EN ISO 11268-2:2015

SLOVENSKI STANDARD
01-oktober-2015
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Soil quality - Effects of pollutants on earthworms - Part 2: Determination of effects on
reproduction of Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
Bodenbeschaffenheit - Wirkungen von Schadstoffen auf Regenwürmer - Teil 2:
Bestimmung der Wirkung auf die Reproduktionsleistung von Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 2: Détermination
des effets sur la reproduction de Eisenia fetida/Eisenia andrei (ISO 11268-2:2012)
Ta slovenski standard je istoveten z: EN ISO 11268-2:2015
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.

EUROPEAN STANDARD
EN ISO 11268-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2015
ICS 13.080.30
English Version
Soil quality - Effects of pollutants on earthworms - Part 2:
Determination of effects on reproduction of Eisenia fetida/Eisenia
andrei (ISO 11268-2:2012)
Qualité du sol - Effets des polluants vis-à-vis des vers de Bodenbeschaffenheit - Wirkungen von Schadstoffen auf
terre - Partie 2: Détermination des effets sur la reproduction Regenwürmer - Teil 2: Bestimmung der Wirkung auf die
de Eisenia fetida/Eisenia andrei (ISO 11268-2:2012) Reproduktionsleistung von Eisenia fetida/Eisenia andrei
(ISO 11268-2:2012)
This European Standard was approved by CEN on 6 August 2015.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11268-2:2015 E
worldwide for CEN national Members.

Contents Page
European foreword .3

European foreword
The text of ISO 11268-2:2012 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 11268-2:2015 by
Technical Committee CEN/TC 345 “Characterization of soils” 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 February 2016, and conflicting national standards shall be withdrawn
at the latest by February 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11268-2:2012 has been approved by CEN as EN ISO 11268-2:2015 without any modification.
INTERNATIONAL ISO
STANDARD 11268-2
Second edition
2012-11-01
Soil quality — Effects of pollutants on
earthworms —
Part 2:
Determination of effects on reproduction
of Eisenia fetida/Eisenia andrei
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 2: Détermination des effets sur la reproduction de Eisenia fetida/
Eisenia andrei
Reference number
ISO 11268-2:2012(E)
©
ISO 2012
ISO 11268-2:2012(E)
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 2012 – All rights reserved

ISO 11268-2:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents and material . 4
6 Apparatus . 5
7 Procedure . 6
7.1 Experimental design . 6
7.2 Preparation of test mixture . 7
7.3 Addition of the earthworms . 8
7.4 Test conditions and measurements . 8
7.5 Reference substance . 9
8 Calculation and expression of results . 9
8.1 Calculation . 9
8.2 Expression of results . 9
9 Validity of the test . 9
10 Statistical analysis . 9
10.1 General . 9
10.2 Single-concentration tests .10
10.3 Multi-concentration tests .10
11 Test report . 11
Annex A (informative) Determination of the chronic toxicity of chemicals on earthworms under
tropical conditions .13
Annex B (informative) Culturing of Eisenia fetida and Eisenia andrei .15
Annex C (informative) Determination of water holding capacity of artificial soil .16
Annex D (informative) Techniques for counting juvenile worms hatched from cocoons.17
Annex E (informative) Performance of the method .18
Bibliography .20
ISO 11268-2:2012(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 11268-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
This second edition cancels and replaces the first edition (ISO 11268-2:1998), which has been technically revised.
ISO 11268 consists of the following parts, under the general title Soil quality — Effects of pollutants on earthworms:
— Part 1: Determination of acute toxicity to Eisenia fetida/Eisenia andrei
— Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei
— Part 3: Guidance on the determination of effects in field situations
iv © ISO 2012 – All rights reserved

ISO 11268-2:2012(E)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and are
[34] [35]
proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616 ). ISO 15799
includes a list and short characterization of recommended and standardized test systems and ISO 17616 gives
guidance on the choice and evaluation of the bioassays. Aquatic test systems with soil eluate are applied to
obtain information about the fraction of contaminants potentially reaching the groundwater by the water path
(retention function of soils), whereas terrestrial test systems are used to assess the habitat function of soils. As
standardized test systems using earthworms as indicator organisms for the habitat function of soil, an acute
test for survival and a chronic test for reproduction are available.
This part of ISO 11268 describes a method that is based on the determination of sublethal effects of contaminated
soils on adult earthworms of the species Eisenia fetida (Savigny 1826) and Eisenia andrei (André 1963).
Optionally, the method can be used for testing chemicals added to standard soils (e.g. artificial soil) for their
sublethal hazard potential to earthworms. Finally, information is provided on how to use this method for testing
chemicals under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular. Background information on the ecology of earthworms and their use in ecotoxicological testing is
available. Other species, e.g. Aporrectodea caliginosa, Lumbricus rubellus and Lumbricus terrestris, have also
been used as test organisms. These or other species have not been proven to be more sensitive in general,
[16][17]
and the database and experience in testing soils is small .
This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the Organization
[27][28] [11]
for Economic Cooperation and Development and by the European Union .
INTERNATIONAL STANDARD ISO 11268-2:2012(E)
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or otherwise harmful
chemicals or infectious microorganisms. Occupational health risks may arise from dust or evaporated
chemicals during handling and incubation. Precautions should be taken to avoid skin contact.
1 Scope
This part of ISO 11268 specifies one of the methods for evaluating the habitat function of soils and determining
the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei by dermal and
alimentary uptake. This chronic test is applicable to soils and soil materials of unknown quality, e.g. from contaminated
sites, amended soils, soils after remediation, agricultural or other sites concerned, and waste materials.
Effects of substances are assessed using a standard soil, preferably a defined artificial soil substrate. For
contaminated soils, the effects are determined in the test soil and in a control soil. According to the objective of the
study, the control and dilution substrate (dilution series of contaminated soil) should be either an uncontaminated
soil comparable to the soil sample to be tested (reference soil) or a standard soil (e.g. artificial soil).
Information is provided on how to use this method for testing chemicals under temperate as well as under
tropical conditions.
The method is not applicable to volatile substances, i.e. substances for which H (Henry’s constant) or the
air/water partition coefficient is greater than 1, or for which the vapour pressure exceeds 0,013 3 Pa at 25 °C.
This method does not take into account the persistence of the substance during the 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method
ISO 11268-2:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
contaminant
substance or agent present in the soil as a result of human activity
[ISO 15176:2002]
3.2
growth
increase in biomass (i.e. the fresh mass of organisms)
NOTE It is expressed as a percentage of the fresh mass of organisms at the start of the test.
3.3
reproduction
mean number of offspring per test vessel after eight weeks’ incubation under the specified test conditions
3.4
ER (effective rate) or EC (effective concentration)
x x
x % effect rate or concentration of the test sample or test substance at which reproduction is reduced by x %
compared to the control
3.5
limit test
single concentration test consisting of at least four replicates each, the test sample without any dilution or the
highest concentration of test substance mixed into the control soil and the control
3.6
lowest observed effect rate (LOER) or effect concentration (LOEC)
lowest tested percentage of a test sample in a control soil or concentration of a substance at which a statistically
significant effect is observed
NOTE The LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test mixtures
above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If this condition cannot be
satisfied, an explanation should be given for how the LOEC and NOEC (3.7) have been selected.
3.7
NOER (no observed effective rate) or NOEC (no observed effect concentration)
test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test substance
which, when compared to the control, has no statistically significant lethal or other effect such as reduced
reproduction or mass alteration (error probability: p < 0,05)
NOTE The NOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass.
3.8
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with similar properties
(nutrient concentrations, pH, organic carbon content and texture) to the test soil
3.9
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are within
a known range
[11] [27] [23]
EXAMPLE Euro-Soils , artificial soil , LUFA standard soil .
NOTE The properties of standard soils can differ from those of the test soil.
2 © ISO 2012 – All rights reserved

ISO 11268-2:2012(E)
3.10
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with test samples or
a reference substance, which fulfils the validity criteria
NOTE In the case of natural soil, it is advisable to demonstrate its suitability for a test and for achieving the test
validity criteria before using the soil in a definitive test.
3.11
test mixture
mixture of contaminated soil or the test substance with a control soil (3.10)
NOTE Test mixtures are given in percent of contaminated soil based on soil dry mass.
3.12
test mixture ratio
ratio of test soil to control soil in a test mixture
NOTE Different ratios may be applied in a dilution series to establish a dose-response relationship.
4 Principle
The effects on reproduction of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed to the test
soil are compared to those observed for samples exposed to a control soil. If appropriate, effects based on
exposure to a dilution range of contaminated soil or range of concentrations of a test substance are determined.
In addition, observations on growth and survival of adult earthworms are recorded. Test mixtures are prepared
at the start of the test and are not renewed within the test period.
After four weeks, adult worms are removed from the test containers and effects on mortality and biomass are
measured by counting and weighing. The effect on reproduction as the definitive end point is measured by
counting the number of offspring hatched from the cocoons after an additional period of four weeks. The results
obtained from the tests are compared with a control soil or, if appropriate, are used to determine the dilutions or
concentrations which cause no effects on biomass, mortality and reproduction (NOER/NOEC) and the dilution
(concentration) resulting in x % reduction of juveniles hatched from cocoons compared to the control (ER /EC ,
x x
56 d), respectively.
If testing a dilution or concentration series, all test dilutions/concentrations above the LOER/LOEC shall have a
harmful effect equal to, or greater than, that observed at the LOER/LOEC. Where there is no prior knowledge
of the dilution/concentration of the test soil/test substance likely to have an effect, then it is useful to conduct
the test in two steps:
— a preliminary test carried out, in accordance with ISO 11268-1, to give an indication of the effect
dilution/concentration and of the dilution/concentration giving no mortality (NOER/NOEC).
Dilutions/concentrations to be used in the definitive test can then be selected.
— the definitive test to determine sublethal effects of (dilutions of) contaminated soil or the concentration of
a chemical which, when evenly mixed into the standard soil, causes no significant effects on numbers of
offspring hatched from cocoons compared with the control (NOER/NOEC), and the lowest concentration
causing effects (LOER/LOEC).
NOTE The use of a reference soil is an essential requirement to demonstrate the present status of the test population,
and to avoid misinterpretation of results.
ISO 11268-2:2012(E)
5 Reagents and material
[15], [19],
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia andrei
[20],
which are between two months and one year old, with a clitellum, and a wet mass between 300 mg and
600 mg (E. fetida) and between 250 mg and 600 mg (E. andrei).
Select worms used for the test to form, as far as is practicable, a homogeneous population from the standpoint
of age, size and mass. Worms should preferably be selected from a synchronized culture with a relatively
homogeneous age structure. Before the test, wash them with potable water.
NOTE An example of culturing Eisenia fetida/Eisenia andrei is given in Annex B.
Condition the selected worms for one day to seven days in standard or control soil before use. The food, which
is also used as a food source in the test (see 5.3), shall be given in sufficient amount (see 7.4).
5.2 Test sample, may consist of field-collected soil or control soil amended by the test mixture.
5.2.1 Field-collected soils, soil or waste materials
The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or waste
materials (e.g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or
manure) under consideration for possible land disposal.
Test samples shall be sieved by 4 mm mesh and thoroughly mixed. If necessary, soil may be air-dried without
heating before sieving. Storage of test samples should be as short as possible. Store the soil in accordance
with ISO 10381-6 using containers that minimize losses of soil contaminants by volatilization and sorption to
the container walls. Soil pH should not be corrected as it can influence bioavailability of soil contaminants.
For interpretation of test results, the following characteristics shall be determined for each soil sampled from
a field site:
— pH in accordance with ISO 10390,
— texture (sand, loam, silt) in accordance with ISO 11277
— water content in accordance with ISO 11465,
— water holding capacity according to Annex C,
— cationic exchange capacity in accordance with ISO 11260,
— organic carbon in accordance with ISO 10694.
NOTE It is important to measure the water holding capacity of all mixtures used in the test.
5.2.2 Control soil, either a) reference soil (3.8) or b) standard soil (3.9) that allows the presence of earthworms.
a) If reference soils from uncontaminated areas near a contaminated site are available, they should be treated
and characterized like the test samples. 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 sample, standard soils
shall be used to prepare the test sample. The properties of the field-collected standard soil shall be reported.
4 © ISO 2012 – All rights reserved

ISO 11268-2:2012(E)
The substrate called artificial soil can be used as a standard soil and has the following composition:
Percentage expressed
on a 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 69 %
than 50 % of particle sizes 0,05 mm to 0,2 mm)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) are necessary to get a
pH of 6,0 ± 0,5.
NOTE 1 Taking the properties of highly non-polar (log K > 2, where K is the octanol/water coefficient) or ionizing
ow ow
substances into account, 5 % of peat have proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that Eisenia fetida can comply with the validity criteria, even as regards reproduction,
[18]
when tested in field soils with lower organic carbon content (e.g. 2,7 %) , and experience shows that this can be achieved in
artificial soil with 5 % peat. It is therefore not necessary, before using such a soil in a definitive test, to demonstrate the suitability
[28].
of the artificial soil in complying with the validity criteria, unless the peat contents lowered more than specified above
Prepare the artificial soil at least three days prior to starting the test, by mixing the dry constituents listed above
thoroughly in a large-scale laboratory mixer. A portion of the deionized water required is added while mixing is
continued. Allowance should be made for any water that is used for introducing the test mixture into the soil.
The amount of calcium carbonate required can vary, depending on the properties of the individual batch of
sphagnum peat and should be determined by measuring sub-samples immediately before the test. Store the
mixed artificial soil at room temperature for at least two days to equilibrate acidity. To determine pH and the
maximum water holding capacity, the dry artificial soil is pre-moistened one or two days before starting the test
by adding deionized water to obtain approximately half of the required final water content of 40 % to 60 % of
the maximum water holding capacity.
The total water holding capacity is determined according to Annex C; the pH is determined according to ISO 10390.
5.3 Feeding, any food of a quality shown to be suitable for at least maintaining worm mass during the test is
[20]
considered acceptable. Experience has shown that oatmeal, mashed potato powder , cow or horse manure
is a suitable food. Checks should be made to ensure that cows or horses from which manure is obtained are not
subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary
products that could adversely affect the worms during the test. Self-collected cow manure is recommended,
since experience has shown that commercially available cow manure used as garden fertilizer can have adverse
effects on the worms. The manure should be air-dried, finely ground and pasteurized before use.
Each fresh batch of food should be fed to a non-test worm culture before use in a test, to ensure that it is of
suitable quality. Growth and cocoon production should not be reduced compared to worms kept in a substrate
[27]
that does not contain the new batch of food (conditions as described in OECD 207 ).
5.4 Boric acid, as a reference substance.
6 Apparatus
Usual laboratory equipment and the following.
6.1 Test containers, made of glass or another chemically inert material, of about one to two litres in capacity,
should be used. The containers should have a cross-sectional area of approximately 200 cm so that a moist
substrate depth of about 5 cm to 6 cm is achieved when 500 g dry mass of substrate are added. 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 earthworms from escaping (e.g. by using
a tape to fix the cover).
ISO 11268-2:2012(E)
6.2 Apparatus to determine the dry mass of the substrate, in accordance with ISO 11465.
6.3 Large-scale laboratory mixer, for the preparation of the test sample (5.2).
6.4 Precision balance, with an accuracy of at least 1 mg.
6.5 Polyethylene-membrane, perforated with small holes allowing exchanges between the sample and
the atmosphere.
6.6 Test environment.
6.6.1 Enclosure, capable of being controlled at a temperature of (20 ± 2) °C.
6.6.2 Light source (e.g. white fluorescent tubes), capable of delivering a constant light intensity of 400 Ix to
800 Ix on the containers at a controlled light/dark cycle of between 12 h:12 h and 16 h:8 h.
7 Procedure
7.1 Experimental design
7.1.1 General
A sample of field-collected test soil can be tested at a single concentration (typically 100 %) or evaluated for
toxicity in a multi-concentration test, whereby a series of dilutions are prepared by mixing measured quantities
with a control soil (5.2.2). When testing substances, a series of concentrations is prepared by mixing quantities
of the test substance with a standard soil (e.g. artificial soil). The concentrations are expressed in milligrams of
test substance per kilogram of dried control soil.
Depending on the knowledge of relevant response levels, a preliminary test may precede the definitive test. Each
definitive test consists of a series of soil mixtures (treatments). Each treatment is replicated at least four times.
7.1.2 Preliminary test
A preliminary test to find the range of mixture ratios affecting earthworms is optional, e.g. 0 %, 1 %, 5 %, 25 %,
50 %, 75 %, 100 %, or of the test substance, e.g. 0 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 1 000 mg/kg.
The preliminary test is conducted without replication.
When no effects are observed, even at 100 % contaminated soil or at concentrations of 1 000 mg test
substance/kg standard soil (dry mass), the definitive test can be designed as a limit test.
7.1.3 Definitive test
The design of the definitive test depends on the test objectives. Typically, the habitat properties of samples of a
field-collected test soil are characterized by comparing the biological effects found in the test soil(s) with those
found in the control soil (3.10) (single-concentration tests). If a reference soil (3.8) to be used as a control is not
available or not appropriate due to toxicity or atypical physicochemical characteristics, effects are compared
to a standard soil instead. If a reference soil is available to be used as a control soil, it is recommended that a
standard soil exhibiting a typical known response be included, and that the results be used to judge the validity
[18]
and acceptability of the test . Results found for the standard soil assist in distinguishing contaminant effects
from non-contaminant effects caused by soil physicochemical properties of the test soil and/or the control soil.
If, for characterization purposes, a test design including a dilution series is required, three designs are possible
(the concentrations shall be spaced by a factor not exceeding 2):
— For the NOEC/NOER approach, at least five concentrations in a geometric series should be used. Four
replicates for each concentration plus eight controls are recommended.
6 © ISO 2012 – All rights reserved

ISO 11268-2:2012(E)
— For the EC approach, 12 concentrations should be used. Two replicates for each concentration plus six
x
controls are recommended. The spacing factor can be variable: smaller at low concentrations and larger
at high concentrations.
— For the mixed approach, six to eight concentrations in a geometric series should be used. Four replicates
for each concentration plus eight controls are recommended. This mixed approach allows a NOEC as well
as an EC evaluation.
x
A limit test can be sufficient if, in the preliminary test, no toxic effect was observed. In the limit test, only the test
soil without any dilution and the control shall be tested with at least four replicates each.
7.2 Preparation of test mixture
7.2.1 Testing of contaminated soil
Mix the test soil with the reference soil or the standard soil thoroughly (either manually or by using a hand
mixer) according to the selected dilution range. Check the homogeneity of the mixture visually. The total mass
of the test soil and the reference soil or the standard soil shall be 500 g to 600 g (dry mass) in each test
container (6.1). Wet the test mixture with deionized water to reach an appropriate water content of usually 40 %
to 60 % of the total water holding capacity determined according to Annex C. In some cases, e.g. when testing
waste materials, higher percentages are required. A rough check of the soil moisture content can be obtained
by gently squeezing the soil in the hand; if the moisture content is correct, small drops of water should appear
between the fingers.
Determine the pH for each test mixture (one container per concentration) according to ISO 10390 at the
beginning and end of the test (when acid or basic substances are tested, do not adjust the pH).
Proceed simultaneously with at least four replicates per concentration and the control(s).
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or otherwise
harmful chemicals or infectious microorganisms. Precautions should be taken to avoid skin contact.
Occupational health risks may arise from dust or evaporated chemicals during handling and incubation.
7.2.2 Testing substances added to the control soil
Control soil (5.2.2) is used to prepare the test sample. For each test container (6.1), the mass of the substrate
used shall be 500 g (dry mass). Add substances to the control soil and mix thoroughly.
For the introduction of test substances, use either method a), b) or c), as appropriate:
a) Water-soluble substance
— Immediately before starting the test, dissolve the quantity of the test substance in the water or a portion of
it required to wet the soil substrate for the replicates of one concentration in order to meet the requirements
of 5.2.2. Mix it thoroughly with the soil substrate before introducing it into the test containers.
b) Substances insoluble in water but soluble in organic solvents
— Dissolve the quantity of test substance required to obtain the desired concentration in a volatile solvent
(such as acetone or hexane) and mix it with a portion of the quartz sand required. After evaporating
the solvent by placing the container under a fume hood, add the remainder of the standard soil and
the water and mix it thoroughly before introducing it into the test containers.
Ultrasonic dispersion, organic solvents, emulsifiers or dispersants can be used to disperse substances with low
aqueous solubility. When such auxiliary substances are used, all test concentrations and an additional control
should contain the same minimum amount of auxiliary substance.
WARNING — Take appropriate precautions when dealing with solvent vapour to avoid danger from
inhalation or explosion, and to avoid damage to extraction equipment, pumps, etc.
ISO 11268-2:2012(E)
c) Substances insoluble in water or organic solvents
— For a substance insoluble in a volatile solvent, prepare a mixture of 10 g of finely ground industrial quartz
sand (see 5.2.2) and the quantity of the test substance required to obtain the desired concentration.
Add that mixture to the remainder of the standard soil and the water and mix thoroughly before
introducing it into the test containers.
— Mix the test substance into the standard soil before the earthworms are added.
Base the concentrations selected to provide the LOEC/NOEC on the results of the preliminary test. Space the
concentrations by a factor not exceeding 2. Substances mixed into the substrate do not need to be tested at
concentrations higher than 1 000 mg/kg mass of test sample. Proceed simultaneously with all replicates per
concentration and the control(s) required according to the selected approach.
Determine the pH for each test sample (one container per concentration) according to ISO 10390 at the
beginning and end of the test.
7.2.3 Preparation of control container
The control container contains the control soil (5.2.2) wetted with deionized water to reach 40 % to 60 % of the
total water holding capacity (determined according to Annex C).
Prepare one control container for the preliminary test and at least four control containers for the definitive test.
Prepare the control containers in the same way as the test containers. If the preparation of the test requires
the use of a solvent (see 7.2.2), use an additional control prepared with solvent but without the test substance.
Cover the containers as indicated in 6.1.
7.3 Addition of the earthworms
For each test container and the control container(s), prepare, wash and gently wipe (using absorbent paper) 10
worms (5.1). Determine the homogeneity of the test population by weighing a sample of 20 worms individually, to
avoid systematic errors in distributing the worms to the test containers. Having ensured homogeneity, batches
of 10 worms shall then be selected, weighed and placed in each test container. Assign batches of worms using
a randomization procedure. The range of mean biomass between vessels should not exceed 100 mg.
Cover the containers as indicated in 6.1 and place them in the test enclosure (6.6.1).
7.4 Test conditions and measurements
One day after addition of the worms, spread 5 g per test container of air-dried finely ground food source (5.3)
on the soil surface and moisten with potable water (about 5 ml to 6 ml per container). Feed once a week during
the test period. If food consumption is low, reduce feeding to a minimum to avoid fungal growth or moulding.
Record feeding activity and the quantity of food applied over the test period for each test container.
Maintain the water content of the soil substrate in the test containers during the test period by reweighing
the test containers periodically and, if necessary, by replenishing lost water. At the end of the test, the water
content shall not differ by more than 10 % from that at the beginning of the test.
Keep the adult worms over a period of four weeks in the test sample. At the end of this period, remove the adults
and, for each container, record the total number and mass of living adult worms. Keep the test containers for
another period of four weeks in the test environment (6.6) to allow offspring to develop. At the beginning of this
period, juveniles are fed once with 5 g of food per test container, carefully mixed by hand into the substrate. After
this period, count the number of offspring per test container hatched from the cocoons using a suitable method.
NOTE Annex D gives examples of two suitable methods, including one which allows counting of cocoons.
8 © ISO 2012 – All rights reserved

ISO 11268-2:2012(E)
7.5 Reference substance
The NOEC and/or the EC of a reference substance shall be determined to provide assurance that the laboratory
x
test conditions are adequate and to verify that the response of the test organism does not change statistically
over time. It is advisable to test a reference substance at least twice a year or, when testing is carried out in a
lower frequency, in parallel to the determination of the toxicity of a test substance.
Boric acid is recommended as a reference substance. If the compound is mixed into the substrate, observe the
effects on reproduction (α = 0,05, where α is the level of significance) at concentrations of between 400 mg and
600 mg of boric acid per kilogram dry mass of substrate. The test report on the performance of the reference
compound shall be completed periodically and if the test conditions have changed.
8 Calculation and expression of results
8.1 Calculation
For each dilution or concentration, determine the percent mortality, the percent loss/increase in biomass of the
adults after four weeks, and the number of offspring produced after another period of four weeks.
8.2 Expression of results
A graphical presentation of the mean values of the end points, including standard deviation of the measured
values against the test soil(s), control soil(s) or the selected series of soil mixture ratios, should be prepared.
This comparison or curve gives an impression of the quality of effects and their magnitudes. Express the
mixture ratio as based on soil dry mass.
If dilution or concentration series were performed, indicate:
— in the EC approach, the percent soil mixture based on dry mass or in milligrams per kilogram of dried soil
x
substrate, the median percent dilution of contaminated soil or median concentration of the test substance,
which reduces the number of juvenile worms to 50 % (EC ) compared to the control within the test period, o
...