Ambient air - Biomonitoring with Higher Plants - Method of the standardised tobacco exposure

This European Standard applies to the determination of the impact of ground-level ozone on a bioindicator plant species (tobacco Nicotiana tabacum cultivars Bel-W3, Bel-B2 and Bel-C) in a given environment. The present document specifies the procedure for the setting-up and use of a system designed to expose these plants to ambient air. It also describes the procedure of leaf injury assessment. Leaf injury caused by ozone appears in the form of necrosis or accelerated leaf aging (senescence) on the leaves of the bioindicator. The macroscopically detectable leaf injury is used as the effect measure bioindicator. The measure is the percentage of dead leaf area on the entire leaf surface. The results of the standardised tobacco exposure indicate ozone-caused injury of the exposed bioindicators and thus enable a spatial and temporal distribution of the impact of ozone on plants to be determined. This Standard applies to the outside atmosphere in all environments but does not apply to the assessment of air quality inside buildings. The method described in this European Standard does not replace modelling or physico-chemical methods of direct measurement of air pollutants, it complements them by demonstrating the biological effect.

Außenluft - Biomonitoring mit Höheren Pflanzen - Verfahren der standardisierten Tabak-Exposition

Diese Europäische Norm gilt für die Bestimmung der Auswirkung von bodennahem Ozon auf eine als Bioindikator verwendete Pflanzenart (Sorten Bel-W3, Bel-B und Bel-C von Tabak Nicotiana tabacum) in einer bestimmten Umgebung.
Das vorliegende Dokument legt das Verfahren für das Einrichten und Anwenden eines Systems fest, das für die Exposition dieser Pflanzen gegenüber Außenluft vorgesehen ist. Es beschreibt außerdem das Verfahren der Beurteilung der Blattschädigung.
Eine durch Ozon hervorgerufene Blattschädigung tritt in Form von Nekrosen oder einer beschleunigten Blattalterung (Seneszenz) an den Blättern des Bioindikators auf. Die makroskopisch erkennbare Blatt-schädigung wird als Wirkungsmessgröße verwendet (siehe Bilder in Anhang A). Das Maß ist der prozentuale Anteil abgestorbener Blattfläche an der gesamten Blatt(ober)fläche.
Die Ergebnisse der standardisierten Tabak-Exposition zeigen ozonbedingte Schädigungen der exponierten Bioindikatoren an und ermöglichen somit eine Bestimmung der räumlichen und zeitlichen Verteilung der Auswirkung von Ozon auf Pflanzen.
Die Norm gilt für die Außenatmosphäre in allen Umgebungen, jedoch nicht für die Beurteilung der Luftqualität innerhalb von Gebäuden.
Das in der vorliegenden Europäischen Norm beschriebene Verfahren ersetzt nicht die Modellierung oder physikalisch-chemische Verfahren der direkten Messung von Luftschadstoffen, sondern ergänzt sie durch das Aufzeigen der biologischen Wirkung.

Air ambiant - Biosurveillance à l'aide de Plantes Majeures - Méthode de l'exposition de tabac standardisée

La présente Norme européenne s’applique à la détermination de l’impact de l’ozone troposphérique sur une espèce de plante bioindicatrice (cultivars de tabac Nicotiana tabacum Bel-W3, Bel-B et Bel-C) dans un environnement donné.
Le présent document spécifie le mode opératoire de mise en place et de suivi d’un dispositif permettant l’exposition de ces végétaux à l’air ambiant. Il décrit également le mode opératoire d’évaluation des lésions foliaires.
Les lésions foliaires causées par l’ozone se traduisent par une nécrose ou un vieillissement accéléré (sénescence) des feuilles du bioindicateur. Les lésions foliaires visibles à l’oeil nu sont utilisées comme mesure de l’effet (voir les illustrations à l’Annexe A). La mesure est le pourcentage de surface foliaire nécrosée sur toute la surface de la feuille.
Les résultats de l’exposition normalisée du tabac indiquent les lésions causées par l’ozone des bioindicateurs exposés et permet ainsi de déterminer la distribution spatio-temporelle de l’impact de l’ozone sur les plantes.
La présente norme s’applique à l’atmosphère extérieure dans tous les environnements mais ne s’applique pas à l’évaluation de la qualité de l’air intérieur des bâtiments.
La méthode décrite dans la présente Norme européenne ne remplace pas la modélisation ou les méthodes physico-chimiques de mesurage direct des polluants atmosphériques, elle les complète en démontrant l’effet biologique.

Zunanji zrak - Biomonitoring z višjimi rastlinami - Metoda standardizirane izpostavljenosti tobaka

Ta evropski standard se uporablja za določanje učinka prizemnega ozona na bioindikatorje rastlinskih vrst (sorte tobaka Nicotiana tabacum Bel-W3, Bel-B2 in Bel-C) v določenem okolju. V tem dokumentu je opredeljen postopek za vzpostavitev in uporabo sistema, načrtovanega za izpostavljanje teh rastlin zunanjemu zraku. Opisuje tudi postopke ovrednotenja poškodb listov. Poškodbe listov, ki jih povzroči ozon, se pojavijo v obliki nekroze ali pospešenega staranja listov (biološko staranje) na listih bioindikatorja. Makroskopske poškodbe listov se uporabljajo kot bioindikator meritve učinka. Meritev je odstotek površine mrtvega lista glede na celotno površino lista. Rezultati standardizirane izpostavljenosti tobaka kažejo škodo, ki jo na izpostavljenih bioindikatorjih povzroča ozon, in s tem omogočajo prostorsko in časovno razporeditev vpliva ozona na rastline. Ta standard se uporablja za zunanjo atmosfero v vseh okoljih, vendar se ne uporablja za ocenjevanje kakovosti zraka v stavbah. Metoda, ki je opisana v tem evropskem standardu, ne nadomešča modeliranja ali fizikalno-kemijskih metod neposrednega merjenja onesnaževal zraka, ampak jih dopolnjuje s prikazom biološkega učinka.

General Information

Status
Published
Public Enquiry End Date
04-Dec-2014
Publication Date
11-Jun-2017
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Feb-2017
Due Date
09-Apr-2017
Completion Date
12-Jun-2017

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Zunanji zrak - Biomonitoring z višjimi rastlinami - Metoda standardizirane izpostavljenosti tobakaAußenluft - Biomonitoring mit Höheren Pflanzen - Verfahren der standardisierten Tabak-ExpositionAir ambiant - Biosurveillance à l'aide de Plantes Majeures - Méthode de l'exposition de tabac standardiséeAmbient air - Biomonitoring with Higher Plants - Method of the standardised tobacco exposure13.040.20Kakovost okoljskega zrakaAmbient atmospheresICS:Ta slovenski standard je istoveten z:EN 16789:2016SIST EN 16789:2017en,fr,de01-julij-2017SIST EN 16789:2017SLOVENSKI
STANDARD



SIST EN 16789:2017



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16789
August
t r s x ICS
s uä r t rä v râ
s uä r v rä t r English Version
Ambient air æ Biomonitoring with Higher Plants æ Method of the standardized tobacco exposure Air ambiant æ Biosurveillance à l 5aide de plantes supßrieures æ Mßthode de l 5exposition normalisße du tabac
Außenluft æ Biomonitoring mit Höheren Pflanzen æ Verfahren der standardisierten TabakæExposition This European Standard was approved by CEN on
s z June
t r s xä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey andUnited Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
9
t r s x CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x y z {ã t r s x ESIST EN 16789:2017



EN 16789:2016 (E) 2 Contents Page European foreword . 3 Introduction . 4 1 Scope . 7 2 Terms and definitions . 7 3 Principle of the method . 8 4 Test method . 9 4.1 Material . 9 4.1.1 Plants . 9 4.1.2 Substrate . 9 4.1.3 Water . 9 4.1.4 Exposure device . 9 4.1.5 Exposure rack . 10 4.2 Cultivation of plants . 11 4.3 Exposure . 15 4.3.1 General . 15 4.3.2 Duration of exposure . 15 4.3.3 Requirements of the exposure locations . 15 5 Visual injury assessment . 16 5.1 Leaf selection . 16 5.2 Identification of ozone-induced injury . 16 5.3 Recognition of injuries not caused by ozone . 16 5.4 Assessment of ozone-induced leaf injury . 17 6 Data handling and data reporting . 17 6.1 General . 17 6.2 Tests of exposure location differences for individual exposure periods . 18 6.2.1 General . 18 6.2.2 Data treatment . 18 6.2.3 Missing value completion . 18 6.2.4 Statistical analysis . 21 6.2.5 Graphical presentation of results . 21 6.3 Tests of differences between exposure locations and between exposure periods 22 7 Performance characteristics . 23 8 Quality assurance and quality control . 23 8.1 Preparation of the plant material. 23 8.2 Requirements for the exposure location . 23 8.3 Requirements for the visual injury assessment . 23 Annex A (informative)
Reference plates and photographs for evaluating the percentage of necrosis on leaf surfaces . 24 Annex B (informative)
Documentation . 28 B.1 General . 28 B.2 Example of the information that shall be recorded at a given exposure location . 28 B.3 Example of the information that shall be recorded for a tobacco plant at a given assessment date . 30 Bibliography . 31 SIST EN 16789:2017



EN 16789:2016 (E) 3 European foreword This document (EN 16789:2016) has been prepared by Technical Committee CEN/TC 264 “Air quality”, the secretariat of which is held by DIN. 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 February 2017, and conflicting national standards shall be withdrawn at the latest by February 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16789:2017



EN 16789:2016 (E) 4 Introduction 0.1 General The impact of air pollution is of growing concern worldwide. Local and regional assessment is necessary as a first step to collect the fundamental information, which can be used to avoid, prevent or minimize harmful effects on human health and the environment as a whole. Biomonitoring can serve as a tool for this purpose. As the effects on indicator organisms are a time-integrated result of complex influences, combining the influences of both air quality and local climatic conditions, this holistic biological approach is considered particularly relevant to human and environmental health end points and thus is of value in air quality management. It is important to emphasize that biomonitoring data differ from those obtained through physico-chemical measurements (ambient concentrations and deposition) and computer modelling (emissions and dispersion data). Biomonitoring provides evidence of the effects that airborne pollutants have on organisms. As such it reveals biologically relevant, field-based, time- and space-integrated indications of environmental health as a whole. Legislation states that there should be no harmful environmental effects from air pollution. This requirement can be met only by investigating the effects at the biological level. The application of biomonitoring in air quality and environmental management requires rigorous standards and a recognized regime so that it can be evaluated as robustly as physico-chemical measurements and modelling in pollution management. Biomonitoring is the way through which environmental changes have historically been detected. Various standard works on biomonitoring provide an overview of the state of the science at the time, e.g. [1; 2; 3]. The first investigations of passive biomonitoring are documented in the middle of the 19th century: by monitoring the development of epiphytic lichens it was discovered that the lichens were damaged during the polluted period in winter and recovered and showed strong growth in summer [4]. These observations identified lichens as important bioindicators. Later investigations also dealt with bioaccumulators. An active biomonitoring procedure with bush beans was first initiated in 1899 [5]. 0.2 Biomonitoring and EU legislation Biomonitoring methods in terrestrial environments address a variety of requirements and objectives within EU environmental policy, primarily in the fields of air quality (Directive 2008/50/EC on ambient air quality and cleaner air for Europe [6]), integrated pollution prevention and control (Directive 2010/75/EU on industrial emissions IED [7]) and conservation (Habitats Directive). It is also relevant to the topics food chain [8] and animal feed [9; 10; 11]. For air quality in Europe, legislators require adequate monitoring of air quality, including pollution deposition as well as avoidance, prevention or reduction of harmful effects. Biomonitoring methods are relevant for both short-term and long-term air quality assessment. Directive 2004/107/EC of 15 December 2004 relating to arsenic, cadmium, mercury, nickel and polycyclic aromatic hydrocarbons in ambient air [12] states that “the use of bio indicators may be considered where regional patterns of the impact on ecosystems are to be assessed”. With respect to IED for industrial installations, the permit procedure includes two particular environmental conditions for setting adequate emission limit values. The asserted concepts of “effects” and “sensitivity of the local environment” open up opportunities for application of biomonitoring methods in relation to the general impact on air quality and the deposition of installation-specific pollutants. The basic properties of biomonitoring methods can be used advantageously for applications such as reference inventories prior to the start of a new installation, mapping of the potential pollution reception areas and (long-term) monitoring of the impact caused by industrial activity. The environmental inspection of installations demands SIST EN 16789:2017



EN 16789:2016 (E) 5 examination against a range of environmental effects. For the competent authority, biomonitoring data contribute to the decision-making process, e.g. concerning the question of tolerance of impacts at the local scale. The Habitats Directive (92/43/EEC on the conservation of natural habitats and of wild fauna and flora [13]) requires competent authorities to assess or adapt planning permission and other activities affecting a site designated at the European level where the integrity of the site could be adversely affected. The Directive also provides for the control of potentially damaging operations, whereby consent may only be granted once it has been shown through appropriate assessment that the proposed operation will not adversely affect the integrity of the site. The responsibility lies with the applicant to demonstrate that there is no adverse effect on such a conservation area. For this purpose, biomonitoring is well suited as a non-intrusive form of environmental assessment. In 2003, as an important element within its integrated environmental policy, the European Commission adopted a European Environment and Health Strategy [14] with the overall aim of reducing diseases caused by environmental factors in Europe. Chapter 5 of this document states that the “community approach entails the collection and linking of data on environmental pollutants in all the different environmental compartments (including the cycle of pollutants) and in the whole ecosystem (bioindicators) to health data (epidemiological, toxicological, morbidity)”. The European Environment and Health Action Plan 2004-2010 [15] which followed the adoption of this strategy focuses on human biomonitoring, but emphasizes the need to “develop integrated monitoring of the environment, including food, to allow the determination of relevant human exposure“. 0.3 Development of the standardized tobacco exposure Ozone is a phytotoxic gas, which is a secondary pollutant formed in the atmosphere. It can lead to growth losses in plants and therefore to reduced yields in agriculture [16; 17; 18; 19; 20; 21; 22; 23]. Ground-level ozone also contributes to the development of forest decline [24; 25; 26; 27]. Effects of ozone on wild plants are the subject of numerous investigations [e.g. 28; 29; 30; 31; 32; 33; 34; 35; 36]. Ozone does not accumulate in plant organs, but can cause visible leaf injury (necrosis). For that reason, the leaf injury of sensitive plants can be used for assessing the effects of ozone [37; 38; 39; 40; 41; 42; 43; 44]. The origins of tobacco cultivars for biomonitoring are described in detail by [45]. They arose as a result of research initiated in 1957 to identify the cause of “weather fleck” in the USA – a mysterious disease which followed periods of hot sunny weather and devastated tobacco crops due to the appearance of extensive foliar lesions. Subsequently it was identified that ground-level ozone was the cause. During the course of a programme of breeding resistance into tobacco a supersensitive individual was identified from which the response indicator cultivar Bel-W3 was developed. In a similar manner the less sensitive Bel-C and tolerant Bel-B were developed. In Europe studies with Bel-W3 commenced in the late 1960s to early 1970s in the UK, Federal Republic of Germany, Belgium and the Netherlands [46; 47; 48; 49; 50]. The extent of the ozone-caused injury to the response indicator plant depends on the ozone dose absorbed. This is partly associated with the ozone concentration measured in the ambient air. High ozone concentrations are usually associated with high temperatures and low relative air humidity which can induce stomatal closure thereby decreasing the absorbed ozone dose. Moreover, high wind speed also decreases the concentration gradient between the ambient air and leaf surface thereby increasing ozone uptake. The tobacco exposure provides a direct measure of the impact of ozone on plants. Significant relationships between the variables of ozone concentration or dose and ozone-induced leaf injury (=bioindicator response) in some species (e.g. wild and cultivated tomato species) have been reported by [51] and [52]. Ozone-induced injury on the extremely sensitive SIST EN 16789:2017



EN 16789:2016 (E) 6 tobacco cultivar Bel-W3, however, cannot directly be translated into impact on native vegetation or crops. Nevertheless, leaf injury in tobacco Bel-W3 can be used as an indicator of the potential vegetation injury, i.e. the maximum vegetation injury to be expected under given pollution and climate conditions [53]. Since 2000, many investigations have employed widespread biomonitoring with Bel-W3 [54; 55; 56; 57; 58; 59; 60]. The largest international survey in Europe was conducted under the auspices of the EuroBionet-programme involving 12 cities in eight countries [61]. SIST EN 16789:2017



EN 16789:2016 (E) 7 1 Scope This European Standard applies to the determination of the impact of ground-level ozone on a bioindicator plant species (tobacco Nicotiana tabacum cultivars Bel-W3, Bel-B and Bel-C) in a given environment. The present document specifies the procedure for setting-up and use of a system designed to expose these plants to ambient air. It also describes the procedure for leaf injury assessment. Leaf injury caused by ozone appears in the form of necrosis or accelerated aging (senescence) on the leaves of the bioindicator. The macroscopically detectable leaf injury is used as the effect measure (see pictures in Annex A). The measure is the percentage of dead leaf area on the entire leaf surface. The results of the standardized tobacco exposure indicate ozone-caused injury of the exposed bioindicators and thus enable a spatial and temporal distribution of the impact of ozone on plants to be determined. This Standard applies to the outside atmosphere in all environments. This standard does not apply to the assessment of air quality inside buildings. The method described in this European Standard does not replace modelling or physico-chemical methods of direct measurement of air pollutants, it complements them by demonstrating the biological effect. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 biomonitoring use of biological systems (organisms and organism communities) to monitor environmental change over space and/or time Note 1 to entry: Biological systems can be further considered as bioindicators. Note 2 to entry: Active biomonitoring refers to deliberate field exposure following a standardized methodology; passive biomonitoring refers to in situ-sampling and/or observation of selected bioindicators currently or previously present in the environment. [SOURCE: EN 16413:2014 [62], 2.1, modified, note 2 to entry added] 2.2 bioindicator organism or part of an organism or an organism community (biocoenosis) which documents environmental impacts Note 1 to entry: It encompasses bioaccumulators and response indicators. [SOURCE: EN 16413:2014 [62], 2.2, modified] 2.3 bioaccumulator organism which can indicate environmental conditions and their modification by accumulating substances present in the environment (air, water or soil) at the surface and/or internally [SOURCE: EN 16413:2014 [62], 2.3] SIST EN 16789:2017



EN 16789:2016 (E) 8 2.4 response indicator effect indicator organism which can indicate environmental conditions and their modification by either showing specific symptoms (molecular, biochemical, cellular, physiological, anatomical or morphological) or by its presence/absence in the ecosystem [SOURCE: EN 16413:2014 [62], 2.4] 2.5 ground-level ozone ozone present in the terrestrial biosphere 2.6 leaf necrosis death of cells or tissues through injury or disease, especially in a localized area of the leaf 2.7 study area geographical area considered by the study Note 1 to entry: It should be described in detail in terms of extent, land use classification and altitudinal range. [SOURCE: EN 16413:2014 [62], 2.9] 2.8 visual injury assessment process of estimating the extent of macroscopically visible injury of the leaf surface 3 Principle of the method The standardized method describes: — the exposure of tobacco plants (cultivars Bel-W3 or Bel-C, and Bel-B) to the ambient air; and — the assessment of the injury caused to the foliage by ground-level ozone. The cultivar Bel-B is more tolerant to ozone pollution than Bel-W3 and Bel-C. It is used as a control to avoid confounding symptoms due to ozone with symptoms resulting from other factors (diseases in particular), to which all three cultivars are equally sensitive. In areas where ozone pollution is expected to be particularly severe, the cultivar Bel-W3 can be too sensitive and can exhibit complete leaf damage. In this case it is better to use the cultivar Bel-C, which is less sensitive to ozone. The repeated exposure of tobacco on several sites enables determination of the temporal and spatial distribution of ozone effects. SIST EN 16789:2017



EN 16789:2016 (E) 9 4 Test method 4.1 Material 4.1.1 Plants Tobacco (Nicotiana tabacum L.) seeds of cultivars Bel-W3, Bel-C and Bel-B are used. Each study should be conducted with seeds derived from the same batch, as these cultivars can exhibit some degree of intra-cultivar variability in their response to ozone. Tobacco seeds can lose their viability over a period of a few years. 4.1.2 Substrate For the cultivation and exposure, a light standardized soil is used. It is important to specify the nutrient content of the soil as this (in particular nitrogen) can modify the response of plants to ozone. Thus the substrate shall contain a basic nitrogen-phosphorus-potassium-content. The range of nutrients is N 200-300 mg/l; P2O5 250-350 mg/l; K2O 300
« x r r mg/l. NOTE The NPK-content of commercial potting soils is frequently given as weight per litre of the product. As such, further fertilisation during cultivation and exposure of the bioindicator plants is not necessary. The substrate should have a pH between 5,5 and 6,5. Before putting the soil into the plant pots, it should be moistened if necessary. 4.1.3 Water For watering the plants drinking water quality (Council Directive 98/83/EC on the quality of water intended for human consumption [63]) is sufficient. If the values given there cannot be complied with, deionised water shall be used. 4.1.4 Exposure device The exposure of the bioindicators takes place in commercially available square plastic plant pots with the dimensions 13 cm × 13 cm (top rim) and a height of 13 cm (volume: ca. 1,25 l to 1,5 l; see Figure 1) or in round pots with comparable soil volume. Four holes are drilled into the base of the pots (if not already present in the purchased pots), through which two moistened glass fibre wicks (diameter: 5 mm to 6 mm, length: 50 cm to 70 cm) or other suitable suction wicks are inserted. The wicks serve as a continuous water supply during cultivation and exposure. At least 7 cm of the wicks should reach into the substrate. The length of loose ends should be chosen in such a way that both ends reach the bottom of the water storage container. As water storage container, a Euro standard stackable plastic container (60 cm × 40 cm × 12 cm) is used, into which an overflow is drilled approximately 2 cm below the upper edge. A white polystyrene block (60 cm × 40 cm × 11 cm) with at least two recesses (11,5 cm × 11,5 cm) into which the plant pots are put is placed onto this tub. In this way, mutual obstruction/shading of the growing plants is avoided. Suitable shaping of its lower edge prevents the block slipping off the tub (Figure 1). For the plants, wooden or bamboo sticks are used as support to prevent wind damage. SIST EN 16789:2017



EN 16789:2016 (E) 10
Key 1: leaf with necrosis 2: polystyrene block 3: water reservoir 4: suction wick Figure 1 — Example of a device for the exposure of tobacco plants 4.1.5 Exposure rack The exposure rack consists of a solid frame construction (Figure 2). The tobacco plants are exposed at a height of 70 cm to 110 cm from ground level to the soil surface in the pots. During the exposure water is supplied by the wicks, which hang from the plant pots into the water reservoir. A filling quantity of 20 l enables two weeks of maintenance-free exposure. The exposure rack is covered with green shading fabric (shading rate 50 %) at the top and at three sides (east, south, west). It is open toward the north. The shaded plants react more sensitively to ground-level ozone than those under direct sunlight as the stomata of the leaves – being the dominating uptake path for ozone – remain open for longer periods of time. One can therefore expect a higher level of leaf injury in shaded plants. SIST EN 16789:2017



EN 16789:2016 (E) 11
Key 1: metal frame construction, consisting of four frame elements (2 pieces 180 cm × 90 cm, 2 pieces 180 cm × 60 cm) 2: shading fabric at three sides (east, south, west) as well as at the top 3: stackable plastic containers (60 cm × 40 cm × 12 cm) as water storage containers 4: example of a polystyrene block (60 cm × 40 cm × 11 cm) with three complete pot-shaped recesses (11,5 cm × 11,5 cm) and recessed edge to prevent slipping 5: plastic pot (13 cm × 13 cm) with suction wicks Figure 2 — Exposure rack [SOURCE: [64]; modified] 4.2 Cultivation of plants The aim of the cultivation is a healthy, vigorous plant. The tobacco plants are cultivated in an environment which minimizes ambient ozone concentration (e.g. greenhouse, open-top chamber, phytotron, if possible supplied with charcoal-filtered air). Efforts should be made to ensure as constant temperature as possible during cultivation. At high temperatures the seedlings should be watered from top down using a sprinkler, in order to avoid overheating; at night the temperature should not drop below 10 °C. The aim is to produce plants at a comparable stage of development, and thus similar sensitivity, for all exposure periods. The tobacco is sown in trays with well-moistened standardized soil (see 4.1.2). The top layer consists of sieved soil, which is smoothed to a flat surface. The seeds are applied evenly and spread very thinly (1 seed/cm2 to 5 seeds/cm2). Since tobacco plants require light for germination, the seeds are not covered with soil. The seed trays are maintained at temperatures between 18 °C and 28 °C, in order to ensure a safe and rapid germination within five to 16 days. If higher temperatures occur, the trays should be kept in a shady place to avoid heat stress. During this time, the surface of the soil shall always be kept moist (e.g. under a transparent cover on the tray). Unused seeds can be stored in a refrigerator (4 °C to 6 °C) for future use (see 4.1.1). After the initial growth, the seedlings (see Figure 3a) should be transferred to a place with ample light. SIST EN 16789:2017



EN 16789:2016 (E) 12
Figure 3a — Seedlings Figure 3b — Two leaf-stage The following cultivation procedure is recommended: Two to three weeks after sowing, the seedling reaches the two leaf-stage (two successive leaves after the two cotyledons; the latter do not count as leaves; see Figure 3b). At this stage, four small bunches of two to six seedlings each are taken from the sowing trays and transferred into the exposure pots (Figure 4). The pots should be kept in a shady place for five to seven days to allow proper root growth without heat and drought stress. Thereafter, a sunnier place is favourable for further growth.
Figure 4 — Tobacco seedlings, immediately after potting The plants are watered carefully the first time using a thin spout to avoid damage to the seedlings. The pots are put into a water storage container without the polystyrene block and set up in a protected, relatively shaded place. The bottom of each container is covered with some water, so that the soil in the pots always remains moist by the use of the suction wicks. In the next two to three days it is checked whether at least one plant from each of the four bunches has established (areas where no pla
...

SLOVENSKI STANDARD
oSIST prEN 16789:2014
01-november-2014
Zunanji zrak - Biomonitoring z višjimi rastlinami - Metoda izpostavljenosti
standardnemu tobaku
Ambient air - Biomonitoring with Higher Plants - Method of the standardised tobacco
exposure
Außenluft - Biomonitoring mit Höheren Pflanzen - Verfahren der standardisierten Tabak-
Exposition
Air ambiant - Biosurveillance à l'aide de Plantes Majeures - Méthode de l'exposition de
tabac standardisée
Ta slovenski standard je istoveten z: prEN 16789
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
oSIST prEN 16789:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 16789:2014

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oSIST prEN 16789:2014

EUROPEAN STANDARD
DRAFT
prEN 16789
NORME EUROPÉENNE

EUROPÄISCHE NORM

September 2014
ICS 13.020.40; 13.040.20
English Version
Ambient air - Biomonitoring with Higher Plants - Method of the
standardised tobacco exposure
Air ambiant - Biosurveillance à l'aide de Plantes Majeures - Außenluft - Biomonitoring mit Höheren Pflanzen - Verfahren
Méthode de l'exposition de tabac standardisée der standardisierten Tabak-Exposition
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 264.

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-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
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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

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16789:2014 E
worldwide for CEN national Members.

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Contents
Page
Introduction .3
1 Scope .6
2 Terms and definitions .6
3 Principle of the method .7
4 Test method .7
5 Visual injury assessment . 14
6 Data handling and data reporting . 15
7 Performance characteristics . 19
8 Quality assurance and quality control. 19
Annex A (informative) Reference plates and photographs for evaluating the percentage of
necrosis on leaf surfaces . 21
Annex B (informative) Supplier information . 25
Annex C (informative) Documentation . 26
Bibliography . 29

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Foreword
This document (prEN 16789:2014) has been prepared by Technical Committee CEN/TC 264 “Air quality”, the
secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
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Introduction
The impact of air pollution is of growing importance worldwide. Local and regional assessment is necessary
as a first step to collect fundamental information, which can be used to avoid, prevent and minimize harmful
effects on human health and the environment as a whole. Biomonitoring may serve as a tool for such a
purpose. As the effects on indicator organisms are a time-integrated result of complex influences combining
both air quality and local climatic conditions, this holistic biological approach is considered particularly close to
human and environmental health end points and thus is relevant to air quality management.
It is important to emphasize that biomonitoring data are completely different from those obtained through
physico-chemical measurements (ambient concentrations and deposition) and computer modelling (emissions
data). Biomonitoring provides evidence of the effects that airborne pollutants have on organisms. As such it
reveals biologically relevant, field-based, time- and space-integrated indications of environmental health as a
whole. Legislation states that there should be no harmful environmental effects from air pollution. This
requirement can be met only by investigating the effects at the biological level. The application of
biomonitoring in air quality and environmental management requires rigorous standards and a recognized
regime so that it can be evaluated in the same way as physico-chemical measurements and modelling in
pollution management.
Biomonitoring is the traditional way through which environmental changes have been detected historically.
Various standard works on biomonitoring provide an overview of the state of the science at the time, e.g. [1],
[2], [3]. The first investigations of passive biomonitoring are documented in the middle of the 19th century: by
monitoring the development of epiphytic lichens it was discovered that the lichens were damaged during the
polluted period in winter and recovered and showed strong growth in summer [4]. These observations
identified lichens as important bioindicators. Later investigations also dealt with bioaccumulators. An active
biomonitoring procedure with bush beans was first initiated in 1899 [5].
Biomonitoring and EU legislation
Biomonitoring methods in terrestrial environments respond to a variety of requirements and objectives of EU
environmental policy primarily in the fields of air quality (Directive 2008/50/EC on ambient air, [6]), integrated
pollution prevention and control (Directive 2008/1/EC, [7], and Directive 2010/75/EU, [8]) and conservation
(Habitats Directive). The topics food chain ([9]) and animal feed ([10], [11], [12]) are alluded to as well.
For air quality in Europe, the legislator requires adequate monitoring of air quality, including pollution
deposition as well as avoidance, prevention or reduction of harmful effects. Biomonitoring methods appertain
to the scope of short and long-term air quality assessment.
Directive 2004/107/EC of 15 December 2004 relating to arsenic, cadmium, mercury, nickel and polycyclic
aromatic hydrocarbons in ambient air ([13]) states that “the use of bio indicators may be considered where
regional patterns of the impact on ecosystems are to be assessed”.
Concerning IPPC from industrial installations, the permit procedure includes two particular environmental
conditions for setting adequate emission limit values. The asserted concepts of "effects" and "sensitivity of the
local environment" open up a broad field for biomonitoring methods, in relation to the general impact on air
quality and the deposition of operational-specific pollutants. The basic properties of biomonitoring methods
can be used advantageously for various applications such as reference inventories prior to the start of a new
installation, the mapping of the potential pollution reception areas and (long-term) monitoring of the impact
caused by industrial activity. The environmental inspection of installations demands the examination of the full
range of environmental effects. For the public authority, biomonitoring data contribute to the decision-making
process, e.g., concerning the question of tolerance of impacts at the local scale.
The Habitat Directive (92/43/EEC on the conservation of natural habitats and of wild fauna and flora, [14])
requires competent authorities to consider or review planning permission and other activities affecting a
European designated site where the integrity of the site could be adversely affected. The Directive also
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provides for the control of potentially damaging operations, whereby consent may only be granted once it has
been shown through appropriate assessment that the proposed operation will not adversely affect the integrity
of the site. The responsibility lies with the applicant to demonstrate that there is no adverse effect on such a
conservation area. For this purpose, biomonitoring is well suited as a non-intrusive form of environmental
assessment.
As an important element within its integrated environmental policy, in 2003 the European Commission
adopted a European Environment and Health Strategy ([15]) with the overall aim of reducing diseases caused
by environmental factors in Europe. In Chapter 5 of this document it is stated that the “community approach
entails the collection and linking of data on environmental pollutants in all the different environmental
compartments (including the cycle of pollutants) and in the whole ecosystem (bio-indicators) to health data
(epidemiological, toxicological, morbidity)”. The European Environment and Health Action Plan 2004-2010
([16]) which followed the adoption of this strategy focusses on human biomonitoring, but emphasizes the need
to “develop integrated monitoring of the environment, including food, to allow the determination of relevant
human exposure“.
Development of the standardised tobacco exposure
Ozone is a phytotoxic gas, which is a secondary pollutant formed in the atmosphere. It can lead to growth
losses in plants and therefore to reduced yields in agriculture [17; 18; 19; 20; 21; 22; 23; 24]. Ground-level
ozone also contributes to the development of forest decline [25; 26; 27; 28]. Effects of ozone on wild plants
are the subject of numerous investigations [e.g. 29; 30; 31; 32; 33; 34; 35; 36; 37].
Ozone does not accumulate in plant organs, but can cause visible leaf injury (necrosis). For that reason, the
leaf injury of sensitive plants can be used for assessing the effects of ozone [38; 39; 40; 41; 42; 43; 44; 45].
The origins of biomonitoring tobacco cultivars are described in detail by [46]. They arose as a result of
research initiated in 1957 to identify the cause of “weather fleck” in the USA – a mysterious disease which
followed periods of hot sunny weather and devastated tobacco crops due to the appearance of extensive foliar
lesions. Subsequently it was identified that ground-level ozone was the cause. During the course of a
programme of breeding resistance into tobacco a supersensitive individual was identified from which the
response indicator cultivar Bel-W3 was developed. In a similar manner the less sensitive Bel-C and tolerant
Bel-B were developed. In Europe studies with Bel-W3 commenced in the late 1960s to early 1970s in the UK,
Federal Republic of Germany, Belgium and the Netherlands [47; 48; 49; 50; 51].
The extent of the ozone-caused injury of the response indicator plant depends on the absorbed ozone dose.
This is partly associated with the ozone concentration measured in the ambient air. High ozone concentrations
are usually associated with high temperatures and low relative air humidity, which can induce stomatal closure
thereby decreasing the absorbed ozone dose. Moreover, high wind speed also decreases the concentration
gradient between the ambient air and leaf surface thereby increasing ozone uptake. As such, the tobacco
exposure provides a direct measure of the impact of ozone on plants.
Significant relationships between the bioindicator response and ozone-induced leaf injury in some species
(e.g., wild and cultivated tomato species) have been reported by [52] and [53]. Ozone-induced injury on the
extremely sensitive tobacco cultivar Bel-W3, however, cannot directly be translated into impact on native
vegetation or crops. However, leaf injury in tobacco Bel-W3 can be used as an indicator of the potential
vegetation injury, i.e. the maximum vegetation injury to be expected under given pollution and climate
conditions [54].
Since 2000, many investigations have employed widespread biomonitoring with Bel-W3 [55; 56; 57; 58; 59;
60; 61]. The largest international survey in Europe was conducted under the auspices of the EuroBionet-
programme involving twelve cities in eight countries [62].
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1 Scope
This European Standard applies to the determination of the impact of ground-level ozone on a bioindicator
plant species (tobacco Nicotiana tabacum cultivars Bel-W3, Bel-B and Bel-C) in a given environment.
The present document specifies the procedure for the setting-up and use of a system designed to expose
these plants to ambient air. It also describes the procedure of leaf injury assessment.
Leaf injury caused by ozone appears in the form of necrosis or accelerated leaf aging (senescence) on the
leaves of the bioindicator. The macroscopically detectable leaf injury is used as the effect measure (see
pictures in Annex A). The measure is the percentage of dead leaf area on the entire leaf surface.
The results of the standardised tobacco exposure indicate ozone-caused injury of the exposed bioindicators
and thus enable a spatial and temporal distribution of the impact of ozone on plants to be determined.
This Standard applies to the outside atmosphere in all environments but does not apply to the assessment of
air quality inside buildings.
The method described in this European Standard does not replace modelling or physico-chemical methods of
direct measurement of air pollutants, it complements them by demonstrating the biological effect.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
biomonitoring
use of biological systems (organisms and organism communities) to monitor environmental change over
space and/or time
Note 1 to entry: Biological systems can be further considered as bioindicators.
Note 2 to entry: Active biomonitoring refers to deliberate field exposure under standardised conditions; passive
biomonitoring refers to in situ-sampling and/or observation of selected biological systems already present in the
environment.
2.2
bioindicator
organism or a part of it or an organism community (biocoenosis) which documents environmental impacts
Note 1 to entry: It encompasses bioaccumulators and response indicators
2.3
bioaccumulator
organism which can indicate environmental conditions and their modification by accumulating substances
present in the environment (air, water or soil) at the surface and/or internally
2.4
response indicator
effect indicator
organism which can indicate environmental conditions and their modification by either showing specific
symptoms (molecular, biochemical, cellular, physiological, anatomical or morphological) or by its
presence/absence in the ecosystem
2.5
ground-level ozone
ozone present in the terrestrial biosphere
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2.6
leaf necrosis
death of cells or tissues through injury or disease, especially in a localized area of the leaf
2.7
study area
geographical area considered by the study.
Note 1 to entry: It should be described in detail in terms of extent, land use classification and altitudinal range.
3 Principle of the method
The method consists of exposing tobacco plants (cultivars Bel-W3 or Bel-C, and Bel-B) to the ambient air and
quantifying the damage caused to the foliage by ground-level ozone.
The cultivar Bel-B is more tolerant to ozone pollution than Bel-W3 and Bel-C. It is used as a control to avoid
confounding the symptoms due to ozone, which are observed essentially on Bel-W3 and Bel-C, with
symptoms resulting from other environmental stresses (diseases in particular), which are observed on all
three cultivars.
In areas where ozone pollution is expected to be particularly severe, the cultivar Bel-W3 may be too sensitive
and exhibit complete leaf damage. In this case it is better to use the cultivar Bel-C, which is less sensitive to
ozone.
The repeated exposure of tobacco on several sites enables the determination of the temporal and spatial
distribution of ozone effects.
4 Test method
4.1 Material
4.1.1 Plants
Tobacco (Nicotiana tabacum L.) seeds of cultivars Bel-W3, Bel-C and Bel-B are used. Supplier information is
provided in Annex B. Each study should be conducted with seeds derived from the same batch, as these
cultivars may exhibit some degree of intra-cultivar variability in their response to ozone. Tobacco seeds may
lose their viability over a period of a few years.
4.1.2 Substrate
For the cultivation and exposure, a light potting soil is used. It is important to specify the nutrient content of the
soil as this (in particular nitrogen) can modify the response of plants to ozone. Thus the substrate shall contain
a basic nitrogen-phosphorus-potassium-content. The range of nutrients is N 200-300 mg/l; P O 250-350 mg/l;
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K O 300 -600 mg/l.
2
NOTE The NPK-content of commercial potting soils is frequently given as weight per litre of the product.
As such, further fertilisation during cultivation and exposure of the bioindicator plants is not necessary. The
substrate should have a pH between 5,5 and 6,5. Before putting the soil into the plant pots, it should be
moistened if necessary.
4.1.3 Water
For watering the plants drinking water quality (Council Directive 98/83/EC on the quality of water intended for
human consumption [63]) is sufficient. If the values given there cannot be complied with, deionised water shall
be used.
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4.1.4 Exposure device
The exposure of the bioindicators takes place in commercially available square plastic plant pots with the
dimensions 13 cm × 13 cm (top rim) and a height of 13 cm (Volume: ca. 1,25 l to 1,5 l; see Figure 1) or in
round pots with comparable soil volume. Four holes are drilled into the base of the pots (if not already present
in the purchased pots), through which two moistened glass fibre wicks (diameter: 5 mm to 6 mm, length:
50 cm to 70 cm) or other suitable suction wicks are inserted. The wicks serve the automatic water supply
during the cultivation and exposure. At least 7 cm of the wicks should reach into the substrate. The length of
loose ends should be chosen in such a way that both ends reach the bottom of the water storage container.
As water storage container, a Euro standard stacking crate (60 cm × 40 cm × 12 cm) is used, into which an
overflow is drilled approximately 2 cm below the upper edge. A white polystyrene block (60 cm × 40 cm ×
11 cm) with two recesses (11,5 cm × 11,5 cm) into which the plant pots are put is placed onto this tub. In this
way, mutual obstruction/shading of the growing plants is avoided. Suitable shaping of its lower edge prevents
the block slipping off the tub (Figure 1). For the plants, wooden or bamboo sticks are used as support to
prevent wind damage.

Figure 1 — Device for the exposure of tobacco plants
Key

1: necrosis
2: polystyrene block
3: water reservoir
4: suctionwick
4.1.5 Exposure rack
The exposure rack consists of a solid frame construction (Figure 2). The tobacco plants are exposed at a
height of 70 cm to 110 cm from ground level to the soil surface in the pots.
During the exposure the water is supplied by the wicks, which hang from the plant pots into the water
reservoir. A filling quantity of 20 l ensures a fortnightly, maintenance-free exposure.
The exposure rack is covered with green shading fabric (shading rate 50 %) at the top and at three sides
(east, south, west). It is open toward the north. The shaded plants react more sensitively to ground-level
ozone than those under direct sunlight as the stomata of the leaves – as the dominating uptake path for ozone
– remain opened for longer. One can therefore expect a higher level of leaf injury in shaded plants.
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Figure 2 — Exposure rack (according to Arndt et al., 1985)
Key:
1: metal frame construction, consisting of four frame elements (2 pieces 180 × 90 cm, 2 pieces 180 × 60 cm)
2: shading fabric at three sides (east, south, west) as well as at the top
3: plastic piling crates (60 cm × 40 cm × 12 cm) as water storage containers
4: polystyrene block (60 cm × 40 cm × 11 cm) with two complete pot-shaped holes (11,5 × 11,5 cm) and
raised edge to prevent slipping
5: plastic pot (13 cm × 13 cm) with suction wicks
4.2 Cultivation of plants
Aim of the cultivation is a healthy, vigorous plant.
The tobacco plants are cultivated in an environment which minimises ambient ozone concentrations (e.g.
greenhouse, open-top chamber, phytotron, if possible supplied with charcoal-filtered air). Efforts should be
made to ensure as constant temperature as possible during cultivation. At high temperatures the seedlings
should be watered from top down using a sprinkler, in order to avoid overheating; at night the temperature
should not drop below 10 °C. The aim is to produce plants at a comparable stage of development and thus
similar sensitivity for all exposure periods.
The tobacco is sown in trays with well-moistened standardized soil (see Subclause 4.1.2). The top layer
consists of sieved soil, which is smoothed to a flat surface. The seeds are applied evenly and spread very
2 2
thinly (1 seed/cm to 5 seeds/cm ). Since tobacco plants require light for germination, the seeds are not
covered with soil. The seed trays are maintained at temperatures between 20 °C to 25 °C, in order to ensure a
safe and rapid germination after four to six days (at temperatures of 18 °C to 20 °C it can take up to ten days
before the seeds germinate). During this time, the surface of the soil should be kept moist (e.g. transparent
cover on the tray).
Unused seeds are stored in a refrigerator (4 °C to 6 °C) for up to two years.
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After the initial growth, the seedlings (see Figure 3a) should be transferred to a cooler place (approx. 18 °C to
20 °C) with ample light.

Figure 3 — a) Seedlings                     b) Two leaf-stage
The following cultivation procedure is recommended: Two to three weeks after sowing, the seedling reaches
the two leaf-stage (two successive leaves after the two cotyledons; the latter do not count as leaves; see
Figure 3b). At this stage, four small bunches of two to six seedlings each are taken from the sowing trays and
transferred into the exposure pots (Figure 4).

Figure 4 — Tobacco seedlings, immediately after potting
The plants are watered carefully the first time using a thin spout to avoid damage to the seedlings. The pots
are put into a water storage container without the polystyrene block and set up in a protected, relatively
shaded place. The bottom of each container is covered with some water, so that the soil in the pots always
remains moist by the use of the suction wicks. In the next two to three days it is checked whether at least one
plant from each of the four bunches has established (areas where no plant has established satisfactorily
should be replanted). One week after the replanting at the latest, four seedlings out of the bunches are
selected in such a way that in each pot remain four plants of different sizes (Figure 5).
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Figure 5 — Tobacco plants after selection to four plants/pot
Five to six weeks after sowing, the plants will have reached a stage at which they begin to compete with each
other. They are therefore thinned to one plant per pot (Figure 6). In doing so, either the largest, middle or
smallest plant of each pot is left for exposure – depending on the general developmental stage (in order to get
a set of plants as homogeneous in size and development as possible).

Figure 6 — Single tobacco plant
Once the plants have reached the stage depicted in Figure 7, they are exposed at the monitoring location (soil
should be well moistened beforehand). Before exposure at the latest, each plant shall be loosely tied to a
wooden or bamboo stick (of at least 40 cm).

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Figure 7 — Tobacco plants at the exposure stage
Key
left: leaf pattern of a tobacco plant; leaves 2 to 8
right: leaf pattern of a tobacco plant; leaves 1 to 8; leaves coloured grey = frame for visual injury assessment;
a = ring
Leaf pattern of a plant ready for exposure (the leaf length refers to the blade only, not including the petiole):
 The two cotyledons have already fallen off and are not counted.
 Leaf 1 has reached a maximum size of approximately 2 cm–3 cm in diameter; it remains round.
 Then follows a likewise round leaf of approximately 3 cm–5 cm maximum diameter (No. 2).
 The next leaf (No. 3) is oval, bluntly pointed and reaches a maximum of hand size when fully grown.
 The next leaf (No. 4) is the first leaf that features the typical tobacco leaf shape (pointed oval) and is
substantially larger than leaf No. 3. Above this leaf a reference ring or a similar indicator is fitted at the
beginning of the exposure. Above this reference is the start of the frame of visual injury assessment (see
Clause 5).
The exposure stage is considered to be reached if leaf 8, thus the fourth leaf above the reference, has a
length of at least 10 cm, while leaf 9 is still shorter than 10 cm in total length. This stage is reached after seven
to ten weeks depending on the local climatic conditions.
A summary of the cultivation is given in Table 1.
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Table 1 — Cultivation pattern
Week 1 Germination of the seeds
Weeks 2–3 Cotyledons and two leaves develop

Weeks 3–4 Select bunches to four different sized plants

Weeks 5–6 Thin to one plant per pot (during selection the
development stage of the individual plant should
be taken into consideration)
Weeks 7–10 Exposure stage is reached. The plants are tied
loosely to a wooden or bamboo stick.


4.3 Exposure
At each exposure date, six tobacco plants per monitoring location are deployed, of which five are the cultivar
Bel-W3 (or Bel-C as appropriate; see Clause 3); one plant of the cultivar Bel-B (cultivation as for Bel-W3) is
exposed (for reasoning see Clause 8).
4.3.1 Duration of exposure
The exposure time is 14 days (±1 day). Exposure should take place in the frost-free periods (e.g. in Central
Europe between mid May until the end of September). Thus approximately ten exposures take place.
Exposures are carried out continuously. Through this it is possible to record the effects of summer peak
concentrations of ground-level ozone.
4.3.2 Requirements of the exposure locations
When selecting the exposure locations the following criteria have to be taken into account in order to obtain
results that are representative of the study area:
The exposure rack
 shall not be too close to buildings (avoid their heat reflection zone),
 shall not be shaded for most of the day,
 shall not be set up where the air flow is
...

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