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) (ISO 17512-1:2008)

ISO 17512-1:2008 specifies a rapid screening method for evaluating the habitat function of soils and the influence of contaminants and chemicals on earthworm behaviour.
The sublethal test is a rapid method that reflects the bioavailability of contaminant mixtures in natural soils and substances spiked into soils to Eisenia fetida and Eisenia andrei. The avoidance behaviour of the worms is the measurement endpoint of the test. This test is not intended to replace the earthworm reproduction test.
Two different designs (a two section unit and a six section unit) have been developed and successfully applied. Both designs are applicable to either single-concentration (e. g. for assessing the quality of a field soil) or multi-concentration (e. g. for assessing the toxicity of a spiked chemical) tests. In both cases, the earthworms are allowed to make the initial choice on which compartment, control and a treatment [in the two section test vessel between right and left side; in the six section test vessel between the (3 + 3) alternating compartments], to enter.

Bodenbeschaffenheit - Vermeidungsprüfung zur Bestimmung der Bodenbeschaffenheit und der Auswirkungen von Chemikalien auf das Verhalten - Teil 1: Prüfung von Regenwürmern (Eisenia fetida und Eisenia andrei) (ISO 17512-1:2008)

Dieser Teil von  ISO 17512 legt ein Screening-Schnellverfahren zur Bewertung der Lebensraumfunktion von Böden und des Einflusses von Verunreinigungen und Chemikalien auf das Verhalten von Regenwürmern fest.
Die subletale Prüfung ist ein Schnellverfahren, das die Bioverfügbarkeit von Gemischen aus Verunreinigungen in natürlichen Böden und von in Böden eingemischten Substanzen für Eisenia fetida und Eisenia andrei wiedergibt. Das Vermeidungsverhalten der Würmer stellt den Endpunkt der Prüfung dar. Die Prüfung ist nicht als Ersatz für den Regenwurm Reproduktionstest vorgesehen.
Zwei unterschiedliche Versuchsanordnungen (ein Zweikammerprüfgefäß und ein Sechskammerprüfgefäß) wurden entwickelt und erfolgreich eingesetzt. Beide Versuchsanordnungen sind sowohl für Prüfungen auf Einzelkonzentrationen (z. B. zur Bewertung der Beschaffenheit eines Feldbodens) als auch für Prüfungen auf Mehrfachkonzentrationen (z. B. zur Bewertung der Toxizität einer zugesetzten Chemikalie) anwendbar. In beiden Fällen ist es den Regenwürmern überlassen anfänglich zu wählen, in welches Segment, ob in den Kontroll  oder Prüfboden, sie eindringen [im Zweikammerprüfgefäß zwischen der linken und der rechten Seite; im Sechskammerprüfgefäß zwischen den (3 + 3) sich abwechselnden Kammern].

Qualité du sol - Essai d'évitement pour contrôler la qualité des sols et les effets des produits chimiques sur le comportement - Partie 1: Essai avec des vers de terre (Eisenia fetida et Eisenia andrei) (ISO 17512-1:2008)

L'ISO 17512-1:2008 spécifie une méthode d'essai biologique de dépistage rapide permettant d'évaluer la fonction d'habitat des sols ainsi que l'influence des contaminants et des produits chimiques sur le comportement des vers de terre.
Cet essai sublétal constitue une méthode rapide qui reflète la biodisponibilité pour les espèces Eisenia fetida et Eisenia andrei des mélanges de contaminants dans les sols naturels et des sols dopés en substances. Le comportement d'évitement des vers constitue le critère d'effet de cet essai. Ce dernier n'est pas destiné à remplacer l'essai de reproduction des vers de terre.
Deux modèles de conception différente (récipient d'essai à deux compartiments et récipient d'essai à six compartiments) ont été mis au point et mis en œuvre avec succès. Les deux modèles conviennent soit aux essais à une seule concentration (par exemple pour évaluer la qualité d'un sol naturel), soit aux essais à plusieurs concentrations (par exemple pour évaluer la toxicité d'un sol dopé par un produit chimique). Dans les deux cas, on laisse aux vers de terre le choix initial du compartiment où pénétrer: sol témoin et un traitement [dans le récipient d'essai à deux compartiments, choix entre le côté droit ou gauche; dans le récipient d'essai à six compartiments, choix entre les compartiments disposés de façon alternée (3 + 3)].

Kakovost tal - Izogibalni preskus za določanje kakovosti tal in učinkov kemikalij na obnašanje - 1. del: Preskus z deževniki (Eisenia fetida in Eisenia andrei) (ISO 17512-1:2008)

General Information

Status
Published
Public Enquiry End Date
02-Feb-2020
Publication Date
23-Sep-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Aug-2020
Due Date
24-Oct-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 17512-1:2020
01-november-2020

Kakovost tal - Izogibalni preskus za določanje kakovosti tal in učinkov kemikalij na

obnašanje - 1. del: Preskus z deževniki (Eisenia fetida in Eisenia andrei) (ISO
17512-1:2008)

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) (ISO

17512-1:2008)
Bodenbeschaffenheit - Vermeidungsprüfung zur Bestimmung der Bodenbeschaffenheit
und der Auswirkungen von Chemikalien auf das Verhalten - Teil 1: Prüfung von
Regenwürmern (Eisenia fetida und Eisenia andrei) (ISO 17512-1:2008)

Qualité du sol - Essai d'évitement pour contrôler la qualité des sols et les effets des

produits chimiques sur le comportement - Partie 1: Essai avec des vers de terre (Eisenia

fetida et Eisenia andrei) (ISO 17512-1:2008)
Ta slovenski standard je istoveten z: EN ISO 17512-1:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 17512-1:2020 en,fr,de

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

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SIST EN ISO 17512-1:2020
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SIST EN ISO 17512-1:2020
EN ISO 17512-1
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 1: Test
with earthworms (Eisenia fetida and Eisenia andrei) (ISO
17512-1:2008)

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 1: Essai avec des vers de Auswirkungen von Chemikalien auf das Verhalten -

terre (Eisenia fetida et Eisenia andrei) (ISO 17512- Teil 1: Prüfung von Regenwürmern (Eisenia fetida und

1:2008) Eisenia andrei) (ISO 17512-1:2008)
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 17512-1:2020 E

worldwide for CEN national Members.
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SIST EN ISO 17512-1:2020
EN ISO 17512-1:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 17512-1:2020
EN ISO 17512-1:2020 (E)
European foreword

The text of ISO 17512-1:2008 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-

1:2020 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 17512-1:2008 has been approved by CEN as EN ISO 17512-1:2020 without any

modification.
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SIST EN ISO 17512-1:2020
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SIST EN ISO 17512-1:2020
INTERNATIONAL ISO
STANDARD 17512-1
First edition
2008-12-15
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)
Qualité du sol — Essai d'évitement pour contrôler la qualité des sols et
les effets des produits chimiques sur le comportement —
Partie 1: Essai avec des vers de terre (Eisenia fetida et Eisenia andrei)
Reference number
ISO 17512-1:2008(E)
ISO 2008
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
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ii © ISO 2008 – All rights reserved
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
Contents Page

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

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

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

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

3 Terms and definitions ...........................................................................................................................2

4 Principle .................................................................................................................................................2

5 Reagents and materials ........................................................................................................................3

6 Apparatus...............................................................................................................................................4

7 Procedure...............................................................................................................................................5

7.1 Appropriate concentration range ........................................................................................................5

7.2 Testing of soils ......................................................................................................................................5

7.3 Testing of chemical...............................................................................................................................6

7.4 Reference substance ............................................................................................................................6

7.5 Validity criteria.......................................................................................................................................6

8 Calculation and expression of results ................................................................................................6

9 Test report..............................................................................................................................................7

Annex A (informative) Test chambers ..............................................................................................................8

Annex B (informative) Example of a breeding technique for Eisenia fetida and Eisenia andrei..............11

Annex C (informative) Further test organisms...............................................................................................12

Annex D (informative) Contaminants that earthworms can detect and avoid in the avoidance test.......13

Annex E (normative) Testing of chemicals in the avoidance test ...............................................................14

Annex F (normative) Determination of water-holding capacity ...................................................................16

Annex G (informative) Comparison of the results obtained in the two section chamber and six

section chamber system ....................................................................................................................17

Annex H (informative) Influence of soil properties on avoidance behaviour — Basis for the

threshold value of 20 % ......................................................................................................................20

Annex I (informative) Data gained in “dual” tests with the same (untreated) control soil on both

sides of the test vessels .....................................................................................................................22

Bibliography......................................................................................................................................................24

© ISO 2008 – All rights reserved iii
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(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 17512-1 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)
The following part is under preparation:
⎯ Part 2: Test with collembolans (Folsomia candida)
iv © ISO 2008 – All rights reserved
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
Introduction

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

are proposed to complement conventional chemical analysis (see ISO 15799). ISO 15799 includes a list and

short characterisation of recommended and standardised test systems. 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. As standardised test systems, a mortality test (ISO 11268-1) and a reproduction test

(ISO 11268-2) exist to investigate the habitat function of a soil with respect to earthworms as representatives

of the soil biocenosis.

The reproduction test with earthworms (ISO 11268-2) is applied to detect effects resulting from sublethal

concentrations. Such endpoints are preferably applied to obtain information on environmental effects.

However, the reproduction test is very labour-intensive and time-consuming, needing long incubation periods

with results obtained only after 56 days. As the test period and the work expense dictate the costs of a given

test, it is preferable to obtain the results within a short test period and at a high level of sensitivity. That is

especially the case for the assessment of remediated soils. This feature is offered by the avoidance test with

Eisenia fetida and Eisenia andrei. Experiences gained in a laboratory comparison test with eight contaminated

soils in three laboratories point out that the avoidance test is as sensitive as the reproduction test (Reference

[5]). However, it is not intended to use this test to replace the earthworm reproduction test.

NOTE The results were compared with those of the earthworm acute and reproduction tests carried out with the

same soils. The results showed that with a criterion of > 80 % avoidance response, a 72 % agreement of the results was

achieved.
© ISO 2008 – All rights reserved v
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SIST EN ISO 17512-1:2020
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SIST EN ISO 17512-1:2020
INTERNATIONAL STANDARD ISO 17512-1:2008(E)
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)
1 Scope

This part of ISO 17512 specifies a rapid screening method for evaluating the habitat function of soils and the

influence of contaminants and chemicals on earthworm behaviour.

The sublethal test is a rapid method that reflects the bioavailability of contaminant mixtures in natural soils and

substances spiked into soils to Eisenia fetida and Eisenia andrei. The avoidance behaviour of the worms is

the measurement endpoint of the test. This test is not intended to replace the earthworm reproduction test.

Two different designs (a two section unit and a six section unit) have been developed and successfully

applied. Both designs are applicable to either single-concentration (e.g. for assessing the quality of a field soil)

or multi-concentration (e.g. for assessing the toxicity of a spiked chemical) tests. In both cases, the

earthworms are allowed to make the initial choice on which compartment, control and a treatment [in the two

section test vessel between right and left side; in the six section test vessel between the (3 + 3) alternating

compartments], to enter.
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 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

chemicals on the emergence and 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 soil and soil materials

© ISO 2008 – All rights reserved 1
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
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

3.2
habitat function

ability of soils/soil materials to serve as habitat for micro-organisms, plants and soil-living animals and their

interactions
[ISO 15799:2003]

NOTE Ecotoxicological tests as indicators for the habitat function provide information concerning the respective test

parameter, e.g. acute test for survival, or chronic tests for reproduction.
3.3
limited habitat function

habitat function (3.2) is limited if on average > 80 % of worms are found in the control soil (indication as an

impact on behaviour)
3.4
effective concentration

concentration at which a specific effect is detected [where x is a percentage (10, 25, 50) of this effect; e.g.

avoidance]

EXAMPLE In this part of ISO 17512, an EC means the concentration of a substance or mixture of substances in

soil that is estimated to cause a behavioural response in 50 % of the test earthworms.

4 Principle

Ten adult earthworms (species Eisenia fetida or Eisenia andrei) are exposed at the same time to a control soil

and a contaminated soil or a soil containing test substances. Test soil and control soil are placed into each

test vessel and the earthworms are thus presented with a choice between the test soil and the control soil.

Two test-vessel designs are available:
a) a two section test vessel; and
b) a six section test vessel.

After an incubation period of two days, the number of worms is determined in all sections of the vessels.

Individual studies (e.g. testing boric acid in one of the two designs in different laboratories) or comparative

investigations (testing the same chemical or soil in the same laboratory, e.g. Reference [8]) have in some

cases shown different results. Recently, both designs were validated in interlaboratory tests in Canada

(Reference [2]) and France; however, no international ring test using both designs in parallel has been

performed so far. Therefore, for the time being, the choice of the design is up to the experimenter. When

doing so, practical considerations like costs of the units as well as the amount of waste produced should also

be taken into consideration.
2 © ISO 2008 – All rights reserved
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
5 Reagents and materials

5.1 Boric acid reference toxicant, recommended. H BO has been used historically as a soil

3 3

chemosterilant and is an effective non-selective biocide (relative molecular mass: 61,81). Earthworms can

detect and avoid sublethal concentrations that adversely affect reproduction. Boric acid satisfies the following

criteria that attest to its suitability as a reference toxicant:

a) it is effective at relatively low concentrations that are not strongly influenced by the nature of the

substrate;

b) it is relatively stable and persistent so that concentrations do not change rapidly over the duration of the

test;

c) it is reasonably water soluble or miscible in water, does not volatilise readily, and can be readily mixed

with soils;
d) there is a standard method for measuring boric acid concentrations in soil;

e) it represents a minimal hazard to technicians and it is free of disposal problems.

5.2 Biological material, consisting of adult earthworms of the species Eisenia fetida or Eisenia andrei

(individual mass: between 300 mg and 600 mg). Synchronisation of breeding of the organisms for this test is

not necessary. An example of how to breed compost worms is given in Annex B.

Condition the selected worms for at least one day in the selected control soil (5.4).

NOTE Eisenia fetida and Eisenia andrei are compost worms. Ecologically, these species are not the most important

in soils (Reference [7]). On the other hand, from a practical point of view, compost worms are much more suitable than

any other lumbricid species due to the fact that they reproduce very quickly and easily in the laboratory (i.e. mass cultures

can be obtained). In addition, the sensitivity of these species is more or less of the same order of magnitude in comparison

to other earthworm species. In most cases, the differences between species are — depending on the chemical or

contaminant mixture tested — not larger than a factor of 10 in acute or chronic tests (References [6], [7]). Despite the fact

that other earthworm species have already successfully been used in avoidance tests (see Annex C), a factor describing

their range of avoidance response is not yet known.

5.3 Test substrate. The soil to be tested should be sieved (size of openings, 2 mm) adjusted to about 60 %

of the maximum water holding capacity. The optimum water content is achieved, if there is no standing water

or free water appearing when the soil is compressed.

NOTE For highly silty and loamy soils, it can be difficult to get the necessary amount of soil sieved to u 2 mm with an

acceptable expenditure of work. The holes of the sieves may 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 u 4 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 F.

5.4 Control soil: three choices are possible (see also ISO 15799). Option a) is preferred, but since such a

soil is often not available either a standard soil, b), or an artificial soil, c), is possible (potential influences of

these soils are covered by the 80 % assessment criterion, see Clause 8).

a) A control soil as similar as the test soil in all characteristics other than the presence of contaminants.

b) A soil with the characteristics according to ISO 11269-2 [C u 1,5 %, sand (0,063 mm to 2 mm) content

org
of 50 % to 75 %, < 20 % of particles less than 0,02 mm; pH of 5 to 7,5].
c) Artificial soil in accordance with ISO 11268-2.
Natural soils should be sieved and the water content adjusted according to 5.3.
© ISO 2008 – All rights reserved 3
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
6 Apparatus
Usual laboratory equipment and in particular the following.
6.1 Containers (see Annex A).

6.1.1 Two section chamber: containers of capacity 1 l to 2 l with a cross-sectional area of about 0,02 m ,

such that a depth of 50 mm to 60 mm of the 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 worms from escaping

(e.g. by using a tape to fix the cover). To avoid lateral effects of light, glass vessels shall be wrapped.

Two section chambers are commercially available .

NOTE Due to the short test period and the proportionally large volume of soil in the vessels, a reduction of chemical

concentration in the soil resulting from sorption to the vessel walls is negligible. Nevertheless, inert material (e.g. glass or

stainless steel) is preferred.
6.1.2 Six section chamber (circular test units or vessels):
1) stainless steel for testing soil contaminated with organic compounds;

2) plastic (high density inert material) for testing soil contaminated with metals or metalloid compounds.

The circular test unit has a central chamber with six cut pie-shaped interconnecting compartments into which

the test soil is placed; interconnecting holes are located along the bottom of the compartment walls (three per

side) and along the bottom of the central chamber (two per side) so that the worms can move freely between

compartments. The plastic test unit should be wrapped in an opaque material (tin foil) to eliminate light.

Provisions to prevent worms from escaping are necessary.

The six section chamber is not commercially available. Therefore all details necessary to construct such

chambers are presented in the figures and in the text.
6.2 Divider (e.g. plastic or thin sheets of metal):

a) for the two section chamber, to divide the containers vertically into two identical sides;

b) for the six section chamber, to slide along the walls of the compartments at the end of a test to isolate

each section.

6.3 Equipment for measuring the water content of a substrate (according to ISO 11465).

6.4 Test environment.

6.4.1 Enclosure or environmental chamber, capable of being maintained at (20 ± 2) °C.

6.4.2 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.

NOTE A day/night cycle was chosen so that the conditions are comparable to the acute and reproduction test.

1) Bellaplast No. 597 is an example of a suitable product available commercially. This information is given for the

convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.

4 © ISO 2008 – All rights reserved
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SIST EN ISO 17512-1:2020
ISO 17512-1:2008(E)
7 Procedure
7.1 Appropriate concentration range

The avoidance test is designed to detect sublethal effects. Therefore, the test is invalid if more than one worm

per vessel (i.e. one out of 10) is dead or missing at the end of the test (see 7.5). In order to avoid mortality, the

performance of a range-finding test is recommended.
7.2 Testing of soils
7.2.1 Two section chamber

At the beginning of the test, the vessels (6.1.1) are divided into two equal sections by means of a vertically

introduced divider. Vessels are filled with sieved soil up to a height of 50 mm to 60 mm. One half of the vessel

is filled with test soil (section A), the other half is filled with control soil (section B). Then the separator is

removed and 10 worms are placed on to the separating line of each test vessel (from there they have the

possibility to dig quickly into the soil, using the slit left by the divider as a starting point). The containers are

covered according to 6.1.1 and placed in the environmental chamber or in the test enclosure (6.4.1).

No feeding of the animals is required during the test.

The test is run with five replicates per treatment (test soils, controls or reference substance). 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

dividers. The dividers shall be inserted before the test units are moved from the environmental chamber. The

number of worms is determined for both sections of the vessels. Worms divided due to the introduction of the

divider are counted as 0,5 independent of the length of the remaining body. Missing worms are considered to

have either escaped from the test chamber or to have died and disintegrated during the test (see 7.1).

7.2.2 Six section chamber
The test soil and control soils are prepared (sieved, hydrated and mixed) and pl
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