Exposure to Oxidized Multi-Walled CNTs Can Lead to Oxidative Stress in the Asian Freshwater Clam Corbicula fluminea (Müller, 1774).

The increasing attention that carbon-based nanomaterials have attracted due to their distinctive properties makes them one of the most widely used nanomaterials for industrial purposes. However, their toxicity and environmental effects must be carefully studied, particularly regarding aquatic biota. The implications of these carbon-based nanomaterials on aquatic ecosystems, due to their potential entry or accidental release during manufacturing and treatment processes, need to be studied because their impacts upon living organisms are not fully understood. In this research work, the toxicity of oxidized multi-walled carbon nanotubes (Ox-MWCNTs) was measured using the freshwater bivalve (Corbicula fluminea) after exposure to different concentrations (0, 0.1, 0.2, and 0.5 mg·L-1 Ox-MWCNTs) for 14 days. The oxidized multi-walled carbon nanotubes were analyzed (pH, Raman microscopy, high-resolution electron microscopy, and dynamic light scattering), showing their properties and behavior (size, aggregation state, and structure) in water media. The antioxidant defenses in the organism's digestive gland and gills were evaluated through measuring oxidative stress enzymes (glutathione-S-transferase, catalase, and superoxide dismutase), lipid peroxidation, and total ubiquitin. The results showed a concentration-dependent response of antioxidant enzymes (CAT and GST) in both tissues (gills and digestive glands) for all exposure periods in bivalves exposed to the different concentrations of oxidized multi-walled carbon nanotubes. Lipid peroxidation (MDA content) showed a variable response with the increase in oxidized multi-walled carbon nanotubes in the gills after 7 and 14 exposure days. Overall, after 14 days, there was an increase in total Ub compared to controls. Overall, the oxidative stress observed after the exposure of Corbicula fluminea to oxidized multi-walled carbon nanotubes indicates that the discharge of these nanomaterials into aquatic ecosystems can affect the biota as well as potentially accumulate in the trophic chain, and may even put human health at risk if they ingest contaminated animals.

Modified Atmosphere Packaging (MAP) for Seaweed Conservation: Impact on Physicochemical Characteristics and Microbiological Activity.

Conventional conservation techniques such as drying, salting or freezing do not allow for preserving the original characteristics of seaweeds. The present work aims to study the impact of minimal processing, in particular "Modified Atmosphere Packaging" (MAP), on the physicochemical characteristics and food safety of two seaweed species, "laver" (Porphyra umbilicalis) and "sea-lettuce" (Ulva lactuca), stored at 6 °C for 15 days. Different parameters were evaluated using analytical methods, namely the composition of headspace gases, color, texture, microorganisms, and volatile organic compounds (VOCs). The main findings of this study were that the MAP treatment was able to inhibit the respiration rate of minimally processed seaweeds, also preserving their color and texture. There was a remarkable reduction in the microbial load for P. umbilicalis treated under modified and vacuum atmospheres, and U. lactuca exhibited relatively steady values with no notable differences between the treatments and the control. Therefore, during the 15-day study period, both seaweeds met the requirements for food safety. GC-TOF-MS allowed to conclude that both MAP and vacuum treatments were more efficient in maintaining the odor characteristics of U. lactuca compared to P. umbilicalis with no significant differences throughout the storage days. Metabolic responses to diverse sources of abiotic stress seemed to account for most of the changes observed.

Hydrophobic DES Based on Menthol and Natural Organic Acids for Use in Antifouling Marine Coatings.

Marine biofouling negatively impacts industries with off-shore infrastructures, such as naval, oil, and aquaculture. To date, there are no ideal sustainable, economic, and environmentally benign solutions to deal with this phenomenon. The advances achieved in green solvents, as well as its application in different industries, such as pharmaceutical and biotechnology, have promoted the emergence of deep eutectic systems (DES). These eutectic systems have applications in various fields and can be revolutionary in the marine-based industrial sector. In this study, the main objective was to investigate the potential use of hydrophobic DES (HDES) based on menthol and natural organic acids for their use as marine antifouling coatings. Our strategy encompassed the physicochemical characterization of different formulations, which allowed us to identify the most appropriate molar ratio and intermolecular interactions for HDES formations. The miscibility of the resulting HDES with the marine coating has been evaluated and proven to be successful. The Men/OL (1:1) system proved to be the most promising in terms of cost-production and thus was the one used in subsequent antifouling tests. The cytotoxicity of this HDES was evaluated using an in vitro cell model (HaCat cells) showing no significant toxicity. Furthermore, the application of this system incorporated into coatings that are used in marine structures was also studied using marine species (Mytilus edulis mussels and Patella vulgata limpets) to evaluate both their antifouling and ecotoxicity effects. HDES Men/OL (1:1) incorporated in marine coatings was promising in reducing marine macrofouling and also proved to be effective at the level of microfouling without viability impairment of the tested marine species. It was revealed to be more efficient than using copper oxide, metallic copper, or ivermectin as antifouling agents. Biochemical assays performed on marine species showed that this HDES does not induce oxidative stress in the tested species. These results are a strong indication of the potential of this HDES to be sustainable and efficiently used in marine fouling control technologies.

Differentiation of the Organoleptic Volatile Organic Compound Profile of Three Edible Seaweeds.

The inclusion of seaweeds in daily-consumption food is a worthy-of-attention challenge due to their high nutritional value and potential health benefits. In this way, their composition, organoleptic profile, and toxicity must be assessed. This work focuses on studying the volatile organic compounds (VOCs) emitted by three edible seaweeds, Grateloupia turuturu, Codium tomentosum, and Bifurcaria bifurcata, with the aim of deepening the knowledge regarding their organoleptic profiles. Nine samples of each seaweed were prepared in glass vials, and the emitted headspace was analyzed, for the first time, with a gas chromatography-ion mobility spectrometry device, a highly sensitive technology. By statistically processing the collected data through PCA, it was possible to accurately differentiate the characteristic patterns of the three seaweeds with a total explained variance of 98%. If the data were pre-processed through PLS Regression, the total explained variance increased to 99.36%. The identification of 13 VOCs was accomplished through a developed database of compounds. These outstanding values in addition to the identification of the main emissions of VOCs and the utilization of a never-before-used technology prove the capacity of GC-IMS to differentiate edible seaweeds based solely on their volatile emissions, increase the knowledge regarding their organoleptic profiles, and provide an important step forward in the inclusion of these highly nutritional ingredients in the human diet.

Impacts of Deoxygenation and Hypoxia on Shark Embryos Anti-Predator Behavior and Oxidative Stress.

Climate change is leading to the loss of oxygen content in the oceans and endangering the survival of many marine species. Due to sea surface temperature warming and changing circulation, the ocean has become more stratified and is consequently losing its oxygen content. Oviparous elasmobranchs are particularly vulnerable as they lay their eggs in coastal and shallow areas, where they experience significant oscillations in oxygen levels. Here, we investigated the effects of deoxygenation (93% air saturation) and hypoxia (26% air saturation) during a short-term period (six days) on the anti-predator avoidance behavior and physiology (oxidative stress) of small-spotted catshark (Scyliorhinus canicula) embryos. Their survival rate decreased to 88% and 56% under deoxygenation and hypoxia, respectively. The tail beat rates were significantly enhanced in the embryos under hypoxia compared to those exposed to deoxygenation and control conditions, and the freeze response duration showed a significant opposite trend. Yet, at the physiological level, through the analyses of key biomarkers (SOD, CAT, GPx, and GST activities as well as HSP70, Ubiquitin, and MDA levels), we found no evidence of increased oxidative stress and cell damage under hypoxia. Thus, the present findings show that the projected end-of-the-century deoxygenation levels elicit neglectable biological effects on shark embryos. On the other hand, hypoxia causes a high embryo mortality rate. Additionally, hypoxia makes embryos more vulnerable to predators, because the increased tail beat frequency will enhance the release of chemical and physical cues that can be detected by predators. The shortening of the shark freeze response under hypoxia also makes the embryos more prone to predation.

Uncovering biodegradability and biocompatibility of betaine-based deep eutectic systems.

Deep eutectic systems (DES) have shown increasing popularity in last decade; however, the number of studies on the potential toxicity towards living organisms remains scarce. These studies are of the utmost importance to infer on the claimed non-toxicity and biocompatibility of DES. Most articles published, at this moment, only evaluate the toxicity towards a cell model or in different strains of bacteria. For this purpose, in this work, the effect of two DES (betaine:sorbitol:water 1:1:3 and betaine:glycerol 1:2) and their individual components were evaluated at different concentrations after administered via intraperitoneal injection in zebrafish (Danio rerio). The total antioxidant capacity, lipoperoxidation, and the activity of various enzymes that work in different antioxidant pathways (superoxide dismutase, glutathione peroxidase, catalase, and glutathione S-transferase) were assessed. The results show no significant toxicity within the tested concentrations: up to 5000 µM and 3000 µM, for the assays using the system betaine:sorbitol:water 1:1:3 and for betaine:glycerol 1:2, respectively. The toxicity of individual components was studied up to 1000 µM. Based on the encouraging results that have been obtained, it is safe to conclude that these two deep eutectic systems can be used as the new class of environmentally friendly solvents.

Proximate Composition, Physicochemical and Microbiological Characterization of Edible Seaweeds Available in the Portuguese Market.

BACKGROUND: The aim of this work was the study of the proximate composition and profile of fatty acids, minerals, and some microbiological aspects of four edible seaweed species (Chondrus crispus, Palmaria palmata, Porphyra sp., and Ulva sp.) available in the Portuguese market for food consumption, and produced in a national Integrated Multi-Trophic System (IMTA). METHODS: Moisture, ash, and total lipids were determined gravimetrically. Crude protein was analysed by Duma's combustion procedures. The total carbohydrate content was assayed by the phenol/sulphuric acid method. The assessment of the fatty acids methyl esters (FAMEs) was determined through GC-MS. Characterization of elemental analysis was performed by ICP-AES. Different standard microbiological methods were applied for microorganisms. Statistics were performed using the non-parametric Mann-Whitney U test to assess significant differences between samples. RESULTS: Lipid contents (n = 3) were very low (1.6-2.3%), particularly in Palmaria palmata, and Chondrus crispus (1.6-1.7%). The protein content (n = 4) varied from 14.4% in P. palmata to 23.7% in Porphyra sp. Carbohydrates (n = 3) were the major constituent of most seaweeds (31-34%), except in Porphyra sp., with higher content in proteins than carbohydrates. Regarding the fatty acid content (n = 4), in general, saturated fatty acids (SFAs) were the most abundant followed by polyunsaturated fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs). Among macro and trace elements (n = 3), Chondrus crispus shows the highest average content in Zn (71.1 mg ⋅ kg - 1 D.W.), Palmaria palmata the highest average content in K (124.8 g ⋅ kg - 1 D.W.), Porphyra sp. the highest average content in P (2.1 g ⋅ kg - 1 D.W.), and Ulva sp. the highest average content of Ca (5.5 g ⋅ kg - 1 D.W.), Mg (55.8 g ⋅ kg - 1 D.W.), and Fe (336.3 mg ⋅ kg - 1 D.W.). In general, Na and K were the most abundant elements among analysed seaweed. Additionally, the microbiological results (n = 4) comply with the Portuguese guidelines (subgroup 2D) on the application of general principles of food hygiene in ready-to-eat foods. CONCLUSIONS: Overall, the results highlight the potential of using these seaweeds as an alternative and sustainable source of elements and bioactive compounds to produce enriched food products with a beneficial potential for human nutrition.

Gadolinium ecotoxicity is enhanced in a warmer and acidified changing ocean as shown by the surf clam Spisula solida through a multibiomarker approach.

Humans have exhaustively combusted fossil fuels, and released pollutants into the environment, at continuously faster rates resulting in global average temperature increase and seawater pH decrease. Climate change is forecasted to exacerbate the effects of pollutants such as the emergent rare earth elements. Therefore, the objective of this study was to assess the combined effects of rising temperature (Δ = + 4 °C) and decreasing pH (Δ = - 0.4 pH units) on the bioaccumulation and elimination of gadolinium (Gd) in the bioindicator bivalve species Spisula solida (Surf clam). We exposed surf clams to 10 µg L-1 of GdCl3 for seven days, under warming, acidification, and their combination, followed by a depuration phase lasting for another 7 days and investigated the Gd bioaccumulation and oxidative stress-related responses after 1, 3 and 7 days of exposure and the elimination phase. Gadolinium accumulated after just one day with values reaching the highest after 7 days. Gadolinium was not eliminated after 7 days, and elimination is further hampered under climate change scenarios. Warming and acidification, and their interaction did not significantly impact Gd concentration. However, there was a significant interaction on clam's biochemical response. The augmented total antioxidant capacity and lipid peroxidation values show that the significant impacts of Gd on the oxidative stress response are enhanced under warming while the increased superoxide dismutase and catalase values demonstrate the combined impact of Gd, warming & acidification. Ultimately, lipid damage was greater in clams exposed to warming & Gd, which emphasizes the enhanced toxic effects of Gd in a changing ocean.

Single and combined ecotoxicological effects of ocean warming, acidification and lanthanum exposure on the surf clam (Spisula solida).

Lanthanum (La) is one of the most abundant emergent rare earth elements. Its release into the environment is enhanced by its use in various industrial applications. In the aquatic environment, emerging contaminants are one of the stressors with the ability to compromise the fitness of its inhabitants. Warming and acidification can also affect their resilience and are another consequence of the growing human footprint on the planet. However, from information gathered in the literature, a study on the effects of ocean warming, acidification, and their interaction with La was never carried out. To diminish this gap of knowledge, we explored the effects, combined and as single stressors, of ocean warming, acidification, and La (15 µg L-1) accumulation and elimination on the surf clam (Spisula solida). Specimens were exposed for 7 days and depurated for an additional 7-day period. Furthermore, a robust set of membrane-associated, protein, and antioxidant enzymes and non-enzymatic biomarkers (LPO, HSP, Ub, SOD, CAT, GPx, GST, TAC) were quantified. Lanthanum was bioaccumulated after just one day of exposure, in both control and climate change scenarios. A 7-day depuration phase was insufficient to achieve control values and in a warming scenario, La elimination was more efficient. Biochemical response was triggered, as highlighted by enhanced SOD, CAT, GST, and TAC levels, however as lipoperoxidation was observed it was insufficient to detoxify La and avoid damage. The HSP was largely inhibited in La treatments combined with warming and acidification. Concomitantly, lipoperoxidation was highest in clams exposed to La, warming, and acidification combined. The results highlight the toxic effects of La on this bivalve species and its enhanced potential in a changing world.

Assessment of deep eutectic solvents toxicity in zebrafish (Danio rerio).

Deep Eutectic Systems (DES) have emerged as a "green alternative" to organic solvents and have been coined as biocompatible and biodegradable. However, the number of studies concerning the real biodegradability and biocompatibility are scarce. Thus, to study the toxicity of certain DES, two different approaches were used: i) zebrafish exposure via water, where the system (DES) was tested at potentially realistic environmental concentrations and ii) via intraperitoneal injection, where the system was tested in different concentrations, relevant to the pharmaceutical industry. These studies were performed using zebrafish, a standardized animal model often used in biomedicine and toxicological assays. The results show low toxicity according to tested concentrations (up to 73.47 µM), when the system CA:T:W, with a 2:1:3 molar ratio, was tested through exposure via water and also in the intraperitoneal injection tests with concentrations up to 6000 µM. The activity of different enzymes involved in antioxidant pathways (glutathione S-transferase, catalase, glutathione peroxidase), the total antioxidant capacity (TAC) and lipoperoxidation (MDA content) were determined suggesting low toxicity of the tested system (DES). The promising results herein presented show that DES present the potential to be used as the new class of green solvents, not only for use in the pharmaceutical industry, but also in cosmetic and chemical engineering processes without causing negative impact on living organisms.

Does Predation Exacerbate the Risk of Endosymbiont Loss in Heat Stressed Hermatypic Corals? Molecular Cues Provide Insights Into Species-Specific Health Outcomes in a Multi-Stressor Ocean.

Ocean warming has been a major driver of coral reef bleaching and mass mortality. Coupled to other biotic pressures, corals' ability for acclimatization and adaptation may become compromised. Here, we tested the combined effects of warming scenarios (26, 30, and 32°C) and predation (wound vs. no wound) in coral health condition (paleness, bleaching, and mortality), cellular stress responses (heat shock protein 70 kDa Hsp70, total ubiquitin Ub, and total antioxidant capacity TAC), and physiological state (integrated biomarker response index, IBR) of seven Scleractinian coral species, after being exposed for 60 days. Results show that although temperature was the main factor driving coral health condition, thermotolerant species (Galaxea fascicularis, Psammocora contigua, and Turbinaria reniformis) displayed increased paleness, bleaching, and mortality in predation treatments at high temperature, whereas thermosensitive species (Acropora tenuis, Echinopora lamellosa, and Montipora capricornis brown and green morphotypes) all died at 32°C, regardless of predation condition. At the molecular level, results show that there were significant main and interactive effects of species, temperature, and predation in the biomarkers assessed. Temperature affected Hsp70, Ub, and TAC, evidencing the role of protein folding and turnover, as well as reactive oxygen species scavenging in heat stress management. Predation increased Hsp70 and Ub, suggesting the activation of the pro-phenoloxidase system and cytokine activity, whereas the combination of both stressors mainly affected TAC during moderate stress and Ub under severe stress, suggesting that redox balance and defense of homeostasis are crucial in tissue repair at high temperature. IBR levels showed an increasing trend at 32°C in predated coral fragments (although non-significant). We conclude that coral responses to the combination of high temperature and predation pressure display high inter-species variability, but these stressors may pose a higher risk of endosymbiont loss, depending on species physiology and stress intensity.

Mapping the distribution of mercury (II) chloride in zebrafish organs by benchtop micro-energy dispersive X-ray fluorescence: A proof of concept.

BACKGROUND: Mercury (Hg) is a globally ubiquitous pollutant and one of the most dangerous metal contaminants, which presents a high risk of bioaccumulation in living organisms. In this study, we mapped the distribution of Hg and other trace elements in zebrafish (Danio rerio), which were exposed to mercury (II) chloride in order to assess its toxicity, bioaccumulation and distribution in fish organs. METHODS: Adult zebrafish were exposed for 7 days to different concentrations of mercury (II) chloride and the elemental distribution was obtained through the micro-energy dispersive X-ray fluorescence technique (µ-EDXRF). RESULTS: The results showed that Hg levels, measured in fish tissues, were indicative of bioaccumulation within some of its organs (e.g. visceral mass, gills), and that the physiological processes of accumulation were highly dose-dependent. In addition, the results showed higher concentrations of Hg in the gills. Moreover, other trace elements (e.g. Fe, Cu and Zn) levels were not altered after fish exposure to mercury(II) chloride. CONCLUSION: The µ-EDXRF results were assessed along with the determination of some oxidative stress biomarkers (e.g. antioxidant enzymes) to understand the effects behind the Hg bioaccumulation and toxicity. These results suggest that the metabolic changes in zebrafish due to the exposure to Hg are consistent with oxidative stress.

Molecular mechanisms linking environmental toxicants to cancer development: Significance for protective interventions with polyphenols.

Human exposure to environmental toxicants with diverse mechanisms of action is a growing concern. In addition to well-recognized carcinogens, various chemicals in environmental and occupational settings have been suggested to impact health, increasing susceptibility to cancer by inducing genetic and epigenetic changes. Accordingly, in this review, we have discussed recent insights into the pathological mechanisms of these chemicals, namely their effects on cell redox and calcium homeostasis, mitochondria and inflammatory signaling, with a focus on the possible implications for multi-stage carcinogenesis and its reversal by polyphenols. Plant-derived polyphenols, such as epigallocatechin-gallate, resveratrol, curcumin and anthocyanins reduce the incidence of cancer and can be useful nutraceuticals for alleviating the detrimental outcomes of harmful pollutants. However, development of therapies based on polyphenol administration requires further studies to validate the biological efficacy, identifying effective doses, mode of action and new delivery forms. Innovative microphysiological testing models are presented and specific proposals for future trials are given. Merging the current knowledge of multifactorial actions of specific polyphenols and chief environmental toxicants, this work aims to potentiate the delivery of phytochemical-based protective treatments to individuals at high-risk due to environmental exposure.

Salinity shapes the stress responses and energy reserves of marine polychaetes exposed to warming: From molecular to functional phenotypes.

Estuarine systems are critical transition zones influenced by sea, land and freshwater. An array of human activities impacts these areas leading to multiple-stressor interactions. Temperature and salinity are among the most relevant drivers in estuaries, shaping species growth, reproduction and distribution. However, few studies provide an overview of cellular rewiring processes under multiple-stressor environments. Here, we tested how salinity could shape the response of ragworms Hediste diversicolor, an important bioindicator and commercial species, to elevated temperature. We exposed polychaetes to three temperatures for a month, simulating control, ocean warming and heatwave conditions (24, 27 and 30 °C, respectively) combined with two salinities (20 and 30). We quantified whole-organism performance (wet weight gain and survival), along with cellular stress response (CSR) and energy reserves of worms after 14 and 28 days of exposure. Significant three-way interactions between temperature, salinity and exposure time show the non-linearity of molecular responses. Worms at a salinity of 20 were more sensitive to warming than worms exposed to a salinity of 30. The combination of high temperature and low salinity can act synergistically to induce oxidative stress and macromolecular damage in worm tissues. This finding was supported by an induction of the CSR, with a concomitant decrease of energy reserves, pointing towards a metabolic compensation strategy. However, under a higher salinity (30), the need for a CSR upon thermal challenge was reduced and energy content increased with temperature, which suggests that environmental conditions were within the optimum range. Heatwaves striking low-salinity areas of estuaries can therefore negatively impact the cellular physiology of H. diversicolor, with greater metabolic costs. However, extreme stress levels were not reached as worms incremented wet weight and survival was high under all conditions tested. Our findings are important for the optimization of ragworm aquaculture and adaptive conservation strategies of estuarine systems.

Conserved fatty acid profiles and lipid metabolic pathways in a tropical reef fish exposed to ocean warming - An adaptation mechanism of tolerant species?

Climate warming is causing rapid spatial expansion of ocean warm pools from equatorial latitudes towards the subtropics. Sedentary coral reef inhabitants in affected areas will thus be trapped in high temperature regimes, which may become the "new normal". In this study, we used clownfish Amphiprion ocellaris as model organism to study reef fish mechanisms of thermal adaptation and determine how high temperature affects multiple lipid aspects that influence physiology and thermal tolerance. We exposed juvenile fish to two different experimental conditions, implemented over 28 days: average tropical water temperatures (26 °C, control) or average warm pool temperatures (30 °C). We then performed several analyses on fish muscle and liver tissues: i) total lipid content (%), ii) lipid peroxides, iii) fatty acid profiles, iv) lipid metabolic pathways, and v) weight as body condition metric. Results showed that lipid storage capacity in A. ocellaris was not affected by elevated temperature, even in the presence of lipid peroxides in both tissues assessed. Additionally, fatty acid profiles were unresponsive to elevated temperature, and lipid metabolic networks were consequently well conserved. Consistent with these results, we did not observe changes in fish weight at elevated temperature. There were, however, differences in fatty acid profiles between tissue types and over time. Liver showed enhanced α-linolenic and linoleic acid metabolism, which is an important pathway in stress response signaling and modulation on environmental changes. Temporal oscillations in fatty acid profiles are most likely related to intrinsic factors such as growth, which leads to the mobilization of energetic reserves between different tissues throughout time according to organism needs. Based on these results, we propose that the stability of fatty acid profiles and lipid metabolic pathways may be an important thermal adaptation feature of fish exposed to warming environments.

Impaired antioxidant defenses and DNA damage in the European glass eel (Anguilla anguilla) exposed to ocean warming and acidification.

The European eel (Anguilla anguilla) has attracted scientific inquiry for centuries due to its singular biological traits. Within the European Union, glass eel fisheries have declined sharply since 1980, from up to 2000 t (t) to 62.2 t in 2018, placing wild populations under higher risk of extinction. Among the major causes of glass eels collapse, climate change has become a growing worldwide issue, specifically ocean warming and acidification, but, to our knowledge, data on physiological and biochemical responses of glass eels to these stressors is limited. Within this context, we selected some representative biomarkers [e.g. glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAC), heat shock proteins (HSP70), ubiquitin (Ub) and DNA damage] to study physiological responses of the European glass eel under distinct laboratory-climate change scenarios, such as increased water temperature (+ 4 °C) and pH reduction (- 0.4 units), for 12 weeks. Overall, the antioxidant enzymatic machinery was impaired, both in the muscle and viscera, manifested by significant changes in CAT, GPx and TAC. Heat shock response varied differently between tissues, increasing with temperature in the muscle, but not in the viscera, and decreasing in both tissues under acidification. The inability of HSP to maintain functional protein conformation was responsible for boosting the production of Ub, particularly under warming and acidification, as sole stressors. The overproduction of reactive oxygen species (ROS), either elicited by warming - due to increased metabolic demand - or acidification - through H+ interaction with O2-, generating H2O2 - overwhelmed defense mechanisms, causing oxidative stress and consequently leading to protein and DNA damage. Our results emphasize the vulnerability of eels' early life stages to climate change, with potential cascading consequences to adult stocks.

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion.

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

Synthesis of glutathione as a central aspect of PAH toxicity in liver cells: A comparison between phenanthrene, Benzo[b]Fluoranthene and their mixtures.

Polycyclic Aromatic Hydrocarbons (PAH) are a class of organic pollutants normally found as mixtures with effects often hard to predict, which poses a major challenge for risk assessment. In this study, we address the effects of Phenanthrene (Phe), benzo[b]fluoranthene (B[b]F) and their mixtures (2 Phe:1 B[b]F; 1 Phe: 1 B[b]F; 1 Phe: 2 B[b]F) over glutathione (GSH) synthesis and function in HepG2 cells. We analyzed the effects on cellular viability, ROS production, glutathione (GSH) levels, protein-S-glutathionylation (PSSG), the activity of glutathione peroxidase (GPx), glutathione-S-transferases (GST) and glutathione reductase (GR). Transcript (mRNA) levels of glutathione synthesis enzymes - glutathione cysteine ligase catalytical (GCLC) and modifying (GCLM) sub-units and glutathione synthetase (GS) - and Nrf2 translocation to the nucleus were analyzed. Phe showed a higher cytotoxicity (IC50 = 130 µM after 24 h) than B[b]F related to a higher ROS production (up-to 50% for Phe). In agreement, GSH levels were significantly increased (up-to 3-fold) by B[b]F and were accompanied by an increase in the levels of PSSG, which is a mechanism that protect proteins from oxidative damage. The upregulation of GSH was the consequence of Nrf2 signaling activation and increased levels of GCLC, GCLM and GS mRNA observed after exposure to B[b]F, but not during exposure to Phe. Most interestingly, all mixtures showed higher cytotoxicity than individual compounds, but intriguingly it was the 1 Phe: 1B[b]F mixture showing the highest cytotoxicity and ROS production. GSH levels were not significantly upregulated not even in the mixture enriched in B[b]F. These results point to the role of GSH as a central modulator of PAH toxicity and demonstrate the idiosyncratic behavior of PAH mixtures even when considering only two compounds in varying ratios.

Different sensitivity to heatwaves across the life cycle of fish reflects phenotypic adaptation to environmental niche.

Predicting responses of marine organisms to global change requires eco-physiological assessments across the complex life cycles of species. Here, we experimentally tested the vulnerability of a demersal temperate fish (Sparus aurata) to long-lasting heatwaves, on larval, juvenile and adult life-stages. Fish were exposed to simulated coastal (18 °C), estuarine (24 °C) summer temperatures, and heatwave conditions (30 °C) and their physiological responses were assessed based on cellular stress response biomarkers (heat shock protein 70 kDa, ubiquitin, antioxidant enzymes, lipid peroxidation) and phenotypic measures (histopathology, condition and mortality). Life-stage vulnerability can be ranked as larvae > adults > juveniles, based on mortality, tissue pathology and the capacity to employ cellular stress responses, reflecting the different environmental niches of each life stage. While larvae lacked acclimation capacity, which resulted in damage to tissues and elevated mortality, juveniles coped well with elevated temperature. The rapid induction of cytoprotective proteins maintained the integrity of vital organs in juveniles, suggesting adaptive phenotypic plasticity in coastal and estuarine waters. Adults displayed lower plasticity to heatwaves as they transition to deeper habitats for maturation, showing tissue damage in brain, liver and muscle. Life cycle closure of sea breams in coastal habitats will therefore be determined by larval and adult stages.

Warming enhances lanthanum accumulation and toxicity promoting cellular damage in glass eels (Anguilla anguilla).

Cumulative and continuing human emissions of greenhouse gases to the atmosphere are causing ocean warming. Rising temperature is a major threat to aquatic organisms and may affect physiological responses, such as acid-base balance, often compromising species fitness and survival. It is also expected that warming may influence the availability and toxicological effects of pollutants, including Rare Earth Elements. These are contaminants of environmental emerging concern with great economic interest. This group comprises yttrium, scandium and lanthanides, being Lanthanum (La) one of the most common. The European eel (Anguilla anguilla) is critically endangered and constitutes a delicacy in South East Asia and Europe, being subject to an increasing demand on a global scale. Considering the vulnerability of early life stages to contaminants, we exposed glass eels to 1.5 µg L-1 of La for five days, plus five days of depuration, under a present-day temperature and warming scenarios (△T = +4 °C). The aim of this study was to assess the bioaccumulation, elimination and specific biochemical enzymatic endpoints in glass eels (Anguilla anguilla) tissues, under warming and La. Overall, our results showed that the accumulation and toxicity of La were enhanced with increasing temperature. The accumulation was higher in the viscera, followed by the head, and ultimately the body. Elimination was less effective under warming. Exposure to La did not impact acetylcholinesterase activity. Moreover, lipid peroxidation peaked after five days under the combined exposure of La and warming. The expression of heat shock proteins was majorly suppressed in glass eels exposed to La, at both tested temperatures. This result suggests that, when exposed to La, glass eels were unable to efficiently prevent cellular damage, with a particularly dramatic setup in a near-future scenario. Further studies are needed towards a better understanding of the effects of lanthanum in a changing world.