SIST EN ISO 11269-2:2013

SLOVENSKI STANDARD
01-maj-2013
1DGRPHãþD
SIST ISO 11269-2:2006
Kakovost tal - Vpliv onesnaževal na talno floro - 2. del: Vpliv onesnaženih tal na
kalitev in rast višjih rastlin (ISO 11269-2:2012)
Soil quality - Determination of the effects of pollutants on soil flora - Part 2: Effects of
contaminated soil on the emergence and early growth of higher plants (ISO 11269-
2:2012)
Bodenbeschaffenheit - Bestimmung der Wirkungen von Schadstoffen auf die Bodenflora
- Teil 2: Wirkung von kontaminierten Böden auf Saatauflauf und frühes Wachstum
höherer Pflanzen (ISO 11269-2:2012)
Qualité du sol - Détermination des effets des polluants sur la flore du sol - Partie 2: Effets
des sols contaminés sur l'émergence et la croissance des végétaux supérieurs (ISO
11269-2:2012)
Ta slovenski standard je istoveten z: EN ISO 11269-2:2013
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 11269-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2013
ICS 13.080.30
English Version
Soil quality - Determination of the effects of pollutants on soil
flora - Part 2: Effects of contaminated soil on the emergence and
early growth of higher plants (ISO 11269-2:2012)
Qualité du sol - Détermination des effets des polluants sur Bodenbeschaffenheit - Bestimmung der Wirkungen von
la flore du sol - Partie 2: Effets des sols contaminés sur Schadstoffen auf die Bodenflora - Teil 2: Wirkung von
l'émergence et la croissance des végétaux supérieurs (ISO verunreinigten Böden auf Saatauflauf und frühes
11269-2:2012) Wachstum höherer Pflanzen (ISO 11269-2:2012)
This European Standard was approved by CEN on 5 February 2013.

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

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11269-2:2013: E
worldwide for CEN national Members.

Contents Page
Foreword . 3
Foreword
The text of ISO 11269-2:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 11269-2:2013 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by August 2013, and conflicting national standards shall be withdrawn at
the latest by August 2013.
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 11269-2:2012 has been approved by CEN as EN ISO 11269-2:2013 without any modification.

INTERNATIONAL ISO
STANDARD 11269-2
Third edition
2012-01-15
Soil quality — Determination of the
effects of pollutants on soil flora —
Part 2:
Effects of contaminated soil on the
emergence and early growth of higher
plants
Qualité du sol — Détermination des effets des polluants sur la flore du
sol —
Partie 2: Effets des sols contaminés sur l’émergence et la croissance
des végétaux supérieurs
Reference number
ISO 11269-2:2012(E)
©
ISO 2012
ISO 11269-2:2012(E)
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Units . 3
5 Principle . 3
6 Test plants . 3
7 Materials . 4
7.1 Test vessels . 4
7.2 Soil . 4
8 Equipment . 6
9 Reference substance . 6
10 Procedure . 6
10.1 Experimental design . 6
10.2 Preparation of the pots . 7
10.3 Preparation of the seeds . 7
10.4 Growth conditions . 8
10.5 Start of the test . 8
10.6 Handling during the test . 8
11 Validity criteria . 9
12 Assessment of the results . 9
12.1 Data presentation . 9
12.2 Expression of the results . 9
13 Statistical analysis . 9
13.1 General . 9
13.2 Range-finding test .10
13.3 Final test .10
14 Test report . 11
Annex A (informative) Additional recommended plant species based on test results gained by applying
[4]
Environment Canada Test Method: EPS 1/RM/45 .13
Annex B (informative) Phytotoxic values for reference compounds: sodium trichloro-acetate
and boric acid .15
Annex C (informative) Recommended method for the measuring of the water-holding capacity
of the soil .16
Annex D (informative) Recommendations for nutrient supply of soils .17
Bibliography .18
ISO 11269-2:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11269-2 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological methods.
This third edition cancels and replaces the second edition (ISO 11269-2:2005), which has been technically revised.
ISO 11269 consists of the following parts, under the general title Soil quality — Determination of the effects of
pollutants on soil flora:
— Part 1: Method for the measurement of inhibition of root growth
— Part 2: Effects of contaminated soil on the emergence and early growth of higher plants
iv © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
Introduction
This part of ISO 11269 describes a procedure for evaluating the quality of soils of different origin carrying
unknown contaminations. The evaluation of the effects on plant growth is based on emergence and inhibitory
effects on early growth of at least two species of higher plants. Guidance for assessing potential effects of
[14]
substances on seedling emergence and growth is given in OECD Guideline 208 .
This part of ISO 11269 refers closely to ISO 22030 and is based on:
a) results from the German research project “Entwicklung eines innovativen und technischen Instrumentariums
zur Optimierung der ökotoxikologischen Bewertung von Böden im Hinblick auf Sanierungsziele und
Schutzerfordernisse”;
b) discussions within the joint project “Ecotoxicological Test Batteries” forming part of the BMBF Joint
[23]
Research Group “Processes for the Bioremediation of Soil” ;
c) results from the BMBF Joint Research Group ERNTE “Erprobung und Vorbereitung einer praktischen
[17]
Nutzung ökotoxikologischer Testsysteme” ;
d) ring-test results of “Ecotoxicological Characterisation of Waste — Results and Experiences from an
[8]
International Ring Test” .
Plant growth can be influenced strongly by soil properties such as texture, pH or levels of nutrients. When
testing natural soils either reference soils (uncontaminated soils with the same properties as the test soil) or
standard soils are used as mixing and control substrate. In the latter case, variations in plant growth can result
from either soil contaminants or differences in soil properties like nutrients and texture. Therefore, results from
soil testing can less easily be interpreted than results from testing of chemicals .
INTERNATIONAL STANDARD ISO 11269-2:2012(E)
Soil quality — Determination of the effects of pollutants on soil
flora —
Part 2:
Effects of contaminated soil on the emergence and early growth
of higher plants
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. Furthermore, test plants might take up
chemicals from the soil and safety measures should also be considered when handling the test plants.
1 Scope
This part of ISO 11269 describes a method to assess the quality of an unknown soil and the soil habitat function
by determining the emergence and early growth response of at least two terrestrial plant species compared
to reference or standard control soils. It is applicable to soils of unknown quality, e.g. from contaminated sites,
amended soils or soils after remediation.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 10381-6, Soil quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil under
aerobic conditions for the assessment of microbiological processes, biomass and diversity in the laboratory
ISO 10390, Soil quality — Determination of pH
ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary analysis)
ISO 11260, Soil quality — Determination of effective cation exchange capacity and base saturation level using
barium chloride solution
ISO 11268-1, Soil quality — Effects of pollutants on earthworms — Part 1: Determination of acute toxicity to
Eisenia fetida/Eisenia andrei
ISO 11268-2, Soil quality — Effects of pollutants on earthworms — Part 2: Determination of effects on
reproduction to Eisenia fetida/Eisenia andrei
ISO 11277, Soil quality — Determination of particle size distribution in mineral soil material — Method by
sieving and sedimentation
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric method
ISO 22030, Soil quality — Biological methods — Chronic toxicity in higher plants
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO 11269-2:2012(E)
3.1
emergence
appearance of the coleoptile or cotyledon above the soil
3.2
contaminant
substance or agent present in the soil as a result of human activity
[28]
[ISO 15176:2002 ]
3.3
hormesis
improvement of seedling emergence, growth or survival (or other response of the test plants) at low concentrations
[1][2]
of chemicals or mixtures of soil that are toxic when applied at higher levels in comparison to the control
3.4
lowest observed effect rate 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 a percentage of the test-soil dry mass per soil-mixture dry mass. All test mixtures
above the LOEC have a harmful effect equal to or greater than that observed at the LOEC. If this condition cannot be
satisfied, an explanation should be given for how the LOEC and NOEC (3.5) have been selected.
3.5
no observed effect concentration
NOEC
test-soil percentage immediately below the LOEC, which when compared to the control has no statistically
significant effect (r < 0,05)
3.6
x % effect concentration
EC
x
x % effect rate
ER
x
percentage of a test soil at which a given endpoint is inhibited by x % compared to the control
3.7
soil mixture ratio
ratio between the test soil and the reference/control soil in a soil mixture, expressed in percent based on
soil dry mass
NOTE Different ratios may be applied in a dilution series to establish a dose-response relationship.
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) as the test soil
2 © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
3.9
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are within
a known range
[11] [14] 1)
EXAMPLE Euro-soils , artificial soil , LUFA soil.
NOTE The properties of standard soils can differ from the test soil.
3.10
control soil
reference or standard soil used as a control and as a medium for preparing dilution series with test soils or a
reference substance
NOTE Both EC and NOEC are expressed in milligrams of test substance per kilogram (dry mass) of the test
substrate. Soil mixtures are given in per cent based on soil dry mass.
4 Units
Emergence is expressed as the percentage of seedlings which emerge as compared with the control pots. The
biomass of the shoots is expressed as dry mass per plant or, if needed, as dry mass per pot.
5 Principle
The test measures emergence and early growth of at least two terrestrial plant species (one monocotyledonous
and one dicotyledonous). The test compares the development of plants in a test soil and/or a series of mixtures
with a control soil. Seeds of the selected plant species are planted in pots containing the soil/soil mixtures
and in control pots containing a reference or standard soil. Pots are kept under growth conditions for the test
species selected. After 50 % of the seedlings in the control have emerged, emergence rates are determined and
plants are thinned out to a specified number. After a period of two weeks to three weeks, the remaining plants
are harvested to determine their biomass. The relative growth inhibition in undiluted test soil is determined to
assess the function of the test soil as a habitat for plants. In addition, NOEC, LOEC or EC and ER values can
x x
be calculated from the dose response curve gained from mixtures of the test soil with control soil.
NOTE An early plant growth test may include additional testing endpoints, e.g. shoot length, root length and root dry
mass. In many instances, root endpoints are more sensitive than shoot dry mass. In almost all cases, emergence is a less
sensitive endpoint.
6 Test plants
One monocotyledonous and one dicotyledonous species are tested in parallel. Oat (Avena sativa) is
recommended as the monocotyledonous and turnip rape (Brassica rapa) and/or wild turnip (Brassica rapa ssp.
rapa) as the dicotyledonous plant species. Oat, turnip rape and wild turnip grow in sandy as well as in loamy
soil with varying water content and a range of pH values from 5,0 to 7,5.
Other species might be selected, e.g. plants with specific physiological characteristics like C-4 plants (corn,
sugar cane, millet), plants in symbiosis with nitrogen-fixing bacteria (e.g. Fabaceae) or plants with ecological or
economical significance in certain regions of the world, provided that these species grow unhindered in control
soil and fulfil the validity criteria of the test (Clause 11). Only plants that tolerate the properties of the test soils
and test conditions (beside their chemical contamination) should be selected. For example, a species sensitive
to low pH values should not be used for testing forest soils with low pH values. Species that do not tolerate wet
soils should not be used in combination with wick watering. Reasons for selecting species other than oat and
wild turnip or turnip rape shall be justified in the test report.
NOTE Additional recommended species including validity criteria and reference toxicant test data for different
endpoints are compiled in Annexes A and B.
1) Euro-soils, artificial soil and LUFA soil are examples of suitable products available commercially. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of these products.
ISO 11269-2:2012(E)
7 Materials
7.1 Test vessels
The test vessels shall be non-porous plastics or glazed pots with a top internal diameter of between 85 mm and
95 mm, taking into account the size. It is recommended to use an automatic watering system, e.g. pots equipped
with glass-fibre wicks, to avoid the time-consuming daily manual adjustment of soil moisture as proposed in
ISO 22030. In this case, one or two glass-fibre wicks (∅ 1 mm) shall be introduced through the bottom of the
vessels. The wicks reach a water reservoir and ensure the water supply during the test. Therefore, at least one
hole shall be prepared to fix the wick. Commercial plant pots often have more than one hole, what might result
in flow back of water. In addition, roots might grow through open holes and circumvent the soil contaminants.
A filter disk can prevent growth of roots through additional holes. Wicks are not used, when the test soil does
not take up water by wicks as shown by a pretest (see 10.2).
7.2 Soil
7.2.1 General
Assessing the toxic potential of field soil from a contaminated site or that of remediated soil, the selected soils
should have pH values after sieving within a range that is not toxic to the test plants, e.g. between 5,0 and 7,5
for Brassica rapa and Avena sativa.
The soil pH should not be corrected. For the time being, pH limits for plant species other than turnip rape and
oat cannot be stated. It is a matter of future research to systematically test more plants on a variety of soils.
Furthermore, tolerance limits for texture, salinity or other soil properties cannot be given for different plant
species so far.
When comparing soils of known and unknown quality, the control soil and field soil under test should be of the
same textural class, and be as similar as practicable in all respects other than the presence of the chemical or
contaminant being investigated. Indeed, significant differences in soil characteristics other than the presence
of contaminants may lead to differences in plant growth and may induce false positive test results.
7.2.2 Test soil
The sample(s) of test soil might be field-collected soil from an industrial, agricultural or other site of concern,
or waste materials (e.g., dredged material, municipal sludge from a sewage sludge treatment plant, composed
material, or manure) under consideration for possible land disposal.
The 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 test soils
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. Soil pH should not be
corrected as it may influence bioavailability of soil contaminants.
For interpretation of test results, the following characteristics should be determined for each soil sampled from
a field site:
a) soil texture (sand, loam, silt) in accordance with ISO 11277,
b) pH (KCl) value in accordance with ISO 10390,
c) water content in accordance with ISO 11465,
d) water-holding capacity (Annex C),
e) cationic exchange capacity in accordance with ISO 11260,
f) organic matter content in accordance with ISO 10694,
g) total and water soluble amounts of potassium, nitrogen and phosphorous.
4 © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
NOTE It is important to measure the water-holding capacity of all mixtures used in the test.
7.2.3 Control soil
Either reference or standard soils can be used as the control soil, if unhindered growth of the test plants in these
soils can be expected. In any case, differences in nutrient levels between a test soil and a control soil can affect
the dose-response pattern. For example, a control soil much richer in nutrients than a test soil might result in a
false positive result (i.e. the test soil appears to have a “toxic” effect on the growth of the test plants). If a control
soil is poorer in nutrients than a test soil, hormesis (3.3) can be expected at low soil mixture ratios or even an
inverse dose response relationship if nutrient supply becomes the main effect. It is therefore recommended to
add nutrients to test and control/reference soils in order to avoid false positive or negative test results (10.6.3).
7.2.3.1 Reference soils
If reference soils from uncontaminated areas near a contaminated site are available, they should be treated
and characterized like the test soils. If a toxic contamination or unusual soil properties cannot be ruled out,
standard control soils should be preferred.
7.2.3.2 Standard soils
Standard soils should be uncontaminated, nutrient-poor natural or artificial soil. If a natural soil is used, its
organic matter content should not exceed 5 %. Fine particles (<20 µm according to ISO 11277) should not
exceed 20 %. Alternatively, artificial soil according to ISO11268-1 and ISO 11268-2 may be used, regardless
of its higher organic matter content. However, the organic matter content of the test and control soil should be
as close to each other as possible.
The substrate called “artificial soil” 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 % 69 %
of particle size 0,05 mm to 0,2 mm)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) is necessary to get a
pH of 6,0 ± 0,5.
NOTE 1 Taking the properties of non-polar (log P > 2) or ionizing substances into account, 5 % of peat have proven
OW
to be sufficient for maintaining the desired structure of the artificial soil. In this case, the respective percentages of the
constituents are modified as follows: peat, 5 %; clay, 20 %; sand 75 %).
NOTE 2 pH (KCl) is measured in a mixed sample in a 1 M solution of potassium chloride (KCl) or a 0,01 M solution of
calcium chloride (CaCl ).
The artificial soil is prepared, at least three days prior to starting the test, by mixing the dry constituents listed
above thoroughly in a large-scale laboratory mixer. A portion of the deionized water required to obtain half of the
final water content of 40 % to 60 % of the maximum water-holding capacity is added while mixing is continued.
The mixed artificial soil shall be stored at room temperature for at least two days to equilibrate acidity. The
amount of calcium carbonate required might vary, depending on properties of the individual batch of sphagnum
peat and should be determined by measuring the pH of subsamples immediately before the test. The total
water-holding capacity is determined according to Annex C, the pH is determined according to ISO 10390.
Allowance should be made for any water that is to be used for introducing the test substance into the soil.
To obtain a dilution series, the test soil is mixed with the control soil thoroughly (either manually or by using a
hand mixer). The homogeneity of the mixture is checked visually.
ISO 11269-2:2012(E)
8 Equipment
Standard laboratory equipment including the following materials is required.
8.1 Controlled environmental chamber, phytotron, plant growth room or greenhouse suitable to maintain
the specified conditions.
8.2 Balance (±0,1 mg).
8.3 Balance for heavier loads (e.g. 10 kg) for preparation of soil mixtures.
8.4 Sieve, stainless steel, with mesh size 4 mm.
8.5 Glass-fibre wicks (∅ 1 mm).
9 Reference substance
It is recommended that a reference substance be tested to demonstrate the uniformity of the laboratory test
conditions and the response of the particular batch of seeds. Sodium trichloroacetate or boric acid is suggested
as the reference substance. A reference test should be carried out regularly and if any major changes in operating
procedures are introduced, for example, change of phytotron/growth room/greenhouse, change of soil or change
of watering regime, etc. Examples of phytotoxic values for two reference compounds are given in Annex B.
10 Procedure
10.1 Experimental design
A sample of field-collected test soil may be tested at a single concentration (typically 100 %) or evaluated for
toxicity in a multi-concentration test whereby a series of concentrations (dilutions) are prepared by mixing
defined quantities with a control soil.
Depending on the knowledge of relevant response levels, a preliminary range-finding test may precede the
final test. Each final test consists of a series of soil mixtures (treatments). Each treatment is replicated at least
four times, i.e. four test pots containing a number of test plants are used.
To avoid any masking of the phytotoxic effect expression by nutrient deficiency, all treatment groups including
control soils are amended by fertilizers (10.6.3) after seedlings have emerged. It has been shown that effects
[6][7][17][22][25]
of pollutants on plant growth are pronounced in soils with optimum nutrient supply .
To assure functioning of the watering system (see 10.6.2), it should be checked whether the test soil sucks
water via wicks sufficiently. Water repellence or poor water transport can occur with very sandy soils, soils
highly contaminated with hydrocarbons or even with soil of high clay content that tend to compact even when
these soils have a high water-holding capacity (determined after initially submerging soils). For this reason, a
pretest including all soils selected for the test and replicated twice should be performed to decide whether wick
watering is applicable or manual watering is required.
10.1.1 Pretest
Two pots equipped with wicks are prepared for each soil, the test soil, the dilutions of the test soil with control
soil, the control soil and (if available) the reference soil. After filling with the sieved test soils, and/or soil
mixtures, the pots are installed above a water reservoir by using a suitable device that avoids direct contact
of the pots with the water supply. The water should reach the soil surface within 24 h. If this is the case, the
soil is expected to be watered successfully by wicks. Otherwise, water should be added manually onto the soil
surface until the soil is wet (but not highly soaked). In many cases, wick watering is successful after such an
initial manual watering. In rare cases, the soils shall be watered manually throughout the whole test period.
6 © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
10.1.2 Range-finding test
A preliminary test to find the range of mixture ratio affecting plant growth is optional. The test soil is mixed with
the reference or a standard control soil by appropriate techniques. Mixture rates of 0 %, 12,5 %, 25 %, 50 %,
and 100 % test soil are suggested. If toxic effects become evident after emergence, the test may be finished
before the end of the growth period of two weeks.
10.1.3 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 a comparison of the biological effects for the test soil(s) with the
effects 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, the results from standard soil shall be used to judge the
[4]
validity and acceptability of the test (see Clause 11) .
If, for characterization purposes, a test design including a dilution series is required, the following three designs
are possible (the concentrations shall be spaced by a factor not exceeding 2).
— 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 ER and EC approach, 12 concentrations should be used. Two replicates for each concentration
x x
plus six controls are recommended. The spacing factor may be variable; smaller at low concentrations,
larger at high concentrations.
— In cases where both threshold levels are required for a mixed approach, 6 concentrations 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 ER /EC evaluation.
x x
A limit test may be sufficient if, in the range-finding 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.
10.2 Preparation of the pots
If soils or soil mixtures have been stored, they should be mixed a second time immediately before use. Pots
either equipped with wicks or without wicks are filled with the soil mixtures to approximately 1 cm below the
upper edge. All the pots of each treatment should contain the same volume of soil. Most soils can be handled
more easily if their water content equals 20 % to 40 % of their maximum water-holding capacity. Wet soils
tend to compact strongly. In addition, seeds might stick to the tweezers when wet. Therefore, it may become
necessary to partially air-dry or rewet the soils before filling the pots. The actual water content of each mixture
should be known to calculate the amount of water needed for initial watering at the start of the test.
The soil should not be compressed strongly. However, if the soil structure appears too loose or incoherent,
settling can be forced by dropping the vessels from a height of less than 5 cm onto a hard surface.
10.3 Preparation of the seeds
Plant 10 uniform undressed seeds of the selected species immediately after filling the pots. If pots other than
the proposed ones are used, the number of seeds may need to be corrected to make equivalent soil volumes
and growth areas available to the plants. Either prepare holes of a depth of 5 mm to 10 mm for Brassica
rapa or 10 mm to 15 mm for Avena sativa, put one seed into each hole and carefully smooth the soil surface.
Alternatively, pick seeds with the tip of tweezers and plant them directly in the required depth.
Oat seeds can be selected by weight. Rejecting very light and heavy seeds can result in a slightly smaller
variation in plant weight between plants. Seeds of Brassica rapa are too small for weight selection. There is
no indication that seeds of varying colour, indicating different stages of maturity, develop differently. Unevenly
shaped seeds should be rejected. If other test species have been chosen, other criteria for selecting seeds
may be appropriate.
ISO 11269-2:2012(E)
10.4 Growth conditions
Temperature, humidity and light conditions shall be suitable for normal growth of the test plants. Tests may be
run in a phytotron, plant-growth room or greenhouse. In addition to daylight (greenhouse), fluorescence tubes,
gas-discharge, metal-halide, high-pressure mercury and high-pressure sodium lamps may be used. Lamps
manufactured for plant growth should be chosen. The lamps should be strong enough to be installed at least
1 m above the soil surface to allow handling of the plants during the test (rearrangement of pots, watering) and
to avoid inhomogeneous temperature. In addition, the lighting rate shall be essentially homogenous across the
area where pots are placed in the test.
For Avena sativa and Brassica rapa, a 16 h illumination period with an intensity of at least 7 000 lx should be
followed by 8 h of darkness.
A temperature of 23 °C ± 3 °C is appropriate for the two species. However, a wider range is acceptable as long
as normal emergence and growth of the plants are occurring.
There should be sufficient ventilation to avoid cross-contamination of volatile toxicants between treatments and
to prevent health hazards.
10.5 Start of the test
In cases where sub-irrigation with glass-fibre wicks can be used (see 10.1), the pots may be installed above
water reservoirs without any additional wetting of the moist soils (see 10.2). Only the wicks are allowed to be
in contact with the water. Only pots from the same treatments may use the same reservoir. Since chemicals
or nutrients might be washed out into the reservoirs, the water volume should be limited (e.g. <0,5 l per pot).
Soil moisture content shall not be adjusted through watering and reweighing but is kept constant at a percentage
close to the maximum water-holding capacity (C ). Pots shall be checked visually for sufficient water
w,max
supply by the wick watering system to maintain good growing conditions.
If, according to the results of the pretest, wick watering is not appropriate, the soil shall be wetted to reach a
water content that is equivalent to 60 % to 80 % of the C immediately after placing the seeds into the moist
w,max
soil (see 10.2).
Soil moisture content shall be adjusted daily to maintain a predetermined percentage of the water-holding
capacity, e.g. 80 % for Avena sativa and 60 % for Brassica rapa. A sufficient check can be made by weighing
several randomly selected pots daily. Anaerobic conditions should be avoided and noted in the test report.
Individual pots or treatment groups should be placed randomly in the incubation area.
10.6 Handling during the test
10.6.1 Number of plants and thinning out
To compensate for non-germinating seeds, a higher number of seeds (typically 10) are planted in each pot
than plants are required for the test. After the emergence assessment within each pot (for Avena sativa about
three days to five days and for Brassica rapa about seven days to eight days after sowing), thin the seedlings
to give a total of five evenly spaced representative specimens of the plants in the pots. It is important that the
density of plants in a test vessel does not limit normal growth. The number of five specimens per pot applies
for Avena sativa and Brassica rapa and the pots specified above, and shall be adjusted if other species or
differing sizes of pots have been used. To withdraw plants, they can be pulled out, or if the soil is very cohesive
or plants grow very close to each other, they can be cut off. When oat is cut, a secondary shoot is sometimes
produced, which shall be cut again later. If necessary, water losses in the reservoirs are compensated with
fertilizer solution (10.6.3). Terminate the test no sooner than 14 days and no later than 21 days after 50 % of the
control seedlings have emerged.
10.6.2 Watering
Demineralized water shall be used to fill the water reservoirs whenever needed. Ensure that the required soil
moisture is being maintained. Therefore, check regularly — for example, visually or by carefully touching the
8 © ISO 2012 – All rights reserved

ISO 11269-2:2012(E)
soil surface — whether the surface is wet. If not, reweigh the pots, and replenish the amount of water needed.
If wick watering fails, carefully pour or spray the volume needed onto the soil surface regularly.
If no wicks are used, the soil moisture should be
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