Testing a new approach to quantify intracellular trace metal contents in microphytoplankton using sequential...

Current methodologies used to quantify intracellular trace metal (TM) contents in microphytoplankton rely on the assumption that samples consist exclusively of phytoplankton cells, with no contribution from inorganic particles. Therefore, the present study aimed to propose a sequential selective extraction protocol for suspended particulate matter that preserve the cellular integrity of phytoplankton until the final Aqua Regia extraction. Seawater was collected in the Arcachon Bay and filtered through polycarbonate membranes (PC; Isopore™, 2 μm pore size). These membranes were put in contact with various extractants to isolate specific TM fractions. A subset of membranes was used for observation with scanning electron microscope (SEM) to assess the impact of the extractants on phytoplankton integrity. Among the tested solutions (i.e. ascorbate, 0.1 M NaOH, hydrogen peroxide, EDTA, 1 M HCl), only hydrogen peroxide was found to be destructive for the phytoplankton cells. Accordingly, a four-step sequential extraction was established: (1) ascorbate solution, (2) 0.1 M NaOH, (3) EDTA, (4) 1 M HCl, in order to selectively remove TM associated with inorganic phases or organic matter. Subsequently, PC membranes were digested by Aqua Regia to break down the phytoplankton cells and quantify the intracellular TM contents. The established protocol proves to be robust and effective to quantification the minimal TM content in diatoms and silicoflagellates cells. Results showed that Al, V, Mn, Fe, Cu, As, Pb, and Th were present in low proportion in the Aqua Regia solution (less than 20 % of the total content), suggesting limited internalization. In contrast, Cr (93 ± 3 %), Co (74 ± 23 %), and Ni (79 ± 17 %) fractions were dominantly present in the intracellular fraction suggesting they play major roles in TM biogeochemical cycles involving phytoplankton.