Chemical characterisation of three haemolytic compounds from the microalgal species Fibrocapsa japonica (Raphidophyceae) Meng Fu a, * , Albert Koulman b , Marion van Rijssel c , Arne Lu¨tzen d , M. Karin de Boer c , Monika R. Tyl c , Gerd Liebezeit a a Research Centre Terramare, Schleusenstasse 1, 26382 Wilhelmshaven, Germany b Department of Pharmaceutical Biology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningnen, The Netherlands c Department of Marine Biology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands d Department of Chemistry, Carl von Ossietzky University Oldenburg, P.O. Box 2503, 26111 Oldenburg, Germany Received 2 June 2003 Abstract The molecular structures of the three main haemolytic compounds (Fj1, Fj2 and Fj3) isolated from the ichthyotoxic microalgal species Fibrocapsa japonica have been investigated by NMR, LC – ESI – MS, ESI – MS – MS, IR, GC – MS and GC – HRMS methods. They are polyunsaturated fatty acids which we identified as: 6,9,12,15-octadecatetraenoic acid (OTA, C18:4v3), 5,8,11,14,17-eicosapentaenoic acid (EPA, C20:5v3) and 5,8,11,14-eicosatetraenoic acid (arachidonic acid AA, C20:4v6). The identity of the latter two was confirmed on the basis of commercial standards (C20:5v3 and C20:4v6). Both displayed the same spectroscopic and chromatographic characteristics as Fj2 and Fj3 and had a similar strong haemolytic effect. We propose that when F. japonica cells accumulate in fish gills during blooms these compounds could be the cause of icthyotoxicity. q 2003 Published by Elsevier Ltd. Keywords: Fibrocapsa japonica; Haemolytic compounds; 6,9,12,15-Octadecatetraenoic acid (OTA, C18:4v3); 5,8,11,14,17-Eicosapentaenoic acid (EPA, C20:5v3); 5,8,11,14-Eicosatetraenoic acid (arachidonic acid AA, C20:4v6); High performance liquid chromatography; Nuclear magnetic resonance; Infrared spectroscopy; Liquid chromatography –electrospray ionisation –mass spectrometry; Gas chromatography –mass spectrometry; Gas chromatography–high resolution mass spectrometry; Erythrocyte lysis assay 1. Introduction In the class Raphidophytes, the species Fibrocapsa japonica, Heterosigma akashiwo and Chattonella spp. have been reported to produce red-tide blooms that induce mass mortality of cultured fish in Southeast Asian coastal waters (Toriumi and Takano, 1973; Nakamura and Watanabe, 1983). The species F. japonica has caused significant damage to coastal fisheries in Japan (Toriumi and Takano, 1973; Yoshimatsu, 1987; Montani et al., 1995). From the beginning of the 1990s F. japonica has been observed in European coastal waters (Billard, 1992; Vrieling et al., 1995; Rademaker et al., 1998). Both molecular and physiological data on F. japonica strains suggest a recent range expansion of this species (Kooistra et al., 2001; De Boer et al., 2002). The mechanism behind the toxicity of F. japonica is still under debate. Oda and co-workers (1997) showed the production of reactive oxygen species by a Japanese F. japonica strain that could damage fish gills. Polyether neurotoxins were found in a European strain (W420) and identified as brevetoxins on the basis of their chromato- graphic behaviour in HPLC (Khan et al., 1996), and brevetoxins were also recently detected using a competitive ELISA technique in American strains (Bridgers et al., 2002). 0041-0101/$ - see front matter q 2003 Published by Elsevier Ltd. doi:10.1016/j.toxicon.2003.09.012 Toxicon 43 (2004) 355–363 www.elsevier.com/locate/toxicon * Corresponding author. Fax: þ 49-4421-944199. E-mail addresses: meng.fu@terramare.de (M. Fu); gerd. liebezeit@terramare.de (G. Liebezeit).