Toxic effects of Heterosigma akashiwo do not appear to be mediated by hydrogen peroxide

The ichthyotoxic red tide organism Heterosigma akashiwo (Raphidophyceae) has been associated with fish kill events within the aquaculture industry for many years. The precise toxicological mechanism involved in these fish kills is unclear; however, much research attention has focused on the production of reactive oxygen species (ROS) by these toxic algae. In this study, we investigated the production of hydrogen peroxide (H2O2) by isolates of H. akashiwo and the nontoxic chlorophyte Tetraselmis apiculata. Subsequently, we tested those concentrations of H2O2 on vertebrate cell lines and the invertebrate Artemia salina (brine shrimp) to investigate mortality. Net production rates for the H. akashiwo isolates ranged from 0.46 to 7.89 pmol H2O2 min−1 (104 cells)−1 while obtaining maximum concentrations between 0.14 and 0.91 µM H2O2. Conversely, T. apiculata produced only 0.03 pmol H2O2 min±1 (104 cells with a maximum level on 0.04 µM. However, toxic effects on UMRߚ106 and HEKߚ293 cells were only induced by acute and protracted exposure to concentrations of H2O2 >= 0.1 mM. Additionally, significant mortality of A. salina in the presence or absence of ferric and ferrous iron was induced by H2O2 levels >= 1 mM. Iron is a redox metal that reduces H2O2 to hydroxy radicals. These data collectively indicate that production of H2O2 by multiple isolates of H. akashiwo is orders of magnitude less than that required for mortality of either the vertebrate cell lines or the invertebrate A. salina. Other nonichthyotoxic roles for extracellular ROS are proposed.