SIST EN ISO 17512-2:2020
(Main)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)
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
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 17512-2:2020
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
SIST EN ISO 17512-2:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 17512-2:2020
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SIST EN ISO 17512-2:2020
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.
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SIST EN ISO 17512-2:2020
EN ISO 17512-2:2020 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 17512-2:2020
EN ISO 17512-2:2020 (E)
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.
3
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SIST EN ISO 17512-2:2020
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SIST EN ISO 17512-2:2020
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
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SIST EN ISO 17512-2:2020
ISO 17512-2:2011(E)
COPYRIGHT PROTECTED DOCUMENT
© 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 copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2011 – All rights reserved
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SIST EN ISO 17512-2:2020
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
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SIST EN ISO 17512-2:2020
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
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SIST EN ISO 17512-2:2020
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
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SIST EN ISO 17512-2:2020
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SIST EN ISO 17512-2:2020
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
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SIST EN ISO 17512-2:2020
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
50
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
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SIST EN ISO 17512-2:2020
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
3
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).
2
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.
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SIST EN ISO 17512-2:2020
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.
7.2 Testing of soils
At the beginning of the test, the containers (6.1) are divided into two equal sections by means of a vertically
introduced divider (6.2). Vessels are filled with sieved soil up to a height of 3 cm to 4 cm (±30 g wet mass per
side; the soil is freshly moistened and should not be pressed). One half of the vessel is filled with test soil
(section A), the other half is filled with control soil (section B). Then the divider is removed and 20 springtails
are placed on top of the line separating the two soils on each test vessel. The containers are covered according
to 6.1 and placed into the environmental chamber or in the enclosure (6.6.1).
No feeding of the animals is required during the test.
The test runs with five replicates. To facilitate checking of the pH and humidity of the test substrates at the end of
the test period, the use of an additional container without animals for each tested combination is recommended.
To obtain a more precise quantification of the behavioural effect, a dilution series may be prepared. For dilution
of the contaminated soil, the control soil should be used.
At the end of the test period (48 h) the control and test soils in each vessel are separated by inserting the
divider. It shall be inserted before the test units are moved from the environmental chamber. One section of the
test container is emptied (alternately the control and the treated soil sections to avoid systematic errors) into a
small vessel followed by the removal of the divider. Once separated, both soils are flooded with water and, after
the addition of a few drops of ink and gentle stirring with a spatula, the animals floating on the water surface
are counted. Missing springtails are considered as dead organisms.
NOTE Although some studies had shown that Folsomia candida can have an avoidance reaction before 48 h of exposure
[4][5][6]
for some substances , the use of 48 h as the test period for avoidance tests with springtails is still recommended.
7.3 Reference substance
Substances having Phenmedipham as the only active ingredient (5.4) are recommended as reference toxicants
for this test to be used in a positive control treatment. Springtails can detect and avoid sublethal concentrations
[4][7]
that adversely affect reproduction . A significant avoidance behaviour response (see Clause 8) should be
obtained in a range of 5 mg to 20 mg reference toxicant (5.4) per kilogram of soil measured on the dry-mass
[2]
basis when artificial soil is used .
4 © ISO 2011 – All rights reserved
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SIST EN ISO 17512-2:2020
ISO 17512-2:2011(E)
Alternatively, boric acid can be used as a reference substance. A significant avoidance-behaviour response
(see Clause 8) should be obtained in a range of 700 mg to 2 800 mg of boric acid per kilogram of soil measured
[10]
on a dry-mass basis when artificial soil is used .
WARNING — When handling these chemicals, appropriate precautions should be taken to avoid ingestion or skin contact.
7.4 Validity criteria
The test is invalid if the number of dead or missing springtails is > 20 % per treatment.
To validate the test set, check the homogeneity of distribution of the springtails. For this purpose, test vessels
with the control soil on both sections shall be prepared and an even distribution of the springtails among both
sections should be observed. On average, the number of springtails in one section should be within the range
of 40 % to 60 % of the total number of animals present.
8 Calculation and expression of results
8.1 General
The mean plus or minus standard deviation of live individuals in the test soil is determined for each treatment
at the end of the test. The results are presented as the number of individuals in the test soil per test vessel.
If the test soil and the control soil differ only regarding the contamination, statistical calculations may be
performed as follows.
8.2 Calculation of the percentage of avoidance
For calculation of the percentage effect per concentration of the tested substance or per soil dilution (in the
case of contaminated natural soil), the number of springtails in the test soil is compared with the number of
springtails in the control soil in accordance with Equation (1).
nn−
ct
x = ×100 (1)
N
where
x is the avoidance, expressed as a percentage;
n is the number of springtails in the control soil (either per vessel or in the control soil of all replicates);
c
n is the number of springtails in the test soil (either per vessel or in the test soil of all replicates);
t
N is the total number of springtails (usually 20 per vessel, or that in all replicates).
A neutral response (0 % avoidance) is considered when an equal number of individuals is found in both soils.
Negative responses (when the springtails prefer the test soil) are considered as 0 % of avoidance.
8.3 NOEC/LOEC calculation
For a single concentration or a single dilution test, the statistical significance of the avoidance response can be
evaluated using the Fisher’s exact test or another statistic appropriate for comparing observed with predicted
[9]
distributions . With this test, comparisons between the observed and a theoretical distribution (assuming
a “no avoidance” situation) of the organisms in the test soil are performed. What is tested is an avoidance
response towards the test soil, and the null hypothesis assumes a situation of no avoidance. Results showing
a statistically significant (p < 0,05) lower number of surviving springtails in the test soil, relative to the expected
number in case of no avoidance, indicate an avoidance response towards the test soil. LOEC and NOEC
values can be evaluated along a gradient of dilutions of a contaminated test soil, or a gradient of concentrations
of a test substance can be determined by analysing the significance of each single Fisher’s exact test.
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SIST EN ISO 17512-2:2020
ISO 17512-2:2011(E)
8.4 EC calculation
x
The desired EC shall be obtained through Probit regressions, taking into account the percentage of
...
SLOVENSKI STANDARD
oSIST prEN ISO 17512-2:2020
01-januar-2020
[Not translated]
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 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: prEN ISO 17512-2
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
oSIST prEN ISO 17512-2:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 17512-2:2020
---------------------- Page: 2 ----------------------
oSIST prEN ISO 17512-2:2020
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
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oSIST prEN ISO 17512-2:2020
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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
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
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)
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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.
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oSIST prEN ISO 17512-2:2020
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
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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
50
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.
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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
3
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).
2
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.
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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.
7.2 Testing of soils
At the beginning of the test, the containers (6.1) are divided into two equal sections by means of a vertically
introduced divider (6.2). Vessels are filled with sieved soil up to a height of 3 cm to 4 cm (±30 g wet mass per
side; the soil is freshly moistened and should not be pressed). One half of the vessel is filled with test soil
(section A), the other half is filled with control soil (section B). Then the divider is removed and 20 springtails
are placed on top of the line separating the two soils on each test vessel. The containers are covered according
to 6.1 and placed into the environmental chamber or in the enclosure (6.6.1).
No feeding of the animals is required during the test.
The test runs with five replicates. To facilitate checking of the pH and humidity of the test substrates at the end of
the test period, the use of an additional container without animals for each tested combination is recommended.
To obtain a more precise quantification of the behavioural effect, a dilution series may be prepared. For dilution
of the contaminated soil, the control soil should be used.
At the end of the test period (48 h) the control and test soils in each vessel are separated by inserting the
divider. It shall be inserted before the test units are moved from the environmental chamber. One section of the
test container is emptied (alternately the control and the treated soil sections to avoid systematic errors) into a
small vessel followed by the removal of the divider. Once separated, both soils are flooded with water and, after
the addition of a few drops of ink and gentle stirring with a spatula, the animals floating on the water surface
are counted. Missing springtails are considered as dead organisms.
NOTE Although some studies had shown that Folsomia candida can have an avoidance reaction before 48 h of exposure
[4][5][6]
for some substances , the use of 48 h as the test period for avoidance tests with springtails is still recommended.
7.3 Reference substance
Substances having Phenmedipham as the only active ingredient (5.4) are recommended as reference toxicants
for this test to be used in a positive control treatment. Springtails can detect and avoid sublethal concentrations
[4][7]
that adversely affect reprodu
...
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