prEN ISO 11267

Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants (ISO/DIS 11267:2022)

Bodenbeschaffenheit - Hemmung der Reproduktion von Collembolen (Folsomia candida) durch Verunreinigungen (ISO/DIS 11267:2022)

Diese Internationale Norm legt eines der Verfahren zur Bewertung der Lebensraumfunktion von Böden und zur Bestimmung der Wirkungen von Bodenverunreinigungen und Substanzen auf die Reproduktion von Folsomia candida Willem bei Aufnahme über die Haut und durch die Nahrung fest. Diese chronische Prüfung ist anwendbar für Böden und Bodenmaterialien unbekannter Beschaffenheit, z. B. von verunreinigten Standorten, beaufschlagten Böden, Böden nach der Sanierung, industriellen, landwirtschaftlichen oder anderen betroffenen Standorten und Abfallmaterialien.
Die Wirkungen der Substanzen werden mit einem Standardboden, vorzugsweise mit einem festgelegten künstlichen Bodensubstrat, bewertet. Bei verunreinigten Böden werden die Wirkungen im zu prüfenden Boden und in einem Kontrollboden bestimmt. Entsprechend der Zielsetzung der Untersuchung sind die Kontrolle und das zur Verdünnung verwendete Substrat (Verdünnungsreihen des verunreinigten Bodens) entweder ein mit dem zu prüfenden Boden vergleichbarer, nicht verunreinigter Boden (Referenzboden) oder ein Standardboden (z. B. künstlicher Boden).
Diese Internationale Norm gibt Informationen, wie dieses Verfahren zur Prüfung von Substanzen unter gemäßigten Bedingungen anzuwenden ist.
Das Verfahren ist nicht auf flüchtige Substanzen anwendbar, d. h. Substanzen, bei denen H (Henry-Konstante) oder der Luft/Wasser-Verteilungskoeffizient größer als 1 ist oder bei denen der Dampfdruck 0,013 3 Pa bei 25 °C überschreitet.
ANMERKUNG   Es kann nicht sichergestellt werden, dass die Prüfsubstanz während der gesamten Versuchsdauer stabil bleibt. Im Prüfverfahren wird die Persistenz der Prüfsubstanz nicht überwacht.

Qualité du sol - Inhibition de la reproduction de Collembola (Folsomia candida) par des contaminants du sol (ISO/DIS 11267:2022)

Kakovost tal - Zaviranje razmnoževanja vrste Folsomia candida iz rodu skakačev (Collembola) zaradi onesnaževal v tleh (ISO/DIS 11267:2022)

General Information

• Status: Not Published
• Publication Date: 03-Sep-2024
• 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: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Start Date: 03-Jul-2023
• Completion Date: 27-Feb-2023
• Ref Project: oSIST prEN ISO 11267:2022 - Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants (ISO/DIS 11267:2022)

Relations

• Revises: EN ISO 11267:2014 - Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants (ISO 11267:2014)
• Effective Date: 13-Nov-2019

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 11267:2022
01-september-2022
Kakovost tal - Zaviranje razmnoževanja vrste Folsomia candida iz rodu skakačev
(Collembola) zaradi onesnaževal v tleh (ISO/DIS 11267:2022)

Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil

contaminants (ISO/DIS 11267:2022)

Bodenbeschaffenheit - Hemmung der Reproduktion von Collembolen (Folsomia candida)

durch Verunreinigungen (ISO/DIS 11267:2022)

Qualité du sol - Inhibition de la reproduction de Collembola (Folsomia candida) par des

contaminants du sol (ISO/DIS 11267:2022)
Ta slovenski standard je istoveten z: prEN ISO 11267
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 11267:2022 en,fr,de

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

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oSIST prEN ISO 11267:2022
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oSIST prEN ISO 11267:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11267
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2022-08-11 2022-11-03
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants

Qualité du sol — Inhibition de la reproduction de Collembola (Folsomia candida) par des contaminants du

sol
ICS: 13.080.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
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NATIONAL REGULATIONS.
ISO/DIS 11267:2022(E)
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11267
ISO/TC 190/SC 4 Secretariat: AFNOR
Voting begins on: Voting terminates on:
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants

Qualité du sol — Inhibition de la reproduction de Collembola (Folsomia candida) par des contaminants du

sol
ICS: 13.080.30
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
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NATIONAL REGULATIONS.
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

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 Range-finding test ............................................................................................................................................................. 6

7.1.3 Definitive test ........................................................................................................................................................................ 7

7.2 Preparation of test mixture ........................................................................................................................................................ 7

7.2.1 Testing contaminated soil .......................................................................................................................................... 7

7.2.2 Testing substances added to the test substrate ..................................................................................... 8

7.2.3 Preparation of control container ......................................................................................................................... 9

7.3 Addition of the biological material ....................................................................................................................................... 9

7.4 Test conditions and measurements ..................................................................................................................................... 9

7.5 Determination of surviving Collembola ........................................................................................................................... 9

8 Calculation and expression of results ........................................................................................................................................... 9

8.1 Calculation .................................................................................................................................................................................................. 9

8.2 Expression of results ........................................................................................................................................... ........................... 10

9 Validity of the test ............................................................................................................................................................................................10

10 Statistical analysis ..........................................................................................................................................................................................10

10.1 General ........................................................................................................................................................................................................ 10

10.2 Single-concentration tests ........................................................................................................................................................ 10

10.3 Multi-concentration tests .......................................................................................................................................................... 11

10.3.1 Range-finding test .......................................................................................................................................................... 11

10.3.2 Definitive test ..................................................................................................................................................................... 11

11 Test report ...............................................................................................................................................................................................................12

Annex A (informative) Techniques for rearing and breeding of Collembola ........................................................13

Annex B (informative) Determination of water-holding capacity ....................................................................................15

Annex C (informative) Guidance on adjustment of pH of artificial soil .......................................................................16

Annex D (informative) Extraction and counting of Collembola ...........................................................................................17

Annex E (informative) Specific information of alternative Collembolan species .............................................18

Bibliography .............................................................................................................................................................................................................................30

iii
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(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.

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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO's adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.

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 11267:2014), which has been technically

revised. The main change to the previous edition is as follows:

— Addition of an annex of specific information when using alternative Collembola species for

reproduction test.
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)
Introduction

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

soil and are proposed to complement conventional chemical analysis (see [2] and [4]). Reference [2]

[4]

includes a list and short characterization of recommended and standardized test systems and 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.

Soil-dwelling Collembola are ecologically relevant species for ecotoxicological testing. Springtails are

prey animals for a variety of endogeic and epigeic invertebrates and they contribute to decomposition

processes in soils. In acidic soils they may be the most important soil invertebrates besides enchytraeids

[19]

with respect to that function, since earthworms are typically absent . Additionally, Collembola

represent arthropod species with a different route and a different rate of exposure compared to

[1] [3]

earthworms and enchytraeids . Various species were used in bioassays of which four species

were used most commonly, Folsomia candida Willem, Folsomia fimetaria L., Onychiurus armatus, and

[20]

Orchesella cincta . Numerous soil toxicity tests supported by Environment Canada (EC) resulted in the

development and standardization of a biological test method for determining the lethal and sublethal

[10]

toxicity of samples of contaminated soil to Collembola . The method prepared by EC includes four

species, Orthonychiurus folsomi, Proisotoma minuta, F. candida, and F. fimetaria. As standardized test

systems using Collembola as indicator organisms for the habitat function of soil, another two methods

exist. One is designed for assessing the effects of substances on the reproductive output of the

[19],[21]

Collembola, F. fimetaria and F. candida in soil, and the other method described here, focuses on

testing contaminated soil. Optionally the method can be used for testing substances added to standard

soils (e.g. artificial soil) for their sublethal hazard potential to Collembola.

This International Standard describes a method that is based on the determination of sublethal

effects of contaminated soils to adult Collembola of the species Folsomia candida Willem. The species

[10],[19]

is distributed worldwide. It plays a similar ecological role to F. fimetari F. candida reproduces

parthenogenetically and is an easily accessible species as it is commercially available and easy to

culture. F. candida is considered to be a representative of soil arthropods and Collembola in particular.

Background information on the ecology of springtails and their use in ecotoxicological testing is

[22]
available.

Distinct Collembolan species inhabit various ecological niches at different soil depths and in different

soil types across the globe. Although considered a surrogate species and therefore frequently used

in ecotoxicological reproduction tests, F. candida is not common in most natural soils (OECD 2016).

Furthermore, species specific morphological adaptations can influence exposure and toxic effects

of chemicals on organisms (Chelinho et al., 2014). Thus, the use of a variety of Collembolan species

representing different morphological adaptations may be advantageous to obtain a broad spectrum

of sensitivities for this group. Therefore, other species like F. fimetaria (euedaphic, distributed

worldwide and found in agricultural soils (OECD 2016)), Onychirus yodai (an euedaphic Asian species

(Janssens 2020), Proisotoma minuta (hemiedaphic, distributed worldwide and inhabiting agricultural

soils (Buch et al. 2016, Janssens 2020)), Protaphorura fimata (euedaphic, occuring through mild

temperate to cold zones (Holmstrup & Slotsbo, 2018; Janssens, 2020)), and Sinella curviseta (epedaphic,

distributed from North America to Europe, Southeast Asia and Japan (Salmon 1964)) were added as

potential alternative test species (Annex E). These species have been used as ecotoxicological test

species before, but available testing experience might be limited.
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oSIST prEN ISO 11267:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 11267:2022(E)
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants
1 Scope

This International Standard specifies one of the methods for evaluating the habitat function of soils

and determining effects of soil contaminants and substances on the reproduction of Folsomia candida

Willem 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, industrial,

agricultural or other sites of concern 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 soil to be tested and in a control soil. According

to the objective of the study, the control and dilution substrate (dilution series of contaminated soil) are

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

(e.g. artificial soil).

This International Standard provides information on how to use this method for testing substances

under temperate 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.

NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the

test method for monitoring the persistence of the substance under test.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

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

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

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

using barium chloride solution

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 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

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)

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 https:// www .electropedia .org/
3.1
contaminant
substance or agent present in the soil as a result of human activity
3.2
ECx
effect concentration for x % effect

concentration (mass fraction) of a test sample or a test substance that causes x % of an effect on a given

end-point within a given exposure period when compared with a control

EXAMPLE An EC50 is a concentration estimated to cause an effect on a test end-point in 50 % of an exposed

population over a defined exposure period.

Note 1 to entry: The ECx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).

When substances are tested, the ECx is expressed as mass of the test substance per dry mass of soil in milligrams

per kilogram.
3.3
ERx
effect rate for x % effect

rate of a contaminated soil that causes x % of an effect on a given end-point within a given exposure

period when compared with a control
3.4
limit test

single concentration test consisting of at least four replicates each, the soil to be tested without any

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

3.5
LOEC
lowest observed effect concentration

lowest test substance concentration that has a statistically significant effect (p < 0,05) when compared

with the control

Note 1 to entry: In this test, the LOEC is expressed as a mass of test substance per dry mass of the soil to be

tested. All test concentrations above the LOEC should usually show an effect that is statistically different from

the control.
3.6
LOER
lowest observed effect rate

lowest rate of a contaminated soil tested in a control soil that has a statistically significant effect

(p < 0,05) when compared with the control
3.7
NOEC
no observed effect concentration

highest test substance concentration immediately below the LOEC at which no statistically significant

effect is observed when compared to the control

Note 1 to entry: In this test, the concentration corresponding to the NOEC has no statistically significant effect

(p < 0,05) within a given exposure period when compared with the control.
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)
3.8
NOER
no observed effect rate

highest rate of a contaminated soil to be tested immediately below the LOER at which no statistically

significant effect is observed when compared to the control
3.9
reference soil

uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic

carbon content and texture) to the soil being studied
3.10
standard soil

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

within a known range
EXAMPLE Euro soils, artificial soil, LUFA standard soil.

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

3.11
control soil

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

be tested 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.
3.12
test mixture

mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil

3.13
test mixture ratio
ratio between the soil to be tested and the control soil in a test mixture
4 Principle

The effects on reproductive output of 10 d to 12 d old Collembola (F. candida) exposed to the soil to be

tested are compared to those observed in a control soil. If appropriate, effects based on exposure to a

test mixture of contaminated soil and control soil or a range of concentrations of a test substance mixed

into control soil are determined. Test mixtures are prepared at the start of the test and are not renewed

within the test period.

The Collembola are incubated until offspring (F ) emerge from eggs laid by mature adults, and the

number of offspring is determined. Usually offspring emerge within 28 d in control experiments. The

results obtained from the tests are compared with a control or, if appropriate, are used to determine

the concentrations which cause no effects on mortality and reproductive output (NOER/NOEC) and the

concentration resulting in x % reduction of juveniles hatched from eggs compared to the control (ERx/

ECx, 28 d) respectively.

If testing a concentration series, all test dilutions/concentrations above the LOER/LOEC have a harmful

effect equal to or greater than that observed at the LOER/LOEC. Where there is no prior knowledge of

the concentration of the soil to be tested or the test substance likely to have an effect, then it is useful to

conduct the test in two steps:

— An acute toxicity test (range-finding test) is carried out, to give an indication of the effect dilution/

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

concentrations to be used in the definitive test can then be selected;
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oSIST prEN ISO 11267:2022
ISO/DIS 11267:2022(E)

— The definitive test on reproductive output to determine sublethal effects of (dilutions of)

contaminated soil or the concentration of a substance which, when evenly mixed into the standard

soil, causes no significant effects on numbers of offspring hatched from eggs 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.
5 Reagents and material

5.1 Biological material, in this test, 10 d to 12 d old juvenile springtails of the species Folsomia

candida Willem are used (see A.1 for details on synchronization of breeding).

5.2 Test mixture, which may consist of field-collected soil, waste material or control soil amended

by the test substance.
5.2.1 Field-collected soil or waste

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.

The field-collected soils used in the test shall be passed through a sieve of 4 mm square mesh to remove

coarse fragments and thoroughly mixed. If necessary, soil may be air-dried without heating before

sieving. Storage of soil to be tested should be as short as possible. The soil shall be stored in accordance

with ISO 18400-206 using containers that minimize losses of soil contaminants by volatilization and

sorption to the container walls. If soils or test mixtures have been stored, they should be mixed a second

time immediately before use. 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:
a) pH in accordance with ISO 10390,
b) texture (sand, loam, silt) in accordance with ISO 11277,
c) water content in accordance with ISO 11465,
d) water-holding capacity according to Annex B,
e) cationic exchange capacity in accordance with ISO 11260,
f) organic carbon in accordance with ISO 10694,
g) percentage of material (mineral and organic) removed by the 4 mm sieve.

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 (3.9) or b) standard soil (3.10) that allows the presence of

Collembola. Control soil and soil used for dilution shall not differ in one test (either a) or b)).

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

be treated and characterized like the soils to be tested. 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, standard soils (e.g. artificial soil, LUFA) shall

be used as test substrate. The properties of the field-collected standard soil shall be reported.

The substrate called artificial soil can be used as a standard soil and has
...