Real-time detection of reactive oxygen species generation by marine phytoplankton using flow injection–chemiluminescence

Angela Milne, Margaret S. Davey, Paul J. Worsfold, Eric P. Achterberg, Alison R. Taylor

Limnol. Oceanogr. Methods 7:706-715 (2009) | DOI: 10.4319/lom.2009.7.706

ABSTRACT: Little is known about the biological production of reactive oxygen species (ROS) such as H2O2 and O2- in the surface waters of the oceans. This article describes two flow injection–chemiluminescence methods to measure H2O2 and O2- production by marine diatoms. These methods each incorporated a polycarbonate filter unit in which live cells were immobilized, followed by downstream determination of H2O2 and O2- in the cell filtrate. The sample analysis rate was ~1 min–1 for H2O2 and continuous for O2- during assays with diatom cells. The respective detection limits for each system were 1.9 nM (H2O2) and 1.6 nM (O2-). An initial examination of the effect of changing light intensity showed that a rapid light-induced production of both O2- and H2O2 by T. weissflogii cells could be readily detected. Moreover, this production was proportional to the biomass present on the flushed filter. These methods enable the monitoring of real-time fluctuations of biological ROS production in response to changing environmental conditions, and therefore facilitate analysis of the biotic component of ROS production and the subsequent impacts on chemical speciation of nutrients and trace metals in aquatic ecosystems.