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In an era of unprecedented environmental changes, understanding the combined effects of multiple stressors on species' performance is urgent. The increasing UV-filter incorporation in daily-life products raises concerns about their potential impact on marine-coastal environments upon release. As stressors rarely act alone, global change-induced factors, such as ocean acidification (OA), can amplify ecological hazards promoted by contaminants in coastal realms. This study investigated the combined impacts of UV-filters 4-methylbenzylidene camphor (4-MBC) and benzophenone-3 (BP-3), at ecologically relevant concentrations (1 and 10 µg/L), under two target pH levels (8.2 and 7.7, reflecting a ∆pH of 0 and -0.3 relative to the average pH at the sampling site), on the biological performance and male reproductive health of the mussel Mytilus galloprovincialis. Using sperm and adult assays alongside a multi-biomarker approach, the study revealed that pH was the primary driver of the decline in mussel physiological and biochemical performances, further intensifying UV-filters' impacts. While sperm cells showed adaptive responses to low pH conditions alone, characterized by reduced lipid peroxidation (LPO) levels and superoxide anion overproduction, adult mussels experienced more pronounced effects, particularly under simultaneous exposure to low pH and UV-filters. Specifically, the adults exhibited distinct bioconcentration patterns under low pH, enhanced cellular metabolic activity and energy-demand compensatory processes, activation of biotransformation pathways, and regulation of antioxidant defenses. Given the ecological and socio-economic importance of M. galloprovincialis and its demonstrated vulnerability to these stressors, these findings highlight the need for further studies on potential transgenerational impacts and evolutionary implications for mussel populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Addressing the impacts of emerging contaminants within the context of climate change is crucial for understanding ecosystem health decline. Among these, the organic UV-filters 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) are widely used in cosmetics and personal care products. Their unique physico-chemical properties, along with their growing commercialization and consumption, have made them ubiquitous in aquatic environments through both direct and indirect releases, raising significant concerns about their potential threats to inhabiting biota. Additionally, increasing surface water temperatures exacerbate ecological risks, making it imperative to understand the implications for non-target species at different biological levels. This study investigated the short- and long-term effects of UV-filters 4-MBC or BP-3, at ecologically relevant concentrations, combined with current and predicted warming scenarios, on the performance and male reproductive health of Mytilus galloprovincialis mussel populations. Using biomarkers across sub-cellular, cellular, tissue, and individual levels, the study revealed significant physiological and biochemical impairments in both sperm cells and adults exposed to UV-filters. Temperature emerged as the primary driver influencing mussel responses and modulating the impacts of 4-MBC/BP-3, emphasizing their sensitivity to temperatures outside the optimal range and interactive effects between stressors. Specifically, sperm motility declined with increasing UV-filter concentrations, while temperature alone influenced ROS production, leading to compromised mitochondrial activity and DNA damage in the presence of combined stressors, indicative of potential reproductive impairments. Adults exhibited high UV-filter bioconcentration potential in whole tissues, compromised physiological status, morphophysiological changes in digestive glands, oxidative stress, and alterations in metabolic capacity, antioxidant defences, and biotransformation mechanisms, correlating with UV-filter exposure and temperature increase. Among the UV-filters tested, 4-MBC was the most detrimental, especially when combined with warming. Overall, this study underscores the vulnerability of M. galloprovincialis to cumulative stressors and highlights the importance of employing a multi-biomarker approach to assess and mitigate the impacts of stressors on coastal ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present study aimed to investigate the effects of europium (Eu) exposure (10 μg/L), warming (a 4 °C increase), and their combination on Mytilus galloprovincialis. Biochemical and histopathological changes in adult mussels were evaluated after a 28-day exposure period. Additionally, biochemical and physiological alterations in sperm were measured following a 30-min exposure period. The overall responses to each treatment were assessed using the Integrated Biological Response index version 2 (IBRv2). In adult mussels, warming elevated metabolism and activated glutathione S-transferases (GSTs), leading to redox imbalance and cellular damage. Europium exposure alone slightly enhanced metabolism and GSTs activity, resulting in cellular damage and histopathological injuries in digestive tubules. The combined exposure to Eu and warming was the most detrimental treatment for adults, as indicated by the highest IBRv2 value. This treatment slightly increased metabolism and uniquely elevated the activity of antioxidant enzymes, as well as GSTs and carboxylesterases. Despite these responses, they were inadequate to prevent redox imbalance, cellular damage, and histopathological injuries in digestive tubules and gills. Regarding sperm, warming reduced reactive oxygen species (ROS) production but raised lipid peroxidation levels. Sperm exposed to this treatment also increased their oxygen consumption and exhibited reduced velocity. The IBRv2 indicated that Eu was the most harmful treatment for sperm, significantly increasing ROS production and notably decreasing sperm velocity. When combined with warming, Eu elevated superoxide anion (O 2 - ) production, lowered sperm velocity, and increased oxygen consumption. This study underscores the importance of investigating the effects of rare earth elements and their interaction with climate change-related factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Being a crucial element for technological development, praseodymium (Pr) has been increasingly used, leading to a rise in its concentration in aquatic systems. However, its potential threats to organisms remain poorly understood. Besides contamination, organisms are also threatened by climate change-related factors, including warming. It is important to evaluate how climate change-related factors may influence the effects of contaminants. To address this, histopathological and biochemical analyses were performed in adult mussels of Mytilus galloprovincialis, following a 28-day exposure to Pr (10 μg/L) and warming (4 °C increase) separately, and in combination. Additionally, biochemical and physiological alterations were analysed in the sperm of mussels after 30-min exposure to the same treatments. Furthermore, it was used the Independent Action model to predict the interaction between Pr and warming. The results showed, in the case of adults exposed to Pr, an increase in superoxide dismutase (SOD) and glutathione S-transferases (GSTs) activities. However, it was insufficient, leading to histopathological injuries, redox imbalance, and cellular damage. In the case of sperm, Pr induced an increase of mitochondrial activity and respiration rate, in response to the increase in systemic metabolic rate and oxygen demand. Warming increased the metabolism, and induced redox imbalance and cellular damage in adults. In sperm, a rise in temperature induced lipid peroxidation and a decrease in velocity. Warming induced some alterations in how adult mussels responded to Pr, activating catalase instead of SOD, and in addition to GSTs, also activated carboxylesterases. However, it was not enough to avoid redox imbalance and cellular damage. In the case of sperm, the combination induced a decrease in H 2 O 2 production, and higher oxygen demand, which prevented the decrease in motility and velocity. This study highlights the limitations of using models and emphasizes the importance of studying the impacts of emerging contaminants, such as rare earth elements, and their combination with climate change-related factors. Under environmental conditions, chronic exposure to the combined effect of different stressors might generate impacts at higher biological levels. This may affect organisms' respiratory and filtration capacity, nutrient absorption, defence capacity against infections or diseases, and sperm viability, ultimately resulting in reduced growth and reproduction, with consequences at the population level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The use of rare earth elements (REEs) has been increasing and one of the most used is neodymium (Nd). Being an emergent contaminant, its negative impacts are poorly understood. Aquatic organisms are also threatened by climate change-related factors, as is the case of warming, which can change the effects of REEs. Thus, the impacts of Nd, warming, and the combination of both stressors were studied in adult mussels and sperm of the species Mytilus galloprovincialis, after an exposure period of 28 days (adults) and 30 min (sperm). The effects were evaluated through the analysis of biochemical and histopathological alterations in adults and biochemical and physiological responses given by sperm. The results showed that mussels only activated their biotransformation capacity when exposed to the stressors acting alone, which was insufficient to avoid lipid peroxidation. Furthermore, warming (alone and combined with Nd) also produces damage to proteins. The digestive gland was the most sensitive organ to Nd, presenting several histopathological alterations. In the case of sperm, all stressors induced lipid peroxidation, a higher oxygen demand, and a decrease in velocity, even if the sperm viability was maintained. It seems that warming influenced the effects of Nd to some extent. The present findings contribute significantly to the field of REEs environmental toxicology by offering valuable insights into the impacts of Nd on various biological levels of mussels. Additionally, within the context of climate change, this study sheds light on how temperature influences the effects of Nd. The obtained results indicate that both stressors can potentially compromise the overall health of mussel populations, thereby affecting other species reliant on them for food and habitat. Moreover, this study highlights impaired sperm health, which could adversely affect their reproductive capacity and ultimately lead to population decline., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The global effort to achieve carbon neutrality has led to an increased demand for renewable energy technologies and their raw materials, namely rare earth elements (REEs). These elements possess unique properties and are used in various applications. However, the increased use of REE-based technologies has resulted in higher amounts of electronic waste, leading to elevated REEs concentrations found in the aquatic environment, with poorly understood threats to wildlife. Praseodymium (Pr) and europium (Eu) are two REEs that, despite their potential environmental risks, have almost unknown effects on aquatic organisms. Therefore, the present study aimed to assess the impacts of different concentrations of Pr and Eu (0, 10, 20, 40, and 80 μg/L) in the mussel species Mytilus galloprovincialis, as well as their ability to recover from exposure to the highest concentration. Mussels accumulated both elements in a dose-dependent manner, with the accumulation of Pr being higher. Accompanying the increase of metabolism, mussels exposed to Pr not only enhanced the activity of the antioxidant enzymes superoxide dismutase (up to 40 μg/L) and glutathione reductase (at 80 μg/L) but also the activity of the biotransformation enzymes carboxylesterases (CbE's) and glutathione S-transferases (GSTs) (at 80 μg/L). Nevertheless, these defence mechanisms were not sufficient to prevent cellular damage. All the Eu concentrations induced cellular damage, despite an increase in the activity of biotransformation enzymes (CbE's and GSTs) in mussel tissue. According to the histopathology assessment, mussels were not able to recover after exposure to both elements and lower concentrations induced higher injuries in digestive tubules. This study highlights that exposure to Pr and Eu had adverse effects on M. galloprovincialis, even at the lowest tested concentration, which may eventually impact mussels' growth, reproductive capacity, and survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The bisindolic alkaloid caulerpin (CAU) is a bioactive compound isolated from green algae of the genus Caulerpa that are highly invasive in the Mediterranean Sea. On the other side, the purine alkaloid caffeine (CAF) is one of the most globally consumed psychoactive substances and a widespread anthropogenic water pollutant. Both compounds display a large panel of biological properties and are well known to accumulate in the tissues of aquatic organisms and, in certain circumstances, co-occur in the human diet. On this premise, the present study aimed to investigate possible synergistic interactions between CAU and CAF by using the bivalve Mytilus galloprovincialis as a model organism. Mussels were exposed to CAF via medium while they were fed with food enriched with CAU. After treatments, biochemical analysis confirmed the toxic potential of CAF, with increased AChE activity and lipid peroxidation. Also, histopathological alterations were observed in the gills and digestive tubules. The NMR-based metabolomics analysis detected higher levels of free amino acids under CAF treatments. Conversely, the food administration of CAU did not affect the above toxicological biomarkers. In addition, we did not observe any cumulative effect between CAF and CAU toward increased cellular damage and neurotoxicity. On the other hand, a possible action of CAU in decreasing CAF toxicity could be hypothesized based on our results. This hypothesis is supported by the activity of CAU as an agonist of peroxisome proliferator-activated receptors (PPARs). PPARs mediate xenobiotic detoxification via cytochromes P450, which is involved in CAF metabolism. Overall, the results obtained not only rule out any cumulative adverse effects of CAF and CAU but also encourage further research to evaluate the possible use of CAU, a compound easily obtained through the valorization of biomass from invasive species, as a food additive to improve the clearance of xenobiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Nowadays, it is of utmost importance to consider climate change factors, such as ocean warming, since the risk of negative impacts derived from increased surface water temperature is predicted to be high to the biodiversity. The need for renewable energy technologies, to reduce greenhouse gas emissions, has led to the increasing use of rare earth elements (REEs). Dysprosium (Dy) is widely used in magnets, motors, electrical vehicles, and nuclear reactors, being considered a critical REE to technology due to its economic importance and high supply risk. However, the increasing use of this element contributes to the enrichment of anthropogenic REEs in aquatic systems. Nevertheless, the information on the potential toxicity of Dy is limited. Moreover, the effects of pollutants can be amplified when combined with climate change factors. Thus, this study aimed to assess the effects of Dy (10 μg/L) in the species Mytilus galloprovincialis under actual (17 °C) and predicted warming conditions (21 °C). The Dy concentration in contaminated mussels was similar between temperatures, probably due to the detoxification capacity in individuals under these treatments. The combined stressors affected the redox balance, but higher impacts were caused by Dy and warming acting alone. In terms of cellular damage, although Dy acting alone was prejudicial to mussels, warming and both stressors acting together induced higher levels of LPO and PC. The histopathological effects of Dy in the digestive tubules were independent of the temperature tested. Regarding effects on sperm, only warming induced cellular damage, while both stressors, alone and together, impaired sperm movement. Overall, this study highlights that warming might influence the effects induced by Dy, but greater impacts were caused by the element. Eventually, the tested stressors may have consequences on mussels' reproduction capacity as well as their growth, abundance, and survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ionic liquids (ILs) have been considered eco-friendly alternatives to conventional organic solvents. However, several studies have reported that ILs exert toxicity towards aquatic invertebrates. Applying in vitro methodology, the aim of the present study was to evaluate the potential effect of three ILs on the biochemical performance of exposed Mytilus galloprovincialis digestive gland and gills cellular fractions. Carboxylesterase might be involved in the derived toxicity mechanism of ILs as activity levels increased significantly in digestive gland exposed fractions. This group of ILs did not seem to induce genotoxicity, except in gills cellular fractions exposed to 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In the literature, in vitro methodology has been suggested as an important complement to animal testing and in silico studies. The present research underlines its efficacy as a quick pre-screening before in vivo testing, particularly with heterogenic groups of substances with high variability in composition, such as ILs and deep eutectic solvents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pharmaceutical active compounds (PhACs) have raised concerns in the last decade due to their increased consumption and inadequate elimination during discharge, resulting in their introduction into water systems and potential significant threats to non-target organisms. However, few studies have investigated the sublethal impacts of PhAC exposure on marine invertebrates. Thus, the present study aimed to assess tissue-specific responses in Mytilus galloprovincialis to sodium lauryl sulfate (SLS), salicylic acid (SA), and caffeine (CAF) (4.0 mg/L, 4.0 mg/L and 2.0 μg/L, respectively). Short-term in vitro exposures with mussel digestive gland and gill tissues were conducted and biochemical responses related to antioxidant and detoxification capacity, cellular damage and neurotoxicity were assessed. The present results clearly showed significant differences in tissue sensitivity and biochemical responses to the contaminants tested. This study highlights the suitability of filter-feeder species as valuable model organisms for studying the sublethal effects of unintended environmental exposures to PhACs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Surfactants are among the most common PPCPs that reach coastal systems, being often used in large quantities in cleaning products such as detergents and soap powders. Sodium lauryl sulfate (SLS) is listed in this group of emerging contaminants. Previous studies have already demonstrated the presence of SLS in aquatic environments and the negative effects on organisms living there. However, with ocean acidification and warming predictions, SLS-induced impacts may differ from those currently known. In this context, the present study aimed to reproduce environmental conditions by assessing the release of substances over a short period and to understand the influence of a rapid increase in temperature on the impacts caused. The marine bivalve Mytilus galloprovincialis was exposed to 2.0 mg/L SLS at 17 °C and 21 °C for 7 days. To assess the possible biochemical changes resulting from the exposure of mussels to SLS, a series of biomarkers related to oxidative stress/damage, detoxification, and metabolic capacity were measured. The SLS accumulation in soft tissues was low (about 0.7 ng/g) at both temperatures. The results evidenced increased metabolic activity, especially in mussels exposed to SLS at 17 °C. An increase in protein content was also observed upon exposure to SLS and increased temperature compared to controls at 17 °C. Although no effects on antioxidant enzymes were observed, protein damage was recorded, especially at 21 °C. These findings confirmed that SLS induces toxic effects and predicted climate change factors may increase the impact on M. galloprovincialis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Personal care products (PCPs) are those compounds used daily (e.g., soaps, shampoos, deodorants, and toothpaste), explaining their frequent detection in aquatic systems. Still, scarce information is available on their effects on inhabiting wildlife. Among the most commonly used PCPs is the surfactant Sodium Lauryl Sulfate (SLS). The present study investigated the influence of temperature (CTL 17 ºC vs 22 ºC) on the effects of SLS (0 mg/L vs 4 mg/L) in the mussel species Mytilus galloprovincialis. Mussels' general health status was investigated, assessing their metabolic and oxidative stress responses. Higher biochemical alterations were observed in SLS-exposed mussels and warming enhanced the impacts, namely in terms of biotransformation capacity and loss of redox homeostasis, which may result in consequences to population maintenance, especially if under additional environmental stressors. These results confirm M. galloprovincialis as an excellent bioindicator of PCPs pollution, and the need to consider actual and predicted climate changes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Marine organisms are constantly exposed to multiple stressors including pollutants released into the environment, such as personal care products (PCPs), and climate change-derived factors, namely warming, which are aggravated by anthropogenic drivers and pose increasing pressure on coastal ecosystems. Avobenzone (AVO) is one of the most used ultraviolet (UV) filters in PCPs which have been increasingly used and, thereby, identified in aquatic environments. However, data regarding the influence of warming on the impacts caused by AVO in bivalves is lacking. Mussels are considered good bioindicators thus being often employed in ecotoxicology studies. Hence, the present study aimed to evaluate the toxic effects of an environmentally relevant concentration of AVO (0.5 μg/L) and warming (21 °C), acting alone or in combination, on sperm and adults of the Mediterranean mussel species Mytilus galloprovincialis, through in vitro and in vivo tests, respectively. AVO and warming effects were evaluated by assessing oxidative status, viability, genotoxicity, motility, and kinetics in sperm, together with the quantification of energy content, metabolic capacity, biological defence mechanisms, cellular damage, and neurotoxicity in adults. AVO induced genotoxicity and increased respiration rate in sperm while enhancing the biotransformation enzymes' activity in adults. Exposure to warming led to an increase in respiration rate, ROS overproduction, cellular damage, and viability decrease in sperm whereas metabolic capacity increased in adults. AVO combined with warming caused oxidative stress, cellular damage, genotoxicity, and decreased motility in sperm, while only antioxidant enzymes' activity was enhanced in adults. Overall, the present study demonstrated that when acting in combination the effects of both stressors were more prominent. Furthermore, considering the multiple-stressor scenario tested, major toxic effects occurred in male gametes in comparison to adults., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The impact of pharmaceuticals on marine invertebrates has been a topic of rising concern, with an increasing number of studies regarding the impacts on bivalves. However, very few investigated the toxicity of mixtures of pharmaceuticals. This knowledge gap was investigated in the present study, where the toxicity of 17α-ethinylestradiol (EE2) and salicylic acid (SA) mixture was evaluated. To this end, Mytilus galloprovincialis mussels were chronically subjected to both pharmaceuticals, acting alone and in combination, and the effects at the cellular level were measured. The Independent Action (IA) model was performed aiming to compare obtained with predicted responses. The integrated biomarker response (IBR) index was used to assess the overall biochemical response given by mussels. The results obtained revealed that the most stressful condition was caused by the combined effect of EE2 and SA, with the highest metabolic capacity, antioxidant (catalase activity) and biotransformation (carboxylesterases activity) activation and cellular damage in organisms exposed to the mixture of both drugs in comparison to responses observed when each drug was acting alone. Predicted responses obtained from the IA model indicate that caution should be paid as frequent deviations to observed responses were found. This study highlights the need for future studies considering the mixture of pollutants, mimicking the actual environmental conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Contaminants of emerging concern (CECs) are a class of chemicals that can spread throughout the environment and may cause adverse biological and ecological effects. While there are many different classes of CECs, one of the most well documented in the aquatic environment are pharmaceutical drugs, such as natural and synthetic estrogens. In particular, the widespread presence of the synthetic estrogen 17 α-Ethinylestradiol (EE2) in water may lead to bioaccumulation in sediment and biota. EE2 is the primary component in contraceptive pills, and is a derivative of the natural hormone estradiol (E2). In this study, the mussel Mytilus galloprovincialis was exposed to EE2 in a semi-static and time-dependent experiment, for a total exposure period of 28 days. Biochemical and transcriptomics analyses were performed on mussel digestive glands after exposure for 14 (T14) and 28 (T28) days. Metabolic and DNA impairments, as well as activation of antioxidant and biotransformation enzymes activation, were detected in T28 exposed mussels. RNA-Seq analysis showed significant differential expression of 160 (T14 compared to controls), 33 (T28 compared to controls) and 79 (T14 compared to T28) genes. Signs of stress after EE2 treatment included up-regulation of gene/proteins involved with immune function, lipid transport, and metabolic and antibacterial properties. This study elucidates the underlying mechanisms of EE2 in a filter feeding organisms to elucidate the effects of this human pharmaceutical on aquatic biota., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Climate change-associated factors and pollutants, such as rare earth elements (REEs), have been identified as contributors to environmental changes. However, the toxicity resulting from the combination of these stressors has received little attention. Neodymium (Nd) is a REE that has been widely used, and this study aimed to evaluate the responses of Mytilus galloprovincialis to Nd exposure (10 µg/L), under actual (17 °C) and predicted warming conditions (21 °C), after fourteen days of exposure followed by fourteen days of recovery (without Nd), analyzing Nd accumulation, histopathological and biochemical alterations. The results showed that increased temperature and Nd exposure caused histopathological injuries in the gills. Contaminated mussels at 17 °C showed cellular damage, while at 21 °C, mussels were able to avoid cellular damage. After the recovery period, no improvements in gill's status were found and cellular damage was still present, highlighting the impacts caused by previous exposure to Nd., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Avobenzone (AVO) is one of the most frequent ultraviolet (UV) filters in personal care products (PCPs). The Mediterranean mussel Mytilus galloprovincialis is a bioindicator often used for ecotoxicological research. Since UV filters reach higher peaks during summer in aquatic bodies, coincident with mussels' spawning period, and bivalves are sessile, both male gametes and adults of this species were used in this experiment. Therefore, the present study aimed to assess how AVO affects M. galloprovincialis at different biological levels. In vitro experiments on sperms (30 min-exposure) and in vivo experiments on adults (28 days-exposure) were carried out at 0.1, 1.0 and 10.0 μg/L of AVO concentrations. The oxidative and physiological status together with genotoxicity in exposed sperms were assessed. Several biochemical parameters related to enzymatic antioxidant defences, biotransformation enzymes, cell membrane damage, energy reserves, and neurotoxicity were evaluated in adult mussels. Results of in vitro sperm exposure to AVO showed significant overproduction of superoxide anions and DNA damages in all treatments and decrease in sperm viability at 1.0 and 10.0 μg/L. AVO exposure also led to complete inhibition of motility of sperms at the highest concentration, while a significant increase of curvilinear velocity and decrease of wobble occurred at 1.0 μg/L. In vivo exposed adults exhibited a significant decrease in metabolic capacity at 0.1 μg/L, a significant increase in the total protein content and enzymatic turnover as superoxide dismutase (antioxidant defence) at 10 μg/L. This study revealed an ecological concern related to the high sensitivity of sperms respectively to adults under environmentally relevant concentrations of AVO, underpinning an hypothesis of male reproductive function impairments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Present in an increasing number of products, UV-filters are continuously discharged into aquatic environments. Despite potential risks for inhabiting organisms are recognized, the effects of UV-filter 4-methylbenzylidenecamphor (4-MBC) on marine invertebrates are poorly investigated. By combining in vitro/in vivo exposures through a multi-biomarker approach on sperms and adults, the present study evaluated how 4-MBC affect the mussel species Mytilus galloprovincialis, providing ecologically relevant information on organisms' responses. From the obtained results, considering mortality as endpoint, sperms revealed a greater sensitivity (EC 50 :347 μg/L) than adults (EC 50 : not calculable). From an ecotoxicological perspective, this resulted in a derived threshold concentration (LOEC) of 100 μg/L and 72 μg/L, respectively. Effects at the cell/molecular level were provided by general redox-status imbalance and oxidative stress. Sperms showed functional and structural impairments, hyperactivation and DNA damage, while adults showed physiological, metabolic/energetic dysfunctions, DNA damage and activation of oxidative and biotransformation enzymes. High 4-MBC bioaccumulation was also observed in exposed mussels (BCFs:14.0-32.0 L/kg). These findings suggest that 4-MBC may impair fitness and survival of the broadcast spawning mussel M. galloprovincialis, affecting reproduction success and population growth., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Marine-coastal systems have been increasingly exposed to multiple stressors, including anthropogenic pollution and variations of Climate Change (CC) related factors, whose coexistence could create associated environmental and ecotoxicological risks. Among emergent stressors, 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) UV-filters are compounds widely used in increasing consumer products, resulting in their ubiquity in aquatic environments and possible pressing challenges on gamete susceptibility. Since most marine invertebrates reproduce by external fertilization, after spawning, gametes may be exposed to several pressures, affecting reproductive success and outcome. The present study focuses on the spermiotoxicity of the environmentally relevant UV-filters 4-MBC and BP-3 combined with salinity shifts, as potential modulators of their harmful effects. For this, Mytilus galloprovincialis male gametes were exposed in vitro to environmentally relevant and slightly higher concentrations (1, 10 and 100 µg/L) of 4-MBC or BP-3 under three different salinities (S 20, 30 and 40). Sperm quality endpoints associated with oxidative status, viability, motility, kinetics, and genotoxicity were evaluated. Similarities and differences in sperm responses among all conditions were highlighted by principal coordinates analysis (PCO). Results showed that salinity acting alone posed greater sperms impairments at the lowest (20) and highest (40) tested levels. When salinity acts as a co-varying stressor, salinity-dominant interactive effects resulted evident, especially for 4-MBC at S 40 and BP-3 at S 20. These findings were pointed out as the worst exposure conditions for M. galloprovincialis sperms, since caused major toxicological effects in terms of: (I) oxidative stress, sperm structural impairments, motility and kinetic alterations in 4-MBC-exposed sperms; (II) DNA damage, compromised mitochondrial activity and hyperactivation in BP-3-exposed ones. Overall, it stands out that salinity influences UV-filter toxicological pathways and, thereby, the potential environmental risk of these contaminants on M. galloprovincialis male gametes, especially in an expected salinity stress scenario., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the recent years, marine heatwaves (MHWs) have caused devastating impacts on marine life. The understanding of the combined effects of these extreme events and anthropogenic pollution is a vital challenge. In particular, the combined effect of MHWs on the toxicity of pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine (CAF) toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed the absence of CAF accumulation in both species. However, the used contaminant generated in both bivalve species alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. The obtained results seemed magnified under a simulated MHWs, suggesting to a climate-induced toxicant sensitivities' response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mixture of contaminants often determine biological responses of marine species, making difficult the interpretation of toxicological data. The pharmaceutical 17 alpha-ethinylestradiol (EE2) and the surfactant Sodium Lauryl Sulfate (SLS) commonly co-occur in the marine environment. This study evaluated the effects of EE2 (125.0 ng/L) and SLS (4 mg/L), acting individually and combined, in the mussel Mytilus galloprovincialis. Contaminated mussels closed their valves for longer periods than control ones, especially in the presence of both contaminants, with longer closure periods immediately after spiking compared to values obtained one day after spiking. Nevertheless, males and females increased their metabolism when in the presence of both contaminants (males) and SLS (females), and independently on the treatment males and females were able to activate their antioxidant and biotransformation defences. Although enhancing defences mussels still presented cellular damage and loss of redox balance, especially noticed in the presence of EE2 for males and SLS for females. Histopathological damage was found at mussel's gills in single and mixture exposure, and qPCR analysis revealed a clear estrogen receptor expression with no additive effect due to combined stressors. The results obtained highlight the harmful capacity of both contaminants but further research on this matter is needed, namely considering different climate change scenarios., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pharmaceutical drugs are Contaminants of Emerging Concern (CECs) and are continuously discharged into the environment. As a result of human and veterinary use, these substances are reaching aquatic coastal systems, with limited information regarding the toxic effects of these compounds towards inhabiting organisms. Among CECs are pharmaceuticals like 17 α-ethinylestradiol (EE2), which is a synthetic hormone with high estrogenic potency. EE2 has been increasingly found in different aquatic systems but few studies addressed its potential toxicity to marine wildlife, in particular to bivalves. Therefore, the aim of the present study was to evaluate the influence of temperature (17 °C-control and 21 °C) on the potential effects of EE2 on the mussel Mytilus galloprovincialis. For this purpose, mussels were exposed to different concentrations of EE2 (5.0; 25.0; 125.0 and 625 ng/L), resembling low to highly polluted sites. Mussels exposed to each concentration were maintained under two temperatures, 17 and 21 °C, which represent actual and predicted warming conditions, respectively. After 28 days, oxidative stress status, metabolism related parameters, neurotoxicity and histopathological alterations were measured. The results obtained clearly showed an interactive effect of increased temperature and EE2, with limited antioxidant and biotransformation capacity when both stressors were acting together, leading to higher cellular damage. The combination of both stressors also enhanced mussels' metabolic capacity and neurotoxic effects. Nevertheless, loss of redox balance was confirmed by the strong decrease of the ratio between reduce glutathione (GSH) and oxidized glutathione (GSSG) in contaminated mussels, regardless the temperature. Histopathological indexes in contaminated mussels were significantly different from the control group, indicating impacts in gills and digestive glands of mussels due to EE2, with higher values observed at 21 °C. Overall, this study demonstrates that of EE2 represents a threat to mussels and predicted warming conditions will enhance the impacts, which in a near future might result in impairments at the population and community levels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The influence of salinity on the effects of sodium lauryl sulfate (SLS) was evaluated using the Mediterranean mussel Mytilus galloprovincialis, exposed for 28 days to SLS (control-0.0 and 4.0 mg/L) under three salinity levels (Control-30, 25 and 35). The effects were monitored using biomarkers related to metabolism and energy reserves, defence mechanisms (antioxidant and biotransformation enzymes) and cellular damage. The results revealed that non-contaminated mussels tended to maintain their metabolic capacity regardless of salinity, without activation of antioxidant defence strategies. On the contrary, although contaminated mussels presented decreased metabolic capacity at salinities 25 and 35, they were able to activate their antioxidant mechanisms, preventing cellular damage. Overall, the present findings indicate that SLS, especially under stressful salinity levels, might potentially jeopardize population survival and reproduction success since reduced metabolism and alterations on mussels' antioxidant mechanisms will impair their biochemical and, consequently, physiological performance., (Copyright © 2021 Elsevier B.V. All rights reserved.)

While many studies have been conducted on drug-inducing alterations in the aquatic environment, little is known about their interaction with climate change, such as rising temperatures. To increase knowledge on this topic, Mytilus galloprovincialis mussels were exposed to two different temperatures 17 ± 1 °C (control) and 21 ± 1 °C in the absence and presence of salicylic acid (SA) (4 mg/L) for 28 days. Salicylic acid in the water and tissues was measured and its impact reported through biomarker responses including: energy metabolism (electron transport system (ETS) activity, glycogen (GLY), protein (PROT) and lipids (LIP) contents), oxidative stress markers (activity of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), glutathione balance between the reduced and the oxidized forms (GSH/GSSG), and damage to membrane lipids (lipid peroxidation - LPO). The mussels responded differently if the stresses imposed were single or combined, with greater impacts when both stressors were acting together. Contaminated mussels exposed to high temperatures were unable to increase their metabolic capacity to restore their defence mechanisms, reducing the expenditure of LIP. In the presence of SA and increased temperature antioxidant defences respond differently, with higher SOD levels and inhibition of CAT. The present study highlights not only the negative impact of warming and SA, but especially how temperature increase will promote the impact of SA in M. galloprovincialis, which under predicted climate change scenarios may greatly impair population maintenance and ecosystem biodiversity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The use of carbon nanotubes (CNTs) is rapidly increasing and several scientific studies have addressed their toxicological properties. However, only a very small number of publications have deal with the interaction between CNTs and other molecules. Triclosan (TCS) is an antibacterial agent used in personal care and household products. Commonly detected in aquatic ecosystems, there is a strong evidence that aquatic biota is sensitive to this compound. Aside from emergent pollutants, aquatic organisms are continuously subjected to abiotic variations including salinities. Therefore, the main goal of the present study was to better understand how physio-chemical interactions of CNTs with TCS under different salinity levels (37, 28 and 19) affect the mussel species Mytilus galloprovincialis through the evaluation of biochemical alterations on gametes (sperms) and adult tissues, providing more ecologically relevant information on organisms' responses. The results showed toxicological effects in terms of sperm metabolic activity and intracellular reactive oxygen species production as well as cellular damage and alteration of metabolic capacity at the adult's stage when exposed to both contaminants acting alone and in combination, under tested salinities. Moreover, when the mussels were exposed to the combination of both contaminants, they showed major toxic impacts on both assessed biological levels (adult tissues and sperms) especially under control salinity. This suggests that toxicity upon mixture exposure compared to single-substance exposure may impair mussels' populations, affecting reproduction success and growth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pharmaceuticals and Personal care products (PPCPs) are frequently released into several marine matrices, representing significant environmental and ecotoxicological risks. Among the widest spread PPCPs in aquatic systems is Salicylic acid (SA), with known negative effects on marine and freshwater species. Nevertheless, the toxicity resulting from these emerging pollutants, including SA, together with climate change has still received little attention up to date. Among climate change related factors salinity is one that most affects aquatic organisms. To better understand the combined impacts of SA and salinity, the present study evaluated the biochemical alterations induced in Mytilus galloprovincialis mussels exposed to SA and different salinity levels, acting individually and in combination. The effects observed clearly highlighted that cellular damages were mainly observed at higher salinity (35), with no additive or synergistic effects derived from the combined presence of SA. Higher antioxidant capacity of mussels in the presence of SA may prevent increased LPO levels in comparison to uncontaminated mussels. Nevertheless, in the presence of SA mussels revealed loss of redox balance, regardless of the salinity level. Furthermore, mussels exposed to SA at control salinity showed increased metabolic capacity which decreased when exposed to salinities 25 and 35. These findings may indicate the protective capacity of mussels towards higher stressful conditions, with lower energy reserves expenditure when in the presence of SA and salinities out of their optimal range. Although limited cellular damages were observed, changes on mussel's redox balance, antioxidant mechanisms and metabolism derived from the combined exposure to SA and salinity changes may compromise mussel's growth and reproduction. Overall, the present study highlights the need to investigate the impacts induced by pollutants under present and future climate change scenarios, towards a more realistic environmental risk assessment., (Copyright © 2020. Published by Elsevier B.V.)

Pharmaceuticals and personal care products (PPCPs) are continuously dispersed into the environment, as a result of human and veterinary use, reaching aquatic coastal systems and inhabiting organisms. However, information regarding to toxic effects of these compounds towards marine invertebrates is still scarce, especially in what regards to metabolic capacity and oxidative status alterations induced in bivalves after chronic exposure. In the present study, the toxic impacts of Sodium lauryl sulfate (SLS), an anionic surfactant widely used as an emulsifying cleaning agent in household and cosmetics, were evaluated in the mussel Mytilus galloprovincialis, after exposure for 28 days to different concentrations (0.0; 0.5; 1.0; 2.0 and 4.0 mg/L). For this, effects on mussels respitation rate, metabolic capacity and oxidative status were evaluated. The obtained results indicate a significant decrease on mussel's respiration rate after exposure to different SLS concentrations, an alteration that was accompanied by a decrease of bioconcentration factor along the increasing exposure gradient, especially at the highest exposure concentration. Nonetheless, the amount of SLS accumulated in organisms originated alterations in mussel's metabolic performance, with higher metabolic capacity up to 2.0 mg/L followed by a decrease at the highest tested concentration (4.0 mg/L). Mussels exposed to SLS revealed limited antioxidant defense mecanhisms but cellular damage was only observed at the highest exposure concentration (4.0 mg/L). In fact, up to 2.0 mg/L of SLS limited toxic impacts were observed, namely in terms of oxidative stress and redox balance. However, since mussel's respiration rate was greatly affected by the presence of SLS, the present study may highlight the potential threat of SLS towards marine bivalves, limiting their filtration capacity and, thus, affecting their global physiological development (including growth and reproduction) and ultimely their biochemical performance (afecting their defense capacity towards stressful conditons)., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A vast variety of substances currently reaches the aquatic environment, including newly developed chemicals and products. Lack of appropriate analytical methods for trace determinations in aquatic ecosystem compartments and lack of information regarding their toxicity explains existing regulation gaps. However, suspicion of their toxicity assigned them as Contaminants of Emerging Concern (CECs). Among CECs are Pharmaceuticals including Salicylic Acid (SA), which is the active metabolite of acetylsalicylic acid (ASA; aspirin). The aim of the present study was to evaluate the potential effects of SA on the mussel Mytilus galloprovincialis. For this, organisms were exposed for 28 days to different concentrations of SA (0.005; 0.05; 0.5 and 5 mg/L), resembling low to highly polluted sites, after which different physiological and biochemical parameters were evaluated to assess organism's respiration rate, neurotoxic, metabolic and oxidative stress status. Our results clearly showed that SA strongly reduced the respiration capacity of mussels. Also, SA inhibited the activity of superoxide dismutase (SOD) and catalase (CAT) enzymes, but increased the activity of glutathione peroxidase (GPx) and glutathione-S-transferases (GSTs), which prevented the occurrence of lipid peroxidation (LPO). Nevertheless, oxidative stress was confirmed by the strong decrease of the ratio between reduce glutathione (GSH) and oxidized (GSSG) glutathione in contaminated mussels. Moreover, neurotoxicity was observed in mussels exposed to SA. Overall, this study demonstrates the metabolic, neurotoxic and oxidative stress impacts of SA in M. galloprovincialis, which may result in negative consequences at the population level., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the present study Mytilus galloprovincialis mussels were exposed for 28 days to three salinities: 30 (control), 25 and 35. Simultaneously, organisms at each salinity were exposed to either the antimicrobial agent Triclosan (TCS) or the pharmaceutical drug Diclofenac (DIC) at 1 μg/L. Salinity alone and exposure to PPCPs changed mussel's metabolic capacity and oxidative status, but no additive or synergetic effects resulting from the combined exposures were observed. Overall, the metabolic capacity of mussels was decreased when exposed to TCS and DIC under control salinity, which was less pronounced at salinities out of the control level. TCS had a notorious effect over glutathione peroxidase activity while DIC exposure enhanced catalase response. Such defence mechanisms were able to prevent cellular damage but still a clear reduction in GSH/GSSG ratio after PPCPs exposures indicates oxidative stress which could compromise bivalve's performance to further stressing events., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Interest on the effects of emerging contaminants over aquatic organisms has increased in the last years. Nonetheless, the toxic action of classical natural and anthropogenically-driven metals has also to be monitored, especially because they reflect real environmental situations. For that, in the present study we focused on the effects on the marine mussel Mytilus galloprovincialis of the personal care product Triclosan (TCS) and Lead (Pb), as toxic metal, under separate and co-exposure situations at environmentally relevant concentrations: TCS (1 μg/L) and Pb (50 μg/L). The consideration of an additional factor such as an increase in ambient temperature was also included to provide a forecasted scenario of climate change: from the ambient temperature at actual conditions (17 °C) to a predicted warming situation (22 °C). Water chemical characterization and some physical properties and bioaccumulation of TCS and Pb in mussels at the end of the experiment (28 days) was considered. The parameters followed up comprise the energy related system production (electron transport system) and glycogen and protein reserves. Antioxidant enzymatic defences towards reactive oxygen species (ROS) and the consequences of ROS damage over endogenous lipids (LPO) and proteins (PC). Overall the results suggested only particular responses to chemical exposures at 17 °C whereas at 22 °C the detoxification machinery was set up and this prevented the occurrence of LPO. Nonetheless, PC formation occurred under Pb and TCS + Pb co-exposure at the highest temperature. Due to the complexity of the study: 4 chemical conditions, 2 temperatures and 10 biomarkers considered, a principal component ordination (PCO) analysis was included. The results of this integrative analysis confirmed a clear effect of the temperature, more responsiveness to drugs at 22 °C and in all likelihood due to Pb presence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recently, anthropogenic enrichment of rare earth elements (REEs) have been reported in natural environments, due to increasing use and discharges of hospital/industrial wastewaters. Gadolinium (Gd), which is mainly used as contrast agent for magnetic resonance imaging in medical exams, may reach concentrations in water up to two orders of magnitude larger than baseline levels. Nevertheless, in marine systems scarce information is available concerning the toxicity of REE towards inhabiting organisms. This study aimed to evaluate the biochemical impact of anthropogenic Gd in the Mediterranean mussel Mytilus galloprovincialis, which is a species of commercial interest and one of the most accepted pollution bioindicator. Organisms were exposed to different concentrations of Gd (0, 15, 30, 60, 120 μg/L) for 28 days. At the end of the experiments, biomarkers related to mussels' metabolic (electron transport system activity and energy reserves content), oxidative stress status (cellular damage and the activity of antioxidant and biotransformation enzymes) and neurotoxic effects (activity of the enzyme Acetylcholinesterase) were measured, as well as Gd bioconcentration in organisms. Results showed a high content of Gd (2.5 ± 0.50 μg/g) in mussels exposed to the highest concentration, contrary to those at control condition and at 15 and 30 μg/L of Gd (levels below 0.38 μg/g). Although no mortality was observed during the experimental period, exposure to Gd strongly affected the biochemical performance of M. galloprovincialis, including the decrease on mussels' metabolism, induction of oxidative stress and neurotoxicity, particularly evidenced at intermediate concentrations. These results may indicate that up to certain stressful levels, although lowering their metabolism, organisms may be able to activate defence strategies to avoid cellular injuries which, on the other hand, may compromise mussels physiological performance such as growth and reproduction success. Nevertheless, our findings support that the widespread utilization of Gd may represent an environmental risk in the future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Little is known about the consequences of exposure to pharmaceuticals and personal care products (PPCPs) in elevated temperatures associated with climate change. To increase the knowledge on this topic, Mytilus galloprovincialis mussels were exposed to 1.0 μg/L of either the antimicrobial Triclosan (TCS) or the anti-inflammatory drug Diclofenac (DIC), at control (17 °C) and 4 °C raised (21 °C) temperatures for 28 days. Triclosan and DIC concentrations in the water and tissues were subsequently measured and related to biomarker responses including: energy metabolism (electron transport system (ETS) activity, glycogen and protein reserves), oxidative stress markers, glutathione balance between the reduced and the oxidised form (GSH/GSSG), and damage to proteins and lipids. Mussels responded to the increase in temperature and drug exposure by lowering their metabolic rate (decreased ETS), increasing their endogenous reserves and antioxidant defences, thus preventing oxidative stress damage, with the exception of DIC exposure at the higher temperature. In all cases, GSH/GSSG ratio was reduced in detriment of the antioxidant form at both PPCPs exposures and elevated temperature with no additive effect due to combined stressors. Overall, either drug exposure or increased temperature could compromise the ability of mussels to withstand further insults., (Copyright © 2019. Published by Elsevier B.V.)

With the increased production and research on nanoparticles, the presence of carbon nanotubes (CNTs) in aquatic systems is very likely to increase. Although it has been shown that CNTs may cause toxicity in marine organisms, to our knowledge, the possible impacts under global temperature increase is still unknown. For this reason, biochemical and physiological impacts induced in Mytilus galloprovincialis due to the presence of functionalized multi-walled CNTs (f-MWCNTs) and increased temperature were investigated in the present study. The mussels exposed to increased temperature alone presented higher metabolic capacity and expenditure of glycogen as an energy resource to fuel up defense mechanisms and thus preventing oxidative damage. Contrarily, organisms exposed to f-MWCNTs alone seemed not stressed enough to demonstrate differences in the metabolism capacity. Furthermore, f-MWCNTs seemed not able to significantly activate their antioxidant and biotransformation enzymes, which in turn may led to oxidative damage in the cells especially when organisms were exposed to a warmer temperature. In fact, at higher temperature, the antioxidant response of organisms exposed to f-MWCNTs was not effective and oxidative damage levels were observed. Nevertheless, no additive or synergetic effects were observed when mussels were exposed to both stressors simultaneously., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intertidal species are frequently exposed to environmental changes associated with multiple stressors, which they must either avoid or tolerate by developing physiological and biochemical strategies. Some of the natural environmental changes are related with the tidal cycle which forces organisms to tolerate the differences between an aquatic and an aerial environment. Furthermore, in these environments, organisms are also subjected to pollutants from anthropogenic sources. The present study evaluated the impacts in Mytilus galloprovincialis exposed to multi-walled carbon nanotubes (0.01 mg/L MWCNTs) when continuously submersed or exposed to tides (5 h of low tide, 7 h of high tide) for 14 days. Our results demonstrated that mussels were physiologically and biochemically affected by MWCNTs, especially when exposed to tides. In fact, when only exposed to the carbon nanoparticles or only exposed to tides, the stress induced was not enough to activate mussels' antioxidant defenses which resulted in oxidative damage. However, when mussels were exposed to the combination of tides and MWCNTs increased metabolism was observed, associated with a possible higher production of reactive oxygen species (ROS), leading to a significant increase in the activities of antioxidant enzymes (superoxide dismutase, SOD and glutathione peroxide, GPx) and oxidized glutathione content (GSSG), preventing the occurrence of cellular damage, expressed as no lipid peroxidation (LPO) or protein carbonylation (PC). Therefore, organisms seemed to be able to tolerate MWCNTs and air exposure during tidal regime; however, the combination of both stressors induced higher oxidative stress. These findings indicate that the increasing presence of carbon nanoparticles in marine ecosystems can induce higher toxic impacts in intertidal organisms compared to organisms continuously submerged. Also, our results may indicate that air exposure can act as a cofounding factor on the assessment of different stressors in organisms living in coastal systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)