The widespread presence of microplastics (MPs) and associated plastic-derived compounds, bisphenols (BPs), in the environment—originating from plastic production or adsorption of environmental pollutants—results in unavoidable human exposure. Therefore, assessing their occurrence in human biological samples is essential for understanding potential health risks. In this study, pyrolysis gas chromatography–mass spectrometry was used to analyse human intraocular fluids—aqueous humour (AH) and vitreous humour (VH)—to identify 11 microplastic polymer clusters. A method based on dansyl chloride derivatisation combined with ultra-performance liquid chromatography–tandem mass spectrometry was employed to screen for five bisphenols (BPs) in blood serum collected from the same patients. Potential MPs isolated from ocular samples were qualitatively assessed by stereomicroscopy. The number of particles ranged from 14 to 69 per gram in AH and from 21 to 34 per gram in VH. Among AH samples, the most frequently detected polymer clusters were C-PVC, C-PMMA, C-PET, and C-PP, with average levels ranging from 0.41 to 0.84 µg/g. The three VH samples contained only C-PMMA, with an average concentration of 0.55 ± 0.12 µg/g. Polymers such as C-PA66 (0.30 ± 0.21 µg/g) and C-PA6 (0.15 ± 0.44 µg/g) were occasionally observed in AH. Analysis of 13 human serum samples demonstrated exposure to bisphenol A (BPA; 4.05–5.18 ng/mL) and bisphenol AF (BPAF; 2.13–4.54 ng/mL), but no clear correlation was observed between serum BP levels and intraocular MPs within the limitations of the present dataset. These findings suggest the presence of MPs and BPs in human biological samples; however, the results should be interpreted with caution owing to potential background contamination and the pilot nature of the study.

Microplastics (MPs) are a ubiquitous marine pollutant, and their presence in bivalves is receiving increasing attention due to the associated risks to human health. The steps of pretreatment, detection, and quantification in the analysis of MPs depend on the type of polymer. Research on MPs is challenging because of the varying characteristics of these materials, such as the size, shape, and polymer type. Consequently, there are no standardized methods for their collection, separation, identification, or quantification. This review specifically examines the available bivalve digestion steps, focusing on efficient and time-reducing methods, such as the microwave-assisted (MAW) procedure and its advantages. Recent achievements in the application of pyrolysis gas chromatography–mass spectrometry (Pyr-GC-MS) are presented for the profiling of polymer mass-related microplastics data in marine bivalves. Here, we provide an overview of the abundance, properties, and polymer types of MPs in bivalve species, highlighting the polymer mass fractions. To date, the available mass-based concentrations have revealed nine types of MPs—polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polystyrene (PS), polymethyl methacrylate (PMMA), polyamide 66 (PA66), polycarbonate (PC), and polyamide 6 (PA6)—with PE, PP, and PVC being the most common. The total MP levels in bivalves were at ppm levels, ranging from 0.26 µg/g to 36.4 µg/g wet weight. The risk of human ingestion of MPs was assessed through the consumption of bivalves as seafood. The overall potential human health risk value (H) for marine bivalves was classified within the moderate to high hazard category.

This study examines the longevity and sensory stability of Dalmatinski pršut, a Protected Geographical Indication dry-cured ham (Commission Implementing Regulation (EU) No 2016/189), under protracted refrigerated vacuum storage. The objective was to elucidate a more empirical basis for shelf-life determination, countering the prevalent practice of arbitrary expiration dating. Over a span of 210 days, at a controlled temperature of 4±2°C, we monitored the ham’s physicochemical properties (water activity water and salt content) and conducted comprehensive sensory evaluation. Findings show the excellent preservation of the ham’s sensory profile, with minimal deviations in flavour intensity and no significant alterations in textural parameters (hardness, solubility) or salinity levels. However, a key finding is the decade-long trend of reduced water content among producers, now ranging between 30–46%, which is below the specified range of 40–55%. Despite this discrepancy, the reduction in water content did not adversely affect the ham’s sensory attributes, indicating a robust resilience to changes in water content within the observed range. Our results challenge the current specifications for dry cured ham Dalmatinski pršut, suggesting a need for revision to reflect the empirical evidence of water content’s insignificant effect on sensory quality. This study highlights the importance of revisiting and potentially adjusting the regulatory standards for water content in dry cured ham Dalmatinski pršut, lowering the established lower limit to 30% and ensuring they are aligned with practical production realities and scientific findings, thereby enhancing the product’s shelf-life determination and maintaining its high-quality standards.

BACKGROUND The diagnosis of West Nile virus (WNV) is challenging due to short-term and low-level viremia, flavivirus cross-reactivity, and long immunoglobulin M (IgM) persistence. AIM To evaluate different methods for WNV detection [reverse transcription-polymerase chain reaction (RT-PCR), IgM/IgG antibodies, IgG avidity] in serum, cerebrospinal fluid (CSF), and urine samples of patients with confirmed WNV infection. METHODS The study included patients with confirmed WNV neuroinvasive infection (n = 62), asymptomatic WNV seropositive individuals (n = 22), and individuals with false-positive WNV IgM antibodies (n = 30). WNV RNA was detected using RT-PCR. A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test (VNT). IgG-positive samples were tested for IgG avidity. RESULTS The WNV-RNA detection rates were significantly higher in the urine (54.5%)/serum (46.4%) than in CSF (32.2%). According to the sampling time, the WNV-RNA detection rates in urine collected within 7 days/8-14/≥ 15 days were 29.4/66.6/62.5% (P = 0.042). However, these differences were not observed in the CSF. The median RT-PCR cycle threshold values were significantly lower in urine (32.5, IQR = 28-34) than in CSF (34.5, IQR = 33-36). The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF. Positive IgM/IgG antibodies were detected in 84.3/9.3% of serum samples collected within 7 days, 100/71.1% of samples collected 8-14, and 100% samples collected after ≥ 15 days. Recent WNV infection was confirmed by low/borderline avidity index (AI) in 13.6% of asymptomatic individuals. A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG. No significant correlation between ELISA IgG and VNT was found. CONCLUSION The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples. IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Praćenjem stanja okoliša putem mjerenja sadržaja različitih onečišćujućih tvari (kontaminanata) u školjkašima ujedno se mogu pratiti biološke promjene i štetni učinci onečišćujućih tvari kojima su ti organizmi izloženi te se može procijeniti utjecaj ljudskih aktivnosti na određeni ekosustav. Akumulacija različitih onečišćujućih tvari u školjkašima namijenjenim za prehranu je stalna opasnost za ljude, kao potrošače na vrhu hranidbenog lanca. Mikroplastika je složeni kontaminant, koji uključuje vrlo širok skup čestica koje u procjeni rizika zbog njihove iznimne raznolikosti (veličina, oblik, topljivost, sastav polimera, adsorbirane kemikalije, biološki materijal, itd.) predstavljaju jedinstveni izazov. Glavni razlog opasnosti konzumacije školjkaša leži u biologiji njihovog organizma budući se oni hrane filtriranjem vode ili mora u kojem se uzgajaju ili prirodno žive, nakupljajući u svom organizmu različite tvari iz okoline. U razumijevanju kemijskog rizika mikroplastike koristi se model profila polimera, odnosno toksičnosti njihovih monomera. Nesumnjivo, analiza mikroplastike uključuje uz kemijsku karakterizaciju i određivanje ukupnog broja čestica kako bi se procijenila izloženost ljudi mikroplastici temeljem podataka o prehrambenim navikama ciljane populacije. U ovom radu dan je pregled literaturnih spoznaja o istraživanjima mikroplastike u dvoljušturnim mekušcima, kemijskoj karakterizaciji mikroplastike i pojavnosti u različitim vrstama školjkaša.

The Mediterranean Sea is particularly endangered by microplastics (MPs), polymers, and additives. These contaminants can be ingested by fishes and, hence, translocate into tissues. We aimed to quantify MPs, polyethylene terephthalate (PET), polycarbonate (PC), bisphenol A (BPA), and p-phthalic acid (PTA) in the edible muscles of swordfish (Xiphias gladius) and bluefin tuna (Thunnus thynnus) caught in the Mediterranean Sea. The MPs were extracted from muscles and characterized by stereomicroscopy and Raman microspectroscopy, while the polymers (PET and PC) and additives (BPA and PTA) were identified by LC-MS/MS. The number of MPs ranged from 140 to 270 no. kg−1 in swordfish and from 160 to 270 no. kg−1 in tuna. The most frequent MP polymer was polypropylene in swordfish (33%) and in tuna (34.7%), while the most abundant pigments were PB115, PB116, PBr101/102. A similar level of plastic contamination was revealed in these two fish species with differences in shapes, colors, pigments and polymers of MPs.

Due to high anthropogenic pressures, science-based coastal management required to ensure the sustainable use of coastal areas highly depends on environmental indicators used for decision-making. In this paper, we argue for the inclusion of Vibrio spp. abundance as a supplemental indicator of water quality for science-based coastal management by examining the environmental and bacterial indicators at a fish farm and a control site in Mali Ston Bay in the Adriatic Sea. Unexpectedly, heterotrophic bacteria, enterococci and Vibrio spp. were more abundant in the cold season, while E. coli and total coliforms, following a more traditional pattern, were more abundant in the warm season. Each of the currently used indicators has a specific purpose: heterotrophic bacteria indicate the presence of both nonpathogenic and pathogenic bacteria, while enterococci are pathogenic bacteria indicating fecal pollution. Vibrio spp. abundance additionally represents a non-fecal bacteria that can cause vibriosis in humans and aquatic organisms. Since vibriosis is the leading cause of disease-related fish mortality in aquaculture, pathogenic Vibrio spp. have large health and economic implications. These implications, as well as the added interpretative value when compared to other bacterial indicators, make Vibrio spp. abundance a good candidate as a water quality indicator. Significant dependence of the abundance on depth further differentiates Vibrio spp. from other indicators, thus bolstering the candidacy - especially in aquaculture areas. Before inclusion of any Vibrio spp. indicators into legislature, further research is needed particularly into (i) abundance thresholds characterizing water quality, and (ii) identification of species whose abundance should be monitored for best estimate of the disease risks.

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks before the disease onset. High titers of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) were found in both serum and cerebrospinal fluid (CSF) samples, indicating acute TBEV infection. West Nile virus (WNV) and Usutu virus (USUV) IgM and/or IgG antibodies were also detected, and a virus neutralization test (VNT) was performed. A high titer of TBEV neutralizing (NT) antibodies (640) was detected, which confirmed acute TBE. However, NT antibodies to WNV and USUV were also detected (titer 80 for both viruses). After TBEV and WNV IgG avidity evaluation, previous flavivirus infection was highly suspected (avidity index 82% and 89%, respectively). Blood, CSF, and urine samples were negative for respective viruses' RNA. The presented case highlights the challenges in flavivirus serodiagnosis. In the published literature, different degrees of cross-reactivity or cross-neutralization between TBEV and dengue, louping ill, Omsk hemorrhagic fever, Langat, and Powassan virus were also observed. Therefore, the serology results should be interpreted with caution, including the possibility of cross-reactivity. In areas where several flaviviruses co-circulate VNT is recommended for disease confirmation.

Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.

This study aimed to identify surface mould species overgrowing the Croatian protected meat products “Istarski pršut” and “Dalmatinski pršut” and their effect on sensory properties. Dry-cured hams were produced in 2018/2019 and obtained from annual fairs. The predominant surface species found on “Dalmatinski pršut” were Aspergillus chevalieri, Penicillium citrinum and Aspergillus cibarius, whereas those overgrowing “Istarski pršut” were Aspergillus proliferans, P. citrinum and Penicillium salamii. The results show species diversity, higher presence, and greater variety of Aspergillus species in “Dalmatinski pršut” in comparison to “Istarski pršut”, and significant variations in 9 of 20 sensory attributes. Principal component analysis revealed a clear distinction between the two, and a large contribution of P. salamii and Penicillium bialowienzense to one principal component. The texture traits, smoky odour, muscle and subcutaneous fatty tissue colour, and mould species found are valuable for product characterisation. The results also indicate that mould species may be responsible for some sensory traits, such as tenderness, juiciness, and lesser freshness.

In the present study, a 4-year surveillance plan for the detection of lipophilic marine biotoxins in Mytilus galloprovincialis and Callista chione from different aquaculture farms and natural beds along the Central Adriatic coasts of Croatia was reported. The samples were analyzed by a validated LC–MS/MS method in accordance with the Regulation (EU) No 2019/627. Lipophilic marine biotoxins belonging to the okadaic acid group, except for azaspiracids, as well as yessotoxins, were found at different concentrations, always below the maximum limits established by the Regulation (EC) No 853/2004. The seasonal distribution showed the highest values in summer months, and an interannual variability was also observed, probably due to environmental conditions aging on the density of harmful algal blooms. The comprehensive data reported in this study should help the future research in making advancing prediction models along the Eastern Adriatic coast.

During the five stages of smoked dry-cured ham processing, proteolysis and protein oxidation were simultaneously detected in the Biceps femoris (BF) and Semimembranosus (SM) muscles. Proteolysis was more advanced in BF than in SM throughout the process of production. The total FAA increased significantly (p < 0.05) throughout the processing, resulting in higher total FAA content in BF than in SM muscle. SDS-PAGE revealed progressive degradation of sarcoplasmic proteins of investigated muscles, with the pronounced changes for the 69.9-41.7 kDa region. SDS-PAGE of BF showed more intense degradation of myofibrillar proteins due to greater proteolysis in BF. Electrophoresis of myofibrillar proteins evidenced the marked degradation of 130 kDa, 96.7 kDa and 27-20.7 kDa bands in both muscles. A similar trend was observed for protein oxidation in BF and SM, with the final values of 26.36 and 23.7 nmol carbonyls/mg proteins, respectively. The Pearson correlation revealed a strong relationship between protein oxidation and proteolysis.

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50; serum median 24.48, IQR 11.93–49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.

Mikroplastika (MP) je prepoznata kao okolišni kontaminant višestrukih opasnosti na temelju fizičkih učinaka zbog veličine čestica, oblika i koncentracije te kemijskih učinaka povezanih s opasnim kemikalijama. Sve je više dokaza o prisutnosti MP u hrani, a posljednjih godina se eksponencijalno povećava i broj publikacija o njezinoj prisutnosti u hrani morskog podrijetla. Međutim, analitičke metode detekcije u hrani morskog podrijetla su unatoč dostupnosti širokog spektra instrumentalnih tehnika za izolaciju i određivanje njezinih fizikalnih svojstava ograničene. Potpuni analitički postupak donosi podatke o kemijskom sastavu i morfološkoj strukturi čestica, ali niti jedna instrumentalna tehnika ne udovoljava svim zahtjevima analize MP. Primjena većeg raspona komplementarnih analitičkih metodologija u pogledu otkrivanja i identifikacije ispunjava zahtjevnost analize ovog okolišnog kontaminanta. Pojava i vrsta unesene MP, kao i njezini zdravstveni učinci koji se trenutno ispituju, uključuju biološke, kemijske i fizičke vrste utjecaja. U svrhu procjene utjecaja MP iz hrane morskog podrijetla na ljudsko zdravlje prvo se mora kvantificirati izloženost, a zatim utvrditi je li ta izloženost dovoljno velika da bi izazvala posljedice sa štetnim učincima. Izloženost ljudi hrani morskog podrijetla izaziva zabrinutosti zbog njezine kontinuirane akumulacije u okolišu. Cilj ovog rada je prikazati dosadašnje spoznaje o prisutnosti MP u hrani morskog podrijetla: školjkašima, ribi i rakovima kao glavnim izvorima. Posebno se analiziraju postojeći analitički pristupi ekstrakcije i instrumentalnog određivanja koncentracije, oblika i vrste materijala MP u zahtjevnim biološkim uzorcima. Istaknuta je potreba daljnje istraživačke aktivnosti o ovom kontaminantu, kao i potreba za stvaranje baze podataka koja je neophodna za uspostavu zakonodavstva u području hrane.

Loggerhead sea turtle ( C. caretta ) is the official European bioindicator of marine litter in the Mediterranean Sea. In 2019, 8 sea turtles, out of 28 specimens loggerhead on the Adriatic coast of Molise, were subjected to necropsy. The intestinal contents were collected and the microplastics until 0.45 μm were extracted. Qualitative and quantitative assessments were performed by stereomicroscope observation and spectroscopic analyses (attenuated total reflection-Fourier transform infrared spectroscopy, ATR-FTIR and Raman microspectroscopy, RMS). Moreover, the analytical quantification of polyethylene terephthalate (PET), polycarbonate (PC), para phthalic acid (PTA) and bisphenol A (BPA) in fat and liver tissues was performed by LC-MS/MS. Microparticles ranging from 0.45 μm to 1 mm were found in all turtles, for a total of 623, while plastic litter greater than 1 mm were found only in 4 specimens (ranging from 0.03 to 0.11 g). Nineteen different polymers and 10 pigments, including polyester (100% of animals), high-density polyethylene (50%) and polypropylene (50%) were identified. BPA, PTA and PET were detected in fat and liver tissues of all animals, while PC was found only in 50%. A major prevalence was registered in the abdominal fat tissue, although only PC compounds were significantly higher in abdominal tissue ( p < 0.05), except for free PTA with liver tissue being the most contaminated ( p < 0.05). Microplastics and additives surely impact the health status of turtles that showed gastrointestinal impairment and an important level of contamination in tissues. Graphical abstract

Characterization of five types of traditional Croatian dry-fermented sausages produced by family farms was performed via identification of superficial mycobiota, physicochemical, sensory, instrumental color, fatty acids & fat quality indices. Detailed characterization of these sausages aimed to achieve standardization of their production and composition and to establish and/or improve their specification protocols. Traditional sausages varied significantly (p < 0.05) in all analyzed parameters except for the number of mold isolates. Sausages coming from eastern Croatia had a greater mold species diversity, with the highest number of isolated mycotoxigenic species in Slavonian domestic sausage. Sensory evaluation showed good acceptability of all sausages. According to health recommendations, Kulenova Seka showed the most representable values for most of fat quality indices. The results suggest the need for certain modifications in fat & fatty acid composition and, to a lesser extent, in salt content, however not at the expense of product safety, quality and acceptability.

Usutu virus (USUV) is an emerging arbovirus isolated in 1959 (Usutu River, Swaziland). Previously restricted to sub-Saharan Africa, the virus was introduced in Europe in 1996. While the USUV has received little attention in Africa, the virus emergence has prompted numerous studies with robust epidemiological surveillance programs in Europe. The natural transmission cycle of USUV involves mosquitoes (vectors) and birds (amplifying hosts) with humans and other mammals considered incidental (“dead-end”) hosts. In Africa, the virus was isolated in mosquitoes, rodents and birds and serologically detected in horses and dogs. In Europe, USUV was detected in bats, whereas antibodies were found in different animal species (horses, dogs, squirrels, wild boar, deer and lizards). While bird mortalities were not reported in Africa, in Europe USUV was shown to be highly pathogenic for several bird species, especially blackbirds (Turdus merula) and great gray owls (Strix nebulosa). Furthermore, neurotropism of USUV for humans was reported for the first time in both immunocompromised and immunocompetent patients. Epizootics and genetic diversity of USUV in different bird species as well as detection of the virus in mosquitoes suggest repeated USUV introductions into Europe with endemization in some countries. The zoonotic potential of USUV has been reported in a growing number of human cases. Clinical cases of neuroinvasive disease and USUV fever, as well as seroconversion in blood donors were reported in Europe since 2009. While most USUV strains detected in humans, birds and mosquitoes belong to European USUV lineages, several reports indicate the presence of African lineages as well. Since spreading trends of USUV are likely to continue, continuous multidisciplinary interventions (“One Health” concept) should be conducted for monitoring and prevention of this emerging arboviral infection.