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.

The use of peracetic acid for estrogen removal from urban wastewaters: E2 as a case study.

17ß-Estradiol (E2) is a natural estrogen produced by the feminine endocrine system. It is excreted mainly through urine and feces. Exposure to E2 may affect the reproductive system of both animals and humans, especially since the removal of E2 in conventional processes and technologies present in the wastewater treatment plants is not sufficient. Chlorine is one of the most studied and used oxidant worldwide. Although there are studies that demonstrate the endocrine disrupting compounds removal like E2, its reaction with organic matter can originate by-products, namely, trihalomethanes, which are known to have high toxic potential. The main aim of the present study was to evaluate the removal of E2 (50 µg E2 L-1-maximum concentration) using peracetic acid (PAA), a seeming cleaner and innocuous alternative to chlorine. To this end, a series of jar tests were performed, using different peracetic acid concentrations (1, 5, 10, and 15 mg L-1) and contact times (10, 15, and 20 min). The results obtained showed that a peracetic acid concentration of 15 mg L-1 with a contact time of 20 min had a removal efficacy of approximately 100%. The second main goal of this study was to evaluate the ecotoxicological potential of the tested treatments on the zebrafish Danio rerio. Several oxidative stress biomarkers were evaluated, namely glutathione S-transferase, lipid peroxidation, and catalase, besides vitellogenin. Both peracetic acid and E2 caused significant increases in the oxidative stress biomarkers, although this did not lead to increased lipid peroxidation levels. In addition, peracetic acid significantly decreased the estrogenic activity of E2, as indicated by decreased vitellogenin levels. Peracetic acid demonstrated to have great potential as an alternative disinfectant for chlorine treatments, and indications for future research are discussed.

Efficacy assessment of peracetic acid in the removal of synthetic 17α-ethinyl estradiol contraceptive hormone in wastewater.

Increasing concerns have been raised on endocrine disrupting chemicals like the sex hormone 17α-ethinylestradiol (EE2), the more since traditional wastewater (WW) treatments appear to be ineffective for their removal. The efficacy of the relatively novel disinfectant peracetic acid (PAA) in EE2 removal was evaluated, as well as its potential effects on WW quality parameters. The treatments tested for EE2 removal were also evaluated in terms of toxicity, through the determination of biochemical responses (antioxidant enzymes, lipid peroxidation and vitellogenin induction) using zebrafish (Danio rerio) as a biological model. PAA contact times less than 20 min appeared insufficient regardless of the PAA dose tested, but a 100% EE2 removal was attained at a PAA concentration of 15 mg/L with a contact time of 20 min. Total suspended solids, chemical oxygen demand and pH in PAA treatments remained well within levels set in European legislation for WW discharge. EE2 induced significant increased vitellogenin (VTG) levels in both female and male fish, indicating increased estrogenic activity, especially in males suggesting an endocrine disruption effect. With the addition of PAA (15 mg/L), however, VTG levels in both sexes returned to control values. Although this PAA treatment showed increased levels of the antioxidant enzyme catalase, the lipid peroxidation levels were similar or even lower than in controls. Overall the results suggest that the use of PAA appears a promising way forward as a less toxic alternative to chlorine disinfection with high efficiency in the removal of EDC like EE2.

Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO2-NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO2-NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO2-NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO2-NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO2-NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO2-NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO2-NP toxicity in D. magna, providing useful information for future research.

Comparing biomarker responses during thermal acclimation: A lethal vs non-lethal approach in a tropical reef clownfish.

Knowledge of thermal stress biology for most tropical fish species in reef ecosystems under climate change is still quite limited. Thus, the objective of this study was to measure the time-course changes of thermal stress biomarkers in the commercially exploited coral reef fish Amphiprion ocellaris, during a laboratory simulated event of increased temperature. Heat shock protein 70kDa (Hsp70) and total ubiquitin (Ub) were determined in the muscle (lethal method) and in the fin (non-lethal alternative method) under two temperature treatments (control - 26°C and elevated temperature - 30°C) throughout one month with weekly samplings. Results suggest that biomarker basal levels are tissue-specific and influence the degree of response under temperature exposure. Responses were highly inducible in the muscle but not in fin tissue, indicating that the latter is not reliable for monitoring purposes. Thermal stress was observed in the muscle after one week of exposure (both biomarkers increased significantly) and Ub levels then decreased, suggesting the animals were able to acclimate by maintaining high levels of Hsp70 and through an effective protein turnover. In addition, the results show that mortality rates did not differ between treatments. This indicates that A. ocellaris is capable of displaying a plastic response to elevated temperature by adjusting the protein quality control system to protect cell functions, without decreasing survival. Thus, this coral reef fish species presents a significant acclimation potential under ocean warming scenarios of +4°C. Monitoring of thermal stress through a non-lethal method, fin-clipping, although desirable proved to be inadequate for this species.

Changes in metabolic pathways of Desulfovibrio alaskensis G20 cells induced by molybdate excess.

The activity of sulfate-reducing bacteria (SRB) intensifies the problems associated to corrosion of metals and the solution entails significant economic costs. Although molybdate can be used to control the negative effects of these organisms, the mechanisms triggered in the cells exposed to Mo-excess are poorly understood. In this work, the effects of molybdate ions on the growth and morphology of the SRB Desulfovibrio alaskensis G20 (DaG20) were investigated. In addition, the cellular localization, ion uptake and regulation of protein expression were studied. We found that molybdate concentrations ranging between 50 and 150 µM produce a twofold increase in the doubling time with this effect being more significant at 200 µM molybdate (five times increase in the doubling time). It was also observed that 500 µM molybdate completely inhibits the cellular growth. On the context of protein regulation, we found that several enzymes involved in energy metabolism, cellular division and metal uptake processes were particularly influenced under the conditions tested. An overall description of some of the mechanisms involved in the DaG20 adaptation to molybdate-stress conditions is discussed.

Effects of carcinogenic versus non-carcinogenic AHR-active PAHs and their mixtures: lessons from ecological relevance.

Polycyclic aromatic hydrocarbons (PAHs) are priority environmental mutagens and carcinogens that occur in the aquatic environment as mixtures rather than the individual compounds for which guidelines are issued. The present work aimed at understanding the interaction effects between carcinogenic and non-carcinogenic PAHs in a model marine fish (Dicentrarchus labrax) in realistic scenarios. Laboratory assays under ecologically-relevant parameters were conducted for 28 days with sediments spiked with low-moderate concentrations (250-800ngg(-1)) of two model PAHs, phenanthrene (non-carcinogenic) and benzo[b]fluoranthene (carcinogenic to experimental animals). Both PAHs induced hepatic histopathological changes that indicate metabolic failure and inflammation, especially in animals exposed to mixtures. Phenanthrene elicited biochemical changes better related to oxidative stress (lipid peroxidation, glutathione and glutathione S-transferase activity) and CYP function, whereas B[b]F disrupted metabolic responses and defences to toxicological challenge. Conversely, mixed PAHs yielded lesions and responses that, altogether, are compatible with the AHR dependent pathway (the basis of PAH mutagenicity), potentially generating supra-additive effects. Nonetheless, the low, ecologically-relevant, concentrations of PAHs diluted dose and time-response relations. Overall, although seemingly predicting the risk of individual PAHs, environmental guidelines may not apply to mixtures by underestimating adverse effects, which calls for a redefinition of standards when determining the true risk of toxicants under realistic circumstances.

Physiological, cellular and biochemical thermal stress response of intertidal shrimps with different vertical distributions: Palaemon elegans and Palaemon serratus.

The ability to cope with high temperature variations is a critical factor in intertidal communities. Two species of intertidal rocky shore shrimps (Palaemon sp.) with different vertical distributions were collected from the Portuguese coast in order to test if they were differentially sensitive to thermal stress. Three distinct levels of biological organization (organismal, biochemical, and cellular) were surveyed. The shrimp were exposed to a constant rate of temperature increase of 1°C x h(-1), starting at 20°C until reaching the CTMax (critical thermal maximum). During heat stress, two biomarkers of protein damage were quantified in the muscle via enzyme-linked immunosorbent assays: heat shock proteins HSP70 (hsp70/hsc70) and total ubiquitin. Muscle histopathological alterations caused by temperature were also evaluated. CTMax values were not significantly different between the congeners (P. elegans 33.4 ± 0.5 °C; P. serratus 33.0 ± 0.5 °C). Biomarker levels did not increase along the temperature trial, but P. elegans (higher intertidal) showed higher amounts of HSP70 and total ubiquitin than P. serratus (lower intertidal). HSP70 and total ubiquitin levels showed a positive significant correlation in both species, suggesting that their association is important in thermal tolerance. Histopathological observations of muscle tissue in P. serratus showed no gross alterations due to temperature but did show localized atrophy of muscle fibers at CTMax. In P. elegans, alterations occurred at a larger scale, showing multiple foci of atrophic muscular fascicles caused by necrotic or autolytic processes. In conclusion, Palaemon congeners displayed different responses to stress at a cellular level, with P. elegans having greater biomarker levels and histopathological alterations.

Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide.

Although the neurotoxic and genotoxic potential of acrylamide has been established in freshwater fish, the full breadth of the toxicological consequences induced by this xenobiotic has not yet been disclosed, particularly in aquatic invertebrates. To assess the effects of acrylamide on a bivalve model, the Mediterranean mussel (Mytilus galloprovincialis), two different setups were accomplished: 1) acute exposure to several concentrations of waterborne acrylamide to determine lethality thresholds of the substance and 2) chronic exposure to more reduced acrylamide concentrations to survey phases I and II metabolic endpoints and to perform a whole-body screening for histopathological alterations. Acute toxicity was low (LC50≈400mg/L). However, mussels were responsive to prolonged exposure to chronic concentrations of waterborne acrylamide (1-10mg/L), yielding a significant increase in lipid peroxidation plus EROD and GST activities. Still, total anti-oxidant capacity was not exceeded. In addition, no neurotoxic effects could be determined through acetylcholine esterase (AChE) activity. The findings suggest aryl-hydrocarbon receptor (Ahr)-dependent responses in mussels exposed to acrylamide, although reduced comparatively to vertebrates. No significant histological damage was found in digestive gland or gills but female gonads endured severe necrosis and oocyte atresia. Altogether, the results indicate that acrylamide may induce gonadotoxicity in mussels, although the subject should benefit from further research. Altogether, the findings suggest that the risk of acrylamide to aquatic animals, especially molluscs, may be underestimated.

Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations.

Acrylamide is an amide used in several industrial applications making it easily discharged to aquatic ecosystems. The toxicity of acrylamide to aquatic organisms is scarcely known, although previous studies with murine models provided evidence for deleterious effects. To assess the effects of acrylamide to freshwater fish, goldfish (Carassius auratus L.) were exposed to several concentrations of waterborne acrylamide and analysed for genotoxic damage, alterations to detoxifying enzymes and histopathology. Results revealed a dose-dependent increase in total DNA strand breakage, the formation of erythrocytic nuclear abnormalities and in the levels of hepatic cytochrome P4501A (CYP1A) and glutathione S-transferase (GST) activity. In addition, acrylamide induced more histopathological changes to pancreatic acini than to the hepatic parenchyma, regardless of exposure concentration, whereas hepatic tissue only endured significant alterations at higher concentrations of exposure. Thus, results confirm the genotoxic potential of acrylamide to fish and its ability to induce CYP1A, probably as a direct primary defence mechanism. This strongly suggests the substance's pro-mutagenic potential in fish, similarly to what is known for rodents. However, the deleterious effects observed in the pancreatic acini, more severe than in the liver, could indicate a specific, albeit unknown toxic mechanism of acrylamide to fish that overran the organism's metabolic defences against a chemical agent rather than causing a general systemic failure.

Bioavailability of cadmium and biochemical responses on the freshwater bivalve Corbicula fluminea--the role of TiO2 nanoparticles.

The increasing and widespread applications of TiO2 engineered nanoparticles (nTiO2) led to the release of these materials into aquatic environments and consequently a change on the assessment of the environmental risk of trace metals. In this work, the role of two commercial nTiO2 with distinct crystalline phases and sizes (nTiO2-P25: 80% anatase+20% rutile, d=20nm; nTiO2-NA: 100% anatase, d=5 nm; 0.1 and 1.0 mg L(-1)) on Cd (112 µg L(-1)) speciation, biouptake and toxicity for the freshwater bivalve Corbicula fluminea was evaluated. The electroanalytical technique 'absence of gradients and Nernstian equilibrium stripping (AGNES)' was used to quantify the free Cd concentrations in the exposure medium in presence of both particles. Despite ca. 30-40% decrease of free Cd in the medium in presence of nTiO2, Cd uptake by C. fluminea was similar in the absence and presence of either of the particles. Superoxide dismutase and glutathione-S-transferase activities remained unchanged for Cd in absence and presence of nTiO2, whereas a significant increase of the catalase activity was obtained at the third day for Cd in presence of both nTiO2. Despite lipid peroxidation data shows that the presence of both nTiO2 seems to exert cells damage, a more quantitative description is not possible with the obtained data. The lack of clear-cut responses by the studied biomarkers, even when only in presence of Cd, do not allow insights into the effect of the presence of nTiO2 on the Cd toxicity to the bivalves. Notwithstanding, morphological changes in the digestive gland were clearly obtained in the presence of Cd, nTiO2 and Cd+nTiO2 indicating an inflammatory response.

Effect of temperature in multiple biomarkers of oxidative stress in coastal shrimp.

Various studies in captivity and in the wild have pointed to the effect of season, and temperature in particular, in the levels of the oxidative stress biomarkers currently used for environmental quality assessment. However, knowledge on how temperature affects the oxidative stress response is unavailable for most species. This study investigated the effect of increasing temperature on lipid peroxidation, catalase activity, superoxide dismutase and glutathione-S-transferase in the shrimps, Palaemon elegans and Palaemon serratus. It was concluded that increasing temperatures significantly affect all the biomarkers tested in both species, with the exception of superoxide dismutase in P. serratus which was not affected by temperature. The oxidative stress response was more intense in P. elegans, than in P. serratus, producing higher peaks of all biomarkers at temperatures between 22°C and 26°C, followed by low levels at higher temperatures. It was concluded that monitoring of ecosystems using oxidative stress biomarkers should take into account the species and thermal history of the organisms. Sampling should be avoided during heat waves and immediately after heat waves.

Development of a New Aggregation Method to Remove Nanoplastics from the Ocean: Proof of Concept Using Mussel Exposure Tests.

The overproduction and mismanagement of plastics has led to the accumulation of these materials in the environment, particularly in the marine ecosystem. Once in the environment, plastics break down and can acquire microscopic or even nanoscopic sizes. Given their sizes, microplastics (MPs) and nanoplastics (NPs) are hard to detect and remove from the aquatic environment, eventually interacting with marine organisms. This research mainly aimed to achieve the aggregation of micro- and nanoplastics (MNPs) to ease their removal from the marine environment. To this end, the size and stability of polystyrene (PS) MNPs were measured in synthetic seawater with the different components of the technology (ionic liquid and chitosan). The MPs were purchased in their plain form, while the NPs displayed amines on their surface (PS NP-NH2). The results showed that this technology promoted a significant aggregation of the PS NP-NH2, whereas, for the PS MPs, no conclusive results were found, indicating that the surface charge plays an essential role in the MNP aggregation process. Moreover, to investigate the toxicological potential of MNPs, a mussel species (M. galloprovincialis) was exposed to different concentrations of MPs and NPs, separately, with and without the technology. In this context, mussels were sampled after 7, 14, and 21 days of exposure, and the gills and digestive glands were collected for analysis of oxidative stress biomarkers and histological observations. In general, the results indicate that MNPs trigger the production of reactive oxygen species (ROS) in mussels and induce oxidative stress, making gills the most affected organ. Yet, when the technology was applied in moderate concentrations, NPs showed adverse effects in mussels. The histological analysis showed no evidence of MNPs in the gill's tissues.

Liver alterations in two freshwater fish species (Carassius auratus and Danio rerio) following exposure to different TiO2 nanoparticle concentrations.

The toxicity of titanium dioxide nanoparticles (TIO2 NPs) and oxidative stress effects were studied in two freshwater fish species (Carassius auratus and Danio rerio) exposed for 21 days to different concentrations (0.01, 0.1, 1, 10, 100/mgL) of TiO2 NPs and to a control (tap water). Additional fish were transferred to clean water for 14 days to assess the ability to recover from exposure to TiO2 NPs. Activities of the enzyme glutathione-S-transferase (GST) and lipid peroxidation (LPO) (malondialdheyde) were measured as indicators of oxidative stress. Histological and ultra-structural changes in livers from both species of fish were evaluated by light and electron microscopy. Results show a general GST activity increase according to TiO2 NPs concentrations, which is in agreement with data from LPO. After 21 days, GST activities decreased possibly caused by suppression of GST synthesis as a result of severe stress. Histological and ultra-structural analysis of livers from exposed fish show degeneration of the hepatic tissue and alterations in hepatocytes such as glycogen depletion and an increase in lipofucsin lysosome-like granules. After a depuration period a partial recovery for biochemical markers and cells was observed. The results suggest that TiO2 promotes alterations in hepatic tissues compatible with oxidative stress.

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.

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.

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.

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.

A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis.

In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).

Assessing the estrogenic potency in a Portuguese wastewater treatment plant using an integrated approach.

The estrogenic potency of a wastewater treatment plant (WWTP) was evaluated using chemical and biological analyses, which showed that after the station treatment processes some of the selected endocrine disruptor compounds (EDCs) were still present in the treated effluent (e.g., bisphenol A, alkylphenols, estrone). Thus, the most common endocrine EDCs were identified and quantified and the overall estrogenicity of the treated effluent assessed by integrating the results. Male goldfish (Carassius auratus) were used as biological indicators in a 28-day experiment. Vitellogenin (Vtg), gonadosomatic and hepatosomatic indices, steroids (17beta-estradiol and 11-ketotestosterone) and histopathology were biomarkers used in fish to evaluate WWTP treated effluent estrogenicity, in combination with instrumental analyses. The results showed a significant increase (P < 0.01) in plasma and liver Vtg, which were significantly correlated (r = 0.66; P < 0.01). The gonadosmatic index was significantly (P < 0.01) reduced in exposed fish. The steroid analyses revealed significant elevations in 17beta-estradiol and depressed 11-ketotestosterone concentrations. The histological examinations show changes in exposed fish gonads, such as regressed testes and in some cases (43% to 75%) the development of ovo-testis in fish exposed to 50% and 100% treated effluent.