Polycyclic aromatic hydrocarbons (PAHs) are persistent and potentially carcinogenic contaminants that accumulate in marine bivalves, raising concerns for ecosystem integrity and seafood safety. However, integrated assessments simultaneously addressing contamination levels, source apportionment, ecological risk and human health implications in commercially traded clams remain scarce. This study quantified 16 priority PAHs in edible clams marketed in Portugal and originating from the North Atlantic coast, the North Sea, and the Mekong Delta (Vietnam), with the aim of characterising contamination patterns and evaluating associated risks. PAHs were extracted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and analysed by gas chromatography-mass spectrometry (GC-MS). Source identification was supported by diagnostic ratios, principal component analysis, and linear discriminant analysis. Ecological risk was estimated through toxicity equivalents and risk quotients, while human exposure was assessed via average daily intake, hazard index, and lifetime carcinogenic risk. Total PAH concentrations ranged from 114.20 to 265.77 μg kg-1 wet weight, with high-molecular-weight PAHs accounting for ≈ 96% of the total burden, indicating dominant combustion-derived inputs. Elevated toxicity equivalents and cumulative risk quotients revealed chronic ecological risk at all sites, particularly in North Sea samples. In contrast, estimated dietary exposure remained below established safety thresholds, and carcinogenic risk values fell within the accepted tolerable range. These findings highlight a contrast between ecological risk and comparatively low human health risk, reinforcing the role of edible bivalves as early-warning bioindicators of coastal PAH contamination and supporting their use in integrated environmental monitoring programmes.

The Lima River basin, located in the northwest of the Iberian Peninsula and influenced by intensive agricultural activity, lacks recent information regarding pesticide occurrence, environmental partitioning, and associated ecological risks. This study investigated the occurrence, distribution, partitioning behaviour, and ecological risks of 56 pesticides in the Lima River through the integrated analysis of dissolved aqueous phase (DAP) and suspended particulate matter (SPM), combined with mixture-based ecological risk assessment and ecotoxicological bioassays.Pesticides were frequently detected in both matrices, with detection frequencies ranging from 59.38–65.63% in DAP and from 78.67–94.79% in SPM, depending on pesticide class. Total insecticide concentrations ranged from 172.25–221.72 ng L-1 in DAP and from 35.72–58.40 μg kg-1 in SPM, depending on the season. Multivariate analyses revealed clear seasonal patterns, with higher pesticide occurrence in the DAP during spring and summer, whereas the SPM preferentially accumulated more hydrophobic and persistent compounds during autumn and winter.Risk assessment identified aquatic invertebrates as the most sensitive trophic group, with insecticides being the main contributors to mixture toxicity. A limited number of insecticides, including dichlorvos, cyfluthrin, and azinphos-methyl, accounted for a substantial proportion of the predicted ecological risk.Overall, the results indicate that pesticide-related ecological risk in the Lima River is strongly influenced by seasonal variability, phase partitioning, and mixture-driven toxicity, highlighting the importance of integrated multi-matrix approaches for environmental monitoring and river basin management.

Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout (Salmo trutta), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.

The brown trout (Salmo trutta) is a commercially and ecologically significant salmonid fish, yet its hepatic cellular and functional dynamics throughout the reproductive cycle remain poorly characterised, particularly in males. This study investigated seasonal and sex-specific liver plasticity across four reproductive stages: spawning capable (December), regressing (March), regenerating (July), and developing (November). We quantified mRNA and protein abundance of key oestrogen-responsive targets—vitellogenin (VtgA) and zona pellucida (ZP) proteins—alongside cell turnover markers, caspase 3 (Casp3) and proliferating cell nuclear antigen (PCNA). These molecular endpoints were integrated with stereological analyses to estimate hepatocyte, nuclear, and cytoplasmic volumes. Results revealed stage-dependent mobilisation and transient hepatic retention of reproductive proteins; females exhibited stronger vitellogenic signatures and more pronounced seasonal shifts than males. Although VtgA and ZP mRNA levels peaked during the developing and spawning-capable stages, males maintained low but consistent levels throughout the cycle, indicating constitutive hepatic oestrogen sensitivity. Regarding cell turnover, PCNA protein data indicated heightened proliferative activity during the spawning-capable and regressing stages. In contrast, while Casp3 mRNA levels remained stable across all stages, protein detection suggested a post-transcriptional increase in apoptotic signalling during the developing phase, consistent with controlled tissue remodelling rather than extensive cell loss. Stereological data confirmed enlarged hepatocyte and nuclear volumes during periods of high secretory and proliferative demand. Overall, these findings demonstrate significant stage-dependent and sex-specific plasticity in brown trout liver, providing a robust reference framework for ecological monitoring, endocrine disruption assessments, and studies of teleost reproductive physiology.

To describe the structural development of the liver in young guppies (Poecilia reticulata) and complement the adult characterisation, we conducted a histological study at 7, 17, and 28 days post-birth. We used serial sections to trace the organisation of blood vessels, bile ducts, and pancreatic tissue within and around the liver. At all ages, the liver was highly cellular and composed mainly of hepatocytes. Pigmented melanomacrophage centres or aggregates were not observed, whereas rodlet cells were rare and restricted to extrahepatic connective tissue. At 7 days, we identified four distinct venous entry points. This multi-afferent pattern persisted at Days 17 and 28. Arterioles remained restricted to the outer hilar region and were not observed within the liver. The gallbladder and common hepatic duct were recognisable at Day 7. In contrast, intrahepatic bile ducts were not discernible at Day 7, were scarce at Day 17, and were more frequent, although still limited, at Day 28. Exocrine pancreatic tissue was mainly extrahepatic at Day 7; intrahepatic pancreatic acini were detected in one fish at this age and were increasingly observed at Days 17 and 28 in association with afferent venous tracts. No vascular-biliary associations comparable to those described in adult guppies were observed. These findings establish a histological baseline for normal post-hatching liver development in guppies and provide a reference for future studies in comparative anatomy and of environmentally associated changes in liver structure during early life stages.

Characterization of sex and biometry of fish donors for primary cell culture is often overlooked. Consequently, the influence of donor biological variability—such as reproductive status—on basal estrogenic expression in fish hepatocytes remains underexplored in toxicological studies. This study examines whether inherent differences among 1-year-old juvenile brown trout (Salmo trutta) are reflected in estrogenic signatures in liver tissue and the corresponding primary hepatocyte cultures from the same individuals. To our knowledge, no previous research has systematically assessed interindividual variation in estrogenic markers in brown trout primary hepatocytes. To address this, we evaluated the gene and protein expression of four classical estrogenic targets: vitellogenin, zona pellucida protein, estrogen receptor alpha, and estrogen receptor beta. No statistically significant differences in basal expression levels were observed across male and female donors in either hepatocytes or liver tissue. Nonetheless, interindividual variability was evident, reflecting natural biological differences in gonadal development and physiological status. The findings highlight the need to document and control donor sex, reproductive status, and individual variability when selecting fish for hepatocyte culture, even when sex differences in basal expression are not statistically significant, thereby providing a crucial baseline for toxicological experiments using primary trout hepatocytes.

Global warming raises surface water temperatures, impacting fish alongside pollutants, such as ubiquitous xenoestrogens. Combined stressor effects are poorly studied but likely to worsen impacts and hinder biota adaptation, warranting further research. Unadapted fish face heightened risks. The liver is a vital metabolic organ, sensitive to temperature and xenoestrogens, eventually adjusting hepatocyte size and number to ensure survival, growth, and reproduction. This study assessed, for the first time, the impact of exposure (45 days) to thermal stress (29 °C versus 26 °C) and ethinylestradiol (EE2, 5 ng/L) on male guppies, primarily on body and quantitative liver morphology. Higher temperature reduced body mass (14%) and standard length (3.6%) gain. EE2 exposure reduced body mass increase (14%), hepatosomatic index (20%), and the volumes of the liver (32%), hepatocytes (16%), and their nuclei (17%). The nucleus-to-cytoplasm ratio and total hepatocyte number remained stable. No histopathological lesions existed. Guppies appear to have adapted to stressors by reducing hepatocyte size and utilizing lipid reserves, yet they exhibited deficits in body growth and hepatosomatic index. Gonadal maturation was unaffected. Only under EE2 at 29 °C did hepatocytes show minimal lipid droplet content (less vacuolation). This indicated exhausted reserves, reinforcing how heat and toxicants interact to exacerbate impacts.

Coastal areas adjacent to urban and industrial regions are vulnerable to pollution from polycyclic aromatic hydrocarbons (PAHs), posing potential risks to both ecosystems and human health. This study quantified 16 priority PAHs in seawater and blue mussels (Mytilus edulis) from four beaches along Portugal's northwest coast over two years for seawater samples and four years for mussel samples. PAHs were measured in the dissolved aqueous phase (DAP), suspended particulate matter (SPM), and mussels using gas chromatography-mass spectrometry (GC-MS). Mean concentrations were 13.3 ng/L (DAP), 134.0 μg/kg dw (SPM) and 136.9 μg/kg ww (mussels). Source identification indicated both pyrogenic (wildfires) and petrogenic (maritime activity) inputs. Toxicity assessment revealed low carcinogenic potential in DAP (TEQ ≈ 0.7 ng/L) and moderate risk in SPM (TEQ ≈ 6 μg/kg dw). Risk coefficients classified DAP as low risk and SPM as moderate risk. In mussels, although the hazard index values were below 1, the estimated lifetime cancer risk (≈ 2.5E-05) exceeded the EU safety threshold of 1.0E-06, indicating a potential carcinogenic risk from human consumption. These findings emphasize the necessity for continuous monitoring and more robust regulatory measures to manage PAH contamination in coastal environments, thereby mitigating associated ecological and public health risks.

Polycyclic aromatic hydrocarbons (PAHs) and global warming impact aquatic ecosystems, eventually interacting. Monolayer (2D) cultures of cell lines, such as the rainbow trout liver RTL-W1, are employed for unveiling toxicological effects in fish. Nonetheless, three-dimensional (3D) models constitute an alternate paradigm, better emulating in vivo responses. Here, ultra-low attachment (ULA) plates were used to generate ten-day-old RTL-W1 spheroids for exposure to a control, a solvent control (0.1% DMSO) and the model PAH benzo[k]fluoranthene (BkF) at 10 and 100 nM and at 18 and 23 °C (thermal stress). After a 4-day exposure, spheroids were analyzed for viability (alamarBlue and lactate dehydrogenase), biometry (area, diameter and sphericity), histocytology (optical and electron microscopy), and mRNA levels of the detoxification-related genes cytochrome P450 (CYP)1A, CYP3A27, aryl hydrocarbon receptor (AhR), glutathione S-transferase (GST), uridine diphosphate–glucuronosyltransferase (UGT), catalase (CAT), multidrug resistance-associated protein 2 (MRP2) and bile salt export protein (BSEP). Immunocytochemistry (ICC) was used to assess CYP1A protein expression. Neither temperature nor BkF exposure altered the spheroids’ viability or biometry. BkF modified the cell’s ultrastructure. The expression of CYP1A was augmented with both BkF concentrations, while AhR’s increased at the higher concentration. The CYP1A protein showed a dose-dependent increase. Temperature and BkF concurrently modelled UGT’s expression, which increased in the 100 nM condition at 23 °C. Conversely, CYP3A27, MRP2, and BSEP expressions lowered at 23 °C. CAT and GST mRNA levels were uninfluenced by either stressor. Overall, BkF and temperature impacted independently or interactively in RTL-W1 spheroids. These seem to be useful novel tools for studying the liver-related effects of temperature and PAHs.

This study investigates the presence of persistent organic pollutants (POPs), specifically polychlorinated biphenyls (PCBs), in edible clams from the North Atlantic Ocean (Portugal and North Sea coastlines) and the South China Sea (Vietnam), available in Portuguese supermarkets. The study unveils the average PCB concentrations in clams, with European specimens containing 64.4 μg/kg wet weight (ww) and Vietnamese having 35.6 μg/kg ww. Both values fall below the European threshold of 75 μg/kg ww, indicating low immediate health risks. However, the study also estimates the daily intake of PCBs, raising concerns about long-term health risks. Moreover, all measured PCB values exceed the international lifetime cancer risk safety limit, with European clams showing a higher cancer risk (1.0E-05) compared to the Vietnamese clams (7.4E-06). The sources of contamination are linked to industrial zones, including oil facilities and recycling plants, particularly in Western Europe, highlighting the need for continuous environmental monitoring.

Aquatic ecosystems are increasingly threatened by multiple anthropogenic stressors, notably climate change and pollution by pharmaceuticals. Global warming is predicted to raise water temperatures by 2–5 °C by the end of the century. As ectotherms, fish are particularly vulnerable due to limited thermal tolerance and temperature-dependent physiology. Pharmaceuticals are introduced into aquatic systems at concentrations ranging from ng·L−1 to µg·L−1, including widely prescribed classes such as antibiotics, hormones, analgesics, antifungals, and neuropsychiatric drugs. This narrative review synthesizes experimental evidence on the interactive effects of warming and pharmaceutical exposure in fish. Thirty-nine peer-reviewed studies published since 2005 were analyzed. The findings indicate that higher temperatures often exacerbate pharmaceutical-induced toxicity, altering oxidative stress, metabolism, reproduction, and behavior. Antibiotic-focused studies showed temperature-dependent acceleration of absorption, distribution, metabolism, and excretion, with shorter half-lives and reduced tissue persistence at higher temperatures. Estrogenic hormones and antifungals have been shown to interact with thermal regimes, disrupting reproductive physiology and skewing sex ratios, particularly in species exhibiting temperature-dependent sex determination. Neuropsychiatric drugs exhibited altered uptake and metabolism under warming conditions, resulting in increased brain bioaccumulation and behavioral alterations affecting ecological fitness. Analgesics and anti-inflammatories remain understudied despite their widespread use, with evidence suggesting synergistic effects on oxidative stress at elevated temperatures. Significant research gaps persist regarding chronic exposures, early developmental stages, ecologically relevant temperature scenarios, and underrepresented or absent drug classes, such as hypolipidemic drugs. Ultimately, broader and integrated approaches are needed to better understand and predict the ecological risks of pharmaceutical pollution in a warming world.

The Ohrid trout (Salmo letnica) is a species endemic to Lake Ohrid (shared by Albania and North Macedonia), which is internationally recognized for its geological longevity and unique natural features. Given that the species has distinctive biological, ecological, and evolutionary characteristics, as well as significant economic value, the decline in this trout’s population is a serious and urgent problem, deserving continuous, scientifically based management. Yet, although it is considered a “Fossil Trout”, research on this species remains limited in relation to science and conservation. To understand the current state of the art, we conducted a systematic review in Web of Science, analyzing 31 indexed articles about the Ohrid trout. These studies primarily focused on the seasonal morphological characteristics of specific organs, phylogenetics, and, to a lesser extent, the impacts of environmental contamination. However, notable gaps exist in understanding sex- and stage-specific physiology, morphotype diversity, and pollutant bioaccumulation. To address these limitations, integrative strategies that combine multi-omics biomarker development, genetic screening of broodstock, and systematic monitoring of pollution and climate-related stressors are crucial. Regional authorities should work with international organizations to establish long-term monitoring of S. letnica. This review aims to provide a critical foundation for overcoming the “Living Fossil Left Behind by Science” paradigm and to foster global initiatives to preserve the long-term survival and evolutionary legacy of this endangered species.

Polycyclic aromatic hydrocarbons (PAHs), prevalent aquatic contaminants, arise from burning fossil fuels, a major source of greenhouse gases driving global warming. PAHs and warmer temperatures individually exert diverse negative effects on aquatic organisms. However, the effects of PAH exposure and/or rising temperature remain largely unknown. Liver in vitro models, like the rainbow trout (Oncorhynchus mykiss) RTL-W1 liver cell line, have been employed to unravel PAH-exposure effects, primarily on cell viability and enzymatic activity. Here, monolayer-cultured (2D) RTL-W1 cells were used to assess the co-exposure effects of temperature (18 and 21 °C) and two PAHs, benzo[a]pyrene (B[a]P) and benzo[k]fluoranthene (B[k]F), at 10 and 100 nM. After a 72 h exposure, the cell density and viability were evaluated using the trypan blue and LDH assays. The mRNA levels of the detoxification-associated genes aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP)1A, CYP3A27, glutathione S-transferase omega 1 (GSTO1), uridine diphosphate-glucuronosyltransferase (UGT), catalase (CAT), and multidrug resistance-associated protein 2 (MRP2) were measured by RT-qPCR. Temperature influenced cell viability and LDH leakage. Both PAHs reduced the cell density and upregulated the mRNA levels of AhR, CYP1A, CYP3A27, and UGT, while GSTO1 and MRP2 were only augmented after the higher B[k]F concentration. Temperature influenced CAT and UGT expression. There was no interaction between temperature and the PAHs. Overall, the results show that B[k]F has more effects on detoxification targets than B[a]P, whereas a temperature increase mildly affects gene expression. The RTL-W1 in 2D seems useful for unravelling not only the liver effects of PAH but also the impact of temperature stress.

The concentrations and spreading of eight synthetic and two natural progestins (PGs) were investigated in surface waters from ten sites at the Douro River Estuary. Samples were filtrated and subjected to solid-phase extraction (SPE) to isolate and concentrate the target PGs. The extracts were cleaned by silica cartridges and analyzed by LC-MS/MS. The finding of biologically relevant amounts of gonanes (22.3 ± 2.7 ng/L), progesterone derivatives (12.2 ± 0.5 ng/L), drospirenone (4.1 ± 0.8 ng/L), and natural PGs (9.4 ± 0.9 ng/L) support the possibility of these compounds acting as endocrine disruptors. Despite the absence of significant differences amongst sampling sites and seasons, the principal component analysis (PCA) and the linear discriminant analysis (LDA) approaches reveal that spring and summer have different patterns of PG distribution compared to autumn and winter. The assessment of risk coefficients (RQs) and the potential concentrations of synthetic progestins in fish blood sustains that all tested compounds pose a significant risk to local biota (RQs > 1). Additionally, three progestins—norethindrone, norethindrone acetate, and medroxyprogesterone acetate—should reach human-equivalent therapeutic levels in fish plasma. Overall, the current data show PGs’ presence and potential impacts in one of the most important estuaries of the Iberian Peninsula.