SIST EN ISO 11267:2024
(Main)Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants (ISO 11267:2023)
Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil contaminants (ISO 11267:2023)
This document 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 document also provides information on how to use this method for testing substances under temperate conditions.
The chronic test described 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.
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 300 Pa at 25 °C.
Bodenbeschaffenheit - Hemmung der Reproduktion von Collembolen (Folsomia candida) durch Verunreinigungen (ISO 11267:2023)
Dieses Dokument 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. Dieses Dokument enthält ebenfalls Informationen darüber, wie diese Methode für die Prüfung von Stoffen unter gemäßigten Bedingungen anzuwenden ist.
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.
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 300 Pa bei 25 °C überschreitet.
Qualité du sol - Inhibition de la reproduction de Collembola (Folsomia candida) par des contaminants du sol (ISO 11267:2023)
Le présent document 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 substances sur la reproduction de Folsomia candida Willem par absorption cutanée et ingestion. Le présent document fournit des informations sur la manière d’utiliser cette méthode pour évaluer des substances dans des conditions tempérées.
L’essai chronique décrit s’applique aux sols et matériaux du sol de qualité inconnue, par exemple provenant de sites pollués, de sols amendés, de sols après remédiation, de sites industriels, agricoles ou d’autres sites d’intérêt et de déchets.
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 excède 300 Pa à 25 °C.
Kakovost tal - Zaviranje razmnoževanja vrste Folsomia candida iz rodu skakačev (Collembola) zaradi onesnaževal v tleh (ISO 11267:2023)
Ta dokument določa eno od metod za ocenjevanje funkcije tal habitata ter določevanje učinkov onesnaževal in snovi v tleh na razmnoževanje vrste Folsomia candida iz rodu skakačev (Collembola) z dermalnim in prehranskim vnosom. Prav tako zagotavlja informacije o uporabi te metode za preskušanje snovi v zmernih pogojih.
Opisan kronični preskus se uporablja za tla in talne materiale neznane kakovosti, na primer tla iz onesnaženih območij, obogatena tla, tla po sanaciji, industrijska, kmetijska in druga problematična mesta ter odpadne materiale.
Metoda se ne uporablja za hlapljive snovi, npr. snovi, pri katerih je H (Henryjeva konstanta) ali koeficient deleža zraka/vode večji od 1 oziroma parni tlak pri 25 °C presega 300 Pa.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-junij-2024
Nadomešča:
SIST EN ISO 11267:2014
Kakovost tal - Zaviranje razmnoževanja vrste Folsomia candida iz rodu skakačev
(Collembola) zaradi onesnaževal v tleh (ISO 11267:2023)
Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil
contaminants (ISO 11267:2023)
Bodenbeschaffenheit - Hemmung der Reproduktion von Collembolen (Folsomia candida)
durch Verunreinigungen (ISO 11267:2023)
Qualité du sol - Inhibition de la reproduction de Collembola (Folsomia candida) par des
contaminants du sol (ISO 11267:2023)
Ta slovenski standard je istoveten z: EN ISO 11267:2023
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 11267
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2023
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 11267:2014
English Version
Soil quality - Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants (ISO
11267:2023)
Qualité du sol - Inhibition de la reproduction de Bodenbeschaffenheit - Hemmung der Reproduktion
Collembola (Folsomia candida) par des contaminants von Collembolen (Folsomia candida) durch
du sol (ISO 11267:2023) Verunreinigungen (ISO 11267:2023)
This European Standard was approved by CEN on 30 July 2023.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11267:2023 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 11267:2023) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “Environmental characterization of
solid matrices” 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 2024, and conflicting national standards
shall be withdrawn at the latest by February 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 11267:2014.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 11267:2023 has been approved by CEN as EN ISO 11267:2023 without any modification.
INTERNATIONAL ISO
STANDARD 11267
Third edition
2023-08
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
Reference number
ISO 11267:2023(E)
ISO 11267:2023(E)
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 11267:2023(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 . 8
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 . . 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 Range-finding test . 11
10.3.2 Definitive test . 11
11 Test report .12
Annex A (informative) Techniques for rearing and breeding Folsomia candida.13
Annex B (normative) 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 other than
Folsomia candida .18
Bibliography .32
iii
ISO 11267:2023(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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
cautioned that this may not represent the latest information, which may be obtained from the patent
database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all
such patent rights.
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, in collaboration with the European Committee for Standardization (CEN)
Technical Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with
the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 11267:2014), which has been technically
revised.
The main change is as follows:
— addition of an annex to provide specific information when using alternative Collembola species for
reproduction test.
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.
iv
ISO 11267:2023(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 References [2] and [4]).
Reference [2] includes a list and short characterization of recommended and standardized test systems
and Reference [4] 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 are probably the most important soil invertebrates besides
[19]
enchytraeids 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
[1] [3]
compared to earthworms and enchytraeids. Various species were used in bioassays of which
four species were used most commonly, Folsomia candida Willem, Folsomia fimetaria L., Onychiurus
[20]
armatus, and 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
[10]
lethal and sublethal 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
[19],[21]
output of the 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 document 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 is distributed worldwide.
[10],[19]
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 available in Reference [22].
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
[28]
ecotoxicological reproduction tests, F. candida is not common in most natural soils. Furthermore,
species specific morphological adaptations can influence exposure and toxic effects of chemicals on
[102]
organisms. Thus, the use of a variety of Collembolan species representing different morphological
adaptations can be advantageous to obtain a broad spectrum of sensitivities for this group. Therefore,
[28]
other species like F. fimetaria (euedaphic, distributed worldwide and found in agricultural soils ),
[31]
Onychirus yodai (an euedaphic Asian species, Proisotoma minuta (hemiedaphic, distributed
[31],[36]
worldwide and inhabiting agricultural soils ), Protaphorura fimata (euedaphic, occuring through
[31],[37]
mild temperate to cold zones ), and Sinella curviseta (epedaphic, distributed from North America
[42]
to Europe, Southeast Asia and Japan ) were added as potential alternative test species (Annex E).
These species have been used as ecotoxicological test species before, but available testing experience is
limited.
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).
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.
v
INTERNATIONAL STANDARD ISO 11267:2023(E)
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants
1 Scope
This document 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 document also provides information on how to use this method for testing
substances under temperate conditions.
The chronic test described 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.
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 300 Pa at
25 °C.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 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 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.
ISO and IEC maintain terminology 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
ISO 11267:2023(E)
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 (3.11) 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 (3.11) 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 (3.5) 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.
3.8
NOER
no observed effect rate
highest rate of a contaminated soil to be tested immediately below the LOER (3.6) 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
ISO 11267:2023(E)
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 soil (3.9) or standard soil (3.10) 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 (3.11)
3.13
test mixture ratio
ratio between the soil to be tested and the control soil (3.11) in a test mixture (3.12)
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.
— A definitive test on the reproductive output determines sublethal effects of (dilutions of) the
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).
A reference soil shall be used to demonstrate the appropriate status of the test population, and to avoid
misinterpretation of results.
ISO 11267:2023(E)
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.
The water holding capacity of all mixtures used in the test should also be measured.
5.2.2 Control soil, either a reference soil or a standard soil that allows the presence of Collembola.
Control soil and soil used for dilution shall not differ in one test (either a reference soil or a standard
soil).
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 the following composition:
Percentage expressed on dry mass basis
— Sphagnum peat finely ground [a particle size of 10 %
(2 ± 1) mm is acceptable] and with no visible plant
remains
ISO 11267:2023(E)
— Kaolinite clay containing not less than 30 % kaolinite 20 %
— Industrial quartz sand (dominant fine sand with more 69 %
than 50 % of particle size 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. Further guidance on how to proceed on the adjustment of pH of artificial soil is
available in Annex C.
NOTE 1 Taking the properties of highly non-polar (log Kow > 2) or ionizing substances into account, 5 % of
peat has proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that F. candida can conform to the validity criteria even on reproductive
output when tested in field soils with lower organic carbon content (e.g. 2,7 %), and there is experience that
this can be achieved in artificial soil with 5 % peat. Therefore, it is not necessary, before using such a soil in a
definitive test, to demonstrate the suitability of the artificial soil for allowing the test to conform to the validity
criteria unless the peat content is lower than that specified above.
Prepare the artificial soil at least three days prior to the start of 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 during mixing. Allowance should be made for any water that is used for introducing the test
substance into the soil. The amount of calcium carbonate required can vary, depending on 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 the 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 shall be determined according to Annex B; the pH shall be determined
according to ISO 10390.
5.3 Food
A sufficient amount, for example, 2 mg to 10 mg, of granulated dried baker’s yeast, commercially
available for household use, is added to each container as a suitable food source, at the beginning of the
test and after about two weeks.
5.4 Reference substance
To ensure the quality of the test system, tests should be performed regularly (once or twice a year) with
a reference substance.
1)
Boric acid and the plant protection product Betosip (a.i. 157 g/l phenmedipham) have been tested in
an interlaboratory test and are recommended as reference substances.
WARNING — When handling these substances, appropriate precautions should be taken to avoid
ingestion or skin contact.
NOTE 1 Boric acid: Effects on reproductive output (i.e. EC50) are observed at concentrations of 147 mg boric
acid per kilogram of artificial soil (dry mass), 111 mg boric acid per kilogram of artificial soil with 5 % peat, and
[5],[21].
169 mg boric acid per kilogram of clay loam soil for F. candida. Taking into consideration these data and due
to the variability of organism sensitivity, an EC50 value between 50 mg and 175 mg boric acid/kg dry mass of
[103],[104]
artificial soil is acceptable based on current laboratory experience and in previous studies .
NOTE 2 Betosip: Effects on reproductive output (α = 0,05) are observed at concentrations between 100 mg
and 200 mg of the product per kilogram of the substrate (dry mass).
1) Betosip is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products
may be used if they can be shown to lead to the same results.
ISO 11267:2023(E)
6 Apparatus
Use laboratory equipment and the following apparatus:
6.1 Test containers made of glass or other chemically inert material of about 100 ml capacity and
with a diameter of about 5 cm, with lids (e.g. plastic, glass discs or parafilm, able to be closed tightly).
6.2 Apparatus to determine the dry mass of the substrate in accordance with ISO 11465.
6.3 Large scale laboratory mixer for the preparation of the test mixture (5.2).
6.4 Suitable accurate balances.
6.5 Apparatus capable of measuring pH.
6.6 Apparatus to determine water-holding capacity of the substrate (see B.2).
6.7 Exhauster for transfer of springtails (see A.2).
6.8 Test environment.
6.8.1 Enclosure, capable of being controlled to a temperature of (20 ± 2) °C.
6.8.2 Light source, capable of delivering a constant light intensity of 400 Ix to 800 Ix at the substrate
surface at a controlled light: dark cycle of between 12 h:12 h and 16 h:8 h.
7 Procedure
7.1 Experimental design
7.1.1 General
A sample of field-collected soil can be tested at a single concentration (typically 100 %) or evaluated
for toxicity in a multi-concentration test whereby a series of concentrations (dilutions) is prepared
by mixing measured quantities with a control soil (5.2.2). When testing substances, a series of
concentrations is prepared by mixing quantities of the test substance with a standard soil (e.g. artificial
soil). The concentrations being expressed in milligrams of test substance per kilogram of dried control
soil (5.2.2). Depending on the knowledge of relevant response levels a range-finding test may precede
the definitive test. Each definitive test consists of a series of soil mixtures (treatments).
7.1.2 Range-finding test
A test to find the range of contaminated soil mixture ratios (e.g. 0 %, 1 %, 5 %, 25 %, 50 %, 75 %, 100 %)
or concentrations of the test substance (e.g. 0 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, 1 000 mg/kg)
affecting Collembola is optional. The range-finding test is conducted without replication.
When no effects are observed, even at 100 % contaminated soil or at concentrations of 1 000 mg test
substance per kilogram of standard soil (dry mass), the definitive test can be designed as a limit test.
Each test container (replicate) is filled with 30 g wet mass of the test sample. To ensure easy migration
of springtails, the substrate in the test container should not be compressed.
Use 10 specimens of 10 d to 12 d old Collembola per container. Prepare the test containers as indicated
in 7.2.1. Place the test containers in the test enclosure (6.8.1) with the light source (6.8.2).
ISO 11267:2023(E)
At the beginning of the test, add about 2 mg of granulated dry yeast (5.3) to each test container, and
cover the containers tightly (e.g. using plastic, glass discs or parafilm). Open the test containers briefly
twice a week to allow aeration.
After 14 d, count the live Collembola in each container, and determine the percentage mortality for each
test substance concentration. Also, observe surviving Collembola and record any symptoms. Due to the
rapid degradation of dead Collembola, missing Collembola are assumed to have died during the test
period.
NOTE To obtain additional information for the determination of the concentration range for the final test,
the test period can be extended to four weeks to allow qualitative determination of effects at concentrations at
which effects on reproductive output can be expected.
7.1.3 Definitive test
The design of the definitive test depends on the test objectives. Typically, the habitat properties of
samples of a field-collected soil are characterized by comparison of the biological effects found in the
soil to be tested with those found in a reference soil, or if not available or not appropriate due to toxicity
or atypical physicochemical characteristics, in a standard soil. Results for the standard soil assist in
distinguishing contaminant effects from non-contaminant effects caused by soil physicochemical
properties. Regardless of whether a reference soil or standard soil is used for the statistical comparisons,
[20]
the results from standard soil shall be used to judge the validity and acceptability of the test .
If for characterization purposes a test design including dilution series is required, three designs are
possible (the concentrations shall be spaced by a factor not exceeding 2).
— For the NOEC/NOER approach, at least five concentrations in a geometric series should be used.
Four replicates for each concentration plus eight controls are recommended.
— For the ERx/ECx approach, 12 concentrations should be used. Two replicates for each concentration
plus six controls are recommended. The spacing factor can be variable; smaller at low concentrations,
larger at high concentrations.
— For the mixed approach, six concentrations to eight concentrations in a geometric series should
be used. Four replicates for each concentration plus eight controls are recommended. This mixed
approach allows a NOEC as well as an ERx/ECx evaluation.
A limit test can be sufficient if no toxic effect is observed in the range-finding test.
To facilitate checking of the pH and humidity of the test sample, use of additional containers for each
concentration and for the control is recommended.
Each test container (replicate) is filled with 30 g wet mass of the test sample. To ensure easy migration
of Collembola, the substrate in the test container should not be compressed.
7.2 Preparation of test mixture
7.2.1 Testing contaminated soil
According to the selected dilution range, the soil to be tested is mixed with the reference soil or the
standard soil thoroughly (either manually or by using a hand mixer). The homogeneity of the mixture is
checked visually. The total mass of the soil to be tested and the reference soil or the standard soil shall
be 30 g (wet mass) in each test container (6.1). The test mixture shall be wetted with deionized water
to reach 40 % to 60 % of the total water holding capacity determined according to Annex B. In some
cases, for example, when testing waste materials, higher percentages are required. A rough check of the
soil moisture content can be obtained by gently squeezing the soil in the hand; if the moisture content is
correct, small drops of water should appear between the fingers.
Determine the pH for each test mixture (one container per concentration) according to ISO 10390 at the
beginning and end of the test (when acid or basic substances are tested, do not adjust the pH).
ISO 11267:2023(E)
Proceed simultaneously with at least four replicates per concentration and the control(s).
WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic, or
otherwise harmful substances or infectious microorganisms. Occupational health risks can
arise from dust or evaporated substances as well as via dermal contact during handling and
incubation.
7.2.2 Testing substances added to the test substrate
A standard soil (5.2.2) is used to prepare the test sample. For each test container (6.1), the mass of
the substrate used shall be 30 g (wet mass). Substances are added to the test substrate and are mixed
thoroughly.
For the introduction of test substances use either method a), b) or c), as appropriate:
a) Water-soluble substance
— Immediately before starting the test, dissolve the quantity of the test substance in the water or
a portion of it required to wet the soi
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