Comparative biochemical and molecular responses of biotransformation and antioxidant systems in three species of Crassostrea (Sacco, 1897) oysters exposed to chrysene.

Chrysene (CHR) is among the most persistent polycyclic aromatic hydrocarbons (PAH) in water and a priority compound for pollutants monitoring, due to its carcinogenic, mutagenic and genotoxic potential. Aquatic animals exposed to CHR may present alterations of biomarkers involved in the biotransformation and oxidative stress-related parameters. The aim of this study was to investigate differences in antioxidant and biotransformation (phase I and II) systems of Crassostrea gigas, C. gasar and C. rhizophorae and its effects resulting from CHR exposure. Adult oysters of these species were exposed to 10 µg L-1 of CHR for 24 h and 96 h. In gills, the transcripts CYP1-like, CYP2-like, CYP2AU1-like, GSTO-like, MGST-like, SULT-like were evaluated after 24 h of exposure. The activity of SOD, CAT, GPx, GR and G6PDH were analyzed in gills and digestive glands after 96 h of exposure. CHR bioaccumulated in tissues. Differences in the remaining levels of CHR in water after 96 h were observed in aquaria containing C. gigas or C. gasar oysters and may be associated to the different filtration rates between these species. Downregulate of biotransformation genes were observed in gills of C. gasar (CYP2AU1-like and GSTO-like) and C. rhizophorae (CYP1-like1, CYP2-like, MGST-like and SULT-like), suggesting that biotransformation responses may be species-specific. Differential activity of antioxidant enzymes were observed in gills and digestive gland of oysters exposed to CHR. Biochemical responses suggested that C. gigas and C. gasar are more responsive to CHR. Differential responses observed among the three Crassostrea species can be related to evolutionary differences, ecological niches and adaptation to environment.

Molecular changes in oysters Crassostrea gigas (Thunberg, 1793) from aquaculture areas of Santa Catarina Island bays (Florianópolis, Brazil) reveal anthropogenic effects.

Anthropogenic activities in coastal regions cause risks to the environmental and human health. Due to the carcinogenic and mutagenic potential, polycyclic aromatic hydrocarbons (PAH) are considered priority for monitoring. Most of the Brazilian production of Crassostrea gigas oysters are placed in the Bays of Santa Catarina Island. The aim of this study was to evaluate molecular responses (phase I and II of biotransformation and antioxidant defense) of C. gigas from six oyster farming areas potentially contaminated by sanitary sewage in Florianópolis Metropolitan (SC, Brazil): Santo Antônio de Lisboa, Sambaqui, Serraria, Caieira, Tapera, Imaruim. We evaluated the transcript levels of CYP1A1-like, CYP2-like, CYP2AU2-like, CYP356A1, GSTA1A-like, GSTO.4A-like, SULT-like, SOD-like and CAT-like by qRT-PCR. Only oysters from Caieira showed levels of thermotolerant coliforms allowed by the law. Chemicals analyses in soft tissues of oysters showed low to average levels of PAH in all monitored areas. Enhanced transcript levels of phase I (CYP1A1-like, CYP3564A1-like, CYP2-like and CYP2AU2-like) were observed in oysters from Serraria and Imaruí, suggesting higher biotransformation activity in these farming areas. Regarding phase II of biotransformation, GSTO.4A-like was up-regulated in oysters from Imaruí compared to Caieira and Santo Antônio de Lisboa. An upregulation of SOD-like and CAT-like were observed in oysters from Imaruí and Serraria, suggesting that oysters from these sites are facing higher prooxidant conditions compared to other areas. By integrating the biological and chemical data it is suggested that human-derived contaminants are affecting the oyster metabolism in some farming areas.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I.

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 µM) and fluorene (0.6 and 1.2 µM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 µM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

Biochemical and molecular biomarkers in integument biopsies of free-ranging coastal bottlenose dolphins from southern Brazil.

Adverse effects of exposure to persistent organic pollutants (POPs) threaten the maintenance of odontocete populations. In southern Brazil, coastal bottlenose dolphins from the Laguna Estuarine System (LES) and Patos Lagoon Estuary (PLE) were sampled using remote biopsies during the winter and summer months. Levels of bioaccumulated POPs were measured in the blubber. The activities of glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were also quantified, as were the mRNA transcript levels of aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), cytochrome P450 1A1-like (CYP1A1), metallothionein 2A (MT2A), GST-π, GPx-4, GR, interleukin 1 alpha (IL-1α), and major histocompatibility complex II (MHCII) in the skin. In general, levels of POPs were similar among sites, sexes, ages and seasons. For most animals, total polychlorinated biphenyl (ΣPCBs) levels were above the threshold level have physiological effects and pose risks to cetaceans. The best-fitting generalized linear models (GLMs) found significant associations between GR, IL-1α and GPx-4 transcript levels, SOD and GST activities, and total polybrominated diphenyl ether (ΣPBDEs) and pesticide levels. GLMs and Kruskal-Wallis analyses also indicated that there were higher transcript levels for most genes and lower GST activity in the winter. These results reinforce the need to consider the influence of environmental traits on biomarker values in wildlife assessments.

Depositional history of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and polycyclic aromatic hydrocarbons (PAHs) in an Amazon estuary during the last century.

The sources and depositional history of OCPs (organochlorine pesticides), PCBs (polychlorinated biphenyls) and PAHs (polycyclic aromatic hydrocarbons) over the last 100years were determined in two sediment cores collected in the Amazon region. It was possible to distinguish two depositional periods along the cores. The first period occurred before extensive anthropogenic effects were registered in the sediments. During this time interval, the concentrations of all OCPs and PCBs were below the detection limits (DL), and the PAH concentrations were low and essentially constant (58.19-124.28ngg-1). The second period starts in the mid-1960s and reflects the increased human influence in the area. The concentrations of OCPs, PCBs, and PAHs increased towards the top of the cores, varying between

Colour spectrum and resin-type determine the concentration and composition of Polycyclic Aromatic Hydrocarbons (PAHs) in plastic pellets.

This study assessed the concentration and composition of Polycyclic Aromatic Hydrocarbons (PAHs) in plastic pellets, collected from sandy beaches and considered different resin and colour tones. Results showed that polyethylene pellets, while displaying a greater range of total PAH concentrations did not differ significantly from polypropylene pellets. More importantly, both resin types demonstrated predictable increases in total PAH across a spectrum of darkening colour tones. Multivariate comparisons of 36 PAH groups, further showed considerable variability across resin type and colour, with lighter coloured pellets comprising lower molecular weight, while darker pellets contained higher weight PAHs. Overall, we show predictable variation in PAH concentrations and compositions of plastic pellets of different ages and resin types that will directly influence the potential for toxicological effects. Our findings suggest that monitoring programs should take these attributes into account when assessing the environmental risks of microplastic contamination of marine and coastal habitats.

Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in sediments from an urban- and industrial-impacted subtropical estuary (Babitonga Bay, Brazil).

Babitonga Bay is a large estuary, which has been under pressure from anthropogenic activities coexisting with a natural area of Atlantic rainforest and mangrove systems. The concentration of persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) was determined to evaluate the contamination status and the determine possible pollution sources in the estuary. The ∑DDT (sum of DDT, DDE and DDD concentrations) was the predominant OCP group, with concentrations ranging from

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC.

Validating the use of biopsy sampling in contamination assessment studies of small cetaceans.

Remote biopsy sampling is the most common technique for acquiring samples from free-ranging marine mammals. However, such techniques may result in variable sampling being sometimes superficial skin and blubber biopsies. For decades, blubber has been used to monitor the exposure of marine mammals to persistent organic pollutants (POPs), but little is known regarding the variability of POPs as a function of blubber depth in small cetaceans and the available literature offers variable results. Thus, the aim of the present study was to validate biopsy sampling for monitoring contaminant concentrations in small, free-ranging cetaceans. Samples from the dorsal blubber of 10 incidentally captured Atlantic spotted dolphins (Stenella frontalis) were separated into two different layers (outer and inner) to investigate the influence of sampling depth on POP concentrations. POP concentrations were compared to those of the full blubber layer. The results revealed no significant differences in lipid content between males and females or among the inner, outer and full blubber layers (p>0.05). Moreover, the wet and lipid weight concentrations of all POP classes analysed [i.e. polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), polybrominated diphenyl ethers (PBDEs), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), chlordanes (CHLs) and mirex] did not differ significantly with blubber depth (p>0.05). POP classes followed the same decreasing order of wet weight concentrations in blubber layers and full blubber: PCBs>DDTs>PBDEs>mirex>HCB>HCHs>CHLs. Moreover, there was a low degree of differentiation in the accumulation of POP congeners. The present findings indicated that the distribution of contaminants was homogenous with blubber depth, which validates the use of biopsy sampling for the assessment of contaminants in small cetaceans.

Virus, protozoa and organic compounds decay in depurated oysters.

AIMS: (1) Evaluate the dynamic of the depuration process of Crassostrea gigas oysters using different ultraviolet doses with different amounts of contaminants (virus, protozoa and organic contaminants) and (2) investigate the morphological changes in the oysters' tissues produced by the depuration procedures. METHODS: The oysters were allocated in sites with different degrees of contamination and analyzed after 14 days. Some animals were used as positive controls by artificial bioaccumulation with HAdV2 and MNV1 and subjected to depuration assays using UV lamps (18 or 36 W) for 168 h. The following pollutants were researched in the naturally contaminated oysters, oysters after 14 days in sites and oysters during the depuration processes: virus (HAdV, HAV, HuNoV GI/GII and JCPyV), by (RT) qPCR; protozoa (Cryptosporidium and Giardia species), by immunomagnetic separation and immunofluorescence; and organic compounds (AHs, PAHs, LABs, PCBs and organochlorine pesticides-OCs), by chromatography. Changes in the oysters' tissues produced by the depuration processes were also evaluated using histochemical analysis by light microscopy. In the artificially bioaccumulated oysters, only HAdV2 and MNV1 were investigated by (RT) qPCR before the depuration procedures and after 96 and 168 h of these procedures. RESULTS: At 14 days post-allocation, HAdV was found in all the sites (6.2 × 105 to 4.4 × 107 GC g(-1)), and Giardia species in only one site. Levels of PCBs and OCs in the oyster's tissues were below the detection limit for all samples. AHs (3.5 to 4.4 µg g(-1)), PAHs (11 to 191 ng g(-1)) and LABs (57 to 751 ng g(-1)) were detected in the samples from 3 sites. During the depuration assays, we found HAdV, Giardia and Cryptosporidium species until 168 h, independent of UV treatment. AHs, PAHs and LABs were found also after 168 h of depuration (36 W and without UV lamp). The depuration procedures did not produce changes in the oysters' tissues. In the artificially contaminated and depurated oysters, we detected HAdV until 168 h and MNV1 until 96 h of depuration. CONCLUSION: The applied depuration treatments were unable to eliminate the protozoa or to degrade the HAdV genomes but were able to degrade the MNV1 genomes. Similarly, the UV water treatment was not efficient for aliphatic hydrocarbons, PAHs and LABs, as their concentrations were equivalent or higher to the concentrations of the control samples and samples from depuration tanks without UV treatment.

Spatial distribution and historical input of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in sediments from a subtropical estuary (Guaratuba Bay, SW Atlantic).

This study evaluated the occurrence and distribution of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in 22 surface sediment samples and one core collected in Guaratuba Bay, a large and well-preserved estuary in Southern Atlantic, Brazil. The concentration of PCBs in the superficial sediment samples ranged from

Persistent organic pollutants and stable isotopes in pinnipeds from King George Island, Antarctica.

In the present work, fat, skin, liver and muscle samples from Leptonychotes weddellii (Weddell seal, n=2 individuals), Lobodon carcinophagus (crabeater seal, n=2), Arctocephalus gazella (Antarctic fur seal, n=3) and Mirounga leonina (southern elephant seal, n=1) were collected from King George Island, Antarctica, and analysed for POPs (PCBs, organochlorine pesticides and PBDEs) and stable isotopes (δ¹³C and δ¹5N in all tissues but fat). PBDEs could be found in only one sample (L. weddellii fat). Generally, PCBs (from 74 to 523 ng g⁻¹ lw), DDTs (from 14 to 168 ng g⁻¹ lw) and chlordanes (from 9 to 78 ng g⁻¹ lw) were the prevailing compounds. Results showed a clear stratification in accordance with ecological data. Nonetheless, stable isotope analyses provide a deeper insight into fluctuations due to migrations and nutritional stress. Correlation between δ(15)N and pollutants suggests, to some degree, a considerable ability to metabolize and/or excrete the majority of them.

Evaluation of tropical water sources and mollusks in southern Brazil using microbiological, biochemical, and chemical parameters.

Florianópolis, a city located in the Santa Catarina State in southern Brazil, is the national leading producer of bivalve mollusks. The quality of bivalve mollusks is closely related to the sanitary conditions of surrounding waters where they are cultivated. Presently, cultivation areas receive large amounts of effluents derived mainly from treated and non-treated domestic, rural, and urban sewage. This contributes to the contamination of mollusks with trace metals, pesticides, other organic compounds, and human pathogens such as viruses, bacteria, and protozoan. The aim of this study was to perform a thorough diagnosis of the shellfish growing areas in Florianópolis, on the coast of Santa Catarina. The contamination levels of seawater, sediments, and oysters were evaluated for their microbiological, biochemical, and chemical parameters at five sea sites in Florianópolis, namely three regular oyster cultivation areas (Sites 1, 2, and oyster supplier), a polluted site (Site 3), and a heavily polluted site (Site 4). Samples were evaluated at day zero and after 14 days. Seawater and sediment samples were collected just once, at the end of the experiment. Antioxidant defenses, which may occur in contaminated environments in response to the increased production of reactive oxygen species (ROS) by organisms, were analyzed in oysters, as well as organic compounds (in oysters and sediment samples) and microbiological contamination (in oysters and seawater samples). The results showed the presence of the following contaminants: fecal coliforms in seawater samples (four sites), human adenovirus (all sites), human noroviruses GI and GII (two sites), Hepatitis A viruses (one site), JC Polyomavirus in an oyster sample from the oyster supplier, Giardia duodenalis cysts, and Cryptosporidium sp oocysts (one site). Among organochlorine pesticides, only DDT (dichlorodiphenyltrichloroethane) and HCH (hexachlorocyclohexane) were detected in some sediment and oysters samples in very low levels; site 4 had the highest concentrations of total aliphatic hydrocarbons, PAHs, and linear alkylbenzenes (LABs) found either in oysters or in sediment samples. The major concentration of fecal sterol coprostanol was found at site 4, followed by site 3. After 14 days of allocation in the four selected sites, there was a significant difference in the enzymes analyzed at the monitored spots. The detection of different contaminants in oysters, seawater, and sediment samples in the present study shows the impact untreated or inadequately treated effluents have on coastal areas. These results highlight the need for public investment in adequate wastewater treatment and adequate treatment of oysters, ensuring safe areas for shellfish production as well as healthier bivalve mollusks for consumption.

Contamination by chlorinated pesticides, PCBs and PBDEs in Atlantic spotted dolphin (Stenella frontalis) in western South Atlantic.

Chlorinated pesticides, PCBs and PBDEs were analysed in nine blubber samples of Atlantic spotted dolphins, Stenella frontalis, incidentally captured during fishing operations in southern and southeastern Brazil between 2005 and 2007. The majority of compounds analysed were detected, suggesting widespread contamination over the region. Although the samples came from a location far from main coastal industrial areas, the results revealed an influence from such sources. Therefore, levels of PCBs (774-23659 ng g(-1) lipid wt.) and PBDEs (23-1326 ng g(-1) lipid wt.) detected seem to be related to the movement of individuals throughout near-shore and offshore waters. The sample from a lactating female exhibited a lower level of contamination and a distinct pattern, indicating selective transfer favouring less lipophilic compounds.

Polychlorinated biphenyls and organochlorine pesticides in plastics ingested by seabirds.

The occurrence of plastic objects in the digestive tract was assessed in eight species of Procellariiformes collected in southern Brazil and the occurrence of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in the ingested plastics pellets and plastic fragments was evaluated. PCBs were detected in plastic pellets (491 ng g(-1)) and plastic fragments (243-418 ng g(-1)). Among the OCPs, p,p'-DDE had the highest concentrations, ranging from 68.0 to 99.0 ng g(-1). The occurrence of organic pollutants in post-consumer plastics supports the fact that plastics are an important source carrying persistent organic pollutants in the marine environment. Although transfer through the food chain may be the main source of exposure to POPs to seabirds, plastics could be an additional source for the organisms which ingest them, like Procellariiformes which are the seabirds most affected by plastic pollution.

Development of a static headspace gas chromatographic/mass spectrometric method to analyze the level of volatile contaminants biodegradation.

Volatile compound biodegradation analysis usually requires the time-consuming step of extraction of the analytes from the matrix using organic solvents or costly radioactive-compounds. Thus, it is desirable to have a simple and fast technique to generate a good evaluation of bacterial biodegradation. The goal of this research was to develop a methodology on the basis of static headspace-gas chromatography/mass spectrometry (HS-GC/MS) to evaluate the level of volatile contaminant biodegradation. The effects of the following parameters were studied: temperature and time of equilibration. The biodegradation experiments were carried out with bacteria inoculation in mineral media in presence of volatile hydrocarbon compounds (toluene, p-xylene, nonane and naphthalene). Autoclaved inoculates were used as control and reference sample. The optimal headspace conditions were observed when the vials were heated at 80 degrees C for 20 min, the syringe at 81 degrees C and an injection volume of 0.4 mL was used. This methodology has the advantage of being relative free from matrix effects.