UNIVERSITÄT HOHENHEIM Jürgen Franzaring, Andreas Klumpp and Heike Beismann

Establishing a European Standard for Biomonitoring The Grass Culture Method

Background Recently, the European Committee for Standardization (CEN) established within the Technical Committee Air Quality a working group entitled "Biomonitoring methods with flowering plants (CEN/TC 264/WG 30)

Background Two proposals were adopted by CEN TC 264 so far. WG 30 deals with: - Developing a European standard for the bioindication of ozone effects (exposure of BelW3 tobacco) - Developing a European standard for grass cultures WG 30 is chaired by Ludwig Radermacher (LANUV Essen), the secretariat is hold by KRdL (Heike Beismann) WG 31 will develop biomonitoring methods with mosses and lichens WG 31 is chaired by Chantal van Haluwyn (France), the secretariat is hold by AFNOR (Sébastien Louis-Rose)

Background Two meetings took place in 2008 (Düsseldorf and London) with participants from B, F, UK, SLO, GR, PL and D. Work is currently dealing with merging the French and German grass culture guidelines, i.e. AFNOR (2008): NF X43-901. Biomonitoring of air quality - Active biomonitoring of air quality by ray-grass: from crops to sample preparation. Biosurveillance de l'air - Biosurveillance active de la qualité de l'air à l'aide de ray-grass : des cultures à la préparation des échantillons. VDI (2003): VDI 3957 Part 2: Biological measuring techniques for the determination and evaluation of effects of air pollutants on plants (bioindication) - Method of standardised grass exposure. VDI/DIN Handbuch Reinhaltung der Luft, Band 1a, 28 pp. Richtlinie VDI 3957 Blatt 2: Biologische Messverfahren zur Ermittlung und Beurteilung der Wirkung von Luftverunreinigungen auf Pflanzen (Bioindikation) Verfahren der standardisierten Graskultur.

The method Pre-grown ryegrass (Lolium multiflorum cv. Lema) is exposed for periods of four weeks in self-watering containers at a standard height of 1.5 m at various locations around specific emitters or in a regional network. Thereafter the grass is harvested and chemical analyses are performed on the dried samples. This active biomonitoring approach enables the clear identification of spatio-temporal impacts of air pollution and can be widely used in environmental impact studies or by industries proving that they operate efficient emission reduction systems.

The method Studies using grass cultures for the biomonitoring of air pollution impacts have been performed in many countries, including Austria, Belgium, France, Germany, Netherlands and Spain. However, methods have not been fixed EU wide. In Germany, where the method was originally developed in the 1960s, investigations using standardised grass cultures were compulsive in local and regional air quality action plans (Luftreinhalteplan) in the 1980s and 1990s. While the method is not much used in such programs any more, it is still applied from time to time in polluter-specific studies. Such studies are usually paid for by the industry (polluters-pay-principle) instead of the public.

Evaluation of element concentrations in grass cultures Compare element concentrations to reference values defined in Animal Feed Directive EU, 2002. Directive 2002/32/EC on undesirable substances in animal feed. Official Journal, 30.05.2002, L 140/10-21. EU, 2005. Commission Directive 2005/87/EC amending Annex I to Directive 2002/32/EC on undesirable substances in animal feed as regards, lead, fluorine and cadmium. Official Journal, 06.12.2005, L 318/19-24. Foodstuff Directive EU, 2006. Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal, 20.12.2006, L 364/5-24. Other EU guidelines, in which biomonitoring is mentioned as a control tool EU, 2004. Directive 2004/107/EC (air quality criteria), COM (2003) 338 (health), COM (2004) 416 (Environment & Health Action Plan), Directive 2008/1/EC (IPPC), BREF: PJ/EIPPCB/WI_BREF_FINAL (BAT, waste incineration), BREF: JP/EIPPCB/MON_BREF_FINAL (Monitoring)

Users Countries: www.eurobionet.com D, UK, F, E, I, A, DK, GR Universität Hohenheim aklumpp@uni-hohenheim.de European bioindicator network (EuroBionet) S; As, Cd, Cr, Cu, Fe, Ni, Pb, Sb, V, Zn; Pt 2000-2002 Abstract: Within a European biomonitoring programme, Italian ryegrass (Lolium multiflorum Lam.) was employed as accumulative bioindicator of airborne trace elements (As, Cd, Cr, Cu, Fe, Ni, Pb, Sb, V, Zn) in urban agglomerations. Applying a highly standardised method, grass cultures were exposed for consecutive periods of four weeks each to ambient air at up to 100 sites in 11 cities during 2000 2002. Results of the 2001 exposure experiments revealed a clear differentiation of trace element pollution within and among local monitoring networks. Pollution was influenced particularly by traffic emissions. Especially Sb, Pb, Cr, Fe, and Cu exhibited a very uneven distribution within the municipal areas with strong accumulation in plants from traffic-exposed sites in the city centres and close to major roads, and moderate to low levels in plants exposed at suburban or rural sites. Accumulation of Ni and V was influenced by other emission sources. The biomonitoring sites located in Spanish city centres featured a much higher pollution load by trace elements than those in other cities of the network, confirming previously reported findings obtained by chemical analyses of dust deposition and aerosols. At some heavily trafficked sites, legal thresholds for Cu, Pb, and V contents in foodstuff and animal feed were reached or even surpassed. The study confirmed that the standardised grass exposure is a useful and reliable tool to monitor and to assess environmental levels of potentially toxic compounds of particulate matter. Reference: Klumpp, A., Ansel, W., Klumpp, G., Breuer, J., Vergne, P., Sanz, M. J., Rasmussen, S., Ro-Poulsen, H., Ribas Artola, A., Peñuelas, J., Shang, H., Garrec, J. P., Calatayud, V. (2008): Airborne trace element pollution in 11 European cities assessed by exposure of standardised ryegrass cultures. Atmospheric Environment, in press. Web address: www.eurobionet.com

Users Country: Germany Universität Hohenheim Franzari@uni-hohenheim.de Bioindication in the vicinity of a specific polluter HF 2005 Abstract: In order to study the pollution gradient in the vicinity of an HF producing factory, a biomonitoring programme was performed employing VDI standardised grass cultures. Specimen plants of Lolium multiflorum cv. Lema were exposed at 11 sites over five monthly periods and the biomass produced was used for subsequent F-analyses. Meteorological data from the study region confirmed that wind direction accounted for changes in the pollution pattern over periods of time. Fluoride concentrations in the grass cultures, however, were unrelated to temperature and precipitation sums during the exposures. The biomass production of the grass cultures proved to be unrelated to these parameters as well but, with the enhanced growth of the plants, the fluoride concentrations were lower due to the dilution of the element with higher biomass accumulation. Because the contribution of particulate fluoride was unknown, both the washed grass cultures and the washing water were analysed in order to determine the amount of external fluoride. Washing reduced the fluoride concentrations by 22% on average, indicating that most of the element was internal fluoride stemming from stomatal uptake. Larger amounts of fluoride, however, could be washed off from grass cultures exposed at sites close to the factory indicating that dust emissions played a greater role at these locations. Because particulate emissions were supposed to arise from CaF2 and the waste-product anhydrite, grass cultures were also analysed for calcium and sulphur. While calcium concentrations were generally high but unrelated to fluoride, sulphur concentrations showed a slight relationship to the F concentrations determined in the unwashed plants. Latter findings indicate the co-deposition of the two elements as surface bound, external loads, but bioindication could not clarify to what extent both elements were partitioned in the gas-toparticle phase. We therefore recommend using the grass culture method in air quality programmes to identify any exceedances of European feeding stuff standards in the vicinity of large emitters. Reference: Franzaring, J., Klumpp, A., Fangmeier, A. (2007): Active biomonitoring of airborne fluoride near an HF producing factory using standardised grass cultures. Atmospheric Environment 41: 4828 4840.

Users Country: Germany Bioindication in the vicinity of a specific polluter HF 2005 Universität Hohenheim Franzari@uni-hohenheim.de Abstract: In order to study the pollution gradient in the vicinity of an HF producing factory, a biomonitoring programme was performed employing VDI standardised grass cultures. Specimen plants of Lolium multiflorum cv. Lema were exposed at 11 sites over five monthly periods and the biomass produced was used for subsequent F-analyses. Meteorological data from the study region confirmed that wind direction accounted for changes in the pollution pattern over periods of time. Fluoride concentrations in the grass cultures, however, were unrelated to temperature and precipitation sums during the exposures. The biomass production of the grass cultures proved to be unrelated to these parameters as well but, with the enhanced growth of the plants, the fluoride concentrations were lower due to the dilution of the element with higher biomass accumulation. Because the contribution of particulate fluoride was unknown, both the washed grass cultures and the washing water were analysed in order to determine the amount of external fluoride. Washing reduced the fluoride concentrations by 22% on average, indicating that most of the element was internal fluoride stemming from stomatal uptake. Larger amounts of fluoride, however, could be washed off from grass cultures exposed at sites close to the factory indicating that dust emissions played a greater role at these locations. Because particulate emissions were supposed to arise from CaF2 and the waste-product anhydrite, grass cultures were also analysed for calcium and sulphur. While calcium concentrations were generally high but unrelated to fluoride, sulphur concentrations showed a slight relationship to the F concentrations determined in the unwashed plants. Latter findings indicate the co-deposition of the two elements as surface bound, external loads, but bioindication could not clarify to what extent both elements were partitioned in the gas-toparticle phase. We therefore recommend using the grass culture method in air quality programmes to identify any exceedances of European feeding stuff standards in the vicinity of large emitters. Reference: Franzaring, J., Klumpp, A., Fangmeier, A. (2007): Active biomonitoring of airborne fluoride near an HF producing factory using standardised grass cultures. Atmospheric Environment 41: 4828 4840. BACKGROUND THE METHOD USERS OUTLOOK

Users Country: Germany www.lubw.de Bioindication at motorways Noble metal emissions from catalytic converters 2003-2008 Standardised grass cultures were used to identify trends of noble metals (Platinum, Rhodium and Palladium) in the vicinity of motorways. References: LUBW (2003): Signale aus der Natur. 20 Jahre biologische Umweltbeobachtung. http://www.lubw.badenwuerttemberg.de/servlet/is/14556/signale_natur.pdf?command=downloadco ntent&filename=signale_natur.pdf http://www.eobc.eu/journal/u232-dbw03-de.pdf http://www.lubw.baden-wuerttemberg.de/servlet/is/38938/

Users Country: Germany Bioindication at Various environmental specific sources pollutants Dr. R. Kostka-Rick http://www.biomonitoring.de/ d/aktuell1999.html Standardised Grass Culture (VDI 3957 / 2) for assessing the impact by inorganic sulfur, fluoride, chloride and metals, and also polychlorinated dioxins and furans (PCDD/PCDF). One of the major strengths are the high resolution in time and space, which can be achieved by this method. Assessment of air pollution by traffic-related metals near a motorway using bioindicators. In order to assess the recent impact by traffic-related metals close to a busy motorway and to evaluate the efficiency of standardized biomonitoring procedures to survey pollutants typically emitted from road traffic, a biomonitoring study is being conducted since 1997 at the Autobahn A 8 Stuttgart-Ulm, Baden-Württemberg, Germany, 75 000 cars/day. In lichens (Hypogymnia physodes, VDI 3799 Part 2) exposedfor 1 year, and in standardized grass exposures (Lolium multiflorum, VDI 3957 Part 2) at both sides of the 6-lane motorway, contents of antimony (Sb), platinum (Pt), copper (Cu) and lead (Pb) are higher at site North downwind from the motorway (prevailing winds from southwest) compared to site South, but not so for cadmium (Cd). Lichens accumulate higher metal concentrations (up to 35fold for Pb, 10fold for Pt) than grass. Decreasing metal contents over time are found in both bioindicators for Pb and Cd, but increasing trends for Sb. For Sb and Pt, metal contents are much higher (about 10-times) close to the motorway than at reference sites (rural background) or in grass grown in filtered air. In contrast to findings from former years, Pb content is only slightly elevated and is most likely attributable to resuspended lead deposits from the past rather than to recent emissions. Antimony has taken over the role as a typical tracer metal for traffic pollution from lead. The use of biomonitoring plants using standardized procedures provides reliable results and allows conclusive interpretation in case robust reference data are available and quality assurance is strictly implemented in instrumental trace analysis. Reference: http://www.biomonitoring.com/e/biom_aktiv.html Kostka-Rick, R. (2008): Standardisierte Biomonitoringverfahren in der langjährigen Routineüberwachung einer Sonderabfalldeponie - Teil 1: Standardisierte Graskultur. Gefahrstoffe 6: 235-243. Nobel, W., Kostka-Rick, R., Bartholmeß, H. (2008): Assessment of air pollution by traffic-related metals near a motorway using bioindicators. Gefahrstoffe 6: 245-250.

Users Country: Germany Bayerisches Landesamt für Umwelt, Bürgermeister- Ulrich-Straße 160 86179 Augsburg Dr. Ludwig Peichl Aktives Biomonitoring mit Weidelgras Bioindication at specific sources Various environmental pollutants Since 1990s Die direkte Wirkung von Schwermetall-Einträgen auf Pflanzen wird durch aktives Biomonitoring nach der VDI- Richtlinie 3957 Blatt 2 mit dem Welschen Weidelgras (Lolium multiflorum) untersucht. Hierzu wird eine "Standardisierte Graskultur" mit Welschem Weidelgras mit einer automatischen Topfbewässerung vier Wochen lang im Freiland exponiert (siehe Abb.). Die große Oberfläche der Grasbüschel wirkt wie eine Bürste, an der die luftgetragenen Schadstoffe hängen bleiben. Untersucht werden an den acht Dauerbeobachtungsstationen des LfU die Anreicherungen der Metalle Aluminium, Arsen, Wismut, Cadmium, Kobalt, Chrom, Kupfer, Eisen, Quecksilber, Mangan, Molybdän, Nickel, Blei, Antimon, Titan, Thallium, Vanadium und Zink. Reference: http://www.lfu.bayern.de/themenuebergreifend/fachinformationen/umweltmon itoring_schwermetalle/aktives_bm_weidelgras/index.htm

Users Country: Germany Landesumweltamt Nordrhein-Westfalen, Postfach 10 23 63, 45023 Essen Dr. Radermacher Bioindication at Various environmental specific sources pollutants Das Verfahren der standardisierten Graskultur wird in der VDI-Richtlinie 3957 Blatt 2 beschrieben. Als Akzeptor wird die Grasart Lolium multiflorum verwendet, die luftverunreinigende Stoffe anreichert. Das Verfahren der Standardisierten Graskultur ist neben der Beprobung von Nahrungs- und Futterpflanzen als bedeutendes Wirkungsmessverfahren anzusehen. Es erlaubt, mit vertretbarem Aufwand Schadstoffgehalte in den Graskulturen mit hoher zeitlicher und räumlicher Auflösung zu erfassen. Die Ergebnisse liefern u.a. einen direkten Wirkungsbezug im Hinblick auf die Kontamination von Weideaufwuchs und ermöglichen, z.b. unter Einbeziehung zuvor experimentell abgeleiteter Transferfaktoren, eine Kontamination von Lebensmitteln tierischer Herkunft der Größenordnung nach abzuschätzen bzw. vorsorgend Nutzungsempfehlungen auszusprechen. Since 1960s Reference: http://www.lanuv.nrw.de/veroeffentlichungen/handbhandzettel/standardgrask ultur.pdf

Users Country: µg g -1 Pb (DM) Germany Landesumweltamt Nordrhein-Westfalen, Postfach 10 23 63, 45023 Essen Dr. Radermacher Das Verfahren der standardisierten Graskultur wird in der VDI-Richtlinie 3957 Blatt 2 beschrieben. Als Akzeptor wird die Grasart Lolium multiflorum verwendet, die luftverunreinigende Stoffe anreichert. Das Verfahren der Standardisierten Graskultur ist neben der Beprobung von Nahrungs- und Futterpflanzen als bedeutendes Wirkungsmessverfahren anzusehen. Es erlaubt, mit vertretbarem Aufwand Schadstoffgehalte in den Graskulturen mit hoher zeitlicher und räumlicher Auflösung zu erfassen. Die Ergebnisse liefern u.a. einen direkten Wirkungsbezug im Hinblick auf die Kontamination von Weideaufwuchs und ermöglichen, z.b. unter Einbeziehung zuvor experimentell abgeleiteter Ruhr Transferfaktoren, eine Kontamination von Lebensmitteln tierischer Herkunft der Größenordnung nach abzuschätzen bzw. vorsorgend Nutzungsempfehlungen auszusprechen. Reference: http://www.lanuv.nrw.de/veroeffentlichungen/handbhandzettel/standardgrask ultur.pdf Bioindication at Various environmental specific sources pollutants Lead concentrations in grass cultures Rural site Since 1960s

Users Country: Austria Dr. Richard Öhlinger AGES GmbH, CC Cluster Chemie Linz, Wieningerstr. 8, 4020 Linz richard.oehlinger@ages.at Bioindication around industrial emission sources and in urban areas S, F, heavy metals, PAH Since 1990s Aktives und passives Biomonitoring Messung von Luftschadstoffen mit pflanzlichen Bioindikatoren: Der Eintrag von Luftschadstoffen auf die Vegetation und deren Einbindung in die Nahrungskette z.b. als Tierfutter haben große Bedeutung auf dem Gebiet der praktischen Luftreinhaltung. Mittels aktivem und passivem Biomonitoring können kostengünstig schutzzielbezogene Angaben über eine etwaige Gefährdung oder Beeinträchtigung gegeben werden. Als Schutzziele gelten Mensch, Tier, Pflanze und Boden. Angewendete Biomonitoringsysteme zur Schadstoffmessung und Bewertung aus landwirtschaftlicher Sicht: Aktives Biomonitoring mit der standardisierten Graskultur: Welsches Weidelgras wird ins Untersuchungsgebiet gebracht, dort auf einer Trägervorrichtung exponiert und nach definierten Perioden der Aufwuchs auf Schadstoffe untersucht. Passives Biomonitoring mit Wiesengras und Baumblätter: Die Pflanzenproben (Wiesengras, Baumblätter) werden direkt vom Untersuchungsgebiet entnommen und auf Schadstoffe untersucht. Anwendungsbeispiele: Überwachung von diversen Industrieanlagen, Einsatz be Umweltverträglichkeits-prüfungen (z.b. Straßenbau, Errichtung von potentiellen Emittenten) References: http://www.ages.at/ernaehrungssicherheit/rueckstaendekontaminanten/biomonitoring/

Users Country: Spain Ecología, Facultad de Biología, Univ. Santiago de Compostela, 15706 Santiago de Compostela, Spain Bioindication around industrial emission sources and in urban areas F 2000 The aim of the present study was to develop a standardised protocol for the use of Lolium perenne cultures to biomonitor the surroundings of an aluminium smelter. Five ecological stations, which corresponded to air quality network monitoring sites pertaining to the smelter, were selected for study. Cultures of L. perenne (one at each site) were exposed for approximately 28 days, at different times between August 1999 and October 2000. At the end of the exposure periods, the fluoride concentrations in the cultures were determined. The concentrations of fluoride determined in the L. perenne cultures varied from 4.32 µg g 1 to 912.22 µg g 1 and followed a similar trend to concentrations of atmospheric fluoride. However, a wider range of fluoride concentration was observed in the L. perenne cultures than in the air surrounding the smelter. The background levels of fluoride in L. perenne cultures were also calculated, thereby allowing classification of fluoride air pollution in the area under study. References: Rey-Asensio, A., Carballeira, A. (2007): Lolium perenne as a biomonitor of atmospheric levels of fluoride. Environment International 33: 583-588.

Users Country: Brazil Instituto de Botânica São Paulo, Brazil Universität Hohenheim Bioindication around industrial emission S, F, heavy metals, sources and in urban PAH areas Abstract: The Atlantic Rain Forest near the industrial complex of Cubatão, south-eastern Brazil, suffers from the impact of severe air pollution. Gaseous and particulate fluorides which are emitted by fertiliser plants are assumed to play an important role in the forest decline. In order to obtain information on the air pollution situation and the spatial and temporal distribution of pollution effects, standardised bioindication methods from temperate climates were introduced and applied during a three-year study. A sensitive Gladiolus cultivar developed typical fluoride-induced leaf lesions in areas affected by airborne fluorides, and Lolium multiflorum cultures accumulated large quantities of fluoride. Highly significant linear regression was found between foliar fluoride contents of both species as well as between leaf damage and fluoride accumulation in Gladiolus plants. By using these indicator species, four different areas of the region were identified with respect to vegetation risk by airborne fluorides. Since 1990 Abstract: This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in São Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment. References: Klumpp, A., Domingos, M., Klumpp, G. (1996): Assessment of the vegetation risk by fluoride emissions from fertiliser industries at Cubatão, Brazil. Science of the Total Environment 192, 219-228. Sandrin, C. Z., Figueiredo-Ribeiro, R. C. L., Machado de Carvalho, M. A., Delitti, W. B. C., Domingos, M. (2008): Subtropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema. Environmental Pollution, in press.

Interested people should contact For participating in WG 30: For participating in WG 30 you will have to be nominated by your national standardisation institution, for more information contact Dr. Heike Beismann For performing own studies using the standardised grass culture: Technical support by the University of Hohenheim, Dr. Jürgen Franzaring or Dr. Andreas Klumpp For financial support: Your national, regional and local Environmental Agencies and of course, also the potential pollutant emitters.