prEN ISO 11268-1

SLOVENSKI STANDARD
01-julij-2026
Kakovost tal - Učinki onesnaževal na deževnike - 1. del: Določanje akutne
strupenosti za Eisenia fetida/Eisenia andrei in druge vrste deževnikov (ISO/DIS
11268-1:2026)
Soil quality - Effects of pollutants on earthworms - Part 1: Determination of acute toxicity
to Eisenia fetida/Eisenia andrei and other earthworm species (ISO/DIS 11268-1:2026)
Bodenbeschaffenheit - Auswirkungen von Schadstoffen auf Regenwürmer - Teil 1:
Bestimmung der akuten Toxizität für Eisenia fetida/Eisenia andrei (ISO/DIS 11268-
1:2026)
Qualité du sol - Effets des polluants vis-à-vis des vers de terre - Partie 1: Détermination
de la toxicité aiguë vis-à-vis d'Eisenia fetida/Eisenia andrei et d’autres espèces de vers
de terre (ISO/DIS 11268-1:2026)
Ta slovenski standard je istoveten z: prEN ISO 11268-1
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.

DRAFT
International
Standard
ISO/DIS 11268-1
ISO/TC 190/SC 4
Soil quality — Effects of pollutants
Secretariat: AFNOR
on earthworms —
Voting begins on:
Part 1: 2026-05-05
Determination of acute toxicity to
Voting terminates on:
2026-07-28
Eisenia fetida/Eisenia andrei and
other earthworm species
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 1: Détermination de la toxicité aiguë vis-à-vis de Eisenia
fetida/Eisenia andrei et d’autres espèces de vers de terre
ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
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Reference number
ISO/DIS 11268-1:2026(en)
DRAFT
ISO/DIS 11268-1:2026(en)
International
Standard
ISO/DIS 11268-1
ISO/TC 190/SC 4
Soil quality — Effects of pollutants
Secretariat: AFNOR
on earthworms —
Voting begins on:
Part 1:
Determination of acute toxicity to
Voting terminates on:
Eisenia fetida/Eisenia andrei and
other earthworm species
Qualité du sol — Effets des polluants vis-à-vis des vers de terre —
Partie 1: Détermination de la toxicité aiguë vis-à-vis de Eisenia
fetida/Eisenia andrei et d’autres espèces de vers de terre
ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2026
TECHNOLOGICAL, COMMERCIAL AND
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Published in Switzerland Reference number
ISO/DIS 11268-1:2026(en)
ii
ISO/DIS 11268-1:2026(en)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents and material . 4
5.2 Test sample, which may consist of field-collected soil, waste material or control soil
amended by the test substance. .4
6 Apparatus: Usual laboratory equipment and the following . 6
6.6 Test environment .6
7 Procedure . 6
7.1 Experimental design .6
7.1.1 General .6
7.1.2 Range-finding test (preliminary test) .6
7.1.3 Definitive test .7
7.2 Preparation of test mixture .7
7.2.1 Testing of contaminated soil and waste materials .7
7.2.2 Testing substances added to the control soil .7
7.2.3 Preparation of control container .8
7.3 Addition of the earthworms .8
7.4 Test conditions and measurements .9
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 .10
10 Statistical analysis .10
10.1 General .10
10.2 Single-concentration tests .10
10.2.1 Quantal effects .10
10.2.2 Quantitative effects .10
10.3 Multi-concentration tests .11
10.3.1 General .11
10.3.2 EC approach .11
x
10.3.3 NOEC approach .11
11 Test report .12
Annex A (informative) Assessment of the habitat function of soils and determination of the
acute toxicity of soil contaminants and chemicals under tropical test 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) Background information on the acute effect of boric acid on earthworms . 17
Annex E (informative) Culturing and testing procedures for assessing the effects of
contaminated soil, waste materials and chemicals on adult mortality using the
earthworm, Dendrodilus rubidus .18

iii
ISO/DIS 11268-1:2026(en)
Annex F (informative) Culturing and testing procedures for assessing the effects of
contaminated soils, waste materials and chemicals on adult mortality using the
earthworm, Aporrectodea caliginosa s.s. .21
Annex G (informative) Test design to acquire data for TKTD modelling .25
Bibliography .27

iv
ISO/DIS 11268-1:2026(en)
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.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement) .
This third edition cancels and replaces the second edition (ISO 11268-1:2012), which has been technically
revised.
The main changes are as follows:
— correction of the vapour pressure value to update Clause 1 according to the recommendations of ECHA/
OECD;
— inclusion of additional technical information on the testing of waste materials;
— inclusion of alternative species of earthworms – Dendrodrilus rubidus, Aporrectodea caliginosa – in
informative annexes; information on their taxonomy and ecology as well as their specific testing
requirements have also been added;
— inclusion of a test design to acquire data for toxicokinetic-toxicodynamic (TKTD) modelling in an
informative annex.
A list of all the parts in the ISO 11268 series can be found on the ISO website.

v
ISO/DIS 11268-1:2026(en)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil and
[1] [2]
are proposed to complement conventional chemical analysis (see ISO 15799 and ISO 17616 ). ISO 15799
[1]
includes a list and short characterization of recommended and standardized test systems, and ISO 17616
[2]
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 laboratory 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 the acute toxicity of
contaminated soils or waste materials to 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 a standard soil
(e.g. artificial soil) for their acute toxic 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. However, these species do not occur regularly in agricultural lands (crop sites and grasslands)
or forests in these regions. In addition, they are not representative of boreal or tropical regions. Therefore,
other species such as Dendrodilus rubidus (an epigeic litter inhabitant in boreal regions) and Aporrectodea
caliginosa (an endogeic mineral dweller in temperate regions) have been added as potential alternative test
species (see Annex E and Annex F). Other species, e.g. 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,
[3][4][5]
but the data basis and experience in testing soils are small.
This part of ISO 11268 has been drawn up taking into consideration test procedures adopted by the
[6] [7]
Organization for Economic Cooperation and Development (OECD 207 , OECD 222 ).

vi
DRAFT International Standard ISO/DIS 11268-1:2026(en)
Soil quality — Effects of pollutants on earthworms —
Part 1:
Determination of acute toxicity to Eisenia fetida/Eisenia
andrei and other earthworm species
WARNING — 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 acute toxicity of soil contaminants, waste materials and chemicals to Eisenia fetida/Eisenia
andrei by dermal and alimentary uptake. It 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 on survival 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) can 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 substances for which the air/soil partition coefficient is greater than one, or
to substances with vapour pressure exceeding 300 Pa at 25 °C.
This method does not take into account the possible degradation of the substances or contaminants during
the test. This method also includes technical information on how to use it with other environmentally
relevant earthworm species, i.e. Dendrodrilus rubidus and Aporrectodea caliginosa (see Annex E and Annex F)
as well as a test design to acquire data for toxicokinetic-toxicodynamic (TKTD) modelling (Annex G).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements 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, treated biowaste and sludge – 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 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/DIS 11268-1:2026(en)
ISO 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic
conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
EN 14735, Characterization of waste - Preparation of waste samples for ecotoxicity tests
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
contaminant
substance or agent present in the soil as a result of human activity
[8]
[SOURCE: ISO 15176:2002 ]
3.2
survival
percentage of living worms at each measurement time
3.3
mortality
percentage of dead or missing worms at the end of the test period
3.4
lethal concentration
LC
median lethal percentage of a test sample in a reference (3.8) or a standard soil (3.9), or concentration of a
substance in the test sample, which kills 50 % of the test animals within the test period
Note 1 to entry: The LC is expressed as a percentage of test-soil dry mass per test mixture (3.11) dry mass.
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 (3.10) and the control
3.6
LOER
LOEC
lowest observed effect rate or effect concentration
lowest tested percentage of a test sample in a control soil (3.10) or concentration of a substance at which a
statistically significant effect is observed
Note 1 to entry: The LOER/LOEC is expressed as a percentage of test soil dry mass per test mixture (3.11) dry mass. All
test mixtures above the LOER/LOEC have a harmful effect equal to or greater than that observed at the LOER/LOEC. If
this condition cannot be satisfied, an explanation should be given for how the LOER/LOEC and NOER/NOEC (3.7) have
been selected.
ISO/DIS 11268-1:2026(en)
3.7
NOEC
no observed effect rate or effect concentration
NOER
test soil percentage immediately below the LOER/LOEC (3.6) or highest tested concentration of a test
substance which, when compared to the control, has no statistically significant lethal or other effect such as
mass alteration (error probability: p < 0,05)
Note 1 to entry: The NOER/NOEC is expressed as a percentage of test soil dry mass per test mixture (3.11) 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
[9] [6] [10]
EXAMPLE Euro-Soils, artificial soil (OECD 207 ), LUFA Standard Soil.
Note 1 to entry: The properties of standard soils can differ from those of the test soil.
3.10
control soil
reference (3.8) or standard soil (3.9) 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 1 to entry: 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, waste material or the test substance with a control soil (3.10)
Note 1 to entry: Test mixtures are given in percentage of contaminated soil based on soil dry mass.
3.12
test mixture ratio
ratio of test soil to control soil (3.10) in a test mixture (3.11)
Note 1 to entry: Different ratios may be applied in a dilution series to establish a dose-response relationship.
4 Principle
The percent mortality of adult earthworms (species: Eisenia fetida or Eisenia andrei) exposed to the test soil
or test mixture are compared to those exposed to a control soil. If appropriate, effects based on exposure
to a dilution range of contaminated soil, waste material or range of concentrations of a test substance are
determined after 7 days and 14 days. Test mixtures are prepared at the start of the test and are not renewed
within the test period.
The results obtained from the tests are compared with a control and are used to determine the dilutions or
concentrations which cause no effects on biomass and survival (NOER/NOEC) and the mortality of 50 % of
earthworms (LC , 14 days).
50 (3.4)
The test is conducted in two steps:
— a preliminary test, which gives an approximate indication of the dilutions (concentrations) responsible
for total mortality and for the absence of mortality, which serves to determine the range of concentrations
for the definitive test;
ISO/DIS 11268-1:2026(en)
— the definitive test to determine the dilutions (concentrations) causing between 10 % and 90 % mortality,
which yields the test result.
If the preliminary test shows no mortality, a limit test (see 7.1.3) may be performed as the definitive test.
NOTE The use of a reference soil is essential to demonstrate the present status of the test population, and to avoid
misinterpretation of results.
Alternative species of earthworms and their respective requirements as well as a test design to acquire data
for TKTD modelling are added as specific annexes in this document:
— Dendrodrilus rubidus (see Annex E);
— Aporrectodea caliginosa (see Annex F);
— Test design to acquire data for TKTD modelling (see Annex G).
5 Reagents and material
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia
[11][12][13]
andrei at least three months 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). It is recommended to check the identity of the strain
[14]
used to avoid species misidentifications. DNA barcoding described in ISO 21286 is suitable for that
purpose.
[14]
NOTE 1 During the ring test performed to validate ISO 21286 , only 17 of the 28 laboratories involved (61
%) provided correct identification of their laboratory culture of compost worms. Most laboratories with wrong or
[15]
unknown assignments had E. andrei in culture, or a mixture of both species .
Select worms used for the test to form, as far as is practicable, a homogeneous population from the
standpoint of size and mass. Worms should preferably be selected from a synchronized culture with a
relatively homogeneous age structure. Before the test, wash them with deionized or potable water.
NOTE 2 An example of culturing Eisenia fetida/Eisenia andrei is given in Annex B.
5.2 Test sample, which may consist of field-collected soil, waste material or control soil
amended by the test substance.
5.2.1 Field-collected soil or waste material
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, the soil or waste material
may be air-dried without heating before sieving. Storage of the soil or waste material to be tested should be
as short as possible. The test sample shall be stored in accordance with ISO 18400-206 (soils) and EN 14735
(waste materials), using containers that minimize losses of contaminants by volatilization and sorption
to the container walls. If soils or test mixtures have been stored, they should be mixed a second time
immediately before use. Soil pH should not be corrected as it can influence bioavailability of contaminants.
For interpretation of test results, the following characteristics shall be determined for each soil sampled
from a field site:
a) pH in accordance with ISO 10390;
b) texture (sand, loam, silt) in accordance with ISO 11277;
c) water content in accordance with ISO 11465;
d) water holding capacity according to Annex C;

ISO/DIS 11268-1:2026(en)
e) cationic exchange capacity in accordance with ISO 11260;
f) organic carbon in accordance with ISO 10694;
g) percentage of material removed by the 4 mm sieve.
According to the objective of the study, the control and dilution substrate (dilution series of contaminated
soil or waste material) should be either an uncontaminated soil comparable to the soil sample (reference soil)
to be tested or standard soil, preferably the artificial soil substrate.
NOTE It is not practical to measure the water holding capacity of all mixtures used in the test. 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 appears between the fingers.
5.2.2 Control soil, either a) reference soil or b) standard soil 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 soil should be preferred.
b) For testing the effects of substances mixed into soil or making dilutions of the test sample, standard soil
shall be used to prepare the test sample. The properties of the field-collected standard soil shall be
reported.
The substrate called artificial soil can be used as a standard soil and has the following composition (Table 1):
Table 1
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 than 50 % of particle sizes 69 %
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 As there are fewer and fewer peatlands in Europe and most of them are in a poor ecological state, peat
extraction is to be restricted in future. To continue ecotoxicological testing, the development of a peat alternative as
an organic component in artificial soils is needed. Research activities to address this issue are currently under way.
NOTE 2 Taking the properties of highly non-polar [log K > 2, where K is the partitioning coefficient (octanol/
ow ow
water)] or ionizing substances into account, 5 % of peat and 74 % of quartz sand have proven to be sufficient for
maintaining the desired structure of the artificial soil.
NOTE 3 It has been demonstrated that Eisenia fetida can comply with the validity criteria, even as regards
[12]
reproduction, 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 and 74 % of quartz sand. It is therefore not needed, before using
such a soil in a definitive test, to demonstrate the suitability of the artificial soil in complying with the validity criteria,
[7]
unless the peat contents lowered more than specified above.OECD 222
Prepare the artificial soil at least three days prior to starting the test, by mixing the dry constituents listed
above, e.g. in a large-scale laboratory mixer. The pH is determined and if necessary adjusted. The amount
of calcium carbonate required can vary, depending on the properties of the individual batch of sphagnum
peat. The maximum water holding capacity is determined (see Annex C). A portion of the deionized water
required is added two to seven days before starting the test to obtain approximately half of the required
final water content of 40 % to 60 % of the maximum water holding capacity. Allowance should be made for
any water that is used for introducing the test substance into the soil. Store the mixed artificial soil at room
temperature for at least two days until starting the test to equilibrate acidity.

ISO/DIS 11268-1:2026(en)
The total water holding capacity shall be determined in accordance with Annex C, the pH shall be determined
according to ISO 10390.
5.2.3 Boric acid, as a reference substance (see Annex D).
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).
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 as lid to cover the test vessels, 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 or waste material can be tested at a single concentration (typically 100 %
for field-collected soil or 25 % for waste material when ratios above 25 % are usually not practical, e.g.
sludge or manure) or evaluated for toxicity in a multi-concentration test, whereby a series of concentrations
(dilutions) is 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 (5.2.2).
Depending on the knowledge of relevant response levels, a preliminary test may precede the definitive test.
7.1.2 Range-finding test (preliminary test)
A preliminary test to find the range of test mixture ratios affecting earthworms is optional, e.g. 0 %, 1 %,
5 %, 25 %, 50 %, 75 %, 100 % for field-sampled soil and 0 %, 1,56 %, 3,12 %, 6,25 %, 12,5 %, 25 % for waste
material (e.g. sludge or manure), 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.

ISO/DIS 11268-1:2026(en)
When no effect is observed, even at 100 % contaminated soil, 25 % waste material 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 (single-concentration tests). If a reference soil to be used as a control is not
available or not appropriate due to toxicity or atypical physico-chemical 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
[12]
the validity and acceptability of the test. Results found for the standard soil assist in distinguishing
contaminant effects from non-contaminant effects caused by soil physico-chemical properties.
If, for characterization purposes, a test design including a dilution series is required, a minimum of five
test mixtures shall be prepared. Therefore, a geometric series of test mixture ratios with a factor not
exceeding 2 shall be selected based on the preliminary test. At least four replicates of each treatment are
prepared. 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 soil shall be tested with at least four replicates each.
When testing chemicals added to standard soil, perform the definitive test on five concentrations of the
test substance, providing a geometric progression between the highest concentration causing no mortality
and the lowest concentration causing total mortality. Proceed simultaneously with four replicates per
concentration and for a control without the test substance and, if necessary, for another control with solvent,
placing each container in the test environment (6.6).
To increase the precision and power of statistical testing when analysing quantitative single-concentration
tests (e.g. effects on biomass development), it is recommended that eight replicates be prepared for the
control.
7.2 Preparation of test mixture
7.2.1 Testing of contaminated soil and waste materials
Mix the test soil or waste material 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 test mixture
visually. The total mass of the test mixture 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 or lower percentages are required. A rough check
of the test mixture moisture content can be obtained by gently squeezing the test mixture 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 test mixture and the control(s).
WARNING — Contaminated soils and waste materials can contain unknown mixtures of toxic,
mutagenic, or otherwise harmful chemicals or infectious microorganisms. Take precautions to avoid
skin contact. Occupational health risks can 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.

ISO/DIS 11268-1:2026(en)
For the introduction of test substances, use either method a), b) or c), as appropriate:
a) Water-soluble substance
1) 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 samples 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
1) 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 (up to 10 %) 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 may 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 — 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.
c) Substances insoluble in water or organic solvents
1) For a substance insoluble in a volatile solvent, separate a portion (up to 10 %) of the quartz sand
required (see 5.2.2). Prepare a mixture of an appropriate quantity of quartz sand and the quantity
of the test substance required to obtain the desired concentration. Add that mixture and any
unused quartz sand to the remainder of the standard soil and the water and mix thoroughly before
introducing it into the test containers.
2) 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 at least four
replicates per concentration and the control(s).
Determine the pH for each test sample (one c
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