Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds

Abstract

Ten natural bloom samples of cyanobacteria from the Danish lakes Knud sø (5), Ravn sø (4), and Salten Langsø (1) collected during 1993-1995 were assayed for toxicity by mouse bioassay, for acetylcholinesterase inhibiting activity by a colorimetric method, and for microcystins by enzyme-linked immunosorbent assay. In the mouse bioassay, seven samples were neurotoxic, two were non-toxic and one gave a protracted toxic response. One of the non-toxic and the single protracted toxic sample both contained anticholinesterase activity equivalent to 4 micrograms anatoxin-a(s) g-1. The neurotoxic samples contained equivalents to 20-3300 micrograms anatoxin-a(s) g-1. The highest anticholinesterase activities (equivalent to 2300 and 3300 micrograms anatoxin-a(s) g-1, respectively) were found in samples collected from Lake Knud sø in connection with bird-kills in 1993 and 1994. Small amounts of microcystins (0.1-0.9 microgram g-1) were detected in all samples but one. All Lake Knud sø and Lake Ravn sø samples were dominated by Anabaena lemmermannii, and the Lake Salten Langsø sample by several species of Anabaena. Gel filtration profiles indicated similarity between the toxic component from the Lake Knud sø 1994 bloom with registered bird-kills and anatoxin-a(s) isolated from Anabaena flos-aquae NRC-525-17. Anticholinesterase-producing cultures of A. lemmermannii were isolated from the Lake Knud sø 1993 bloom. These laboratory cultures produced anatoxin-a(s) equivalents of 29-743 micrograms g-1. Other cultures of A. lemmermannii isolated from Lake Knud sø and Lake Ravn sø were hepatotoxic or non-toxic. Dead birds collected from Lake Knud sø during the neurotoxic 1993 Anabaena bloom possibly died from cyanobacterial toxicosis. The stomach contents contained colonies and single trichomes of Anabaena, and anticholinesterase activities equivalent to 2.1-89.7 micrograms anatoxin-a(s) kg-1 body weight and microcystins (53-95 ng kg-1) were also detected.