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Azaspiracids (AZAs) are marine polyether biotoxins produced by dinoflagellates that accumulate in filter-feeding organisms and pose a threat to human health and seafood safety. This study presents the first comprehensive analysis of azaspiracid analogs in shellfish from the Adriatic Sea with the use of high-resolution mass spectrometry. AZA-2 was quantified in samples collected from Šibenik Bay between January and May 2024, with the highest concentrations observed in early January. In addition to AZA-2, several known analogs (AZA-6, AZA-9, AZA-10, AZA-19, AZA-41, and AZA-43) and a potentially new analog (m/z 884.4928) were also detected. The fragmentation patterns of this new analog indicate a structural similarity to AZA-19 with a possible double bond modification. Potential pitfalls regarding the misinterpretation of spectra derived from molecules containing 13C atoms were recognized and addressed. The presence of multiple analogs, some of which have high toxic potential, suggests that regulatory practice should consider including more than three analogs in the monitoring program.

The marine diatoms Pseudo-nitzschia spp. are globally distributed primary producers, with certain species capable of producing neurotoxin domoic acid (DA), causing amnesic shellfish poisoning (ASP). This study investigates the toxicity and growth rates of Pseudo-nitzschia species isolated from aquaculture areas in the Central Adriatic Sea. A total of 54 strains from eight species were analysed, with strains originating from four different study sites. Growth rates and toxin production were examined across different growth phases in other species and strains. Most species, including P. allochrona, P. calliantha, P. delicatissima, P. fraudulenta, P. galaxiae, P. mannii, and P. multistriata, did not produce DA at any growth phase. The only species that showed toxin production, at 18 °C was P. pseudodelicatissima (0.0007–0.0250 pg cell−1). Notably, the majority of P. pseudodelicatissima strains were toxic in the decay phase and some strains showed continuous toxin production throughout all growth phases. The highest growth rates for the analysed species were recorded in strains of P. delicatissima, which also exhibited the highest cell abundance (8.19 × 105 cell mL−1), followed by the species P. allochrona, P. mannii and P. pseudodelicatissima.

Pufferfishes are among the best-known marine organisms that accumulate marine biotoxins such as Tetrodotoxin (TTX). In the Mediterranean Sea, the silver-cheeked toadfish Lagocephalus sceleratus is the most reported TTX-bearer, causing many fatal and non-fatal cases. In Lebanon, no previous studies have measured TTX levels although the possibility of TTX-poisoning is high since L. sceleratus is caught in different sizes and can be mistaken with other small fishes. Hence, this study reports TTX and its analogue 4,9-anhydro TTX in L. sceleratus collected from Lebanese waters in the Eastern Mediterranean Sea. The results show that TTX concentrations in fish tissues varied between 0.10 and 252.97 µg/g, while those of 4,9-anhydro TTX oscillated between 0.01 and 43.01 µg/g. Internal organs of L. sceleratus were the most toxic parts of its body, with the highest TTX levels found in gonads (mainly ovaries) and liver, followed by the muscles and skin with concentrations always exceeding the safety level. Toxicity fluctuations of L. sceleratus, its expansion, ecological and economic effects were also elucidated. Based on the present findings, it has been confirmed that L. sceleratus constitutes a health, ecological and economic risks, and therefore its trade in seafood markets should be banned to avoid any potential intoxication.

Over the last decade, bisphenol A (BPA) has become a chemical of concern in the marine environment. There is little data on BPA levels in the eastern Adriatic Sea, Croatian waters. This study provides concentrations of BPA in marine sediments and suspended matter sampled from the Kaštela Bay (the central part of the Adriatic Sea) for two years. The results obtained show that BPA in sediment samples ranged from 1.05 to 46.31 µg kg−1, while they were higher in the suspended matter, ranging from 1.84 to 81.39 µg kg−1. To demonstrate a possible correlation between BPA concentrations and other parameters in sediment and suspended matter, the granulometric composition and organic matter content were determined. There was no correlation between BPA and investigated sediment characteristics. To assess the possible effects of BPA on marine biota, its possible estrogenic effect was also investigated by calculating estradiol equivalent concentration (EEQ, µg kg−1), the maximum value of which, in this study, was 0.0181 µg kg−1 in the sediment samples and 0.0317 µg kg−1 in suspended matter samples. Based on the presented results, it is unlikely that BPA could cause significant endocrine disruption to marine life in the study area.

This is the first study that presents concentrations of domoic acid detected in the whole shellfish tissue from breeding and harvesting areas along the Croatian coast of the Adriatic Sea during the period 2006 to 2008. Shellfish sample analyses after SAX cleaning procedures, using a UV-DAD-HPLC system, showed the presence of domoic acid in four species. The most prevalent of those species were the blue mussel (Mytilus galloprovincialis), followed by European flat oyster (Ostrea edulis), Mediterranean scallop (Pecten jacobaeus) and proteus scallop (Flexopecten proteus). Domoic acid, a potentially lethal phycotoxin that causes amnesic shellfish poisoning (ASP), was detected for the first time in January 2006 with the highest value of 6.5486 μg g-1 in whole shellfish tissue. Pseudo-nitzschia spp. bloom events preceded these high domoic acid concentrations. According to this study, retention of domoic acid in the blue mussel M. galloprovincialis is more than 42 days. This investigation indicates the first presence of domoic acid in Croatian shellfish, but in concentrations under the regulatory limit (20 μg g-1), therefore shellfish consumption was not found to endanger human health.

The Croatian National Monitoring Program revealed the presence of Diarrhetic Shellfish Poisoning (DSP) toxicity in Mediterranean blue mussel (Mytilus galloprovincialis) from breeding farms in southern Adriatic Sea through January to June 2011. The mouse bioassay tests (MBA; at the time the official method for DSP toxins) were accompanied by atypical symptomatology in the animals and this caused doubts about the assay results. Consequently, in parallel studies reported here, the concentration of Cd, Cr, Cu, Ni, Pb and Zn in soft tissue of DSP positive and negative mussels samples was determined. Cd, Cr, Zn and Ni show higher values in approximately 75% of the DSP positive samples, whereas for Pb and Cr the values were 26% and 34%, respectively. This trend was unchanged during the whole observation period.

This paper deals with Croatian counties cluster analysis as the base for developping a proactive demographic policy. Unfortunately, Croatia has no national demographic strategy and no national population policy is carried out. Some local governments are taking isolated policy measures but due to an unsystematic and distressed network at the national level it has to date given no significant effects. The Croatian nation is currently experiencing the initial process of demographic extinction. This process began even before the great emigration wave that started about a year and half ago. Since there are no financial resources for the simultaneous and complete implementation of an active demographic policy across the entire Croatian territory, this paper proposes a new approach. Namely, the main demographic indicators have been calculated and analyzed for each Croatian county. After that, using a multivariate methodology, fifteen demographic indicators that significantly differ from county to county were selected as criteria for clustering Croatian counties by k-means method. Clustering output defines several clusters consisting of a smaller number of counties with similar demographic characteristics. These clusters form a spatial county unit in which appropriate measures of an active demographic policy should be urgently implemented. In this way the process of active demographic policy can start with less financial resources and can be limited maybe only to spaces with poorest demographic characteristics. Moreover, the results of this study might very well stimulate \"richer\" government units to carry out the appropriate active demographic policy measures in their areas without waiting for the adoption of laws and regulations at the national state level.

Vertical profiles of trace metal (Cd, Pb, Zn, Cu, Ni) concentrations, organic matter content, carbonate content and granulometric composition were determined in two sediment cores from the submarine pit Dragon Ear (Middle Adriatic). Concentrations of the analyzed metals (Cd: 0.06-0.12 mg kg-1, Pb: 28.5-67.3 mg kg-1, Zn: 17.0-65.4 mg kg-1, Cu: 21.1-51.9 mg kg-1, Ni: 27.8-40.2 mg kg-1) were in usual range for Adriatic carbonate marine sediments. Nevertheless, concentrations of Cu, Zn, and especially Pb in the upper layer of sediments (top 12 cm) were higher than in bottom layer, while Cd and Ni concentration profiles were uniform. Regression analysis and principal component analysis were used to interpret distribution of trace metals, organic matter and carbonate content in sediment cores. Results of both analysis showed that concentrations of all trace metals in the core below the entrance to the pit were significantly positively correlated with organic matter and negatively correlated with carbonate, while in the core more distant from the entrance only Pb showed significant positive correlation with organic matter. Obtained results indicated that, except for lead which was enriched in surface sediment, in the time of sampling (before the building of the nautical marina) investigated area belonged to unpolluted areas. (PUBLICATION ABSTRACT)

Dinophysis spp. blooms and related shellfish toxicity events of diarrhetic shellfish poisoning (DSP) have been the most reported toxicity event through the Croatian National monitoring program. With the aim to characterize the DSP toxin profile in shellfish farmed in Croatia, for the first time a complete analysis of the toxin profile of Croatian mussels has been carried out using the LC-MS/MS technique. The obtained results showed okadaic acid (OA) as the main toxin contaminating Croatian mussels at that time. The maximum concentration of OA in shellfish tissue was recorded 12 days after the Dinophysis fortii bloom, thus suggesting that rapid growth of the toxin level in the shellfish occurred in the first week after the bloom while it was slower in the second week. Furthermore, the presence of only OA at concentrations which could endanger human health suggests D. fortii as the main organism responsible for the toxic event that occurred in Lim Bay. The presence of gymnodimine and spirolides in Croatian mussel has been detected for the first time, while the presence of yessotoxin and pectenotoxin-2 is confirmed.

With the aim of investigating whether yessotoxin (YTX) is responsible for diarrhetic shellfish poisoning (DSP) events in Croatian waters, three different methods were combined: a modified mouse bioassay (MBA) that discriminates YTX from other DSP toxins, the enzyme-linked immunosorbent assay method (ELISA) and liquid chromatography-mass spectrometry (LC-MS/MS). Among 453 samples of mussels and seawater analyzed in 2007, 10 samples were DSP positive. Results obtained by the modified MBA method revealed that most of the samples were positive for YTX, with the exception of samples from Lim Bay (LB 1) The ELISA method also identified the presence of YTX in these samples. DSP toxin profiles showed the presence of okadaic acid (OA) in three, and YTX in four out of nine samples that were analyzed by LC-MS/MS. The phytoplankton community structure pattern revealed Lingulodinium polyedrum (Stein) Dodge, which was present in the water prior to and/or during toxicity events at low concentrations (80 to 1440 cells L-1), as a potential YTX producing species. It is proposed that L. polyedrum cells accumulated in mussels and the subsequently observed toxicity may be related to metabolism after ingestion, resulting in carboxy YTX as the major analog in the mussel.