Antioxidant defense responses in Mytella guyanensis (Lamarck, 1819) exposed to an experimental diesel oil spill in Paranaguá Bay (Paraná, Brazil).

We evaluated the effects of diesel oil on the bivalve Mytella guyanensis using biomarkers of oxidative stress (glutathione S-transferase, glutathione peroxidase, and reduced glutathione) after an experimental in situ spill in a mangrove area in southern Brazil. A linear model was developed for the Multiple Before-After Control-Impact (MBACI) experimental design to assess the significance of biological responses. Control and impacted sites were sampled seven and two days before as well as two and seven days after the spill. With the exception of a late response of reduced glutathione (GSH) levels on day seven, none of the biomarkers were significantly altered by the impact. This result was attributed to the high environmental variability of the experimental sites combined with a low sensitivity of Mytella guyanensis to diesel oil at short time-scales. The high resistance of M. guyanensis suggests that its antioxidant response is triggered only after a medium- to long-term exposure to contaminants.

Synthesis, Characterization, and Low-Toxicity Study of a Magnesium(II) Complex Containing an Isovanillate Group.

The beneficial effect of polyphenols and magnesium(II) against oxidative stress motivated our research group to explore the antioxidant activity of phenMgIso, an aqueous soluble magnesium(II) complex containing 1,10-phenanthroline (phen) and isovanillic acid (Iso) as ligands. Combined electrospray ionization-mass spectrometry and DOSY-NMR techniques identified two complexes in methanolic solution: hexacoordinated [Mg(phen)2(Iso)]+ and tetracoordinated [Mg(phen)(Iso)]+. The cyclic voltammogram of phenMgIso in the anodic region showed a cyclic process that interrupts the isovanillic acid degradation, probably by stabilization of the corresponding phenoxyl radical via complexation with Mg(II), which is interesting for antioxidant applications. phenMgIso competes with 2,2,6,6-tetramethylpiperidine by 1O2 with IC50(1O2) = 15 µg m-1 and with nitrotetrazolium blue chloride by superoxide ions (IC50(O2 •-) = 3.6 µg mL-1). Exposure of both zebrafish (2 mg L-1) and wistar male rats (3 mg kg-1 day-1 dose for 21 days) to phenMgIso does not cause mortality or visual changes compared with the respective control groups, thus phenMgIso could be considered safe under the conditions of this study. Moreover, no significant changes in comparison to both control groups were observed in the biochemical parameters on the brain-acetylcholinesterase activity, digestive tract enzyme catalase, and glutathione-S-transferase. Conversely, the performance of superoxide dismutase activity in wistar male rats increased in the presence of a complex, resulting in enhanced capacity of rats for superoxide radical enzymatic scavenging. The synergistic action of phenMgIso may be explained by the strong electrostatic interaction between Mg(II) and the O,O(phenolate) group, which makes the Iso ligand easier to oxidize and deprotonate, generating a cyclic stable species under oxidative conditions.

Multivariate and integrative approach to analyze multiple biomarkers in ecotoxicology: A field study in Neotropical region.

Aquatic pollution has dramatically worsened in developing countries, due to the discharge of a mixture of pollutants into water bodies, to the lack of stringent laws, and the inadequate treatment of effluents. In this study, the Neotropical fish Astyanax aff. paranae was sampled from three sites with different pollution levels: 1) a Biological Reserve (Rebio), protected by the Brazilian government; 2) an agricultural area in one of the most productive regions of Brazil, upstream of an urban zone; and 3) a site downstream from urban zone, characterized by the influx of different effluents, including wastes from industry, a sewer treatment plant, and agricultural areas. We assess biomarkers at multiple levels, such as the comet assay, hepatic histopathological analysis, brain and muscle acetylcholinesterase (AChE) and the hepatic enzymes glutathione-S-transferase (GST), catalase (CAT), and lipoperoxidation (LPO), during winter and summer. The interpretation of field results is always a very complex operation, since many factors can influence the variables analyzed in uncontrollable conditions. For this reason, we apply an integrative multivariate analysis. The results showed that the environmental risk of the three sites was significantly different. We can see a gradient in data distribution in discriminant analysis: separating, from one side, the fish of Rebio; in the middle are the fish from agricultural area and, in the other side are the animals from downstream site. Overall, the biomarkers responses were more greatly altered in the downstream site, whereas fish from the agricultural area showed an intermediate level of damage. The greatest changes were likely caused by agriculture, industrial chemical effluents and ineffective sewage treatments, in a synergic interaction in downstream site. In conclusion, the use of multiple biomarkers at different response levels to assess the toxic effects of mixed pollutants in a natural aquatic environment is an important tool for monitoring polluted regions.

Evaluation of the water quality of the upper reaches of the main Southern Brazil river (Iguaçu river) through in situ exposure of the native siluriform Rhamdia quelen in cages.

Increase in industrial growth, urban and agricultural pollution, with consequent impacts on aquatic ecosystems are a major focus of research worldwide. Still, not many studies assess the impacts of contamination through in situ studies, using native species, also considering the influence of seasonality on their responses. This study aimed to evaluate the water quality of the basin of the Upper Iguaçu River, the main source of water supply to Curitiba, a major capital of Southern Brazil, and its Metropolitan area. Several biomarkers were evaluated after in situ exposure of the native catfish Rhamdia quelen inside cages for 7 days. Ten study sites were chosen along the basin, based on a diffuse gradient of contamination, corresponding to regions upstream, downstream, and within "great Curitiba". In each site, fish were exposed in Summer and Winter. The complex mixture of contaminants of this hydrographic basin generated mortality, and ion-, osmoregulatory and respiratory disturbances in the catfish as, for example, reduction of plasma osmolality and ionic concentrations, increased hematocrit levels and gill water content, altered branchial and renal activities of the enzyme carbonic anhydrase, as well as raised levels of plasma cortisol and glucose. Biomarkers were mostly altered in fish exposed in Great Curitiba and immediately downstream. There was a notable influence of season on the responses of the jundiá. A multivariate redundancy analysis revealed that the best environmental variables explained 30% of the variation in biomarkers after controlling for spatial autocorrelation. Thus, this approach and the chosen parameters can be satisfactorily used to evaluate contamination environments with complex mixtures of contaminants, in other urban basins as well.

Biomarkers of waterborne copper exposure in the Neotropical fish Prochilodus lineatus.

The main goal of the present study was to investigate the effects of acute exposure to copper (Cu) using a Neotropical freshwater fish as sentinel species through multi biomarkers analysis at different biological levels. Juveniles of Prochilodus lineatus were kept under control condition (no Cu addition in the water) or exposed to environmentally relevant concentrations of waterborne Cu (5, 9 and 20µgL(-1)) for 96h. These concentrations were selected to bracket the current Brazilian water quality criteria for Cu in fresh water (9 and 13µgL(-1) dissolved copper). Endpoints analyzed included ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, reduced glutathione (GSH) and metallothionein-like protein (MT) concentration, lipid peroxidation (LPO) level, tissue damage index, and incidence of free melano-macrophages (FMM) and melano-macrophage centers (MMC) in the liver. They also included DNA damage (frequency of nucleoids per comet class, number of damaged nucleoids per fish and DNA damage score) in erythrocytes, as well as muscle and brain acetylcholinesterase (AChE) activity and behavioral parameters (swimming distance and velocity, time spent swimming and swimming activity in the upper and lower layers of the water column). Fish exposed to any of the Cu concentrations tested showed increased liver MT concentration and LPO level, higher number of damaged nucleoids in erythrocytes per fish, and inhibited muscle AChE activity. Also, increased liver SOD activity was observed in fish exposed to 9 and 20µgL(-1) Cu. Fish exposed to 5 and 9µgL(-1) Cu spent lower amount of time swimming. Fish exposed to 9µgL(-1) Cu showed increased swimming distance and velocity while those exposed to 20µgL(-1) Cu had lower swimming distance and velocity, as well as, spent less time swimming in the lower layer of the water column when compared to those kept under control condition. These findings indicate that Cu exposure at environmentally relevant concentrations (below or close to the current Brazilian water quality criteria) induced significant biological (histological, biochemical and genetic) and ecological (swimming and exploratory abilities) damages in the Neotropical fish P. lineatus. They also suggest that MT concentration, DNA damage (comet assay), LPO (TBARS method), SOD and AChE activity, together with swimming behavior analyses are potential biomarkers to assess and monitor areas impacted by Cu in fresh water.

Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae)

ABSTRACT Changes in physiology of the nervous system and metabolism can be detected through the activity of acetylcholinesterase (AChE), alpha esterase (EST-a) and beta esterase (EST-ß) in chironomids exposed to pollutants. However, to understand the real effect of xenobiotics on organisms, it is important to investigate how certain factors can interfere with enzyme activity. We investigated the effects of different temperatures, food stress and two steps of the enzymatic protocol on the activity of AChE, EST-a and EST-ß in Chironomus sancticaroli. In experiment of thermal stress individuals from the egg stage to the fourth larval instar were exposed to different temperatures (20, 25 and 30 °C). In food stress experiment, larvae were reared until IV instar in a standard setting (25 °C and 0.9 g weekly ration), but from fourth instar on they were divided into groups and offered different feeding regimes (24, 48 and 72 h with/without food). In sample freezing experiment, a group of samples was processed immediately after homogenization and another after freezing for 30 days. To test the effect of centrifugation on samples, enzyme activity was quantified from centrifuged and non-centrifuged samples. The activity of each enzyme reached an optimum at a different temperature. The absence of food triggered different changes in enzyme activity depending on the period of starvation. Freezing and centrifugation of the samples significantly reduced the activity of three enzymes. Based on these results we conclude that the four factors studied had an influence on AChE, EST-a and EST-ß and this influence should be considered in ecotoxicological approaches.

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests

Low malathion concentrations influence metabolism in Chironomus sancticaroli (Diptera, Chironomidae) in acute and chronic toxicity tests. Organophosphate compounds are used in agro-systems, and in programs to control pathogen vectors. Because they are continuously applied, organophosphates often reach water sources and may have an impact on aquatic life. The effects of acute and chronic exposure to the organophosphate insecticide malathion on the midge Chironomus sancticaroli are evaluated. To that end, three biochemical biomarkers, acetylcholinesterase (AChE), alpha (EST-α) and beta (EST-β) esterase were used. Acute bioassays with five concentrations of malathion, and chronic bioassays with two concentrations of malathion were carried out. In the acute exposure test, AChE, EST-α and EST-β activities declined by 66, 40 and 37%, respectively, at 0.251 µg L-1 and more than 80% at 1.37, 1.96 and 2.51 µg L-1. In chronic exposure tests, AChE and EST-α activities declined by 28 and 15% at 0.251 µg L-1. Results of the present study show that low concentrations of malathion can influence larval metabolism, indicating high toxicity for Chironomus sancticaroli and environmental risk associated with the use of organophosphates.