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The influence of salinity and a number of nitrogen (N) and phosphorus (P) levels on growth and haemolytic activity of the harmful algal bloom (HAB) species Fibrocapsa japonica were studied simultaneously. The F. japonica strain studied had a euryhaline growth profile with an optimum growth rate at 26 psu. Measurements of the minimum cell quota of N and P revealed an optimum N:P ratio of 24.5 and a relatively high requirement of nutrients (q 0 N = 7.1 pmol cell ). These characteristics fit the habitat of this species, among which is the P-controlled eutrophied Dutch coastal waters. Salinity had a significant effect on the growth rate of F. japonica and on its haemolytic activity, even on the basis of equal cell volume. The highest haemolytic activity was found at 16 psu, the lowest salinity condition tested. The EC 50 values that were expressed on a per cell basis revealed that F. japonica had a high haemolytic activity when compared with other HAB species. Under N limitation, cells of F. japonica were less haemolytic than cells not limited for N, but this could be caused by a slight decrease in cell volume only. Remarkably, cells not limited for N appeared to form aggregates, which in combination with the elevated levels of haemolytic compounds per cell may result in obstruction of gills, causing fish kills during F. japonica blooms. So, despite the absence of brevetoxin in our strain of F. japonica, this strain may be lethal to fish due to its haemolytic activity.KEY WORDS: Cell quota · N:P ratio · Brevetoxins · Toxicity · Harmful algal blooms · Phytoplankton Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 37: 171-181, 20041972, 1989, Toriumi & Takano 1973; since 1990 ichthyotoxicity of F. japonica has been reported from the German coast and the Mediterranean Sea (European Commission 2003). The fish kills caused by F. japonica have been ascribed to the production of brevetoxins (Khan et al. 1996, Bridgers et al. 2004, reactive oxygen species (ROS;Oda et al. 1997), haemolytic compounds (Fu et al. 2004a,b), mucocyst ejection (de Boer et al. 2004b), and possibly combinations of these. Raphidophyte species are not always toxic to fish, as was shown for H. akashiwo and Chattonella spp. (Ishimatsu et al. 1996, Twiner & Trick 2000. Therefore, it is likely that different strains produce different toxic compounds or sometimes no toxins at all; this may be the case for F. japonica as well. The production of toxic compounds by raphidophytes could also be affected by environmental conditions such as light (Ono et al. 2000, Khan et al. 2001, Marshall et al. 2002, temperature (Twiner & Trick 2000, Khan et al. 2001, salinity (Haque & Onoue 2002a,b) and the presence of bacteria (Carrasquero-Verde 1999). For other HAB algae, it is known that algal species react differently to altered nutrient conditions with respect to toxin production (Granéli et al. 1998).So far, toxin production by raphidophytes has not been studied in relation to nutrient conditions. In this study,...

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The marine microalga Fibrocapsa japonica Toriumi and Takano (Raphidophyceae) produces haemolysins, neurotoxins and reactive oxygen species (ROS). To quantify potential effects of such bioactive compounds on surrounding organisms the marine bacterium Vibrio fischeri was exposed to F. japonica culture samples. Inhibition of V. fischeri 's natural luminescence, indicative of impaired metabolism, was related to the number of F. japonica cells added. The effect was fast, within 15 min. It was caused by one, possibly several, excreted substances that were less active after heating. Freezing of culture supernatant partly inactivated these substances, but ROS-scavenging enzymes had no effect. Light enhanced the V. fischeri luminescence inhibition in two ways. The direct effect of light on the action of F. japonica luminescence inhibiter(s) could be described by a saturation curve with maximum effect above 20 mmol photons m À2 s À1 . Light also had an indirect effect: biomass production, dependent on light availability, was closely related to the amount of inhibiting compound(s) produced by the alga. Algal growth rate, rather than its cell density, determined the bacterial luminescence inhibition per F. japonica cell, resulting in a 5-fold stronger inhibition at maximum growth rates compared to cells that barely grew during the stationary growth phase. The bioassay with F. japonica and V. fischeri has allowed quantification of the negative effects on bacteria in the microalgal microenvironment. The results presented here suggest that at favourable growth conditions F. japonica releases bioactive compounds that improve its competitive abilities.