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. 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.
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.
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. Dieses Verfahren berücksichtigt nicht den möglichen Abbau der Substanz während der Prüfung.
WARNUNG — Verunreinigte Böden können unbekannte Gemische von toxischen, mutagenen oder auf andere Art schädlichen Chemikalien oder infektiösen Mikroorganismen enthalten. Berufsbedingte Gesundheitsrisiken können durch Staub oder flüchtige Chemikalien während der Handhabung und Inkubation der verunreinigten Böden auftreten. Es sollten Vorkehrungen getroffen werden, um den Hautkontakt mit ihnen zu vermeiden.
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)
Kakovost tal - Učinki onesnaževal na deževnike - 2. del: Določanje učinkov na reprodukcijo Eisenia fetida/Eisenia andrei (ISO 11268-2:2023)
General Information
Relations
Standards Content (Sample)
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 11268-2ICS:
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
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: 13.080.30This 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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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
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POTENTIAL TO BECOME STANDARDS TO
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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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
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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 effectsof 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 effectsof 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.© ISO 2021 – All rights reserved
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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 pHISO 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 solutionISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity
to Eisenia fetida/Eisenia andrei© ISO 2021 – All rights reserved
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ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentationISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method3 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]
3.2growth
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.
3.3reproduction
mean number of offspring per test container after eight weeks’ incubation under the specified test
conditions3.4
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 control3.5
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
3.6lowest 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 observedNote 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.
3.7NOER (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
3.9standard 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.
3.10control 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 criteriaNote 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.
3.12test 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 PrincipleThe 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 xIf 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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