Cell-by-cell estimation of PAH sorption and subsequent toxicity in marine phytoplankton

Polycyclic Aromatic Hydrocarbons (PAHs) have elicited increasing concern due to their ubiquitous occurrence in coastal marine environments and resultant toxicity in organisms. Due to their lipophilic nature, PAHs tend to accumulate in phytoplankton cells and thus subsequently transfer to other compartments of the marine ecosystem. The intrinsic fluorescence properties of PAHs in the ultraviolet (UV)/blue spectral range have recently been exploited to investigate their uptake modes, localization, and aggregation in various biological tissues. Here, we quantitatively evaluate the sorption of two model PAHs (phenanthrene and pyrene) in three marine phytoplankton species (Chaetoceros tenuissimus, Thalassiosira sp. and Proteomonas sp.) using a combined approach of UV excitation flow cytometry and fluorescence microscopy. Over a 48-h exposure to a gradient of PAHs, Thalassiosira sp. showed the highest proportion of PAH-sorbed cells (29% and 97% of total abundance for phenanthrene and pyrene, respectively), which may be attributed to its relatively high total lipid content (33.87 percent dry weight). Moreover, cell-specific pulse amplitude modulation (PAM) microscope fluorometry revealed that PAH sorption significantly reduced the photosynthetic quantum efficiency (F_v/F_m) of individual phytoplankton cells. We describe a rapid and precise hybrid method for the detection of sorption of PAHs on phytoplankton cells. Our results emphasize the ecologically relevant sub-lethal effects of PAHs in phytoplankton at the cellular level, even at concentrations where no growth inhibition was apparent. This work is the first study to address the cell-specific impacts of fluorescent toxicants in a more relevant toxicant-sorbed subpopulation; these cell-specific impacts have to date been unidentified in traditional population-based phytoplankton toxicity assays.