ISO 11268-2:2023
(Main)Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei and other earthworm species
Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on reproduction of Eisenia fetida/Eisenia andrei and other earthworm species
This document 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. This method is designed mainly for determining the effects of soil contaminants and chemicals on the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method also includes technical information on how to use it with other environmentally relevant earthworm species: e.g. Dendrodrilus rubidus and Aporrectodea caliginosa (see Annexes B and C). This method does not apply 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 persistence of the substance during the test.
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 et d’autres espèces de vers de terre
Le présent document spécifie l’une des méthodes permettant d’évaluer la fonction d’habitat des sols et de déterminer les effets de polluants et de produits chimiques présents dans le sol sur la reproduction d’Eisenia fetida/Eisenia andrei par absorption cutanée et ingestion. Cet essai chronique s’applique aux sols et aux matériaux de type sol de qualité inconnue, provenant par exemple de sites pollués, de sols amendés, de sols après dépollution, de sols agricoles ou autres sites d’intérêt et de déchets. Cette méthode est principalement destinée à déterminer les effets de polluants et de produits chimiques présents dans le sol sur la reproduction d’Eisenia fetida/Eisenia andrei. Des informations techniques sont également fournies sur la manière d’utiliser Eisenia fetida/andrei pour évaluer des produits chimiques dans des conditions tropicales (voir l’Annexe A). Enfin, cette méthode intègre également des informations techniques sur la manière de l’utiliser avec d’autres espèces de vers de terre pertinentes sur le plan environnemental: par exemple, Dendrodrilus rubidus et Aporrectodea caliginosa (voir les Annexes B et C). Cette méthode ne s’applique ni aux substances pour lesquelles le coefficient de partage air/sol est supérieur à un, ni aux substances pour lesquelles la pression de vapeur à 25 °C excède 300 Pa. Cette méthode ne tient pas compte de la rémanence de la substance pendant l’essai.
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 11268-2
Third edition
2023-01
Soil quality — Effects of pollutants on
earthworms —
Part 2:
Determination of effects on
reproduction of Eisenia fetida/Eisenia
andrei and other earthworm species
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 et d’autres espèces de vers de terre
Reference number
© ISO 2023
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Published in Switzerland
ii
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 . 6
7 Procedure .6
7.1 Experimental design . 6
7.1.1 General . 6
7.1.2 Preliminary test . 7
7.1.3 Definitive test . 7
7.2 Preparation of test mixtures . 7
7.2.1 Testing of contaminated soil . 7
7.2.2 Testing substances added to the control soil . 8
7.2.3 Preparation of control container . 8
7.3 Addition of the earthworms . 9
7.4 Test conditions and measurements . 9
7.5 Reference substance . 9
8 Calculation and expression of results .10
8.1 Calculation . 10
8.2 Expression of results . . 10
9 Validity of the test .10
10 Statistical analysis .10
10.1 General . 10
10.2 Single-concentration tests . 10
10.3 Multi-concentration tests . 11
10.3.1 Preliminary test . 11
10.3.2 Definitive test . 11
11 Test report .12
Annex A (informative) Determination of the chronic toxicity of chemicals on Eisenia fetida/
Eisenia andrei under tropical conditions .13
Annex B (informative) Culturing and testing procedures for assessing the effects
of contaminants on juvenile production using the earthworm, Dendrodilus rubidus .15
Annex C (informative) Culturing and testing procedures for assessing the effects
of contaminants on adult survival and cocoon production and hatchability using
the earthworm, Aporrectodea caliginosa s.s. .19
Annex D (informative) Culturing of Eisenia fetida and Eisenia andrei .23
Annex E (normative) Determination of water holding capacity .24
Annex F (informative) Techniques for counting juvenile worms hatched from cocoons.25
Annex G (informative) Experience with performance criteria of the test .26
Bibliography .33
iii
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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
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 11268-2:2012), which has been
technically revised.
The main changes are as follows:
— modification of the concentration for the reference substance (boric acid);
— 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.
A list of all the parts in the ISO 11268 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
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 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.
This document 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 or test soil under tropical conditions (see Annex A).
Eisenia fetida and Eisenia andrei are considered to be representatives of soil fauna and earthworms in
particular in temperate regions. Background information on their earthworm ecology 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 Annexes B and C). These alternative earthworm
species have been used as ecotoxicological test species for some time, however, testing experience has
been limited to specific countries.
This document has been drawn up taking into consideration test procedures adopted by the
[45][46] [29]
Organization for Economic Cooperation and Development and by the European Union .
v
INTERNATIONAL STANDARD ISO 11268-2:2023(E)
Soil quality — Effects of pollutants on earthworms —
Part 2:
Determination of effects on reproduction of Eisenia fetida/
Eisenia andrei and other earthworm species
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 document 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.
This method is designed mainly for determining the effects of soil contaminants and chemicals on
the reproduction of Eisenia fetida/Eisenia andrei. Technical information is also provided on how to use
Eisenia fetida/andrei for testing chemicals under tropical conditions (see Annex A). Finally, this method
also includes technical information on how to use it with other environmentally relevant earthworm
species: e.g. Dendrodrilus rubidus and Aporrectodea caliginosa (see Annexes B and C).
This method does not apply 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 persistence of the substance during the test.
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 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
contaminant
substance or agent present in the soil as a result of human activity
[SOURCE: ISO 15176:2019, 3.2.6]
3.2
growth
increase in biomass (i.e. the fresh mass of organisms)
Note 1 to entry: 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 container after eight weeks’ incubation under the specified test
conditions
3.4
ER
x
EC
x
effective rate
effective concentration
x % effect rate or concentration of the test sample or test substance at which reproduction (3.3) 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 (3.10) and the control
3.6
LOER
LOEC
lowest observed effect rate
lowest observed 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 LOEC is expressed as a percentage of test-soil dry mass per test-mixture dry mass. All test
mixtures (3.11) 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
NOEC
no observed effective rate
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 (3.3) or mass alteration (error probability: p < 0,05)
Note 1 to entry: 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
[29] [46] [40]
EXAMPLE Euro-Soils, artificial soil, 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 or the test substance with a control soil (3.10)
Note 1 to entry: Test mixtures are given in percent of contaminated soil based on soil dry mass.
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 , 56 d), respectively.
x x
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 recommended 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;
— a 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 A reference soil is used to demonstrate the appropriate status of the test population, and to avoid
misinterpretation of results.
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).
Alternative species of earthworms and their respective requirements are added as specific annexes in
this document:
— Dendrodrilus rubidus (see Annex B);
— Aporrectodea caliginosa (see Annex C).
5 Reagents and material
5.1 Biological material, consists of adult earthworms of the species Eisenia fetida or Eisenia
[32][36][37]
andrei , which are between three 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). It is recommended
to check the identity of the strain used to avoid species misidentifications. DNA barcoding described in
ISO 21286 is suitable for that purpose.
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
[57]
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 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 2 An example of culturing Eisenia fetida/Eisenia andrei is given in Annex D.
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 5.4), 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.
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, composted
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 18400-206 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 E;
— cationic exchange capacity in accordance with ISO 11260;
— organic carbon in accordance with ISO 10694.
The water holding capacity of all mixtures used in the test should also be measured.
5.3 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 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.
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 %
— I ndu s t r i a l qu a r t z s a nd (dom i n a nt f i ne s a nd w it h mor e 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
ow ow
ionizing 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 for adult survival and
[20]
juvenile 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. It is therefore not necessary, before using such a
soil in a definitive test, to demonstrate the suitability of the artificial soil in complying with the validity criteria,
[30].
unless the peat content is lower than 5 %
Prepare the artificial soil at least three days prior to starting the test, by mixing the dry constituents
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 the 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 water holding capacity is determined according to Annex E; the pH is determined according to
ISO 10390.
5.4 Feeding, any food of a quality shown to be suitable for at least maintaining worm mass during
[37]
the test is 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 can 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
[45]
substrate that does not contain the new batch of food (conditions as described in OECD 207 ).
5.5 Boric acid, as reference substance.
6 Apparatus
The usual laboratory equipment and the following shall be used.
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, 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 lx to 800 lx on the containers at a controlled light/dark cycle of between 12 h:12 h and 16 h:8 h.
7 Procedure
7.1 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. 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).
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 (single-concentration tests). If a reference soil 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
[35]
included, and that the results be used to judge 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 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 two (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.
— For the EC approach, 12 concentrations should be used. Two replicates for each concentration
x
plus six control replicates 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 control replicates are recommended. This mixed
approach allows a NOEC as well as an EC estimate to be calculated.
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 mixtures
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 E. 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 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 5.3. 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.
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 5.3) 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 wetted with deionized water to generally reach 40 %
to 60 % of the total water holding capacity (determined according to Annex E) and consistent with the
rate used in the soil to be tested.
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.
Attention should be paid to the variation of mean biomass between containers. It is recommended that
this variation does not exceed 150 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
(5.4) on the soil surface and moisten with potable water (about 5 ml to 10 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. It is
recommended that the water content does not differ by more than 10 % from that of 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 F gives examples of two suitable methods, including one which allows counting of cocoons.
7.5 Reference substance
The NOEC and/or the EC of a reference substance shall be determined to provide assurance that the
x
laboratory 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, effects
on reproduction should be observed (α = 0,05, where α is the level of significance) at concentrations of
between 200 mg and 600 mg of boric acid per kilogram dry mass of substrate (see Annex G).
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
x
dried soil 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, or
— in the
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