SIST ISO 22030:2006
(Main)Soil quality -- Biological methods -- Chronic toxicity in higher plants
Soil quality -- Biological methods -- Chronic toxicity in higher plants
This International Standard describes a method for determining the inhibition of the growth and reproductive capability of higher plants by soils under controlled conditions. Two species are recommended: a rapid-cycling variant of turnip rape (Brassica rapa CrGC syn. Rbr) and oat (Avena sativa). The duration of test should be sufficient to include chronic endpoints that demonstrate the reproductive capability of the test plants. By using natural test soils, e.g. from contaminated sites or remediated soils, and by comparing the development of the test plants in these soils with reference or standard control soils, the test can be used to assess soil quality, especially the function of the soil as a habitat for plants. Annex A describes modifications allowing use of the chronic plant assay for the testing of chemicals incorporated into soil. By preparing a dilution series of a test substance in standard control soils, the same endpoints can be measured to assess the chronic toxicity of chemicals. This 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.
Qualité du sol -- Méthodes biologiques -- Toxicité chronique sur les plantes supérieures
L'ISO 22030:2005 décrit une méthode de détermination de l'inhibition de la croissance et de la capacité de reproduction des plantes supérieures par les sols dans des conditions contrôlées. Deux espèces sont recommandées: une variante de colza à cycle rapide (Brassica rapa CrGC syn. Rbr) et l'avoine (Avena sativa). Il convient que la durée de l'essai soit suffisante pour inclure les points de mesure chroniques qui apportent la preuve de la capacité de reproduction des plantes soumises à l'essai.
En utilisant des sols d'essai naturels provenant, par exemple, de sites contaminés ou de sols réhabilités, et en comparant le développement des plantes testées dans ces sols à celui des plantes testées dans les sols de référence ou les sols témoins standards, l'essai peut permettre d'évaluer la qualité du sol, notamment sa fonction d'habitat des plantes.
Cette méthode peut être modifiée pour permettre d'exploiter l'analyse chronique des plantes pour les essais de substances ou de produits chimiques incorporés dans le sol. En préparant une série de dilutions d'une substance dans les sols témoins standard, il est possible de déterminer les mêmes points de mesure afin d'évaluer la toxicité chronique de ces substances ou de ces produits chimiques.
Cette méthode n'est pas applicable aux substances volatiles, c'est-à-dire celles pour lesquelles la constante de Henry ou le coefficient de partage air/eau est supérieur à 1, ou pour lesquelles la pression de vapeur à 25 °C est supérieure à 0,013 3 Pa à 25 °C.
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INTERNATIONAL ISO
STANDARD 22030
First edition
2005-02-01
Soil quality — Biological methods —
Chronic toxicity in higher plants
Qualité du sol — Méthodes biologiques — Toxicité chronique sur les
plantes supérieures
Reference number
ISO 22030:2005(E)
©
ISO 2005
---------------------- Page: 1 ----------------------
ISO 22030:2005(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2005
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 2005 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22030:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 2
4 Principle. 4
5 Materials. 4
5.1 Test plants. 4
5.2 Soil and soil storage . 4
5.2.1 General. 4
5.2.2 Test soil. 5
5.2.3 Control soil. 5
5.3 Reference substance. 6
6 Apparatus. 6
7 Methods. 8
7.1 Experimental design. 8
7.2 Pre-test. 8
7.3 Preliminary test (range-finding). 8
7.4 Final test. 9
7.5 Preparation of the pots. 9
7.6 Preparation of the seeds . 9
7.7 Growth conditions. 9
7.8 Start of the test. 10
7.9 Handling during the test. 10
7.9.1 Number of plants and thinning out . 10
7.9.2 Watering. 10
7.9.3 Rearrangement of test vessels. 10
7.9.4 Pollination. 10
7.9.5 Reporting. 11
7.10 Endpoint measurements. 11
7.10.1 Seedling emergence. 11
7.10.2 Harvest at day 14. 11
7.10.3 Final harvest. 11
7.11 Summary and timetable of the test . 12
8 Validity criteria. 13
9 Assessment of the results . 13
9.1 Presentation of measured data . 13
9.2 Statistical analysis. 14
9.2.1 Preliminary test. 14
9.2.2 Final test. 14
9.2.3 NOEC (no observed effect concentration) approach . 14
9.2.4 EC (effect concentration) approach. 14
x
10 Test report. 15
Annex A (informative) Testing of chemicals in soil . 16
Bibliography . 17
© ISO 2005 – All rights reserved iii
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ISO 22030:2005(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 22030 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
iv © ISO 2005 – All rights reserved
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ISO 22030:2005(E)
Introduction
This International Standard describes a procedure for evaluating the quality of soils of different origin carrying
unknown contaminations. The method, slightly modified, can also be used to measure the toxicity of known
chemicals incorporated into soil.
The evaluation of the inhibition and chronic toxicity is based on emergence, vegetative growth and
reproductive capacity of at least two species of higher plants.
This International Standard is based on:
a) results of the research project “Development of a chronic bioassay using higher plants”, sponsored by the
[3]
German Ministry for Education and Research (BMBF), Bonn , and
b) discussions within the joint project “Ecotoxicological Test Batteries” forming part of the BMBF Joint
[10]
Research Group “Processes for the Bioremediation of Soil” .
© ISO 2005 – All rights reserved v
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INTERNATIONAL STANDARD ISO 22030:2005(E)
Soil quality — Biological methods — Chronic toxicity in higher
plants
WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic or otherwise
harmful chemicals or infectious microorganisms. Occupational health risks can arise from dust or
evaporated chemicals during handling and incubation. Furthermore, test plants can absorb chemicals
from the soil and safety measures should also be considered when handling these test plants.
1 Scope
This International Standard describes a method for determining the inhibition of the growth and reproductive
capability of higher plants by soils under controlled conditions. Two species are recommended: a rapid-cycling
variant of turnip rape (Brassica rapa CrGC syn. Rbr) and oat (Avena sativa). The duration of test should be
sufficient to include chronic endpoints that demonstrate the reproductive capability of the test plants.
By using natural test soils, e.g. from contaminated sites or remediated soils, and by comparing the
development of the test plants in these soils with reference or standard control soils, the test can be used to
assess soil quality, especially the function of the soil as a habitat for plants.
Annex A describes modifications allowing use of the chronic plant assay for the testing of chemicals
incorporated into soil. By preparing a dilution series of a test substance in standard control soils, the same
endpoints can be measured to assess the chronic toxicity of chemicals. This 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.
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 11268-1:1993, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 1: Determination
of acute toxicity using artificial soil substrate
ISO 11268-2:1998, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 2: Determination
of effects on reproduction
ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
chemicals on the emergence and growth of higher plants
ISO 15176:2002, Soil quality — Characterization of excavated soil and other soil materials intended for re-use
ISO 15799, Soil quality — Guidance on the ecotoxicological characterization of soils and soil materials
ASTM D1076:2002, Standard Specification for Rubber-Concentrated, Ammonia Preserved, Creamed, and
Centrifuged Natural Latex
© ISO 2005 – All rights reserved 1
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ISO 22030:2005(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
artificial soil
mixture of sand, kaolinite, peat and calcium carbonate
NOTE ISO 11268-1 describes such a soil for toxicity tests using earthworms. Pure quartz sand, mineral wool,
vermiculite or other synthetic substrates should not be used.
3.2
biomass
total mass of shoots, flowers and seed pods
NOTE 1 Biomass is expressed as dry mass per plant or, if needed, as dry mass per pot.
NOTE 2 During the test period, some of the test plants can reach different growth stages and their water content can
differ when the plants are harvested. Thus the dry mass better represents the biomass produced during the growth period.
3.3
concentration
mass of test substance per amount of soil
NOTE Concentration is expressed as a mass fraction, in milligrams per kilogram (mg/kg) of dry soil.
3.4
contaminant
substance or agent present in the soil as a result of human activity
[ISO 15176:2002]
3.5
control soil
uncontaminated substrate, used as a control and as medium for preparing dilution series with test soils or
chemicals, that allows the growth of healthy plants
NOTE Either artificial or natural standard or reference soils can be used, 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 can 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 (see 3.9) can be expected at low soil-mixture ratios, or even an inverse dose response
relationship, if nutrient supply becomes the main effect. This International Standard does not provide numerical values for
the nutrients.
3.6
effect concentration
EC
x
concentration (mass fraction) of a test chemical or the percentage (mass fraction) of a test soil at which a
given endpoint is inhibited by x % compared to the control
NOTE The effect concentration is expressed in milligrams per kilogram. When chemicals are tested, the EC is
x
expressed as mass of the test substance per dry mass of soil; when soils are tested, the EC is expressed as a
x
percentage of test soil dry mass per soil mixture dry mass.
3.7
emergence
development of a seedling contained within a seed, ending the latent period
NOTE It is expressed as the percentage of seedlings which emerge from test pots as compared with the control pots.
2 © ISO 2005 – All rights reserved
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ISO 22030:2005(E)
3.8
habitat function
ability of soils/soil materials to serve as a habitat for microorganisms, plants, soil-living animals and their
interactions (biocenosis)
[ISO 15799]
3.9
hormesis
improvement of seedling emergence, growth or survival (or other response of the test plants) at low
concentrations of chemicals or mixtures of soil that are toxic when applied at higher levels in comparison to
[1]
the control
3.10
lowest observed effect concentration
LOEC
lowest tested concentration (mass fraction) of a test substance in soil at which a statistically significant effect
on a given endpoint (p < 0,05) compared with the control is observed
cf. NOEC (3.11)
NOTE Analogously, the term LOEC is used for the lowest tested mixture ratio of a test soil in a reference or a
standard control soil at which a statistically significant effect is observed. The LOEC is expressed as mass of the test
substance per mass of dry soil or, in the latter case, as percentage of test-soil dry mass per soil-mixture dry mass. All test
concentrations above the LOEC have a harmful effect equal 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 have been selected.
3.11
no observed effect concentration
NOEC
test substance concentration (mass fraction) or soil mixture ratio immediately below the LOEC, which when
compared to the control has no statistically significant effect (p < 0,05)
cf. LOEC (3.10)
3.12
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with properties (nutrient
concentrations, pH, organic carbon content and texture) similar to the test soil
3.13
soil mixture ratio
ratio of the dry mass of test soil to the dry mass of reference/control soil
NOTE It is expressed as a percentage.
3.14
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are within a
known range
EXAMPLES Euro soils, artificial soil.
NOTE The properties of standard soils may differ from those of the test soil.
© ISO 2005 – All rights reserved 3
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ISO 22030:2005(E)
4 Principle
This International Standard describes a plant test that includes both acute and chronic endpoints. The test
measures emergence, early growth and reproduction of two terrestrial plant species (Avena sativa and a
rapid-cycling variety of Brassica rapa are recommended). The test compares responses of plants in a test soil
and/or a series of dilutions with a control soil. This test method may also be used for the testing of chemicals
by applying various concentrations of the test substance to a standard control soil. Seeds of both plant
species are planted in pots containing the soil/soil mixtures and in control pots containing a reference or
standard soil. Pots are placed in a temperature- and light-controlled room or growth chamber. They are
watered via wicks. After emergence of the plants, emergence rates are determined and plants are thinned out
to a specified number. After two weeks, some of the plants are harvested to determine their biomass. After
another period of three weeks to four weeks (rapid-cycling Brassica rapa) or five weeks to 6 weeks (Avena
sativa), the remaining plants are harvested for measuring additional endpoints characterizing their
reproductive potential. In all cases, the test duration should be sufficient to determine reproductive endpoints
(e.g. number or biomass of flowers or seeds or fruit).
Typically, 10 seeds are sown in four replicate test pots each. Plants are thinned out to 8 per pot, and four
plants each are harvested at day 14 and at the end of the test. If in any pot less than 8 plants have emerged,
the number of plants harvested at day 14 shall be reduced such that four plants remain for the final harvest.
The relative inhibition in undiluted test soils is determined to assess the suitability of the soil for plants. In
addition, based on a dilution series, NOEC, LOEC and EC values can be calculated from the dose response
x
curves. The latter is required when chemicals are tested.
5 Materials
5.1 Test plants
One monocotyledonous and one dicotyledonous species are tested in parallel. Oat (Avena sativa) is
recommended as the monocotyledonous and Brassica rapa as the dicotyledonous plant species.
To shorten the test period, a rapid-cycling variety of turnip rape (Brassica rapa CrGC syn. Rbr) is strongly
1)
recommended . Flowering starts after two weeks and seed production can be determined after approximately
five weeks.
Other species may be selected, e.g. from the list given in ISO 11269-2 or plants with specific physiological
characteristics such as C-4 plants (corn, sugar cane, millet), plants in symbiosis with nitrogen-fixing bacteria (e.g.
Fabaceae) or plants with ecological or economic significance in certain regions of the world. These plants shall
grow unhindered in control soil under the conditions specified. Only plants that tolerate the properties of the test
soils and test conditions (including 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. Oat and rapid-cycling
turnip rape grow in sandy as well as loamy soil with varying water content and a range of pH values from 5,0 to
7,5. Species that do not tolerate wet soils should not be used in combination with wick watering. Reasons for
selecting species other than oat and turnip rape shall be justified in the test report.
5.2 Soil and soil storage
5.2.1 General
The description of methods for representative sampling of soils from contaminated sites is not within the
[13]
Scope of this International Standard. A suitable sampling method is given in ISO 10381-6 .
1) Seeds and Wisconsin Fast Plants kits are suitable products supplied by the Carolina Biological Supply Company,
Burlington, NC, USA. This information is given for the convenience of users of this International Standard 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.
4 © ISO 2005 – All rights reserved
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ISO 22030:2005(E)
The chronic plant test can be used to assess the toxic potential of natural soils from a variety of contaminated
sites. It can also be used to assess the quality of remediated soils. 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. Test soils shall be passed through a sieve of mesh 4 mm to 5 mm square to remove coarse fragments,
and mixed thoroughly. If necessary, soil may be air-dried without heating before sieving. Storage of test soils
should be as short as possible. Storage at approximately 4 °C using containers that minimize losses of soil
contaminants by volatilization and sorption to the container walls is recommended.
5.2.2 Test soil
The water-holding capacity shall be measured for all soil mixtures used in the test. Additionally, test soils
should be characterized by:
texture (sand, silt, clay);
pH value;
salinity;
organic carbon;
total and water-soluble amounts of potassium, nitrogen and phosphorus.
Soil pH should not be corrected. Measurements of soil contaminants (heavy metals, hydrocarbons, pesticides,
explosives, PCBs and others) are not mandatory.
It should be checked whether the test soil sucks water via wicks sufficiently (see 5.4). Water repellency or
poor water transport can occur with very sandy soils, soils highly contaminated with hydrocarbons or even
with soils of high clay content that tend to compact even when these soils have a high water-holding capacity
(determined after initially submerging the soils). To assure functioning of the watering system, a pre-test
including all soils selected for the test and replicated twice should be performed to decide whether wick
watering is sufficient or manual watering is required.
NOTE For the time being, pH limits for plant species other than turnip rape and oat cannot be given. It is 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 yet be given for different plant species.
5.2.3 Control soil
Either artificial, reference or standard soils may be used as control substrate.
If reference soils from uncontaminated areas near a contaminated site are available, they should be treated
and characterized like the test soils. In addition, to verify that a reference soil does not carry toxic
contaminants, chemical analysis of the expected contaminants shall be carried out. If toxic contamination or
unusual soil properties cannot be ruled out, standard control soils should be given preference.
Standard soils should be uncontaminated, nutrient-poor natural or artificial soils. If a natural soil is used, its
organic matter content should not exceed 5 %. Fine particles (< 20 µm) should not exceed 20 %.
Alternatively, artificial soil in accordance with reference [6] and ISO 11268-2 may be used, regardless of its
higher organic matter content. However, the organic matter contents of the test and control soil should be as
close to each other as possible. The artificial soil consists of the following components (percentage based on
dry mass):
10 % sphagnum peat [air-dried and finely ground (2 mm ± 1 mm)];
20 % kaolin clay (kaolinite content preferably above 30 %);
approximately 69 % (depending on the amount of CaCO needed) air-dried industrial quartz sand
3
(predominantly fine sand with more than 50 % mass fraction of particle size 0,05 mm to 0,2 mm).
© ISO 2005 – All rights reserved 5
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ISO 22030:2005(E)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) is necessary to obtain
3
a pH of 6,0 ± 0,5. The amount of calcium carbonate required can vary, depending on properties of the
individual batch (mainly the peat) and should be determined by measuring sub-samples immediately before
the test.
The artificial soil is prepared by mixing the dry constituents listed above thoroughly in a large-scale laboratory
mixer approximately one week before starting the test.
The mixed artificial soil shall be stored 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 premoistened one or two days
before starting the test by adding enough deionized water to obtain approximately half of the required final
water content of 40 % to 60 % of the maximum water-holding capacity (corresponding to 50 % ± 10 %
moisture dry mass). The pH value is measured by mixing the soil with 1 mol/l KCl in a ratio of 1 to 5 (in
accordance with Annex C of ISO 11268-2:1998). If the measured pH is not within the required range, a
sufficient amount of CaCO shall be added or a new batch of artificial soil shall be prepared. Parallel to
3
determining the pH, the maximum water-holding capacity of the artificial soil shall be determined.
Afterwards, the artificial soil is divided into as many batches as the number of concentrations plus controls that
is used in the test. Evaporation from the test substrate shall be avoided until the start of the test. The final
moisture content is reached by adding water together with, or in parallel to, the application of the test
substance. The moisture contents at the beginning and end of the test are determined by drying small
samples at 105 °C overnight and reweighing.
If the control soil is richer in nutrients than the test soil, growth inhibition may become apparent from the dose
(mixture) response curves. It is recommended to use nutrient-poor control soils or to add nutrients in order to
avoid false-positive test results.
Addition of nutrients can reduce the relative difference between control and test soils. Nevertheless, nutrient
additions may result in
slower ripening of the plants,
strong vegetative growth (larger test containers needed),
adverse effects on the test plants, when a test soil already has high levels of nutrients or salts.
None of the soils used in the chronic plant test needs to be sterilized.
To obtain a dilution series, the test soil is thoroughly mixed with the reference or standard control soil (either
manually or by using a hand mixer). The homogeneity of the mixture is checked visually.
5.3 Re
...
SLOVENSKI STANDARD
SIST ISO 22030:2006
01-december-2006
.DNRYRVWWDO±%LRORãNHPHWRGH±.URQLþQDWRNVLþQRVW]DYLãMHUDVWOLQH
Soil quality -- Biological methods -- Chronic toxicity in higher plants
Qualité du sol -- Méthodes biologiques -- Toxicité chronique sur les plantes supérieures
Ta slovenski standard je istoveten z: ISO 22030:2005
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST ISO 22030:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST ISO 22030:2006
---------------------- Page: 2 ----------------------
SIST ISO 22030:2006
INTERNATIONAL ISO
STANDARD 22030
First edition
2005-02-01
Soil quality — Biological methods —
Chronic toxicity in higher plants
Qualité du sol — Méthodes biologiques — Toxicité chronique sur les
plantes supérieures
Reference number
ISO 22030:2005(E)
©
ISO 2005
---------------------- Page: 3 ----------------------
SIST ISO 22030:2006
ISO 22030:2005(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2005
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 2005 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 22030:2006
ISO 22030:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 2
4 Principle. 4
5 Materials. 4
5.1 Test plants. 4
5.2 Soil and soil storage . 4
5.2.1 General. 4
5.2.2 Test soil. 5
5.2.3 Control soil. 5
5.3 Reference substance. 6
6 Apparatus. 6
7 Methods. 8
7.1 Experimental design. 8
7.2 Pre-test. 8
7.3 Preliminary test (range-finding). 8
7.4 Final test. 9
7.5 Preparation of the pots. 9
7.6 Preparation of the seeds . 9
7.7 Growth conditions. 9
7.8 Start of the test. 10
7.9 Handling during the test. 10
7.9.1 Number of plants and thinning out . 10
7.9.2 Watering. 10
7.9.3 Rearrangement of test vessels. 10
7.9.4 Pollination. 10
7.9.5 Reporting. 11
7.10 Endpoint measurements. 11
7.10.1 Seedling emergence. 11
7.10.2 Harvest at day 14. 11
7.10.3 Final harvest. 11
7.11 Summary and timetable of the test . 12
8 Validity criteria. 13
9 Assessment of the results . 13
9.1 Presentation of measured data . 13
9.2 Statistical analysis. 14
9.2.1 Preliminary test. 14
9.2.2 Final test. 14
9.2.3 NOEC (no observed effect concentration) approach . 14
9.2.4 EC (effect concentration) approach. 14
x
10 Test report. 15
Annex A (informative) Testing of chemicals in soil . 16
Bibliography . 17
© ISO 2005 – All rights reserved iii
---------------------- Page: 5 ----------------------
SIST ISO 22030:2006
ISO 22030:2005(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 22030 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
iv © ISO 2005 – All rights reserved
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SIST ISO 22030:2006
ISO 22030:2005(E)
Introduction
This International Standard describes a procedure for evaluating the quality of soils of different origin carrying
unknown contaminations. The method, slightly modified, can also be used to measure the toxicity of known
chemicals incorporated into soil.
The evaluation of the inhibition and chronic toxicity is based on emergence, vegetative growth and
reproductive capacity of at least two species of higher plants.
This International Standard is based on:
a) results of the research project “Development of a chronic bioassay using higher plants”, sponsored by the
[3]
German Ministry for Education and Research (BMBF), Bonn , and
b) discussions within the joint project “Ecotoxicological Test Batteries” forming part of the BMBF Joint
[10]
Research Group “Processes for the Bioremediation of Soil” .
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SIST ISO 22030:2006
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SIST ISO 22030:2006
INTERNATIONAL STANDARD ISO 22030:2005(E)
Soil quality — Biological methods — Chronic toxicity in higher
plants
WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic or otherwise
harmful chemicals or infectious microorganisms. Occupational health risks can arise from dust or
evaporated chemicals during handling and incubation. Furthermore, test plants can absorb chemicals
from the soil and safety measures should also be considered when handling these test plants.
1 Scope
This International Standard describes a method for determining the inhibition of the growth and reproductive
capability of higher plants by soils under controlled conditions. Two species are recommended: a rapid-cycling
variant of turnip rape (Brassica rapa CrGC syn. Rbr) and oat (Avena sativa). The duration of test should be
sufficient to include chronic endpoints that demonstrate the reproductive capability of the test plants.
By using natural test soils, e.g. from contaminated sites or remediated soils, and by comparing the
development of the test plants in these soils with reference or standard control soils, the test can be used to
assess soil quality, especially the function of the soil as a habitat for plants.
Annex A describes modifications allowing use of the chronic plant assay for the testing of chemicals
incorporated into soil. By preparing a dilution series of a test substance in standard control soils, the same
endpoints can be measured to assess the chronic toxicity of chemicals. This 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.
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 11268-1:1993, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 1: Determination
of acute toxicity using artificial soil substrate
ISO 11268-2:1998, Soil quality — Effects of pollutants on earthworms (Eisenia fetida) — Part 2: Determination
of effects on reproduction
ISO 11269-2, Soil quality — Determination of the effects of pollutants on soil flora — Part 2: Effects of
chemicals on the emergence and growth of higher plants
ISO 15176:2002, Soil quality — Characterization of excavated soil and other soil materials intended for re-use
ISO 15799, Soil quality — Guidance on the ecotoxicological characterization of soils and soil materials
ASTM D1076:2002, Standard Specification for Rubber-Concentrated, Ammonia Preserved, Creamed, and
Centrifuged Natural Latex
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SIST ISO 22030:2006
ISO 22030:2005(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
artificial soil
mixture of sand, kaolinite, peat and calcium carbonate
NOTE ISO 11268-1 describes such a soil for toxicity tests using earthworms. Pure quartz sand, mineral wool,
vermiculite or other synthetic substrates should not be used.
3.2
biomass
total mass of shoots, flowers and seed pods
NOTE 1 Biomass is expressed as dry mass per plant or, if needed, as dry mass per pot.
NOTE 2 During the test period, some of the test plants can reach different growth stages and their water content can
differ when the plants are harvested. Thus the dry mass better represents the biomass produced during the growth period.
3.3
concentration
mass of test substance per amount of soil
NOTE Concentration is expressed as a mass fraction, in milligrams per kilogram (mg/kg) of dry soil.
3.4
contaminant
substance or agent present in the soil as a result of human activity
[ISO 15176:2002]
3.5
control soil
uncontaminated substrate, used as a control and as medium for preparing dilution series with test soils or
chemicals, that allows the growth of healthy plants
NOTE Either artificial or natural standard or reference soils can be used, 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 can 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 (see 3.9) can be expected at low soil-mixture ratios, or even an inverse dose response
relationship, if nutrient supply becomes the main effect. This International Standard does not provide numerical values for
the nutrients.
3.6
effect concentration
EC
x
concentration (mass fraction) of a test chemical or the percentage (mass fraction) of a test soil at which a
given endpoint is inhibited by x % compared to the control
NOTE The effect concentration is expressed in milligrams per kilogram. When chemicals are tested, the EC is
x
expressed as mass of the test substance per dry mass of soil; when soils are tested, the EC is expressed as a
x
percentage of test soil dry mass per soil mixture dry mass.
3.7
emergence
development of a seedling contained within a seed, ending the latent period
NOTE It is expressed as the percentage of seedlings which emerge from test pots as compared with the control pots.
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ISO 22030:2005(E)
3.8
habitat function
ability of soils/soil materials to serve as a habitat for microorganisms, plants, soil-living animals and their
interactions (biocenosis)
[ISO 15799]
3.9
hormesis
improvement of seedling emergence, growth or survival (or other response of the test plants) at low
concentrations of chemicals or mixtures of soil that are toxic when applied at higher levels in comparison to
[1]
the control
3.10
lowest observed effect concentration
LOEC
lowest tested concentration (mass fraction) of a test substance in soil at which a statistically significant effect
on a given endpoint (p < 0,05) compared with the control is observed
cf. NOEC (3.11)
NOTE Analogously, the term LOEC is used for the lowest tested mixture ratio of a test soil in a reference or a
standard control soil at which a statistically significant effect is observed. The LOEC is expressed as mass of the test
substance per mass of dry soil or, in the latter case, as percentage of test-soil dry mass per soil-mixture dry mass. All test
concentrations above the LOEC have a harmful effect equal 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 have been selected.
3.11
no observed effect concentration
NOEC
test substance concentration (mass fraction) or soil mixture ratio immediately below the LOEC, which when
compared to the control has no statistically significant effect (p < 0,05)
cf. LOEC (3.10)
3.12
reference soil
uncontaminated site-specific soil (e.g. collected in the vicinity of a contaminated site) with properties (nutrient
concentrations, pH, organic carbon content and texture) similar to the test soil
3.13
soil mixture ratio
ratio of the dry mass of test soil to the dry mass of reference/control soil
NOTE It is expressed as a percentage.
3.14
standard soil
field-collected soil or artificial soil whose main properties (e.g. pH, texture, organic matter content) are within a
known range
EXAMPLES Euro soils, artificial soil.
NOTE The properties of standard soils may differ from those of the test soil.
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ISO 22030:2005(E)
4 Principle
This International Standard describes a plant test that includes both acute and chronic endpoints. The test
measures emergence, early growth and reproduction of two terrestrial plant species (Avena sativa and a
rapid-cycling variety of Brassica rapa are recommended). The test compares responses of plants in a test soil
and/or a series of dilutions with a control soil. This test method may also be used for the testing of chemicals
by applying various concentrations of the test substance to a standard control soil. Seeds of both plant
species are planted in pots containing the soil/soil mixtures and in control pots containing a reference or
standard soil. Pots are placed in a temperature- and light-controlled room or growth chamber. They are
watered via wicks. After emergence of the plants, emergence rates are determined and plants are thinned out
to a specified number. After two weeks, some of the plants are harvested to determine their biomass. After
another period of three weeks to four weeks (rapid-cycling Brassica rapa) or five weeks to 6 weeks (Avena
sativa), the remaining plants are harvested for measuring additional endpoints characterizing their
reproductive potential. In all cases, the test duration should be sufficient to determine reproductive endpoints
(e.g. number or biomass of flowers or seeds or fruit).
Typically, 10 seeds are sown in four replicate test pots each. Plants are thinned out to 8 per pot, and four
plants each are harvested at day 14 and at the end of the test. If in any pot less than 8 plants have emerged,
the number of plants harvested at day 14 shall be reduced such that four plants remain for the final harvest.
The relative inhibition in undiluted test soils is determined to assess the suitability of the soil for plants. In
addition, based on a dilution series, NOEC, LOEC and EC values can be calculated from the dose response
x
curves. The latter is required when chemicals are tested.
5 Materials
5.1 Test plants
One monocotyledonous and one dicotyledonous species are tested in parallel. Oat (Avena sativa) is
recommended as the monocotyledonous and Brassica rapa as the dicotyledonous plant species.
To shorten the test period, a rapid-cycling variety of turnip rape (Brassica rapa CrGC syn. Rbr) is strongly
1)
recommended . Flowering starts after two weeks and seed production can be determined after approximately
five weeks.
Other species may be selected, e.g. from the list given in ISO 11269-2 or plants with specific physiological
characteristics such as C-4 plants (corn, sugar cane, millet), plants in symbiosis with nitrogen-fixing bacteria (e.g.
Fabaceae) or plants with ecological or economic significance in certain regions of the world. These plants shall
grow unhindered in control soil under the conditions specified. Only plants that tolerate the properties of the test
soils and test conditions (including 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. Oat and rapid-cycling
turnip rape grow in sandy as well as loamy soil with varying water content and a range of pH values from 5,0 to
7,5. Species that do not tolerate wet soils should not be used in combination with wick watering. Reasons for
selecting species other than oat and turnip rape shall be justified in the test report.
5.2 Soil and soil storage
5.2.1 General
The description of methods for representative sampling of soils from contaminated sites is not within the
[13]
Scope of this International Standard. A suitable sampling method is given in ISO 10381-6 .
1) Seeds and Wisconsin Fast Plants kits are suitable products supplied by the Carolina Biological Supply Company,
Burlington, NC, USA. This information is given for the convenience of users of this International Standard 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.
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The chronic plant test can be used to assess the toxic potential of natural soils from a variety of contaminated
sites. It can also be used to assess the quality of remediated soils. 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. Test soils shall be passed through a sieve of mesh 4 mm to 5 mm square to remove coarse fragments,
and mixed thoroughly. If necessary, soil may be air-dried without heating before sieving. Storage of test soils
should be as short as possible. Storage at approximately 4 °C using containers that minimize losses of soil
contaminants by volatilization and sorption to the container walls is recommended.
5.2.2 Test soil
The water-holding capacity shall be measured for all soil mixtures used in the test. Additionally, test soils
should be characterized by:
texture (sand, silt, clay);
pH value;
salinity;
organic carbon;
total and water-soluble amounts of potassium, nitrogen and phosphorus.
Soil pH should not be corrected. Measurements of soil contaminants (heavy metals, hydrocarbons, pesticides,
explosives, PCBs and others) are not mandatory.
It should be checked whether the test soil sucks water via wicks sufficiently (see 5.4). Water repellency or
poor water transport can occur with very sandy soils, soils highly contaminated with hydrocarbons or even
with soils of high clay content that tend to compact even when these soils have a high water-holding capacity
(determined after initially submerging the soils). To assure functioning of the watering system, a pre-test
including all soils selected for the test and replicated twice should be performed to decide whether wick
watering is sufficient or manual watering is required.
NOTE For the time being, pH limits for plant species other than turnip rape and oat cannot be given. It is 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 yet be given for different plant species.
5.2.3 Control soil
Either artificial, reference or standard soils may be used as control substrate.
If reference soils from uncontaminated areas near a contaminated site are available, they should be treated
and characterized like the test soils. In addition, to verify that a reference soil does not carry toxic
contaminants, chemical analysis of the expected contaminants shall be carried out. If toxic contamination or
unusual soil properties cannot be ruled out, standard control soils should be given preference.
Standard soils should be uncontaminated, nutrient-poor natural or artificial soils. If a natural soil is used, its
organic matter content should not exceed 5 %. Fine particles (< 20 µm) should not exceed 20 %.
Alternatively, artificial soil in accordance with reference [6] and ISO 11268-2 may be used, regardless of its
higher organic matter content. However, the organic matter contents of the test and control soil should be as
close to each other as possible. The artificial soil consists of the following components (percentage based on
dry mass):
10 % sphagnum peat [air-dried and finely ground (2 mm ± 1 mm)];
20 % kaolin clay (kaolinite content preferably above 30 %);
approximately 69 % (depending on the amount of CaCO needed) air-dried industrial quartz sand
3
(predominantly fine sand with more than 50 % mass fraction of particle size 0,05 mm to 0,2 mm).
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ISO 22030:2005(E)
Approximately 0,3 % to 1,0 % calcium carbonate (CaCO , pulverized, analytical grade) is necessary to obtain
3
a pH of 6,0 ± 0,5. The amount of calcium carbonate required can vary, depending on properties of the
individual batch (mainly the peat) and should be determined by measuring sub-samples immediately before
the test.
The artificial soil is prepared by mixing the dry constituents listed above thoroughly in a large-scale laboratory
mixer approximately one week before starting the test.
The mixed artificial soil shall be stored 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 premoistened one or two days
before starting the test by adding enough deionized water to obtain approximately half of the required final
water content of 40 % to 60 % of the maximum water-holding capacity (corresponding to 50 % ± 10 %
moisture dry mass). The pH value is measured by mixing the soil with 1 mol/l KCl in a ratio of 1 to 5 (in
accordance with Annex C of ISO 11268-2:1998). If the measured pH is not within the required range, a
sufficient amount of CaCO shall be added or a new batch of artificial soil shall be prepared. Parallel to
3
determining the pH, the maximum water-holding capacity of the artificial soil shall be determined.
Afterwards, the artificial soil is divided into as many batches as the number of concentrations plus controls that
is used in the test. Evaporation from the test substrate shall be avoided until the start of the test. The final
moisture content is reached by adding water together with, or in parallel to, the application of the test
substance. The moisture c
...
NORME ISO
INTERNATIONALE 22030
Première édition
2005-02-01
Qualité du sol — Méthodes
biologiques — Toxicité chronique sur les
plantes supérieures
Soil quality — Biological methods — Chronic toxicity in higher plants
Numéro de référence
ISO 22030:2005(F)
©
ISO 2005
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ISO 22030:2005(F)
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ii © ISO 2005 – Tous droits réservés
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ISO 22030:2005(F)
Sommaire Page
Avant-propos. iv
Introduction . v
1 Domaine d'application. 1
2 Références normatives. 1
3 Termes et définitions . 2
4 Principe. 4
5 Matériaux. 4
5.1 Plantes testées. 4
5.2 Sol et stockage du sol . 5
5.2.1 Généralités. 5
5.2.2 Sol d’essai. 5
5.2.3 Sol témoin. 6
5.3 Substance de référence. 7
6 Équipement. 7
7 Méthodes. 9
7.1 Montage expérimental. 9
7.2 Essai préalable. 9
7.3 Essai préliminaire (détermination de la plage de concentrations) . 9
7.4 Essai final. 9
7.5 Préparation des pots . 9
7.6 Préparation des graines . 10
7.7 Conditions de croissance . 10
7.8 Début de l’essai. 10
7.9 Manipulation au cours de l’essai. 11
7.9.1 Nombre de plantes et éclaircissage. 11
7.9.2 Arrosage. 11
7.9.3 Nouvelle disposition des récipients d’essai . 11
7.9.4 Pollinisation. 11
7.9.5 Compte rendu. 11
7.10 Mesure des points de mesure . 12
7.10.1 Germination de plantules. 12
ème
7.10.2 Récolte au 14 jour . 12
7.10.3 Récolte finale. 12
7.11 Récapitulatif et calendrier de l’essai. 13
8 Critères de validité . 14
9 Évaluation des résultats. 14
9.1 Présentation des mesures . 14
9.2 Analyse statistique. 15
9.2.1 Essai préliminaire. 15
9.2.2 Essai final. 15
9.2.3 Approche CSEO (concentration sans effet observé) . 15
9.2.4 Approche CE (concentration avec effet) . 15
x
10 Rapport d'essai. 16
Annexe A (informative) Essais des produits chimiques dans le sol. 17
Bibliographie . 18
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ISO 22030:2005(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée
aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du
comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,
Partie 2.
La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur
publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne
pas avoir identifié de tels droits de propriété et averti de leur existence.
L'ISO 22030 a été élaborée par le comité technique ISO/TC 190, Qualité du sol, sous-comité SC 4, Méthodes
biologiques.
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ISO 22030:2005(F)
Introduction
La présente Norme internationale décrit une méthode d’évaluation de la qualité des sols d’origine diverse
présentant des contaminations inconnues. Légèrement modifiée, la méthode peut également être utilisée pour
mesurer la toxicité de produits chimiques connus incorporés dans le sol.
L’évaluation de l’inhibition et de la toxicité chronique repose sur la germination, la croissance végétative et la
capacité de reproduction d’au moins deux espèces de plantes supérieures.
La présente Norme internationale se fonde sur
a) les résultats du projet de recherche «Développement d’un bio-essai chronique à l’aide de plantes
[3]
supérieures» parrainé par le Ministère allemand de l’éducation et de la recherche (BMBF) , et
b) les discussions menées dans le cadre du projet commun «Batteries d’essai écotoxicologiques» au sein
[10]
du Groupe de recherche commun du BMBF «Procédés de bioréhabilitation du sol» .
© ISO 2005 – Tous droits réservés v
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NORME INTERNATIONALE ISO 22030:2005(F)
Qualité du sol — Méthodes biologiques — Toxicité chronique
sur les plantes supérieures
AVERTISSEMENT — Les sols contaminés peuvent contenir des mélanges inconnus de produits
chimiques toxiques, mutagènes ou de nocivité autre ou des micro-organismes infectieux. La
poussière ou les produits chimiques qui en émanent au cours de la manipulation et de l’incubation
peuvent engendrer des risques pour la santé sur les lieux de travail. De plus, les plantes testées
pourraient absorber des produits chimiques provenant du sol et il convient également d’envisager de
prendre des mesures de sécurité lors de la manipulation de ces plantes.
1 Domaine d'application
La présente Norme internationale décrit une méthode de détermination de l’inhibition de la croissance et de la
capacité de reproduction des plantes supérieures par les sols dans des conditions contrôlées. Deux espèces
sont recommandées: une variante de colza à cycle rapide (Brassica rapa CrGC syn. Rbr) et l’avoine (Avena
sativa). Il convient que la durée de l’essai soit suffisante pour inclure les points de mesure chroniques qui
apportent la preuve de la capacité de reproduction des plantes soumises à l'essai.
En utilisant des sols d’essai naturels provenant, par exemple, de sites contaminés ou de sols réhabilités, et en
comparant le développement des plantes testées dans ces sols à celui des plantes testées dans les sols de
référence ou les sols témoins standard, l’essai peut permettre d’évaluer la qualité du sol, notamment sa
fonction d’habitat des plantes.
L’Annexe A décrit des modifications permettant d’exploiter l’analyse chronique des plantes pour les essais de
substances ou de produits chimiques incorporés dans le sol. En préparant une série de dilutions d’une
substance dans des sols témoins standard, il est possible de déterminer les mêmes points de mesure afin
d’évaluer la toxicité chronique de ces substances ou de ces produits chimiques. Cette méthode n’est pas
applicable aux substances volatiles, c’est-à-dire celles pour lesquelles H (constante de Henry) ou le
coefficient de partage air/eau est supérieur à 1, ou pour lesquelles la pression de vapeur à 25 °C est
supérieure à 0,013 3 Pa à 25 °C.
2 Références normatives
Les documents de référence suivants sont indispensables pour l'application du présent document. Pour les
références datées, seule l'édition citée s'applique. Pour les références non datées, la dernière édition du
document de référence s'applique (y compris les éventuels amendements).
ISO 11268-1:1993, Qualité du sol — Effets des polluants vis-à-vis des vers de terre (Eisenia fetida) —
Partie 1: Détermination de la toxicité aiguë en utilisant des substrats de sol artificiel
ISO 11268-2:1998, Qualité du sol — Effets des polluants vis-à-vis des vers de terre (Eisenia fetida) —
Partie 2: Détermination des effets sur la reproduction
ISO 11269-2, Qualité du sol — Détermination des effets des polluants sur la flore du sol — Partie 2: Effets
des substances chimiques sur l'émergence et la croissance des végétaux supérieurs
ISO 15176:2002, Qualité du sol — Caractérisation de la terre excavée et d'autres matériaux du sol destinés à
la réutilisation
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ISO 22030:2005(F)
ISO 15799, Qualité du sol — Lignes directrices relatives à la caractérisation écotoxicologique des sols et des
matériaux du sol
ASTM D1076:2002, Standard Specification for Rubber-Concentrated, Ammonia Preserved, Creamed, and
Centrifuged Natural Latex
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
3.1
sol artificiel
mélange de sable, de kaolinite, de tourbe et de carbonate de calcium
NOTE L’ISO 11268-1 décrit ce type de sol pour des essais de toxicité utilisant des vers de terre. Il convient de ne
pas utiliser de sable de quartz pur, de laine minérale, de vermiculite ou d'autres substrats de synthèse.
3.2
biomasse
ensemble des pousses, des fleurs et des gousses
NOTE 1 La biomasse est exprimée en masse sèche par plante ou, si nécessaire, en masse sèche par pot.
NOTE 2 Au cours de la période d’essai, certaines plantes testées peuvent atteindre différents stades de croissance et
leur teneur en eau peut varier lors de leur récolte. La masse sèche représente donc mieux la biomasse produite durant la
croissance.
3.3
concentration
masse de substance expérimentée par quantité de sol
NOTE La concentration est exprimée, en fraction massique, en milligrammes par kilogramme (mg/kg) de sol sec.
3.4
contaminant
substance ou agent présent(e) dans le sol et résultant de l’activité humaine
[ISO 15176:2002]
3.5
sol témoin
substrat non contaminé utilisé comme témoin et comme milieu de préparation des dilutions sériées avec les
sols ou les produits chimiques d’essai et permettant la croissance de plantes saines
NOTE Des sols standard artificiels et naturels ou de référence peuvent être utilisés s’il est possible d’y escompter
une croissance sans entrave des plantes testées. En tout état de cause, les différences de niveau nutritif entre un sol
d’essai et un sol témoin peuvent avoir une incidence sur le profil dose-réponse. Un sol témoin beaucoup plus riche en
substances nutritives qu’un sol d’essai peut, par exemple, donner un résultat positif erroné (c’est-à-dire que le sol d’essai
semble avoir un effet «toxique» sur la croissance des plantes testées). Dans le cas d’un sol témoin plus pauvre en
substances nutritives qu’un sol d’essai, on peut s’attendre à un phénomène d’hormesis (voir 3.9) à des concentrations
faibles de mélange, voire même à une relation dose/effet inversée, si l’apport en substances nutritives devient le principal
effet. La présente Norme internationale ne donne pas de valeurs numériques pour les substances nutritives.
3.6
concentration des effets
CE
x
concentration (fraction massique) d’une substance ou d’un produit chimique d’essai ou pourcentage (fraction
massique) d’un sol d’essai pour lequel un effet de x % par rapport au témoin est observé
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NOTE La concentration des effets (CE ) est exprimée en milligrammes par kilogramme. Dans le cas d’essais de
x
substances ou de produits chimiques, CE est exprimée en masse de substance expérimentée par masse sèche de sol et,
x
dans le cas d’essais de sols, en pourcentage de masse sèche de sol d’essai par masse sèche de mélange de sols.
3.7
germination
développement du germe contenu dans une graine, mettant fin à la période de latence
NOTE La germination est exprimée en pourcentage de plantules qui germent par rapport aux pots témoins.
3.8
fonction habitat
aptitude des sols et des matériaux du sol à servir d’habitat aux micro-organismes, aux plantes, aux animaux
vivants dans le sol et leurs interactions (biocénose)
[ISO 15799]
3.9
hormesis
amélioration de la germination, de la croissance ou de la survie des plantules (ou autre réponse des plantes
testées), à des concentrations faibles de substances ou de produits chimiques ou de mélanges de sols
[1]
toxiques en cas d’application à des niveaux plus élevés que celui du témoin
3.10
concentration minimale avec effet observé
CMEO
concentration (fraction massique) testée la plus faible d’une substance expérimentée dans le sol pour laquelle
on observe un effet statistiquement significatif pour un point de mesure donné (p < 0,05) par rapport au témoin
Voir CSEO (3.11)
NOTE Par analogie, le terme CMEO est employé pour la concentration de mélange la plus faible soumise à essai
dans un sol de référence ou un sol témoin standard, à laquelle on observe un effet statistiquement significatif. La CMEO
est exprimée en masse de substance expérimentée par masse de sol sec ou, dans le dernier cas, en pourcentage de
masse sèche de sol d’essai par masse sèche de mélange de sols. Toutes les concentrations d’essai supérieures à la
CMEO ont un effet nocif égal ou supérieur à celui observé à la CMEO. S’il n’est pas possible de respecter cette condition,
il convient de donner une explication sur le mode de choix de la CMEO et de la concentration sans effet observé.
3.11
concentration sans effet observé
CSEO
concentration (fraction massique) de substance expérimentée ou proportion de mélanges de sols
immédiatement inférieure à la CMEO qui n’a pas d’effet statistiquement significatif, comparé au témoin
(p < 0,05)
Voir CMEO (3.10)
3.12
sol de référence
sol non contaminé spécifique d’un site (par exemple collecté à proximité d’un site contaminé), ayant des
propriétés analogues (concentrations en substances nutritives, pH, teneur en carbone organique et texture) à
celles du sol d’essai
3.13
concentration de mélange de sols
rapport entre la masse sèche de sol d’essai et la masse sèche de sol de référence/témoin
NOTE Elle est exprimée en pourcentage.
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3.14
sol standard
sol collecté sur le terrain ou sol artificiel dont les principales propriétés (par exemple pH, texture, teneur en
matières organiques) se situent dans une plage connue
EXEMPLE Sols euro, sol artificiel.
NOTE Les propriétés des sols standard peuvent différer de celles du sol d’essai.
4 Principe
La présente Norme internationale décrit un essai de plantes comportant des points de mesure aigus et
chroniques. L’essai mesure la germination, la croissance précoce et la reproduction de deux espèces de
plantes terrestres (Avena sativa et une variété à cycle rapide de Brassica rapa sont recommandées). L’essai
compare les réponses des plantes dans un sol d’essai et/ou celles d’une série de dilutions avec un sol témoin.
Cette méthode peut également être utilisée pour les essais de substances ou de produits chimiques en
appliquant diverses concentrations de la substance expérimentée à un sol témoin standard. Les graines de
deux espèces de plantes sont plantées dans des pots contenant le sol/les mélanges de sols et dans des pots
témoins contenant un sol de référence ou standard. Les pots sont placés dans un local à température et
lumière contrôlées ou chambre de croissance. Ils sont humidifiés à l’aide de mèches. À l’issue de la
germination des plantes, on détermine les taux de germination et les plantes sont éclaircies pour n’en
conserver qu’un nombre spécifié. Après deux semaines, une partie des plantes est récoltée afin de
déterminer leur biomasse. Trois à quatre semaines (Brassica rapa à cycle rapide) ou cinq à six semaines plus
tard (Avena sativa), les plantes restantes sont récoltées afin de mesurer les effets caractéristiques de leur
potentiel de reproduction. Dans tous les cas, il convient que la durée de l’essai soit suffisante pour déterminer
les points de mesure de reproduction (par exemple, le nombre ou la biomasse des fleurs ou des graines ou
encore des fruits).
Généralement, 10 graines par pot sont semées dans quatre pots d’essai en réplicat. Les plantes sont
ème
éclaircies à raison de 8 par pot et, dans chacun, quatre plantes sont récoltées le 14 jour puis à la fin de
ème
l’essai. Si moins de 8 plantes ont levé dans un pot, le nombre de plantes récoltées au 14 jour doit être
réduit de manière à laisser quatre plantes pour la récolte finale.
L’inhibition relative dans les sols d’essai non dilués est déterminée afin d’évaluer si le sol convient aux plantes.
En se basant sur une série de dilutions, il est en outre possible de calculer les valeurs de CSEO, CMEO et
CE à partir des courbes dose/effet, la dernière étant requise en cas d’essai de substances ou produits
x
chimiques.
5 Matériaux
5.1 Plantes testées
Une espèce monocotylédone et une espèce dicotylédone sont soumises à essai en parallèle. L’avoine (Avena
sativa) est recommandée comme espèce monocotylédone et Brassica rapa comme espèce dicotylédone.
Il est vivement recommandé d’utiliser une variété de colza à cycle rapide (Brassica rapa CrGC syn. Rbr) afin
1)
de raccourcir la période d’essai . La floraison débute au bout de deux semaines et la production de graines
peut être déterminée au bout de cinq semaines environ.
1) Des kits de graines et de plantes à croissance rapide, dans le cadre du «Wisconsin Fast Plants Program»
(Programme de croissance rapide de plantes du Wisconsin) sont des produits appropriés distribués par la société
«Carolina Biological Supply Company». Cette information est donnée à l’intention des utilisateurs de la présente Norme
internationale et ne signifie nullement que l’ISO approuve ou recommande l’emploi exclusif du produit ainsi désigné. Des
produits équivalents peuvent être utilisés s’il est démontré qu’ils conduisent aux même résultats.
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D’autres espèces pourraient être sélectionnées, par exemple dans la liste donnée dans l’ISO 11269-2, de
même que des plantes ayant des caractéristiques physiologiques spécifiques comme les plantes C-4 (maïs,
canne à sucre, millet), des plantes en symbiose avec les bactéries fixant l’azote (par exemple Fabaceae) ou
des plantes ayant une importance écologique ou économique dans certaines régions du monde. Ces plantes
doivent croître sans entrave dans le sol témoin, dans les conditions spécifiées. Il convient de sélectionner
uniquement les plantes qui supportent les propriétés des sols d’essai et les conditions d’essai (outre leur
contamination chimique). Il convient, par exemple, de ne pas utiliser une espèce sensible à de faibles valeurs
du pH pour les essais de sols forestiers à faible pH. L’avoine et le colza à cycle rapide poussent dans un sol
sablonneux ou argileux, ayant une teneur en eau variable et un pH dans une plage de valeurs comprises
entre 5,0 et 7,5. Il convient de ne pas associer à une hydratation par mèche les espèces qui ne supportent
pas les sols humides. Les motifs de sélection d’espèces autres que l’avoine et le colza doivent être justifiés
dans le rapport d’essai.
5.2 Sol et stockage du sol
5.2.1 Généralités
La description des méthodes d’échantillonnage représentatif de sols provenant de sites contaminés n’entre
pas dans le domaine d’application de la présente Norme internationale. Une méthode d'échantillonnage
[13]
appropriée est donnée dans l’ISO 10381-6 .
L’essai chronique sur plantes peut être réalisé pour évaluer le potentiel toxique de sols naturels provenant de
divers sites contaminés. Il peut également permettre d’évaluer la qualité de sols réhabilités. Après tamisage, il
convient que les sols sélectionnés aient un pH qui se situe dans une plage non toxique pour les plantes
testées, par exemple entre 5,0 et 7,5 pour Brassica rapa et Avena sativa. Les sols d’essai doivent être passés
au tamis de 4 mm à 5 mm d’ouverture de maille afin d’éliminer les fragments grossiers puis soigneusement
homogénéisés. Si nécessaire, le sol peut être séché à l’air sans chauffage avant tamisage. Il convient que le
stockage des sols d’essai soit aussi court que possible. Il est recommandé de stocker les sols à 4 °C environ,
dans des récipients réduisant les pertes de contaminants du sol par volatilisation et sorption dans les parois
du récipient.
5.2.2 Sol d’essai
La capacité de rétention d’eau doit être mesurée pour tous les mélanges utilisés lors de l’essai.
Il convient également que les sols d’essai soient caractérisés par:
la texture (sable, limon, argile);
le pH;
la salinité;
le carbone organique;
les quantités totales et solubles dans l’eau de potassium, d'azote et de phosphore.
Il convient de ne pas corriger le pH du sol. La mesure des contaminants du sol (métaux lourds, hydrocarbures,
pesticides, explosifs, PCB, etc.) n’est pas obligatoire.
Il convient de vérifier que le sol d’essai absorbe suffisamment l’eau par les mèches (voir 5.4). Une
hydrophobicité ou un mauvais transport de l’eau peuvent apparaître avec les sols sablonneux, les sols
fortement contaminés par les hydrocarbures, voire même les sols à haute teneur en argile qui ont tendance à
se compacter, même s’ils ont une capacité élevée de rétention d’eau (déterminée après avoir préalablement
noyé les sols). Afin de garantir le fonctionnement du système d’arrosage, il convient de procéder sur deux
réplicats à un essai préalable portant sur tous les sols sélectionnés pour l’essai afin de déterminer si
l’arrosage par mèche est suffisant ou si un arrosage manuel est nécessaire.
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NOTE Il n’est pas possible, pour l’instant, d’indiquer des limites de pH pour des espèces autres que le colza et
l’avoine. Il appartiendra à la recherche ultérieure de soumettre à essai un plus grand nombre de plantes sur une variété de
sols. En outre, les limites de tolérance en matière de texture, de salinité ou d'autres propriétés des sols ne peuvent
actuellement être indiquées pour différentes espèces.
5.2.3 Sol témoin
Des sols artificiels, de référence ou standard peuvent être utilisés comme substrats témoins.
Si l’on dispose de sols de référence provenant de zones non contaminées proches d’un site contaminé, il
convient de les traiter et de les caractériser comme les sols d’essai. Une analyse chimique des contaminants
toxiques attendus doit en outre être effectuée sur le sol de référence afin de vérifier qu’il en est exempt. S’il
n’est pas possible d’exclure la présence d’une contamination toxique ou d’autres propriétés inhabituelles, il
convient de préférer des sols témoins standard.
Il convient que les sols standard soient des sols naturels ou artificiels, non contaminés et pauvres en
substances nutritives. En cas d’utilisation d’un sol naturel, il convient que sa teneur en matières organiques ne
dépasse pas 5 %. Il convient que le pourcentage de particules fines (< 20 µm) ne dépasse
...
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