First report of detection of microcystins in farmed Mediterranean mussels Mytilus galloprovincialisin Thermaikos gulf-Greece, using ELISA and liquid chromatography high-resolution mass spectrometry

Abstract Background Microcystins are emerging marine biotoxins, produced by potentially toxic cyanobacteria. Their presence has been reported in aquatic animals in Greek internal waters, while data are few in marine environments. Since the climate change induce eutrophication and harmful algal blooms in coastal marine ecosystems affecting the public health, further research on microcystins’ presence in marine waters is required. The aim of this study was to detect the potential presence of microcystins in mussels Mytilus galloprovincialis in the largest farming areas in Thermaikos gulf, in Northern Greece, and to investigate their temporal and spatial distribution, adding to the knowledge of microcystins presence in Greek Mediterranean mussels. Results A three years microcystins’ assessment was conducted from 2013 to 2016, in farmed Mediterranean mussels Mytilus galloprovincialis, in five sampling areas in Thermaikos gulf, in northern Greece, where the 90% of mussels’ farming activities is located. The isolation of potentially toxic cyanobacteria was confirmed by molecular methods. An initial screening was performed with a qualitative and quantitative direct monoclonal (DM) ELISA and results above 1ng/g were confirmed for the occurrence of the most common microcystins -RR, -LR and -YR, by Ultra High Performance Liquid Chromatography (UHPLC) coupled with a high- resolution mass spectrometer (HRMS) (Orbitrap analyzer). Microcystin -RR and microcystin -LR were detected, while the intensity of microcystin-YR was below the method detection limit. Most samples that exhibited concentrations above 1ng/g were detected during the warm seasons of the year and especially in Spring. Results indicated an overestimation of the ELISA method, since concentrations ranged between 0.70 ± 0.15 ng/g and 53.90 ± 3.18 ng/g, while the confirmation declared that the levels of microcystins were 6 to 22 times lower. Conclusions Microcystin RR and microcystin LR were detected for the first time in mussel Mytilus galloprovincialis, harvested from farms in Thermaikos gulf, in Central Macedonia, in Greece. Their presence was linked to potentially toxic cyanobacteria. Bioaccumulation in digestive gland was observed, while the concentrations in muscles were found extremely low. Samples with levels above 1ng/g were observed mostly during spring, confirming the seasonal distribution of microcystins. The comparison of the results by the ELISA and the LC-Orbitrap MS method indicated an overestimation of concentration by the ELISA method.