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Atmospheric Environment 40 (2006) 7437–7448

Ozone pollution and ozone biomonitoring inEuropean cities Part II. Ozone-induced plant injury and its

relationship with descriptors of ozone pollution

Andreas Klumppa,�, Wolfgang Ansela, Gabriele Klumppa, Phillippe Vergneb,Nicolas Sifakisc, Marıa Jose Sanzd, Stine Rasmussene, Helge Ro-Poulsene,

Angela Ribasf, Josep Penuelasf, Harry Kambezidisc, Shang Heg,1,Jean Pierre Garrecg, Vicent Calatayudd

aInstitute for Landscape and Plant Ecology and Life Science Center, University of Hohenheim, 70599 Stuttgart, GermanybENS Lyon and Lyon Botanical Garden, 46 Allee d’Italie, 69364 Lyon Cedex 07, France

cInstitute for Space Applications & Remote Sensing and Institute of Environmental Research & Sustainable Development, National

Observatory of Athens (NOA), P.O. Box 20048, 11810 Athens, GreecedFundacion CEAM, Parque Tecnologico, c/ Charles Darwin 14, 46980 Paterna (Valencia), Spain

eBotanical Institute, University of Copenhagen, Øster Farimagsgade 2D, 1353 Copenhagen K, DenmarkfUnitat d’Ecofisiologia CSIC-CEAB-CREAF, CREAF (Centre de Recerca Ecologica i Aplicacions Forestals), Universitat Autonoma de

Barcelona, 08193 Bellaterra (Barcelona), SpaingINRA Nancy, Laboratoire Pollution Atmospherique, 54280 Champenoux, France

Received 16 December 2005; received in revised form 4 July 2006; accepted 4 July 2006

Abstract

Within the scope of a biomonitoring study conducted in twelve urban agglomerations in eight European countries, the

ozone-sensitive bioindicator plant Nicotiana tabacum cv. Bel-W3 was employed in order to assess the occurrence of

phytotoxic ozone effects at urban, suburban, rural and traffic-exposed sites. The tobacco plants were exposed to ambient

air for biweekly periods at up to 100 biomonitoring sites from 2000 to 2002. Special emphasis was placed upon

methodological standardisation of plant cultivation, field exposure and injury assessment. Ozone-induced leaf injury

showed a clearly increasing gradient from northern and northwestern Europe to central and southern European locations.

The strongest ozone impact occurred at the exposure sites in Lyon and Barcelona, while in Edinburgh, Sheffield,

Copenhagen and Dusseldorf only weak to moderate ozone effects were registered. Between-site differences within local

networks were relatively small, but seasonal and inter-annual differences were strong due to the variability of

meteorological conditions and related ozone concentrations.

The 2001 data revealed a significant relationship between foliar injury degree and various descriptors of ozone pollution

such as mean value, AOT20 and AOT40. Examining individual sites of the local monitoring networks separately, however,

yielded noticeable differences. Some sites showed no association between ozone pollution and ozone-induced effects,

whereas others featured almost linear relationships. This is because the actual ozone flux into the leaf, which is modified by

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www.elsevier.com/locate/atmosenv

1352-2310/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.atmosenv.2006.07.001

�Corresponding author. Tel.: +49711 4593043; fax: +49 711 4593044.

E-mail address: aklumpp@uni-hohenheim.de (A. Klumpp).1Present address: Chinese Academy of Forestry, Research Institute of Forest Ecology and Environmental Science, Wan Shou Shan,

Beijing 100091, PR China.

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various environmental factors, rather than ambient ozone concentration determines the effects on plants. The advantage of

sensitive bioindicators like tobacco Bel-W3 is that the impact of the effectively absorbed ozone dose can directly be

measured.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Air quality; Bioindicators; Tobacco Bel-W3; AOT40; Urban air pollution

1. Introduction

Tropospheric ozone is an air pollutant of majorconcern on both the European and global scale,with current concentrations being high enough toharm human health, agricultural productivity andbiodiversity over wide areas. Recently establishedtarget values and long-term objectives for theprotection of human health and vegetation (EU(European Union), 2002) are frequently beingexceeded in large regions of Europe, includingurban agglomerations in many countries (EEA(European Environment Agency), 2003). Ozoneconcentrations and doses in various regions andcities reveal a strong spatial and temporal varia-bility, with a clear north–south gradient and asignificant differentiation between rural, suburbanand urban sites within a given region or municipalarea. This reflects different climatic conditions andemission sources of precursor substances (Sanzet al., 2004; Klumpp et al., 2006).

Strong efforts are being made to reduce ozonepollution, e.g., by cutting down precursor emissionsfrom traffic and industrial sources based on agree-ments established by the Gothenburg Protocol(UNECE (United Nations Economic Commissionfor Europe), 1999) and the NEC Directive (EU(European Union), 2001). Observations and projec-tions point at a positive response to these measures:peak concentrations are declining, but global andsupra-regional developments apparently provoke agradual increase of global or hemispheric back-ground values. This indicates that troposphericozone will remain on the environmental agenda inthe future (Prather et al., 2003; Grennfelt, 2004;Vingarzan, 2004; Ashmore, 2005).

Air quality control fundamentally aims at verify-ing whether compliance with the target or limitvalues set by national laws and European directivesactually avoids harmful effects on humans and theenvironment. Biomonitoring using accumulative orsensitive indicator plants is an appropriate means todetect and monitor air pollution effects becausebioindicators react to the biologically active pro-

portion of air pollution; they therefore display theintegrated response of past and present environ-mental conditions. The extremely ozone-sensitivetobacco (Nicotiana tabacum L.) cultivar Bel-W3 hasbeen used in numerous biomonitoring studiesworldwide for more than four decades (Heggestad,1991; Mulgrew and Williams, 2000).

The different methods applied concerning plantcultivation, age and developmental stage of indica-tor plants, exposure duration, injury assessment,etc. have compromised the comparability of pre-vious results (Heggestad, 1991; Toncelli and Lor-enzini, 1999; Cuny et al., 2004; Saitanis et al., 2004;among others). A strict standardisation of methodsis required to overcome the relatively poor compar-ability of data and the low acceptance of thisbiological monitoring procedure by regulators andpolicy makers. The first such national initiativeswere taken in Germany starting in the 1990s (VDI(Verein Deutscher Ingenieure), 2003). In 1999,EuroBionet, the ‘European Network for the Assess-ment of Air Quality by the Use of BioindicatorPlants’, was established as a network of researchinstitutes and municipal environmental authoritiesfrom twelve urban agglomerations in eight EUMember States. It aimed at promoting environ-mental awareness of the urban population and atassessing and evaluating air quality using highlystandardised bioindication methods. Among var-ious techniques, the ozone-sensitive tobacco cultivarBel-W3 was employed to assess the occurrence ofphytotoxic ozone effects at urban, suburban, ruraland traffic-exposed sites.

The present paper reports on the results ofstandardised exposure of tobacco plants at up to100 biomonitoring sites in urban agglomerationsduring three years. These experiments were afirst field test of the standardised method of tobaccoexposure in such a large geographical area. Thedetailed analysis of foliar injury on exposed tobaccoplants focuses on the 2001 data which are themost complete. We present the intensity as well asspatial and temporal variability of ozone-inducedinjuries and explore the relationship between

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ambient ozone levels and ozone-induced effects onindicator plants.

2. Material and methods

2.1. The EuroBionet programme

The present study was part of the Europeanbioindicator programme EuroBionet (www.eurobionet.com), which aimed at assessing andevaluating air quality in twelve urban agglomera-tions throughout Europe using various bioindicatorspecies from 2000 to 2002 (Klumpp et al., 2002,2004). To this end, a network of municipaladministrations and research institutes was estab-lished under the coordination of the University ofHohenheim. The project started in 1999 with thefollowing cities and regions as participants: Copen-hagen (Denmark), Edinburgh (UK), Klagenfurt(Austria), Greater Lyon (France), Sheffield (UK),and Verona (Italy). The City of Dusseldorf (Ger-many), the City of Ditzingen/Greater Stuttgart(Germany), Greater Nancy (France) and the regio-nal government of Catalonia/Barcelona (Spain)joined the network in 2000, and the cities ofValencia (Spain) and Glyfada/Greater Athens(Greece) in 2001 (cp. Klumpp et al., 2006).

2.2. Local networks

In each city, local bioindicator networks with8–10 exposure sites were implemented, includingone or two reference sites with low levels of primaryair pollutants as well as urban, suburban, industrialand traffic-exposed sites. Overall, about 100 bioin-dicator stations were established and operatedduring up to three years. Various criteria wereconsidered when selecting the monitoring sites. Thefirst was a relatively uniform distribution over thecity area to best represent the pollution burden ofthe conurbation. Proximity to existing air monitor-ing stations, protection from theft and vandalism,and city planning matters also played an importantrole. The ‘Stuttgart’ monitoring network did notinclude biomonitoring sites in the city centre butthree sites in the network’s associate partner, thetownship of Ditzingen northwest of the Stuttgart/Middle Neckar conurbation, as well as four sites onthe university campus and in three municipalities inthe Neckar valley southeast of Stuttgart. They wererun directly by the coordination team and weretreated as one common network. At all the sites,

various bioindicator species were exposed to ambi-ent air in order to assess the effects of ozone,sulphurous compounds, metals, hydrocarbons andmutagenic substances (Klumpp et al., 2002, 2004).

2.3. Cultivation and exposure of tobacco plants

Seeds of the ozone-sensitive tobacco cultivar Bel-W3 were obtained from the State Institute for CropProduction (Landesanstalt fur Pflanzenbau, Rhein-stetten, Germany). The plants were cultivatedbetween April and September each year in the localgreenhouses of the partner cities using a mixture ofcommercially available, standardised soil type ED73and river sand (8:1 by volume), plastic pots (1.5 L)and a semi-automatic watering system made of glassfibre wicks and water containers. The procedurelargely corresponded to a method described in adraft version of the guideline of the GermanAssociation of Engineers (VDI, 2003). All materialnecessary for plant cultivation and field exposurewas distributed to the local teams by the coordina-tion office in order to ensure a high level ofmethodological standardisation.

Plants with six fully expanded leaves were selectedfor exposure. Since cultivation in filtered air was notusually possible in the greenhouses, the plants werechecked prior to exposure for leaf injury that mayhave been caused by elevated ozone concentrationsduring cultivation. Such values were considered inthe final plant assessment after the two-weekexposure period. Per site and series, four–six plantswere exposed to ambient air in exposure racks(exposure height 90 cm, frame height 180 cm) asoriginally described by Arndt et al. (1985). Eachrack was covered with green shading fabric (50%)on the top and at three sides and remained opentowards the northern side. During outdoor expo-sure, plants were irrigated by the above system ofsuction wicks hanging into water reservoirs. Theexposure duration was 1471 days. Thus, up toeight exposure series were carried out between lateMay and mid-September.

2.4. Visual injury assessment

After the two-week exposure period, the tobaccoplants were exchanged with a new set and theozone-induced plant injury on three reference leaves(leaves no. four–six) was recorded as percentage ofdamaged leaf area in relation to whole leaf area.The visual assessment of damaged leaf area was

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pydone in 5% steps, using a photo catalogue withexemplary images of damaged leaves for thepurpose of comparison. Based on the mean injurydegree of each of the three reference leaves, themean leaf injury was calculated for each bioindi-cator station and exposure period. The leaves werealso checked for plant diseases and mechanicaldamage, e.g., due to storms or vandalism. Such kindof leaf damage was occasionally observed, thenleaves or whole plants were not further consideredfor ozone injury assessment.

From the raw data of the injury assessments, themean percentage of leaf injury was computed andclassified into different ozone impact classes accord-ing to a five-step scale (Table 1). At the end of eachexperimental year, the mean percentages of leafinjury from all exposure series were also determinedat each station. This calculation was the basis forthe comparative statistical evaluation within thenetwork that included analysis of variance (ANO-VA) and pairwise comparisons of means using theTukey test.

2.5. Comparative evaluation of ozone pollution and

ozone-induced injury

Hourly or half-hourly ozone concentration mea-surements were obtained from monitoring stationsroutinely operating at or close to the biomonitoringsites (cp. Klumpp et al., 2006). Mean concentrationsper day, biweekly exposure period and wholeexperimental season were computed, and cumula-tive exposure indices (AOT20, AOT40) were calcu-lated as described by the EU Directive (EU, 2002).Details on ozone monitoring and calculation ofozone descriptors are given in Part I of this paper(Klumpp et al., 2006). Ozone exposure parameters(mean, AOT20, AOT40) and degree of ozone-induced injury on tobacco leaves were compara-tively evaluated by Spearman rank correlationand linear regression analysis. All statistical proce-dures were performed using the WinStat softwarepackage.

2.6. Quality assurance and control

The entire project placed special emphasis on thestandardisation of all procedural steps from plantcultivation and exposure at the monitoring sites todata acquisition and processing. This strict harmo-nisation was designed to eliminate potential externalfactors that could influence plant response and toreduce methodological error.

The processes of quality assurance and qualitycontrol therefore covered all aspects of the bioindi-cation procedure:

� All material necessary for cultivation and ex-posure such as seed stocks, plant pots, substrate,fertiliser and exposure facilities was procuredcentrally and dispatched to the cities.� A detailed and comprehensively illustrated hand-

book describing the procedures of cultivation,field exposure and injury assessment in differentlanguages—and designed as a practical guideeven for untrained technicians—was provided toall working groups.� Practical demonstrations of the key operational

steps were organised at the beginning and duringthe course of the project.� Methodological compliance by the local teams

was checked during repeated on-site visits by thecoordination team.

3. Results and discussion

3.1. Ozone-induced leaf injuries

Typical ozone-induced injuries were recorded onthe leaves of exposed tobacco plants in all cities ofthe network and during all three study years.However, while more than 70% of the visualassessments revealed ‘‘very weak’’ to ‘‘moderate’’grades of leaf injury in Sheffield, Edinburgh andCopenhagen, these injury classes appeared muchless frequently in Barcelona (14%) and Lyon (10%).Here, ‘‘strong’’ and ‘‘very strong’’ ozone effectsdominated. The monitoring networks in Dusseldorf,Nancy, Klagenfurt and Verona occupied an inter-mediate position in this respect. Most assessmentshere lay in the range of ‘‘moderate’’ and ‘‘strong’’ozone effects, whereby the extreme classes of 1 and 5(‘‘very weak’’ and ‘‘very strong’’ effects) were ratherrare. In the Stuttgart network, which cannot directlybe compared with the other networks because of its

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Table 1

Five-stepped scale for classifying ozone effects on tobacco plants

Ozoneimpact

Veryweak

Weak Medium Strong Verystrong

Damage class 1 2 3 4 5Leaf 0−5 6−15 16−30 31−60 > 60 injury (%)

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more suburban/rural nature, more than 50% of theassessments revealed ‘‘strong’’ or ‘‘very strong’’ozone damage to the tobacco plants. The pollutionpattern therefore resembled those in Nancy andKlagenfurt, i.e. a higher proportion of extremevalues. In Valencia, data from only four exposureseries during 2001 were available; they showed‘‘very strong’’ ozone injury during one exposureseries and only ‘‘moderate’’ to ‘‘weak’’ ozone injuryduring the other campaigns. For Glyfada, whichjoined the project in late 2001, only data from 2002was available. ‘‘Very strong’’ leaf damage wasrecorded. The latter two cities were not furtherconsidered when comparing local networks.

The average percentage of leaf injury (mean valuefrom eight exposure series) was computed for eachsite. The distribution of these mean site values in theindividual cities is depicted in Fig. 1. In this box-whisker plot the central box shows the lower andupper quartiles and the median, and the whiskersextend to the maximum and minimum values. Thelowest site mean in the entire monitoring network(7%) was recorded at the ‘Tinsley’ site in Sheffield,the highest site mean (83%) at ‘Feyzin’ in Lyon.Within the local monitoring networks, most ofthe site means lay within a relatively small range(cf. 25–75% boxes of the plot). The values are in

broad agreement with the known distribution oftropospheric ozone concentrations over Europe(EEA, 2003; Sanz et al., 2004; Klumpp et al.,2006): a clear gradient with increasing ozone impactfrom northern to central and southern Europe wasobserved. An analysis of variance (ANOVA) andsubsequent comparison of means using the Tukeytest revealed significant differences in ozone effectsbetween the cities. The monitoring networks inLyon and Barcelona in particular differed from theother cities.

3.1.1. Spatial variability of ozone-induced injuries

within the local monitoring networks in 2001

Unlike the comparison between the cities, nosignificant differences between individual stationswithin the local networks were generally observed in2001. This is reflected in the relatively shortwhiskers in Fig. 1. The difference between thehighest and lowest site mean was at most one gradein the five-step evaluation scale. This is confirmedby the mean leaf injury and the most extreme valuesfor the local monitoring networks (Table 2). In mostcities the lowest site means occurred at urbanbioindicator stations due to ozone depletion byhigher NO levels in city centres. Strongest effectstypically occurred in suburban districts and in areas

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Ed Sh Co Dü Na St Kl Ve Ly Ba

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Fig. 1. Distribution of the site means within the local city monitoring networks in 2001 (25–75% box: range including 50% of the site

means in the individual cities), classification of ozone-induced injury degree according to the five-stepped scale (solid horizontal lines and

right y-axis) and results of the Tukey test. Ed ¼ Edinburgh; Sh ¼ Sheffield; Co ¼ Copenhagen; Du ¼ Dusseldorf; Na ¼ Nancy;

St ¼ Stuttgart; Kl ¼ Klagenfurt; Ve ¼ Verona; Ly ¼ Lyon; Ba ¼ Barcelona. Local networks with significant (po0.05) differences to

others are marked by different lowercase letters.

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with comparably low pollution by primary pollu-tants (reference stations). Such distribution patternsof ozone effects in urban agglomerations are knownfrom several other biomonitoring programmes(Mignanego et al., 1992; Godzik, 2000; Nali et al.,2001; Stabentheiner et al., 2004; Kostka-Rick andHahn, 2005). The present study, however, did notverify statistically significant differences betweenurban and traffic-exposed sites on one hand andsuburban and reference sites on the other handwhen pooling data from all local networks.

3.1.2. Seasonal variations of ozone-induced leaf

injuries within the local monitoring networks during

the period 2000– 2002

Contrary to the relatively low spatial variationwithin the local networks, strong seasonal and inter-annual differences in ozone-induced leaf injury werecaused by the variable meteorological conditionsand related ozone concentrations. Figs. 2–4 exem-plarily illustrate ozone effects at individual bioindi-cator stations in Dusseldorf, Klagenfurt and Lyon,encompassing the entire study period between July2000 and July 2002. The relatively long whiskers atmost sites reflect the high variability in plant injurydue to varying ozone concentrations and weatherconditions during the individual exposure periods.In Dusseldorf, the most severe leaf damage during atotal of 15 biweekly exposure series was registered inJuly 2001 (78%, ‘Morsenbroicher Ei’ site), whereasin some periods no injury at all was detected at thesame site. In Klagenfurt, the most severe leafdamage occurred in August 2000, with values ofapproximately 80% at the sites ‘Sattnitz’, ‘Worther-see’ and ‘KoschatstraXe’. Compared with Dussel-

dorf the mean values were higher at all stations.Maximum values (480%) were also recorded inindividual exposure series at all stations in Lyon,particularly during 2001, but variability at all siteswas also very strong between different exposureseries.

3.2. Relationships between ozone pollution and

ozone-induced injury

The relationship between ozone pollution andozone-induced injury was studied by pooling datafrom 16 exposure sites in all cities except for thosenetworks (Barcelona, Valencia) where less than fivedata pairs (tobacco exposure/ozone concentration)were available in 2001. A highly significant correla-tion was found between foliar injury degree andvarious descriptors of ozone pollution such as meanvalue, AOT20 and AOT40, although the relation-ships were not very strong. Table 3 lists thecomputed correlation coefficients for comparisonsbased on single biweekly exposure periods (n ¼ 112)and on the entire exposure time from late May tomid-September (n ¼ 16). The correlation coeffi-cients were similar for all three tested ozoneparameters. For the data pairs based on biweeklyexposure periods, all coefficients were significantlydifferent from 0 (r ¼ 0: no correlation), althoughthey only reached a comparably low value of 0.32.When ozone and tobacco data within the localmonitoring networks were aggregated over theentire study period (late May to mid-September),the correlation coefficients clearly increased(r ¼ 0.56–0.61). When differentiating between ur-ban and suburban sites, correlation coefficients of

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Table 2

Mean leaf injury as well as maximum and minimum biweekly site means within the local monitoring networks in 2001

Mean leaf injury of local network (%) Maximum site mean (%) Minimum site mean (%)

Edinburgh 25 30 suburban/reference 16 urbanSheffield 19 26 suburban 7 urbanCopenhagen 20 26 suburban 13 urbanDusseldorf 33 suburban/reference 16 urbanNancy 38 45 reference 28 urbanStuttgart 36 42 suburban 26 suburban/referenceKlagenfurt 38 42 reference 31 suburbanVerona 32 39 suburban 29 suburban/referenceLyon 72 83 industrial 64 urbanBarcelona 58 72 reference 42 urban

26

Coloured labelling according to the five-step evaluation scale.

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pyurban sites rose to r ¼ 0.43–0.49 for single exposureperiods and to 0.76–0.79 for the whole experimental season. Suburban sites, by contrast, showedweaker correlation for the individual exposureperiods and no significant relationship for the entireperiod.

Ribas and Penuelas (2003), in a biomonitoringstudy in rural Catalonia (Spain), found strongcorrelations (r40.97) between AOT20 and AOT40values and ozone effects on leaves of tobacco Bel-W3 when leaf damage was categorised in 10%intervals. Hence, we reran our statistical analysesconsidering the damage classes proposed by thoseauthors or the five damage classes used by thepresent study instead of the exact percentage ofinjured leaf area. The correlation analyses based oncategorised leaf injury, however, did not producesignificantly different results. Only at suburbansites were slightly stronger relationships betweenAOT values and categorised leaf injury observed

(data not shown). This may be due to thehigher variability in site characteristics (ozoneconcentrations, meteorological conditions) in ourstudy versus in the relatively limited area inCatalonia.

Examining individual sites of the local networksseparately, however, yielded noticeable differencesin the correlation between ozone pollution andozone-induced effects. In Lyon, for example, noassociation was found between different ozonedescriptors and corresponding tobacco data. Al-ready at comparably low AOT20 values of1100 ppb h, very strong ozone injury appeared onexposed tobacco plants. In Dusseldorf, by contrast,descriptors of ozone pollution (AOT20 and meanconcentration) and the corresponding leaf injury at‘Lorick’ site were almost linearly associated (Fig. 5).Similar linear relationships between ozone pollution(as AOT20) and tobacco injury were also found in2000 in Barcelona when only urban sites and only

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Airportsuburban

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Löricksuburban

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Heyestr.suburban

Graf-Adolfstreet

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Fig. 2. Ozone effects on tobacco Bel-W3 in the Dusseldorf monitoring network (2000–2002).

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pyexposure periods with stable weather conditionswere considered (Ribas and Penuelas, 2002).

Efforts to determine relationships between ambientozone concentrations or doses and symptomatology oftobacco and other bioindicator plants have repeatedlybeen undertaken, but with variable success. In small-scale studies and under relatively homogeneous me-teorological conditions, even linear relationships be-tween both pollution and effect criteria may be found(Heggestad, 1991; Biondi et al., 1992; Mignanego et al.,1992; Nali et al., 2001; Kostka-Rick, 2002; Ribas andPenuelas, 2002; Cuny et al., 2004). Variable relation-ships between ozone injury and ozone concentrationsat different sites and in different studies may partly beattributed to the fact that air pollution monitoringstations and bioindicators cannot not always beinstalled side by side (cp. Table 1 in Part I). Thus,actual ozone values affecting the tobacco plants maydiffer from the measured concentrations. Additionally,it should be stated that it normally takes some time(1, 2 days) for the ozone injury to develop, andtherefore the periods used for calculating ozone

concentrations and those responsible for ozone injurydevelopment are not exactly the same.

In large-area networks like those presented here,however, it is of major importance that the ozoneconcentration may account for only a small part ofthe variance in symptomatology: this is especiallytrue under strong spatial and temporal meteorolo-gical variations. It is widely accepted that the ozoneflux through the stomata into the leaf and theconsequential cumulative ozone uptake rather thanambient ozone concentrations determine ozone-induced effects on plants. This flux, however, ismodified by various environmental factors such astemperature, humidity and wind speed (Penuelas etal., 1999; Pihl Karlsson et al., 2004; Filella et al.,2005). Flux models would therefore be needed toquantitatively link ozone to plant response. Thisapproach was outside the scope of the presentproject. The advantage of exposing sensitive bioin-dicators like tobacco Bel-W3 is that the impact ofthe effective ozone dose on the plant can directlybe measured comparatively simply. Some authors

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Sattnitzindustrial

Wörtherseesuburban

Koschatstreet

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Annabichlsuburban

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Völkermarktstreet

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Fig. 3. Ozone effects on tobacco Bel-W3 in the Klagenfurt monitoring network (2000–2002).

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argue that the degree of ozone-induced injury ontobacco leaves provides a better basis for the riskassessment of incidence or extent of ozone-induced

leaf injury in crops than do any descriptors ofambient ozone concentrations or doses (Kostka-Rick, 2002).

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Fig. 4. Ozone effects on tobacco Bel-W3 in the Lyon monitoring network (2000–2002).

Table 3

Spearman rank correlation coefficients rs between different ozone parameters and ozone effects (assessed as % leaf injury) considering

biweekly exposure periods and entire exposure duration of up to eight biweekly periods

Type of site Ozone parameters Biweekly exposure periods Entire exposure duration

All n ¼ 112 n ¼ 16

Mean concentration 0.32*** 0.56*

AOT40 0.32*** 0.59**

AOT20 0.32*** 0.61**

Urban n ¼ 54 n ¼ 8

Mean concentration 0.43*** 0.76*

AOT40 0.48*** 0.79*

AOT20 0.49*** 0.76*

Suburban n ¼ 58 n ¼ 8

Mean concentration 0.28* 0.33ns

AOT40 0.27* 0.29ns

AOT20 0.29* 0.26ns

*Significant at po0,05.**Significant at po0.01.***Significant at po0.001.ns Not significant.

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4. Final remarks

The highly standardised biomonitoring procedurewith tobacco Bel-W3 based on a draft version of theVDI-Guideline (2003) was successfully applied overa large geographical area ranging from Scotland toGreece. It proved its value in demonstrating andquantifying ozone-induced effects on plants. Instrongly ozone-polluted regions with potentially

extreme foliar damage it might be recommendableto expose less sensitive cultivars like Bel-B inparallel and to use younger leaves as referenceleaves for injury assessment. Such modificationshave been incorporated in the finally publishedguideline (VDI, 2003), which now serves as astarting point for the Europe-wide standardisationof this methodology (Nobel et al., 2005). In thiscontext, a further outcome of the EuroBionet

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R2 = 0.76

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R2 = 0.88

Fig. 5. Scatter plot (with regression line and coefficient of determination R2) of the relationship between ozone-induced leaf injury and

AOT20 (above) or mean ozone concentration (below) at the ‘Lorick’ site in Dusseldorf during 2001.

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project is the recommendation to cultivate thebioindicator plants in filtered air as far as possible:how exposure to elevated ozone levels duringgreenhouse cultivation might influence plant sensi-tivity during subsequent outdoor exposure remainsto be clarified (Heagle and Heck, 1974; Schraudneret al., 1998). Such an additional standardisation stepmay help reduce the variability of plant response toambient ozone.

Our studies demonstrated that ozone pollutionand ozone-induced effects generally increased alonga gradient from northern Europe to central andsouthern Europe in the study year 2001, but thattopographic characteristics and the distribution ofair pollutant emissions may strongly influenceozone pollution and its impact on the local scale(Ribas and Penuelas, 2003; Klumpp et al., 2006).Although highest ozone burdens and strongestozone-induced plant injuries occurred in rural andsuburban sites, we show that ozone pollution mayreach high levels also at central locations and evenat street sites. The widespread occurrence andgeographical pattern of ozone-induced plant injuryin Europe, as determined here through tobaccoexposure mostly at urban and suburban sites, widelycorresponded to the findings of the UNECEnetwork, which uses differentially sensitive whiteclover clones to assess ozone-induced injury mostlyin rural areas (Harmens et al., 2004). Ozone-inducedinjury on sensitive indicator plants cannot directlybe translated into impact on native vegetation orcrops. Nonetheless, the relationships between thesensitivity of bioindicators and other plant speciesunderline the value of the former as an indicator ofpotential vegetation damage under given pollutionand climate conditions (Kostka-Rick and Hahn,2005). Finally, our studies demonstrated thattobacco plants are outstanding tools for environ-mental communication and education: they makethe noxious effects of ozone pollution visible tocitizens in their everyday life (Klumpp et al., 2004).

Acknowledgements

This study was supported by the LIFE Environ-ment Programme of the European Commission,DG Environment, under the Grant LIFE/99/ENV/D/000453. We thank the following local andregional authorities and their respective projectleaders and co-workers for their valuablesupport: Landeshauptstadt Dusseldorf, Umweltamt(H.-W. Hentze, M. Wiese), Communaute urbaine

de Lyon, Ecologie urbaine (O. Laurent), Comune diVerona, Servizio Ecologia (T. Basso, N. Belluzzo,S. Oliboni, S. Pisani, R. Tardiani), The City ofEdinburgh Council, Air Quality Section (T. Stir-ling), Sheffield City Council, Environment &Regulatory Services (G. McGrogan, N. Chaplin),Landeshauptstadt Klagenfurt, Abt. Umweltschutz(H.-J. Gutsche), City of Copenhagen, EPA (J. DahlMadsen), Generalitat de Catalunya, Dept. MediAmbient, Barcelona (X. Guinart), CommunauteUrbaine du Grand Nancy (F. Perrollaz), City ofGlyfada (G. Kolovou), and Ayuntamiento deValencia, Oficina Tecnica de la Devesa-Albufera(A. Vizcaino, A. Quintana) as well as the munici-palities of Ditzingen, Plochingen, Deizisau andAltbach (Germany). Gratitude is also expressed tothe staff of all institutions involved in the presentstudies, and to M. Stachowitsch for proof readingthe English manuscript.

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