Toxicity, transfer and depuration of anatoxin-a (cyanobacterial neurotoxin) in medaka fish exposed by single-dose gavage

Abstract

The proliferations of cyanobacteria are increasingly prevalent in warm and nutrient-enriched waters and occur in many rivers and water bodies due especially to eutrophication. The aim of this work is to study in female medaka fish the toxicity, the transfer and the depuration of the anatoxin-a, a neurotoxin produced by benthic cyanobacterial biofilms. This work will provide answers regarding acute toxicity induced by single gavage by anatoxin-a and to the risks of exposure by ingestion of contaminated fish flesh, considering that data on these aspects remain particularly limited. The oral LD50 of a single dose of ({+/-})-anatoxin-a was determined at 11.50 {micro}g.g-1. First of all, lethal dose (100% from 20 {micro}g.g-1) provokes rapid respiratory paralysis (in 1-2 min) of the fish inducing the death by asphyxia. Noticeably, no death nor apparent neurotoxicologic effect occurred during the experimentation period for the 45 fish exposed to a single sub-acute dose of ({+/-})-anatoxin-a corresponding to the no observable adverse effect level (NOAEL = 6.67 {micro}g.g-1). Subsequently, the toxico-kinetics of the ({+/-})-anatoxin-a was observed in the guts, the livers and the muscles of female medaka fish for 10 days. In parallel, a protocol for extraction of anatoxin-a has been optimized beforehand by testing 3 different solvents on several matrices, the extraction with 75% methanol + 0.1% formic acid appearing to be the most efficient. Anatoxin-a was quantified by high-resolution qTOF mass spectrometry coupled upstream to a UHPLC chromatographic chain. The toxin could not be detected in the liver after 12 h, and in the gut and muscle after 3 days. The mean clearance rates of ({+/-})-anatoxin-a calculated after 12 h are above 58%, 100% and 90% for the guts, the livers and the muscles, respectively. Non-targeted metabolomics investigations performed on the fish liver indicates that the single sub-acute exposure by gavage induces noticeable metabolome dysregulations, including important phospholipid decreases, with an organism recovery period of above 12-24h. Overall, the medaka fish do not appear to accumulate ({+/-})-anatoxin-a and to largely recover after 24h following a single sub-acute oral liquid exposure at the NOAEL.