SIST EN ISO 23266:2021

SLOVENSKI STANDARD
SIST EN ISO 23266:2021
01-julij-2021
Kakovost tal - Preskus zaviranja razmnoževanja pršic (Oppia nitens) zaradi
izpostavljenosti onesnaževalom v tleh (ISO 23266:2020)
Soil quality - Test for measuring the inhibition of reproduction in oribatid mites (Oppia
nitens) exposed to contaminants in soil (ISO 23266:2020)
Bodenbeschaffenheit - Verfahren zur Bestimmung der Wirkungen von verunreinigten
Böden auf das Überleben und die Reproduktion von Hornmilben (Oppia nitens) (ISO
23266:2020)
Qualité du sol - Essai de détermination de l'inhibition de la reproduction chez les
acariens oribates (Oppia nitens) exposés aux contaminants dans le sol (ISO
23266:2020)
Ta slovenski standard je istoveten z: EN ISO 23266:2021
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 23266:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 23266:2021

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SIST EN ISO 23266:2021


EN ISO 23266
EUROPEAN STANDARD

NORME EUROPÉENNE

April 2021
EUROPÄISCHE NORM
ICS 13.080.30
English Version

Soil quality - Test for measuring the inhibition of
reproduction in oribatid mites (Oppia nitens) exposed to
contaminants in soil (ISO 23266:2020)
Qualité du sol - Essai de détermination de l'inhibition Bodenbeschaffenheit - Verfahren zur Bestimmung der
de la reproduction chez les acariens oribates (Oppia Wirkungen von verunreinigten Böden auf das
nitens) exposés aux contaminants dans le sol (ISO Überleben und die Reproduktion von Hornmilben
23266:2020) (Oppia nitens) (ISO 23266:2020)
This European Standard was approved by CEN on 5 April 2021.

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, Turkey 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 23266:2021 E
worldwide for CEN national Members.

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SIST EN ISO 23266:2021
EN ISO 23266:2021 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 23266:2021
EN ISO 23266:2021 (E)
European foreword
The text of ISO 23266:2020 has been prepared by Technical Committee ISO/TC 190 "Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 23266:2021 by
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 October 2021, and conflicting national standards shall
be withdrawn at the latest by October 2021.
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.
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, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 23266:2020 has been approved by CEN as EN ISO 23266:2021 without any modification.

3

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SIST EN ISO 23266:2021

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SIST EN ISO 23266:2021
INTERNATIONAL ISO
STANDARD 23266
First edition
2020-06
Soil quality — Test for measuring the
inhibition of reproduction in oribatid
mites (Oppia nitens) exposed to
contaminants in soil
Qualité du sol — Essai de détermination de l'inhibition de la
reproduction chez les acariens oribates (Oppia nitens) exposés aux
contaminants dans le sol
Reference number
ISO 23266:2020(E)
©
ISO 2020

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 4
5 Reagents and material . 4
6 Apparatus . 7
7 Procedure. 7
7.1 Experimental design . 7
7.1.1 General. 7
7.1.2 Range-finding test (preliminary test) . 7
7.1.3 Definitive test . 8
7.2 Preparation of test mixture . 8
7.2.1 Testing contaminated soil . 8
7.2.2 Testing substances added to the test substrate . 9
7.2.3 Preparation of control containers .10
7.3 Addition of the mites .10
7.4 Test conditions and measurements .10
7.5 Determination of adult survival and reproductive output .10
8 Calculation and expression of results .11
8.1 Calculation .11
8.2 Expression of results .11
9 Validity of the test .11
10 Statistical analysis .12
10.1 General .12
10.2 Single-concentration tests .12
10.3 Multi-concentration tests.12
10.3.1 Range-finding test .12
10.3.2 Definitive test .12
11 Test report .13
Annex A (informative) Techniques for rearing and breeding of Oppia nitens .15
Annex B (normative) Determination of water holding capacity .18
Annex C (informative) Guidance on adjustment of pH of artificial soil .19
Annex D (informative) Extraction and counting of Oppia nitens .20
Annex E (informative) Performance of the method .24
Bibliography .29
© ISO 2020 – All rights reserved iii

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SIST EN ISO 23266:2021
ISO 23266:2020(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 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.
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 2020 – All rights reserved

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil
[1] [2]
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 with regards to supporting soil biota and interactions within.
Mites (Acari) are a world-wide and diverse group of arthropods belonging to a sub-class of Arachnida
with over 40 000 species recorded, divided into two super-orders (Acariformes and Parasitiformes).
Due to their relative small size (a few μm to a few cm), they occupy specific ecological niches on plants
[5]
as well as in soils .
In recent years, there has been an increase in the development of biological test methods for assessing
contaminated soil, which has historically lagged behind that for other media (e.g., water and sediment).
Ecotoxicology tests for soil are challenged, among other things, by the complexity of soil systems
(e.g., lack of homogeneity) and the variety of exposure routes (e.g., via alimentary uptake, exposure
to pore water or soil vapours, or direct contact with soil particles). A recently developed method
[3]
(ISO 21285 ) assesses the effects of contaminated soil on the reproduction of the predatory mite
(Hypoaspis aculeifer), mainly through alimentary uptake. Oribatid mites represent a different but
essential ecological niche than H. aculeifer within soil, contributing to carbon mineralization and soil
formation, as well as nitrogen and phosphorous release through grazing activities. Oribatid mites are
among the most diverse and abundant micro-arthropod species within soil, however, their slower
metabolism and development, coupled with low fecundity, long life span, and limited dispersal capacity
[6]
increase the potential for susceptibility and sensitivity to short- and long-term disturbances . The
[7][8][9]
use of oribatid mites in the context of soil ecotoxicology testing has been thoroughly reviewed
[10][11]
. Recent research using Oppia nitens for soil testing has demonstrated applicability and relative
sensitivity of the species for the assessment of contaminated soils from both agronomic regions, and
[6][12][13][14][15]
those from the boreal and taiga ecozones . Research has also demonstrated its sensitivity
[16][17][18][19] [20][21] [16][17][22]
to metals , pesticides , and organic compounds . Oppia nitens is an oribatid
mite, inhabiting the upper organic layer of soil, and is a member of the largest oribatid family (Oppiidae)
with approximately 1 000 species in 129 genera widely distributed throughout Holarctic and Antarctic
[23]
regions . They are sexually reproducing, polyphagous fungivores that can be easily reared in the
[10]
laboratory in soil or on plaster of Paris, and on a diet of Baker’s yeast .
This method outlines procedures for conducting 28-day tests for determining the effects of
contaminated soils on the survival and reproduction of the oribatid mite, Oppia nitens. Optionally, the
method can be used for testing chemicals added to standard soils (e.g., artificial soil) for their lethal
and sublethal hazard potential to oribatid mites. The performance of this method has been assessed
[15]
in an international inter-laboratory investigation , as summarized in Annex E. Mites represent
communities that cannot be omitted from environmental hazard assessment. This species is considered
to be representative of non-predatory soil mites.
© ISO 2020 – All rights reserved v

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SIST EN ISO 23266:2021

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SIST EN ISO 23266:2021
INTERNATIONAL STANDARD ISO 23266:2020(E)
Soil quality — Test for measuring the inhibition of
reproduction in oribatid mites (Oppia nitens) exposed to
contaminants in soil
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
effects of soil contaminants and individual chemical substances on the reproduction of the oribatid
mite Oppia nitens by dermal and alimentary uptake. This chronic (28-day) test is applicable to soils
and soil materials of unknown quality (e.g., contaminated sites, amended soils, soils after remediation,
agricultural or other sites under concern and waste materials). This method is not intended to replace
the earthworm or Collembola tests since it represents another taxonomic group (= mites; i.e., arachnids),
nor the predatory mite test since this species represents a different trophic level and ecological niche.
Effects of substances are assessed using 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 with similar properties to the soil sample to be tested (reference soil) or
a standard soil (e.g., artificial soil).
Information is provided on how to use this method for testing substances under temperate conditions.
This document is not applicable to substances for which the air/soil partition coefficient is greater than
1, or to substances with vapour pressure exceeding 300 Pa at 25 °C.
NOTE The stability of the test substance cannot be assured 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 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 quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level
using barium chloride solution
ISO 11265, Soil quality — Determination of the specific electrical conductivity
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 2020 – All rights reserved 1

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

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 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
substance or agent present in the soil as a result of human activity
3.2
effect concentration
ECx
concentration (mass fraction) of a test sample or 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
effect rate
ERx
rate of a soil to be tested that causes an x % of an effect on a given end-point within a given exposure
period when compared with a control
3.4
lethal concentration
LCx
concentration (mass fraction) of a test sample or test substance that causes x % mortality within a
given exposure period when compared with a control
EXAMPLE An LC50 is a concentration estimated to cause mortality in 50 % of an exposed population over a
defined exposure period.
Note 1 to entry: The LCx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).
When substances are tested, the LCx is expressed as mass of the test substance per dry mass of soil in milligrams
per kilogram.
3.5
limit test
single concentration test consisting of at least five replicates each, the test soil (3.14) without any
dilution or the highest concentration of test substance mixed into the control soil and the control
2 © ISO 2020 – All rights reserved

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

3.6
lowest observed effect concentration
LOEC
lowest tested concentration (mass fraction) of a test sample or test substance that has a statistically
significant effect (probability p < 0,05)
Note 1 to entry: In this test, the LOEC is expressed as a percentage of soil to be tested (dry mass) per soil mixture
(dry mass) or 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.
3.7
lowest observed effect rate
LOER
lowest rate of a soil to be tested in a control soil at which a statistically significant effect is observed
3.8
no observed effect concentration
NOEC
highest tested concentration (mass fraction) of a test sample or test substance immediately below the
LOEC at which no effect is observed
Note 1 to entry: In this test, the concentration corresponding to the NOEC, has no statistically significant effect
(probability p < 0,05) within a given exposure period when compared with the control.
3.9
no observed effect rate
NOER
lowest rate of a soil to be tested immediately below the LOER which, when compared to the control, has
no statistically significant effect (probability p < 0,05) within a given exposure period
3.10
reproduction
mean number of offspring per test vessel after a 28-day incubation under the specified test conditions
3.11
reference soil
uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic
carbon content and texture) to the test soil (3.14)
3.12
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 those of the test soil.
3.13
control soil
reference or standard soil (3.12) used as a control and as a medium for preparing dilution series with
test soils/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.
3.14
test soil
sample of field-collected soil or chemical-spiked soil to be evaluated for toxicity to mites
© ISO 2020 – All rights reserved 3

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SIST EN ISO 23266:2021
ISO 23266:2020(E)

3.15
test mixture
mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil (3.13)
Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.
3.16
test mixture ratio
ratio of test soil to control soil in a test mixture (3.15)
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, laboratory-cultured mites, Oppia nitens, exposed to the test soil
are compared to those observed for organisms in 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 during the test period.
The test is started with 15 adult mites from age-synchronized cultures (aged 8 to 10 d after ecdysis
(i.e., moult) to adult form) per test vessel. The test is performed in 30 mL glass vessels with a wet-
weight equivalent to a volume of ~20 mL of soil, and a minimum of five replicates are prepared for
each treatment. The test runs for 28 d at (20 ± 2) °C by which time offspring (F ) have emerged from
1
eggs laid by the adults and the number of offspring produced is determined. Survival of the adults is
also determined at the end of the test. The results obtained from the tests are compared with a control
or, where a serial dilution design is used, to determine the concentration resulting in x % reduction
of juveniles produced compared to the control (ECx, 28 d), depending on the experimental design. An
estimate of the test conc
...

SLOVENSKI STANDARD
oSIST prEN ISO 23266:2020
01-december-2020
Kakovost tal - Preskus zaviranja razmnoževanja pršic (Oppia nitens) zaradi
izpostavljenosti onesnaževalom v tleh (ISO 23266:2020)
Soil quality - Test for measuring the inhibition of reproduction in oribatid mites (Oppia
nitens) exposed to contaminants in soil (ISO 23266:2020)
Bodenbeschaffenheit - Verfahren zur Bestimmung der Wirkungen von verunreinigten
Böden auf das Überleben und die Reproduktion von Hornmilben (Oppia nitens) (ISO
23266:2020)
Qualité du sol - Essai de détermination de l'inhibition de la reproduction chez les
acariens oribates (Oppia nitens) exposés aux contaminants dans le sol (ISO
23266:2020)
Ta slovenski standard je istoveten z: prEN ISO 23266
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 23266:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 23266:2020

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oSIST prEN ISO 23266:2020
INTERNATIONAL ISO
STANDARD 23266
First edition
2020-06
Soil quality — Test for measuring the
inhibition of reproduction in oribatid
mites (Oppia nitens) exposed to
contaminants in soil
Qualité du sol — Essai de détermination de l'inhibition de la
reproduction chez les acariens oribates (Oppia nitens) exposés aux
contaminants dans le sol
Reference number
ISO 23266:2020(E)
©
ISO 2020

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oSIST prEN ISO 23266:2020
ISO 23266:2020(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2020
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

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oSIST prEN ISO 23266:2020
ISO 23266:2020(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 4
5 Reagents and material . 4
6 Apparatus . 7
7 Procedure. 7
7.1 Experimental design . 7
7.1.1 General. 7
7.1.2 Range-finding test (preliminary test) . 7
7.1.3 Definitive test . 8
7.2 Preparation of test mixture . 8
7.2.1 Testing contaminated soil . 8
7.2.2 Testing substances added to the test substrate . 9
7.2.3 Preparation of control containers .10
7.3 Addition of the mites .10
7.4 Test conditions and measurements .10
7.5 Determination of adult survival and reproductive output .10
8 Calculation and expression of results .11
8.1 Calculation .11
8.2 Expression of results .11
9 Validity of the test .11
10 Statistical analysis .12
10.1 General .12
10.2 Single-concentration tests .12
10.3 Multi-concentration tests.12
10.3.1 Range-finding test .12
10.3.2 Definitive test .12
11 Test report .13
Annex A (informative) Techniques for rearing and breeding of Oppia nitens .15
Annex B (normative) Determination of water holding capacity .18
Annex C (informative) Guidance on adjustment of pH of artificial soil .19
Annex D (informative) Extraction and counting of Oppia nitens .20
Annex E (informative) Performance of the method .24
Bibliography .29
© ISO 2020 – All rights reserved iii

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oSIST prEN ISO 23266:2020
ISO 23266:2020(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 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.
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 2020 – All rights reserved

---------------------- Page: 6 ----------------------
oSIST prEN ISO 23266:2020
ISO 23266:2020(E)

Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil
[1] [2]
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 with regards to supporting soil biota and interactions within.
Mites (Acari) are a world-wide and diverse group of arthropods belonging to a sub-class of Arachnida
with over 40 000 species recorded, divided into two super-orders (Acariformes and Parasitiformes).
Due to their relative small size (a few μm to a few cm), they occupy specific ecological niches on plants
[5]
as well as in soils .
In recent years, there has been an increase in the development of biological test methods for assessing
contaminated soil, which has historically lagged behind that for other media (e.g., water and sediment).
Ecotoxicology tests for soil are challenged, among other things, by the complexity of soil systems
(e.g., lack of homogeneity) and the variety of exposure routes (e.g., via alimentary uptake, exposure
to pore water or soil vapours, or direct contact with soil particles). A recently developed method
[3]
(ISO 21285 ) assesses the effects of contaminated soil on the reproduction of the predatory mite
(Hypoaspis aculeifer), mainly through alimentary uptake. Oribatid mites represent a different but
essential ecological niche than H. aculeifer within soil, contributing to carbon mineralization and soil
formation, as well as nitrogen and phosphorous release through grazing activities. Oribatid mites are
among the most diverse and abundant micro-arthropod species within soil, however, their slower
metabolism and development, coupled with low fecundity, long life span, and limited dispersal capacity
[6]
increase the potential for susceptibility and sensitivity to short- and long-term disturbances . The
[7][8][9]
use of oribatid mites in the context of soil ecotoxicology testing has been thoroughly reviewed
[10][11]
. Recent research using Oppia nitens for soil testing has demonstrated applicability and relative
sensitivity of the species for the assessment of contaminated soils from both agronomic regions, and
[6][12][13][14][15]
those from the boreal and taiga ecozones . Research has also demonstrated its sensitivity
[16][17][18][19] [20][21] [16][17][22]
to metals , pesticides , and organic compounds . Oppia nitens is an oribatid
mite, inhabiting the upper organic layer of soil, and is a member of the largest oribatid family (Oppiidae)
with approximately 1 000 species in 129 genera widely distributed throughout Holarctic and Antarctic
[23]
regions . They are sexually reproducing, polyphagous fungivores that can be easily reared in the
[10]
laboratory in soil or on plaster of Paris, and on a diet of Baker’s yeast .
This method outlines procedures for conducting 28-day tests for determining the effects of
contaminated soils on the survival and reproduction of the oribatid mite, Oppia nitens. Optionally, the
method can be used for testing chemicals added to standard soils (e.g., artificial soil) for their lethal
and sublethal hazard potential to oribatid mites. The performance of this method has been assessed
[15]
in an international inter-laboratory investigation , as summarized in Annex E. Mites represent
communities that cannot be omitted from environmental hazard assessment. This species is considered
to be representative of non-predatory soil mites.
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oSIST prEN ISO 23266:2020
INTERNATIONAL STANDARD ISO 23266:2020(E)
Soil quality — Test for measuring the inhibition of
reproduction in oribatid mites (Oppia nitens) exposed to
contaminants in soil
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
effects of soil contaminants and individual chemical substances on the reproduction of the oribatid
mite Oppia nitens by dermal and alimentary uptake. This chronic (28-day) test is applicable to soils
and soil materials of unknown quality (e.g., contaminated sites, amended soils, soils after remediation,
agricultural or other sites under concern and waste materials). This method is not intended to replace
the earthworm or Collembola tests since it represents another taxonomic group (= mites; i.e., arachnids),
nor the predatory mite test since this species represents a different trophic level and ecological niche.
Effects of substances are assessed using 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 with similar properties to the soil sample to be tested (reference soil) or
a standard soil (e.g., artificial soil).
Information is provided on how to use this method for testing substances under temperate conditions.
This document is not applicable to substances for which the air/soil partition coefficient is greater than
1, or to substances with vapour pressure exceeding 300 Pa at 25 °C.
NOTE The stability of the test substance cannot be assured 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 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 quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level
using barium chloride solution
ISO 11265, Soil quality — Determination of the specific electrical conductivity
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
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ISO 23266:2020(E)

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 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
substance or agent present in the soil as a result of human activity
3.2
effect concentration
ECx
concentration (mass fraction) of a test sample or 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
effect rate
ERx
rate of a soil to be tested that causes an x % of an effect on a given end-point within a given exposure
period when compared with a control
3.4
lethal concentration
LCx
concentration (mass fraction) of a test sample or test substance that causes x % mortality within a
given exposure period when compared with a control
EXAMPLE An LC50 is a concentration estimated to cause mortality in 50 % of an exposed population over a
defined exposure period.
Note 1 to entry: The LCx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).
When substances are tested, the LCx is expressed as mass of the test substance per dry mass of soil in milligrams
per kilogram.
3.5
limit test
single concentration test consisting of at least five replicates each, the test soil (3.14) without any
dilution or the highest concentration of test substance mixed into the control soil and the control
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3.6
lowest observed effect concentration
LOEC
lowest tested concentration (mass fraction) of a test sample or test substance that has a statistically
significant effect (probability p < 0,05)
Note 1 to entry: In this test, the LOEC is expressed as a percentage of soil to be tested (dry mass) per soil mixture
(dry mass) or 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.
3.7
lowest observed effect rate
LOER
lowest rate of a soil to be tested in a control soil at which a statistically significant effect is observed
3.8
no observed effect concentration
NOEC
highest tested concentration (mass fraction) of a test sample or test substance immediately below the
LOEC at which no effect is observed
Note 1 to entry: In this test, the concentration corresponding to the NOEC, has no statistically significant effect
(probability p < 0,05) within a given exposure period when compared with the control.
3.9
no observed effect rate
NOER
lowest rate of a soil to be tested immediately below the LOER which, when compared to the control, has
no statistically significant effect (probability p < 0,05) within a given exposure period
3.10
reproduction
mean number of offspring per test vessel after a 28-day incubation under the specified test conditions
3.11
reference soil
uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic
carbon content and texture) to the test soil (3.14)
3.12
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 those of the test soil.
3.13
control soil
reference or standard soil (3.12) used as a control and as a medium for preparing dilution series with
test soils/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.
3.14
test soil
sample of field-collected soil or chemical-spiked soil to be evaluated for toxicity to mites
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3.15
test mixture
mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil (3.13)
Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.
3.16
test mixture ratio
ratio of test soil to control soil in a test mixture (3.15)
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, laboratory-cultured mites, Oppia nitens, exposed to the test soil
are compared to those observed for organisms in 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 during the test period.
The test is started with 15 adult mites from age-synchronized cultures (aged 8 to 10 d after ecdysis
(i.e., moult) to adult form) per test vessel. The test is performed in 30 mL glass vessels with a wet-
weight equivalent to a volume of ~20 mL of soil, and a minimum of five replicates are prepared for
each treatment. The test runs for 28 d at (20 ± 2) °C by which time offspring (F ) have emerged from
1
eggs laid by the adults and the number of offspring produced is determined. Survival of the adults is
also determined at the end of the test. The results obtained from the tests are compared with a control
or, where a serial dilution design is used, to determine the concentration resulting in x % reduction
of juveniles produced compared to the control (ECx, 28 d), depending on the experimental design. An
estimate of the test concentration resulting in x % mortality (LCx, 28 d) is an optional test endpoint. If a
multi-concentration hypothesis test design is used, the reproductive output of the mites exposed to the
test mixtures is compared to that of the controls in order to determine the concentration which causes
no effects on mortality and reproduction (NOEC).
In cases where there is no prior knowledge of the dilution/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:
— a range-finding test is carried out to give an indication of the effect dilution/concentration, and
the dilution/concentration resulting in no mortality. Dilutions/concentrations to be used in the
definitive test can then be selected; and
— the definitive test to determine lethal and sub-lethal effects of (dilutions of) contaminated soil or
the concentration of a substance which, when evenly mixed into the standard soil, results in: 1)
x % inhibition of reproduction, ECx (e.g., EC10, EC20, or EC50), or 2) causes no significant effects
on numbers of offspring hatched from eggs compared with the control for estimation of the NOEC
and LOEC.
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, adult mites, Oppia nitens C.L. Koch 1836, aged 8 to 10 d (i.e., 8 to 10-d post-ecdysis to adult
form), established from newly emerged adults collected over a 1- to 3-d period, are required to start the
test. The mites shall be selected from an age-synchronised culture. A method for culturing Oppia nitens
and for obtaining age-synchronised test organisms is provided in Annex A.
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Adult Oppia nitens are obtained from laboratory cultures maintained under conditions of temperature,
photoperiod, and food similar to those in the test. Species identification should be confirmed by
qualified personnel experienced in identifying soil mites using the distinguishing taxonomic features,
[26]
described in taxonomic keys , or using DNA-based taxonomic identification (i.e., barcoding) as
[4]
outlined in ISO 21286 . All mites used in a test shall be derived from the same population and source.
Sources of animals to be used to establish cultures include government or private laboratories that are
[24]
culturing Oppia nitens for soil toxicity tests, or commercial biological suppliers .
5.2 Test mixture can consist of field-collected soil or control soil mixed with the test soil or spiked
with 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 works, plant-
derived compost, or manure) under consideration for possible land disposal.
The field-collected soils used in this test shall be passed through a sieve of 4 to 10 mm square mesh
to remove coarse fragments and thoroughly mixed. If necessary, soil may be air-dried without
heating before sieving. Storage of the test soil 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.
NOTE A 4-mm mesh sieve is appropriate for use with any mineral-based soil with relatively low organic
matter (e.g., agricultural soil), however for forest soils or wetland soils with higher organic matter content, sieves
with larger mesh sizes (e.g., 6 to 8 mm for forest soils and 8 to 10 mm for wetland soils) could be requ
...