Ambient air - Biomonitoring with Higher Plants - Method of the standardized 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-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 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. 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.
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 Alterung (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. Die Norm gilt 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 supérieures - Méthode de l'exposition normalisée du tabac

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.

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Publication Date
16-Aug-2016
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
17-Aug-2016
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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

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

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