Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish.

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Evaluation of Different Techniques and Materials for Filling in 3-dimensional Printed Teeth Replicas with Perforating Internal Resorption by Means of Micro-Computed Tomography.

INTRODUCTION: The aim of this study was to evaluate the filling ability of 2 obturation techniques in 3-dimensional (3D) printed teeth with perforating internal resorption (PIR). METHODS: A maxillary central incisor was instrumented and scanned by micro-computed tomographic (micro-CT) imaging. The 3D model was exported in the stereolithographic format and, with the aid of OrtogOnBlender software (Cícero Moraes, Sinop, SP, Brazil), a PIR in the middle third of the root canal was designed. Thirty-two replicas were printed in surgical resin and distributed into 4 groups (n = 8) according to the obturation technique and the material used: 2 groups used the hybrid technique, 1 with Bio-C Sealer (BCS; Angelus, Londrina, PR, Brazil)/gutta-percha (GP; VDW GmbH, Munich, Germany) + Bio-C Repair (BCR; Angelus, Londrina, PR, Brazil) and the other with BioRoot (BR; Septodont, Saint Maur des Fosses, France)/gutta-percha (GP) + Biodentine (BD; Septodont, Saint Maur des Fosses, France), and 2 groups used the incremental technique, 1 with BCR and the other with BD. Postobturation micro-CT imaging was performed to measure the percentage volume of voids and laser confocal microscopy to measure the surface roughness (µm) of the repair cements. Data were compared using analysis of variance and Kruskal-Wallis tests. RESULTS: Regarding the filling volume in the apical third, the BCS/GP + BCR (89.70 ± 5.15), BR/GP + BD (87.70 ± 8.43), and BCR (84.20 ± 9.00) groups showed the highest percentages compared with the BD group (69.70 ± 6.88) (P < .05). In the area of internal resorption, the BCS/GP + BCR (96.00 ± 2.64) and BCR (95.30 ± 2.93) groups showed the highest percentages compared with the BR/GP + BD group (91.50 ± 1.35) (P < .05). The BD group showed intermediate values that were sometimes similar to the BCS/GP + BCR and BCR groups and similar to the BR/GP + BD group (P > .05). Regarding the quality of the filling in the perforation area, the BCR group showed better results compared with the BD group (P < .001). Regarding roughness, the BCR group (1.66 ± 0.65) showed lower surface roughness compared with the BD group (2.51 ± 0.89) (P < .05). CONCLUSIONS: The capacity and quality of the filling in teeth with PIR were superior with the incremental technique with BCR and the hybrid technique with BCS/GP + BCR.

Co-exposure effects of lead and TiO2 nanoparticles in primary kidney cell culture from the freshwater fish Hoplias malabaricus.

This study evaluated the effects of Lead (Pb) and titanium dioxide nanoparticles (TiO2 NPs) alone or in combination in anterior kidney macrophages of the freshwater fish Hoplias malabaricus, naïve or stimulated with 1 ng.mL-1 lipopolysaccharide (LPS). Pb (1 ×10-5 to 1 ×10-1 mg.mL-1) or TiO2 NPs (1.5 ×10-6 to 1.5 ×10-2 mg.mL-1) reduced cell viability despite LPS stimulation, especially Pb 10-1 mg.mL-1. In combination, lower concentrations of NPs intensified Pb-induced cell viability reduction while higher concentrations restored the cell viability independently of LPS stimulation. Basal and LPS- induced NO production was reduced by both TiO2 NPs and Pb isolated. The combination of both xenobiotics avoided this reduction of NO production by the isolated compounds at lower concentrations but the protective effect was lost as the concentrations increased. None xenobiotic increase DNA fragmentation. Therefore, at specific conditions, TiO2 NPs may have a protective effect over Pb toxicity, may also provide additional toxicity at higher concentrations.

Can Carbodiimide (EDC) and Chitosan Cross-linking Agents Effect the Longevity of Fiberglass Posts Luted with Different Types of Composite Cements to Root Dentin?

PURPOSE: To evaluate the effect of carbodiimide (EDC) and chitosan (CHI) on the enzymatic activity (EA) and bond strength (BS) of different composite cements to root dentin. MATERIALS AND METHODS: Ninety (90) maxillary canines were sectioned, standardizing the length of the roots. The roots were endodontically treated, prepared, divided into 3 groups according to dentin treatment (distilled water [DW], CHI 0.2 wt%, or EDC 0.5M), and further subdivided into 3 subgroups according to composite cement (RelyX ARC [3M Oral Care], Panavia F 2.0 [Kuraray Noritaki], or RelyX U200 [3M Oral Care]). Of the slices obtained by sectioning, the most cervical of each third were subjected to a push-out test and the most apical were subjected to in-situ zymography. Half of the slices were analyzed immediately, and the other half after 6 months. The results were analyzed with ANOVA or the chi-squared test. RESULTS: RelyX ARC showed higher BS associated with CHI, while RelyX U200 showed higher BS associated with EDC (p = 0.044). For Panavia F 2.0, the treatment did not influence BS (p > 0.05). For the cervical and middle thirds, no differences were observed between the cements, while the apical third revealed higher BS for RelyX U200 (p < 0.001). The highest percentage of adhesive-to-dentin failures was observed for Panavia F 2.0. EDC showed the lowest percentage of adhesive-to-dentin failures. According to zymographic analysis, DW and CHI showed greater fluorescence for RelyX ARC, while EDC exhibited the lowest fluorescence of all cements (p > 0.05). CONCLUSION: The different mechanisms of action of solutions for pre-treatment of intraradicular dentin yielded different results depending on the adhesive used. EDC resulted in higher bond strength and higher enzyme inhibition for RelyX U200, while the treatment with chitosan resulted in higher bond strength and lower enzymatic activity for RelyX ARC. Although EDC and chitosan treatments did not influence the bond strength for Panavia F 2.0, both resulted in higher enzyme inhibition for this composite cement.

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.

Evaluation of Resistance Management for the Oriental Fruit Moth (Lepidoptera: Tortricidae) to Insecticides in Brazilian Apple Orchards.

The oriental fruit moth, Grapholita molesta (Busk, 1916), is one of the most important pests in apple orchards in southern Brazil. Chemical control is still the most commonly used strategy for pest control. The aim of this study was to determine the resistance evolution of seven South Brazilian populations to four insecticides (chlorantraniliprole, lufenuron, chlorpyrifos, and fenitrothion). Bioassays were paired with metabolic analyses of glutathione-S-transferase (GST), carboxylesterases (α-NA and ß-NA), and acetylcholinesterase (AChE) to understand the possible role with phytosanitary strategies in the insects' susceptibility. Insect populations were collected in the municipalities of Campo do Tenente, Lapa and Porto Amazonas, Fraiburgo, São Joaquim, and Vacaria and multiplied in the laboratory. Two susceptible and two resistant populations were used as references. The bioassays showed that five populations were considered more resistant to organophosphates, six to lufenuron and two to chlorantraniliprole when compared with the sensitive population. None of the field populations had greater resistance than the resistant laboratory population. The enzymatic activity of AChE and GST was elevated in most of the populations that were less susceptible to organophosphates and lufenuron. The populations originating from orchards that used sexual confusion techniques had the greatest susceptibility based on toxicological and biochemical bioassays. Populations under pressure from various compounds had high GST, α and ß-NA activity. There is evidence that a diversity of control strategies can provide better resistance management.

Visible-light reduced silver nanoparticles' toxicity in Allium cepa test system.

Silver nanoparticles (AgNPs) are widely used in consumer products due to their antibacterial property; however, their potential toxicity and release into the environment raises concern. Based on the limited understanding of AgNPs aggregation behavior, this study aimed to investigate the toxicity of uncoated (uc-AgNP) and coated with polyvinylpyrrolidone (PVP-AgNP), at low concentrations (0.5-100 ng/mL), under dark and visible-light exposure, using a plant test system. We exposed Allium cepa seeds to both types of AgNPs for 4-5 days to evaluate several toxicity endpoints. AgNPs did not cause acute toxicity (i.e., inhibition of seed germination and root development), but caused genotoxicity and biochemical alterations in oxidative stress parameters (lipid peroxidation) and activities of antioxidant enzymes (superoxide dismutase and catalase) in light and dark conditions. However, the light exposure decreased the rate of chromosomal aberration and micronuclei up to 5.60x in uc-AgNP and 2.01x in PVP-AgNP, and 2.69x in uc-AgNP and 3.70x in PVP-AgNP, respectively. Thus, light exposure reduced the overall genotoxicity of these AgNPs. In addition, mitotic index alterations and morphoanatomical changes in meristematic cells were observed only in the dark condition at the highest concentrations, demonstrating that light also reduces AgNPs cytotoxicity. The light-dependent aggregation of AgNPs may have reduced toxicity by reducing the uptake of these NPs by the cells. Our findings demonstrate that AgNPs can be genotoxic, cytotoxic and induce morphoanatomical and biochemical changes in A. cepa roots even at low concentrations, and that visible-light alters their aggregation state, and decreases their toxicity. We suggest that visible light can be an alternative treatment to remediate AgNP residues, minimizing their toxicity and environmental risks.

Cytotoxicity and enzymatic biomarkers as early indicators of benthic responses to the soluble-fraction of diesel oil.

Xenobiotics from oil tanker leaks and industrial discharges are amongst the main human impacts to confined coastal areas. We assessed the genotoxic responses to the water-soluble fraction of diesel oil in the polychaete Laeonereis culveri and the bivalve Anomalocardia flexuosa, two widespread benthic species in subtropical estuaries from the Southwestern Atlantic. We hypothesized that the highest responsiveness would be expressed by significantly different biomarkers responses between control and oil-impacted treatments. Responsiveness to diesel oil was investigated using an experimental design with two fixed factors (contaminant percentages and times of exposure). After exposure, we monitored the responses of the oxidative stress enzymes and performed micronuclei tests. Results were congruent for both species. Antioxidant defense of glutathione S-transferase and the induction of micronuclei and nuclear buds, the latter just for the bivalve, were significantly affected by polycyclic aromatic hydrocarbons, with significant increases on the seventh day of exposure and in the higher concentrations, compared to controls groups. We assessed the benefits and drawbacks of using each biomarker in laboratory experiments. Both species are indicators of early, and rapid responses to genotoxic contaminants in subtropical estuarine habitats. We suggest that the micronuclei frequency in A. flexuosa is a simple, fast and cheap test for genotoxicity in oil-impacted areas. Such early biomarkers are needed to develop better protocols for impact assessment and monitoring under real field conditions.

Cloning, partial sequencing and expression analysis of the neural form of P450 aromatase (cyp19a1b) in the South America catfish Rhamdia quelen.

Brain aromatase is a key enzyme exclusively expressed in fish radial glial cells that convert androgens into estrogens, thus controlling neuroendocrine functions and neurogenesis. As an important step in characterizing the neuroendocrine systems of Rhamdia quelen (jundiá), a partial cDNA sequence (1045 bp) of brain aromatase (cyp19a1b) was cloned and sequenced. At the nucleotide level the cDNA sequence was found to be 88% identical to cyp19a1b of two species of catfish, Ictalurus punctatus and Silurus meridionalis. The predicted amino acid sequence was between 80 and 91% similar to other teleosts. Real-time RT-qPCR analysis revealed that cyp19a1b was detected in pituitary, hypothalamus, telencephalon, head and posterior kidneys, liver and gonads (testis and ovary) of both males and females. The effects of E2 on cyp19a1b expression are sexually dimorphic in R. quelen. The injection of 17ß-estradiol (E2) decreased head kidney mRNA levels of cyp19a1b in males and increased cyp19a1b mRNA levels in the pituitary and head kidney of females. This study demonstrated that the R. quelen cyp19a1b gene is expressed in brain, pituitary and peripheral tissues in both males and females.

Toward sustainable environmental quality: Identifying priority research questions for Latin America.

The Global Horizon Scanning Project (GHSP) is an innovative initiative that aims to identify important global environmental quality research needs. Here we report 20 key research questions from Latin America (LA). Members of the Society of Environmental Toxicology and Chemistry (SETAC) LA and other scientists from LA were asked to submit research questions that would represent priority needs to address in the region. One hundred questions were received, then partitioned among categories, examined, and some rearranged during a workshop in Buenos Aires, Argentina. Twenty priority research questions were subsequently identified. These research questions included developing, improving, and harmonizing across LA countries methods for 1) identifying contaminants and degradation products in complex matrices (including biota); 2) advancing prediction of contaminant risks and effects in ecosystems, addressing lab-to-field extrapolation challenges, and understanding complexities of multiple stressors (including chemicals and climate change); and 3) improving management and regulatory tools toward achieving sustainable development. Whereas environmental contaminants frequently identified in these key questions were pesticides, pharmaceuticals, endocrine disruptors or modulators, plastics, and nanomaterials, commonly identified environmental challenges were related to agriculture, urban effluents, solid wastes, pulp and paper mills, and natural extraction activities. Several interesting research topics included assessing and preventing pollution impacts on conservation protected areas, integrating environment and health assessments, and developing strategies for identification, substitution, and design of less hazardous chemicals (e.g., green chemistry). Finally, a recurrent research need included developing an understanding of differential sensitivity of regional species and ecosystems to environmental contaminants and other stressors. Addressing these critical questions will support development of long-term strategic research efforts to advance more sustainable environmental quality and protect public health and the environment in LA. Integr Environ Assess Manag 2018;14:344-357. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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.

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.

Pseudoreplication and the usage of biomarkers in ecotoxicological bioassays.

Pseudoreplication is a widely discussed topic in the scientific community. Its principal critique concerns the lack of independence in flawed experimental designs and the use of inferential statistics to test the hypothesis of such experiments. Thirty years after its appearance, it remains misunderstood by many researchers, including ecotoxicologists. In the present study, we try to clarify some of its concepts by filling in what seems to be a gap in the terminology of manipulative experiments. We propose the term "experimental medium" to refer strictly to the relevant spatial scale of the experiment to preserve the specificity of the experimental and observational units and to display pseudoreplication as a kind of misinterpretation and/or misanalysis of inferential statistics. A classification of the types of experimental designs in ecotoxicology is offered, and the problems in using inferential statistics in suboptimal designs are discussed. We hope to shed some light on such a classic topic for ecotoxicologists. Environ Toxicol Chem 2017;36:2868-2874. © 2017 SETAC.

Inhibition of immune responses and related proteins in Rhamdia quelen exposed to diclofenac.

Nonsteroidal anti-inflammatory drugs are among the most widely detected pharmaceuticals in surface water worldwide. The nonsteroidal anti-inflammatory drug diclofenac is used to treat many types of pain and inflammation. Diclofenac's potential to cause adverse effects in exposed wildlife is a growing concern. To evaluate the effects of waterborne diclofenac on the immune response in Rhamdia quelen (South American catfish), fish were exposed to 3 concentrations of diclofenac (0.2, 2.0, and 20.0 µg/L) for 14 d. Some of the exposed fish were also given an intraperitoneal injection on day 14 of 1 mg/kg of carrageenan to evaluate cell migration to the peritoneum. Total blood leukocyte count and carrageenan-induced leukocyte migration to the peritoneal cavity, particularly of polymorphonuclear cells, were significantly affected for all diclofenac exposure groups. Nitric oxide production was significantly reduced in the diclofenac-treated fish. Plasma and kidney proteins were analyzed by means of liquid chromatography-tandem mass spectrometry in a shotgun proteomic approach. In both plasma and kidney of diclofenac-exposed R. quelen, the expression of 20 proteins related to the inflammatory process, nitric oxide production, leukocyte migration, and the complement cascade was significantly altered. In addition, class I major histocompatibility complex was significantly decreased in plasma of diclofenac-treated fish. Thus, waterborne exposure to diclofenac could lead to suppression of the innate immune system in R. quelen. Environ Toxicol Chem 2017;36:2092-2107. © 2017 SETAC.

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.

Antioxidant responses in estuarine invertebrates exposed to repeated oil spills: Effects of frequency and dosage in a field manipulative experiment.

We have experimentally investigated the effects of repeated diesel spills on the bivalve Anomalocardia brasiliana, the gastropod Neritina virginea and the polychaete Laeonereis culveri, by monitoring the responses of oxidative stress biomarkers in a subtropical estuary. Three frequencies of exposure events were compared against two dosages of oil in a factorial experiment with asymmetrical controls. Hypotheses were tested to distinguish between (i) the overall effect of oil spills, (ii) the effect of diesel dosage via different exposure regimes, and (iii) the effect of time since last spill. Antioxidant defense responses and oxidative damage in the bivalve A. brasiliana and the polychaete L. culveri were overall significantly affected by frequent oil spills compared to undisturbed controls. The main effects of diesel spills on both species were the induction of SOD and GST activities, a significant increase in LPO levels and a decrease in GSH concentration. N. virginea was particularly tolerant to oil exposure, with the exception of a significant GSH depletion. Overall, enzymatic activities and oxidative damage in A. brasiliana and L. culveri were induced by frequent low-dosage spills compared to infrequent high-dosage spills, although the opposite pattern was observed for N. virginea antioxidant responses. Antioxidant responses in A. brasiliana and L. culveri were not affected by timing of exposure events. However, our results revealed that N. virginea might have a delayed response to acute high-dosage exposure. Experimental in situ simulations of oil exposure events with varying frequencies and intensities provide a useful tool for detecting and quantifying environmental impacts. In general, antioxidant biomarkers were induced by frequent low-dosage exposures compared to infrequent high-dosage ones. The bivalve A. brasiliana and the polychaete L. culveri are more suitable sentinels due to their greater responsiveness to oil and also to their wider geographical distribution.

Effects of trophic exposure to diclofenac and dexamethasone on hematological parameters and immune response in freshwater fish.

The aim of the present study was to evaluate the effects of diclofenac and dexamethasone on hematological parameters and immune response in the fish species Hoplias malabaricus after trophic exposure. Fish were fed twice every week with Astyanax sp., which were given an intraperitoneal inoculation with diclofenac (0 µg/kg, 0.2 µg/kg, 2.0 µg/kg, or 20.0 µg/kg) or dexamethasone (0.03 µg/kg, 0.3 µg/kg, or 3.0 µg/kg). After 12 doses, the hematological parameters and lipopolysaccharide-induced nitric oxide production by head kidney monocytic lineage were evaluated. Exposed fish also received 1 mg/kg of carrageenan intraperitoneal, and cell migration to the peritoneal cavity was evaluated after 4 h. Diclofenac and dexamethasone altered the red blood cell count, as well as hematocrit and hemoglobin levels. The total blood leukocyte count decreased in all groups. A significantly reduced carrageenan-induced leukocyte migration to the peritoneal cavity, particularly of polymorphonuclear cells, was observed at all tested doses, suggesting a possible immunosuppressive effect. The basal nitric oxide synthesis of head kidney cell cultures was reduced at the highest dose of diclofenac and was increased at the highest dose of dexamethasone. The lipopolysaccharide-stimulated nitric oxide production was reduced in all treatments, thus corroborating the immunosuppressive effect. Although some fish responses were variable for different drugs, the results suggested that trophic exposure to diclofenac and dexamethasone can lead to hematological changes and immunotoxic effects, causing negative impacts in aquatic organisms.

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.

Photochemical degradation increases polycyclic aromatic hydrocarbon (PAH) toxicity to the grouper Epinephelus marginatus as assessed by multiple biomarkers.

The effects of halogen-light-irradiated and non-irradiated PAHs on the grouper Epinephelus marginatus were assessed through biomarkers including morphometric parameters, liver histopathology, biliary PAH concentration, genetic alterations, and enzyme activity modulation. E. marginatus juveniles were divided into three groups: control (C), non-irradiated PAHs (PAHs1), and irradiated PAHs (PAHs2). Test groups were exposed for 14 days to a 0.5 ppm PAH solution in the semi-static system. After this period, fish were anesthetized with benzocaine (2%) and peripheric blood was collected by caudal puncture. Blood smears were prepared and stained with propidium iodide. Fish livers were collected, fixed in McDowell's solution, embedded in paraplast, thin-sectioned, and stained with hematoxylin-eosin (H&E). For biochemical analyses including superoxide dismutase, catalase, and glutathione S-transferase activities, fish livers were collected and preserved in liquid nitrogen. Water samples were analyzed using gas chromatography-mass spectrometry (GC-MS) and bile synchronous fluorescence spectroscopy. Fish in the PAHs2 group had micronuclei (MN) in blood cells, as well as significant differences in nuclear morphology (NMA). Significant morphological alterations were observed in the livers from fish exposed to PAHs as well as inhibition of the catalase activity. Our results show that irradiation altered the bioavailability of PAHs, especially benzanthracene, which has great impact in aquatic ecosystems. Among the consequences of physical and chemical changes to PAHs, we observed a significant increase in NMA and MN incidence in E. marginatus erythrocytes, indicating the potential initiation of mutagenic and carcinogenic processes.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil.

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.