Acute exposure of embryo, larvae and adults of Danio rerio to fipronil commercial formulation reveals effects on development and motor control.

The insecticide fipronil, one of the main pesticides used in Brazil, is often detected in natural aquatic environments, and causes neuronal hyperexcitation by inhibiting GABAergic neurotransmission, leading to putative alterations in behaviour and development. This work sought to analyse the toxicity of formulated Regent® 800WG (80% fipronil) on development (fish embryo toxicity test, FET), morphology, and swimming behaviour of larvae and adults of zebrafish (Danio rerio). FET was performed following OECD236 guidelines at concentrations ranging from 0.002 to 1600 µg.L-1 of formulated Regent® 800WG. Adults were exposed to 0.2, 2 and 20 µg.L-1 of the product for 24 and 96 h, and were submitted to the light-dark, novel tank and swimming endurance tests No lethal parameters were observed in larvae, but in concentrations above 400 µg.L-1, there was shortening of the body axis and decreased swimming behavior. In adults, exposure to the pesticide did not lead to changes in free swimming parameters. However, a marked decrease of swimming endurance was observed at all experimental treatments, although probably not in consequence of energetic depletion, since baseline blood glucose levels and condition factor were similar at all conditions. Furthermore, zebrafish adults did not show their natural preference for the dark environment. The pesticide likely has anxiolytic effects on zebrafish, as well as a compromising effect on locomotor control, illustrating that behavioural changes, which could affect activities on the natural environment, such as escape and predation, may occur even in environmentally relevant concentrations of this pollutant.

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.

Bioaccumulation of nickel and its biochemical and genotoxic effects on juveniles of the neotropical fish Prochilodus lineatus.

Juveniles of the freshwater fish Prochilodus lineatus were exposed to three concentrations of nickel (Ni): 25, 250 and 2500 µg L(-1) or water only for periods of 24 and 96 h to test for Ni bioaccumulation, its effects on antioxidant defenses and metallothioneins, and the occurrence of DNA damage. After exposure, the fish were sampled and tissue removed from the gills, liver, kidney and muscle to test for Ni accumulation and conduct biochemical (gills and liver) and genotoxic (blood cells and gills) analyses. The results showed that Ni accumulates in the organs in different proportions (kidney>liver>gills>muscle) and accumulation varied according to exposure time. Metallothionein (MT) levels increased in the liver and gills after exposure to Ni, implying that the presence of Ni in these tissues could induce MT synthesis. We also observed that Ni exposure affected antioxidant defenses, increasing lipid peroxidation in the liver of fish exposed to Ni for 96 h at the highest concentration tested. DNA damage increased in both blood cells and gills of fish exposed to all Ni concentrations, indicating the genotoxic potential of Ni on fish. We therefore concluded that Ni accumulates in various tissues and results in oxidative and DNA damage in P. lineatus, and that the maximum permitted Ni concentration set in Brazilian legislation (25 µg L(-1)) for freshwaters is not safe for this species.

Physiological effects of gasoline on the freshwater fish prochilodus lineatus (characiformes: prochilodontidae)

The purpose of this work was to evaluate the effects of the water-soluble fraction of gasoline (WSFG) on the Neotropical freshwater fish Prochilodus lineatus. The WSFG was prepared by mixing gasoline in water (1:4) and animals were exposed for 6, 24 and 96h to 5% diluted WSFG or only to water. After exposure, blood was collected from the caudal vein and the gills were removed. The following parameters were analyzed: hematological (hemoglobin, hematocrit, number of red blood cells), osmo-ionic (plasma Na+, Cl- and K+ and plasma osmolarity), metabolic (total plasma proteins and glucose), endocrine (cortisol), density and distribution of chloride cells [CC] in the gills (immunohistochemistry), and branchial Na+/K+-ATPase (NKA) activity. Hemolysis was found to occur after 96h exposure to WSFG, as indicated by the decrease in the hematological parameters analyzed, followed by an increase in plasma K+. Secondary stress response was revealed by the occurrence of hyperglycemia in the three periods of exposure, despite the absence of significant increases in the plasma cortisol. The exposure to WSFG also caused an increase in the quantity of CC and in plasma Na+, after 24h, as well as in the enzymatic activity of NKA and plasma osmolarity, after 24h and 96h. These results indicate that fish exposed to the WSFG showed physiological adjusts to maintain their osmotic balance. However, the increase in the quantity of CC in the lamellae may interfere in the gas exchange impairing respiration.

O objetivo deste trabalho foi avaliar os efeitos da fração solúvel da gasolina (FSG) em Prochilodus lineatus. A FSG foi preparada misturando-se a gasolina à água (1:4) e os animais foram expostos por 6, 24 e 96h à FSG diluída 5% ou apenas à água. Após a exposição, o sangue foi coletado pela veia caudal e as brânquias foram retiradas. Os seguintes parâmetros foram analisados: hematológicos (hemoglobina, hematócrito, quantidade de células vermelhas), osmo-iônicos (concentrações plasmáticas de Na+, Cl-, K+ e osmolaridade), metabólicos (concentrações plasmáticas de proteínas totais e glicose), endócrino (cortisol plasmático), densidade e distribuição de células-cloreto [CC] nas brânquias (imunohistoquímica) e a atividade da enzima Na+/K+-ATPase (NKA) branquial. Hemólise foi observada após 96h de exposição à FSG, indicada pela diminuição dos parâmetros hematológicos analisados, seguido pelo aumento do K+ plasmático. Houve resposta secundária de estresse visualizada pela ocorrência da hiperglicemia nos três tempos de exposição, apesar da ausência de diferenças significativas na concentração plasmática do cortisol. A exposição à FSG também provocou aumento na quantidade das CC e na concentração de Na+, após 24h, e na atividade enzimática da NKA e osmolaridade, após 24h e 96h. Esses resultados indicam que peixes expostos à FSG apresentaram ajustes fisiológicos para manter o equilíbrio osmótico. Entretanto, o aumento na quantidade de CC nas lamelas pode ter interferido nas trocas gasosas prejudicando a respiração.

Gasoline effects on biotransformation and antioxidant defenses of the freshwater fish Prochilodus lineatus.

Biochemical biomarkers in the Neotropical freshwater fish Prochilodus lineatus were evaluated following acute exposures to the water-soluble fraction of gasoline (WSFG). Fish were exposed to the WSFG diluted to 5% in water (WSFG group) or only to water (Control group) for 6, 24 and 96 h and the gills and liver were removed for the biochemical analyses. Fish exposed to WSFG for 24 and 96 h showed significant increase in the activity of 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) both in liver and gills, pointing toward phase I and phase II biotransformation of the compounds present in the WSFG. The results also indicated the activation of antioxidant defenses in both the liver and gills after fish exposure to WSFG. The liver showed activation of catalase (CAT) and glutathione peroxidase (GPx) after 96 h exposure. An increase in hepatic content of reduced glutathione (GSH) together with decreased glutathione reductase (GR) activity was observed after 24 and 96 h of exposure to WSFG. In the gills, only catalase (CAT) activity augmented after 6 and 24 h of exposure and GSH content increased after 24 h of WSFG exposure. However, in both the organs, activation of the antioxidant defenses was not enough to prevent oxidative damage since they showed lipid peroxidation (LPO) at one of the experimental times: the liver after 6 h and the gills only after 96 h of exposure to WSFG. This may indicate better adaptation of the liver to longer exposures, starting from 24 h. As the gills are the first organ to be exposed to xenobiotics, the antioxidant defenses were triggered immediately upon exposure to WSFG and were able to prevent the occurrence of LPO during the initial times.