Mazzeo, Antonia (2016) Quali-quantitative determination and structural characterization of microalgal toxins in environmental matrices. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Quali-quantitative determination and structural characterization of microalgal toxins in environmental matrices
Date: 2016
Number of Pages: 227
Institution: Università degli Studi di Napoli Federico II
Department: Farmacia
Scuola di dottorato: Scienze farmaceutiche
Dottorato: Scienza del farmaco
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
D'Auria, Maria
Dell'Aversano, CarmelaUNSPECIFIED
Date: 2016
Number of Pages: 227
Uncontrolled Keywords: Palytoxin, Ostreopsis, DSP
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 11 Apr 2016 07:10
Last Modified: 10 May 2017 01:00


“Harmful Algal Blooms” (HABs) are massive proliferations of marine microalgae producing biotoxins. These natural phenomena, occurring under particular climatic and environmental conditions, represent a worldwide problem since they pose serious threats to human health and heavily affect economy of coastal areas. Humans may be affected by marine biotoxins through three main exposure routes: the oral route through consumption of contaminated seafood, the respiratory route through inhalation of aerosolized toxins, and the dermal route through direct contact with microalgae and/or contaminated seawater. However, for some marine biotoxins the mechanism through which they exert their toxicity has not been clarified yet. The Ostreopsis phenomenon currently represents the major HAB-related threat to humans in the Mediterranean area. Since the late 1990s, blooms of Ostreopsis cf. ovata have been repetitively recorded all along the Italian coastlines as well as along the Mediterranean coasts of Spain, France, Croatia, Greece and North Africa. Concomitantly, a severe respiratory syndrome has been observed in humans requiring in some cases hospitalization. Previous studies have demonstrated that O. cf. ovata produces congeners of palytoxin (PLTX), (ovatoxins and an isobaric PLTX), one of the most potent marine toxins so far known, originally isolated from soft corals belonging to the genus Palythoa. However, inhalatory toxicity of PLTX and its congeners was completely unknown and the mechanism through which they harm humans through inhalation is still matter of speculation. The increasing spread of the Ostreopsis phenomenon and the ever-growing number of ovatoxins (OVTXs) being discovered makes the need of evaluating their toxicity urgent. The availability of sufficient amounts of well characterized reference material is the cornerstone for the achievement of toxicity data. Beside PLTX congeners, a wide array of marine biotoxins present serious concern for humans. As a result, National and Local authorities in charge of safeguarding public health have enacted monitoring and risk management programs of toxin with the purpose of limiting adverse impacts of HABs; these regulations require routine monitoring of shellfish for toxins and the analysis of water samples for the presence of toxin-producing microalgae. However, the analysis of shellfish and the analysis of microalgae presents number of technical and practical limitations; for these reasons, in recent years, passive sampling (Solid Phase Adsorption Toxins Tracking, SPATT) has been investigated as an alternative mean to highlight the presence of toxic microalgae directly in seawater. In this frame, the aim of this thesis was: • to investigate the mechanism through which O. cf. ovata and Palythoa spp. and the toxins they produce exert their toxicity; • to determine toxin profile and content of Ostreopsis spp. strains from South-East Mediterranean Sea (Cyprus Island and Lebanon), New Zealand and Australia to serve the double purpose of evaluating the risk they present to humans and investigating the presence of novel PLTX analogues by LC-HRMSn; • to use SPATT methods for detection of marine biotoxins; • to perform preliminary studies toward isolation of PLTX congeners. Chapter 1 presents a general introduction. In Chapter 2 the potentially active role of the mucilaginous matrix produced by O. cf. ovata, has been for the first time investigated. In order to better elucidate toxicity dependence on direct/indirect contact, the role of the mucilaginous matrix and the potential differences in toxicity along the growth curve of O. cf. ovata, a toxic bioassay during exponential, stationary and late stationary phases was carried out. Simultaneously, a molecular assay was performed to quantify intact cells or to exclude the presence of the cells. Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) analyses were also carried out to evaluate toxin profile and content in the different treatments. As a result, a higher mortality of model organism (Artemia salina nauplii) was also observed, especially during the late stationary phase, when direct contact between the model organism and intact microalgal cells occurred (LC50-48h < 4 cells/ml on A. salina). Also growth medium devoid of microalgal cells but containing O. cf. ovata mucilage caused significant toxic effects. This finding was also supported by chemical analyses which showed the highest toxin content in pellet extract (95%) and around 5% of toxins in the growth medium holding mucous, while the growth medium devoid of both cells and mucilage did not contain any detectable toxins. Additionally, the connection between mucilaginous matrix and the thecal plates, pores and trychocysts was explored by way of atomic force microscopy (AFM) to investigate the cell surface at a sub-nanometer resolution, providing a pioneering description of cellular features. In Chapter 3 LC-HRMS analysis of both soft coral and seawater from a home marine aquarium involved in a whole family poisoning is reported. Several anecdotal reports exist of aquarium hobbyists that experienced severe respiratory distress and/or skin injury following cleaning operation of home aquaria containing Palythoa sp. soft corals as well as hundreds of cases of respiratory illness and/or dermatitis have been recorded in proximity to the sea concomitantly with algal blooms of Ostreopsis spp. in the Mediterranean area. Both Palythoa spp. and Ostreopsis spp. contain congeners of PLTX whose inhalation hazard is however unknown. In this study, we demonstrate the presence of high levels of PLTXs (PLTX and hydroxy-PLTX) in both soft coral and seawater collected in the marine aquarium. Due to the high toxin levels found in seawater, a procedure for a rapid and efficient determination of palytoxin in seawater was developed. A comparison of symptoms of Palythoa- and Ostreopsis-related inhalatory poisonings showed many similarities including fever, respiratory distress, nausea, and flu-like symptoms. From the chemical and symptomatological data reported herein it is reasonable to hold PLTXs responsible for respiratory disorders following inhalation. Although the exact mechanism through which PLTX congeners exert their inhalatory toxicity is still unknown, this represents a step toward demonstrating that they exert toxic effects through inhalation both in natural environments and in the surroundings of private and public aquaria. In Chapter 4 the investigation of the toxin profile and content of six strains of Ostreopsis sp. from Cyprus Island is reported. The samples were analyzed through an integrated approach based on molecular, chemical and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced OVTX-a, OVTX-d, OVTX-e and isobaric PLTX, so far found only in O. cf. ovata, the other three strains produced only new PLTX-like compounds, that we named OVTX-i, -j1, -j2, and -k. The new OVTXs had the same carbon skeleton as OVTX-a differing primarily for an additional C2H2O2 moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j1 and -k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and -j1). Toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg/cell, definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, eco-toxicological test on A. salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island pose a relatively low risk to humans. Chapter 5 reports the chemical analysis of five strains of Ostreopsis spp. collected from Lebanon coasts and of six strains of Ostreopsis spp. (ovata, siamensis and Ostreopsis sp.) from New Zealand and Australia. LC-HRMS of Lebanese extracts showed that three strains produced OVTX-a, OVTX-d, OVTX-e in very minute amounts (0.28-0.94 pg/cell), thus presentig a low risk to humans. The toxin profile of these strains quali-quantitatively matched with that of other Ostreopsis sp. strains from Cyprus Island. The other extracts did not contain any PLTX congener. Molecular analyses showed that Ostreopsis sp. from Lebanon and from Cyprus Island are actually the same species that we named Ostreopsis fattorussoi. LC-HRMS analysis of Ostreopsis spp. strains from New Zealand and Australia showed that none of the known PLTX congeners so far known were contained in any of the extracts. Only a new analogue, was present in one of the Ostreopsis sp. extracts. It presented a C5H7ON moiety as well as 2 unsaturation less than OVTX-a. An extensive LC-HRMSn study, allowed to locate such modification in the very limited region stretching from the A-side terminal to C-8. This compound presented a characteristic fragmentation behavior: indeed, a formaldehyde loss could be observed from the precursor ion as well as from most of the A-side fragment ions. Formaldehyde losses may occur when a CH2OH moiety is present in the molecule following a Mac Lafferty-like rearrangement. We located the CH2OH moiety on the terminal amide. Chapter 6 reports on a survey of phytoplankton and algal toxins in Nigerian coastal waters. Seawater samples were obtained from four sites for phytoplankton identification (Bar Beach and Lekki in Lagos State, Port Harcourt in Rivers State and Uyo in Akwa Ibom State), on three occasions between the middle of October 2014 and the end of February 2015. The phytoplankton community was generally dominated by diatoms and cyanobacteria; however, several species of dinoflagellates were also identified: Dinophysis caudata, Lingulodinium polyedrum and two benthic species of Prorocentrum. Passive samplers (containing Diaion® HP-20 resin) were deployed for several 1-week periods on the same four sites to obtain profiles of algal toxins present in the seawater. Quantifiable amounts of OA and PTX-2, as well as traces of DTX-1 were detected at several sites. The highest toxin concentrations (60 ng OA g/HP-20 resin) were found at Lekki and Bar Beach stations, which had also the highest salinities. Non-targeted analysis using full-scan HRMS showed that algal metabolites differed from site to site and for different sampling occasions. Screening against a marine natural products database indicated the potential presence of cyanobacterial compounds in the water column, which was also consistent with phytoplankton analysis. Chapter 7 reports another application of passive samplers in the French lagoon of Ingril. Seawater portions of 30 L were collected and pre concentrated by passive sampling with HP-20 resin over a 48 h period. Detection of lipophilic toxins in the extracts of the resin was carried out using liquid chromatography coupled to tandem mass spectrometry. This combination allowed the detection of sub-ppb levels of dissolved toxins and would permit further studies for accurate modelling of the toxins adsorption behavior by passive sampling devices. In particular, we determined the levels of OA, DTX-1, PTX-2 and PnTX-G in seawater from Ingril lagoon. OA was the most concentrated compound with ca. 8.6 ng/L, followed by DTX-1 with ca. 1.4 ng/L, and both PTX-2 and PnTX-G at ca. 0.2-0.3 ng/L. This is one of the first direct analyses of lipophilic dinoflagellate toxins in seawater. However, these concentrations were observed in a lagoon and should be confirmed in open coastal waters. Chapter 8 describes preliminary studies aiming to quantitative isolation of palytoxin. Previous reported isolation procedure, provided very poor recoveries of palytoxin congeners as pure compound, with no indication whether such low recoveries were due to instability of the compounds in solution or to irreversible adsorption to materials or to other uncontrolled factors. In view of a large scale isolation work aimed to preparation of PLTX reference material, the causes underlying the huge PLTX loss were investigated. We focused on the evaporation of PLTX under various experimental conditions namely the use of different evaporation systems (Centrifugal Vacuum Concentrator and N2 stream) versus freeze drying, complete drying versus concentration, the influence of various solvents (aqueous or pure organic), the effect of most common materials (normal and silanized glass vials, polypropylene and Teflon tubes) and of the re-dissolution solvent (nature and quantity) on toxin recovery. Preliminary results on stability of PLTX under various acidic conditions were also obtained. PLTX behaved differently when it was simply concentrated or completely dried down. Recoveries were strongly dependent on solubility of PLTX in the mixtures used as well as on the materials in which evaporation was carried out. We found that, in order to enhance PLTX recovery, freeze drying is an appropriate procedure to be avoided. In general, the highest recoveries were obtained when PLTX was completely dried down in Teflon carrying out evaporation in aqueous blends or concentration in pure organic solvent. The worst recoveries were obtained using glass materials probably due to the sticking of PLTX on the surface of the vials; in order to reduce this phenomenon water should be used in the mixture. Experiments on stability of PLTX in acid blends demonstrated that PLTX is an acid-sensitive molecule and that, depending on pH, it rapidly degradates forming mainly a PLTX methyl-ester by cleavage of the enamide functionality contained in the A-side terminal of the molecule. Structural insights on the PLTX methyl-ester were gained by LC-HRMS/MS.

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