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EuroBionet European Network for the Assessment of Air Quality by the Use of Bioindicator Plants LIFE99 ENV/D/000453 Final Report

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  • t

    EAt L

    EuroBioneuropean Network for the ssessment of Air Quality by

    he Use of Bioindicator Plants

    IFE99 ENV/D/000453

    Final Report

  • University of Hohenheim 2004 Compilation and Project Coordination: PD Dr. Andreas Klumpp

    Editors: PD Dr. Andreas Klumpp Dipl.-Biol. Wolfgang Ansel Dr. Gabriele Klumpp Institute for Landscape and Plant Ecology Section Plant Ecology and Ecotoxicology University of Hohenheim D-70593 Stuttgart, Germany

    Photos Cover: Ansel, Arndt, Calatayud, Klumpp, Laurent, Audiovisual Library European Commission

  • CONTENTS

    Key Words / Schlagworte / List of Abbreviations....................................................... IV

    1 Zusammenfassung...................................................................................................... 1

    2 Summary........................................................................................................ 3

    3 Introduction................................................................................................................. 5

    4 LIFE Project Framework............................................................................................. 4.1 Project structure......................................................................................................... 4.2 Work plan and time schedule..................................................................................... 4.3 Project meetings............................................................................... 4.4 Deviations from the project proposal..........................................................................

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    5 Procedures for Bioindication of Air Pollution........................................... 5.1 Air pollution and bioindicators in urban agglomerations................ 5.2 Establishment of the local bioindicator networks....................................................... 5.3 The bioindication methods: cultivation, exposure and assessment of effects............

    5.3.1 Standardised tobacco exposure........................................................................ 5.3.2 Exposure of poplar clones..............................................................................5.3.3 Tradescantia micronucleus test......................................................................... 5.3.4 Standardised grass culture................................................................................ 5.3.5 Standardised curly kale exposure......................................................................5.3.6 Bioindicator stations...........................................................................................

    5.4 Exposure programme................................................................................................. 5.5 Data evaluation and presentation of the results......................................................... 5.6 Quality assurance and quality control..................................................................... 5.7 Comparative evaluation of air pollution and effect data...............................

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    6 Results from Bioindication of Air Pollutants.......................................... 6.1 The procedures manual: one step towards a Europe-wide standardisation.............. 6.2 Ozone pollution in the EuroBionet and its effects on tobacco and poplar...........

    6.2.1 Characterisation of ozone pollution in the partner cities of the EuroBionet....6.2.2 Ozone effects on the tobacco variety Bel-W3....................................................6.2.3 Ozone effects on poplar clones......................................................................... 6.2.4 Comparative assessment of ozone pollution and ozone-induced injury........

    6.3 Micronucleus formation in Tradescantia as a means for detecting genotoxic substances at urban monitoring stations.................................................................6.3.1 Results of measuring campaigns in the EuroBionet network 2000 2002....... 6.3.2 Final evaluation of the Tradescantia experiments........................

    6.4 SO2 pollution and sulphur enrichment in grass cultures......................................... 6.4.1 Characterisation of SO2 pollution in the partner cities of the EuroBionet........... 6.4.2 Sulphur accumulation in grass cultures 6.4.3 Comparative analysis of SO2 pollution and sulphur accumulation in grass cultures.................................................................................................

    6.5 Heavy metals and trace elements in grass cultures and poplar leaves..................6.5.1 Background and maximum values for heavy metal and trace element content in grass cultures during 2000 and 2001................................................ 6.5.2 Metal pollution of grass cultures during 2001.................................................... 6.5.3 Statistical analyses of the emission source groups...........................................

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  • 6.5.4 Heavy metal pollution in the Glyfada monitoring network during 2002.............. 6.5.5 Industrial heavy metal pollution in Sheffield....................................................... 6.5.6 Reduction of traffic-related lead emissions in Spain........................... 6.5.7 Demonstration of small-scale pollution differences using the example of a street profile in Copenhagen....................................................................... 6.5.8 Random sampled studies of platinum contents in grass cultures...................... 6.5.9 Enrichment of heavy metals and trace elements in exposed poplar clones.....................................................................................................

    6.6 Accumulation of polycyclic aromatic hydrocarbons (PAH) in curly kale..................... 6.6.1 Overview of PAH content in curly kale in the monitoring networks of the EuroBionet during the exposure periods 2000/2001 and 2001/2002......... 6.6.2 Level and composition of the PAH content in the local monitoring networks.... 6.6.3 Statistical analyses of the emission source groups........................................... 6.6.4 Content of the tracer substance benzo(a)pyrene in curly kale..........................

    6.7 Comparison of pollution types amongst the cities of the network..............................

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    7 Activities Related to the Dissemination of the Results: the Communication Concept of the EuroBionet............................................................................................ 7.1 Deficits in environmental communication................................................................... 7.2 The communication concept of the EuroBionet......................................................... 7.3 The idea and implementation of the Green Box....................................................... 7.4 Target group general public............................................................................

    7.4.1 Implementation of publicity drives and events................................................... 7.4.2 Production and distribution of material for public relations work........................ 7.4.3 Intensive press campaigns.............................................................................

    7.5 Target group scientists........................................................................................... 7.6 Target group decision makers and stakeholders from local politics, public

    administration and NGOs...................................................................... 7.7 Target group users from private enterprises.............................................................. 7.8 Target group children and teenagers.........................................................................

    7.8.1 The pilot school project in Ditzingen.................................................................. 7.8.2 School projects in the partner cities...................................................................

    7.9 Activities extending beyond the target groups........................................................... 7.9.1 Project conferences........................................................................................... 7.9.2 The Internet as an information platform.............................................................

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    8 Evaluation of the Results and Conclusions............................................... 8.1 Evaluation of the procedures employed for bioindication...........................................

    8.1.1 Exposure of tobacco Bel-W3 for the proof of ozone effects.................... 8.1.2 Exposure of poplar clones for monitoring of ozone and

    heavy metal pollution........................................................................................ 8.1.3 Exposure of Tradescantia #4430 for the proof of mutagenic effects...... 8.1.4 Exposure of standardised grass culture as an accumulative indicator for metal and sulphur pollution.............................................. 8.1.5 Exposure of curly kale for monitoring pollution with polycyclic aromatic hydrocarbons............................................. 8.1.6 General procedural aspects...........................................................................

    8.2 Project management.................................................................................................. 8.3 Reproducibility and economic feasibility....................................................................

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    8.4 Useful outcomes for potential target groups.................................................... 8.5 Comparison with the original project goals......................................................... 8.6 Environmental benefits............................................................................................... 8.7 Applicability to other areas at a local and supraregional level................................... 8.8 Innovative aspects.....................................................................................................

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    II

  • 8.9 Effectiveness of the mechanisms for dissemination of the results............................. 8.10 Potential for job creation.............................................................. 8.11 Relevancy for EU legislation and EU funding programmes..................................

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    9 The Hohenheim Recommendations.......................................................................... 133

    10 References................................................................................................................. 135

    11 Annex I....................................................................................................................... 11.1 Figures.............................................................................................................. 11.2 Tables.......................................................................................................................

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    12 Annex II (Materials and Documents).................................................................... 168

    III

  • EuroBionet Key Words/Abbreviations

    Schlagworte: Luftqualitt Luftverunreinigungen Bioindikatoren Umweltberwachung Ballungsrume Stdtenetzwerk Standardisierung Kommunikationskonzept ffentlichkeitsarbeit Umweltkommunikation Umweltbewusstsein Umwelterziehung

    Key Words: Air Quality Air Pollution Bioindicators Environmental Monitoring Urban Areas City Network Standardisation Communication Concept Publicity Campaigns Environmental Communication Environmental Awareness Environmental Education

    List of Abbreviations: AOT40: Accumulated exposure Over a Threshold of 40 ppb BCR: Community Bureau of Reference CEN: Comit Europen de Normalisation CLRTAP: Convention on Long-Range Transboundary Air Pollution COST: European Co-operation in the field of Scientific and Technical Research CRM: Certified Reference Material EEA: European Environment Agency FMV: Futtermittelverordnung / German Feeding Stuff Regulation GC/MS: Gas Chromatography/Mass Spectrometry ICP Integrated Cooperative Programme ICP/MS: Inductively-Coupled-Plasma/Mass-Spectrometry SME: Small and Medium-sized Enterprises MCN: Micronuclei MID: Maximum Immission Dosis NOx: Nitrogen Oxides NGO: Non-governmental Organisation O3: Ozone PAB: Project Advisory Board PAH: Polycyclic Aromatic Hydrocarbons PCB: Polychlorinated Biphenyls PCDD/PCDF: Polychlorinated Dibenzodioxins/-furans SO2: Sulphur Dioxide SRM: Standard Reference Material TVO: Trinkwasserverordnung / German Drinking Water Regulation UNECE: United Nations Economic Commission for Europe US-EPA: United States Environmental Protection Agency VDI: Verein Deutscher Ingenieure / Association of German Engineers WHO: World Health Organisation

    IV

  • EuroBionet Zusammenfassung

    1 ZUSAMMENFASSUNG Obwohl sich in den letzten Jahrzehnten die Luftqualitt in den Mitgliedsstaaten der EU deutlich verbessert hat, ist Luftverschmutzung nach berzeugung von Behrden und Brgern weiterhin eines der drngendsten Umweltprobleme. Diese Einschtzung kommt auch im 6. Umweltakti-onsprogramm der Europischen Kommission zum Ausdruck, in dem ein besonderer Schwer-punkt auf die Verbesserung der Luftqualitt gelegt wird. Anders als in frheren Jahrzehnten sind es heutzutage als Folge des stetig anwachsenden Kraftfahrzeugverkehrs vor allem Photooxi-dantien, aber auch Schwebstube und Kohlenwasserstoffe, die die Luftqualitt beeintrchtigen. Die Einhaltung von Richt- und Grenzwerten fr Luftverunreinigungen wird mit Hilfe von automa-tisch arbeitenden Immissionsmessstationen berwacht. Die mit chemisch-physikalischen Me-thoden gewonnenen Informationen ber die Schadstoffkonzentrationen ermglichen jedoch keine unmittelbaren Rckschlsse auf ihre Wirkungen auf Lebewesen, da diese von einer Viel-zahl interner und externer Faktoren beeinflusst werden. Der Nachweis schdlicher Wirkungen kann nur mittels lebender Organismen, so genannten Bioindikatoren, erfolgen, die empfindlich auf bestimmte Schadstoffe reagieren oder toxische Substanzen anreichern. Bioindikatoren wer-den in einigen EU-Lndern zwar schon seit vielen Jahren zur Umweltberwachung eingesetzt, ihre Akzeptanz bei Behrden, Entscheidungstrgern und der ffentlichkeit ist aber auf europi-scher Ebene auf Grund der unzureichenden Standardisierung der Methoden und einer daraus resultierenden geringen Vergleichbarkeit der Ergebnisse insgesamt noch sehr gering. Bioindika-torpflanzen weisen auch eine Reihe von Eigenschaften auf, die sie nicht nur fr die wirkungsbe-zogene berwachung der Luftqualitt, sondern in besonderem Mae fr die Umweltkommuni-kation und Umwelterziehung geeignet erscheinen lassen. Hufig zeigen sie die schdlichen Wirkungen der Luftverunreinigungen unmittelbar, d. h. mit bloem Auge erkennbar, indem sie mit sichtbaren Schadsymptomen auf die Umweltbelastung reagieren. Sie machen damit ein Problem, das sonst nur in abstrakten Zahlen wahrgenommen wird, direkt und in der Alltagswelt der Brger wahrnehmbar. Vor diesem Hintergrund wurde 1999 mit finanzieller Untersttzung durch das LIFE Umwelt Pro-gramm der Europischen Kommission das Europaweite Netzwerk zur Beurteilung der Luftqua-litt mit Bioindikatorpflanzen (EuroBionet) gegrndet. In diesem Netzwerk aus 12 Stdten und Regionen in acht EU-Lndern wurden unter der wissenschaftlich-technischen Koordination der Universitt Hohenheim Bioindikatorpflanzen zur berwachung der Luftqualitt und zur Frde-rung des Umweltbewusstseins eingesetzt. An dem Projekt nahmen Kommunalverwaltungen und Forschungsinstitute aus Edinburgh (GB), Sheffield (GB), Kopenhagen (DK), Dsseldorf (D), Nancy (F), Lyon (F), Barcelona (E), Valencia (E), Ditzingen (D), Klagenfurt (A), Verona (I) und Glyfada (GR) teil. In den Partnerstdten wurden lokale Bioindikatormessnetze mit insgesamt mehr als 100 Stationen installiert und ber drei Jahre betrieben. An diesen Stationen wurden nach hoch standardisierten Verfahren angezogene Bioindikatorpflanzen (Tabak, Pappel, Wei-delgras, Spinnwurz/Tradescantia und Grnkohl) gegenber der Umgebungsluft exponiert und anschlieend auf Schadwirkungen durch Ozon, Schwefelverbindungen, Metalle, Kohlenwas-serstoffe und mutagene Substanzen untersucht. Die wissenschaftlichen Untersuchungen wur-den von einem intensiven Programm zur ffentlichkeitsarbeit und Umwelterziehung begleitet.

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  • EuroBionet Zusammenfassung

    Die Versuchsserien lieferten zahlreiche Daten zur rumlich/zeitlichen Verteilung der Wirkungen von Luftschadstoffen innerhalb der stdtischen Messnetze sowie im europaweiten Vergleich. So wurde mit Hilfe von Tabakpflanzen ein deutlicher Gradient der Ozonwirkungen von Nord- und Nordwesteuropa nach Sd- und Mitteleuropa erkennbar. Die strksten ozoninduzierten Blatt-schdigungen wurden an den Stationen in Lyon, Barcelona und Hohenheim beobachtet, wh-rend in Edinburgh, Sheffield, Kopenhagen und Dsseldorf nur schwache bis mige Ozonwir-kungen auftraten. Die Auswertung der Immissionsmessungen ergab, dass in der Mehrzahl der Stdte die internationalen Richt- und Zielwerte zum Schutz der Vegetation berschritten wur-den. Der Tradescantia-Kleinkerntest zum Nachweis mutagener Wirkungen wurde erstmals er-folgreich in einer derart groen geographischen Ausdehnung eingesetzt. Hinweise auf ein er-hhtes genotoxisches Potenzial wurden vor allem an Standorten mit hoher Verkehrsbelastung gefunden. Die Experimente zeigten, dass mit der Standardisierten Graskultur lokale Brennpunk-te mit deutlich erhhter Schwermetallbelastung nachgewiesen werden knnen, es aber auch mglich ist, die kleinrumige Verteilung der Belastung und kurzfristige Vernderungen der Emissionssituation zu dokumentieren. Insgesamt wurde die Belastung mit Schwefel und Schwermetallen als niedrig bis mig hoch eingestuft, Grenzwerte fr Futtermittel wurden an den meisten Stationen eingehalten. Auffllig war die hohe Schwermetallbelastung in den spani-schen Grostdten, wobei jedoch whrend der Projektlaufzeit ein deutlicher Rckgang der Bleiwerte zu verzeichnen war. Antimon erwies sich als besonders geeignet, Messpunkte bezg-lich des Einflusses von Verkehrsemissionen zu charakterisieren. Die Anreicherung verschiede-ner Polyzyklischer Aromatischer Kohlenwasserstoffe in Grnkohl zeigte eine deutliche Differen-zierung zwischen stdtischen Stationen einerseits und Referenzstationen andererseits. Insge-samt lagen die Gehalte in einem mittleren, fr Stdte typischen Konzentrationsbereich. Fr das Projekt wurde eigens ein zentrales, modular aufgebautes Kommunikationskonzept zur dezentralen Umsetzung in den Partnerstdten entwickelt. Im Mittelpunkt des Erlebnis-vor-Ort-Gedankens der Kommunikationsstrategie stand der auf zentralen Pltzen der Stdte installierte Informationspavillon in Form eines grnen Wrfels (Green Box), der zum Zentrum der ffent-lichkeitsarbeit und zum Ausgangspunkt fr zahlreiche Veranstaltungen wurde. Infolge der ber-zeugenden Auendarstellung des Projekts wurde in Fernsehen, Rundfunk und Presse hufig ber die Aktivitten berichtet. Die Themen Luftqualitt, Bioindikation und Umweltschutz erregten Aufmerksamkeit in der Bevlkerung und boten Ansatzpunkte fr die Kommunikation zwischen Verwaltung und Brgern. EuroBionet war besonders attraktiv fr Schulen. Die direkte Konfron-tation mit den Folgen der Luftbelastung fhrte zu einer persnlichen Betroffenheit der Jugendli-chen. Der Umgang mit dem lebenden Objekt Pflanze erwies sich als geeignet, ber die Zu-sammenhnge zwischen Verkehr, Luftbelastung und Umweltschden zu informieren. Die erfolgreiche Verwendung von Bioindikatorpflanzen im EuroBionet leistete einen wichtigen Beitrag auf dem Weg zur europaweiten Standardisierung von Bioindikationsmethoden als Vor-aussetzung fr ihre Etablierung als Verfahren zur wirkungsbezogenen Umweltberwachung. Gleichzeitig wurde demonstriert, dass Bioindikatoren in besonderem Mae fr die Umwelterzie-hung und fr die ffentlichkeitsarbeit der Kommunen geeignet sind und so zu einer effizienteren Kommunikation zwischen Behrden und Brgern auf dem Umweltsektor beitragen knnen.

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  • EuroBionet Summary

    2 SUMMARY Although air quality in the EU Member States has improved considerably over the last decades, air pollution still represents one of the most urgent environmental problems in the eyes of both the public and government authorities. This judgement is also reflected in the 6th Environment Action Programme of the European Commission, where a particular emphasis is placed on im-proving air quality. In contrast to earlier decades it is now photo-oxidants, suspended particulate matter and hydrocarbons that primarily affect air quality because of the ever growing road traf-fic. Compliance with target values and thresholds for air pollutants is checked with the help of automated air pollution monitoring stations. However, the information gathered using chemical-physical procedures regarding pollutant concentrations does not allow any direct inferences to be drawn regarding pollutant effects on living beings since these are influenced by a great num-ber of intrinsic and extrinsic factors. Confirmation of harmful effects can only be made with the help of living organisms, so called bioindicators, that either react sensitively towards specific pollutants, or accumulate toxic substances. Bioindicators have already been used for many years in some EU countries for environmental monitoring, but their acceptance by authorities, decision-makers and the public is still low at the European level due to the insufficient stan-dardisation of procedures and the poor comparability of the results derived from that. Bioindica-tor plants feature a range of properties that make them suitable not only for effect-related moni-toring of air quality, but particularly for environmental communication and education. Often they reveal the noxious effects of air pollution directly, i.e. visibly to the naked eye, when they re-spond with apparent injury symptoms to environmental pollution. They can indicate a problem in a way that is obvious to the everyday life of the citizen and that might otherwise only be per-ceived as abstract numbers. With this in mind, the "European Network for the Assessment of Air Quality by the Use of Bioin-dicator Plants" (EuroBionet) was set up in 1999 with financial assistance from the LIFE Envi-ronment Programme of the European Commission. In this network of twelve cities and regions in eight EU countries, bioindicator plants have been used under the scientific and technical co-ordination of the University of Hohenheim for monitoring air quality and promoting environ-mental awareness. Communal administrations and research institutes from Edinburgh (GB), Sheffield (GB), Copenhagen (DK), Dsseldorf (D), Nancy (F), Lyon (F), Barcelona (E), Valencia (E), Ditzingen (D), Klagenfurt (A), Verona (I) and Glyfada (GR) took part in the project. Within these partnered cities, local bioindicator networks with more than 100 monitoring stations in all were established and operated over three years. At these stations bioindicator plants (tobacco, poplar, rye grass, spiderwort/Tradescantia and curly kale) cultivated according to highly stan-dardised procedures were exposed to ambient air in order to assess and to demonstrate the effects of ozone, sulphurous compounds, metals, hydrocarbons and mutagenic substances. The scientific investigations were accompanied by an intensive programme of public relations work and environmental education.

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  • EuroBionet Summary

    The experiments provided numerous data on the spatial and temporal distribution of the effects of air pollutants both within local networks and at the European level. A clear gradient of ozone-induced effects from northern and northwest Europe to southern and central Europe became evident using tobacco plants. The strongest ozone-induced leaf injuries were observed at the exposure sites in Lyon, Barcelona and Hohenheim, while in Edinburgh, Sheffield, Copenhagen and Dsseldorf only weak to moderate ozone impact was registered. Evaluation of data on pol-lutant concentrations revealed that in the majority of the cities the international threshold and target values for protecting vegetation were exceeded. The Tradescantia micronucleus test for assessing mutagenic effects was carried out for the first time successfully over such a large geographic area. Signs of a raised genotoxic potential were found particularly at sites with high levels of traffic. The experiments showed that with standardised grass culture, local hotspots of heavy metal pollution could be verified, and that it was also possible to document the small-scale distribution of the pollution load and short-term changes in emission status. Overall, im-pact by sulphur and heavy metals was classified as low to moderate, and limit values for feed-ingstuffs were adhered to at most stations. A comparably high heavy metal pollution was no-ticed in Spanish cities, but a clear drop in lead levels was registered during the course of the project. Antimony proved to be particularly characteristic of traffic-influenced sites. The enrich-ment of different polycyclic aromatic hydrocarbons in curly kale revealed a clear differentiation between urban and reference stations. Overall the contents lay at an intermediate concentration level typical for urban agglomerations. A centralised, modularly constructed communication concept was developed for decentralised implementation in the participating cities. At the centre of this local experience idea of the communication strategy were information pavilions in the form of green cubes ("Green Box") which became the foci of public relations activities and the starting points for numerous events. Because of the convincing presentation of the project, activities were frequently reported on television, radio and in the press. The issues of air quality, bioindication and environmental pro-tection aroused the attention of the public and offered starting points for better communication between municipal administration and the citizens. EuroBionet was particularly attractive for the schools. The direct confrontation with the harmful consequences of air pollution led to a per-sonal involvement of younger individuals. Dealing with plants as living objects proved to be suit-able for informing younger people about the associations between traffic, air pollution and envi-ronmental damage. The successful use of bioindicator plants in the EuroBionet represented an important milestone on the road to a Europe-wide standardisation of bioindication; this represents an absolute pre-condition for its establishment as a procedure for effect-related environmental monitoring. Si-multaneously, it was also proven that bioindicators are highly suitable for environmental educa-tion and municipal public relations activities, and in this way they can contribute to a more effi-cient communication in the environmental sector between municipal authorities and the citizens they serve.

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  • EuroBionet Introduction

    3 INTRODUCTION More than two thirds of the European population currently live in urban agglomerations. Be-cause of the clustering of stationary and mobile emission sources over comparatively small ar-eas, the specific conditions of city climates and pollution dispersion, and the high population density, a particularly large impairment of air quality results in those areas that also entails negative effects on human health, animals, plants and material goods. According to a study by the European Environmental Agency in 1995, 25 million inhabitants in 115 European cities were found to be exposed to SO2 and dust (winter smog) that exceeded WHO guidelines. As many as 37 million in the summer months suffered from photochemical smog (EEA 1998). In most cities of the European Union the air quality has substantially improved over the last decades due to stricter legal regulations, the adoption of less-polluting technologies and the transfer of industry from the city centres. Pollution from winter smog in particular has decreased over this period. Nevertheless, air pollution remains one of the most urgent European environmental problems. Steadily increasing motor traffic plays a leading role here. For this reason pollution with ozone and other photo-oxidants (summer smog), suspended particulate matter and poten-tially carcinogenic organic air pollutants now represent central topics of discussion. Even after expected improvements in air quality following the implementation of additional EU programmes for traffic-related pollutant reduction such as Auto-Oil II, infringements of permissible concentra-tions of some air pollutants are still to be expected. According to opinion polls in the frame of Eurobarometer, air pollution is also in the view of the population one of the most impending en-vironmental problems in Europe. A large percentage of citizens expressed their worries about air pollution (EC 1999a, 2002). Like the availability of capital, workforce and traffic infrastructure, air quality in the coming years might also become a factor affecting economic growth and de-velopment in entire regions. Bioindicators and environmental monitoring National laws and European Directives oblige the Member States of the European Union to establish air quality monitoring networks and to record the ambient concentrations of the most important air pollutants continuously. Such measurements are made with the aid of physical-chemical procedures in automated monitoring stations. In this way it is checked whether the threshold and target values for the protection of human health and ecosystems set in the so-called Daughter Directives are adhered to. How-ever, measurements of pollutant concentrations do not provide any direct information regarding their possible effects on man and the environment since the reaction of an organism not only depends on the concentration and duration of action of a pollutant, but it is also influenced by a range of predisposing or accompanying factors including weather, nutrition, age, developmental state and the simultaneous action of other stressors. In particular, the consequences of chronic exposure to low concentrations of several pollutants can hardly be evaluated by physical-chemical measurements since thresholds and target values usually relate to individual compo-nents or at best interactions with at most one other component, and the dearth of knowledge allows no multi-factorial pollution situations to be considered. Demonstration of noxious effects can only proceed using living organisms that either react sensitively to specific pollut-ants/pollutant mixtures or concentrate toxic substances in their tissues. Such bioindicators also integrate the effects of all environmental factors including interactions with other pollutants or

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  • EuroBionet Introduction

    climatic conditions. In this way risks from complex pollutant mixtures and even chronic stress below pollutant thresholds can be recorded. Bioindicator plants have already been used for many years in scientific studies to demonstrate pollutant effects. In some countries such as Germany, Austria, the Netherlands and Italy, some methods have found their way into the routine monitoring of industrial plants and conurbations by private firms and environmental authorities. At a Europe-wide level the use of bioindicator plants has not yet been established (with a few notable exceptions). One reason for the lacking acceptance amongst decision makers in politics, public administration and private enterprises is the insufficient standardisation of the procedures, a fact making it difficult to compare different results obtained from their use. Bioindicators and environmental communication The ongoing reduction of the current pollution burden is complicated by the fact that there are now more small, mobile pollutant sources compared to the relatively few large emission sources of old. Those who have to under-take steps to reduce environmental pollution caused by individual motor vehicle usage are not just automobile manufacturers. Consumers who use these vehicles must also look at steps to adapt their life-style and especially mobility behaviour. An increased environmental awareness of the urban population in particular, which should lead to an increased acceptance for meas-ures to decrease pollutant release from traffic and promote alternative mobility concepts, is therefore in the immediate interests of environmental authorities at a local, regional and supraregional level. Apart from these means-oriented considerations, the obligations of authorities and other public institutions to inform the public about the state of the environment is growing in importance. Since the World Summit in Rio de Janeiro 1992, the rights of citizens to free access to environ-mental information has now been assured. This right was reaffirmed in the rhus Convention of the UNECE in 1998 and most recently in EC-Directive 2003/4/EC. Free access for citizens to environmental data and the obligation of public institutions to provide adequate and compre-hensive information raises questions as to how complex facts can be conveyed to the public in general. Despite the high costs associated with the operation of automated air pollution monitor-ing networks, the state of knowledge regarding the causes and effects of air pollution amongst technically uneducated laypersons remains surprisingly small. Europe-wide surveys conducted within the Eurobarometer programme have revealed that a large percentage of the population does not feel adequately informed on air quality matters despite the variety of information avail-able, although the interest in environmental problems remains as high as ever (EC 1999a, 2002). An increased requirement for efficient and attractive communication strategies from the environmental sector can therefore be deduced from that. Bioindicator plants feature a range of properties that appear to make them suitable not just for effect-related monitoring of air quality, but also for environmental communication and education. They often reveal the noxious effects of air pollution directly, i.e. they are apparent to the naked eye, when they respond with visible injury symptoms to environmental pollution. They therefore

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  • EuroBionet Introduction

    render a problem that would otherwise only be observed in abstract form as being directly visi-ble to the everyday life of the citizens. They address man at an emotional level since clear damage to plants can evoke a personal involvement and make it possible to draw conclusions about the effects of air pollution on ones own health. Aims and goals With all this in mind the LIFE Project EuroBionet (European Network for the Assessment of Air Quality by the Use of Bioindicator Plants) was established in 1999. Euro- Bionet is a network of municipal administrations and research institutes from twelve cities and regions in eight EU countries which under the coordination of the University of Hohenheim has applied bioindicator plants for monitoring air quality and increasing the environmental aware-ness of the urban population. The objectives of the experiments scheduled over three years were, to contribute to a Europe-wide implementation of bioindicator plants for monitoring air qual-

    ity, to assess and evaluate air quality in the participating cities and regions, to compare potentially different pollution types amongst the participating cities, to contribute to a standardisation of methods applied at a European level, to provide data for the development of remedial actions, to promote a transfer of know-how and knowledge about bioindication both within and out-

    side the network, to demonstrate the noxious effects of air pollutants on living beings in a clear and sustain-

    able manner, to sensitise the urban population towards environmental quality issues, to stimulate initiatives in schools, companies, authorities and households, and to provide an environmental communication and urban marketing tool for the participating

    municipal authorities. Local bioindicator networks were established for this purpose in the participating cities and op-

    erated over three years. Bioindicator plants cultivated under standardised conditions were ex-

    posed to ambient air at these sites in order to assess and to demonstrate the effects of different

    air pollutants. The scientific and technical investigations were accompanied by an intense pro-

    gramme of public relations and environmental education on the basis of a communication con-

    cept developed within the scope of the project.

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  • EuroBionet LIFE Project Framework

    4 LIFE PROJECT FRAMEWORK 4.1 Project structure Under the scientific, technical and administrative coordination of the University of Hohenheim, twelve local and regional governments with their local scientific partners participated in the EuroBionet city network. The implementation and progress of the project were accompanied by a "Project Advisory Board". In order to exploit synergies, an intensive as possible linking of sci-entific bodies, local government departments, citizens and especially schools was striven for during the establishment of the network. The Institute for Landscape and Plant Ecology (Section Plant Ecology and Ecotoxicology) of the University of Hohenheim was applicant, beneficiary, coordinator and partner of the project. Under the leadership of the project coordinator Dr. Andreas Klumpp a coordination office was set up at the University of Hohenheim (scientific and technical coordination: Dipl.-Biol. W. Ansel/Dr. G. Klumpp, administrative coordination: D. Wahnelt, analytical service: B. Meier, as well as other scientific and technical staff). City network (partners): The project was carried out with the following local and regional au-thorities (cf. Fig. 4.1):

    Generalitat de Catalunya/Barcelona (E) City of Copenhagen (DK) Stadt Ditzingen (D) Landeshauptstadt Dsseldorf (D) City of Edinburgh (GB) City of Glyfada (GR) Landeshauptstadt Klagenfurt (A) Communaut urbaine de Lyon (F) Communaut urbaine du Grand Nancy (F) City of Sheffield (GB) Ayuntamiento de Valencia (E) Comune di Verona (I)

    The local project management in the cities was performed by the local and regional authorities as partners of the network. In order to realise the tasks within the project, cooperation between different departments of the respective local authorities was usually necessary. In addition, a close cooperation with scientific institutions as local partners was entered into frequently or an informal cooperation with other institutions was agreed upon. The project partners and co-operating institutions with their respective local project leaders or contacts are listed in Table 11.1 (Annex I).

    9

  • EuroBionet LIFE Project Framework

    Hohenheim

    Fig. 4.1: Map of Europe with the partners of the network. At the start of the project a "Project Advisory Board (PAB)" was set up. Its tasks consisted of

    supporting the coordinator and project leader in his work,

    providing for a coordinated and structured running of the project,

    promoting the exchange of information amongst the partners,

    pursuing the implementation of the communication concept in the participating cities,

    if needed, modifying or amending the work programme defined in the project proposal with the approval of the European Commission,

    determining procedures for publications and press communications,

    settling discrepancies amongst the partners or with the coordinator, and

    taking measures with a participating party wherever any agreements are broken.

    10

  • EuroBionet LIFE Project Framework

    Three members of the PAB were designated by the coordinator, a further member was chosen by the participants of the 1st Technical Project Workshop as a representative of the city partners. The PAB consisted of the following individuals:

    Dr. Jrgen Husler, Interbrand Zintzmeyer & Lux GmbH, Cologne/Zurich Dr. Georg H. M. Krause, Landesamt fr Umwelt Nordrhein-Westfalen, Essen Gary McGrogan, Environment and Regulatory Services, Sheffield City Council Dr. Karl Theo von der Trenck (until May 2000 Dr. Harald Gebhardt), Landesanstalt fr

    Umweltschutz Baden-Wrttemberg, Karlsruhe. An organisational chart of the project structure is provided in Fig. 11.1 (Annex I). 4.2 Work plan and time schedule The distribution of tasks as well as the rights and obligations of the partners in the network were determined in cooperation contracts that were agreed upon at project onset between the Uni-versity of Hohenheim and the various municipal and regional administrations. Table 4.1 pro-vides an overview of the distribution of tasks between project coordination and the city partners in the project. Table 4.1: Distribution of tasks between project partners and project coordination

    University of Hohenheim (Coordinator, partner) City partners Scientific, technical and administrative coordination

    Establishment of local teams, cooperation with local partners

    Definition of the scientific and technical methodologies

    Installation and maintenance of local bio-indicator networks

    Procedural training, monitoring, quality assurance Cultivation and exposure of the bioindicator plants

    Material procurement and distribution Assessment of visible plant injuries Central sample analyses Sampling of plant material for analyses Organisation and execution of bioindication of mutagenic substances (Tradescantia programme)

    Provision of climate and air pollution data

    Development of a communication concept Implementation of the communication concept

    Data evaluation, discussion, appraisal Evaluation of local data sets Organisation of workshops and conferences Environmental education programmes Press work, project presentations Public relations work, project presentations Preparation of reports for the European Commission

    Preparation of reports at the local level

    The project work is divided into three, partly overlapping phases. Phase 1 involved the prepara-tory work such as the establishment of the city network, recruitment of the local teams, the

    11

  • EuroBionet LIFE Project Framework

    elaboration of a procedures manual, the procurement and dispatch of materials, the choice of monitoring sites and the development of a communication concept. Phase 2 dealt with the per-formance of studies on air quality using bioindicators over three experimental periods, the evaluation of data as well as the implementation of the communication concept and the organi-sation of publicity campaigns. Phase 3 mainly involved the final evaluation and interpretation of the results, the preparation and realisation of the final conference, and the preparation of the final reports. The individual tasks were realised largely according to the time schedule in the project proposal and the application for the extension of the project (Table 11.2 Annex I). 4.3 Project meetings During the project, four internal project meetings were organised. The Technical Workshop from the 12th to the 14th January 2000 at the University of Hohenheim dealt with the establish-ment of the network, preparation of the scientific and technical work programme, and presenta-tion of the communication concept for the EuroBionet. After the general description of the pro-ject concept and make-up, the local participants were introduced to a provisional version of the bioindication procedures manual which was provided both in German and English. The individ-ual methods for bioindication were presented by colleagues from the coordination team in single lectures as well as in the form of practical demonstrations. The managing director of the brand-marketing company Interbrand Zintzmeyer & Lux, Dr. Jrgen Husler, presented a draft for the communication concept of the EuroBionet. In a closing discussion involving the workshop par-ticipants, detailed questions regarding the project course, the communication concept and methodological/financial aspects were addressed. In three more project meetings on March 7th 2001 in Altbach near Stuttgart, April 22nd and 23rd 2002 and November 4th 2002 in Hohenheim, the reports of the project leader and the coordination team regarding the scientific project re-sults, organisational and financial aspects, the realisation of the communication concept, dis-cussions about the results achieved as well as information exchange between the project par-ticipants took the centre stage. The implementation of the scientific and communicative tasks of the project was facilitated by direct contacts between the representatives of the participating cities and their scientific partners, which also allowed a better application of synergies in the fields of public relations and environmental education. The constitutional meeting of the Project Advisory Board was held on January 13th 2000 in Hohenheim during the Technical Workshop. Further meetings of the PAB members with the coordinator on May 17th 2000 in Hohenheim, on October 26th 2000 in Cologne and March 7th 2001 in Altbach near Stuttgart served to fix the project structures during the starting phase, to implement the communication concept, to assist the planning of the public presentation of the project, and to prepare presentations for local political and scientific events. During the further course of the project, agreements were brokered between the members of the PAB and the coordinator both in written form and by telephone.

    12

  • EuroBionet LIFE Project Framework

    4.4 Deviations from the project proposal The cities Arnhem (NL), Hamm (D), Karlsruhe (D) and Toulouse (F) that had originally under-signed the initial project proposal to LIFE could not fulfil their intentions for financial as well as organisational reasons. Dsseldorf (D) and Grand Nancy (F) in 2000 as well as Glyfada (GR) and Valencia (E) in 2001 were then confirmed as substitute partners. The town of Ditzingen (in the District of Ludwigsburg/D) participated from 2000 as an associate partner within the scope of a school project in cooperation with local schools and the University of Hohenheim. The city of Plochingen as well as the municipalities of Altbach and Deizisau (both in the District of Esslingen/D) also cooperated on a looser basis. At the same time as the interim report was produced a cost-neutral, three-month prolongation of the project term was applied for to LIFE so that a third series of experiments with bioindicator plants could be carried out in the summer half of 2002 as an addition to the initial work sched-ule. In this way it was hoped that a broader database for evaluating air quality as well as a more comprehensive utilisation of the communication concept could be guaranteed. Because of lim-ited personnel and financial resources from the participating cities, however, and the limited time available for analysis and evaluation, only a (both temporally and materially) reduced bio-indication programme was implemented in the year 2002.

    13

  • EuroBionet Procedures for Bioindication of Air Pollution

    5 PROCEDURES FOR BIOINDICATION OF AIR POLLUTION 5.1 Air pollution and bioindicators in urban agglomerations Today, the air quality in many urban agglomerations within Europe is characterised by a large number of differing types of air pollutants occurring in concentrations and combinations that vary both over time and space. Unlike the situation between the 1950s and the 1970s, acidic air pollutants are playing an ever decreasing role, while oxidative substances, organic pollutants, suspended particulate matter and potentially genotoxic substances are growing in importance. On the basis of these developments in air pollution and considering the limited availability of applicable biomonitoring methods, bioindicator plants were selected for the following pollutants or pollutant groups:

    Ozone,

    Sulphurous compounds,

    Heavy metals and trace elements,

    Polycyclic aromatic hydrocarbons (PAH),

    Mutagenic (genotoxic) substances. According to the definition of bioindicators as "organisms or communities of organisms which react to environmental factors by changing their vital function and/or their chemical composition thus permitting to infer the state of their environment" (ARNDT et al. 1996), one needs to differ-entiate between so-called sensitive indicators, that respond relatively sensitively with visible injury symptoms to specific pollutants or pollutant groups, and accumulative indicators that are able to accumulate large amounts of toxic substances in their tissues without expressing visible injuries. Both types of bioindicators can be applied in the form of an active biomonitoring, i.e. a largely standardised exposure in the monitoring area (VDI 1999a). The following bioindication procedures widely used in routine programmes and as such suitable for application in a Europe-wide demonstration project were selected (Table 5.1): Table 5.1: Bioindication methods and effect criteria used in the EuroBionet.

    Bioindicator Species Air Pollutants Effect Criteria Tobacco (Nicotiana tabacum cv. Bel-W3)

    Ozone (photooxidants) Visible leaf injuries

    Poplar (Populus nigra) clone Brandaris

    Ozone (photooxidants), heavy metals, trace elements

    Visible leaf injuries Accumulation

    Spiderwort (Tradescantia sp.) clone #4430

    Genotoxic substances Chromosome damage (micronuclei)

    Italian rye grass (Lolium multi-florum Lema)

    Sulphurous compounds, heavy metals, trace elements

    Accumulation

    Curly kale (Brassica oleracea Hammer/Grsa)

    Polycyclic aromatic hydro-carbons (PAH)

    Accumulation

    15

  • EuroBionet Procedures for Bioindication of Air Pollution

    5.2 Establishment of the local bioindicator networks Within each participating city, local bioindicator networks with 8 - 10 stations were installed. Dif-ferent criteria were considered when selecting the monitoring sites. The stations should have been distributed relatively uniformly over the city area so that the pollution burden of that par-ticular agglomeration could be represented as best as possible. In order to register local pollu-tion differences within the cities, some stations had to be set up close to vehicle traffic hubs or industrial emitters. One to two sites in areas with comparably low pollution by primary pollutants served as reference stations. The monitoring sites were classified according to their location as either urban, suburban, street, industrial or reference stations. The proximity to existing air monitoring stations, protection from theft and vandalism, city planning matters and in particular communicative aspects also played an important role in establishing the local monitoring net-works. After a preselection by the local teams the final definition of the monitoring stations and the exact localisation of the exposure systems were performed on the occasion of joint on-site visits of co-workers of the coordination team and the local project groups. During this process a range of factors including topography, subsoil type and free air circulation were to be consid-ered, all of which are explained in detail in the methods manual (cf. 6.1). Fig. 5.1 illustrates the monitoring network in Grand Nancy for exemplary purposes. Maps of the other monitoring net-works and a tabular listing of the measuring sites can be found in Annex I (Fig. 11.2 and Table 11.3). Fig. 5.1: The bioindicator network in Grand Nancy.

    16

  • EuroBionet Procedures for Bioindication of Air Pollution

    The monitoring network in the associate town of Ditzingen in the northwest of the Stutt-gart/Middle Neckar conurbation was operated directly by the University of Hohenheim (in coop-eration with the municipality and local schools). Five stations on school property in different dis-tricts as well as a sixth station at the A81 motorway from Stuttgart to Heilbronn were established for this purpose. Four more stations were also set up by the university at the Hohenheim cam-pus, and as a continuation to an earlier bioindicator programme in the vicinity of the coal-fired power plant Altbach/Deizisau in the municipalities of Altbach, Deizisau and Plochingen in the Neckar valley southwest of Stuttgart. 5.3 The bioindication methods: cultivation, exposure and assessment of effects 5.3.1 Standardised tobacco exposure The high ozone sensitivity of the tobacco cultivar Nicotiana tabacum Bel-W3 has been compre-hensively documented since the fundamental studies published by HEGGESTAD & MENSER (1962) in the USA. The exposure of Bel-W3 today numbers amongst the world-wide most fre-quently employed procedures for bioindication (HEGGESTAD 1991). If elevated ozone concentra-tions occur in a study area, the first point-shaped, silvery to tan tissue destructions (necroses) already appear after 2-3 days on the leaf surface of the exposed tobacco plants. These can then transform into extended brown necroses and lead ultimately to death of the leaves at high ozone doses (Fig. 5.2). In comparative studies with different wild and cultivated plant species (e.g. Medicago lupulina, Trifolium repens, Impatiens parviflora etc.), important inferences could also be drawn from the results of tobacco exposure regarding the potential risk for the natural vegetation (VDI 2000a). Because of the obvious, largely pollutant-specific reaction, tobacco plants are suitable not only for monitoring air quality, but also as demonstration objects in the fields of environmental communication and education.

    Fig. 5.2: Symptoms of ozone effects on tobacco (left an undamaged leaf; centre and right a moderately or strongly damaged leaf).

    The procedure employed in the project corresponds to the method of standardised tobacco ex-posure described in VDI-Guideline 3957/6 (draft 1999) (see procedures manual, chapter 2). The plants were cultivated in the period between April and September in the local greenhouses from uniform seeds and using a standardised substrate. In order to be able to select uniform as pos-sible plant material of a predefined developmental state at the start of exposure, two selection steps occurred during the cultivation regarding the size of the plantlets. The duration of cultiva-

    17

  • EuroBionet Procedures for Bioindication of Air Pollution

    tion per series was between 6 and 8 weeks depending on geographic location and seasonal weather conditions. Plants with six fully expanded leaves were selected for exposure. The pattern of leaves on an exposure-ready plant is illustrated in Fig. 5.3. Leaves 4 6 served as reference leaves for the injury assessment. Since cultivation in filtered air was not usually possible in the greenhouses, prior checking of the plants was carried out at exposure onset in order to register any leaf injury that may have been caused by raised ozone concentrations during cultivation. Such values were considered in the final assessment of the plants at the end of the two-week exposure pe-riod. Per site and series, 4 6 plants were exposed to ambient air in shaded exposure racks (expo-sure height 90 cm, frame height 180 cm) for 14 1 days (Fig. 5.3). During the outdoor expo-sure, irrigation of the plants was performed automatically using a wick system supplied by water reservoirs. At the end of the two-week exposure period the extent of the ozone-induced injuries was recorded on the reference leaves and the tobacco plants were exchanged with a new se-ries. Assessment of foliar injuries was performed visually by colleagues from the local teams in 5% steps and using a photo catalogue with exemplary images of damaged plants. In order to ensure the highest possible level of quality for assessment, the assessment technique was demonstrated and practiced with the help of on-site visits beforehand. Using the percentage leaf injury degrees of the reference leaves, the mean percentage of leaf injury was calculated for each of the bioindicator stations.

    Fig. 5.3: Exposure stage of a tobacco plant (left; from VDI 2000a) and exposure facility (right). From the raw data of the injury assessments, mean percentage of leaf injury was computed and classified into different ozone impact classes according to a five-step scale (Table 5.2). At the end of the experimental year the mean percentages of leaf injury from all exposure series were also determined at each station. This calculation was the basis for the comparative statistical evaluation within the network that included a variance analysis and pairwise comparisons of means.

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  • EuroBionet Procedures for Bioindication of Air Pollution

    Table 5.2: Five-stepped scale for classifying ozone effects on tobacco.

    > 60 % 31-60 % 16-30 % 6-15 % 0-5 %

    Ozone effects Very weak 1 Weak

    2Medium

    3Strong

    4 Very strong

    5

    % Leaf injury

    In the monitoring networks of Hohenheim and Ditzingen the ozone-resistant tobacco variety Bel-B was also employed for comparative purposes (in addition to the Bel-W3), as recommended in a more recent version of the VDI-Guideline (VDI 2000). Cultivation and assessment was per-formed in the same way as that already described for Bel-W3. At the Hohenheim station the differently ozone-sensitive NC-S and NC-R biotypes of white clover (Trifolium repens) were also exposed during the study years 2001 and 2002. These were cultivated and assessed according to the instructions of the UNECE ICP Vegetation (UNECE 2001). 5.3.2 Exposure of poplar clones Since as long ago as the 1970s poplar plants have been employed as sensitive and accumula-tive bioindicators for various air pollutants. The special advantages of the poplar procedure lie in their simple cultivation technique and the fact that genetically homogenous plant material can be produced by means of shoot propagation (BALLACH 1997). In addition, a number of poplar hybrids are available that reveal specific sensitivities/resistances towards individual air pollut-ants (e.g. sulphur dioxide, nitrogen oxides or ozone). Ozone-sensitive poplar clones respond to raised ozone concentrations with leaf injuries (point-shaped chloroses and necroses, discolour-ation), premature senescence, premature defoliation and stunted growth (Fig. 5.4).

    Fig. 5.4: Typical ozone effects on poplar clones in controlled fumigation experiments.

    19

  • EuroBionet Procedures for Bioindication of Air Pollution

    No VDI-Guideline exists yet for the poplar procedure. The cultivation and exposure of the poplar plants for the EuroBionet project is described in the procedures manual (chapter 3) and is based on the methodological work of ARNDT et al. (1992). In spring 2000 and 2001, certified cuttings of the ozone-sensitive poplar clone Populus nigra Brandaris were acquired from a tree nursery in the Netherlands and dispatched to the partner cities. From the start of April to the end of May the cuttings were precultivated in greenhouses (Fig. 5.5). During this period two shoots usually developed per cutting. A few days prior to exposure onset one shoot was selected per plant according to the prevailing developmental stage regarding sprout length or number of leaves before it was scored for visible injury symptoms. The surplus shoot was removed. The exposure of the poplar plants at the bioindicator sites started at the same time as the first tobacco series. For each site 4 poplar plants were exposed in shaded exposure frames (expo-sure height 60 cm, frame height 250 cm) to ambient air (Fig. 5.5). Irrigation was carried out us-ing glass fibre wicks emanating from water tanks. Unlike the tobacco procedure the poplar plants remained at the stations during the entire experimental period (from the end of May to the beginning of September: i.e. 14 weeks). Weaker, chronic ozone effects should also be able to be recorded over this longer period of exposure.

    Fig. 5.5: Cultivation and exposure of poplar clones. The assessment of ozone-induced effects was done biweekly in the same rhythm as the injury assessment of tobacco plants. The following parameters were recorded: Total number of leaves formed Still attached and exfoliated leaves (as a percentage of all leaves formed) Number of leaves with typical ozone-induced injuries Shoot length Relative growth rate (RGR)*

    * Calculation RGR: (ln (x2)-ln(x1))/(t2-t1); x: Shoot length, t: time of assessment, unit: [mm/mm*d]

    20

  • EuroBionet Procedures for Bioindication of Air Pollution

    The arithmetic means of the effect parameters were calculated from the individual values from each of the four plants at a site. During the first study period in 2000, numerous and sometimes similar looking symptoms ap-peared on the leaves due to the effects of viruses, fungal diseases and insects in addition to the typical forms of ozone injury. Since a differential diagnosis of these symptoms by the local staff was not possible because of a lack of training and time, the separate recording of leaves with typical ozone injuries was refrained from in the study year 2001. In order to obtain extra infor-mation on chronic pollution from heavy metals, the heavy metal and trace element content of leaf samples from the exposed poplar plants were studied in the second experimental year. Af-ter the last assessment in September 2001, 10 older but still unyellowed leaves were harvested for this purpose from all four plants at a location before they were pooled in a mixed sample and dispatched for centralised analysis to the EuroBionet laboratory in Hohenheim. The preparation and analysis of the sample material was performed using the same methods applied for the grass cultures (see Chapter 5.3.5). 5.3.3 Tradescantia micronucleus test The Tradescantia micronucleus test dates back to the studies of MA et al. (1978) and is now employed globally using a meanwhile largely standardised procedure (MA et al. 1994) in labora-tory and field experiments for the assessment of the genotoxic effects e.g. of chemicals, radia-tion, waste water and air pollution. The principle of the bioassay is based on the registration of chromosome defects in the pollen mother cells of the Tradescantia clone #4430. Meiotic divi-sion in the buds of this plant reacts very sensitively to different mutagenic substances. As a re-sult, chromosome breakage or non-disjunction can occur. The DNA fragments arising manifest themselves in a later cell stage ("early tetrad stage") in the pollen mother cells as micronuclei that can be easily counted microscopically (Fig. 5.6). The frequency of micronuclei formation represents a measure of the mutagenic potential of the environment, i.e. impact by genotoxic substances. Pickl

    Fig. 5.6: Cultivation of Tradescantia in the greenhouse (le(centre) and micronuclei in the pollen mother cell

    21Micronucleift), buds in the exposure stage s (right).

  • EuroBionet Procedures for Bioindication of Air Pollution

    The methodology employed in the EuroBionet is based on test protocol of the International Programme of Chemical Safety (MA et al. 1994) and is described in detail in the procedures manual (chapter 4). Because of the specific conditions necessary for plant cultivation, the Tradescantia plants for all experimental series were grown in the greenhouses in Hohenheim (Fig. 5.6). Two days before experimental onset, cuttings of a suitable developmental stage were dispatched by express-courier to the participating cities. After arrival and one day of acclimatisa-tion, cuttings were exposed for 12 - 30 h in the monitoring network. In order to guarantee stan-dardised exposure conditions for the Tradescantia experiment within the EuroBionet project, a special exposure frame was developed. The simple construction of the frame allowed a flexible attachment to different types of supports such as traffic signals and street-lamps (Fig. 5.7). Ex-tra measurement points could therefore be selected for studies at heavily-used streets and traf-fic hubs in the direct vicinity of the roadways.

    Fig. 5.7: Exposure frame for Tradescantia (left) and street stations in Stuttgart (centre) and Valencia (right).

    Upon completion of exposure, Tradescantia inflorescences were harvested and fixed on site. They were then dispatched by express delivery to Hohenheim where the samples were con-served and microscopically examined for micronuclei in a specialised laboratory (kotox Ltd. Stuttgart). For each monitoring point 5 buds with pollen mother cells in the early tetrad stage were prepared from the collected sample material. For each preparation 300 tetrads were scored at 400-fold magnification for the presence of micronuclei. The micronuclei rates were then recorded as the number of micronuclei (MCN) per 100 tetrads (i.e. percentages). Compari-sons were carried out between differently polluted stations within the local monitoring network as well as with control values from parallel-run greenhouse experiments. Statistical evaluation for significant differences within a study area was performed using the non-parametric Kruskal-Wallis test (H-test). For subsequent pairwise comparisons of site means the Tukey-Kramer test was employed.

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  • EuroBionet Procedures for Bioindication of Air Pollution

    5.3.4 Standardised grass culture The procedure for standardised grass culture dates back to studies on fluoride and lead pollu-tion in the state of North Rhine-Westphalia at the end of the 1960s (SCHOLL 1971) and was the first standardised bioindication procedure to be established as a VDI-Guideline (VDI 1978). It is based on the ability of grass cultures to accumulate a large number of inorganic and organic air contaminants without incurring even visible damage. The procedure used in the project with Italian rye grass (Lolium multiflorum LAM. cv. Lema) corresponds to that described in VDI-Guideline 3957/2 (VDI 2003) and is described in detail in the procedures manual (chapter 5). Cultivation was performed in the greenhouses of the par-ticipating cities from a uniform seed stock. The use of a standardised soil substrate as well as a controlled fertilisation were of special importance for ensuring the comparability of the results. Regular cutting during cultivation ensured a good tillering of the cultures. Glass fibre wicks in the substrate ensured a regular water supply from water tanks during cultivation and field expo-sure. After an approx. 6 7 week cultivation, the grass cultures were exposed at the bioindicator sta-tions in special appliances consisting of an iron rod, an attached steel basket and a water sup-ply vessel. One grass culture was exposed for each series and site. The cultures were set up in such a way that the upper edges of the pots were 1.5 m above the ground (Fig. 5.8). After an exposure of 28 days the grass cultures were replaced with new cultures. The plant material grown during the exposure period was harvested and pre-dried. Then the samples were dis-patched for centralised analysis in the EuroBionet laboratory in Hohenheim.

    Fig. 5.8: Exposure system for standardised grass cultures (left: schematic construction of the plant pot; centre, right: exposure at stations in Nancy and Ditzingen).

    23

  • EuroBionet Procedures for Bioindication of Air Pollution

    For measuring elemental content the samples were further dried at 80 C and ground with an agate mill. The sulphur concentrations were determined using a CS analyser, the BCR standard CRM 129 (hay powder) served as reference material. For determining metal contents a wet digestion was performed in a microwave oven. Measurement was performed using an atomic absorption spectrometer in the flame (iron/Fe, copper/Cu, zinc/Zn) or in the graphite furnace (lead/Pb, cadmium/Cd, chromium/Cr, nickel/Ni). The concentrations of the trace elements anti-mony (Sb), arsenic (As) and vanadium (V) were determined at the State Institute for Agricultural Chemistry at the University of Hohenheim using an ICP-MS. The standards CRM 281 (rye grass') and SRM 1515 (apple leaves') served as standards for quality control with the metal analyses, where the recovery rate was usually > 95 %. In selected samples from the year 2000, measurement of platinum content by Differential Pulse Cathodic Stripping Voltametry after a three-step digestion procedure was performed by the Steinbeis Transfer Centre of Applied and Environmental Chemistry at the University of Applied Sciences FH Reutlingen. The statistical evaluation of the data was done in several steps. First of all, elemental contents were evaluated using a method developed by ERHARDT et al. (1996). In this procedure, the high number of low values from samples of the study area which show only a small pollution impact are used as reference values. The mean value of these reference values forms the basis level or background value for the study area and describes the general pollution baseline. A value is then regarded as elevated or distinctly elevated when the elemental content of the sample ex-ceeds the background value by 3 or 6 standard deviations, respectively. With several exposure series the background values are first computed for each series, and from these values the mean background value is then determined for each element and each monitoring network. This procedure was carried out in two steps:

    1. Evaluation of data from each individual city 2. Combined evaluation of data from all monitoring stations within the EuroBionet and cal-

    culation of a European background value (background value bvEurope) for each element. Using the background values (Bv) and their standard deviations (s) the monitoring sites were grouped into four classes for graphical representation according to their elemental concentra-tions (x) (Table 5.3). Table 5.3: Subclassification into pollution classes according to heavy metal or sulphur content of

    the exposed grass cultures.

    Classification Heavy metal resp. S content Measured value x

    1 Very low x < bvEurope

    2 Low bvEurope < x < bvEurope + 3s

    3 Elevated bvEurope + 3s < x < bvEurope + 6s

    4 Distinctly elevated bvEurope + 6s < x

    24

  • EuroBionet Procedures for Bioindication of Air Pollution

    Based on this, the mean ranks of the individual sites relating to the different elements were in-cluded for comparative analysis of the monitoring networks. Relationships between the contents of individual elements as well as between the elemental contents of grass cultures and poplars were tested using Spearman rank correlation coefficients. With cluster analysis using squared Euclidean distance as a measurement of distance and the Ward algorithm as an agglomeration procedure, the monitoring points were subclassified according to their relative similarity into groups ("clusters") which were graphically illustrated using so-called dendrograms. 5.3.5 Standardised curly kale exposure The procedure for standardised curly kale exposure is based on studies by HETTCHE (1975) on the accumulation of polycyclic aromatic hydrocarbons (PAH) in curly kale leaves. The special suitability of curly kale is based on the large surface area of the extensively crimpled leaf sur-faces and the prominent wax layer that favours the accumulation of lipophilic substances. Curly kale has in the meantime become widespread as an indicator for organic air pollution by con-taminants such as PAH, PCB, PCDD/PCDF as well as those of an inorganic nature. The procedure applied in the project corresponds largely to that of the VDI-Guideline 3957/3 (VDI 2000b) and is described in detail in the procedures manual (chapter 6). The cultivation of curly kale plants (Brassica oleracea acephala cv. "Hammer/Grsa") was carried out from August to October in the greenhouses of the participating cities also with a standardised substrate, con-trolled fertilisation and glass fibre wicks for water supply. Before exposure the plants were brought into the open air for one week for hardening in a rain protected location that was also as little polluted as possible. A uniform plant size as well as a precisely defined sampling are basic preconditions for ensuring comparability of the results. At exposure onset the plants should have 10 + 2 leaves with a minimum length of 15 cm (including the leaf stalk) (Fig. 5.9). The youngest leaf with this mini-mum length was marked and served as a reference leaf for sampling at the end of the expo-sure. The exposure of 4 plants per site was performed in the same frame employed for tobacco, but without shading. After an exposure time of 8 weeks, 8 leaves were harvested from each plant (reference leaf + 2 older + 5 younger) (Fig. 5.9). The leaves of the four plants from one site were pooled into a mixed sample. Pre-cleaned aluminium trays were used for packing the samples. These were dispatched directly to the central laboratory responsible for PAH analysis in cooled, thermally insulated containers by express courier. In this way there was a maximum of 24 hours between sampling and the arrival of the plant material in the laboratory. The tests were analysed for 20 PAH components in the laboratory of the Agricultural Research Institute (LUFA) in Karlsruhe. Measurement was performed after the eluate was purified several times by GC/MS using internal standards.

    25

  • EuroBionet Procedures for Bioindication of Air Pollution

    Fig. 5.9: Curly kale plant ready for exposure (left), exposure facility (right) and harvesting

    scheme (below).

    For a better comparability of the results with other studies, the sum values of those 16 sub-stances classified as "priority pollutants" according to the US-EPA (16 EPA-PAH), 12 low-volatile PAH (* in Table 5.4) as well as the 6 components listed in the German drinking water regulations from 1986 (TVO-PAH) were also provided in addition to the sum of all substances examined (Table 5.4). Since the measurement of the four highly volatile PAH does not as a rule produce reproducible results, only the values of the 12 less volatile PAH were used as a basis for comparative con-siderations of the PAH profiles in plants and for statistical tests. Background values describing general basic pollution levels were computed for each monitoring network, just as they were for the standardised grass cultures. Because of the influence of cli-matic factors and the differing PAH profiles amongst the various cities, calculation of a Euro-pean background value was not performed. Using the Spearman rank correlation coefficients it was checked whether significant relationships existed between individual PAH components or between heavy metal contents in Italian rye grass and PAH accumulation in curly kale. Cluster analyses were carried out for the monitoring networks in the individual cities. By comparing PAH profiles and forming ratios between source-specific components and generally stable compo-nents it was attempted to differentiate different locations according to the emission sources that predominated there.

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  • EuroBionet Procedures for Bioindication of Air Pollution

    Table 5.4: Details about the structure, volatility and carcinogenicity of the examined PAH com-ponents.

    PAH component Code Number of rings

    16 EPA-PAH *: low volatility

    6 TVO-PAH Carcinogenity1

    Naphthalene NAP 2 x Acenaphthylene ACY 3 x Acenaphthene ACE 3 x Fluorene FLU 3 x Phenanthrene PHE 3 x* + Anthracene ANT 3 x* + Fluoranthene FLUA 4 x* x + Pyrene PYR 4 x* + Benz(a)anthracene BaA 4 x* ++ Triphenylene+Chrysene CHR 4 x* ++ Benzo(b)fluoranthene BbF 5 x* x +++ Benzo(k)fluoranthene BkF 5 x* x +++ Benzo(e)pyrene BeP 5 + Benzo(a)pyrene BaP 5 x* x +++ Perylene PER 5 + Indeno(1.2.3.cd)pyrene IP 6 x* x Dibenz(ah)anthracene DahA 5 x* ++ Benzo(ghi)perylene BghiP 6 x* x ++ Anthanthrene AN 6 Coronene COR 7 1 classification according to KONTEYE (1988): + weakly, ++ moderately, +++ strongly carcino-genic 5.3.6 Bioindicator stations After selection of the sites for the local bioindicator stations, the construction of the exposure facilities was started from the spring of 2000. At each station one exposure system for standard-ised grass culture, one metal frame for poplar, one metal frame for tobacco and curly kale as well as one exposure container for Tradescantia were installed according to instructions pro-vided in the procedures manual (Fig. 5.10, Fig. 11.3 Annex I). Depending on local conditions the stations were either freely accessible or protected against unauthorised entry and vandalism by fencing. Placards with information about the project and the use of bioindicator plants in envi-ronmental monitoring were erected at most of the monitoring points. In all, up to 100 stations were in operation between the study years of 2000 - 2002. 5.4 Exposure programme The procedures manual contains time schedules for the cultivation and exposure of the bioindi-cator plants adapted to the regionally different climatic conditions (Table 11.4 Annex I). Cultiva-tion of the plants in the greenhouses was started as a rule in the beginning/middle of April (Ital-ian rye grass, poplar and tobacco) or the beginning/middle of August (curly kale). While the start and duration of the cultivation of the individual plant species varied depending on local climate and prevailing weather conditions, the outdoor exposure of the plants was mostly synchronised over the entire city network in order to ensure that the individual experimental series were com-

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  • EuroBionet Procedures for Bioindication of Air Pollution

    parable with one another. The exposure of grass cultures began from the 20th calendar week (CW), poplar and tobacco from the 22nd CW, and kale from the 42nd CW. The short-term ex-periments with Tradescantia could not be carried out simultaneously in all the participating cities for organisational reasons. In addition, the Tradescantia exposure had to be adapted to the most favourable weather conditions in order to avoid the disruptive influence of climatic factors. Fig. 5.10: Design of a bioindicator station with exposure facilities for grass cultures, tobacco and

    poplar in Klagenfurt (left) and an information placard at a station in Valencia. In all during the summer halves of 2000 and 2001, 5 four-week exposure periods with Italian rye grass, 8 two-week exposures with tobacco, 1 eight-week exposure with curly kale, one experi-ment with poplar over 14 weeks as well as 1-2 monitoring periods with Tradescantia were car-ried out (Table 5.5). In the cities that only joined the network later, fewer exposure series could be performed. In southern European cities additional experiments could be done during the au-tumn wherever favourable weather conditions permitted. In the summer half of 2002 only an abridged test programme could be realised with tobacco, Italian rye grass and Tradescantia. Table 5.5: Overview of the monitoring campaigns carried out between 2000 and 2002 with the

    Tradescantia micronucleus test. City 2000 2001 2002 Edinburgh 30./31.08. (12h) 01./02.08.* Sheffield 01./02.08.* Copenhagen 30./31.08. (12h) 08./09.08.* Dsseldorf 16./17.08.** 22./23.08.* 19./20.06. Nancy 23./24.08. 28./29.09. (12h) Klagenfurt 09./10.08. 18./19.07. Verona 06./07.09.

    27./28.06.*

    26./27.09. (12h) 24./25.07.

    Lyon 13./14.09. 13./14.09. Barcelona

    20./21.06.* 03/04.10.

    15./16.05.

    Valencia 17./18.10. 12./13.06. Glyfada 11./12.07.*

    * could not be evaluated for methodological reasons; ** only partly assessable; (12h) only a 12 hour exposure due to bad weather conditions.

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  • EuroBionet Procedures for Bioindication of Air Pollution

    5.5 Data evaluation and presentation of the results The decentralised data acquisition (assessment of leaf injuries in tobacco and poplar, growth measurements with poplar, sampling of Italian rye grass, curly kale, poplar and Tradescantia) by the local teams was followed by the centralised analysis and evaluation at the University of Hohenheim. This procedure was designed to guarantee a maximum possible standardisation of the bioindication procedures and to eliminate methodological errors as far as possible, so that a better comparability of the results from the different participating cities could be assured. After checking and validation of the raw data, the results of the individual exposure series were clas-sified for better intelligibility into four-five damage classes, as already described with the individ-ual procedures. The aggregated and subclassified data was then conveyed via e-mail to the local teams. The results were publicised as rapidly as possible on the project website as well as locally on the indicator columns of the green boxes (see chapter 7). 5.6 Quality assurance and quality control A strength of the entire bioindication programme lay in the extensive standardisation of all pro-cedural steps from plant cultivation, exposure at the monitoring sites, to data acquisition and processing. With this strict harmonisation it was intended to eliminate as far as possible any risk of methodological error that was considered to be particularly great taking into account the co-operation that had to be achieved between the differently trained and geographically removed working teams. In this way it was supposed to achieve a greater acceptance of the methodology amongst authorities, decision makers and the public in general. The processes of quality assurance and quality control therefore covered all aspects of the bio-indication procedure: All materials necessary for cultivation and exposure, such as the seed stocks, plant pots,

    substrates, fertilisers and exposure facilities, were procured centrally and dispatched to the participating cities. This basic equipment for the first two study years contained approx. 3 tonnes of material per monitoring network which was also complemented by replenishment deliveries in the springs of the following years.

    A high value was set particularly on the very detailed definition of procedures for cultivation and exposure of the indicator plants. The primary tools for this purpose were 1) the detailed and comprehensively illustrated procedures manual developed for the project in different languages, that was designed as a practical guide also for untrained workers (cf. 6.1), and 2) the practical demonstrations of the most important operational steps provided at the technical workshop held at the start of the project.

    In regularly dispatched circulars, important, currently pending steps relating to the cultiva-tion and experimental procedures were addressed and information was provided about any potential sources of errors.

    The compliance to the methodology by the local teams was checked by repeated on-site visits by colleagues from the coordination office, at which aspects relating to cultivation were clarified and procedures for recording pollutant effects were trained.

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  • EuroBionet Procedures for Bioindication of Air Pollution

    A further important contribution to quality assurance was the centralised chemical and mi-croscopic analysis of the plant samples by specialised laboratories under the supervision of the coordination team. In this way potential sources of error due to the use of different ana-lytical devices and procedures could be excluded and data scattering could be minimised.

    5.7 Comparative evaluation of air pollution and effect data In order to enable a comparative evaluation of air quality using air pollution and biological effect data within the local networks, some bioindicator stations were established in all participating cities in close proximity to automated air monitoring stations (Tab. 5.6). Measurement of air pol-lutants in the stations was performed using automated analysers according to the state-of-the-art. Concentrations of the most important pollutants were provided as hourly means. Depending on the technical equipping of the air monitoring stations and the bioindicator plants employed (tobacco and poplar as sensitive indicators for ozone, grass culture as an accumula-tive indicator for sulphur), emphasis was placed both on the comparative study of pollution with the air pollutants ozone and SO2 and the effects caused by them (leaf injury to tobacco as well as sulphur enrichment in grass cultures). For the period between May - July as well as April - September of the years 2000 and 2001, various cumulative exposure indices (AOT40+) were calculated according to the Directive 2002/3/EC of the European Parliament and of the Council of 12 February 2002 relating to ozone in ambient air that are of special importance for vegeta-tion protection (Table 5.7). Since the tobacco variety Bel-W3 already develops characteristic ozone-induced injuries at low ambient ozone levels (< 30ppb), the AOT20 was also calculated (accumulated ozone concen-tration above the threshold of 20 ppb, analogous to the AOT40 calculation). In addition, the fol-lowing parameters were computed: Average daily profile of ozone concentrations, Number of hourly means > 90 ppb (information threshold, health protection), Number of hourly means > 120 ppb (alert threshold, health protection). For evaluating pollution with SO2, the means of the SO2-concentrations for the individual four-week exposure periods of the grass cultures as well as the mean value over the entire exposure period per year were computed. Evaluation was performed according to the Council Directive relating to limit values for sulphur dioxide, nitrogen dioxide, and oxides of nitrogen, particulate + AOT40 (accumulated exposure over a threshold of 40 ppb) means the sum of the differences between hourly concentrations greater than 40 ppb and the base value 40 ppb over a given period using only the 1 hour values measured between 8:00 and 20:00 Central European Time each day. k AOT40 = hi (Ci 40 ppb) i=1 K = number of hours in the investigation period Ci= Mean ozone concentration (ppb) in hour i hi= 1 if Ci > 40 ppb, hi = 0 if Ci < 40 ppb

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  • EuroBionet Procedures for Bioindication of Air Pollution

    matter and lead in ambient air (1999/30/EC). There, compliance to a value of 7.5 ppb (~ 20 g SO2/m3) is required as an annual mean or a winter mean value for the protection of ecosystems. Table 5.6: Locations of the air pollution monitoring stations for O3 and SO2 and the correspond-

    ing bioindicator sites.

    Network Air monitoring station / bioindicator station Site

    Ozone

    SO2

    Edinburgh Princes Street / Donaldsons College urban x* x Sheffield City Center / Heeley Farm urban x* x

    Jaegersborg / ~ suburban x -- Jagtvej / Assistens Kirkegard urban x* x (2000)Copenhagen Lille Valby / ~ suburban x x (2000)Lrick / Strandbad Lrick suburban x* x Mrsenbroicher Ei/ ~ urban, street -- x Dsseldorf#

    Further Strae/ ~ suburban -- x Brabois / Airlor suburban x x Tomblaine / Mto France suburban x -- CUGN / Parc Ste. Marie urban x* x

    Nancy

    Fleville/ ~ suburban x x Hohenheim / ~ suburban x -- Hohenheim Plochingen / ~ suburban x x Koschatstr. / ~ urban x* x Klagenfurt Vlkermarktstrae / ~ urban -- x St. Just / ~ urban x x Croix Luizet / ~ urban x x Lyon Gerland / ~ urban x* x Torricelli / San Mattia suburban x x Verona#ZAI / Liceo Galilei suburban x* x Bellaterra / ~ suburban x -- Gracia / ~ urban x x Barcelona Sants / ~ urban x* x Avd. Aragn / ~ urban x x Prof. Beltrn Bguena / ~ urban x* x Pl. Granero/ ~ urban x x

    Valencia

    Catlico/ ~ urban x x x: Measured components. (2000): Only data from 2000 *: Data employed for the comparative evaluation of ozone pollution and ozone-induced plant injury. #: No O3 concentrations from urban stations available. Table 5.7: Target values and long-term objectives reg