Divalent Mercury in Dissolved Organic Matter Is Bioavailable to Fish and Accumulates as Dithiolate and Tetrathiolate Complexes.

The freshwater cyprinid Tanichthys albonubes was used to assess the bioavailability of divalent mercury (Hg(II)) complexed in dissolved organic matter (DOM) to fish. The fish acquired 0.3 to 2.2 µg Hg/g dry weight after 8 weeks in aquaria containing DOM from a Carex peat with complexed mercury at initial concentrations of 14 nM to 724 nM. Changes in the relative proportions of dithiolate Hg(SR)2 and nanoparticulate ß-HgS in the DOM, as quantified by high energy-resolution XANES (HR-XANES) spectroscopy, indicate that Hg(SR)2 complexes either produced by microbially induced dissolution of nanoparticulate ß-HgS in the DOM or present in the original DOM were the forms of mercury that entered the fish. In the fish with 2.2 µg Hg/g, 84 ± 8% of Hg(II) was bonded to two axial thiolate ligands and one or two equatorial N/O electron donors (Hg[(SR)2+(N/O)1-2] coordination), and 16% had a Hg(SR)4 coordination, as determined by HR-XANES. For comparison, fish exposed to Hg2+ from 40 nM HgCl2 contained 10.4 µg Hg/g in the forms of dithiolate (20 ± 10%) and tetrathiolate (23 ± 10%) complexes, and also Hg xS y clusters (57 ± 15%) having a ß-HgS-type local structure and a dimension that exceeded the size of metallothionein clusters. There was no evidence of methylmercury in the fish or DOM within the 10% uncertainty of the HR-XANES. Together, the results indicate that inorganic Hg(II) bound to DOM is a source of mercury to biota with dithiolate Hg(SR)2 complexes as the immediate species bioavailable to fish, and that these complexes transform in response to cellular processes.

Mercury(II) Binding to Metallothionein in Mytilus edulis revealed by High Energy-Resolution XANES Spectroscopy.

Of all divalent metals, mercury (HgII ) has the highest affinity for metallothioneins. HgII is considered to be enclosed in the α and ß domains as tetrahedral α-type Hg4 Cys11-12 and ß-type Hg3 Cys9 clusters similar to CdII and ZnII . However, neither the four-fold coordination of Hg nor the existence of Hg-Hg atomic pairs have ever been demonstrated, and the HgII partitioning among the two protein domains is unknown. Using high energy-resolution XANES spectroscopy, MP2 geometry optimization, and biochemical analysis, evidence for the coexistence of two-coordinate Hg-thiolate complex and four-coordinate Hg-thiolate cluster with a metacinnabar-type (ß-HgS) structure in the α domain of separate metallothionein molecules from blue mussel under in vivo exposure is provided. The findings suggest that the CXXC claw setting of thiolate donors, which only exists in the α domain, acts as a nucleation center for the polynuclear complex and that the five CXC motifs from this domain serve as the cluster-forming motifs. Oligomerization is driven by metallophilic Hg⋅⋅⋅Hg interactions. Our results provide clues as to why Hg has higher affinity for the α than the ß domain. More generally, this work provides a foundation for understanding how metallothioneins mediate mercury detoxification in the cell under in vivo conditions.

Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish.

Nature-based tourism is gaining extensive popularity, increasing the intensity and frequency of human-wildlife contacts. As a consequence, behavioral and physiological alterations were observed in most exposed animals. However, while the majority of these studies investigated the effects of punctual exposure to tourists, the consequences of constant exposition to humans in the wild remains overlooked. This is an important gap considering the exponential interest for recreational outdoor activities. To infer long-term effects of intensive tourism, we capitalized on Odontostilbe pequira, a short-lived sedentary Tetra fish who spends its life close to humans, on which it feeds on dead skin. Hence, those fish are constantly exposed to tourists throughout their lifecycle. Here we provide an integrated picture of the whole phenomenon by investigating, for the first time, the expression of genes involved in stress response and neurogenesis, as well as behavioral and hormonal responses of animals consistently exposed to tourists. Gene expression of the mineralocorticoid (and cortisol) receptor (mr) and the neurogenic differentiation factor (NeuroD) were significantly higher in fish sampled in the touristic zone compared to those sampled in the control zone. Additionally, after a simulated stress in artificial and controlled conditions, those fish previously exposed to visitors produced more cortisol and presented increased behavioral signs of stress compared to their non-exposed conspecifics. Overall, nature-based tourism appeared to shift selection pressures, favoring a sensitive phenotype that does not thrive under natural conditions. The ecological implications of this change in coping style remain, nevertheless, an open question.

Chemical Forms of Mercury in Human Hair Reveal Sources of Exposure.

Humans are contaminated by mercury in different forms from different sources. In practice, contamination by methylmercury from fish consumption is assessed by measuring hair mercury concentration, whereas exposure to elemental and inorganic mercury from other sources is tested by analysis of blood or urine. Here, we show that diverse sources of hair mercury at concentrations as low as 0.5 ppm can be individually identified by specific coordination to C, N, and S ligands with high energy-resolution X-ray absorption spectroscopy. Methylmercury from seafood, ethylmercury used as a bactericide, inorganic mercury from dental amalgams, and exogenously derived atmospheric mercury bind in distinctive intermolecular configurations to hair proteins, as supported by molecular modeling. A mercury spike located by X-ray nanofluorescence on one hair strand could even be dated to removal of a single dental amalgam. Chemical forms of other known or putative toxic metals in human tissues could be identified by this approach with potential broader applications to forensic, energy, and materials science.

Uptake, accumulation and metabolization of the antidepressant fluoxetine by Mytilus galloprovincialis.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) antidepressant, is among the most prescribed pharmaceutical active substances worldwide. This study aimed to assess its accumulation and metabolization in the mussel Mytillus galloprovincialis, considered an excellent sentinel species for traditional and emerging pollutants. Mussels were collected from Ria Formosa Lagoon, Portugal, and exposed to a nominal concentration of fluoxetine (75 ng L(-1)) for 15 days. Approximately 1 g of whole mussel soft tissues was extracted with acetonitrile:formic acid, loaded into an Oasis MCX cartridge, and fluoxetine analysed by liquid chromatography with tandem mass spectrometry (LC-MSn). After 3 days of exposure, fluoxetine was accumulated in 70% of the samples, with a mean of 2.53 ng g(-1) dry weight (d.w.) and norfluoxetine was only detected in one sample (10%), at 3.06 ng g(-1) d.w. After 7 days of exposure, the accumulation of fluoxetine and norfluoxetine increased up to 80 and 50% respectively, and their mean accumulated levels in mussel tissues were up to 4.43 and 2.85 ng g(-1) d.w., respectively. By the end of the exposure period (15 days), both compounds were detected in 100% of the samples (mean of 9.31 and 11.65 ng g(-1) d.w., respectively). Statistical analysis revealed significant accumulation differences between the 3rd and 15th day of exposure for fluoxetine, and between the 3rd and 7th against the 15th day of exposure for norfluoxetine. These results suggest that the fluoxetine accumulated in mussel tissues is likely to be metabolised into norfluoxetine with the increase of the time of exposure, giving evidence that at these realistic environmental concentrations, toxic effects of fluoxetine in mussel tissues may occur.

Occurrence of pharmaceutical compounds and pesticides in aquatic systems.

This paper deals with the detection and quantification of APIs and other priority substances in the Arade River estuary (Portugal) providing the usefulness of Polar Organic Compound Integrative Samplers (POCIS). Thirteen APIs were detected whose variation was site and time dependent. Caffeine was at the highest concentration (804±209 ng/L) followed by theophylline (184±44 ng/L). Other APIs were analgesic, anticonvulsant, non-steroidal anti-inflammatory drugs, anti-lipidemic, anxiolytic and antidepressants. Twenty pesticides comprising atrazine, diuron, isoproturon, terbutryn and simazine included in the Water Framework Directive priority list were also site and time dependent. Carbendazim occurred at the highest concentration (45±18 ng/L at site 1) but atrazine, diuron, isoproturon and simazine levels were below the Environmental Quality Standards. Although the summer impact was unclear, the results highlighted POCIS suitability for profiling these contaminants. This is to our knowledge the first study concerning APIs and pesticides in this area.

Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.

Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 µg L(-1). Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-π-like), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock protein-like (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 µg L(-1). A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COX-like, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 µg L(-1) for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 µg L(-1)) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.

Effects of non-steroidal anti-inflammatory drug (NSAID) diclofenac exposure in mussel Mytilus galloprovincialis.

In recent years, research studies have increasingly focused on assessing the occurrence of active pharmaceutical ingredients (APIs) in ecosystems. However, much remains unknown concerning the potential effects on APIs on non-target organisms due to the complexity of the mode of action, reactivity and bioconcentration potential for each specific drug. The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is one of the most frequently detected APIs in surface waters worldwide and has recently been included in the list of priority substances under the European Commission. In this study, mussels (Mytilus galloprovincialis) were exposed to an environmentally relevant nominal concentration of DCF (250 ng L(-1)) over 15 days. The responses of several biomarkers were assessed in the mussel tissues: condition index (CI); superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and phase II glutathione-S-transferase (GST) activities, lipid peroxidation levels (LPO) associated with oxidative stress, acetylcholinesterase (AChE) activity related to neurotoxic effects and vitellogenin-like proteins linked to endocrine disruption. This study demonstrated significant induction of SOD and GR activities in the gills in addition to high CAT activity and LPO levels in the digestive gland. Phase II GST remained unaltered in both tissues, while the up-regulation of the AChE activity was directly related to the vitellogenin-like protein levels in exposed females, indicating an alteration in the estrogenic activity, rather than a breakdown in cholinergic neurotransmission function. This study confirmed that DCF at a concentration often observed in surface water induces tissue-specific biomarker responses. Finally, this study also revealed the importance of a multi-biomarker approach when assessing the potentially deleterious effects in a species that may be vulnerable to the continuously discharge of APIs into the ecosystems; this approach provides crucial new information regarding the unknown effects of DCF.

Does selective serotonin reuptake inhibitor (SSRI) fluoxetine affects mussel Mytilus galloprovincialis?

Fluoxetine (FLX) the active pharmaceutical ingredient (API) in Prozac(®) is a widely prescribed psychoactive drug which ubiquitous occurrence in the aquatic environment is associated to a poor removal rate in waste-water treatment plant (WWTP) systems. This API acts as a selective serotonin reuptake inhibitor (SSRI) frequently reported to cause disrupting effects in non-target species. The objective of this study includes a multibiomarker response evaluation on mussel Mytilus galloprovincialis during two weeks exposure to 75 ng L(-1) FLX assessing antioxidant enzymes activities--superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST); lipid peroxidation (LPO), acetylcholinesterase (AChE) neurotoxic response and endocrine disruption through alkali-labile phosphates (ALP) indirect measurement of vitellogenin-like proteins. Results show transient tissue-specific enzymatic responses and damage affecting mostly mussel gills. However, the clear ALP levels inhibition throughout time in both sex-differentiated gonads gives evidence to FLX reinforced action as an endocrine disruptor rather than an oxidative or neurotoxic inducer.

Does non-steroidal anti-inflammatory (NSAID) ibuprofen induce antioxidant stress and endocrine disruption in mussel Mytilus galloprovincialis?

Ibuprofen (IBU) is one of the most sold over-the-counter non-steroidal anti-inflammatory drugs (NSAID) and widely detected in the aquatic ecosystems. Nevertheless, the information regarding IBU effects in biota is still sparse. The goal of this study was to assess IBU potential effect as oxidative stress and endocrine disruption inducer in mussel Mytilus galloprovincialis applying a battery of biomarkers. Over two weeks of exposure to IBU (250 ngL(-1)), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), phase II glutathione S-transferase (GST) activities and lipid peroxidation (LPO) levels were determined in the digestive gland and alkali-labile phosphates (ALP) were carried out in sex-differentiated mussels' gonads. The results confirm a transitory induction of antioxidant activities responses concomitant to lipid peroxide formation outline and an increase of ALP levels over time, particularly in exposed males which may lead to mussels' reproductive fitness impairment highlighting a higher impact of IBU as an endocrine disruptor than as a short-term reactive oxygen species (ROS)-generator.

Non-steroidal anti-inflammatory drug (NSAID) ibuprofen distresses antioxidant defense system in mussel Mytilus galloprovincialis gills.

Active pharmaceutical ingredients (APIs) are presently considered an emergent class of environmental contaminants. Ibuprofen (IBU) is one of the most applied non-steroidal anti-inflammatory drugs (NSAIDs) in the world. Several authors report the occurrence of IBU in influents and effluents of waste water treatment plants (WWTPs), surface, river and public tap water in numerous countries. However, very little is known about the risks and chronic effects of IBU exposure in non-target organisms. This approach undertakes the assessment of several oxidative stress biomarkers responses through the analysis of antioxidant enzymes activities (superoxide dismutase - SOD, catalase - CAT, glutathione S-transferase - GST, glutathione reductase - GR) and lipid peroxidation (LPO) levels in sentinel species mussel Mytilus galloprovincialis gills exposed for 2 weeks to an environmental realistic concentration of IBU. Results clearly show the significant induction and positive correlation between SOD activity and LPO in exposed gills, concomitant to an antioxidant defense depletion of CAT, GR and GST compared to controls. The integration of all biomarkers in mussels' gills separates non- and exposed groups supporting the breakdown of the redox defense system and IBU's pro-oxidant action. Further studies are needed to test possible endocrine disruption effects in mussels' reproduction fitness as IBU is involved on prostaglandins biosynthesis inhibition.

Detoxification mechanisms in shrimp: comparative approach between hydrothermal vent fields and estuarine environments.

Hydrothermal vents are extreme deep-sea habitats that, due to their singular features, still intrigue scientific communities. Swift growth rates and profuse biomass of biological communities can be observed, despite of their inherently unstable physical-chemical and toxic conditions, indicating that organisms inhabiting this environment must be well adapted to these inhospitable conditions. The caridean shrimp, Chorocaris chacei, Mirocaris fortunata and Rimicaris exoculata, together with bathymodiolid mussels, dominate the vent fauna along the Mid-Atlantic Ridge (MAR). Crustacean species are widely used as biological indicators of environmental alterations, since they play a key ecological role as planktivorous grazers, epibenthic scavengers or as prey species. The biological consequences of the hydrothermal metal-rich environment in shrimp species are still largely unknown. Therefore, the aim of this study was the determination of the metal levels (Ag, Cd, Cu, Fe, Mn and Zn), metallothioneins (MT) and lipid peroxidation (LPO) in shrimp species collected in Rainbow, Lucky Strike and Menez-Gwen vent sites, in order to evaluate their different adaptation strategies toward metals when compared with two common coastal shrimp species (Palaemon elegans and Palaemonetes varians) from a fairly unpolluted estuarine system in south Portugal (Ria Formosa). Results show significant differences in metal concentrations, MT levels and lipid peroxidation between vent and coastal shrimp and also between shrimp species from the same site. This indicates that biochemical responses in both vent and coastal shrimp are affected not only by the environmental characteristics but also by inter-specific differences. Nevertheless, these responses apparently grant a successful adaptation for the survival in a metal-extreme environment.