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Sediment contamination and seawater warming are two major stressors to macrobenthos in estuaries. However, little is known about their combined effects on infaunal organisms. Here we investigated the responses of an estuarine polychaete Hediste diversicolor to metal-contaminated sediment and increased temperature. Ragworms were exposed to sediments spiked with 10 and 20 mg kg -1 of copper at 12 and 20 °C for three weeks. No considerable changes were observed in the expression of genes related to copper homeostasis and in the accumulation of oxidative stress damage. Dicarbonyl stress was attenuated by warming exposure. Whole-body energy reserves in the form of carbohydrates, lipids and proteins were little affected, but the energy consumption rate increased with copper exposure and elevated temperature, indicating higher basal maintenance costs of ragworms. The combined effects of copper and warming exposures were mostly additive, with copper being a weak stressor and warming a more potent stressor. These results were replicable, as confirmed by two independent experiments of similar settings conducted at two different months of the year. This study suggests the higher sensitivity of energy-related biomarkers and the need to search for more conserved molecular markers of metal exposure in H. diversicolor., 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 Ltd. All rights reserved.)

Zinc oxide nanoparticles are released into marine environments from industrial, medical and consumer uses sparking concerns about their potential ecotoxicological effects. Ecological hazard assessment of nZnO in marine ecosystems is hindered by the lack of understanding of the potential interactive effects of nZnO toxicity with other common abiotic stressors, such as salinity fluctuations, in marine organisms. To close this gap in our knowledge, we carried out a comprehensive biomarker-based assessment of the combined effects of salinity and nZnO in a sentinel marine bivalve, the blue mussels Mytilus edulis. The mussels were exposed for 21 days to clean seawater (control), an environmentally relevant concentration (100 μg Zn l -1 ) of nZnO or dissolved Zn (to identify the toxic effects attributable to Zn 2+ toxicity) under the normal (15), low (5) and fluctuating (5-15) salinity regimes. The selected molecular and biochemical markers focused on the oxidative stress, apoptosis, detoxification system and inflammation in the gills and the digestive gland of the mussels. Biomarker analysis showed different effects of nZnO and dissolved Zn on biomarkers of oxidative stress, xenobiotic detoxification and apoptosis but similar effects of both pollutants on the levels of metallothioneins and inflammatory markers. Exposure to nZnO led to elevated levels of lipid peroxidation, upregulation of p53 and p38 stress kinases and apoptosis-related genes, most notably in the gills. Exposure to dissolved Zn led to accumulation of protein carbonyls and activated redox-sensitive detoxification enzymes (NADPH-P450 reductase and glutathione-S-transferase) in the mussels. The ambient salinity had significant effects the cellular adverse effects of nZnO in the mussels. The nZnO-induced cellular stress was detectable under the normal (15) and fluctuating (5-15) salinity conditions in the studied brackish water population of the mussels. At low salinity (5), nZnO toxicity signal was almost completely dampened. These findings indicate that chronic osmotic stress close to the tolerance limits of M. edulis prevails over the effects of the environmentally relevant nZnO and dissolved Zn concentrations in combined exposures. These stressor interactions might ameliorate the cellular toxicity of nZnO in the mussels but limit applicability of cellular stress biomarkers for detecting the toxic effects of nanopollutants in low salinity habitats., 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.)

Organic UV filters have emerged as a new threat to marine organisms, but ecotoxicological studies have so far focused on only a few substances despite the chemical diversity of these synthetic sunscreen agents. Here we examined the responses of blue mussels Mytilus edulis to ensulizole, a non-lipophilic UV filter commonly found in the Baltic Sea. Mussels were exposed for three weeks to five ensulizole concentrations of 10, 10 2 , 10 3 , 10 4 , and 10 5  ng/L. Stress on stress response was evaluated by subjecting mussels to air exposure. A battery of biomarkers related to detoxification and antioxidant defense, oxidative stress damage, energy reserves and metabolism, autophagy, apoptosis, inflammation, and DNA damage was measured in the gills and the digestive gland. In general, ensulizole affected the antioxidant response, energy storage, and cell death-related processes in mussel tissues. Mussels exposed to low, environmentally relevant concentrations of ensulizole had a shorter air survival time than the control. Ensulizole often showed the non-monotonic concentration-response curves, suggesting the complex effects of this UV filter at molecular, biochemical, and organismal levels., 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.)

Temperature is an important abiotic factor that modulates all aspects of ectotherm physiology, including sensitivity to pollutants. Nanoparticles are emerging pollutants in coastal environments, and their potential to cause toxicity in marine organisms is a cause for concern. Here we studied the interactive effects of temperature (including seasonal and experimental warming) on sublethal toxicity of ZnO nanoparticles (nano-ZnO) in a model marine bivalve, the blue mussel Mytilus edulis. Molecular markers were used to assess the pollutant-induced cellular stress responses in the gills and the digestive gland of mussels exposed for 21 days to 10 μg l -1 and 100 μg l -1 of nano-ZnO or dissolved Zn under different temperature regimes including ambient temperature (10 °C and 15 °C in winter and summer, respectively) or experimental warming (+5 °C). Exposure to high concentration (100 μg l -1 ) of nano-ZnO caused oxidative injury to proteins and lipids and induced a marked apoptotic response indicated by increased transcript levels of apoptosis-related genes p53, caspase 3 and the MAPK pathway (JNK and p38) and decreased mRNA expression of anti-apoptotic Bcl-2. No significant induction of inflammatory cytokine-related response (TGF-β and NF-κB) of tissues was observed in nano-ZnO exposed-mussels. Furthermore, the oxidative injury and apoptotic response could differentiate the effects of nano-ZnO from those of dissolved Zn in the mussels. This study revealed that oxidative stress and stress-related transcriptional responses to nano-ZnO were strongly modified by warming and season in the mussels. No single biomarker could be shown to consistently respond to nano-ZnO in all experimental groups, which implies that multiple biomarkers are needed to assess nano-ZnO toxicity to marine organisms under the variable environmental conditions of coastal habitats., 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 © 2021 Elsevier B.V. All rights reserved.)

The global occurrence of organic UV filters in the marine environment is of increasing ecotoxicological concern. Here we assessed the toxicity of UV filters ensulizole and octocrylene in the blue mussels Mytilus edulis exposed to 10 or 100 μg l -1 of octocrylene and ensulizole for two weeks. An integrated battery of biochemical and molecular biomarkers related to xenobiotics metabolism and cellular toxicity (including oxidative stress, DNA damage, apoptosis, autophagy and inflammation) was used to assess the toxicity of these UV filters in the mussels. Octocrylene (but not ensulizole) accumulated in the mussel tissues during the waterborne exposures. Both studied UV filters induced sublethal toxic effects in M. edulis at the investigated concentrations. These effects involved induction of oxidative stress, genotoxicity (indicated by upregulation of DNA damage sensing and repair markers), upregulation of apoptosis and inflammation, and dysregulation of the xenobiotic biotransformation system. Octocrylene induced cellular stress in a concentration-dependent manner, whereas ensulizole appeared to be more toxic at the lower (10 μg l -1 ) studied concentration than at 100 μg l -1 . The different concentration-dependence of sublethal effects and distinct toxicological profiles of ensulizole and octocrylene show that the environmental toxicity is not directly related to lipophilicity and bioaccumulation potential of these UV filters and demonstrate the importance of using bioassays for toxicity assessment of emerging pollutants in coastal marine 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 © 2021 Elsevier B.V. All rights reserved.)

Engineered nanoparticles including ZnO nanoparticles (nZnO) are important emerging pollutants in aquatic ecosystems creating potential risks to coastal ecosystems and associated biota. The toxicity of nanoparticles and its interaction with the important environmental stressors (such as salinity variation) are not well understood in coastal organisms and require further investigation. Here, we examined the interactive effects of 100 μg l -1 nZnO or dissolved Zn (as a positive control for Zn 2+ release) and salinity (normal 15, low 5, and fluctuating 5-15) on bioenergetics and intermediate metabolite homeostasis of a keystone marine bivalve, the blue mussel Mytilus edulis from the Baltic Sea. nZnO exposures did not lead to strong disturbances in energy or intermediate metabolite homeostasis regardless of the salinity regime. Dissolved Zn exposures suppressed the mitochondrial ATP synthesis capacity and coupling as well as anaerobic metabolism and modified the free amino acid profiles in the mussels indicating that dissolved Zn is metabolically more damaging than nZnO. The environmental salinity regime strongly affected metabolic homeostasis and altered physiological and biochemical responses to nZnO or dissolved Zn in the mussels. Exposure to low (5) or fluctuating (5-15) salinity affected the physiological condition, energy metabolism and homeostasis, as well as amino acid metabolism in M. edulis. Generally, fluctuating salinity (5-15) appeared bioenergetically less stressful than constantly hypoosmotic stress (salinity 5) in M. edulis indicating that even short (24 h) periods of recovery might be sufficient to restore the metabolic homeostasis in this euryhaline species. Notably, the biological effects of nZnO and dissolved Zn became progressively less detectable as the salinity stress increased. These findings demonstrate that habitat salinity must be considered in the biomarker-based assessment of the toxic effects of nanopollutants on coastal organisms., 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 © 2021 Elsevier B.V. All rights reserved.)

Input of ZnO nanoparticles (nZnO) from multiple sources have raised concerns about the potential toxic effects on estuarine and coastal organisms. The toxicity of nZnO and its interaction with common abiotic stressors (such as elevated temperature) are not well understood in these organisms. Here, we examined the bioenergetics responses of the blue mussel Mytilus edulis exposed for 21 days to different concentrations of nZnO or dissolved zinc (Zn 2+ ) (0, 10, 100 μg l -1 ) and two temperatures (ambient and 5 °C warmer) in winter and summer. Exposure to nZnO had little effect on the protein and lipid levels, but led to a significant depletion of carbohydrates and a decrease in the electron transport system (ETS) activity. Qualitatively similar but weaker effects were found for dissolved Zn. In winter mussels, elevated temperature (15 °C) led to elevated protein and lipid levels increasing the total energy content of the tissues. In contrast, elevated temperature (20 °C) resulted in a decrease in the lipid and carbohydrate levels and suppressed ETS in summer mussels. These data indicate that moderate warming in winter (but not in summer) might partially compensate for the bioenergetics stress caused by nZnO toxicity in M. edulis from temperate areas such as the Baltic Sea., 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 Ltd. All rights reserved.)

Pollution by toxic metals including cadmium (Cd) and hypoxia are important stressors in estuaries and coastal waters which may interactively affect sessile benthic organisms, such as oysters. We studied metabolic responses to prolonged hypoxic acclimation (2 weeks at 5% O2) in control and Cd-exposed (30 d at 50 μg L(-1) Cd) oysters Crassostrea virginica, and analyzed the effects of these stressors on abundance of Vibrio spp. in oysters. Hypoxia-acclimated oysters retained normal standard metabolic rates (SMR) at 5% O2, in contrast to a decline of SMR observed during acute hypoxia. However, oysters spent more time actively ventilating in hypoxia than normoxia resulting in enhanced Cd uptake and 2.7-fold higher tissue Cd burdens in hypoxia. Cd exposure led to a significant decrease in tissue glycogen stores, increase in free glucose levels and elevated activity of glycolytic enzymes (hexokinase and aldolase) indicating a greater dependence on carbohydrate catabolism. A compensatory increase in activities of two key mitochondrial enzymes (citrate synthase and cytochrome c oxidase) was found during prolonged hypoxia in control oysters but suppressed in Cd-exposed ones. Cd exposure also resulted in a significant increase in abundance of Vibrio parahaemolyticus and Vibrio vulnificus levels during normoxia and hypoxia, respectively. Overall, Cd- and hypoxia-induced changes in metabolic profile, Cd accumulation and bacterial flora of oysters indicate that these stressors can synergistically impact energy homeostasis, performance and survival of oysters in polluted estuaries and have significant consequences for transfer of Cd and bacterial pathogens to the higher levels of the food chain., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Marine organisms are exposed to periodical oxygen deficiency and pollution stress in estuarine and coastal zones which may strongly affect their performance and survival. We studied the combined effects of exposure to a common pollutant, cadmium (Cd), and intermittent anoxia on anaerobic metabolism, energy status and mRNA expression of 13 genes involved in and/or controlled by the hypoxia inducible factor-1 (HIF-1) pathway in hepatopancreas of an intertidal bivalve, the eastern oyster Crassostrea virginica. In control oysters, prolonged anoxia resulted in a selective suppression of nitric oxide synthase (NOS) and upregulation of cytochrome c oxidase subunit IV (COX4) while the levels of other transcripts remained unchanged. During post-anoxic recovery, mRNA expression of hypoxia inducible factor-1alpha (HIF-1alpha) was elevated, phosphoenolpyruvate carboxykinase (PEPCK), NOS and LON protease suppressed, and mRNA expression of other studied genes not changed. Notably, most of the key glycolytic genes that are stimulated by HIF-1 in mammals, either remained unchanged or were downregulated in anoxic oysters suggesting a different mechanism of molecular response to oxygen deficiency. Patterns of transcriptional response during anoxia and reoxygenation were significantly altered by Cd exposure in a gene-specific manner. Anaerobic metabolism (indicated by accumulation of l-alanine, succinate and acetate during anoxia) was also suppressed in Cd-exposed oysters. In control oysters, ATP turnover rate (M(ATP)) during anoxia was mostly sustained by anaerobic glycolysis with negligible contributions from ATP and PLA breakdown. In contrast, in Cd-exposed oysters ATP breakdown contributed significantly to anaerobic M(ATP). Thus, while control oysters could efficiently defend the ATP levels and tissue energy status during prolonged anoxia, Cd-exposed oysters experienced a disturbance in tissue energy balance indicated by the depletion of ATP, a rapid decline in adenylate energy charge and increase in ADP/ATP ratios. This energy deficiency combined with suppression of anaerobic metabolism may strongly affect performance and survival of oysters in polluted estuaries where metal pollution may co-occur with "dead zones"., (Copyright 2010 Elsevier B.V. All rights reserved.)

Temperature and heavy metals such as cadmium (Cd) are important stressors which can strongly affect physiology of marine ectotherms in polluted estuaries. Mitochondria are among the key intracellular targets for these stressors, but the mechanisms of Cd-induced mitochondrial damage are not fully understood. In this study we determined the effects of acclimation temperature (12, 20 and 28 degrees C) and Cd exposure (0 or 50 microg L(-1) Cd) in vivo on activity and mRNA expression of a key mitochondrial enzyme, aconitase, which is known as a sensitive marker of oxidative stress, and on mRNA expression of LON protease involved in the degradation of oxidatively damaged mitochondrial proteins, in eastern oysters Crassostrea virginica. Sensitivity of mitochondrial aconitase to exposure to Cd in vitro (0 or 50 microM) was also determined in oysters acclimated to different temperatures and Cd levels. Acclimation at 28 degrees C resulted in a strong decrease in activity of mitochondrial aconitase as well as mRNA expression of aconitase and LON protease suggesting mitochondrial dysfunction at elevated temperatures. Exposure of isolated mitochondria to 50 microM Cd in vitro resulted in a 20-25% inhibition of mitochondrial aconitase reflecting oxidative damage of this enzyme. However, long-term (3-6 weeks) exposure of whole oysters to Cd had no effect on mitochondrial aconitase activity suggesting that this enzyme is well protected against Cd-induced oxidative stress in vivo. Aconitase mRNA expression was positively correlated with the enzyme activity within control and Cd-exposed groups; however, this correlation was strikingly different when compared between control and Cd-exposed oysters. The level of aconitase transcript was considerably lower (3-13-fold) in Cd-exposed oysters while the specific aconitase activities were similar in control and Cd-exposed oysters indicating regulation at the post-transcriptional level. LON protease expression was upregulated by 2-4-fold in Cd-exposed oysters suggesting an increase in mitochondrial protein degradation as a novel protective mechanism against Cd-induced mitochondrial stress. Our data indicate that mitochondrial aconitase is not a good biomarker for Cd-induced oxidative stress in oysters in vivo, because of its complex regulation at transcriptional and post-transcriptional levels, low sensitivity to Cd effects in vivo but high sensitivity to acclimation temperature that can potentially mask effects of other stressors under the field conditions.