Luciana Tartaglione

Complex palytoxin-like profile of Ostreopsis ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry.

Over the past decades, Italian coastlines have been plagued by recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies have confirmed the presence of ovatoxin-a (OVTX-a) as the major toxin of the algal toxin profile together with small amounts of putative palytoxin (PLTX). In our ongoing research on O. ovata toxins we report herein on in-depth investigation of an O. ovata culture carried out by high-resolution (HR) liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS(2)). Particularly, the presence of putative PLTX and OVTX-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, OVTX-b, -c, -d, and -e, was highlighted. Elemental formulae have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that the whole of the new ovatoxins represents about 46% of the total toxin content and, thus, their presence has to be taken into account when LC/MS-based monitoring programs of either plankton or contaminated seafood are carried out.

Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas.

Massive blooms of the benthic dinoflagellate Ostreopsis ovata Fukuyo have recently occurred along the whole Italian coastlines, both Tyrrhenian and Adriatic, resulting sometimes in benthonic biocenosis sufferings and, occasionally, in human health problems. In this work, two strains of O. ovata collected in 2006 along the Adriatic and Tyrrhenian coastlines and grown in culture were studied to characterize their growth and toxin profile. The two strains showed different cell volumes, the Adriatic strain being nearly twice bigger than the Tyrrhenian, but they had similar slow growth rates. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that both strains produce putative palytoxin (pPLTX) and ovatoxin-a (OVTX-a), a palytoxin-like compound presenting 2 oxygen atoms less than palytoxin. Toxin content was determined at the end of the stationary and exponential growth phases and reached the highest value in the Adriatic strain at the end of the stationary phase, with concentrations of 353.3 microg l(-1) for OVTX-a and 30.4 microg l(-1) for pPLTX. Toxin released in the growth medium was also measured and resulted to be the highest at the end of the stationary phase, suggesting that a long lasting bloom could enhance the toxin content in the water and cause toxic effects in people inhaling the aerosol.

Isolation and Structure Elucidation of Ovatoxin-a, the Major Toxin Produced by Ostreopsis ovata

Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata -related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-as complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity.

LC-MS of palytoxin and its analogues: State of the art and future perspectives.

The state of the art of LC-MS of palytoxin and its analogues is reported in the present review. MS data for palytoxin, 42-hydroxy-palytoxin, ostreocin-D, mascarenotoxins, and ovatoxins, obtained using different ionization techniques, namely fast-atom bombardment (FAB), matrix assisted laser desorption ionization (MALDI), and electrospray ionization (ESI), are summarized together with the LC-MS methods used for their detection. Application of the developed LC-MS methods to both plankton and seafood analysis is also reported, paying attention to the extraction procedures used and to limits of detection (LOD) and quantitation (LOQ) achieved. In a research setting, LC-MS has shown a good potential in determination of palytoxin and its analogues from various sources, but, in a regulatory setting, routine LC-MS analysis of palytoxins is still at a preliminary stage. The LOQ currently achieved in seafood analysis appears insufficient to detect palytoxins in shellfish extract at levels close to the tolerance limit for palytoxins (30 μg/kg) proposed by the European Food Safety Authority (EFSA, 2009). In addition, lacking of certified reference standard of palytoxins as well as of validation studies for the proposed LC-MS methods represent important issues that should be faced for future perspectives of LC-MS technique.

Unique Toxin Profile of a Mediterranean Ostreopsis cf. ovata Strain: HR LC-MSn Characterization of Ovatoxin-f, a New Palytoxin Congener

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

Influence of temperature and salinity on Ostreopsis cf. ovata growth and evaluation of toxin content through HR LC-MS and biological assays

In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts.

Stereostructure and Biological Activity of 42-Hydroxy-palytoxin: A New Palytoxin Analogue from Hawaiian Palythoa Subspecies

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

Ostreopsis cf. ovata bloom in the northern Adriatic Sea during summer 2009: Ecology, molecular characterization and toxin profile

Intense blooms of the benthic dinoflagellate Ostreopsis cf. ovata have occurred in the northern Adriatic Sea since 2006. These blooms are associated with noxious effects on human health and with the mortality of benthic organisms because of the production of palytoxin-like compounds. The O. cf. ovata bloom and its relationships with nutrient concentrations at two stations on the Conero Riviera (northern Adriatic Sea) were investigated in the summer of 2009. O. cf. ovata developed from August to November, with the highest abundances in September (1.3×10(6) cells g(-1) fw corresponding to 63.8×10(3) cells cm(-2)). The presence of the single O. cf. ovata genotype was confirmed by a PCR assay. Bloom developed when the seawater temperature was decreasing. Nutrient concentrations did not seem to affect bloom dynamics. Toxin analysis performed by high resolution liquid chromatography-mass spectrometry revealed a high total toxin content (up to 75 pg cell(-1)), including putative palytoxin and all the ovatoxins known so far.

Toxin Levels and Profiles in Microalgae from the North-Western Adriatic Sea—15 Years of Studies on Cultured Species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.

First finding of Ostreopsis cf. ovata toxins in marine aerosols.

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome.