Impact of inorganic mercury on carotenoids in freshwater algae: Insights from single-cell resonance Raman spectroscopy

The influence of inorganic mercury (Hg(II)) exposure on photosynthetic microorganisms and their pigments remains understudied. Here, we employed resonance Raman (RR) spectroscopy to investigate the responses of two freshwater phytoplankton species, the green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana to Hg(II) exposure. We selectively recorded the spectral RR signature of carotenoids in intact cells exposed to concentrations of 10 nM and 100 nM of Hg(II), representative for contaminated environment and unexposed control cells. A two-hour exposure of C. reinhardtii resulted in a slight decrease in lutein and β-carotene levels, while total carotenoids RR band broadening, as revealed by the FWHM of the υ₁(C=C) stretching mode from averaged RR spectra, suggested conformational changes in pigments. Higher Hg(II) concentration induced more pronounced conformational changes. Similarly, a two-hour exposure of C. meneghiniana resulted in slight decreased level of the fucoxanthin, while diadinoxanthin showed an opposite trend compared to control: when fucoxanthin decreased, diadinoxanthin increased under 10 nM Hg (II) exposure. At higher concentrations, the decrease in fucoxanthin was less pronounced, accompanied by a broadening of the band area, (with FHHM increased), indicating possible conformer occurrence in response to Hg-induced stress. The changes in the main carotenoid species of the two algae are species-specific, Hg(II) concentration-specific, and dependent on exposure time. The calculated spectral differences in absorbances from UV-VIS spectra of methanol extracts from each group supported the main findings obtained by RR, though with caution due to the selective extraction efficiency of the respective carotenoids. This study highlighted for a first time the capability of single-cell RR spectroscopy as a valuable tool for toxicity assessment and for comprehending early-stage alterations in carotenoid metabolism due to toxic metal exposure in vivo.