1. Introduction In Azores, the young fronds usually emerge naturally from the soil between the end of January and the beginning of February. If the fronds are cut as a prevention or control measure, the bracken can grow up any time along the year, because the frond are cut but the rhizome still stay into the soil. A study carried out during 2004 in a FCT project, demonstrated the highest concentration of PTA appears at the beginning of the growth season, generally in spring with the first emerging fronds, and decrease along the year (Leitão, et al., unpublished) as the results obtained by Rasmussen and Hansen (2002) in Denmark, and studies made in New Zealand by Smith et al. (1989). The aim of this research was the determination of the geographic variation in ptaquiloside content of bracken fronds which grow in pastures or pasture margins. The study was carried out during July 2005, in the dry season, when cows do not have enough grass to eat and the bracken fern is one of the last green plants in the fields, fact that increase the possibility of bracken ingestion by the cattle (Pinto, 2001). It is also the season when bracken reaches its highest development in terms of biomass. Discussion and Conclusions Ptaquiloside mean content varied from 38 to 1526 µg/g dry matter, with significant differences between sites, with a maximum of 2872 µg/g and a global mean of 348 µg/g (standard deviation, 552 µg/g). A considerable variation was also found within each stand. In a study carried out in Denmark the amount of ptaquiloside varied from 213 to 2145 µg/g, with significant differences between sites (Rasmussen et al., 2003a). In general, sub-sites were quite similar, with the exception of sites with high ptaquiloside content, where variation between sub-sites was higher. Our results were similar to those obtained by Leitão (2006) in a seasonal study of ptaquiloside content in São Miguel, where values between 700 and 300 µg/g were found in July/August. No relationship was detected with altitude or soil type, instead other studies confirmed a reduction in ptaquiloside content for lower altitudes, particularly in caudatum variety (Alonso-Amelot et al, 1999). Ptaquiloside loads per sample varied from 0 to more than 500 mg/m2, common values for São Miguel (Leitão, 2006), but high values when comparing with a study carried out in Denmark, where the load varied from 10 to 260 mg/m2 (Rasmussen et al., 2003b). The wide distribution of P. aquilinum and the ptaquiloside concentrations and loads detected are in agreement with the frequent occurrence of Bovine Enzootic Haematuria in São Miguel Island. Methods Study Sites. We sampled 20 bracken stands mainly in pastures and pasture margins, distributed throughout São Miguel Island, from 26 to 584 m of altitude. The sites were selected with GIS, using Arcview 3.2 (ESRI, 1992-1999) trying to find the most different conditions (slope, aspect and altitude) around the island. At each site we sampled three sub-sites with a few meters separation. Sampling. At each sub-site, the number of fronds per square meter was counted, 10 fronds were measured (stipe length and frond height) and the phenological stage recorded. Four bracken fronds were collected at each sub-site to determine biomass and ptaquiloside content. A kilogram of soil was sampled at each sub-site and sent to Soil Analysis Laboratory (Universidade dos Açores, DCA). Once in the laboratory, bracken samples were dried (50ºC during 5 days), milled and stored at – 20ºC till analysis. Laboratory analysis. Ptaquiloside analyses were made in the Department of Natural Sciencies of The Royal Veterinary and Agricultural University in Copenhagen. Ptaquiloside from dried samples of fronds was extracted with deionized water by shaking. The suspension was centrifuged before an aliquot was passed through a 1.0x10 cm I.D.glass Econo-Column® (BIO-RAD, New York, USA) drypacked with Polyamide 6 resin (Fluka, Steinheim, Switzerland). Ptaquiloside was converted to Pterosin B by base-acid treatment following the method of Agnew and Lauren (1991), and Pterosin B determined by UV-absorption using a Merck Hitachi HPLC system (L-4200 UV-VIS Detector; 655A-40 Auto-Sampler; D-6000A Interface; L- 6200 Intelligent Pump) equipped with Merck LiChroCART® column (125x4 mm) packed with LiChrospher® 100 RP-8, 4μm. The separations were performed at isocratic mode with a water-acetonitril eluent (77:23, volume:volume).The column was flushed after each analysis, and the total time of analysis was 20 min, with a retention time for Pterosin B of approx. 7 min. Sample injection-volumes of 30-100μl were used for analysis, and all samples were analysed in duplicate. The limit of detection was 0.25 ppm for fronds. Pterosin B was quantified by using external standards made from pure ptaquiloside. Statistical analysis. A one-way analysis of variance (ANOVA) was used to calculate the variation within and between the 20 sites. P. aquilinum sampling sites at São Miguel Island Geographic variation of ptaquiloside content in bracken from São Miguel Island (Azores) Illas 1, X., Silva 1, L., & Rasmussen 2, L. H. 1 Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Portugal; 2 Centre for Advanced Food Studies, KVL; Rolighedsvej 30, 1; DK-1958 Frederiksberg C, Denmark Aknowledgements This work is part of a project supported by FCT, POCTI AGG/48737/2002. We thank Carlos Pinto and Manuel Leitão for their valuable cooperation. References Alonso-Amelot, M. E.,Calcagno, M. P., Pérez-Inojosa, M. (1999) Growth and selective defensive potencial in relation to altitude in neotropical Pteridium aquilinum var. caudatum. In: Taylor, J.A. and Smith, R. T., (Eds.) Bracken Fern: Toxicity, Biology and Control. Proceedings of the international bracken group conference, Manchester 1999. IBG, Aberystwyth: 43-47. Smith, B.L., Lauren, D.R., Prakash, A.S. (1999). Bracken Fern (Pteridium) : Toxicity in Animal and Human Health. In: Bracken Fern: Toxicity, Biology and Control, pp.76-85. Agnew, M.P., Lauren, D.R. (1991). Determination of Ptaquiloside in Bracken Fern (Pteridium Esculentum). In: Journal of Chromatography 538 (2): 462-468. Smith, B.L., Lauren, D.R., Embling, P.P., Agnew, M.P. (1989). Ptaquiloside in Australian and New Zealand ferns as a cause of neoplasia. In: Bracken Biology and Management, pp. 241-246. Pinto, A.C., Louzã, V., Almeida, M., Melo, Y., Vaz, M.C., Peleteiro, Smith, B.L. (2001). Epidemiological characterisation of urinary bladder tumours occurence in São Miguel Island (Azores) dairy cattle population. Revista Portuguesa de Ciencias Veterinárias 11-19. Rasmussen, L.H., Jensen, L.S., Hansen, H.C.B. (2003a). Distribution of the carcinogenic terpene ptaquiloside in Bracken fronds, rhizomes (Pteridium aquilinum), and litter in Denmark. Journal of Chemical Ecology, 29: 771-778. Rasmussen, L.H., Kroghsbo, Stine, Frisvad, J.C., Hansen, H.C.B. (2003b) Ocurrence of the carcinogenic Bracken constituent ptaquiloside in fronds, topsoils and organic soil layers in Denmark. Chemosphere 51 (2003) 117-127. Dairy cow in direct contact with bracken fern Results Variation of Ptaquiloside content with altitude. No relationship was found Variation of Ptaquiloside content with frond height. No relationship was found. Variation of Ptaquiloside content with frond density. No relationship was found. VariationSum of SquaresdfMean SquareF Between Groups18492825,119973306,65,8 Within Groups15602699,893167771,0p Total34095524,9112 3,0E-09 Variation of Ptaquiloside load with altitude. No relationship was found. Bracken fern, Pteridium aquilinum (L.) Kuhn is a common plant in the Azores Islands, where it is found in different habitats, but mainly in marginal areas and pastures. The carcinogenic potential of some of its compounds, namely ptaquiloside, are known. Dairy farm industry is an important part of the Azorean economy, affected by a relatively high incidence of urinary bladder tumours in cattle.