SIST EN ISO 11267:2014

SLOVENSKI STANDARD
01-maj-2014
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Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil
contaminants (ISO 11267:2014)
Bodenbeschaffenheit - Hemmung der Reproduktion von Collembolen (Folsomia candida)
durch Verunreinigungen (ISO 11267:2014)
Qualité du sol - Inhibition de la reproduction de Collembola (Folsomia candida) par des
contaminants du sol (ISO 11267:2014)
Ta slovenski standard je istoveten z: EN ISO 11267:2014
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 11267
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2014
ICS 13.080.30
English Version
Soil quality - Inhibition of reproduction of Collembola (Folsomia
candida) by soil contaminants (ISO 11267:2014)
Qualité du sol - Inhibition de la reproduction de Collembola Bodenbeschaffenheit - Hemmung der Reproduktion von
(Folsomia candida) par des contaminants du sol (ISO Collembolen (Folsomia candida) durch Verunreinigungen
11267:2014) (ISO 11267:2014)
This European Standard was approved by CEN on 4 January 2014.

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
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11267:2014 E
worldwide for CEN national Members.

Contents Page
Foreword .3
Foreword
This document (EN ISO 11267:2014) has been prepared by Technical Committee ISO/TC 190 “Soil quality” in
collaboration with 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 August 2014, and conflicting national standards shall be withdrawn at
the latest by August 2014.
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 11267:2014 has been approved by CEN as EN ISO 11267:2014 without any modification.

INTERNATIONAL ISO
STANDARD 11267
Second edition
2014-02-15
Soil quality — Inhibition of
reproduction of Collembola (Folsomia
candida) by soil contaminants
Qualité du sol — Inhibition de la reproduction de Collembola
(Folsomia candida) par des contaminants du sol
Reference number
ISO 11267:2014(E)
©
ISO 2014
ISO 11267:2014(E)
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
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Published in Switzerland
ii © ISO 2014 – All rights reserved

ISO 11267:2014(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Reagents and material . 4
6 Apparatus . 6
7 Procedure. 6
7.1 Experimental design . 6
7.2 Preparation of test mixture . 7
7.3 Addition of the biological material . 9
7.4 Test conditions and measurements . 9
7.5 Determination of surviving Collembola . 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
10.3 Multi-concentration tests.11
11 Test report .11
Annex A (informative) Techniques for rearing and breeding of Collembola .13
Annex B (informative) Determination of water-holding capacity .15
Annex C (informative) Guidance on adjustment of pH of artificial soil .16
Annex D (informative) Extraction and counting of Collembola .17
Bibliography .18
ISO 11267:2014(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.
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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
This second edition cancels and replaces the first edition (ISO 11267:1999), which has been technically
revised.
iv © ISO 2014 – All rights reserved

ISO 11267:2014(E)
Introduction
Ecotoxicological test systems are applied to obtain information about the effects of contaminants in soil
and are proposed to complement conventional chemical analysis (see [2] and [4]). Reference [2] includes
a list and short characterization of recommended and standardized test systems and [4] 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.
Soil-dwelling Collembola are ecologically relevant species for ecotoxicological testing. Springtails are
prey animals for a variety of endogeic and epigeic invertebrates and they contribute to decomposition
processes in soils. In acidic soils they may be the most important soil invertebrates besides enchytraeids
[19]
with respect to that function, since earthworms are typically absent. Additionally, Collembola
represent arthropod species with a different route and a different rate of exposure compared to
[1] [3]
earthworms and enchytraeids. Various species were used in bioassays of which four species were
used most commonly, Folsomia candida, Folsomia fimetaria, Onychiurus armatus, and Orchesella cincta.
[20]
Numerous soil toxicity tests supported by Environment Canada (EC) resulted in the development
and standardization of a biological test method for determining the lethal and sublethal toxicity of
[10]
samples of contaminated soil to Collembola. The method prepared by EC includes three species,
Orthonychiurus folsomi, Folsomia candida, and Folsomia fimetaria. As standardized test systems using
Collembola as indicator organisms for the habitat function of soil, another two methods exist. One is
designed for assessing the effects of substances on the reproductive output of the Collembola, Folsomia
[19] [21]
fimetaria L. and Folsomia candida Willem in soil, , , and the other method described here, focuses on
testing contaminated soil. Optionally the method can be used for testing substances added to standard
soils (e.g. artificial soil) for their sublethal hazard potential to Collembola.
This International Standard describes a method that is based on the determination of sublethal effects
of contaminated soils to adult Collembola of the species Folsomia candida Willem. The species is
[10] [19]
distributed worldwide. It plays a similar ecological role to Folsomia fimetaria. , Folsomia candida
reproduces parthenogenetically and is an easily accessible species as it is commercially available and
easy to culture. Folsomia candida is considered to be a representative of soil arthropods and Collembola
in particular. Background information on the ecology of springtails and their use in ecotoxicological
[22]
testing is available.
INTERNATIONAL STANDARD ISO 11267:2014(E)
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil contaminants
1 Scope
This International Standard specifies one of the methods for evaluating the habitat function of soils and
determining effects of soil contaminants and substances on the reproduction of Folsomia candida Willem
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, industrial, agricultural or
other sites of concern 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 soil to be tested and in a control soil. According to
the objective of the study, the control and dilution substrate (dilution series of contaminated soil) are
either an uncontaminated soil comparable to the soil to be tested (reference soil) or a standard soil (e.g.
artificial soil).
This International Standard provides information on how to use this method for testing substances
under temperate 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.
NOTE The stability of the test substance cannot be ensured over the test period. No provision is made in the
test method for monitoring the persistence of the substance under test.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 10390, Soil quality — Determination of pH
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO 11267:2014(E)
3.1
contaminant
substance or agent present in the soil as a result of human activity
3.2
ECx
effect concentration for x % effect
concentration (mass fraction) of a test substance that causes x % of an effect on a given end-point within
a given exposure period when compared with a control
EXAMPLE An EC50 is a concentration estimated to cause an effect on a test end-point in 50 % of an exposed
population over a defined exposure period.
Note 1 to entry: The ECx is expressed as a percentage of soil to be tested (dry mass) per soil mixture (dry mass).
When substances are tested, the ECx is expressed as mass of the test substance per dry mass of soil in milligrams
per kilogram.
3.3
ERx
effect rate
rate of a soil to be tested that causes an x % of an effect on a given end-point within a given exposure
period when compared with a control
3.4
limit test
single concentration test consisting of at least four replicates each, the soil to be tested without any
dilution or the highest concentration of test substance mixed into the control soil and the control
3.5
LOEC
lowest observed effect concentration
lowest test substance concentration that has a statistically significant effect (probability p < 0,05)
Note 1 to entry: In this test, the LOEC is expressed as a mass of test substance per dry mass of the soil to be
tested. All test concentrations above the LOEC should usually show an effect that is statistically different from
the control.
3.6
LOER
lowest observed effect rate
lowest rate of a soil to be tested in a control soil at which a statistically significant effect is observed
3.7
NOEC
no observed effect concentration
highest test substance concentration immediately below the LOEC at which no effect is observed
Note 1 to entry: In this test, the concentration corresponding to the NOEC has no statistically significant effect
(probability p < 0,05) within a given exposure period when compared with the control.
3.8
NOER
no observed effect rate
lowest rate of a soil to be tested immediately below the LOER which, when compared to the control, has
no statistically significant effect (probability p < 0,05) within a given exposure period
3.9
reference soil
uncontaminated soil with comparable pedological properties (nutrient concentrations, pH, organic
carbon content and texture) to the soil being studied
2 © ISO 2014 – All rights reserved

ISO 11267:2014(E)
3.10
standard soil
field-collected soil or artificial soil whose main properties (pH, texture, organic matter content) are
within a known range
EXAMPLE Euro soils, artificial soil, LUFA Standard soil.
Note 1 to entry: The properties of standard soils can differ from the soil to be tested.
3.11
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with soils to
be tested 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.
3.12
test mixture
mixture of contaminated soil or the test substance (e.g. chemical, biosolid, waste) with control soil
3.13
test mixture ratio
ratio between the soil to be tested and the control soil in a test mixture
4 Principle
The effects on reproduction of 10 d to 12 d old Collembola (Folsomia candida) exposed to the soil to be
tested are compared to those observed in a control soil. If appropriate, effects based on exposure to a
test mixture of contaminated soil and control soil or a range of concentrations of a test substance mixed
into control soil are determined. Test mixtures are prepared at the start of the test and are not renewed
within the test period.
The Collembola are incubated until offspring (F ) emerge from eggs laid by mature adults, and the
number of offspring is determined. Usually offspring emerge within 28 d in control experiments. The
results obtained from the tests are compared with a control or, if appropriate, are used to determine
the concentrations which cause no effects on mortality and reproduction (NOER/NOEC) and the
concentration resulting in x% reduction of juveniles hatched from eggs compared to the control
(ERx/ECx, 28 d) respectively.
If testing a concentration series, all test dilutions/concentrations above the LOER/LOEC have a harmful
effect equal to or greater than that observed at the LOER/LOEC. Where there is no prior knowledge of
the concentration of the soil to be tested or the test substance likely to have an effect, then it is useful to
conduct the test in two steps:
— An acute toxicity test (range-finding test) is carried out, to give an indication of the effect
dilution/concentration, and the dilution/concentration giving no mortality (NOER/NOEC).
Dilutions/concentrations to be used in the definitive test can then be selected;
— the definitive test on reproduction to determine sublethal effects of (dilutions of) contaminated
soil or the concentration of a substance which, when evenly mixed into the standard soil, causes
no significant effects on numbers of offspring hatched from eggs 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 11267:2014(E)
5 Reagents and material
5.1 Biological material, in this test, 10 d to 12 d old juvenile springtails of the species Folsomia candida
Willem are used (see A.1 for details on synchronization of breeding).
5.2 Test mixture, which may consist of field-collected soil or control soil amended by the test substance.
5.2.1 Field-collected soil or waste
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.
The field-collected soils used in the test shall be passed through a sieve of 4 mm square mesh to remove
coarse fragments and thoroughly mixed. If necessary, soil may be air-dried without heating before
sieving. Storage of soil to be tested should be as short as possible. The soil shall be stored in accordance
with ISO 10381-6 using containers that minimize losses of soil 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 soil
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 B,
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
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 (3.9) or b) standard soil (3.10) that allows the presence of
Collembola. Control soil and soil used for dilution shall not differ in one test (either a) or b)).
a) If reference soils from uncontaminated areas near a contaminated site are available, they should
be treated and characterized like the soils to be tested. 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, standard soils (e.g. artificial soil, LUFA) shall be
used as test substrate. The properties of the field-collected standard soil shall be reported.
4 © ISO 2014 – All rights reserved

ISO 11267:2014(E)
The substrate called artificial soil can be used as a standard soil and has the following composition:
Percentage expressed on dry mass basis
− Sphagnum peat finely ground [a particle size of          10 %
(2 ± 1) mm is acceptable] and with no visible plant remains
− Kaolinite clay containing not less than 30 % kaolinite          20 %
− Industrial quartz sand (dominant fine sand with more          69 %
than 50 % of particle size 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) or ionizing substances into account, 5 % of peat
ow
have proven to be sufficient for maintaining the desired structure of the artificial soil.
NOTE 2 It has been demonstrated that Folsomia candida can comply with the validity criteria even on
reproduction when tested in field soils with lower organic carbon content (e.g. 2,7 %), and there is experience
that this can be achieved in artificial soil with 5 % peat. Therefore, it is not necessary before using such a soil in a
definitive test to demonstrate the suitability of the artificial soil for allowing the test to comply with the validity
criteria unless the peat contents is lower than specified above.
Prepare the artificial soil at least three days prior to the start of 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 during mixing. Allowance should be made for any water that is used for introducing the test
substance into the soil. The amount of calcium carbonate required can vary, depending on 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 Food
A sufficient amount, e.g. 2 mg to 10 mg, of granulated dried baker’s yeast, commercially available for
household use, is added to each container as a suitable food source, at the beginning of the test and after
about two weeks.
5.4 Reference substance
To ensure the quality of the test system, tests should be performed regularly (once or twice a year) with
a reference substance.
1)
Boric acid and the plant protection product Betosip (a.i. 157 g/l phenmedipham) have been tested in a
ring test, and are recommended as reference substances.
WARNING — When handling these substances, appropriate precautions should be taken to avoid
ingestion or skin contact.
NOTE 1 Boric acid: Effects on reproduction were observed at concentrations (EC50) of 147 mg boric acid/kg
[5] [21]
dry mass of artificial soil and 169 mg boric acid/kg clay-loam soil dry mass). ,
1) Betosip is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products
may be used if they can be shown to lead to the same results.
ISO 11267:2014(E)
NOTE 2 Betosip: Effects on reproduction (α = 0,05) were observed at concentrations between 100 mg and
200 mg of the product per kilogram dry mass of the substrate.
6 Apparatus
Use laboratory equipment and the following apparatus.
6.1 Test containers made of glass or other chemically inert material of about 100 ml capacity and with
a diameter of about 5 cm, with lids (e.g. plastic, glass discs or parafilm, able to be closed tightly).
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 mixture (5.2).
6.4 Suitable accurate balances.
6.5 Apparatus capable of measuring pH and water content of the substrate.
6.6 Exhauster for transfer of springtails (see A.2).
6.7 Test environment.
6.7.1 Enclosure, capable of being controlled to a temperature of (20 ± 2) °C.
6.7.2 Light source, capable of delivering a constant light intensity of 400 Ix to 800 Ix at the substrate
surface 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 soil can be tested at a single concentration (typically 100 %) 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 being expressed in milligrams of test substance per kilogram of dried control soil (5.2.2).
Depending on the knowledge of relevant response levels a range-finding test may precede the definitive
test. Each definitive test consists of a series of soil mixtures (treatments).
7.1.2 Range-finding test
A test to find the range of mixture ratio affecting Collembola 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 range-finding 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.
Each test container (replicate) is filled with 30 g wet mass of the test sample. To ensure easy migration
of springtails, the substrate in the test container should not be compressed.
6 © ISO 2014 – All rights reserved

ISO 11267:2014(E)
Use 10 specimens of 10 d to 12 d old Collembola per container. Prepare the test containers as indicated
in 7.2.1. Place the test containers in the test enclosure (6.7.1) with the light source (6.7.2).
At the beginning of the test, add about 2 mg of granulated dry yeast (5.3) to each test container, and
cover the containers tightly (e.g. using plastic, glass discs or parafilm). Open the test containers briefly
twice a week to allow aeration.
After 14 d, count the live Collembola in each container, and determine the percentage mortality for each
test substance concentration. Also observe surviving Collembola and record any symptoms. Due to the
rapid degradation of dead Collembola, missing Collembola are assumed to have died during the test
period.
NOTE To obtain additional information for the determination of the concentration range for the final test, the
test period can be extended to four weeks to allow qualitative determination of effects at concentrations at which
effects on reproduction could be expected.
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 soil are characterized by comparison of the biological effects found in the soil
to be tested(s) with those found in a reference soil, or if not available or not appropriate due to toxicity
or atypical physicochemical characteristics, in a standard soil. Results for the standard soil assist in
distinguishing contaminant effects from non-contaminant effects caused by soil physicochemical
properties. Regardless of whether a reference soil or standard soil is used for the statistical comparisons,
[20]
the results from standard soil shall be used to judge the validity and acceptability of the test.
If for characterization purposes a test design including 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.
— For the ECx approach, 12 concentrations should be used. Two replicates for each concentration plus
six controls are recommended. The spacing factor can be variable; smaller at low concentrations,
larger at high concentrations.
— For the mixed approach, six concentrations 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 ECx evaluation.
A limit test can be sufficient if in the range-finding test no toxic effect was observed.
To facilitate checking of the pH and humidity of the test sample, use of additional containers for each
concentration and for the control is recommended.
Each test container (replicate) is filled with 30 g wet mass of the test sample. To ensure easy migration
of Collembola, the substrate in the test container should not be compressed.
7.2 Preparation of test mixture
7.2.1 Testing contaminated soil
According to the selected dilution range the soil to be tested is mixed with the reference soil or the
standard soil thoroughly (either manually or by using a hand mixer). The homogeneity of the mixture is
checked visually. The total mass of the soil to be tested and the reference soil or the standard soil shall
be 30 g (wet mass) in each test container (6.1). The test mixture shall be wetted with deionized water to
reach 40 % to 60 % of the total water holding capacity determined according to Annex B. 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.
ISO 11267:2014(E)
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 can contain unknown mixtures of toxic, mutagenic, or otherwise
harmful substances or infectious microorganisms. Occupational health risks can arise from dust
or evaporated substances as well as via dermal contact during handling and incubation.
7.2.2 Testing substances added to the test substrate
Standard 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 30 g (wet mass). Substances are added to the test substrate and mixed 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 samples for the replicates of one concentration in order
to meet the requirements of 5.2.2, and mix it thoroughly with the soil 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) 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
soil and the water and mix it thoroughly before introducing it into the test containers.
NOTE 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 5.2.2) and the quantity of the test substance required to obtain the
desired concentration. Add that mixture to the remainder of the soil and the water and mix
thoroughly before introducing it into a test container.
Base the concentrations selected to provide the LOEC/NOEC on the results of the range-finding 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 mixture (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 B).
8 © ISO 2014 – All rights reserved

ISO 11267:2014(E)
Perform one control container for the range-finding 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 biological material
Ten juvenile Collembola (10 d to 12 d old) are placed in each test container.
Collembola are tapped or sucked from the breeding containers to transfer them to the test containers.
This can easily be done using an exhaustor as described in A.2. Before they are transferred to the test
containers, organisms are counted and checked for damage both to reduce control mortality and to
avoid systematic trial errors.
7.4 Test conditions and measurements
At the beginning of the test and after a period of 14 d, add about 2 mg of granulated dry yeast (5.3) to
each test container, and cover the containers tightly (e.g. using plastic, glass discs or parafilm). Open the
test containers briefly twice a week to allow aeration.
Determine the water content and the pH of the soil at the beginning and end of the test for one vessel of
each test conditions. When acidic or basic substances are tested, do not adjust the pH.
After two weeks, check the water content by reweighing the additional test containers, and compensate
for water loss if it exceeds 2 % of the initial water content.
7.5 Determination of surviving Collembola
Determine the number of Collembola present four weeks after introducing the parental Collembola onto
the test and control substrates. Pour the test sample into a 500 ml to 600 ml container and add water.
After gentle stirring of the suspension with a spatula, Collembola drift to the water surface. Count adults
and juveniles, if present, by a suitable procedure (see Annex D) and report the numbers.
NOTE Other extraction methods (e.g. high-gradient extraction) may be used if they have proved to be
effective.
8 Calculation and expression of results
8.1 Calculation
For each dilution or concentration, determine the percent mortality and number of offspring produced
after a 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 soil(s) to be tested, control soil(s) or the selected series of test 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 ECx/ ERx -approach the % soil to be tested based on dry mass o
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