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
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei and other earthworm species
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.
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
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.
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Standards Content (Sample)
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 2023
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Published in Switzerland
ii
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
iii
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
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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.
iv
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 .
v
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
...
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