Detection of intracellular Reactive Oxygen Species (ROS) by flow cytometry on microalgae: studies on laboratory monoalgal and natural mixed cultures

Abstract

Levels of intracellular reactive oxygen species (ROS), such as H2O2 and oxygen radicals, increase in stress conditions. Thus, they can be considered as a biomarker for toxic stress in marine microalgal populations. Three specific fluorochromes for intracellular ROS ((Bis-(1,3-Dibutylbarbituric Acid)TrimethineOxonol (DiBAC4(3)); Dihidrorhodamine 123 (DHR123) andHydroetidine (HE)) as well as a fluorochrome for cellular viability (Fluorescein diacetate(FDA)) were assayed on monolagal cultures of the diatom Phaeodactylumtricornutum under copper stress in order to select a suitable fluorochrome for detecting intracellular ROS. Among all fluorochromes assayed, DiBAC4(3) offered a better response than the others. Then, wild populations obtained from natural locations (sampling stations at the Guadalete River estuary) were exposed to sub-lethal copper concentrations and compared with controls free of added metal. A decrease in the fluorescent DiBAC4(3) signal for FL1 (515-545 nm), which is associated to less cellular stress, was found for the samples with higher turbidity. The hypothesis is that copper adsorbs to silt-sized suspended sediment and it is not bioavailable for growing microalgae, decreasing thus metal toxicity.