EN 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

• Status: Published
• Publication Date: 30-May-2023
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444/WG 4 - Biological characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 01-Feb-2023
• Completion Date: 01-Feb-2023
• Ref Project: SIST EN 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)

Relations

• Replaces: EN ISO 11268-2:2015 - Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
• Effective Date: 18-Jan-2023

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 11268-2
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 11268-2:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 11268-2:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11268-2
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-11-16 2022-02-08
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —

Partie 2: Détermination des effets sur la reproduction de Eisenia fetida/Eisenia andrei

ICS: 13.080.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
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NOTIFICATION OF ANY RELEVANT PATENT
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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 effects
of contaminants on juvenile production using the earthworm, Dendrodilus rubidus,

representative of boreal and taiga eco-zones in northern temperate regions ..............................15

Annex C (informative) Culturing and testing procedures for assessing the effects
of contaminants on adult survival and cocoon production and hatchability using
the earthworm, Aporrectodea caliginosa s.s., representative of temperate regions

and agricultural soils ...................................................................................................................................................................................19

Annex D (informative) Culturing of Eisenia fetida and Eisenia andrei ..........................................................................23

Annex E (informative) Determination of water holding capacity .....................................................................................24

Annex F (informative) Techniques for counting juvenile worms hatched from cocoons.........................25

Annex G (informative) Experience with performance criteria of the test ...............................................................26

Bibliography .............................................................................................................................................................................................................................33

iii
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Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see

www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,

Biological characterization.

This third edition cancels and replaces the second edition (ISO 11268-2:2012), which has been

technically revised. The main changes compared to the previous edition are as follows:

— Modification of the concentration for the reference substance (boric acid);

— Inclusion of alternative species of earthworms – Dendrodrilus rubidus, Aporrectodea caliginosa – In

informative annexes. Information on their taxonomy and ecology as well as their specific testing

requirements have also been added.
A list of all the parts in the ISO 11268 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
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Introduction

Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil

[2] [3]

and are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616 ).

ISO 15799 includes a list and short characterization of recommended and standardized test systems

and ISO 17616 gives guidance on the choice and evaluation of the bioassays. Aquatic test systems with

soil eluate are applied to obtain information about the fraction of contaminants potentially reaching

the groundwater by the water path (retention function of soils), whereas terrestrial test systems are

used to assess the habitat function of soils. As standardized test systems using earthworms as indicator

organisms for the habitat function of soil, an acute test for survival and a chronic test for reproduction

are available.

This document describes a method that is based on the determination of sublethal effects of

contaminated soils on adult earthworms of the species Eisenia fetida (Savigny 1826) and Eisenia andrei

(André 1963). Optionally, the method can be used for testing chemicals added to standard soils (e.g.

artificial soil) for their sublethal hazard potential to earthworms. Finally, information is provided on

how to use this method for testing chemicals or test soil under tropical conditions (see Annex A).

Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in

particular in temperate regions. Background information on their earthworm ecology and their use in

ecotoxicological testing is available. However, these species do not occur regularly in agricultural lands

(crop sites and grasslands) or forests in these regions. In addition, they are not representative of boreal

or tropical regions. Therefore, other species such as Aporrectodea caliginosa (an endogeic mineral

dweller in temperate regions) and Dendrodilus rubidus (an epigeic litter inhabitant in boreal regions)

have been added as potential alternative test species (Annexes B, C). These alternative earthworm

species have been used as ecotoxicological test species for some time, but testing experience has been

limited to specific countries.

This document has been drawn up taking into consideration test procedures adopted by the

[43][44] [27]

Organization for Economic Cooperation and Development and by the European Union .

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oSIST prEN ISO 11268-2:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 11268-2:2021(E)
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei

WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or

otherwise harmful chemicals or infectious microorganisms. Occupational health risks may

arise from dust or evaporated chemicals during handling and incubation. Precautions should be

taken to avoid skin contact.
1 Scope

This document specifies one of the methods for evaluating the habitat function of soils and determining

the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by

dermal and alimentary uptake. This chronic test is applicable to soils and soil materials of unknown

quality, e.g. from contaminated sites, amended soils, soils after remediation, agricultural or other sites

concerned, and waste materials.

Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate.

For contaminated soils, the effects are determined in the test soil and in a control soil. According to the

objective of the study, the control and dilution substrate (dilution series of contaminated soil) should be

either an uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard

soil (e.g. artificial soil).

This method is designed mainly for determining the effects of soil contaminants and chemicals on

the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use

Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method

also includes technical information on how to use it with other environmentally relevant earthworm

species: e.g., Aporrectodea caliginosa and Dendrodrilus rubidus (see Annexes B, C).

This method does not apply to substances for which the air/soil partition coefficient is greater than

one, or to substances with vapour pressure exceeding 300 Pa, at 25 °C. This method does not take into

account the persistence of the substance during the test.
2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.

ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic

conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

ISO 10390, Soil, treated biowaste and sludge – Determination of pH

ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary

analysis)

ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level

using barium chloride solution

ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity

to Eisenia fetida/Eisenia andrei
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ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by

sieving and sedimentation

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric

method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
contaminant
[1]

substance or agent present in the soil as a result of human activity[ISO 15176:2002]

3.2
growth
increase in biomass (i.e. the fresh mass of organisms)

Note 1 to entry: It is expressed as a percentage of the fresh mass of organisms at the start of the test.

3.3
reproduction

mean number of offspring per test container after eight weeks’ incubation under the specified test

conditions
3.4
ER (effective rate) or EC (effective concentration)
x x

x % effect rate or concentration of the test sample or test substance at which reproduction is reduced

by x % compared to the control
3.5
limit test

single concentration test consisting of at least four replicates each, the test sample without any dilution

or the highest concentration of test substance mixed into the control soil and the control

3.6
lowest observed effect rate (LOER) or effect concentration (LOEC)

lowest tested percentage of a test sample in a control soil or concentration of a substance at which a

statistically significant effect is observed

Note 1 to entry: The LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test

mixtures above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If this condition

cannot be satisfied, an explanation should be given for how the LOEC and NOEC (3.7) have been selected.

3.7
NOER (no observed effective rate) or NOEC (no observed effect concentration)

test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test

substance which, when compared to the control, has no statistically significant lethal or other effect

such as reduced reproduction or mass alteration (error probability: p < 0,05)

Note 1 to entry: The NOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass.

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3.8
reference soil

uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar

properties (nutrient concentrations, pH, organic carbon content and texture) to the test soil

3.9
standard soil

field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are

within a known range
[27] [43] [38]
EXAMPLE Euro-Soils , artificial soil , LUFA standard soil .

Note 1 to entry: The properties of standard soils can differ from those of the test soil.

3.10
control soil

reference or standard soil used as a control and as a medium for preparing dilution series with test

samples or a reference substance, which fulfils the validity criteria

Note 1 to entry: In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving

the test validity criteria before using the soil in a definitive test.
3.11
test mixture
mixture of contaminated soil or the test substance with a control soil (3.10)

Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.

3.12
test mixture ratio
ratio of test soil to control soil in a test mixture

Note 1 to entry: Different ratios may be applied in a dilution series to establish a dose-response relationship.

4 Principle

The effects on reproduction of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed

to the test soil are compared to those observed for samples exposed to a control soil. If appropriate,

effects based on exposure to a dilution range of contaminated soil or range of concentrations of a test

substance are determined. In addition, observations on growth and survival of adult earthworms are

recorded. Test mixtures are prepared at the start of the test and are not renewed within the test period.

After four weeks, adult worms are removed from the test containers and effects on mortality and

biomass are measured by counting and weighing. The effect on reproduction as the definitive end

point is measured by counting the number of offspring hatched from the cocoons after an additional

period of four weeks. The results obtained from the tests are compared with a control soil (3.10) or, if

appropriate, are used to determine the dilutions or concentrations which cause no effects on biomass,

mortality and reproduction (NOER/NOEC) and the dilution (concentration) resulting in x % reduction

of juveniles hatched from cocoons compared to the control (ER /EC , 56 d), respectively.

x x

If testing a dilution or concentration series, all test dilutions/concentrations above the LOER/LOEC

shall have a harmful effect equal to, or greater than that observed at the LOER/LOEC. Where there is

no prior knowledge of the dilution/concentration of the test soil/test substance likely to have an effect,

then it is useful to conduct the test in two steps:

— a preliminary test carried out, in accordance with ISO 11268-1, to give an indication of the effect

dilution/concentration and of the dilution/concentration giving no mortality (NOER/NOEC).

Dilutions/concentrations to be used in the definitive test can then be selected.

— the definitive test to determine sublethal effects of (dilutions of) contaminated soil or the

concentration of a chemical which, when evenly mixed into the standard soil, causes no significant

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effects on numbers of offspring hatched from cocoons compared with the control (NOER/NOEC),

and the lowest concentration causing effects (LOER/LOEC).

NOTE The use of a reference soil is an essential requirement to demonstrate the present status of the test

population, and to avoid misinterpretation of results.

Alternative species of earthworms and their respective requirements are added as specific annexes in

the document:
— Dendrodrilus rubidus (see Annex B);
— Aporrectodea caliginosa (see Annex C).
5 Reagents and material

5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia

[30], [34], [35],

andrei which are between two months and one year old, with a clitellum, and a wet mass

between 300 mg and 600 mg (E. fetida) and between 250 mg and 600 mg (E. andrei). It is recommended

to check the identity of the strain used to avoid species misidentifications. ISO 21286 is suitable for that

purpose.

NOTE During the ring test performed to validate ISO 21286, only 17 of the 28 laboratories involved (61 %)

provided correct identification of their laboratory culture of compost worms. Most laboratories with wrong or

unknown assignments had E. andrei in culture, or a mixture of both species (REF?].

Select worms used for the test to form, as far as is practicable, a homogeneous population from the

standpoint of age, size and mass. Worms should preferably be selected from a synchronized culture

with a relatively homogeneous age structure. Before the test, wash them with potable water.

NOTE An example of culturing Eisenia fetida/Eisenia andrei is given in Annex D.

Condition the selected worms for one day to seven days in standard or control soil before use. The food,

which is also used as a food source in the test (see 5.4), shall be given in sufficient amount (see 7.4).

5.2 Test sample, may consist of field-collected soil or control soil amended by the test mixture.

The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or

waste materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed

material, or manure) under consideration for possible land disposal.

Test samples shall be sieved by 4 mm mesh and thoroughly mixed. If necessary, soil may be air-dried

without heating before sieving. Storage of test samples should be as short as possible. Store the soil

in accordance with ISO 18400-206 using containers that minimize losses of soil contaminants by

volatilization and sorption to the container walls. Soil pH should not be corrected as it can influence

bioavailability of soil contaminants.

For interpretation of test results, the following characteristics shall be determined for each soil sampled

from a field site:
— pH in accordance with ISO 10390,
— texture (sand, loam, silt) in accordance with ISO 11277,
— water content in accordance with ISO 11465,
— water holding capacity according to Annex E,
— cationic exchange capacity in accordance with ISO 11260,
— organic carbon in accordance with ISO 10694.
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NOTE It is important to measure the water holding capacity of all mixtures used in the test.

5.3 Control soil, either a) reference soil (3.8) or b) standard soil (3.9) that allows the presence of

earthworms.

a) If reference soils from uncontaminated areas near a contaminated site are available, they should be

treated and characterized like the test samples. If a toxic contamination or unusual soil properties

cannot be
...