SIST EN ISO 11268-2:2015
(Main)Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
This part of ISO 11268 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).
Information is provided on how to use this method for testing chemicals under temperate as well as under
tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry’s constant) or the
air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 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 (ISO 11268-2:2012)
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 11268-2:2012)
L'ISO 11268-2:2012 spécifie l'une des méthodes permettant d'évaluer la fonction d'habitat des sols et de déterminer les effets de contaminants du sol et de produits chimiques 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 contaminés, de sols amendés, de sols après remédiation, de sols agricoles ou autres sites d'intérêt et de déchets.
Les effets des substances sont évalués à l'aide d'un sol standard, de préférence un substrat de sol artificiel défini. Pour les sols contaminés, les effets sont déterminés dans le sol soumis à essai et dans un sol témoin. Selon l'objectif de l'étude, le substrat témoin et de dilution (gamme de dilutions d'un sol contaminé) peut être soit un sol non contaminé comparable à l'échantillon de sol à évaluer (sol de référence), soit un sol standard (par exemple un sol artificiel).
Des informations sont fournies sur la manière d'utiliser cette méthode pour évaluer des produits chimiques dans des conditions tempérées ainsi que dans des conditions tropicales.
La méthode ne s'applique pas aux substances volatiles, c'est-à-dire aux substances pour lesquelles H (constante de Henry) ou le coefficient de partage air/eau est supérieur à 1, ou pour lesquelles la pression de vapeur à 25 °C excède 0,013 3 Pa.
Cette méthode ne tient pas compte de la stabilité de la substance pendant l'essai.
Kakovost tal - Učinki onesnaževal na deževnike - 2. del: Določanje učinkov na reprodukcijo Eisenia fetida/Eisenia Andrei (ISO 11268-2:2012)
Ta del standarda EN-ISO 11268 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.
Vpliv snovi se določi z uporabo standardnih tal, najbolje z določenim umetnim talnim substratom. Za kontaminirana tla se učinki določijo v preskušenih in kontrolnih tleh. Glede na cilj raziskave naj bi bila kontrolni in zredčeni substrat (serija redčenja kontaminiranih tal) neokužena tla, primerljiva s tlemi, ki se jih preskuša (referenčna tla), ali standardna tla (npr. umetna tla). Zagotovljene so informacije o uporabi te metode za preskušanje kemikalij v zmernih in tropskih pogojih.
Ta metoda ni primerna za hlapljive snovi, npr. snovi, pri katerih je H (Henryjeva konstanta) ali koeficient deleža zraka/vode večji od 1 ali pri katerih parni tlak pri 25 °C presega 0,0133 Pa. Pri tej metodi se ne upošteva obstojnost snovi med preskusom.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11268-2:2015
01-oktober-2015
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UHSURGXNFLMR(LVHQLDIHWLGD(LVHQLD$QGUHL ,62
Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2:
Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
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 11268-2:2012)
Ta slovenski standard je istoveten z: EN ISO 11268-2:2015
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 11268-2:2015 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11268-2:2015
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SIST EN ISO 11268-2:2015
EUROPEAN STANDARD
EN ISO 11268-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2015
ICS 13.080.30
English Version
Soil quality - Effects of pollutants on earthworms - Part 2:
Determination of effects on reproduction of Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
Qualité du sol - Effets des polluants vis-à-vis des vers de Bodenbeschaffenheit - Wirkungen von Schadstoffen auf
terre - Partie 2: Détermination des effets sur la reproduction Regenwürmer - Teil 2: Bestimmung der Wirkung auf die
de Eisenia fetida/Eisenia andrei (ISO 11268-2:2012) Reproduktionsleistung von Eisenia fetida/Eisenia andrei
(ISO 11268-2:2012)
This European Standard was approved by CEN on 6 August 2015.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11268-2:2015 E
worldwide for CEN national Members.
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SIST EN ISO 11268-2:2015
EN ISO 11268-2:2015 (E)
Contents Page
European foreword .3
2
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SIST EN ISO 11268-2:2015
EN ISO 11268-2:2015 (E)
European foreword
The text of ISO 11268-2:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 11268-2:2015 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by February 2016, and conflicting national standards shall be withdrawn
at the latest by February 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11268-2:2012 has been approved by CEN as EN ISO 11268-2:2015 without any modification.
3
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SIST EN ISO 11268-2:2015
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SIST EN ISO 11268-2:2015
INTERNATIONAL ISO
STANDARD 11268-2
Second edition
2012-11-01
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
Reference number
ISO 11268-2:2012(E)
©
ISO 2012
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(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 . 5
7 Procedure . 6
7.1 Experimental design . 6
7.2 Preparation of test mixture . 7
7.3 Addition of the earthworms . 8
7.4 Test conditions and measurements . 8
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 . 9
10 Statistical analysis . 9
10.1 General . 9
10.2 Single-concentration tests .10
10.3 Multi-concentration tests .10
11 Test report . 11
Annex A (informative) Determination of the chronic toxicity of chemicals on earthworms under
tropical conditions .13
Annex B (informative) Culturing of Eisenia fetida and Eisenia andrei .15
Annex C (informative) Determination of water holding capacity of artificial soil .16
Annex D (informative) Techniques for counting juvenile worms hatched from cocoons.17
Annex E (informative) Performance of the method .18
Bibliography .20
© ISO 2012 – All rights reserved iii
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11268-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
This second edition cancels and replaces the first edition (ISO 11268-2:1998), which has been technically revised.
ISO 11268 consists of the following parts, under the general title Soil quality — Effects of pollutants on earthworms:
— Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei
— Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei
— Part 3: Guidance on the determination of effects in field situations
iv © ISO 2012 – All rights reserved
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and are
[34] [35]
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 part of ISO 11268 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 under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular. Background information on the ecology of earthworms and their use in ecotoxicological testing is
available. Other species, e.g. Aporrectodea caliginosa, Lumbricus rubellus and Lumbricus terrestris, have also
been used as test organisms. These or other species have not been proven to be more sensitive in general,
[16][17]
and the database and experience in testing soils is small .
This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the Organization
[27][28] [11]
for Economic Cooperation and Development and by the European Union .
© ISO 2012 – All rights reserved v
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SIST EN ISO 11268-2:2015
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SIST EN ISO 11268-2:2015
INTERNATIONAL STANDARD ISO 11268-2:2012(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 part of ISO 11268 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).
Information is provided on how to use this method for testing chemicals under temperate as well as under
tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry’s constant) or the
air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
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 2012 – All rights reserved 1
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
contaminant
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 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 vessel 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 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 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
[11] [27] [23]
EXAMPLE Euro-Soils , artificial soil , LUFA standard soil .
NOTE The properties of standard soils can differ from those of the test soil.
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
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 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 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 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 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 ,
x x
56 d), respectively.
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 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.
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
5 Reagents and material
[15], [19],
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia andrei
[20],
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).
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 B.
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 5.3), 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.
5.2.1 Field-collected soils, soil or waste materials
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 10381-6 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 C,
— cationic exchange capacity in accordance with ISO 11260,
— organic carbon in accordance with ISO 10694.
NOTE It is important to measure the water holding capacity of all mixtures used in the test.
5.2.2 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 ruled
out, standard control soils should be preferred.
b) For testing the effects of substances mixed into soil or making dilutions of the test sample, standard soils
shall be used to prepare the test sample. The properties of the field-collected standard soil shall be reported.
4 © ISO 2012 – All rights reserved
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SIST EN ISO 11268-2:2015
ISO 11268-2:2012(E)
The substrate called artificial soil can be used as a standard soil and has the following composition:
Percentage expressed
on a dry-mass basis
— Sphagnum peat finely ground and with no visible plant remains 10 %
— Kaolinite clay containing not less than 30 % kaolinite 20 %
— Industrial quartz sand (dominant fine sand with more 69 %
than 50 % of particle sizes 0,05 mm to 0,2 mm)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) are necessary to get a
3
pH of 6,0 ± 0,5.
NOTE 1 Taking the properties of highly non-polar (log K > 2, where K is the octanol/water coefficient) or ionizing
ow ow
substances into account, 5 % of peat have proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that Eisenia fetida can comply with the validity criteria, even as regards reproduction,
[18]
when tested in field soils with lower organic carbon content (e.g. 2,7 %) , and experience shows that this can be achieved in
artificial soil with 5 % peat. It is therefore not necessary, before using such a soil in a definitive test, to demonstrate the suitability
[28].
of the artificial soil in complying with the validity criteria, unless the peat contents lowered more than specified above
Prepare the artificial soil at least three days prior to starting the test, by mixing the dry constituents listed above
thoroughly in a large-scale laboratory mixer. A portion of the deionized water required is added while mixing is
continued. Allowance should be made for any water that is used for introducing the test mixture into the soil.
The amount of calcium carbonate required can vary, depending on the properties of the individual batch of
sphagnum peat and should be determined by measuring sub-samples immediately before the test. Store the
mixed artificial soil at room temperature for at least two days to equilibrate acidity. To determine pH and the
maximum water holding capacity, the dry artificial soil is pre-moistened one or two days before starting the test
by adding deionized water to obtain approximately half of the required final water content of 40 % to 60 % of
the maximum water holding capacity.
The total water holding capacity is determined according to Annex C; the pH is determined according to
...
SLOVENSKI STANDARD
kSIST FprEN ISO 11268-2:2015
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Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2:
Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
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 11268-2:2012)
Ta slovenski standard je istoveten z: FprEN ISO 11268-2
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
kSIST FprEN ISO 11268-2:2015 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN ISO 11268-2:2015
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kSIST FprEN ISO 11268-2:2015
EUROPEAN STANDARD
FINAL DRAFT
FprEN ISO 11268-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2015
ICS 13.080.30
English Version
Soil quality - Effects of pollutants on earthworms - Part 2:
Determination of effects on reproduction of Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
Qualité du sol - Effets des polluants vis-à-vis des vers de Bodenbeschaffenheit - Wirkungen von Schadstoffen auf
terre - Partie 2: Détermination des effets sur la reproduction Regenwürmer - Teil 2: Bestimmung der Wirkung auf die
de Eisenia fetida/Eisenia andrei (ISO 11268-2:2012) Reproduktionsleistung von Eisenia fetida/Eisenia andrei
(ISO 11268-2:2012)
This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical
Committee CEN/TC 345.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which
stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language
made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprEN ISO 11268-2:2015 E
worldwide for CEN national Members.
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kSIST FprEN ISO 11268-2:2015
FprEN ISO 11268-2:2015 (E)
Contents Page
Foreword .3
2
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kSIST FprEN ISO 11268-2:2015
FprEN ISO 11268-2:2015 (E)
Foreword
The text of ISO 11268-2:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as FprEN ISO 11268-2:2015 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This document is currently submitted to the Unique Acceptance Procedure.
Endorsement notice
The text of ISO 11268-2:2012 has been approved by CEN as FprEN ISO 11268-2:2015 without any
modification.
3
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kSIST FprEN ISO 11268-2:2015
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kSIST FprEN ISO 11268-2:2015
INTERNATIONAL ISO
STANDARD 11268-2
Second edition
2012-11-01
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
Reference number
ISO 11268-2:2012(E)
©
ISO 2012
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(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 . 5
7 Procedure . 6
7.1 Experimental design . 6
7.2 Preparation of test mixture . 7
7.3 Addition of the earthworms . 8
7.4 Test conditions and measurements . 8
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 . 9
10 Statistical analysis . 9
10.1 General . 9
10.2 Single-concentration tests .10
10.3 Multi-concentration tests .10
11 Test report . 11
Annex A (informative) Determination of the chronic toxicity of chemicals on earthworms under
tropical conditions .13
Annex B (informative) Culturing of Eisenia fetida and Eisenia andrei .15
Annex C (informative) Determination of water holding capacity of artificial soil .16
Annex D (informative) Techniques for counting juvenile worms hatched from cocoons.17
Annex E (informative) Performance of the method .18
Bibliography .20
© ISO 2012 – All rights reserved iii
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11268-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
This second edition cancels and replaces the first edition (ISO 11268-2:1998), which has been technically revised.
ISO 11268 consists of the following parts, under the general title Soil quality — Effects of pollutants on earthworms:
— Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei
— Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei
— Part 3: Guidance on the determination of effects in field situations
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and are
[34] [35]
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 part of ISO 11268 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 under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular. Background information on the ecology of earthworms and their use in ecotoxicological testing is
available. Other species, e.g. Aporrectodea caliginosa, Lumbricus rubellus and Lumbricus terrestris, have also
been used as test organisms. These or other species have not been proven to be more sensitive in general,
[16][17]
and the database and experience in testing soils is small .
This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the Organization
[27][28] [11]
for Economic Cooperation and Development and by the European Union .
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kSIST FprEN ISO 11268-2:2015
INTERNATIONAL STANDARD ISO 11268-2:2012(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 part of ISO 11268 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).
Information is provided on how to use this method for testing chemicals under temperate as well as under
tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry’s constant) or the
air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
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 2012 – All rights reserved 1
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
contaminant
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 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 vessel 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 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 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
[11] [27] [23]
EXAMPLE Euro-Soils , artificial soil , LUFA standard soil .
NOTE The properties of standard soils can differ from those of the test soil.
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
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 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 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 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 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 ,
x x
56 d), respectively.
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 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.
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
5 Reagents and material
[15], [19],
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia andrei
[20],
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).
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 B.
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 5.3), 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.
5.2.1 Field-collected soils, soil or waste materials
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 10381-6 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 C,
— cationic exchange capacity in accordance with ISO 11260,
— organic carbon in accordance with ISO 10694.
NOTE It is important to measure the water holding capacity of all mixtures used in the test.
5.2.2 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 ruled
out, standard control soils should be preferred.
b) For testing the effects of substances mixed into soil or making dilutions of the test sample, standard soils
shall be used to prepare the test sample. The properties of the field-collected standard soil shall be reported.
4 © ISO 2012 – All rights reserved
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kSIST FprEN ISO 11268-2:2015
ISO 11268-2:2012(E)
The substrate called artificial soil can be used as a standard soil and has the following composition:
Percentage expressed
on a dry-mass basis
— Sphagnum peat finely ground and with no visible plant remains 10 %
— Kaolinite clay containing not less than 30 % kaolinite 20 %
— Industrial quartz sand (dominant fine sand with more 69 %
than 50 % of particle sizes 0,05 mm to 0,2 mm)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) are necessary to get a
3
pH of 6,0 ± 0,5.
NOTE 1 Taking the properties of highly non-polar (log K > 2, where K is the octanol/water coefficient) or ionizing
ow ow
substances into account, 5 % of peat have proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that Eisenia fetida can comply with the validity criteria, even as regards reproduction,
[18]
when tested in field soils with lower organic carbon content (e.g. 2,7 %) , and experience shows that this can be achieved in
artificial soil with 5 % peat. It is therefore not necessary, before using such a soil in a definitive test, to demonstrate the suitability
[28].
of the artificial soil in complying with the validity criteria, unless the peat contents lowered more than specified above
Prepare the artificial soil at least three days prior to starting the test, by mixing the dry constituents listed above
thoroughly in a large-scale laboratory mixer. A portion of the deionized water required is added while mixing is
continued. Allowance should be made for any water that is used for introducing the test mixture into the soil.
The amount of calcium carbonate required can vary, depending on the properties of the individual batch of
sphagnum peat and should be determined by measuring sub-samples immediately before the test. Store the
mixed artificial soil at room temperature for at least two days to equilibrate acidity. To determine pH and the
maximum water holding capacity, the dry artificial soil is pre-moistened one or two days before starting the test
by adding deionized water to obtain approximately half of the required final water content of 40 % to 60 % of
the maximum water holding capacity.
The total water holding capacity is determined according to Annex C; the pH is determined according to ISO 10390.
5.3 Feeding, any food of a quality shown to be suitable for at least maintaining worm mass during the test is
[20]
considered acceptable. Experience has shown that oatmeal, mashed potato powder , cow or horse manure
is a suitable food. Checks should be made to ensure that cows or horses from which manure is obtained are not
subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary
products that could adversely affect the worms durin
...
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