SIST EN ISO 22030:2011
(Main)Soil quality - Biological methods - Chronic toxicity in higher plants (ISO 22030:2005)
Soil quality - Biological methods - Chronic toxicity in higher plants (ISO 22030:2005)
ISO 22030:2005 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 is applicable to assess soil quality, especially the function of the soil as a habitat for plants.
This method can be modified to allow 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 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.
Bodenbeschaffenheit - Biologische Verfahren - Chronische Toxizität in höheren Pflanzen (ISO 22030:2005)
Diese Internationale Norm beschreibt ein Verfahren zur Bestimmung der Hemmwirkung von Böden auf das
Wachstum und die Fortpflanzungsfähigkeit von höheren Pflanzen unter geregelten Bedingungen. Zwei
Pflanzenarten werden empfohlen: eine schnell wachsende Sorte der Stoppelrübe (Brassica rapa CrGC oder
Rbr) sowie Hafer (Saathafer) (Avena sativa). Die Prüfdauer sollte ausreichend bemessen sein, so dass sie
chronische Endpunkte umfasst, durch die die Fortpflanzungsfähigkeit der Prüfpflanzen nachgewiesen werden
kann.
Durch die Verwendung natürlicher Prüfböden, z. B. von verunreinigten Standorten oder sanierten Böden, und
durch Vergleich der Entwicklung der Prüfpflanzen in diesen Böden mit deren Entwicklung in Referenz- oder
Standard-Kontrollböden kann diese Prüfung dazu dienen, die Bodenqualität, insbesondere die Funktion des
Bodens als Lebensraum für Pflanzen, zu bewerten.
In Anhang A sind die Modifikationen beschrieben, die vorzunehmen sind, um die chronische Pflanzenprüfung
für die Prüfung von in den Boden eingemischten Chemikalien zu verwenden. Durch Herstellung einer Reihe
von Verdünnungen einer Prüfsubstanz in Standard-Kontrollböden können dieselben Endpunkte zur Messung
der chronischen Toxizität von Chemikalien verwendet werden. Dieses Verfahren ist nicht auf flüchtige
Substanzen anwendbar, d. h. auf Substanzen, bei denen H (Henrysche Konstante) oder der Luft-Wasser-
Verteilungskoeffizient über 1 liegt oder bei denen der Dampfdruck bei 25 °C 0,013 3 Pa überschreitet.
Qualité du sol - Méthodes biologiques - Toxicité chronique sur les plantes supérieures (ISO 22030:2005)
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.
Kakovost tal - Biološke metode - Kronična strupenost za višje rastline (ISO 22030:2005)
ISO 22030:2005 opisuje metodo za določanje zaviranja rasti in razmnoževalne zmožnosti višjih rastlin na tleh v nadzorovanih pogojih. Priporočata se dve vrsti: varianta oljne repice s hitrim ciklom (Brassica rapa CrGC syn. Rbr) in oves (Avena sativa). Preskus naj traja dovolj dolgo, da vključi kronične končne točke, ki dokazujejo razmnoževalno zmožnost preskusnih rastlin.
Z uporabo naravnih preskusnih tal, npr. iz onesnaženih lokacij ali saniranih tal, in s primerjavo razvoja preskusnih rastlin na teh tleh in na referenčnih oziroma standardnih kontrolnih tleh se preskus lahko uporabi za ocenjevanje kakovosti tal, predvsem funkcije tal kot habitata za rastline.
Ta metoda se lahko modificira, da omogoči uporabo kroničnega poskusa na rastlinah za preskušanje kemikalij, vključenih v tla. S pripravo serije razredčitev preskusne snovi v standardnih kontrolnih tleh se lahko merijo iste končne točke za oceno kronične toksičnosti kemikalij.
Ta metoda ne velja za hlapne snovi, tj. snovi, pri katerih je Henryjeva konstanta ali porazdelitveni koeficient zrak/voda večji od 1 ali pri katerih parni tlak presega 0.013 3 Pa pri 25 °C.
General Information
Relations
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Bodenbeschaffenheit - Biologische Verfahren - Chronische Toxizität in höheren Pflanzen (ISO 22030:2005)Qualité du sol - Méthodes biologiques - Toxicité chronique sur les plantes supérieures (ISO 22030:2005)Soil quality - Biological methods - Chronic toxicity in higher plants (ISO 22030:2005)13.080.30Biološke lastnosti talBiological properties of soilsICS:Ta slovenski standard je istoveten z:EN ISO 22030:2011SIST EN ISO 22030:2011en,fr,de01-november-2011SIST EN ISO 22030:2011SLOVENSKI
STANDARD
SIST EN ISO 22030:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN ISO 22030
June 2011 ICS 13.080.30 English Version
Soil quality - Biological methods - Chronic toxicity in higher plants (ISO 22030:2005)
Qualité du sol - Méthodes biologiques - Toxicité chronique sur les plantes supérieures (ISO 22030:2005)
Bodenbeschaffenheit - Biologische Verfahren -Chronische Toxizität in höheren Pflanzen (ISO 22030:2005) This European Standard was approved by CEN on 10 June 2011.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 22030:2011: ESIST EN ISO 22030:2011
EN ISO 22030:2011 (E) 2 Contents Page Foreword .3 SIST EN ISO 22030:2011
EN ISO 22030:2011 (E) 3 Foreword The text of ISO 22030:2005 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 22030:2011 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 December 2011, and conflicting national standards shall be withdrawn at the latest by December 2011. 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 22030:2005 has been approved by CEN as a EN ISO 22030:2011 without any modification.
SIST EN ISO 22030:2011
SIST EN ISO 22030:2011
Reference numberISO 22030:2005(E)© ISO 2005
INTERNATIONAL STANDARD ISO22030First edition2005-02-01Soil quality — Biological methods — Chronic toxicity in higher plants Qualité du sol — Méthodes biologiques — Toxicité chronique sur les plantes supérieures
SIST EN ISO 22030:2011
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© ISO 2005 – All rights reserved
SIST EN ISO 22030:2011
ISO 22030:2005(E) © ISO 2005 – All rights reserved
iii 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 ECx (effect concentration) approach.14 10 Test report.15 Annex A (informative)
Testing of chemicals in soil.16 Bibliography.17
SIST EN ISO 22030:2011
ISO 22030:2005(E) iv
© ISO 2005 – All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 22030 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods. SIST EN ISO 22030:2011
ISO 22030:2005(E) © ISO 2005 – All rights reserved
v 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 German Ministry for Education and Research (BMBF), Bonn [3], and b) discussions within the joint project “Ecotoxicological Test Batteries” forming part of the BMBF Joint Research Group “Processes for the Bioremediation of Soil” [10].
SIST EN ISO 22030:2011
SIST EN ISO 22030:2011
INTERNATIONAL STANDARD ISO 22030:2005(E) © ISO 2005 – All rights reserved
1 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 SIST EN ISO 22030:2011
ISO 22030:2005(E) 2
© ISO 2005 – All rights reserved 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
ECx 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 ECx is expressed as mass of the test substance per dry mass of soil; when soils are tested, the ECx is expressed as a 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. SIST EN ISO 22030:2011
ISO 22030:2005(E) © ISO 2005 – All rights reserved
3 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 the control [1] 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. SIST EN ISO 22030:2011
ISO 22030:2005(E) 4
© ISO 2005 – All rights reserved 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 ECx values can be calculated from the dose response 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 recommended1). 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 Scope of this International Standard. A suitable sampling method is given in ISO 10381-6 [13].
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. SIST EN ISO 22030:2011
ISO 22030:2005(E) © ISO 2005 – All rights reserved
5 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, rega
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SLOVENSKI STANDARD
kSIST FprEN ISO 22030:2011
01-april-2011
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Soil quality - Biological methods - Chronic toxicity in higher plants (ISO 22030:2005)
Bodenbeschaffenheit - Biologische Verfahren - Chronische Toxizität in höheren Pflanzen
(ISO 22030:2005)
Qualité du sol - Méthodes biologiques - Toxicité chronique sur les plantes supérieures
(ISO 22030:2005)
Ta slovenski standard je istoveten z: FprEN ISO 22030
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
kSIST FprEN ISO 22030:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN ISO 22030:2011
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kSIST FprEN ISO 22030:2011
EUROPEAN STANDARD
FINAL DRAFT
FprEN ISO 22030
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2010
ICS 13.080.30
English Version
Soil quality - Biological methods - Chronic toxicity in higher
plants (ISO 22030:2005)
Qualité du sol - Méthodes biologiques - Toxicité chronique Bodenbeschaffenheit - Biologische Verfahren - Chronische
sur les plantes supérieures (ISO 22030:2005) Toxizität in höheren Pflanzen (ISO 22030:2005)
This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical
Committee CEN/TC 345.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
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© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprEN ISO 22030:2010: E
worldwide for CEN national Members.
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kSIST FprEN ISO 22030:2011
FprEN ISO 22030:2010 (E)
Contents Page
Foreword .3
2
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kSIST FprEN ISO 22030:2011
FprEN ISO 22030:2010 (E)
Foreword
The text of ISO 22030:2005 has been prepared by Technical Committee ISO/TC 190 “Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as FprEN ISO 22030:2010 by
Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is held by NEN.
This document is currently submitted to the Unique Acceptance Procedure.
Endorsement notice
The text of ISO 22030:2005 has been approved by CEN as a FprEN ISO 22030:2010 without any
modification.
3
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kSIST FprEN ISO 22030:2011
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kSIST FprEN ISO 22030:2011
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
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kSIST FprEN ISO 22030:2011
ISO 22030:2005(E)
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ii © ISO 2005 – All rights reserved
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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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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kSIST FprEN ISO 22030:2011
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 fo
...
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