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Essential nutrients are critical for physiological processes of organisms. In fish, they are obtained primarily from the diet, and their transfer and accumulation are known to be impacted by environmental variables such as water temperature, pH and salinity, as well as by diet composition and matrices. Yet, prey items consumed by fish may also contain toxic compounds such as marine toxins associated with harmful algae. These biotoxins have the potential to affect essential trace element assimilation in fish through chemical interactions such as the formation of trace element-toxin complexes or by affecting general fish physiology as in the modification of ion-specific transport pathways. We assessed the influence of dietary exposure to brevetoxins (PbTxs), ichthyotoxic neurotoxins produced by the dinoflagellate Karenia brevis, on trophic transfer of two essential trace elements, Mn and Zn, in a fish model. Using ecologically relevant concentrations of PbTxs and trace elements in controlled laboratory conditions, juvenile turbots Scophthalmus maximus were given food containing PbTxs before or at the same time as a feeding with radiotracers of the chosen essential elements ( 54 Mn and 65 Zn). Treatments included simultaneous exposure (PbTxs +  54 Mn +  65 Zn) in a single-feeding, 3-week daily pre-exposure to dietary PbTx followed by a single feeding with 54 Mn and 65 Zn, and a control ( 54 Mn and 65 Zn only). After a 21-day depuration period, turbot tissue brevetoxin levels were quantified and assimilation efficiencies of 54 Mn and 65 Zn were assessed. PbTxs were found in turbot tissues in each exposure treatment, demonstrating dietary trophic transfer of these toxins; yet, no differences in assimilation efficiencies of Mn or Zn were found between treatments or the control (p > 0.05). These results indicate that, in our experimental conditions, PbTx exposure does not significantly affect the trophic transfer of Mn and Zn in fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nitrite (NO2(-)) is commonly present as contaminant in aquatic environment and toxic to aquatic organisms. In the present study, we investigated the effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Fish were exposed to various concentrations of nitrite (0, 0.02, 0.08, 0.4 and 0.8mM) for 96 h. Fish blood and gills were collected to assay haematological parameters, oxidative stress and expression of genes after 0, 24, 48 and 96 h of exposure. In blood, the data showed that the levels of methemoglobin (MetHb), triglyceride (TG), potassium (K(+)), cortisol, heat shock protein 70 (HSP70) and glucose significantly increased in treatments with higher concentrations of nitrite (0.4 and/or 0.8mM) after 48 and 96 h, while the levels of haemoglobin (Hb) and sodium (Na(+)) significantly decreased in these treatments. In gills, nitrite (0.4 and/or 0.8mM) apparently reduced the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH), increased the formation of malondialdehyde (MDA), up-regulated the mRNA levels of c-jun amino-terminal kinase (JUK1), p53, caspase-3, caspase-7 and caspase-9 after 48 and 96 h of exposure. The results suggested caspase-dependent and JUK signaling pathways played important roles in nitrite-induced apoptosis in fish. Further, this study provides new insights into how nitrite affects the physiological responses and apoptosis in a marine fish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Laboratory tests with marine flatfish were conducted to investigate associations among gene expression, higher biological responses and wastewater effluent exposure. In the present study, male hornyhead turbot (Pleuronichthys verticalis) were exposed to environmentally realistic (0.5%) and higher (5%) concentrations of chemically enhanced advanced-primary (PL) and full-secondary treated (HTP) effluents from two southern California wastewater treatment plants (WWTP). Hepatic gene expression was examined using a custom low-density microarray. Alterations in gene expression (vs. controls) were observed in fish exposed to both effluent types. Fish exposed to 0.5% PL effluent showed changes in genes involved in the metabolism of xenobiotics, steroids, and lipids, among other processes. Fish exposed to 5% PL effluent showed expression changes in genes involved in carbohydrate metabolism, stress responses, xenobiotic metabolism, and steroid synthesis, among others. Exposure to 5% HTP effluent changed the expression of genes involved in lipid, glutathione and xenobiotic metabolism, as well as immune responses. Although no concentration-dependent patterns of response to effluent exposure were found, significant Spearman correlations were observed between the expression of 22 genes and molecular and/or higher biological responses. These results indicate that microarray gene expression data correspond to higher biological responses and should be incorporated in studies assessing fish health after exposure to complex environmental mixtures., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Coastal habitats play a major role as nurseries for many fish species; however, they are also submitted to pollutants and oxygen fluctuations. Fry's concept of metabolic scope for activity was used to evaluate the effect of polychlorinated biphenyls (PCBs) on the aerobic metabolism in juvenile common sole (0-1 year old). Aerobic metabolic scope (AMS) in control and PCB-contaminated fish via food pathway was determined using respirometry techniques. Furthermore, the hypoxia tolerance in control and PCB-contaminated fish was evaluated by assessing their critical oxygen concentration (O(2crit)). Our results showed that while PCB-contaminated fish were able to maintain a constant AMS and O(2crit), PCBs tend to affect their aerobic metabolism by acting on maximal oxygen consumption (MO(2max)) in hypoxia and standard metabolic rate, but only at the highest PCB concentration between 30 and 60 days of exposure. In conclusion, we can hypothetise that the tested PCB-exposures may not impair the tolerance to hypoxia and the survival of common sole in their natural environment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The fluoranthene (Fluo) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in human food and in marine compartments. However, the existing data on its genotoxicity is poor and controversial. The aim of this study was to assess in vitro the potential genotoxicity of Fluo in sole and its possible effect on CYP450 modulation. Freshly isolated hepatocytes were exposed for 24 h to a range of Fluo concentrations from 0.5 to 50 μM in both culture flasks and microplate wells. The ethoxyresorufin-O-deethylase (EROD) activity was measured as an indicator of the activity of the cytochrome P450 1A1 (CYP1A1). The genotoxic effects were evaluated by measuring both DNA strand breaks and DNA adducts by the alkaline comet assay and the postlabeling technique respectively. Calf thymus DNA was also exposed to Fluo in the presence of sole liver microsomes in order to check for Fluo DNA adduct formation. In sole hepatocytes, Fluo was shown to induce a decrease in the EROD activity in a concentration-dependent manner. A significant genotoxic effect was observed in terms of DNA strand breakage from an exposure concentration of 5 μM: despite a concentration-dependent effect was observed, it did not follow a linear dose-response. The response was similar whatever the way of exposure in flasks or in wells. One reproducible adduct was detected in the hepatocytes exposed to the highest concentrations of Fluo. The formation of Fluo adducts was confirmed by the detection of one reproducible adduct following in vitro exposure of calf thymus DNA to 100 and 200 μM of Fluo in the presence of sole microsomes. These results demonstrate the potential of sole hepatocytes to metabolize Fluo in 24 h into reactive species, able to induce genotoxicity by DNA strand breakage and DNA adduct formation. Moreover, a miniaturized cell exposure system was validated for further experiments using fewer amounts of hepatocytes and contaminants, and allowing exposure to PAH metabolites., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ozone is frequently used for water treatment and disinfection in recirculating aquaculture systems. However, due to the fragmentary data on chronic toxicity of ozone produced oxidants (OPO) and its safe concentrations, the daily application of ozone in aquaculture is challenging. To evaluate the chronic effects of sublethal OPO concentrations, juvenile turbot (Psetta maxima, L.) were exposed to OPO concentrations of 0.06, 0.10 and 0.15 mg/l for 21 days. Gills were analysed for histopathological alterations and mRNA expression of heat shock protein 70 (hsp70), hsp90 as well as glutathione-S-transferase (gst) were determined in the gills and the liver after 1d, 7d and 21 d. Histopathologic findings confirmed adverse effects at 0.10-0.15 mg/l, but these (necrosis, lamellar clubbing, hypertrophy, hyperplasia) could only be observed after an extended exposure (mostly 21 d), and were considered as irreversible tissue damage. Hsp70 expression in the gills was only significantly increased at the highest OPO concentration (0.15 mg/l) on 1d and 7d, and returned to basic levels until day 21. Hsp90 mRNA was already increased at 0.10mg/l after 1 and 7 days of exposure, and again was comparable to the control group on day 21. In contrast, elevated gst mRNA expression was only observed on day 7 at 0.10mg and 0.15 mg/l. Although similar trends were observed in the liver for all markers, differences were only significant in exceptional cases due to the high individual variation observed. Thus, mRNA expression in the gills rather than in the liver is recommended as a marker to characterize OPO-induced oxidative stress in turbot. It has to be noted that mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration, suggesting a collapse of adaptive mechanisms as a possible explanation for the observed tissue damage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Several biomarker responses were determined in three fish species, Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps, from two estuaries of the Portuguese coast, Ria de Aveiro and Tejo. Both estuaries have significant anthropogenic influences from multiple sources (industrial, agricultural and shipping activities), which was evident from sediment chemical characterization concerning metal (copper, zinc, nickel, lead and chromium) and polycyclic aromatic hydrocarbon (PAH) concentrations. Spatial variability in fish responses was observed across species for most biomarkers of exposure [the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST), and metallothionein concentrations (MT)] and effect biomarkers [lipid peroxidation (LPO), RNA to DNA ratio (R:D), protein and lipid content]. In general, the interspecific differences in biomarker responses were greater than the spatial differences, due to differences in the behavior and habitat use of the species. Nevertheless, similarities were also observed considering both chemical load and biomarker responses. In highly polluted sites fish showed in general a significant antioxidant enzyme induction, associated with decreased R:D values, while fish from the least impacted site had little enzyme induction and better condition indices (high R:D and low LPO values). EROD activity was also higher for all species in the Tejo than Ria de Aveiro estuary, despite the generally higher total PAH measured in Ria de Aveiro, most likely due to a higher proportion of 4 and 6-ring PAHs, considered more toxic than low molecular weight PAHs, in the Tejo. In conclusion, this multi-biomarker approach considering multiple species provided improved understanding of the diverse responses and effects of exposure to contaminants and the effective risk it poses for different fish species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The long term consequences of oil exposure upon marine populations are still poorly evaluated. One particular missing piece of information relates to the link between oil exposure, individuals' ability to face environmental contingencies and populations' production and dynamics. In that context, the present paper investigates the impact of oil exposure upon fish cardio-respiratory performance, this performance being viewed as a key determinant of individual fitness. Experimental conditions replicated the contamination conditions observed during the weeks that immediately followed the Erika oil spill (west coast of France; December 1999). Sole (Solea solea), were exposed to number-2 oil for 5 days and were then challenged with an acute rise in temperature (from 15 to 30 degrees C at 1.5 degrees Ch(-1)). Oxygen consumption, cardiac output, heart rate and stroke volume were monitored throughout. Experimental results showed that compared to uncontaminated control animals, oil-exposed sole displayed impaired cardio-respiratory responses and were unable to meet the temperature-driven increase in tissues oxygen demand. The relationship between oxygen consumption and cardiac output indicated that oil-exposed fish had recourse to venous oxygen stores very early in the thermal challenge (20 degrees C). In control fish there was no evidence for depletion of venous oxygen store until above 25 degrees C.

Solea senegalensis is a commercially relevant aquaculture species that remains largely unexplored at the genomic level. The aim of this study was to identify novel genomic responses to lipopolysaccharide and copper sulphate challenges using suppression subtractive hybridization (SSH) and real-time RT-PCR. Forward- and reverse-subtractive libraries were generated for the identification of genes whose transcription is altered in response to lipopolysaccharide (LPS) (immunomodulator) in head kidney (immunologically important organ) and to CuSO(4) (common algacide) in liver (central metabolic organ and important source of immune transcripts). A total of 156 genes involved in major physiological functions were identified by SSH, the identified sequences representing a significant increase in the number of sole ESTs in public databases. Fifteen genes represented in the subtracted libraries were selected for further tissue, temporal and inducible transcriptional profiling by real-time RT-PCR. A rigorous quantification of transcript copy numbers was performed for this purpose in both pooled and individual samples from two independent experiments. More than half of the investigated mRNAs encode proteins that deal with different aspects of the immune response, like NCCRP1 (non-specific cytotoxic cell receptor), C3 and C7 (complement components), and ferritin M, HP and TF (iron homeostasis), or play a crucial role in its regulation, like TRAF3. Other mRNAs studied encode proteins involved in metabolism, like TKT and NDUFA4, the response to stimulus, like CEBPB (transcription factor) and CIRBP (RNA-binding protein), and other cell processes. Highly abundant (>500 molecules/pg total RNA) and rare (< or =1 molecules/pg) mRNA species were quantified in each sole organ examined, and outstanding differences were also recorded in the comparison between the two organs, e.g. C3 and TF mRNAs were largely overexpressed in liver (>5000 molecules/pg) as compared to head kidney (<5 molecules/pg). Most investigated mRNAs displayed significant alterations in their steady-state copy number following LPS and/or CuSO(4) stimulation, i.e. they were (i) up-regulated in response to both treatments in at least one of the two organs (NCCRP1, CEBPB, SQSTM1, NDUFA4, C7 and HP), (ii) up-regulated (TF, CIRBP, TRFA, C3) or down-regulated (TKT) by LPS, their levels remaining essentially unchanged upon CuSO(4) challenge, or (iii) down-regulated by LPS, though up-regulated by CuSO(4) (ferritin M). Quantifications in individual fish were consistent with those in pooled samples with respect to both the direction and the absolute changes in transcript abundance.

Cytochrome P4501A (CYP1A), benzo(a)pyrene (B(a)P) activation and biliary elimination, phase II activities, and peroxisomal and antioxidant activities of turbot (Scophthalmus maximus) were studied in a long-term controlled experiment. Fish were serially exposed in water on day 1 and on completion of months 3, 6 and 9 to 0.1, 0.2, 0.1 and 0.1mg B(a)P/l, respectively, while another group was identically treated with additional PCB77 (3,3',4,4'-tetrachlorobiphenyl) at 1% of concomitant B(a)P (w/w). Temporally persistent responses were obtained by sampling on week 3 and 3 months from each latest exposure. Serial exposure to B(a)P+PCB77 progressively induced liver 7-ethoxyresorufin O-deethylase (EROD) activity and CYP1A protein levels (ELISA, western blotting) towards months 9, 12 and gill EROD activity on month 12. It associated with an apparent increase in liver benzo(a)pyrene diol epoxide (BPDE)-DNA adduct levels (ultrasensitive enzyme radioimmunoassay), and elevated bile B(a)P metabolite levels on month 9 females as compared to males. In contrast, B(a)P alone did not cause (p>0.05) comparable effects on liver EROD, CYP1A, adducts nor on bile metabolites. Both exposed groups demonstrated evidence for lasting oxidative stress as hepatic superoxide dismutase, catalase and glutathione peroxidase activities were significantly altered (p<0.05) with symptomatic pro-oxidant associations among them. Both treatments affected liver somatic index similarly (increase on month 3, decrease on month 9 in males). Continued exposure on month 18 (0.2mg B(a)P/l, 1% PCB77) followed by sampling 6 months later showed sustained induction (p<0.001) of hepatic EROD in B(a)P+PCB77 group, which was not seen in B(a)P alone treatment. Thus, PCB77 co-exposure prolonged CYP1A induction and contributed to a persistent oxidative challenge in B(a)P-exposed turbot. The results indicate synergistic effects of polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) exposure in the aquatic environment.

Medetomidine is proposed as a candidate antifouling compound proven effective against barnacles. It is routinely used as a sedative in veterinary medicine. It is therefore important to also investigate possible adverse effects on non-target organisms. Thus, sublethal effects on two different ages of juvenile turbot (Psetta maxima) exposed to a wide concentration range of medetomidine (0.063-420nM) were assessed after exposure under semistatic as well as flow-through conditions, for a maximum of 96h. Effects on respiration frequency and amount of oxygen consumed were assessed, as well as the ability of turbot to adapt to a dark background. A significant decrease was seen both in respiration frequency with a lowest observed effect concentration (LOEC) of 2.1nM as well as in amount of oxygen consumed (LOEC=420nM) and colour adaptation (LOEC=4.2nM). Colour adaptation was also evaluated in a short exposure experiment, 1h, where significant effects were observed (LOEC=2.1nM). Reversibility, when fish were incubated in clean seawater following exposure, was seen for all observed effects. Ecological relevance of the observed effects is discussed.

We have characterized hepatic phenol sulfotransferase (SULT) activities in two benthic fish species, Mullus barbatus and Lepidorhombus boscii, using p-nitrophenol, dopamine, 17beta-estradiol, 4-nonylphenol, and 1-naphthol as substrates. High affinity sulfation of 17beta-estradiol was observed in both species (Km=28-75 nM), suggesting the presence of a specific estrogen sulfotransferase that catalyzes the formation of estradiol-3 sulfate. Among the tested compounds, 1-naphthol was the most effective substrate for sulfation, with Vmax/Km ratios several hundred-fold higher than the other substrates examined. Both species sulfated the tested compounds, but only M. barbatus was able to sulfate dopamine. We also tested the inhibitory effects of common marine pollutants with estrogenic (4-nonylphenol) and androgenic (tributyltin, triphenyltin) properties on p-nitrophenol and 17beta-estradiol SULT activities. 4-Nonylphenol and triphenyltin inhibited sulfation of both substrates at micromolar concentrations in both species. However, tributyltin was only effective against SULTs from L. boscii, again at micromolar concentrations. The data indicate that M. barbatus and L. boscii are able to sulfate a range of xenobiotics and endogenous compounds, and inhibition of these activities by environmental pollutants may contribute to the known toxic effects of these compounds.

Cellular biomarkers of exposure and biological effects were measured in hepatocytes of turbot exposed to either Cd, Cu or Zn at concentrations of 1 and 10 mg/l seawater for 7 days and after depuration for 14 days. Metal content in hepatocyte lysosomes was determined by image analysis after autometallography (AMG) as volume density of autometallographed black silver deposits (Vv(BSD)). Metallothionein (MT) levels were quantified on liver sections by microdensitometry after immunohistochemical staining with a polyclonal anti cod-MT antibody (MT-OD), and in the cytosolic fraction of hepatocytes by difference pulse polarography (MT-DPP). Lysosomal structural changes (lysosomal volume, surface and numerical densities--Vv(LYS), Sv(LYS) and Nv(LYS-), and surface-to-volume ratio S/V(Lys)) were quantified by image analysis after demonstration of beta-glucuronidase activity on liver cryotome sections. Vacuolisation produced by metal-exposure in hepatocytes was quantified by stereology as volume density of vacuoles (Vv(VAC)). Exposure time and metal concentrations significantly affected Vv(BSD) in lysosomes, MT levels and the degree of vacuolisation after 1 h and 1 day exposure to the three metals. The highest Vv(BSD), MT and Vv(VAC) values were recorded after 7 days exposure in all cases. MT-OD and MT-DPP were significantly correlated with Vv(BSD). Vv(LYS) in hepatocytes increased significantly after exposure to the metals. Exposure biomarkers returned to control values after depuration with the exception of those turbots that had been exposed to 10 mg Cd/l. Alike, Vv(LYS) and Sv(lys) (Cu exposure) and Nv(LYS) (Cd and Zn exposures) returned to control values after depuration. It has been therefore demonstrated that the biomarkers used are reversible and return towards control levels once metal exposure ceases. Overall, it is concluded that Vv(BSD), MT-levels and lysosomal responses are valuable biomarkers to assess metal exposure and its effects in turbot, although in quantitative terms the biomarker response varied between metals.

Concentrations of serum/plasma estradiol, biliary fluorescent aromatic compounds (FACs), levels of hepatic CYP1A expression, and DNA damage were measured in sexually mature hornyhead turbot (Pleuronichthys verticalis) exposed in the laboratory for 7 days to a gradient of sediments collected from a natural petroleum seep in the Santa Barbara Channel. Coal oil point (COP) sediments were homogenized and divided into four treatments containing 0 (sediment from the Orange County Sanitation District's reference location), 33, 66, and 100% (COP) sediments. Sediment concentrations of 20 PAHs ranged from below the detection limit for the 0% COP sediment treatments to 105 microg/g in the 100% treatments with lower molecular weight compounds predominating. Concentrations of biliary FACs were not linear with COP treatment but levels of hepatic DNA damage increased linearly with increasing concentrations of high molecular weight PAHs. Hepatic CYP1A expression was elevated only in the 100% treatments. A reduction of plasma estradiol in male and female fish was observed in all COP exposures. These results demonstrate that acute sediment-only exposure of flatfish to naturally-derived PAHs elicits alterations in biochemical endpoints indicative of PAH bioavailability and adverse effects with different sensitivities.