| Abstract | In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL⁻¹ of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu¹]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS², which slowly converted to [D-Leu¹,Glu(OMe)⁶]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS² in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS² were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g⁻¹) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu¹]MC-LR was quantitated in tissues at 25–67 ng g⁻¹ (LC–MS²). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present. |
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