EN ISO 17512-1:2020

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
• Publication Date: 05-May-2020
• Withdrawal Date: 29-Nov-2020
• ICS: 13.080.10 - Chemical characteristics of soils
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444 - Environmental characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 06-May-2020
• Due Date: 07-Mar-2022
• Completion Date: 06-May-2020
• Ref Project: SIST EN ISO 17512-1:2020 - 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)

Overview

EN ISO 17512-1:2020 (CEN adoption of ISO 17512-1:2008) specifies a rapid, sublethal avoidance test to assess soil quality and the effects of chemicals on earthworm behaviour. The standard uses the earthworms Eisenia fetida and Eisenia andrei as test organisms and measures their avoidance behaviour as an endpoint to indicate bioavailability of contaminants in natural or spiked soils. It is a screening method to evaluate the habitat function of soils and is not intended to replace the earthworm reproduction test.

Key topics and technical requirements

• Test principle: Ten adult earthworms are offered a choice between control soil and test soil (contaminated or spiked) inside a test vessel; distribution of worms between compartments is the measurement endpoint.
• Test designs: Two validated chamber designs are specified:
  • Two-section unit (control vs treatment; right/left)
  • Six-section unit (three alternating compartments per side)
• Test modes:
  • Single-concentration-e.g., assessing field soil quality
  • Multi-concentration-e.g., dose–response for a chemical
• Endpoints and criteria:
  • Avoidance behaviour quantified and expressed as effective concentrations (ECx) where applicable
  • A soil is considered to have a limited habitat function if >80% of worms are found in the control soil
• Procedural elements covered: terms/definitions, reagents and materials, apparatus, test procedure (including appropriate concentration ranges), validity criteria, calculation & reporting requirements
• Annexes: guidance on test chambers, breeding techniques, water-holding capacity, comparison of chamber systems, and lists of contaminants earthworms can detect

Applications

• Rapid screening of soil contamination and soil remediation outcomes
• Ecotoxicological assessment for environmental impact studies and site characterization
• Pre-screening to prioritize soils or chemicals for more detailed chronic tests (e.g., reproduction tests)
• Laboratory quality control and regulatory screening where quick evaluation of bioavailability and behavioural effects is needed

Who uses this standard

• Environmental and ecotoxicology laboratories
• Soil scientists and agronomists
• Remediation consultants and contractors
• Regulatory agencies and environmental monitoring bodies
• Research institutions studying soil-fauna interactions and contaminant bioavailability

Related standards

• ISO 11268-2: earthworm reproduction test (longer-term, chronic endpoint)
• ISO 15799: guidance on ecotoxicological characterization of soils
• ISO 11465, ISO 10390, ISO 11269-2: referenced methods for soil properties and plant testing

Keywords: soil quality, avoidance test, earthworm, Eisenia fetida, Eisenia andrei, bioavailability, soil ecotoxicology, ISO 17512-1, habitat function, soil contamination.

Standards Content (Sample)

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

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.

Contents Page
European foreword . 3

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.
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
ISO 17512-1:2008(E)
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ii © ISO 2008 – All rights reserved

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 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

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.
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 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
EC
x
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

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 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.
1)
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

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 placed to a depth of 50 mm to
60 mm (350 ml soil) in each of three compartments in an alternating pattern (e.g. compartments 1, 3, and 5
have test soil and compartments 2, 4, and 6 have control soil) (see also Annex E). There is no soil in the
central chamber. Ten earthworms are added to the central chamber, one at a time, and the compartment
entered by each individual is recorded. The containers are covered (6.1.2) and placed in an environmental
chamber (6.4.1).
No feeding of the animals is performed during the test.
The test is run with five replicates for a single concentration test and at least with duplicates for a multi-
concentration test. For multi-concentration tests, the test soil consists of the site soil diluted with the
appropriate control soil.
At the end of the test period (48 h) the dividers are positioned to prevent further movement of the earthworms
between compartments. The dividers shall be inserted before the test units are moved from the environmental
chamber. The number of worms in each compartment is recorded and the total number in each treatment
within a test unit determined. Individual earthworms sliced inadvertently by the dividers are to be recorded 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).
NOTE The hypothesis tested is that at the beginning of the test the worms are randomly distributed among sections
and at the end of the test for a true avoidance response the earthworms are not distributed randomly among the sections
in a vessel. If, at the beginning of the test, the worms are non-randomly distributed, then there might not be an avoidance
response. Alternatively, there might be an avoidance response at the beginning of the test by worms refusing to enter
sections with contaminated soil that they instantly avoid. This rarely happens when the levels in soil are sublethal.
ISO 17512-1:2008(E)
7.3 Testing of chemical
While the main use of avoidance tests is testing of potentially contaminated soils, it is also possible to use this
test for the assessment of the effects of single chemicals after they have spiked into a soil (examples of
chemicals detected by earthworms are given Annex D). Modifications to test single chemicals (including
statistical procedures) are specified in Annex E.
7.4 Reference substance
Boric acid is recommended as the reference toxicant. An avoidance behaviour response should be obtained
at a concentration of 750 mg H BO per kilogram of soil measured on the dry mass basis when artificial soil or
2 3
another control soil is used. Testing by the soil toxicology laboratory of Environment Canada generated a
boric acid EC of 618 mg/kg in a six section chamber test for avoidance behaviour using a chernozem clay
loam control soil spiked with boric acid (Reference [8]). When reporting EC values, also state the main soil
properties (i.e. pH, texture and organic matter content).
7.5 Validity criteria
The test is invalid if the number of dead or missing worms is > 10 % per treatment.
To validate the test set, check the homogeneity of distribution of the worms. For this purpose, fill the whole
test vessel with the same soil and ensure that the orientation of the test vessels in the room is the same. On
average, the ratio of worms should be within the range 60 % : 40 % for a two section chamber. More
information concerning the distribution of worms in such dual tests using different soils is provided in Annex I.
8 Calculation and expression of results
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. For tests using the two section vessel, as well as for the six section vessel, 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.
For a single concentration test, the mean number of individuals at the end of the test in the test soil is
compared to the mean of the control soil treatment using Fisher's exact test or another statistic appropriate for
pairwise comparisons (Reference [15]). Results showing a significantly lower mean number of surviving
worms in the test soil, relative to those in the control soil, indicate an avoidance response (or preference for
the control soil) to the test soil. This result suggests that the habitat function of the test soil is limited.
For calculation of the percentage effect of a substance concentration, the mean number of worms in the test
soil is compared with the mean number of worms in the control soil [negative responses (= the worms prefer
the test soil) are considered as 0 % of avoidance]) in accordance with Equation (1).
nn−
⎛⎞
ct
x=×100 (1)
⎜⎟
N
⎝⎠
where
x is avoidance, expressed as a percentage;
n is the number of worms in the control soil (either per vessel or in the control soil of all replicates);
c
n is the number of worms in the test soil (either per vessel or in the test soil of all replicates);
t
N is the total number of worms (usually 10; either per vessel or in the control soil of all replicates).
6 © ISO 2008 – All rights reserved

ISO 17512-1:2008(E)
Using these data, any median effective concentration, EC , for a specified percentage effect (EC or EC )
x 50 20
and its associated confidence limits can be calculated.
For statistical analysis of ecotoxicity data see Reference [15].
A comparison of results obtained in two and six chamber systems is given in Annex G.
If control soil and test soil differ in more of the main properties than just contaminants, statistical calculations
are not appropriate. In this case, the application of a fixed threshold value instead of a statistical significant
difference between the number of worms in the control and the test soil is recommended. Test soils with less
than 20 % of the total number of worms are classified as having limited habitat function.
Data on the influence of soil properties on avoidance behaviour are given in Annex H.
If an attraction of > 80 % by the test soil is observed, the presence of chemical substances cannot be
excluded. The result should also be assessed as an effect.
9 Test report
The test report shall contain at least the following information:
a) a reference to this part of ISO 17512;
b) the results expressed in accordance with Clause 8;
c) detailed description of the characteristics of the test soil and of the control soil;
d) if chemicals are tested, a detailed description of the test substance and method of application or
incorporation;
e) complete description of the biological material employed (species, mass range, breeding conditions,
supplier);
f) full description of the experimental design and procedure;
g) description of the test conditions, including moisture content and pH value;
h) number of adults in the test soil and in the control soil at the beginning (only relevant for the six section
chamber test) and end of the test;
i) mortality of the adults;
j) description of obvious morphological symptoms observed in the test organisms;
k) assessment with respect to habitat function limited and not limited, respectively or statistically calculated
values (lowest observed effective concentration, no observed effective concentration and/or EC )
x
including 95 % confidence limits, method of calculation, plot of the exposure concentration-response
relationship;
l) discussion of the results;
m) all details not specified in this part of ISO 17512 or considered as optional, as well as any effect which
may have affected the results.
ISO 17512-1:2008(E)
Annex A
(informative)
Test chambers
A.1 Two section chamber
Round or rectangular containers (glass or plastic) of capacity 1 l to 2 l with a cross-sectional area of about
0,02 m .
Figure A.1 — Example of a two section chamber
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ISO 17512-1:2008(E)
A.2 Six section chamber
Circular six section vessel with a central soil-free chamber. The design of the test unit is circular with an outer
diameter of 230 mm. Each test unit is partitioned into six cut pie-shaped compartments that surround a
central, circular (54 mm diameter) compartment. A series of holes of diameter 10 mm (two per compartment)
connect the central compartment to each of the six compartments. The six compartments are also connected
to adjacent compartments by three holes along the bottom of the section walls separating the compartments.
The test unit is constructed of either high-quality stainless steel (1 mm to 4 mm thick) for use with soils
contaminated with organic compounds, or plastic (5 mm to 6 mm thick) for use with soils contaminated with
metal compounds.
Figure A.2 — Example of a six section chamber
ISO 17512-1:2008(E)
Dimensions in millimetres
Key
1 removable partition (six per test unit) 6 holes between compartments
2 inner chamber without soil where worms are placed at start of test 7 wooden support stand
3 lid 8 cut pie-shaped test compartments
4 outer wall of test unit 9 wall separating compartments
5 holes between inner chamber and compartment 10 steel base of test unit
Figure A.3 — Details of a six section chamber
10 © ISO 2008 – All rights reserved

ISO 17512-1:2008(E)
Annex B
(informative)
Example of a breeding technique for Eisenia fetida and Eisenia andrei
This method is in accordance with ISO 11268-1:1993, Annex A and ISO 11268-2:1998, Annex A.
Both species can be bred in a wide range of animal wastes. The recommended breeding medium is a mixture
of one volume of horse or cattle manure and one volume of peat. The medium should have a pH value of
about 6 to 7 (regulated with calcium carbonate), a low ionic conductivity (less than 6 mg/kg soil dry mass or
less than 0,5 % common salt concentration) and should not be contaminated excessively with ammonia or
animal urine. The substrate should be moist, but not too wet. In cases of doubt, the moisture should be
checked as follows. When the soil is gently squeezed by hand, only small drops of water should appear
between the fingers. Breeding boxes of capacity 10 l to 50 l are suitable.
To obtain worms of standard age and mass, it is best to start the culture with cocoons. Therefore adult worms
are put into a breeding box with fresh substrate to produce cocoons and remove them after 14 days to
28 days. These individuals can be used for further breeding batches. The earthworms hatched from the
cocoons are used for testing when mature (after at least two months, but less than 12 months).
Breeding is preferably carried out in an environmental chamber or enclosure at (20 ± 2) °C. At this
temperature, worms become mature after two months to three months.
ISO 17512-1:2008(E)
Annex C
(informative)
Further test organisms
The test has also been performed with Lumbricus terrestris, an organism with a high ecological relevance. In
this case some modifications of the described test design are necessary.
Individual mass 3 g to 10 g
Incubation period 72 h
Temperature (18 ± 2) °C
Reference substance Boric acid
so far, the test has only been performed in the six section chamber; although L.
Test vessels terrestris is larger than E. fetida, the same test vessels were successfully applied
(Reference [10])
In addition, mineral soil dwelling species like Aporrectodea caliginosa (Reference [3]) or Aporrectodea
tuberculata, (References [19], [20]) as well as epigeic species like Lumbricus rubellus and Dendrobaena
octaedra were used in two section chambers (References [19], [20]). Other test species might be suitable.
Necessary modifications (e.g. size of test chamber, test duration, temperature) should be defined for each of
the additional test species.
For breeding methods of common soil dwelling earthworm species, see Reference [18].
12 © ISO 2008 – All rights reserved

ISO 17512-1:2008(E)
Annex D
(informative)
Contaminants that earthworms can detect
and avoid in the avoidance test
The avoidance test is suitable for assessing those contaminants that can be detected by earthworms (e.g.
Eisenia fetida) via sensory receptors (e.g. chemoreceptors). This seems to apply for a broad range of
contaminants. Up to now it has been shown that the test is suitable for mineral oil, polyaromatic hydrocarbons,
2,4,6-trinitrotoluene (TNT), manganese, zinc, copper sulfate, petroleum hydrocarbons (crude oil), and
mixtures consisting of several heavy metals, KCl, NH Cl, benomyl, carbendazim, lambda-cyhalothrin,
mancozeb and complex hydrocarbon mixtures such as amines and glycol products and condensate
(References [4], [5], [8], [9], [10], [11], [12]).
If testing volatile compounds such as low-molecular petroleum hydrocarbons (BTEX, C to C ), they may
5 10
impact the results of the avoidance test due to transport to the control soil (section B) via the gaseous phase.
ISO 17512-1:2008(E)
Annex E
(normative)
Testing of chemicals in the avoidance test
E.1 General
To test chemicals, modifications to the test procedures are required.
E.2 Test substrate
If the test is used for substance testing, prepare artificial soil according to ISO 11268-2 or use a sandy soil
with the characteristics specified in ISO 11269-2 (organic carbon content u 1,5 % mass fraction, sand content
of 50 % to 75 %, < 20 % in the fine particle fraction; pH of 5 to 7,5). The sandy soil should be sieved to
u 2 mm. Adjust the water content according to 5.3. The incubation corresponds to the testing of soils.
E.3 Control soil
Use the same soil as control soil and as test soil (see 5.3). However, do not add test substance to the control
soil. Adjust the water content according to 5.3.
E.4 Procedure
E.4.1 General. For the testing of chemicals use one of the methods specified in E.4.2 to E.4.5.
E.4.2 Water-soluble tes
...