Biochemical responses in Pacific oysters Magallana gigas (Thunberg, 1793): Tools to evaluate the environmental quality of aquaculture areas.

The discharge of sanitary sewage into the bays of the Florianópolis Metropolitan Area (Southern Brazil), has led to the contamination of oyster farms. Consequently, linear alkylbenzenes (LABs) were quantified in the sediment, and the biochemical responses in gills and digestive gland of oysters from six farms were assessed. Our findings revealed elevated levels of LABs in the sediment of the Imaruim and Serraria farms. Additionally, alterations were observed in the antioxidant enzymes: catalase, glutathione peroxidase and superoxide dismutase in both oyster tissue from the Serraria, Santo Antonio de Lisboa and Sambaqui farms. Furthermore, correlation analyses indicated strong and moderate associations between biochemical responses, organic contaminants, and certain physicochemical parameters. Consequently, our results demonstrated the activation of the antioxidant system in oysters, representing a protective response to the presence of sanitary sewage and other contaminants. Therefore, we propose the utilization of biochemical biomarkers for monitoring the environmental quality of farms.

Gender influences molecular and histological biomarkers in mature oysters Crassostrea gasar (Adanson, 1757) after pyrene exposure.

Oysters are frequently used as sentinel organisms for monitoring effects of contaminants due to their sessile, filtering habits and bioaccumulation capacity. These animals can show elevated body burden of contaminants, such as pyrene (PYR). PYR can be toxic at a molecular level until the whole oyster, which can show reproductive and behavioral changes. Considering that biologic parameters, such as gender or reproductive stage can interfere in the toxic effects elicited by contaminants uptake, the aim of this study was to evaluate some molecular and histological responses in females and males of oyster Crassostrea gasar exposed to PYR (0.25 and 0.5 µM) for 24 h at the pre-spawning stage. PYR concentrations were analyzed in water and in tissues of female and male oysters. Gene transcripts related to biotransformation (CYP3475C, CYP2-like, CYP2AU1, CYP356A, GSTO-like, GSTM-like, SULT-like), stress (HSP70), and reproduction (Vitellogenin, Glycoprotein) were quantified in gills. In addition, histological analysis and histo-localization of CYP2AU1 mRNA transcripts in gills, mantle and digestive diverticulum were carried out. Females and males in pre-spawning stage bioconcentrated PYR in their tissues. Males were more sensitive to PYR exposure. CYP2AU1 transcripts were higher in males (p < 0.05), as well as tubular atrophy was observed only in males exposed to PYR (p < 0.05). As expected, vitellogenin transcripts were lower in males (p < 0.05). Given these results, it is suggested that levels of CYP2AU1 be a good biomarker of exposure to PYR in oyster C. gasar and that it is important to consider the gender for the interpretation of biomarker responses.

Polycyclic aromatic hydrocarbons in marine mammals: A review and synthesis.

Most marine mammal species and populations are listed as endangered, threatened, or depleted under the Endangered Species Act and the Marine Mammal Protection Act. Organic contaminants such as polycyclic aromatic hydrocarbons from anthropogenic activities are part of the threat to marine mammals. The evaluation of the potential bioaccumulation of these compounds by marine mammals is a tool for adoption of policies to reduce polycyclic aromatic hydrocarbons discharges to the marine environment, where important players such as the oil and gas industries, maritime transport and sewage companies operate. This review seeks to present a bibliographic survey covering all published peer reviewed works of the contents of polycyclic aromatic hydrocarbons in biological tissues of marine mammals. It intended to compare the sampling protocols, procedures for preservation of the tissues, and the analytical method applied to quantify the polycyclic aromatic hydrocarbons, no to criticize any of them but to review the data and discuss how they can be compared.

Historical deposition of PAHs in mud depocenters from the Southwestern Atlantic continental shelf: The influence of socio-economic development and coal consumption in the last century.

Polycyclic aromatic hydrocarbon (PAH) concentrations were determined in four dated sediment cores collected in mud depocenters of the southern Brazilian continental shelf. Core dating results covered the interval between 1925 and 2017. The total PAH concentrations (ΣPAHs) ranged from 44.69 ng g-1 to 305.43 ng g-1 and were similar between the analysed cores. Fine-grained sediments and total organic carbon (TOC) results did not correlate with the ΣPAHs, indicating that the variations in PAH concentrations are mostly related to variations in sources and emissions. PAH source appointment indicated a high input of a natural compound (perylene) and the predominance of anthropogenic PAHs from coal, biomass, and fuel combustion. Alkylated PAHs presented high contributions throughout all cores. The historical deposition of PAHs was associated with different periods of the socio-economic and industrial development of near coastal cities and reflected very well the history of coal production and consumption in the southern region of Brazil. The low levels of ΣPAHs before 1945 in all analysed cores may be related to the beginning of the industrialization process and the lower urbanization degree in the region. Between 1945 and 1965, the gradual ΣPAHs increase reflects the establishment and enlargement of the southern Brazilian industrial sector. The interval between 1965 and 1990 corresponded to the highest ΣPAHs in three of the four analysed cores. After 1990, a relative decrease in the ΣPAHs was observed in most cores and may be related the multiple cuts of incentives to the industrial usage of coal, as well as to Brazil's efforts in environmental regulation for coal extraction and consumption.

Insights into leaded gasoline registered in mud depocenters derived from multivariate statistical tool: southeastern Brazilian coast.

Lead has been widely used since antiquity, but its uses drastically increased during the Industrial Revolution. The global emission of Pb into the environment was mainly due to tetraethyl lead added to gasoline as an antiknock additive. Because of its toxicity and neurological effects, the compound was phased out in the 1980s. Isotopic signatures are widely applied to differentiate sources of Pb; however, this is an expensive and sophisticated analysis compared to elemental analysis. Thus, this study aims to gain insight into leaded gasoline registered in mud depocenters from the southeastern Brazilian coast using multivariate statistical tools on elemental analysis data of trace elements. Seven multiple cores were collected on board the Research Vessel Alpha Crucis. Al, As, Ba, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, P, Pb, Sc, Sr, V and Zn were analyzed by acid digestion and quantified by ICP-OES. Levels and enrichment factors of Pb resulted in homogeneous columns, indicating that small variations in concentrations can be attributed to grain size differences, not presenting contaminated levels. From statistical results, the highest contribution on the first component was represented by a lithogenic source with the leaching of continental rocks. Lead content was notable in its high loadings in other components, which suggests atmospheric deposition. An increase in these components in subsurface samples from vertical profiles between 1935 and 1996 could represent a fingerprint of the consumption of leaded gasoline in Brazil between 1923 and 1989. Thus, statistical analysis of elemental data enabled to infer possible sources and pathways of Pb to the environment, without isotopic analysis.

Dredging impacts on the toxicity and development of sediment quality values in a semi-arid region (Ceará state, NE Brazil).

Sediment dredging impacts coastal environments by promoting the resuspension of fine particles and remobilization of contaminants that may trigger toxic effects. In this study, we evaluated the sediment quality in harbor areas of Mucuripe bay, a semi-arid ecosystem located in Ceará state (Brazil), which is subject to dredging activities. A sampling survey was conducted right after dredging operations and data compared to another survey performed prior dredging. Sediments were analyzed for fine particles, organic carbon, nutrients, metals, hydrocarbons, and tributyltin (TBT). Toxicity of whole-sediment and liquid phase exposures were also determined. The concentrations of Cd, Cr, Cu, and Zn decreased after dredging, which was confirmed by the geoaccumulation index. Levels of TBT dropped while phosphorus, aliphatic and polycyclic aromatic hydrocarbons increased. Toxic effects persisted, indicating a post-dredging recontamination combined with other sources such as urban runoff, wastewater discharges, harbor activities, and antifouling particles. Data from Mucuripe and Pecém harbors were compiled and site-specific sediment quality values (SQVs) were developed by using multivariate methods. The threshold values proposed by our study were lower and more effective to predict toxicity compared to international guidelines, indicating levels of contamination for this tropical region in which toxic effects may occur. Considering the large geographic area with different sediment characteristics of the Brazilian coast, this study represents a significant contribution to sediment toxicity assessment of dredging activities in semi-arid environments.

Data on hydrocarbons in sediment samples, and its body burden levels in tissues of Anomalocardia flexuosa from toxicity testing.

The analysis of hydrocarbons in terms of individual compounds is relevant to understand the origin and source of these substances, as well as its distribution in environmental compartments, including sediments and biota. Hydrocarbons concentrations were determined in sediments and in whole-body soft tissues of the tropical clam Anomalocardia flexuosa in sediment toxicity testing using samples of Mucuripe bay (Ceará State, NE Brazil) collected in 2011 during dredging events [1]. Data of target compounds included aliphatic (AHs) and aromatic hydrocarbons (PAHs), and linear alkylbenzenes (LABs). AHs compounds were determined on gas chromatography with flame ionization detector (GC-FID), while PAHs and LABs were determined on gas chromatography coupled to a mass spectrometer (GC/MS) in a selected ion mode (SIM). The potential of this dataset is baseline information on hydrocarbons contamination in sediments from a semi-arid region and the bioaccumulation of organic contaminants in marine organisms that can be used as models in ecotoxicological studies.

Differential responses in the biotransformation systems of the oyster Crassostrea gigas (Thunberg, 1789) elicited by pyrene and fluorene: Molecular, biochemical and histological approach - Part II.

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 µM) and FLU (0.6 and 1.2 µM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 µM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 µM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.

Bioavailability of polycyclic aromatic hydrocarbons to penguins on the coast of southeastern Brazil.

Bioavailability of polycyclic aromatic hydrocarbons on São Paulo state coast (southeastern Brazil) was evaluated through the analysis of biliary metabolites in Spheniscus magellanicus (n = 79). The animals analyzed in present study were either found dead during beach monitoring procedures or died at rehabilitation centers. Analyses of naphthalene (NAP), phenanthrene (PHE) and benzo[a]pyrene (BaP) metabolites were performed using a high-performance liquid chromatograph equipped with fluorescence detectors. Total metabolite (TM) concentrations ranged from below the method quantification limit to 270 µg g-1 of bile. TM concentrations were mainly composed of NAP metabolites, followed by PHE metabolites. BaP metabolites were detected in only two samples. This is the first study using PAHs metabolites in S. magellanicus to assess the bioavailability of these compounds in coastal regions.

Biomarkers responses of the clam Anomalocardia flexuosa in sediment toxicity bioassays using dredged materials from a semi-arid coastal system.

Few test organisms are employed for sediment toxicity assessments in Tropical regions, including Brazil. We assessed the ability of the clam Anomalocardia flexuosa to respond to contamination in sediment bioassays using dredging materials of a semi-arid region (Ceará State, NE Brazil), with attention to sublethal responses. Sediments were collected during and after dredging (survey 1 and 2, respectively) and animals exposed in laboratory over 28 days, with responses measured at 7 days. Bioaccumulation of contaminants was determined in whole-body soft tissues as a metric of bioavailability, and biomarkers' changes were monitored in terms of enzymes of phase I and II metabolism, acetylcholinesterase (AChE), and antioxidant responses, lipid peroxidation (LPO) and DNA damage (strand breaks). Clams accumulated aliphatic (AHs) and aromatic hydrocarbons (PAHs), and linear alkylbenzenes (LABs) compared to control conditions (day 0), with increased amounts of As, Cd, Cu, and Zn observed in some samples. The enzyme glutathione S-transferase was enhanced in animals exposed to samples, indicating activation of phase II metabolism. Changes observed in glutathione peroxidase (GPx), glutathione reductase (GR), LPO and strand breaks were related to oxidative stress. AChE enzymatic activity also changed, as an indicator of neurotoxicity caused by sediment exposure. The computed integrated biomarker response index (IBR) ranked sites according to the contamination status and proximity to its sources. Correlations found for biomarkers and bioaccumulation of hydrocarbons indicated the influence of harbor activities, effluent discharges, and urban runoff on the sediment pollution of Mucuripe Bay. Data also showed that SQGs are unable to predict bioaccumulation and subchronic effects. Based on our results we consider that biomarkers responses in A. flexuosa are important endpoints to be applied in sediment toxicity bioassays in tropical regions.

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.

Evaluation of macroalgae and amphipods as bioindicators of petroleum hydrocarbons input into the marine environment.

The brown alga Sargassum furcatum and three families of amphipods (Ampithoidae, Caprellidae and Hyalidae) associated to that algae were evaluated as bioindicators of petroleum hydrocarbons input into the marine environment of São Sebastião Channel, in southeastern region of Brazil. The n-alkanes pattern were mainly associated with the natural composition of the macroalgae and amphipods, although some indicatives of petroleum hydrocarbons such as unresolved complex mixture and the no predominance of odd over even n-alkanes have been observed in some samples. Total PAHs ranged from 33.4 to 2010 ng g-1 dry weight with the predominance of low molecular weight PAHs, mostly of naphthalene and alkyl-naphthalenes, which also suggested petroleum input. Even in low concentration, Sargassum furcatum and amphipods species studied seems to be good indicators of the introduction of petroleum hydrocarbons.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Stress responses in Crassostrea gasar exposed to combined effects of acute pH changes and phenanthrene.

Ocean acidification is a result of the decrease in the pH of marine water, caused mainly by the increase in CO2 released in the atmosphere and its consequent dissolution in seawater. These changes can be dramatic for marine organisms especially for oysters Crassostrea gasar if other stressors such as xenobiotics are present. The effect of pH changes (6.5, 7.0 and 8.2) was assessed on the transcript levels of biotransformation [cytochromes P450 (CYP2AU1, CYP2-like2) and glutathione S-transferase (GSTΩ-like)] and antioxidant [superoxide dismutase (SOD-like), catalase (CAT-like) and glutathione peroxidase (GPx-like)] genes, as well as enzyme activities [superoxide dismutase, (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferases transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH)] and lipid peroxidation (MDA) in the gills of Crassostrea gasar exposed to 100 µg·L-1 of phenanthrene (PHE) for 24 and 96 h. Likewise, the PHE burdens was evaluated in whole soft tissues of exposed oysters. The accumulation of PHE in oysters was independent of pH. However, acidification promoted a significant decrease in the transcript levels of some protective genes (24 h exposure: CYP2AU1 and GSTΩ-like; 96 h exposure: CAT-like and GPx-like), which was not observed in the presence of PHE. Activities of GST, CAT and SOD enzymes increased in the oysters exposed to PHE at the control pH (8.2), but at a lower pH values, this activation was suppressed, and no changes were observed in the G6PDH activity and MDA levels. Biotransformation genes showed better responses after 24 h, and antioxidant-coding genes after 96 h, along with the activities of antioxidant enzymes (SOD, CAT), probably because biotransformation of PHE increases the generation of reactive oxygen species. The lack of change in MDA levels suggests that antioxidant modulation efficiently prevented oxidative stress. The effect of pH on the responses to PHE exposure should be taken into account before using these and any other genes as potential molecular biomarkers for PHE exposure.

Organic contamination of beached plastic pellets in the South Atlantic: Risk assessments can benefit by considering spatial gradients.

Microplastics are important vectors for the transport and accumulation of persistent organic contaminants in coastal and marine environments. We determined the concentration of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) associated with microplastic pellets collected along a 39-km stretch of Brazil's South Atlantic coastline to understand the spatial dynamics and potential risk posed by these contaminants. Total PAH concentrations ranged from 1,454 to 6,002 ng g-1 and regularly exceeded the threshold effect level (TEL) for sediments defined by the United States Environmental Protection Agency. Sampling stations, spaced evenly along the coastline (i.e., 3-km intervals) exhibited a general north-to-south decline in the concentrations of some PAHs, but this spatial gradient was complicated by small-scale differences in the concentrations and composition of associated contaminants. Similarly, analysis of individual isomer ratios revealed further complexity driven by differences in the contribution of petrogenic versus pyrolytic inputs which pose different levels of risk to marine organisms. PCB concentrations ranged from 0.8 to 104.6 ng g-1 and were dominated by low chlorinated congeners likely to have originated from major industrial areas to the north. Overall, this study highlights the challenge of directly linking microplastic pollution with the potential toxicological effects of organic contaminants in coastal waters. We recommend that monitoring programs should explicitly consider both the origin (i.e., pellet sources and dispersal pathways) and nature of organic contamination (i.e., concentration and composition) when assessing the risks for biota and human health.

Sub-lethal Responses of the Polychaete Armandia agilis in Whole-sediment Toxicity Testing.

The present study assessed biochemical responses as sublethal endpoints in the polychaete Armandia agilis exposed to contaminated sediments to in order to assess its potential use as a test organism. Sediment samples from several locations at a dredging site were obtained and used in whole-sediment exposures. Samples were tested with A. agilis to determine the 10-day toxicity of the 100% sample and the enzymatic activity of catalase (CAT), glutathione-S-transferase (GST) and acetylcholinesterase (AChE) biochemical measurements made in whole-body homogenates of a subset of the surviving organisms. Biochemical responses reported in A. agilis were not statistically different from the reference site sediment, however, the integrated analysis demonstrated that contaminants bound to sediment samples influenced the sublethal effects.

Biochemical and molecular responses in oysters Crassostrea brasiliana collected from estuarine aquaculture areas in Southern Brazil.

Biochemical and molecular responses were evaluated in oysters Crassostrea brasiliana collected from three oyster farms, at Guaratuba Bay, southern Brazil, forming a pollutant gradient: Farm 1 (reference site - farther from the urban area), Farm 2 (intermediate site) and Farm 3 (nearest to the urban area). Oxidative stress markers, DNA damage and transcript levels of CYP2AU1, CYP2-like1, CYP2-like2, SULT-like, GPx-like, SOD-like, CAT-like, GSTmicrosomal-like, GSTomega-like, FABP-like and ALAd-like genes were analyzed in the gills. The levels of polycyclic aromatic hydrocarbons, linear alkylbenzenes and polychlorinated biphenyls were also evaluated in the soft tissues of the oysters and in the sediment of the Farms. Higher GSTomega-like, CYP2AU1 and FABP-like transcript levels, GR and G6PDH activities and lipid peroxidation levels were observed in oysters from Farms 2 and 3, suggesting pollutant effects on oysters. Alterations in oxidative stress markers also suggest a response against a prooxidant condition in C. brasiliana due to pollutant effects.

Molecular and cellular effects of temperature in oysters Crassostrea brasiliana exposed to phenanthrene.

Exposure of aquatic organisms to polycyclic aromatic hydrocarbons (PAH), such as phenanthrene (PHE), may increase the production of reactive oxygen species (ROS) and cause changes in the biotransformation systems. In addition, changes in water temperature can cause adverse effects in the organisms. Estuarine species, like the oyster Crassostrea brasiliana, can adapt and tolerate temperature variation. To evaluate the influence of temperature on biological responses of C. brasiliana exposed to PHE, oysters were maintained at three temperatures (18, 24 and 32 °C) for 15 days and co-exposed afterwards to 100 µg.L-1 of PHE for 24 and 96 h. Levels of PHE in the water and oyster tissues were determined, respectively after 24 and 96 h. In addition, thermal stress, biotransformation and oxidative stress-related genes were analyzed in oyster gills, together with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferases (GST) and levels of lipid peroxidation. Oyster accumulated significant levels of PHE. HSP70-like transcripts were affected by PHE exposure only at 32 °C. Transcript levels of cytochrome P450 isoforms (CYP2-like2 and CYP2AU1) were down-regulated in oysters exposed to PHE for 24 h at 32 °C. GSTΩ-like transcript levels were also down-regulated in the PHE-exposed group at 32 °C. After 96 h, CYP2-like2 transcripts were higher in the PHE exposed groups at 32 °C. Oysters kept at 18 °C showed higher levels of SOD-like transcripts, together with higher GST, GPx and G6PDH activities, associated to lower levels of lipoperoxidation. In general the biological responses evaluated were more affected by temperature, than by co-exposure to PHE.

Gas Chromatography Coupled to High Resolution Time-of-Flight Mass Spectrometry as a High-Throughput Tool for Characterizing Geochemical Biomarkers in Sediments.

The performance of gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GC-HRTofMS) for characterizing geochemical biomarkers from sediment samples was evaluated. Two approaches to obtain the geochemical biomarkers were tested: (1) extraction with organic solvent and subsequent derivatization and (2) in-situ derivatization thermal desorption. Results demonstrated that both approaches can be conveniently applied for simultaneous characterization of many geochemical biomarkers (alkanes, alkanols, sterols, and fatty acids), avoiding conventional time-consuming purification procedures. GC-HRTofMS reduces both sample preparation time and the number of chromatographic runs compared to traditional methodologies used in organic geochemistry. Particularly, the approach based on in-situ derivatization thermal desorption represents a very simple method that can be performed in-line employing few milligrams of sediment, eliminating the need for any sample preparation and solvent use. The high resolving power (m/Δm 50% 25,000) and high mass accuracy (error ≤ 1 ppm) offered by the "zig-zag" time-of-flight analyzer were indispensable to resolve the complexity of the total ion chromatograms, representing a high-throughput tool. Extracted ion chromatograms using exact m/z were useful to eliminate many isobaric interferences and to increase significantly the signal to noise ratio. Characteristic fragment ions allowed the identification of homologous series, such as alkanes, alkanols, fatty acids, and sterols. Polycyclic aromatic hydrocarbons were also identified in the samples by their molecular ions. The characterization of geochemical biomarkers along a sedimentary core collected in the area of Valo Grande Channel (Cananéia-Iguape Estuarine-Lagunar System (São Paulo, Brazil)) provided evidences of environmental changes. Sediments deposited before opening of channel showed dominance of biomarkers from mangrove vegetation, whereas sediments of the pos-opening period showed an increase of biomarkers from aquatic macrophyte (an invasive vegetation).

Evaluation of polycyclic aromatic hydrocarbons bioavailability on Santos Bay (Brazil) through levels of biliary metabolites.

This study evaluated the PAH bioavailability from Santos Bay (Brazil) in 4 species of fish, using PAH biliary metabolites. The collection was done monthly, between July and December, in three different regions of Santos Bay. The metabolites were analyzed through a high performance liquid chromatograph with fluorescence detectors. Total metabolites concentrations ranged from 65.5 to 589µgg-1 of bile, evidencing PAH bioavailability on Santos Bay. Levels of phenanthrene and benzo[a]pyrene metabolites were in the classification range of areas moderate contaminated. Those concentrations were lower in Nebris microps and higher in Sphoeroides testudineus (p<0.05). Naphthalene metabolites concentrations did not differ significantly among fish species and were in the classification range of low contaminated areas. There were no significant spatial and temporal differences in levels among sampled areas. These results are environmentally important given the high levels of urbanization and the absence of biomonitoring data in this area.