Preprint volume - SIBM

Pre-print Volume – Posters
PLANKTON COMMITEE
C. FACCA, D. BILANICOVA, G. POJANA, A. MARCOMINI, A. SFRISO
Dep. of Environmental Sciences, University Ca’ Foscari Venice,
Calle Larga Santa Marta, 2137 - 30123 Venezia, Italia.
facca@unive.it
COUPLING TAXONOMIC AND CHEMICAL ANALYSES
TO MONITOR HARMFUL ALGAE OCCURRENCE IN LAGOONS
(VENICE AND PO DELTA SYSTEM, ITALY)
ANALISI TASSONOMICHE E CHIMICHE PER IL MONITORAGGIO
DELLE ALGHE TOSSICHE NELLE LAGUNE (VENEZIA E DELTA DEL PO)
Abstract – Harmful algae occurrence was monitored in the Venice lagoon and in the Po Delta lagoons by
taxonomic identification with optical microscopy, and chemical determination of toxins by HPLC coupled
with High Resolution Time-Of-Flight Mass Spectrometry (HR-TOF-MS). These techniques were coupled in
order to verify the potential of a developing protocol overcoming the current anti-ethical and expensive
Yasumoto test on mice. The abundance of potentially toxic cells resulted negligible, but toxins were anyway
analytically detected and structurally confirmed by the high accuracy of the MS detector employed.
Key-words: Dinophysis, harmful algae, biotoxins, lagoons.
Introduction – The risks due to Harmful Algae (HA) proliferation are well known
worldwide and numerous research and sanitary institutions are currently involved in
the monitoring of their occurrence. The methods dedicated to the identification of HA
are numerous and involve different approaches (taxonomy, genetics, toxicology,
chemistry, biochemistry). However, the method, the one recognized by national
regulations to prevent risk to human health, is the Yasumoto’s test on mice. It is often
coupled with taxonomic identification, but it results anti-ethic, poorly accurate and
rather expensive. To overcome such limitations our research group is proposing to
integrate the taxonomic investigation with analytical chemistry analysis in order to
correctly identify and quantify the toxin presence and occurrence. As already reported
by literature, the presence of such harmful species is not necessarily correlated to the
occurrence of poisoning events, so this approach wants to combine routine taxonomic
monitoring activity with accurate chemical identification of toxins (Okadaic Acid,
DTX1, DTX2, Domoic Acid, PTX2) to highlight the real risk for human health due to
the microalgal proliferations.
Materials and methods – Seawater samples were collected 12 times from June to
August 2009 in the Venice lagoon and in a couple of lagoons in the Po Delta system,
where clam farming is the main economical resource. Phytoplankton cell abundance
and taxonomic composition were determined under the inverted light microscope
according to the Utermöhl’s method (1958), settling 25 ml of sample in order to
identify also species-specific low abundances. Qualitative observations were carried
out on 20 µm mesh net samples. Seawater was filtered on GF/F Whatman filters to
extract toxins from microalgal cells. Solvent mixture (80:20 vol methanol:water) was
sonicated, and then 10-times concentrated to 1 ml by evaporation and centrifugation
(Blanco et al., 2007; Fernández et al., 2006). Aliquots of 10 µl of the supernatant were
injected in an Agilent 1200 High Performance Liquid Chromatography system using an
Agilent G1329B autosampler. The chromatographic separation of lipophilic toxins
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