SIST EN ISO 17512-2:2020

Soil quality - Avoidance test for determining the quality of soils and effects of chemicals on behaviour - Part 2: Test with collembolans (Folsomia candida) (ISO 17512-2:2011)

ISO 17512-2:2011 specifies a rapid screening method for evaluating the habitat function of soils based on the avoidance behaviour of springtails.
The test is a rapid method that reflects the bioavailability of contaminants in natural soils and substances spiked into soils to Folsomia candida. In both cases, it is possible to establish a dose-response-relationship. The avoidance behaviour of the springtails is the measurement endpoint of the test. This test is not intended to replace the Collembola reproduction test.

Bodenbeschaffenheit - Vermeidungsprüfung zur Bestimmung der Bodenbeschaffenheit und der Auswirkungen von Chemikalien auf das Verhalten - Teil 2: Prüfung mit Collembolen (Folsomia candida) (ISO 17512-2:2011)

In diesem Teil von ISO 17512 wird ein Screening-Schnellverfahren zur Bewertung der Lebensraumfunktion von Böden auf der Grundlage des Vermeidungsverhaltens von Collembolen festgelegt.
Die Prüfung ist ein Schnellverfahren, das die Bioverfügbarkeit von Verunreinigungen in natürlichen Böden und von Böden zugesetzten Substanzen für Folsomia candida wiedergibt. Der gemessene Parameter der Prüfung ist das Vermeidungsverhalten der Collembolen. In beiden Fällen ist es möglich, eine Dosis-Wirkungs-Beziehung herzustellen. Es besteht nicht die Absicht, mit dieser Prüfung den Collembolen-Reproduktionstest zu ersetzen.

Qualité du sol - Essai d'évitement pour contrôler la qualité des sols et les effets des produits chimiques sur le comportement - Partie 2: Essai avec des collemboles (Folsomia candida) (ISO 17512-2:2011)

La présente partie de l'ISO 17512 spécifie une méthode de criblage rapide permettant d'évaluer la fonction d'habitat des sols sur la base du comportement d'évitement des collemboles.
Cet essai correspond à une méthode rapide qui reflète la biodisponibilité des polluants dans les sols naturels et des substances introduites dans le sol vis-à-vis de Folsomia candida. Dans les deux cas, il est possible d'établir une relation dose-réponse. Le comportement d'évitement des collemboles constitue le critère d'effet de cet essai. Cet essai n'est pas destiné à remplacer l'essai de reproduction des collemboles.

Kakovost tal - Izogibalni preskus za določanje kakovosti tal in učinkov kemikalij na obnašanje - 2. del: Preskus s Folsomia candida iz rodu skakačev (Collembola) (ISO 17512-2:2011)

General Information

• Status: Published
• Public Enquiry End Date: 02-Feb-2020
• Publication Date: 23-Sep-2020
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 17-Aug-2020
• Due Date: 22-Oct-2020
• Completion Date: 24-Sep-2020
• Ref Project: EN ISO 17512-2:2020 - Soil quality - Avoidance test for determining the quality of soils and effects of chemicals on behaviour - Part 2: Test with collembolans (Folsomia candida) (ISO 17512-2:2011)

Overview

EN ISO 17512-2:2020 - Soil quality - Avoidance test with collembolans (Folsomia candida) provides a rapid screening method to evaluate the habitat function of soils by measuring the avoidance behaviour of springtails. The test reflects the bioavailability of contaminants in natural soils or substances spiked into soils and allows establishment of dose–response relationships. It is a screening tool for soil ecotoxicology and is not intended to replace the Collembola reproduction test.

Key topics and technical requirements

• Test organism: Folsomia candida (springtails) from synchronized laboratory cultures; typical test ages are 10–12 days (juveniles) or 20–22 days (adults).
• Principle: Individuals are given a choice between control soil and test soil in the same vessel; after an incubation period of 2 days the distribution is recorded and avoidance quantified.
• Endpoint: Avoidance behaviour (percentage choosing control vs test soil). Limited habitat function is indicated when >70% of animals are in the control soil.
• Metrics: Calculation of NOEC / LOEC and effective concentrations (ECx, e.g., EC50 for 50% avoidance) and dose–response relationships.
• Test substrate preparation: Natural soils sieved (typically ≤2 mm), moisture-adjusted to about 40–60% of maximum water-holding capacity (WHC); pH and water content determined per ISO methods.
• Apparatus & procedure: Two-section vessels or chambers, standardised rearing techniques, reference substances, and validity criteria are defined. Annexes provide guidance on rearing, chamber design, testing of chemicals, and WHC determination.
• Scope limits: Rapid screening only-does not replace chronic reproduction assays (ISO 11267).

Applications and users

This standard is used for:

• Rapid screening of contaminated or remediated soils for habitat suitability.
• Assessing bioavailability of pollutants (metals, organic contaminants, pesticides) in environmental monitoring.
• Preliminary hazard assessment in contaminated site investigation and risk-based remediation.
• Laboratory ecotoxicology studies to prioritise samples for more detailed chronic testing.

Typical users:

• Environmental testing laboratories and contract ecotoxicology facilities
• Soil scientists and ecotoxicologists
• Remediation consultants and contaminated land specialists
• Regulatory agencies and risk assessors evaluating soil quality and chemical impacts

Related standards

• ISO 17512-1: Avoidance test with earthworms (Eisenia spp.)
• ISO 11267: Collembola reproduction test (Folsomia candida)
• ISO 11268-2: Earthworm reproduction effects
• ISO 11465: Dry matter and water content (gravimetric)
• ISO 15799: Guidance on ecotoxicological characterization of soils

Keywords: EN ISO 17512-2:2020, soil quality, avoidance test, Folsomia candida, collembolans, bioavailability, soil ecotoxicology, NOEC, LOEC, EC50.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2020
Kakovost tal - Izogibalni preskus za določanje kakovosti tal in učinkov kemikalij na
obnašanje - 2. del: Preskus s Folsomia candida iz rodu skakačev (Collembola)
(ISO 17512-2:2011)
Soil quality - Avoidance test for determining the quality of soils and effects of chemicals
on behaviour - Part 2: Test with collembolans (Folsomia candida) (ISO 17512-2:2011)
Bodenbeschaffenheit - Vermeidungsprüfung zur Bestimmung der Bodenbeschaffenheit
und der Auswirkungen von Chemikalien auf das Verhalten - Teil 2: Prüfung mit
Collembolen (Folsomia candida) (ISO 17512-2:2011)
Qualité du sol - Essai d'évitement pour contrôler la qualité des sols et les effets des
produits chimiques sur le comportement - Partie 2: Essai avec des collemboles
(Folsomia candida) (ISO 17512-2:2011)
Ta slovenski standard je istoveten z: EN ISO 17512-2:2020
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 17512-2
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Avoidance test for determining the quality of
soils and effects of chemicals on behaviour - Part 2: Test
with collembolans (Folsomia candida) (ISO 17512-2:2011)
Qualité du sol - Essai d'évitement pour contrôler la Bodenbeschaffenheit - Vermeidungsprüfung zur
qualité des sols et les effets des produits chimiques sur Bestimmung der Bodenbeschaffenheit und der
le comportement - Partie 2: Essai avec des collemboles Auswirkungen von Chemikalien auf das Verhalten -
(Folsomia candida) (ISO 17512-2:2011) Teil 2: Prüfung mit Collembolen (Folsomia candida)
(ISO 17512-2:2011)
This European Standard was approved by CEN on 13 April 2020.

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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17512-2:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 17512-2:2011 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 17512-
2:2020 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 November 2020, and conflicting national standards
shall be withdrawn at the latest by November 2020.
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 17512-2:2011 has been approved by CEN as EN ISO 17512-2:2020 without any
modification.
INTERNATIONAL  ISO
STANDARD 17512-2
First edition
2011-10-01
Soil quality — Avoidance test for
determining the quality of soils and
effects of chemicals on behaviour —
Part 2:
Test with collembolans (Folsomia candida)
Qualité du sol — Essai d’évitement pour contrôler la qualité des sols et
les effets des produits chimiques sur le comportement —
Partie 2: Essai avec des collemboles (Folsomia candida)
Reference number
ISO 17512-2:2011(E)
©
ISO 2011
ISO 17512-2:2011(E)
©  ISO 2011
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2011 – All rights reserved

ISO 17512-2:2011(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure . 4
7.1 Appropriate concentration/dilution range . 4
7.2 Testing of soils . 4
7.3 Reference substance . 4
7.4 Validity criteria . 5
8 Calculation and expression of results . 5
8.1 General . 5
8.2 Calculation of the percentage of avoidance . 5
8.3 NOEC/LOEC calculation . 5
8.4 EC calculation . 6
x
9 Test report . 6
Annex A (informative) Techniques for rearing and breeding of Collembola . 7
Annex B (informative) Test chambers . 9
Annex C (normative) Testing of chemicals in the avoidance test .10
Annex D (normative) Determination of the water-holding capacity .12
Bibliography .13
© ISO 2011 – All rights reserved  iii

ISO 17512-2:2011(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 17512-2 was prepared by Technical Committee ISO/TC 190,  Soil quality, Subcommittee SC 4,
Biological methods.
ISO 17512 consists of the following parts, under the general title Soil quality — Avoidance test for determining
the quality of soils and effects of chemicals on behaviour:
—  Part 1: Test with earthworms (Eisenia fetida and Eisenia andrei)
—  Part 2: Test with collembolans (Folsomia candida)
iv © ISO 2011 – All rights reserved

ISO 17512-2:2011(E)
Introduction
The use of the avoidance behaviour of soil invertebrates as an indicator of unfavourable conditions allows
a preliminary assessment of contaminated soils in a short period of time, with a high degree of sensitivity.
Being rapid, cost-effective and ecologically relevant, the avoidance tests with earthworms were proposed to
complement conventional chemical analysis. Supporting the results obtained in the chronic tests, the avoidance
bioassays can be used as a first screening tool in the assessment of the habitat function of soils. Considering
the fact that the avoidance response of soil invertebrates differs between species due to their distinct sensitivity
to contaminants and modes of exposure, it is recommended to standardize a second rapid cost-effective and
ecologically relevant avoidance bioassay.
Springtails have shown a distinct sensitivity towards several contaminants when compared with earthworms,
[1][2]
complementing the information obtained in the avoidance tests with earthworms . Until now, the species
Folsomia candida has been the most commonly used collembolan test species due to a great facility to keep
[3]
laboratory cultures and due to their high locomotor ability . Folsomia candida is considered to be a hemi-
edaphic species, meaning that it lives mainly in the soil. Furthermore, this species is already used in ISO 11267.
© ISO 2011 – All rights reserved  v

INTERNATIONAL STANDARD ISO 17512-2:2011(E)
Soil quality — Avoidance test for determining the quality
of soils and effects of chemicals on behaviour —
Part 2:
Test with collembolans (Folsomia candida)
1 Scope
This part of ISO 17512 specifies a rapid screening method for evaluating the habitat function of soils based on
the avoidance behaviour of springtails.
The test is a rapid method that reflects the bioavailability of contaminants in natural soils and substances
spiked into soils to Folsomia candida. In both cases, it is possible to establish a dose-response-relationship.
The avoidance behaviour of the springtails is the measurement endpoint of the test. This test is not intended to
replace the Collembola reproduction test.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 10390, Soil quality — Determination of pH
ISO 11267:1999, Soil quality — Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants
ISO 11268-2:1998, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 2: Determination
of effects on reproduction
ISO 11269-2,  Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
contaminated soils on the emergence and early growth of higher plants
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method
ISO 15799, Soil quality — Guidance on the ecotoxicological characterization of soils and soil materials
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
avoidance behaviour
tendency (of an organism) to avoid the test soil while preferring the control soil
[ISO 17512-1:2008]
3.2
test soil
either a natural or an artificial clean soil that is spiked with the test substance or a contaminated natural soil
(a site soil)
3.3
control soil
natural or artificial uncontaminated soil
© ISO 2011 – All rights reserved  1

ISO 17512-2:2011(E)
See 5.3.
3.4
limited habitat function
habitat function is limited if, on average, > 70 % of springtails are found in the control soil (indication of an
impact on behaviour) after they were allowed to choose between the control soil and tested soil
3.5
effective concentration
EC
x
concentration at which a specific effect is detected [where x is a percentage (10, 25, 50) of this effect; e.g. avoidance]
NOTE  In this part of ISO 17512-2, an EC  means the concentration of a test substance, or dilution of a contaminated
test soil, that is estimated to cause an avoidance response of 50 %.
EXAMPLE  An avoidance of 50 % occurs when the number of springtails in the test soil is 50 % of the number that
should be there in the case of no avoidance: no avoidance (control soil = 10 individuals; test soil = 10 individuals); 50 %
avoidance (control soil = 15 individuals; test soil = 5 individuals).
3.6
lowest observed effect concentration
LOEC
lowest tested concentration of a test substance, or dilution of a contaminated test soil, that is observed to cause
a statistically significant avoidance response (p ≤ 0,05)
NOTE 1  In the case of a test substance, the concentration is expressed as mass of the test substance per dry mass of test
substrate; in the case of a contaminated test soil, the concentration is expressed as the percentage dilution of the test soil.
NOTE 2  All tested concentrations/dilutions above the LOEC should have a harmful effect equal to or greater than those
observed at the LOEC. When this condition is not observed, a full explanation should be given for how the LOEC (and
hence the NOEC) has been selected.
3.7
no observed effect concentration
NOEC
test concentration/dilution tested immediately below the LOEC, which causes a not statistically significant
avoidance response (p > 0,05)
4 Principle
Springtails (Folsomia candida) are exposed at the same time to the control soil and the test soil. These soils are
filled into the two sections of the same vessel. After an incubation period of two days, the number of springtails
is determined in each section of the vessels.
5 Reagents and materials
5.1 Biological material, only springtails of the species Folsomia candida (Willem) (see A.2.6) coming from
synchronized cultures should be used.
NOTE  Typically, in this test springtails can be used when they are 10 d to 12 d old (or alternatively adult, e.g. 20 d
to 22 d old).
5.2 Test substrate, in the case of a natural soil, the substrate to be tested should be sieved (2 mm) and
moisture-adjusted to about 40 % to 60 % of the maximum water-holding capacity. If standing water or free water
appears when the soil is compressed before achieving the desired percentage of maximum WHC, a lower
percentage might be used. The optimum water content is achieved if there is no standing water or free water
appearing when the soil is compressed.
2 © ISO 2011 – All rights reserved

ISO 17512-2:2011(E)
NOTE  For highly silty and loamy soils, it can be difficult to get the necessary amount of soil sieved to ≤ 2 mm with
an acceptable expenditure of work. The holes of the sieves might plug up within several minutes. Frequent cleaning is
necessary. In this case, it is acceptable to sieve the amount of soil needed for the test to ≤ 5 mm.
Determine the water content and the pH in the presence of 1 mol/l KCl, in accordance with ISO 11465 and
ISO 10390, respectively, immediately before the start of the test. In addition, the maximum water-holding
capacity shall be determined according to Annex D.
If testing a substance, a different procedure should be followed (see Annex C).
5.3 Control soil, two choices are possible (see also ISO 15799). Either a) a reference soil or b) a standard
soil that allows the presence of springtails.
a)  If reference soils from uncontaminated areas near a contaminated site are available, they should be treated
and characterized like the test soils. If a toxic contamination or unusual soil properties cannot be ruled out,
standard control soils should be preferred.
b)  For testing the effects of substances mixed into soil or making dilutions of the test soil, standard soils (e.g.
LUFA 2.2) shall be used as the 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 and with no visible plant remains 10 %
—  Kaolinite clay containing not less than 30 % Kaolinite 20 %
—  Industrial quartz sand (dominant fine sand with more than 50 % of particle  69 %
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.
Natural soil should be sieved and the water content should be adjusted according to 5.2.
5.4 Reference substance, having Phenmedipham as the only active ingredient.
6 Apparatus
Usual laboratory equipment and the following.
6.1 Containers (see Annex B).
Cylindrical containers of capacity 200 ml to 300 ml with a cross-sectional area of about 50 cm, such that a
depth of 3 cm to 4 cm of soil is achieved.
Test containers shall permit gaseous exchange between the medium and the atmosphere and access of
light (e.g. by means of a perforated transparent cover), and shall have provisions to prevent springtails from
escaping (e.g. by using a tape to fix the cover). To avoid lateral effects of light, test vessels should be made of
opaque material, otherwise they should be wrapped with aluminium foil.
NOTE  Due to the short test period and the proportionally large volume of soil in the vessels (considering the small
amount of soil needed by the springtails), a reduction of the chemical concentration in the soil resulting from sorption to
the vessel walls is negligible. Therefore, plastic vessels can be used, although, when available, the use of inert material
(e.g. glass or stainless steel) is preferred.
6.2 Divider, made of plastic or thin sheets of metal. It shall divide the test containers vertically into two
identical sections.
© ISO 2011 – All rights reserved  3

ISO 17512-2:2011(E)
6.3 Equipment for measuring the water content of a substrate (in accordance with ISO 11465).
6.4 Apparatus, for measuring the pH of the substrate.
6.5 Exhaustor, to transfer the springtails (see A.3).
6.6 Test environment.
6.6.1 Enclosure or environmental chamber, capable of being maintained at (20 ± 2) °C.
6.6.2 Light source, capable of delivering a constant light intensity of 400 lx to 800 lx on the containers at a
controlled light/dark cycle of between 12 h:12 h and 16 h:8 h.
7 Procedure
7.1 Appropriate concentration/dilution range
The avoidance test is designed to detect sublethal effects. Therefore, the test is invalid if more than 20 % of
the total number of springtails are dead or missing at the end of the test (see 7.4). In order to avoid mortality,
a range-finding test should be performed in those cases when testing a substance or a dilution gradient of
contaminated natural soil.
...