Stefania Gorbi

Microplastics are increasingly recognized as pervasive contaminants in remote mountain freshwater ecosystems, yet evidence from high-altitude regions remains limited. This study investigated microplastics occurrence and characteristics in surface water, sediments, and fish from the Braldu-Shigar-Skardu freshwater system in Gilgit-Baltistan, northern Pakistan. Samples were collected from remote headwaters, tributaries, main river channels, and the Satpara Lake-Dam system. Fish originated from wild populations, farming facilities, and local markets to provide a broader assessment of microplastic exposure. Matrix-specific extraction protocols were applied, followed by stereomicroscope sorting and polymer identification by µFT-IR spectroscopy. Microplastics were detected in nearly all sites and matrices, with concentrations ranging from 1 to 17 MPs L ^-1 in water, 55–537 MPs kg ^-1 in sediments, and 1–11 MPs individual ^-1 in fish gastrointestinal tracts. Notably, highest frequency of microplastics occurrence was measured in farmed individuals (78%–83%), suggesting that aquaculture-related practices may represent an additional source of microplastics. Wild fish showed the lowest occurrence (25%), whereas those obtained from local markets revealed intermediate values (62%), indicating variable exposure across different pathways. Surface waters were dominated by small fragments and polypropylene, indicative of recent inputs and origin from fragmentation. Sediments showed a broader size distribution, and greater polymer diversity, consistent with their role as long-term sinks. The highest polymer diversity was highlighted in fish, indicating multiple exposure pathways. Spatial patterns did not show a geographical downstream increase, but rather a patchy distribution shaped by local anthropogenic input, hydrological connectivity, and depositional dynamics. Glacier-fed rivers primarily act as transport corridors, whereas low-energy environments such as Satpara Lake function as sinks promoting microplastics accumulation. Overall, the study provides the first integrated multi-compartment baseline for microplastics in Gilgit-Baltistan and supports future monitoring and management actions for these high altitude critical freshwater resources.

Nanoplastics (NPs) pose significant risks due to their small size and ability to penetrate biological tissues. However, the molecular pathways and cellular mechanisms affected by NP exposure in marine teleosts remain poorly understood, especially in tropical reef fishes. This study examined the impact of short-term (7 days) waterborne exposure of 100 nm-carboxyl-modified polystyrene NPs on the false clownfish (Amphiprion ocellaris) exposed at two daily concentrations: low (20 µg/L, environmentally relevant) and high (2000 µg/L). A multidisciplinary approach, including biochemical and transcriptomic analyses, was conducted to assess toxic effects. Biochemical assays revealed limited changes in antioxidant defenses (CAT, GR, GST, TOSC). However, the Integrated Biomarker Response index (IBRv2i) suggested a compromised physiological condition, supported by transcriptomic data. Transcriptomic profiling revealed 409 significantly differentially expressed genes (DEGs) in the high-concentration and 354 DEGs in the low-concentration groups, with 120 shared DEGs mostly upregulated and indicative of a core molecular response. Collectively, the transcriptional profile of the low-concentration group resembled an early-warning, energy-reallocation strategy aimed at preserving essential sensory functions while minimizing expendable functions. The high-concentration group amplified the shared stress signature and recruited an additional 289 unique genes, resulting in pronounced enrichment of Gene Ontology terms related to “muscle contraction”, “oxygen transport”, “hydrogen-peroxide catabolism”, and “extracellular-matrix”. This study demonstrates that PS-NP exposure can alter gene expression and physiology in juvenile reef fish, even at environmentally relevant concentrations. Molecular responses varied with concentrations highlighting the role of exposure level in influencing biological systems and potential long-term impacts of NP pollution in marine environments.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin’s origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg⁻¹) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg⁻¹) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a “potential vector” should therefore be further investigated.

Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg⁻¹ and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg⁻¹) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg⁻¹). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.