Toxicity evaluation of Fibrocapsa japonica from the Northern Adriatic Sea through a chemical and toxicological approach

Since the 1990s red-tide blooms of Fibrocapsa japonica have frequently been observed in European waters; despite their recurrence also in the Adriatic coastal areas, they have never been conclusively linked to ichthyotoxic events. Since the toxicity of F. japonica is still under debate and its effects differ among the strains, in this study all the compounds previously postulated as involved in the toxic mechanism, such as brevetoxins, fatty acids and ROS, were screened for Adriatic strains. Whole algal extracts were analyzed for brevetoxin PbTx-2, which was not found, and for a qualitative and quantitative analysis of fatty acids. The fatty acid profile evidenced the presence of PUFAs, with considerable amounts of 18:4n-3, 20:4n-6, and 20:5n-3, as already found in different strains, but with a lower amount of arachidonic acid (20:4n-6). None of the PUFAs was released in the extracellular medium. Different toxicological assays (Vibrio fischeri, Artemia sp., haemolysis of fish erythrocytes) were performed using algal cellular, extracellular or subcellular samples as well as fractions of the algal extract, in order to identify the toxic compounds. The tested cultures were found to inhibit V. fischeri bioluminescence, to affect Artemia nauplii viability and to have haemolytic effects comparable to those previously observed in different strains. The solid phase extraction (SPE) of the Adriatic F. japonica extracts resulted in a high concentration of PUFAs in two main fractions, which caused mortality in Artemia nauplii and inhibition of V. fischeri bioluminescence. Fish (Dicentrarchus labrax) assays were also performed, reporting mortality after long exposure times and the cause of fish death was investigated. A significant increase of H2O2 in the tanks where sea basses were exposed to F. japonica was obtained, indicating that the presence of fish stimulates H2O2 production by algal cells and leading to the hypothesis of H2O2 involvement in F. japonica toxicity. The presence of oxidative stress in the exposed fish was confirmed by the increased malondialdehyde concentration in their gills. Overall these results indicate that since F. japonica cells can easily cling to fish gills, due to their high polysaccharide production, it is presumable that high PUFA amounts are released in loco, as a result of cell breakage, and that exert their toxicity together with ROS. This study shows that the Adriatic strains of F. japonica can be harmful to higher level organisms. Nevertheless a high cell density and a long lasting bloom are necessary to cause severe damage to fish gills or death for crustaceans.