A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation

Abstract

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20-280 μm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg^-1 by day 13, exceeding the European Food Safety Authority recommended concentration of 44 μg kg^-1 in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.

Keywords: bioaccumulation; feeding experiment; humic acid; marine toxin; shellfish.

Figure 1

Agar-gelatin microcapsules containing Alexandrium minutum cells ((A); black arrows) and humic acid (the brown solids; (B–D)). The black arrow in (D) shows a partially digested capsule containing humic acid that was found in the digestive gland of a Paphies australis.

Figure 2

Tetrodotoxin (TTX) concentrations in experimental controls (CTL) and treatment samples (TTX) for the two tissue types (combined digestive gland and siphon, and everything else “rest”) and “whole” Paphies australis (n = 5) individuals after being fed controlled amounts of TTX every three days for 7 (A) and 13 days (B). The solid black line shows median, box shows 1st and 3rd quartiles, whiskers extend to the last data point within 1.5 times the inter-quartile range. Dots outside the whiskers are considered as outliers.