Identification and occurrence of microcystins in freshwaters and fish from a eutrophic dam through LC-HRMS

Microcystins (MCs) are cyclic heptapeptides originating from various cyanobacteria in eutrophic aquatic environments. Their potential consequences on ecosystems and public health underscores the need to explore MCs' occurrence. In this study, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) analysis and a suspect screening workflow supported by open-source tools were employed for the determination of MCs in freshwater and biota samples from a eutrophic dam in Uruguay. MS² spectra were acquired using higher-energy collisional dissociation (HCD), data-dependent acquisition (DDA), and stepped NCE of [M + H]⁺ and [M + 2H]²⁺ ions. In addition to the confirmation of 3 MCs in the samples, a comprehensive study of the acquired spectra led to the tentative identification of 30 MCs, including 2 partially described variants not previously reported. 33 MCs were determined in freshwaters, exhibiting a maximum MC concentration in a sample of 12731 μg/L. Regarding fish, the 61 % of the samples exhibited at least a positive determination. 8 MCs were detected and [D-Leu¹]MC-LR, [seco-4/5]MC-LR, MC-LR, MC-RR, MC-WR, and [D-Asp³]MC-RR could be semi-quantified (3-127 μg/kg, w.w). In 2 samples, the MC-LR content per 100 g of fish was found to be close to 80 % the tolerable daily intake for chronic exposure recommended by the WHO. The identification of [seco-4/5]MC-LR in biota highlights the labile byproducts of MCs and the need for wide-scope analytical approaches. This study emphasizes the extensive range of MCs present in eutrophic freshwater environments, their accumulation in exposed biota, and their potential entry into the food web.