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Blooms of the toxic red tide phytoplankton Heterosigma akashiwo (Raphidophyceae) are responsible for substantial losses within the aquaculture industry. The toxicological mechanisms of H.akashiwo blooms are complex and to date, heavily debated. One putative type of ichthyotoxin includes the production of reactive oxygen species (ROS) that could alter gill structure and function, resulting in asphyxiation. In this study, we investigated the potential of H.akashiwo to produce extra- cellular hydrogen peroxide, and have investigated which cellular processes are responsible for this production. Within all experiments, H.akashiwo produced substantial amounts of hydrogen peroxide (up to 7.6 pmol min -1 10 4 cells -1 ), resulting in extracellular concentrations of ~0.5 µmol l -1 H2O2. Measured rates of hydrogen peroxide production were directly proportional to cell density, but at higher cell densities, accuracy of H2O2 detection was reduced. Whereas light intensity did not alter H2O2 production, rates of production were stimulated when temperature was elevated. Hydrogen peroxide production was not only dependent on growth phase, but also was regulated by the avail- ability of iron in the medium. Reduction of total iron to 1 nmol l-1 enhanced the production of H2O2 relative to iron replete conditions (10 µmol l-1 iron). From this, we collectively conclude that produc- tion of extracellular H2O2 by H.akashiwo occurs through a metabolic pathway that is not directly linked to photosynthesis.

Possible physiological mechanisms for production of hydrogen peroxide by the ichthyotoxic flagellate Heterosigma akashiwo