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« Soil quality – Biological methods» ISO/TC 190/SC 4
Date:
2011-06-14
Doc. Number:
N 533
Assistant:
Françoise BERTHOU
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Your contact:
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Monique.pottevin@afnor.org
Subject : ISO/CD 11267 – Inhibition of reproduction of Collembola
(Folsomia candida) by soil pollutants
Comments :
This draft is submitted to the CD vote until the 12th
of September
2011.
Follow up :
Please vote only via the ISO Livelink CIB
© ISO 2011 – All rights reserved
Document type: International Standard
Document subtype:
Document stage: (30) Committee
Document language: E
STD Version 2.4a
M:\dms\dgie\domD099\iso\ISO 11267 Revision\ISO_CD_11267_(E)_2011-06.doc
ISO/TC 190/SC 4 N
Date: 2011-06-14
ISO/CD 11267
ISO/TC 190/SC 4/WG 2
Secretariat: DIN
Soil quality — Inhibition of reproduction of Collembola (Folsomia
candida) by soil pollutants
Qualité du sol — Inhibition de la reproduction de Collembola (Folsomia candida) par des polluants du sol
Warning
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to
change without notice and may not be referred to as an International Standard.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of
which they are aware and to provide supporting documentation.
ISO/CD 11267
ii © ISO 2011 – All rights reserved
Copyright notice
This ISO document is a working draft or committee draft and is copyright-protected by ISO. While the
reproduction of working drafts or committee drafts in any form for use by participants in the ISO standards
development process is permitted without prior permission from ISO, neither this document nor any extract
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Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
ISO/CD 11267
© ISO 2011 – All rights reserved iii
Contents Page
Foreword ............................................................................................................................................................iv
Introduction........................................................................................................................................................iv
1 Scope......................................................................................................................................................1
2 Normative references............................................................................................................................1
3 Terms and definitions ...........................................................................................................................2
4 Principle .................................................................................................................................................3
5 Reagents ................................................................................................................................................3
6 Apparatus...............................................................................................................................................5
7 Procedure...............................................................................................................................................6
8 Calculation and expression of results ................................................................................................9
9 Validity of the test..................................................................................................................................9
10 Statistical analysis ..............................................................................................................................10
11 Test report............................................................................................................................................11
Annex A (informative) Techniques for rearing and breeding of Collembola..............................................13
Annex B (informative) Techniques for counting juvenile springtails..........................................................16
Annex C (informative) Determination of water-holding capacity of artificial soil ......................................17
Annex D (informative) Guidance on adjustment of pH of artificial soil.......................................................18
Bibliography......................................................................................................................................................19
ISO/CD 11267
iv © ISO 2011 – All rights reserved
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11267 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
This second/third/... edition cancels and replaces the first/second/... edition (), [clause(s) / subclause(s) /
table(s) / figure(s) / annex(es)] of which [has / have] been technically revised.
ISO/CD 11267
© ISO 2011 – All rights reserved v
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 characterisation 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.
Soil dwelling Collembola are relevant ecologically 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 with respect to
that function, since earthworms are typically absent [23]. Additionally, collembolans represent arthropod
species with a different route and a different rate of exposure compared to earthworms [3] and enchytraeids
[8]. Various species were used in bioassays of which four species were used most commonly, Folsomia
candida, Folsomia fimetaria, Onychiurus armatus, and Orchesella cincta [24]. Numerous soil toxicity tests
supported by Environment Canada (EC) resulted in the development and standardization of a a biological test
method for determining the lethal and sublethal toxicity of samples of contaminated soil to collembolans [14].
The method prepared by EC includes three species, Orthonychiurus folsomi, Folsomia candida, and Folsomia
fimetaria. As standardized test systems using springtails as indicator organisms for the habitat function of soil,
another two methods exist. One is designed for assessing the effects of chemicals on the reproductive output
of the collembolans, Folsomia fimetaria L. and Folsomia candida Willem in soil [23], [25], and the other
method being described here, focusses on testing contaminated soil. Optionally the method can be used for
testing chemicals added to standard soils (e.g. artificial soil) for their sublethal hazard potential to
collembolans.
This International Standard describes a method that is based on the determination of sublethal effects of
contaminated soils to adult springtails of the species Folsomia candida (Willem). The species is distributed
world wide. It plays a similar ecological role as Folsomia fimetaria [14], [23]. Folsomia candida reproduces
partheogenetically and is an easy accessible species as it is commercially available and easy to culture.
Folsomia candida is considered to be a representative of soil arthropods and collembolans in particular.
Background information on the ecology of springtails and their use in ecotoxicological testing is available [26].
COMMITTEE DRAFT ISO/CD 11267
© ISO 2011 – All rights reserved 1
Soil quality — Inhibition of reproduction of Collembola
(Folsomia candida) by soil pollutants
1 Scope
This International Standard specifies one of the methods for evaluating the habitat function of soils and
determining effects of soil contaminants and chemicals to 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, agricultural or other sites under 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 test soil and in a control soil. According to the objective
of the study, the control and dilution substrate (dilution series of contaminated soil) should be either an
uncontaminated soil comparable to the soil sample to be tested (reference soil) or a standard soil (e. g.
artificial soil).
Information is provided how to use this method for testing chemicals. 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. The stability of the test substance cannot be
assured over the test period. No allowance is made in the test method described for possible degradation of
the test substance over the course of the experiment.
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or otherwise
harmful chemicals or infectious micro-organisms. Occupational health risks may arise from dust or
evaporated chemicals during handling and incubation. Precautions should be taken to avoid skin
contact.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil
under aerobic conditions for the assessment of microbial processes, biomass and diversity in the laboratory
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary
analysis)
ISO 10390:2005, Soil quality — Determination of pH
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO/DIS 11268-2:2011, Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects
on reproduction to Eisenia fetida/Eisenia andrei
ISO 11274, Soil quality — Determination of the water retention characteristic — Laboratory methods
ISO/CD 11267
2 © ISO 2011 – All rights reserved
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11461, Soil quality — Determination of soil water content as a volume fraction using coring sleeves —
Gravimetric method
ISO 11465, Soil Quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
ISO 15176:2002, Soil quality — Characterization of excavated soil and other soil materials intended for re-use
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
contaminant
substance or agent present in the soil as a result of human activity
[ISO 15176:2002].
3.2
ERx (effective rate) or ECx (effective concentration)
x % effect rate or concentration of the test soil or test substance at which reproduction is reduced by x %
compared to the control
3.3
lowest observed effect rate (LOER) or effect concentration (LOEC)
lowest tested percentage of a test soil in a reference or a standard control soil or concentration of a substance
at which a statistically significant effect is observed
NOTE The LOEC is expressed as percentage of test soil dry mass per test mixture dry mass. All test mixtures above
the LOEC have a harmful effect equal or greater than observed at the LOEC. If this condition cannot be satisfied, an
explanation should be given for how the LOEC and NOEC (3.4) have been selected.
3.4
NOER (No Observed Effective Rate) or NOEC (No Observed Effect Concentration)
test soil percentage immediately below the LOER/LOEC or, highest tested concentration of a test substance
which when compared to the control has no statistically significant lethal or other effect such as reduced
reproduction or mass alteration (p < 0,05)
NOTE The NOEC is expressed as percentage of test soil dry mass per test mixture dry mass.
3.5
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) as the test soil
3.6
standard soil
field collected soil or artificial soil whose main properties (e. g. pH, texture, organic matter content) are within a
known range
EXAMPLE Euro-Soils [20 ], artificial soil [22], LUFA Standard Soil [21].
NOTE The properties of standard soils can differ from the test soil.
ISO/CD 11267
© ISO 2011 – All rights reserved 3
3.7
control soil
reference or standard soil used as a control and as medium for preparing dilution series with test soils or a
reference substance, that allows the presence of earthworms
3.8
test mixture
mixture of contaminated soil or the test substance with control soil (3.9)
NOTE Test mixtures are given in per cent contaminated soil based on soil dry mass.
3.9
test mixture ratio
ratio between the test soil and the control soil in a test mixture
NOTE Different ratios may be applied in a dilution series to establish a dose-response relationship.
4 Principle
The effects on reproduction of 10- to 12-day old springtails (Folsomia candida) exposed to the test soil are
compared to those observed to samples exposed to a control soil. If appropriate, effects based on exposure to
a dilution range of contaminated soil or a range of concentrations of a test substance are determined. Test
mixtures are prepared at the start of the test and are not renewed within the test period.
The springtails are incubated until offspring (F1) emerge from eggs laid by mature adults, and the number of
offspring is determined. Normally offspring emerge within 28 days in control experiments. 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 mortality and reproduction (NOER/NOEC) and the dilution
(concentration ) resulting in x % reduction of juveniles hatched from eggs compared to the control (ERx/ECx,
28 d) respectively.
In case of testing a dilution or concentration series of 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/of the test substance likely to have an effect, then it is
useful to conduct the test in two steps:
⎯ a preliminary 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 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 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.
5 Reagents
5.1 Biological material
In this test, 10- to 12-day old juvenile springtails of the species Folsomia candida (Willem) are used (see A.1
for details on synchronization of breeding).
5.2 Test substrate may consist of field-collected soil or control soil amended by the test substance.
ISO/CD 11267
4 © ISO 2011 – All rights reserved
5.2.1 Field-collected soils, soil or waste materials
The sample(s) can be field-collected soil from an industrial, agricultural or other site of concern, or waste
materials (e. g. dredged material, municipal sludge from a wastewater treatment plant, composed material, or
manure) under consideration for possible land disposal.
Test soils shall be sieved by 4 mm mesh and thoroughly mixed. If necessary, soil may be air dried without
heating before sieving. Storage of test soils should be as short as possible. Store the soil in accordance with
ISO 10381-6 using containers that minimise losses of soil contaminants by volatilisation and sorption to the
container walls. If soils or soil 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 D,
e) cationic exchange capacity in accordance with ISO 11260,
f) organic carbon in accordance with ISO 10694,
NOTE It is important to measure the water holding capacity of all mixtures used in the test.
5.2.2 Control soil, either a) reference soil (3.5) or b) standard soil (3.7) that allows the presence of
earthworms.
g) If reference soils from uncontaminated areas near a contaminated site are available, they should be
treated and characterised like the test soils. If a toxic contamination or unusual soil properties cannot be
ruled out, standard control soils should be preferred.
h) For testing the effects of substances mixed into soil or making dilutions of the test soil, standard soils
shall be used as test substrate. 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
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 size 0,05 mm to 0,2 mm)
69 %
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO3, pulverised, analytical grade) are necessary to get a
pH of 6,0 ± 0,5.
NOTE 1 Taking the properties of highly non-polar (log Kow > 2) or 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 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
ISO/CD 11267
© ISO 2011 – All rights reserved 5
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 lowered more than specified above [??].
Prepare the artificial soil at least three days prior to start the test, by mixing the dry constituents listed above
thoroughly in a large-scale laboratory mixer. A portion of the deionized water required is added while mixing is
continued. 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 deionised water to obtain 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 D, the pH is determined according to
ISO 10390.
NOTE 3 Allowance should be made for any water that is used for introducing the test substance into the soil.
5.3 Reference substance
To ensure the quality of the test system, tests should be performed regularly (once or twice a year) with a
reference substance.
The agricultural chemical Betosip (a.i. 157 g/l Phenmedipham) and boric acid have been tested in a ring test,
and are recommended as reference substances.
WARNING — When handling these chemicals, appropriate precautions should be taken to avoid
ingestion or skin contact.
NOTE 1 Betosip: Effects on reproduction (α = 0,05) were observed at concentrations of between 100 mg and 200 mg
of the product per kilogram dry mass of the substrate.
NOTE 2 Boric acid: Effects on reproduction were observed at concentrations (IC50) of 147 mg boric acid/kg dry weight
of artificial soil and 169 mg boric acid/kg clay-loam soil dry weight) [9], [25].
6 Apparatus
Standard laboratory equipment, and:
6.1 Test containers made of glass or other chemically inert material (able to be closed tightly) of about
100 ml capacity and with a diameter of about 5 cm.
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 substrate (5.2).
6.4 Precision balance with an accuracy of at least 1 mg.
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 °C ± 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.
ISO/CD 11267
6 © ISO 2011 – All rights reserved
7 Procedure
7.1 Experimental design
7.1.1 General
A sample of field-collected test soil can be tested at a single concentration (typically 100 %) or evaluated for
toxicity in a multi-concentration test whereby a series of dilutions are prepared by mixing measured quantities
with a control soil (5.2.2). When testing substances a series of concentrations is prepared by mixing quantities
of the test substance with a standard soil (e. g. artificial soil). Depending on the knowledge of relevant
response levels a preliminary test may precede the definitive test. Each definitive test consists of a series of
soil mixtures (treatments). Each treatment is replicated at least four times.
7.1.2 Preliminary test
A preliminary test to find the range of mixture ratio 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 concentrations being expressed in milligrams of test substance per kilogram of dried control soil (5.2.2)
and a control using ten worms per container). 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.
Each test container (replicate) is filled with 30 g wet mass of the test substrate. To ensure easy migration of
springtails, the substrate in the test container should not be compressed.
Use 10 specimens of 10- to 12-day old springtails per mixture ratio or concentration and 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 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 days, count the live springtails in each container, and determine the percentage mortality for each test
substance concentration. Also observe surviving springtails and record any symptoms. Due to the rapid
degradation of dead springtails, missing springtails 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 test soil are characterised by comparison of the biological effects found in the test soil(s) with
those found in the control soil (3.7) (single-concentration tests). If a reference soil (3.5) to be used as 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 control soil, it is
recommended to include additionally a standard soil exhibiting a typical known response, and to use these
results to judge the validity and acceptability of the test [24]. 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 characterisation purposes a test design including dilution series is required, three designs are possible
(the concentrations shall be spaced by a factor not exceeding 2):
ISO/CD 11267
© ISO 2011 – All rights reserved 7
⎯ For the NOEC 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, 6 to 8 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 preliminary test no toxic effect was observed. In the limit test only the test
soil without any dilution and the control (or the test soil vs. the control soil) shall be tested with at least four
replicates each.
To facilitate checking of the pH and humidity of the test substrate, 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 substrate. To ensure easy migration of
springtails, the substrate in the test container should not be compressed.
7.2 Preparation of test mixture
7.2.1 Testing 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 30 g (wet mass) in each test container (6.1).
Wet the test mixture with deionised water to reach an appropriate water content of usually 40 % to 60 % of the
total water holding capacity determined according to Annex C. In some cases, e. g. when testing waste
materials, higher percentages are required. A rough check of the soil moisture content can be obtained by
gently squeezing the soil in the hand, if the moisture content is correct small drops of water should appear
between the fingers.
Determine the pH for each test mixture (one container per concentration) according to ISO 10390 at the
beginning and end of the test (when acid or basic substances are tested, do not adjust the pH).
Proceed simultaneously with at least four replicates per concentration and the control(s).
WARNING — Contaminated soils may contain unknown mixtures of toxic, mutagenic, or otherwise
harmful chemicals or infectious micro-organisms. 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 test substrate
Standard soil (5.2.2) is used as test substrate. For each test container (6.1), the mass of the substrate used
shall be 30 g (wet mass). Add substances to the test substrate 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 required
for the replicates of one concentration in water (or that portion of it necessary to wet the soil
substrate) in order to meet the requirements of 5.2.2. Mix it thoroughly with the soil substrate before
introducing it into a test container.
ISO/CD 11267
8 © ISO 2011 – All rights reserved
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
standard 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 standard soil and the water and mix
thoroughly before introducing it into a test container.
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 substrate. Proceed simultaneously with at least four
replicates per concentration and the control(s).
Determine the pH for each test substrate (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 deionised water to reach 40 % to 60 % of the
total water holding capacity (determined according to Annex C).
Perform 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 Introduction of the test organisms
Ten juvenile springtails (10 to 12 days old) are placed in each test container.
Springtails 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 clause 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 days, add about 2 mg of granulated dry yeast 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.
ISO/CD 11267
© ISO 2011 – All rights reserved 9
Determine the water content and the pH (in the presence of 1 mol/I KCl) of the artificial soil at the beginning
and end of the test. 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 springtails
Determine the number of springtails present four weeks after introducing the parental springtails onto the test
and control substrates. Pour the test substrate into a 500 ml to 600 ml container and add water. After gentle
stirring of the suspension with a spatula, springtails drift to the water surface. Count adults and juveniles, if
present, by a suitable procedure (see Annex B) 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 endpoints 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 ECx-approach the % soil mixture based on dry mass or in milligrams per kilogram of 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 % (EC50) compared to the control
within the test period or
⎯ in the NOEC-approach the soil mixture ratio immediately below the 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 mass alteration and reduction of reproduction (p < 0,05).
9 Validity of the test
The results are considered to be valid if:
⎯ The mortality of the adults in the control(s) should not exceed 20 % at the end of the test.
⎯ The reproduction rate should reach a minimum of 100 instars per control vessel.
⎯ The coefficient of variation of reproduction in the control should not exceed 30 %.
ISO/CD 11267
10 © ISO 2011 – All rights reserved
10 Statistical analysis
10.1 General
Most of test methods with sub-lethal endpoints, e. g. growth, reproduction, involve quantitative effects, e. g.
measuring the weight of the organisms or counting juvenile worms. Quantal effects may also be measured in
the same test, such as mortality after four weeks exposure.
NOTE Guidance given here for statistical evaluation of test results aims to make the investigator aware of problems
that can arise in consequence of a test design selected. Computer programs do not necessarily guard against violations of
rules that can cause erroneous analyses. It is strongly recommended to look for more information in specific guidance
documents (e. g. as provided by [13]) or to contact a statistician.
10.2 Single-concentration tests
Quantitative single-concentration tests (e. g. effects on reproduction or the biomass development) have
different statistical methods. For sampling at several locations with field replication, ANOVA would be a first
step if results were suitable. If the null hypothesis of no difference was rejected, analysis would proceed to
one of several multiple-comparison tests [13].
An example of a single-concentration test for quantitative effects can be counting juvenile worms as endpoint
of effects on reproduction or measuring the average biomass of earthworms after exposure to a sample of
undiluted contaminated soil, compared to numbers of juvenile worms or biomass of earthworms exposed to a
reference or standard soil. If there was only one sample tested, and one control material, without any
replicates, results can be not compared by any statistical test. In a quantitative test with replication for the test
soil (material) and for the control soil, a standard t-test would be suitable for statistical analysis.
Analysis of variance (ANOVA) involving multiple comparisons of endpoint data derived for undiluted test soils
including field replicates of field-collected soil from more than one sampling location is commonly used for
statistical interpretation of the significance of quantitative findings (e. g. biomass) from soil toxicity tests. This
is a hypothesis-testing approach, and is subject to appreciable weaknesses [13]. The parametric analyses
(e. g. ANOVA and multiple comparisons) for such data assume that the data are normally distributed, that the
treatments are independent, and that the variance is homogenous among the different treatments. These
assumptions shall be tested. If the data satisfy these assumptions, analysis may proceed. If not, data may be
transformed and tested again. As parametric tests are reasonably robust in the face of moderate deviations
from normality and equality of variance, parametric analysis should proceed, even if moderate nonconformity
continues after transformation, [13].
10.3 Multi-concentration tests
10.3.1 Preliminary test
If a clear dose-response is obvious, ECx-values can be estimated by using regression techniques like logistic
regression function or probit analysis. In other cases the effect range should be determined by expert
knowledge.
10.3.2 Definitive test
A point estimate (ERx/ECx-approach) is recommended as the best quantitative endpoint. This is usually a
specific degree of reduction in performance compared to the control. Linear and non-linear regression
methods are widely applied for statistical analysis. Operators should be aware of being able to understand the
judgements in selecting appropriate mathematical models.
Hypothesis testing (NOEC-approach) is commonly used to identify dilutions (concentrations) with significant
effects compared to the control. As this method has many flaws it is not recommended for future use,
ISO/CD 11267
© ISO 2011 – All rights reserved 11
Therefore, in cases where various dilutions (concentrations) of each sample of field-collected soil with
negative control soil are tested, data preferably are analysed by the ECx-approach, or if required by
legislation, by the NOEC-approach:
⎯ ERx/ECx (effect concentration)-approach
The ERx/ECx-approach can only be used if a clear dose response relationship is found. Wherever possible,
the R2
should be 0,7 or higher and the test mixtures used encompass 20 % to 80 % effects. If these
requirements are not fulfilled, expert knowledge is necessary for the interpretation of the test results.
To compute an ERx/ECx-value, the treatment means are used for regression analysis after an appropriate
dose-response function has been found (e. g. probit or logistic function). A desired ERx/ECx is obtained by
inserting a value corresponding to x% of the control mean into the equation found by regression analysis.
Since EC50 values have smaller confidence limits compared with smaller effect concentrations (e. g.
ER/EC20), it is recommended to determine ER/EC50 values.
⎯ NOEC (No-observed-effect-concentration)-approach
First of all, a statistical analysis of the homogeneity of the variances shall be made, e. g. by using Cochran´s
test. With homogeneous data, an appropriate statistical analysis, e. g. a „One-Way Analysis of Variance
(ANOVA)“, followed by a one-sided Dunnett test (α = 0,05), should be performed. If the homogeneity
requirement is not fulfilled, it is recommended to evaluate if an appropriate transformation of the data can
solve the problem. Otherwise non-parametric methods, e. g. the U-test by Mann & Whitney or the BonferroniU-Test
can be used.
If a limit test has been performed and the pre-requisites (normality, homogeneity) of parametric test
procedures are fulfilled, the Student-t-test, otherwise the Mann-Whitney-U-test procedure should be used.
In any case the results of the statistical evaluation shall be biologically interpreted.
NOTE Guidance given here for statistical evaluation of test results aims to make the investigator aware of problems
that can arise in consequence of a test design selected. Computer programs do not necessarily guard against violations of
rules that can cause erroneous analyses. It is strongly recommended to look for more information in specific guidance
documents (e. g. as provided by [13]) or to contact a statistician in any case, a special design is used.
11 Test report
The test report shall include the following information:
a) a reference to ISO 11267;
b) the results, expressed as in 8.2;
c) detailed description of the test substance and information on physical and chemical properties if helpful
for the interpretation of the test result;
d) complete description of the biological material employed (species, age, breeding conditions, supplier);
e) method of preparation of the test substrate together with an indication of the auxiliary substances used for
a low-/non-water-soluble substance;
f) results obtained with the reference substance, if performed;
g) detailed conditions of the test environment;
h) a table giving the percent mortality of adults at each concentration and in the control(s);
i) number of dead or missing adults and number of offspring per test container at the end of the test;
ISO/CD 11267
12 © ISO 2011 – All rights reserved
j) depending on the statistical approach selected, list the lowest concentration causing significant effects
(LOEC), the highest concentration causing no observed effects (NOEC), EC10 and EC50 for the inhibition
of reproduction and the method used for calculation (optional);
k) description of any pathological or other symptoms, or distinct changes in behaviour observed in the test
organisms per test container;
l) water content and pH of artificial soil at the start and at the end of the test for the control and for each
concentration;
m) any operating details not specified in this International Standard, as well as any factors that may have
affected the results.
ISO/CD 11267
© ISO 2011 – All rights reserved 13
Annex A
(informative)
Techniques for rearing and breeding of Collembola
A.1 Conditions for rearing and breeding
A.1.1 Breeding substrate
Plaster of Paris (plaster for stucco, pH 6,4) and activated charcoal (pulverized chemically activated charcoal,
pH 6 to 7), are mixed in a mass ratio of 8:1, but higher ratios, (9:1 to 11:1) may also be used. Depending on
the type of plaster, 60 g to 100 g water are added to 100 g of the mixture. This provides a highly moist
substrate, while the charcoal adsorbs waste gases and excretion products. The dark background facilitates
observation.
The presence of water on the saturated substrate surface is essential for breeding springtails, and the pH can
readily be determined by using pH indicators placed on this wet substrate surface.
A.1.2 Breeding containers
Commercial plastic containers with a volume of about 400 ml should be used. Fill the containers to a depth of
about 1 cm with the breeding substrate, and add deionized water to almost saturation. The moisture content
can be maintained automatically by using an absorbent wick, implanted in the substrate and running to a
water bath below the container, or by supplying distilled water with a pipette until the substrate is saturated but
there is no water standing on the substrate surface.
Close the breeding containers tightly using suitable covers, and aerate periodically (e. g. in combination with
feeding) by lifting the cover for a short time.
The covers may also be perforated for aeration by a needle.
CAUTION — Care should be taken that predacious mites do not penetrate the containers.
A.1.3 Climatic conditions
For keeping and breeding the springtails, a climatic chamber with a controlled temperature of 20 °C to 22 °C
and 70 % to 80 % relative humidity with constant lighting at 400 Ix to 800 ix (or light:dark cycle 16 h:8 h) is
most suitable.
A.1.4 Food
For breeding and for the test, use granulated dry yeast as food supply. Feeding the breeding containers once
or twice a week is recommended, but to avoid spoilage by fungi, food should be applied in small amounts at
frequent intervals.
A.1.5 Transfer
After about eight weeks, transfer the springtails to fresh breeding containers by tapping or blowing. The
transfer to fresh containers usually induces oviposition.
ISO/CD 11267
14 © ISO 2011 – All rights reserved
A.1.6 Test organisms of a standard age
To obtain 10- to 12-day old juvenile springtails for the test, transfer egg clusters from breeding containers to a
freshly prepared breeding substrate, using a fine spatula or hair brush. After 48 h, remove the egg clusters
and feed instars hatched from the eggs.
NOTE The egg clusters are easily removed if they are placed on small pieces of breeding substrate or cover glasses
laid on the breeding substrate, and juvenile springtails collected after a further 10 days incubation.
Alternatively, 10- to 12-day old juvenile springtails may be obtained by placing a number of adult springtails in
small containers with plaster of Paris in the base, and allowing them to lay eggs over a two-day period. After
this time remove the adults. Twelve days after the first juveniles have emerged from the eggs, they can be
used for the test. To ensure successful synchronization, it is advisable to check the containers for egg
production before removing the adults. In some cases, the adults do not start laying eggs immediately, and
only few eggs will be produced in two days. If this is observed to be the case, then keep the adults in the
containers for an additional day or more.
For both methods, avoid overcrowding in the containers, as this may lead to reduced growth. As a
consequence, the 10- to 12-day old animals used for the test may be too small and not yet able to produce a
sufficient number of eggs to meet the requirements of the test.
A.2 Transfer of springtails to the test containers
The springtails are easily transferred from the breeding substrate to the test substrate by an exhauster. An
example is shown in Figure A.1.
Key
1 Eppendorf-pipette tip 4 rubber stopper
5 commercial aquarium pump2 cylinder (10 ml volume) with rolled flange
and plastic cover for control of sucked springtails 6 regulator
3 gauze 7 foot switch
Figure A.1 — Low-suction exhaustor apparatus for transfer of springtails
ISO/CD 11267
© ISO 2011 – All rights reserved 15
The springtails are sucked individually through a pipette tip to a small covered container to control damage of
springtails. Care shall be taken to ensure that the suction of the pump is low to avoid damage to the
springtails. After removing the cover, springtails provided for one test container are transferred onto the
substrate surface of the test container.
Alternatively, a manual exhaustor can be hand-made from a small glass tube and a piece of flexible plastic
tube. For this purpose a Pasteur pipette can be used, from which the small end has been removed and the
narrow aperture annealed in a flame to smooth the cut edges. The other end of the tube is covered with a
piece of very fine-mesh gauze, and a piece of flexible plastic tube is fixed to it. With such an exhaustor, the
juveniles can be sucked up by mouth and transferred to the test containers.
Warning: If tests are performed regularly, sucking up the juveniles by mouth should be avoided as
technicians may run the risk of becoming allergic to materials (e.g. fungal spores, faeces, exoskeleton
particles etc.) inhaled.
ISO/CD 11267
16 © ISO 2011 – All rights reserved
Annex B
(informative)
Techniques for counting juvenile springtails
For evaluating effects on reproduction, the juvenile springtails swimming on the surface of the watered
substrate are counted. When reproduction is high, the use of technical devices to facilitate counting is
recommended.
If the swimming juveniles are distributed evenly over the water surface, a counting grid may be used and a
sample for counting taken at random. Aggregations of instars on the water surface can be a problem and can
be prevented by adding a drop of sewing-machine oil.
A culture-counter may also be used to count the animals on a projected picture (slide) of the water surface.
Normal photographic equipment is adequate for this purpose (e. g. single-lens reflex camera, macro-lens or
other device for close-up photography). Film speed has to be adjusted according to light intensity (e. g. flash
or cold light source) and the desired shutter speed. If a cold light source is used, the minimum film speed
value is 400 ASA. A useful relation between a sufficient projection format on a screen and the beaker volume
is met with volumes of 50 ml to 100 ml (lens used: Zeiss S-Planar 1:2,8 f = 60 mm, angular field 39°, min.
focus 0,24 m). To improve the contrast between white springtails and surrounding water surface, water may
be coloured dark with ink.
Overall, the average error of counting should not exceed 10 %.
NOTE To avoid errors in determining mortality of the parental springtails, the number of live adults floating on the
water surface should be counted by using a binocular microscope.
ISO/CD 11267
© ISO 2011 – All rights reserved 17
Annex C
(informative)
Determination of water-holding capacity of artificial soil
C.1 General
The following method-has been found to be appropriate for the purposes of determination of water-holding
capacity (WHC) of artificial soil and may be used as an alternative to ISO 11274.
C.2 Apparatus
C.2.1 Glass tube, approximately 20 mm to 50 mm diameter and at least 100 mm in length.
C.2.2 Water bath, at room temperature.
C.2.3 Filter paper.
C.2.4 Drying oven, set to 105 °C ± 5 °C.
C.2.5 Balance, capable of weighing with an accuracy of ± 0,1 g.
C.3 Procedure
Plug the bottom of the tube with a filter paper, and after filling with the artificial soil substrate to a depth of
5 cm to 7 cm, place the tube on a rack in a water bath. Gradually submerge the tube until the water level is
above the top of the soil. Leave the substrate sample in the water for about 3 h.
As not all water absorbed by the substrate capillary can be retained, the tube containing the sample should be
placed for a period of 2 h on very wet finely ground quartz sand for draining. The same quartz sand as is used
for the soil substrate is satisfactory.
Weigh the sample, dry it to constant mass at 105 °C and reweigh it.
C.4 Calculation of water-holding capacity (WHC)
100WHC ×
−−
=
D
DTS
where
WHC is the water-holding capacity, expressed as a percent of dry mass;
S is the mass of water-saturated substrate + mass of tube + mass of filter paper;
T is the tare (mass of tube + mass of filter paper);
D is the dry mass of substrate.
ISO/CD 11267
18 © ISO 2011 – All rights reserved
Annex D
(informative)
Guidance on adjustment of pH of artificial soil
To estimate how much CaCO3 is needed to obtain the desired pH (6,0 ± 0,5), artificial soil is prepared by
mixing peat, sand, kaolin and water as described in 5.2.1. Small portions are taken and mixed with different
amounts of CaCO3 e.g. corresponding with concentrations of 0,2 %, 0,4 %, 0,6 %, 0,8 % and 1,0 % dry mass.
From these portions, the pH is determined as described in ISO 10390 and the results are plotted as a graph of
pH versus the amount of CaCO3. From this graph, the amount of CaCO3 necessary to obtain a pH of 6,0 ± 0,5
can be estimated.
ISO/CD 11267
© ISO 2011 – All rights reserved 19
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