
dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown
quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites
concerned, and waste materials.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate.
For contaminated soils, the effects are determined in the test soil and in a control soil. According to the
objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be
either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard
soil (e.g. artificial soil).
This method is designed mainly for determining the effects of soil contaminants and chemicals on
the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use
Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method
also includes technical information on how to use it with other environmentally relevant earthworm
species: e.g., Aporrectodea caliginosa and Dendrodrilus rubidus (see Annexes B, C).
This method does not apply to substances for which the air/soil partition coefficient is greater than
one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C. This method does not take into
account the persistence of the substance during the test.
2 Normative references
The following referenced documents are indispensable for the application 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 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic
conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil, treated biowaste and sludge – 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 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity
to Eisenia fetida/Eisenia andrei
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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/
3.1
contaminant
[1]
substance or agent present in the soil as a result of human activity[ISO 15176:2002]
3.2
growth
increase in biomass (i.e. the fresh mass of organisms)
Note 1 to entry: It is expressed as a percentage of the fresh mass of organisms at the start of the test.
3.3
reproduction
mean number of offspring per test container after eight weeks’ incubation under the specified test
conditions
3.4
ER (effective rate) or EC (effective concentration)
x x
x % effect rate or concentration of the test sample or test substance at which reproduction is reduced
by x % compared to the control
3.5
limit test
single concentration test consisting of at least four replicates each, the test sample without any dilution
or the highest concentration of test substance mixed into the control soil and the control
3.6
lowest observed effect rate (LOER) or effect concentration (LOEC)
lowest tested percentage of a test sample in a control soil or concentration of a substance at which a
statistically significant effect is observed
Note 1 to entry: The LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test
mixtures above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If this condition
cannot be satisfied, an explanation should be given for how the LOEC and NOEC (3.7) have been selected.
3.7
NOER (no observed effective rate) or NOEC (no observed effect concentration)
test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test
substance which, when compared to the control, has no statistically significant lethal or other effect
such as reduced reproduction or mass alteration (error probability: p < 0,05)
Note 1 to entry: The NOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass.
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3.8
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar
properties (nutrient concentrations, pH, organic carbon content and texture) to the test soil
3.9
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are
within a known range
[27] [43] [38]
EXAMPLE Euro-Soils , artificial soil , LUFA standard soil .
Note 1 to entry: The properties of standard soils can differ from those of the test soil.
3.10
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with test
samples or a reference substance, which fulfils the validity criteria
Note 1 to entry: In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving
the test validity criteria before using the soil in a definitive test.
3.11
test mixture
mixture of contaminated soil or the test substance with a control soil (3.10)
Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.
3.12
test mixture ratio
ratio of test soil to control soil in a test mixture
Note 1 to entry: Different ratios may be applied in a dilution series to establish a dose-response relationship.
4 Principle
The effects on reproduction of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed
to the test soil are compared to those observed for samples exposed to a control soil. If appropriate,
effects based on exposure to a dilution range of contaminated soil or range of concentrations of a test
substance are determined. In addition, observations on growth and survival of adult earthworms are
recorded. Test mixtures are prepared at the start of the test and are not renewed within the test period.
After four weeks, adult worms are removed from the test containers and effects on mortality and
biomass are measured by counting and weighing. The effect on reproduction as the definitive end
point is measured by counting the number of offspring hatched from the cocoons after an additional
period of four weeks. The results obtained from the tests are compared with a control soil (3.10) or, if
appropriate, are used to determine the dilutions or concentrations which cause no effects on biomass,
mortality and reproduction (NOER/NOEC) and the dilution (concentration) resulting in x % reduction
of juveniles hatched from cocoons compared to the control (ER /EC , 56 d), respectively.
x x
If testing a dilution or concentration series, all test dilutions/concentrations above the LOER/LOEC
shall have a harmful effect equal to, or greater than that observed at the LOER/LOEC. Where there is
no prior knowledge of the dilution/concentration of the test soil/test substance likely to have an effect,
then it is useful to conduct the test in two steps:
— a preliminary test carried out, in accordance with ISO 11268-1, to give an indication of the effect
dilution/concentration and of the dilution/concentration giving no mortality (NOER/NOEC).
Dilutions/concentrations to be used in the definitive test can then be selected.
— the definitive test to determine sublethal effects of (dilutions of) contaminated soil or the
concentration of a chemical which, when evenly mixed into the standard soil, causes no significant
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effects on numbers of offspring hatched from cocoons compared with the control (NOER/NOEC),
and the lowest concentration causing effects (LOER/LOEC).
NOTE The use of a reference soil is an essential requirement to demonstrate the present status of the test
population, and to avoid misinterpretation of results.
Alternative species of earthworms and their respective requirements are added as specific annexes in
the document:
— Dendrodrilus rubidus (see Annex B);
— Aporrectodea caliginosa (see Annex C).
5 Reagents and material
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia
[30], [34], [35],
andrei which are between two months and one year old, with a clitellum, and a wet mass
between 300 mg and 600 mg (E. fetida) and between 250 mg and 600 mg (E. andrei). It is recommended
to check the identity of the strain used to avoid species misidentifications. ISO 21286 is suitable for that
purpose.
NOTE During the ring test performed to validate ISO 21286, only 17 of the 28 laboratories involved (61 %)
provided correct identification of their laboratory culture of compost worms. Most laboratories with wrong or
unknown assignments had E. andrei in culture, or a mixture of both species (REF?].
Select worms used for the test to form, as far as is practicable, a homogeneous population from the
standpoint of age, size and mass. Worms should preferably be selected from a synchronized culture
with a relatively homogeneous age structure. Before the test, wash them with potable water.
NOTE An example of culturing Eisenia fetida/Eisenia andrei is given in Annex D.
Condition the selected worms for one day to seven days in standard or control soil before use. The food,
which is also used as a food source in the test (see 5.4), shall be given in sufficient amount (see 7.4).
5.2 Test sample, may consist of field-collected soil or control soil amended by the test mixture.
The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or
waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed
material, or manure) under consideration for possible land disposal.
Test samples shall be sieved by 4 mm mesh and thoroughly mixed. If necessary, soil may be air-dried
without heating before sieving. Storage of test samples should be as short as possible. Store the soil
in accordance with ISO 18400-206 using containers that minimize losses of soil contaminants by
volatilization and sorption to the container walls. Soil pH should not be corrected as it can influence
bioavailability of soil contaminants.
For interpretation of test results, the following characteristics shall be determined for each soil sampled
from a field site:
— pH in accordance with ISO 10390,
— texture (sand, loam, silt) in accordance with ISO 11277,
— water content in accordance with ISO 11465,
— water holding capacity according to Annex E,
— cationic exchange capacity in accordance with ISO 11260,
— organic carbon in accordance with ISO 10694.
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NOTE It is important to measure the water holding capacity of all mixtures used in the test.
5.3 Control soil, either a) reference soil (3.8) or b) standard soil (3.9) that allows the presence of
earthworms.
a) If reference soils from uncontaminated areas near a contaminated site are available, they should be
treated and characterized like the test samples. If a toxic contamination or unusual soil properties
cannot be
...
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