Analysis of free and metabolized microcystins in samples following a bird mortality event

Amanda J Foss; Christopher O Miles; Ingunn A Samdal; Kjersti E Løvberg; Alistair L Wilkins; Frode Rise; J Atle H Jaabæk; Peter C McGowan; Mark T Aubel

doi:10.1016/j.hal.2018.10.006

Analysis of free and metabolized microcystins in samples following a bird mortality event

Harmful Algae. 2018 Dec:80:117-129. doi: 10.1016/j.hal.2018.10.006. Epub 2018 Nov 3.

Authors

Amanda J Foss¹, Christopher O Miles², Ingunn A Samdal³, Kjersti E Løvberg³, Alistair L Wilkins⁴, Frode Rise⁵, J Atle H Jaabæk⁵, Peter C McGowan⁶, Mark T Aubel⁷

Affiliations

¹ GreenWater Laboratories/CyanoLab, 205 Zeagler Drive, Palatka, FL, 32177, United States. Electronic address: amandafoss@greenwaterlab.com.
² Norwegian Veterinary Institute, P. O. Box 750 Sentrum, N-0106, Oslo, Norway; Measurement Science and Standards, National Research Council, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada.
³ Norwegian Veterinary Institute, P. O. Box 750 Sentrum, N-0106, Oslo, Norway.
⁴ Norwegian Veterinary Institute, P. O. Box 750 Sentrum, N-0106, Oslo, Norway; Chemistry Department, University of Waikato, Private Bag 3105, 3240, Hamilton, New Zealand.
⁵ Department of Chemistry, University of Oslo, P.O. Box 1033, N-0315, Oslo, Norway.
⁶ U.S. Fish and Wildlife Service, Chesapeake Bay Field Office, Annapolis, MD, United States.
⁷ GreenWater Laboratories/CyanoLab, 205 Zeagler Drive, Palatka, FL, 32177, United States.

PMID: 30502804
DOI: 10.1016/j.hal.2018.10.006

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.

Keywords: Adda; ELISA; LC–MS; MMPB; Microcystin; animal intoxication; avian botulism; conjugate; deconjugation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Birds*
Chromatography, Liquid
Environmental Monitoring
Enzyme-Linked Immunosorbent Assay
Harmful Algal Bloom*
Liver / metabolism
Microcystins / chemistry
Microcystins / metabolism*
Microcystins / toxicity
Tandem Mass Spectrometry

Substances

Microcystins