Simplified Monitoring of Anatoxin-A by Reverse-Phase High Performance Liquid Chromatography and the Sub-Acute Effects of Anatoxin-A in Rats

Abstract

Anatoxin-a is a potent neuromuscular blocking agent produced by certain strains of the blue-green algae Anabaena flos-aquae. Its sporadic and unpredictable production during algal blooms, occasionally causing animal deaths, led to a concern that animals and humans may ingest sub-chronic doses of anatoxin-a, resulting in delayed toxicity. A method to detect and monitor anatoxin-a in natural water bodies was therefore a primary concern. Toxic A. flos-aquae was cultivated and the toxin extracted by centrifugation and solvent extraction procedures. Anatoxin-a was then analyzed by reverse-phase high performance liquid chromatography (HPLC) using an isocratic gradient. A useful concentration range where detector response remained linear to toxin concentration went from 1 to 500 ppm. Combined with the extraction procedure, which averaged 90% recoveries, an overall assay sensitivity of 0.1 ppm was achieved.

Anatoxin-a in drinking water was then administered to rats at 0 ppm, 0.5 ppm, and 5 ppm for 7 weeks. (5 ppm equals approximately 1/20 the acute oral LD₅₀). In a second study, anatoxin-a was injected intraperitoneally (i.p.) to rats at 0 mg/kg and 0.062 mg/kg daily for 21 days. (0.062 mg/kg equals approximately 1/4 the acute i.p. LD₅₀). No changes were seen in body weight, food consumption, or serum enzymes (SGPT, GGTP alkaline phosphatase, and cholinesterase) in either study. Gross and microscopic examination of the tissues showed no differences between treated and control groups. This indicates that anatoxin-a does not cause apparent toxic signs in doses less than those causing acute effects.