Silvio Tarou Sasaki

Results from a 15-year study on hydrocarbon concentrations in water and sediment from Admiralty Bay, King George Island, Antarctica

Abstract Admiralty Bay on the King George Island hosts the Brazilian, Polish and Peruvian research stations as well as the American and Ecuadorian field stations. Human activities in this region require the use of fossil fuels as an energy source, thereby placing the region at risk of hydrocarbon contamination. Hydrocarbon monitoring was conducted on water and sediment samples from the bay over 15 years. Fluorescence spectroscopy was used for the analysis of total polycyclic aromatic hydrocarbons (PAHs) in seawater samples and gas chromatography with flame ionization and/or mass spectrometric detection was used to analyse individual n-alkanes and PAHs in sediment samples. The results revealed that most sites contaminated by these compounds are around the Brazilian and Polish research stations due to the intense human activities, mainly during the summer. Moreover, the sediments revealed the presence of hydrocarbons from different sources, suggesting a mixture of the direct input of oil or derivatives and derived from hydrocarbon combustion. A decrease in PAH concentrations occurred following improvement of the sewage treatment facilities at the Brazilian research station, indicating that the contribution from human waste may be significant.

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.

Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction.

Understanding the toxic mechanisms by which organisms cope to environmental stressful conditions is a fundamental question for ecotoxicology. In this study, we evaluated biochemical responses and hydrocarbons bioaccumulation of the mangrove oyster Crassostrea brasiliana exposed for 96 h to four sublethal concentrations of diesel fuel water-accommodated fraction (WAF). For that purpose, enzymatic activities (SOD, CAT, GPx, GR, G6PDH, GST and GGT), HSP60 and HSP90 immunocontent and lipid peroxidation (LPO) levels were determined in the gill and digestive gland of oysters and related to the hydrocarbons accumulated in the whole soft tissues. The results of this study revealed clear biochemical responses to diesel fuel WAF exposure in both tissues of the oyster. The capacity of C. brasiliana to bioaccumulate aliphatic and aromatic hydrocarbons in a dose-dependent manner is a strong indication of its suitability as a model in biomonitoring programs along the Brazilian coast, which was also validated by the response of the antioxidant defenses, phase II biotransformation and chaperones. HSP60 levels and GGT activity were the most promising biomarkers in the gill, while GST and GR activities stood out as suitable biomarkers for the detection of diesel toxicity in the digestive gland. The decrease of SOD activity and HSP90 levels may also reflect a negative effect of diesel exposure regardless the tissue. The present results provide a sound preliminary report on the biochemical responses of C. brasiliana challenged with a petroleum by-product and should be carefully considered for use in the monitoring of oil and gas activities in Brazil.

A multi-molecular marker assessment of organic pollution in shore sediments from the Río de la Plata Estuary, SW Atlantic

Organic pollution was evaluated in surface sediments along the middle portion of the Río de la Plata Estuary, SW Atlantic. A multi-molecular marker approach was performed to identify major sources of organic compounds using diagnostic indices. The relative contribution of different sources of hydrocarbons was quantified by source apportionment employing Principal Component Analysis/Multiple Linear Regression (PCA/MLR) as chemometric technique. All molecular markers indicated high chronic organic pollution in the stations of Montevideo Bay. Main sources of aliphatic and polycyclic aromatic hydrocarbons were petroleum inputs and combustion, due to oil transport and refinement, harbour activities and vehicular emissions. Major sources of linear alkylbenzenes and steroids were industrial and domestic sewage. Although, significant anthropogenic inputs, a natural footprint of terrestrial higher plants contribution was recognized. Multi-molecular marker and comprehensive assessments can improve the establishment of more precise regulation actions to reduce pollution levels.

Effects of harbor activities on sediment quality in a semi-arid region in Brazil

Tropical marine environments are rich in biodiversity and the presence of harbor activities in these areas can harm the coastal ecosystems. In this study, we assessed sediment quality of two harbors from a tropical region in Brazil by applying multiple lines-of-evidence approach. This approach included the integration of results on: (1) grain size, organic matter, organic carbon, nitrogen, phosphorus, trace metals, polycyclic aromatic hydrocarbons, linear alkylbenzenes, and tributyltin; (2) acute toxicity of whole sediments and chronic toxicity of liquid phases; and (3) benthic community descriptors. Our results revealed that the main contaminants detected in sediments from Mucuripe and Pecém Harbors were chromium, copper, nitrogen, zinc, and tributyltin. These toxicants arise from typical harbor activities. However, the changes in benthic composition and structure appear to depend on a combination of physical impacts, such as the deposition of fine sediments and the toxic potential of contaminants, especially in Mucuripe. Thus, apart from toxicants physical processes are important in describing risks. This information may assist in management and conservation of marine coastal areas.

Exposure to phenanthrene and depuration: Changes on gene transcription, enzymatic activity and lipid peroxidation in gill of scallops Nodipecten nodosus

Understanding the mechanism of phenanthrene (PHE) biotransformation and related cellular responses in bivalves can be an important tool to elucidate the risks of polycyclic aromatic hydrocarbons (PAHs) to aquatic organisms. In the present study it was analyzed the transcriptional levels of 13 biotransformation genes related to cytochrome P450 (CYP), glutathione S-transferase (GST), sulfotransferase (SULT), flavin-containing monooxygenase and fatty acid-binding proteins by qPCR in gill of scallops Nodipecten nodosus exposed for 24 or 96h to 50 or 200μgL(-1) PHE (equivalent to 0.28 and 1.12μM, respectively), followed by depuration in clean water for 96h (DEP). Likewise, it was quantified the activity of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), GST and levels of lipid peroxidation. Increased transcriptional levels of CYP2UI-like, CYP2D20-like, CYP3A11-like, GSTomega-like, SULT1B1-like genes were detected in organisms exposed to PHE for 24 or 96h. In parallel, GR and GPX activities increased after 96h exposure to 200μgL(-1) PHE and G6PDH activity increased after 24h exposure to 50μgL(-1) PHE. This enhancement of antioxidant and phase I and II biotransformation systems may be related to the 2.7 and 12.5 fold increases in PHE bioaccumulation after 96h exposure to 50 and 200μgL(-1) PHE, respectively. Interestingly, DEP caused reestablishment of GPX and GR activity, as well as to the transcript levels of all upregulated biotransformation genes (except for SULT1B1-like). Bioaccumulated PHE levels decreased 2.5-2.9 fold after depuration, although some biochemical and molecular modifications were still present. Lipid peroxidation levels remained lower in animals exposed to 200μgL(-1) PHE for 24h and DEP. These data indicate that N. nodosus is able to induce an antioxidant and biotransformation-related response to PHE exposure, counteracting its toxicity, and DEP can be an effective protocol for bivalve depuration after PHE exposure.

Upregulation of biotransformation genes in gills of oyster Crassostrea brasiliana exposed in situ to urban effluents, Florianópolis Bay, Southern Brazil

The release of untreated sanitary sewage, combined with unplanned urban growth, are major factors contributing to degradation of coastal ecosystems in developing countries, including Brazil. Sanitary sewage is a complex mixture of chemicals that can negatively affect aquatic organisms. The use of molecular biomarkers can help to understand and to monitor the biological effects elicited by contaminants. The aim of this study was to evaluate changes in transcript levels of genes related to xenobiotic biotransformation in the gills of oysters Crassostrea brasiliana transplanted and kept for 24h at three areas potentially contaminated by sanitary sewage (Bücheller river, BUC; Biguaçu river, BIG; and Ratones island, RAT), one farming area (Sambaqui beach, SAM) and at one reference site (Forte beach, FOR) in the North Bay of Santa Catarina Island (Florianópolis, Brazil). Transcript levels of four cytochrome P450 isoforms (CYP2AU1, CYP3A-like, CYP356A1-like and CYP20A1-like), three glutathione S-transferase (GST alpha-like, GST pi-like and GST microsomal 3-like) and one sulfotransferase gene (SULT-like) were evaluated by means of quantitative reverse transcription PCR (qRT-PCR). Chemical analysis of the sediment from each site were performed and revealed the presence of aliphatic and polycyclic aromatic hydrocarbons, linear alkylbenzenes and fecal sterols in the contaminated areas (BUC and BIG). Water quality analysis showed that these sites had the highest levels of fecal coliforms and other parameters evidencing the presence of urban sewage discharges. Among the results for gene transcription, CYP2AU1 and SULT-like levels were upregulated by 20 and 50-fold, respectively, in the oysters kept for 24h at the most contaminated site (BUC), suggesting a role of these genes in the detoxification of organic pollutants. These data reinforce that gills possibly have an important role in xenobiotic metabolism and highlight the use of C. brasiliana as a sentinel for monitoring environmental contamination in coastal regions.

Histological responses and localization of the cytochrome P450 (CYP2AU1) in Crassostrea brasiliana exposed to phenanthrene.

Phenanthrene (PHE) is an abundant polycyclic aromatic hydrocarbon (PAH), widely distributed in aquatic environment. The aim of this study was to evaluate the histological and molecular effects in the native oyster Crassostrea brasiliana(Lamarck, 1819) exposed to 100 and 1000 μg L(-1) PHE for 1, 5 and 10 days. Histological and chemical analyses were performed to evaluate, respectively, alterations in oyster tissues and bioaccumulation. In situ hybridization (ISH) was used to assess tissue distribution of CYP2AU1, a gene formerly identified as activated by PHE exposure in this species.Quantitative polymerase chain reaction (qPCR) in mantle was carried out to validate ISH data. Oysters bioaccumulated PHE increasingly along the exposure period in both exposure concentrations. Histologic changes, like tubular atrophy in digestive diverticula (digestive gland) and increased number of mucous cells in the mantle were observed in animals exposed to PHE for 10 days. ISH showed the presence of CYP2AU1transcripts in gills, digestive diverticula, mantle, intestine and gonads, but significant differences in transcript detection by ISH between treatments occurred only in gills, mantle and intestine. A positive and significant correlation between tubular atrophy and CYP2AU1hybridization signal was observed in digestive diverticula, suggesting that this gene product might be involved in energetic metabolism in C. brasiliana. Increased mucous cells and CYP2AU1transcript levels were observed in the mantle, where the inner and middle lobes showed higher intensity of hybridization signal. Mantle should be considered as a target organ for CYP2AU1 transcript evaluation and histological alterations in biomonitoring studies. CYP2AU1 signal in female gonads was observed in all follicular cells from different gonadic stages, while in male only the spermatic follicle cells of the wall in the pre-spawning stage showed this signal. ISH was an effective technique to evaluate the effects of PHE exposure and to locate CYP2AU1 transcripts in different tissues of oyster C. brasiliana.

Transcriptional changes in oysters Crassostrea brasiliana exposed to phenanthrene at different salinities

Euryhaline animals from estuaries, such as the oyster Crassostrea brasiliana, show physiological mechanisms of adaptation to tolerate salinity changes. These ecosystems receive constant input of xenobiotics from urban areas, including polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (PHE). In order to understand the influence of salinity on the molecular responses of C. brasiliana exposed to PHE, oysters were acclimatized to different salinities (35, 25 and 10) for 15days and then exposed to 100μgL-1 PHE for 24h and 96h. Control groups were kept at the same salinities without PHE. Oysters were sampled for chemical analysis and the gills were excised for mRNA quantification by qPCR. Transcript levels of different genes were measured, including some involved in oxidative stress pathways, phases I and II of the xenobiotic biotransformation systems, amino acid metabolism, fatty acid metabolism and aryl hydrocarbon receptor nuclear translocator putative gene. Higher transcript levels of Sulfotransferase-like gene (SULT-like) were observed in oysters exposed to PHE at salinity 10 compared to control (24h and 96h); cytochrome P450 isoforms (CYP2AU1, CYP2-like1) were more elevated in oysters exposed for 24h and CYP2-like2 after 96h of oysters exposed to PHE at salinity 10 compared to control. These results are probably associated to an enhanced Phase I biotransformation activity required for PHE detoxification under hyposmotic stress. Higher transcript levels of CAT-like, SOD-like, GSTm-like (96h) and GSTΩ-like (24h) in oysters kept at salinity 10 compared to organisms at salinities 25 and/or 35 are possibly related to enhaced ROS production. The transcription of these genes were not affected by PHE exposure. Amino acid metabolism-related genes (GAD-like (24h), GLYT-like, ARG-like (96h) and TAUT-like at 24h and 96h) also showed different transcription levels among organisms exposed to different salinities, suggesting their important role for oyster salinity adaptation, which is not affected by exposure to these levels of PHE.

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.