Miele, Valentina (2025) Foodborne diseases due to emerging marine and freshwater toxins. Validation of LC-MS/MS and LC-HRMS methods for toxin determination. [Tesi di dottorato]

Documento PDF PhD_Thesis_Valentina_Miele.pdf Visibile a [TBR] Amministratori dell'archivio Download (9MB) | Richiedi una copia

Abstract

Water is an indispensable resource for life on Earth, representing the primary element that sustains both terrestrial and aquatic ecosystems. Seas, rivers, and lakes not only provide vital resources such as fishery products and drinking water but also serve as habitats for a wide range of species, from aquatic microfauna to more complex life forms. Phytoplankton represents the productive base of marine and freshwater ecosystems and is mainly composed of microalgae and cyanobacteria, microscopic living organisms responsible for producing approximately half of the total oxygen generated by plant organisms on Earth. Among the 5000 known species of marine microalgae, 300 are harmful to humans due to the production of secondary metabolites, namely toxins. Under specific environmental conditions, these organisms can proliferate massively, leading to a phenomenon known as “Harmful Algal Blooms” (HABs), which not only compromises water quality, but also has serious economic and health impacts. The rapid overgrowth of microalgae can lead to oxygen depletion in aquatic systems, resulting in the death of fish and invertebrates. Additionally, HABs can have socio-economic consequences, often requiring the closure of fish and shellfish farms, the treatment of drinking water supplies, and restrictions on tourism in affected coastal regions. The most concerning impact, however, is on human health. Human intoxication can occur through inhalation, skin contact, or consumption of seafood contaminated by toxins, provoking the so called “Foodborne diseases”. Toxins can be divided into two main classes, i.e. regulated and emerging toxins. The European Food Safety Authority (EFSA) defines as “emerging toxins” marine and freshwater toxins that are not yet regulated in Europe, as no maximum permitted levels in seafood have been established. This lack of regulation is typically due to insufficient experimental evidence on human toxicity and/or ecotoxicity, the absence of sufficiently pure toxins for in-vitro and in-vivo toxicity studies, the lack of reference materials for validating analytical methods, and limited information on the distribution of these toxins in the food chain. In this context falls my PhD project, with the aim to meet the EFSA Emerging Risks Guidelines and the World Health Organization (WHO) Guidelines for Drinking Water Quality. My work was focused mainly on the development and validation of analytical methods based on Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) for detection and quantification of some classes of emerging toxins, such as Ciguatoxins, Palytoxins and Cyanobacteria, optimizing different LC-HRMS methods for each of them. These methods were subsequently applied to analyse complex matrices, such as fish tissue or macroalgae, to determine the presence of known compounds, and, using the untargeted analysis, detect new congeners. Chapter 1 offers a broad introduction that addresses marine microalgae and freshwater cyanobacteria, the effects of HABs on human health and environment and an excursus on marine and freshwater toxins, with an in-dept description of emerging toxins. The focus is mainly on three classes of emerging toxins: Ciguatoxins, Palytoxins and Cyanotoxins. Chapter 2 is a description of the analytical techniques used in my PhD project, i.e. UV-Vis and LC-HRMS. The theoretical aspect and the used methodologies are briefly discussed. In addition, the molecular networking workflow is outlined. Chapter 3 describes the investigation of the toxin profile of Gambierdiscus balechii using a two-tiered approach based on LC-HRMS and N2a-bioassay. A bioassay-guided micro-fractionation was performed, allowing to identify three biologically active fractions exhibiting CTX-like and MTX-like toxicity. These fractions were in-dept analysed by LC-HRMS using an ad-hoc developed method and revealed the presence of a putative I/C-CTX congener, identified for the first time in G. balechii, 44-methylgambierone was confirmed at 8.6 pg cell-1, and MTX-like compounds were hypothesized. Chapter 4 describes a preliminary study on the stability of standard samples of palytoxin under various experimental conditions typically encountered during isolation procedures. Two analytical techniques were employed: liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode and UV-Vis spectrophotometry at two different temperatures of analysis, with the aim to evaluate the influence of temperature and time on its stability Chapter 5 reports the optimization and the application of an isolation procedure for ovatoxin-a (OVTX-a) from Ostreopsis cf. ovata cell pellets. The aim of this work was to isolate a sufficient quantity of OVTX-a with a purity grade above 90% to measure, in collaboration with national and foreign partners, toxicity in vivo through different routes of administration, to support preliminary stability studies for the production of OVTX-a certified reference material (CRM) not yet available on the market. The procedure, already developed by MarBioTox lab, is composed of 4 steps, i.e. extraction, Medium Pressure Liquid Chromatography (MPLC), semi-preparative HPLC (HPLC-step1) and preparative HPLC (HPLC-step2). Each step was carefully evaluated, and the application of the overall procedure allowed us to isolate an OVTX-a fraction with a purity grade of 94%. Chapter 6 describes the comparison between two complementary analytical methods in the identification of OVTXs in Ostreopsis samples. In detail, two O. cf. ovata pellets (CBA2-122 and CBA1410) and one O. spp. pellet (CAWD221) were extracted and subjected to micro-scale oxidation. The extracts and the oxidated samples were analysed using two methods, i.e. LC-MS/MS for targeted analyses and LC-HRMSn for targeted and untargeted analyses. The combination of the two allowed us to identify a new putative PLTX-like compound, namely Compound 1 in CAWD221. Chapter 7 reports the activity performed during my period abroad at Institute of Nanoscience and Nanotechnology of the National Centre for Scientific Research “Demokritos”, Athens, Greece. Particularly, the development and validation of an analytical method for identification and quantification of microcystins, anabaenopeptins and nodularin based on SPE coupled to LC-HRMS was carried out. The defined protocol was then applied on environmental samples collected from water sources of the water company “Acqua Bene Comune” (ABC), Naples, for targeted analyses. Moreover, an untargeted approach to investigate the presence of additional contaminants using Feature Based Molecular Networking (FBMN) and dedicated databases was accomplished. In Chapter 8 a description of the activities performed during the 6 months spent at the private company ABC - Acqua Bene Comune Napoli Azienda Speciale, Via Argine 929, 80147 Napoli ABC S.p.a., https://www.abc.napoli.it/ is reported. During this period, I was able to learn how to perform routine test on water samples provided to ABC from different parts of the city.

Downloads

Actions (login required)

Modifica documento