Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)

ISO 11268-2:2012 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

Status
Published
Publication Date
11-Aug-2015
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
12-Aug-2015
Completion Date
12-Aug-2015

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SLOVENSKI STANDARD
SIST EN ISO 11268-2:2015
01-oktober-2015
.DNRYRVWWDO8þLQNLRQHVQDåHYDOQDGHåHYQLNHGHO'RORþDQMHXþLQNRYQD
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

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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.

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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.
2 © ISO 2012 – All rights reserved
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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.
© ISO 2012 – All rights reserved 3
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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

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

...

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