Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae

Background: Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phyto plankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alteratio ns, so we investigated how copper disturbs those cells. Methods: Cytotoxic effects of sublethal and lethal copper concentrations ra nging from 4.2 nM (Control condition) to 3.13 muM estimated labile copper w ere studied in batch cultures of Amphidinium carterae. Cell morphology, mot ility, autofluorescence, and fluorescence diacetate (FDA)-dependent fluores cence generation were evaluated by flow cytometry (FCM) and microscopy. Results: Exposure of A. carterae to toxic levels of copper impaired cell mo bility, delayed cell proliferation, led to increased green autofluorescence , and at 3.13 muM labile copper also induced encystment and death. Chloroph yll fluorescence, however, was not affected. Kinetic FCM assay of FDA-depen dent fluorescence generation showed a dose-dependent enhancement of fluores cein fluorescence immediately after copper addition and in cultures with su stained exposure to this toxicant. Conclusions: Our data suggest that copper toxicity occurs quickly at the me mbrane level in relation to oxidative stress generation. Based on fluoresce nce kinetic studies, the Na+/H+ antiporter seemed to be affected by copper, thereby affecting intracellular pH. (C) 2001 Wiley-Liss, Inc.