Paulo S.M. Carvalho

Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site

Abstract A comparison between some pro- and antioxidant parameters in erythrocyte, gill, liver and kidney of Nile tilapia, O. niloticus from a fish farm (Control group) and from a polluted site in the Billings Reservoir (Reservoir group) was done. No hematological alterations were observed in blood of Billings fish. The erythrocyte oxidative stress was characterized by increased oxygen uptake and decreased induction time (induced by t-butyl hydroperoxide, t-BHP) evidencing a higher susceptibility to oxidative damage. Moreover, beside the augmented erythrocyte superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase activities, a decrease in both catalase (CAT) activity and total glutathione content (GSH), in erythrocytes of Reservoir fish was observed. The higher cytochrome b5 levels observed in the gill microsomal fraction is likely to be the cause of the enhanced oxyradical production. This fact, associated with the diminished CAT and G6PDH activities, accounts for a gill oxidative stress of Reservoir fish. Except for the NADPH-cytochrome P-450 (c) reductase activity, the other liver pro-oxidant parameters were greatly increased in the Reservoir fish. These results together with the increase in SOD activity and decrease in CAT, glutathione reductase (GR) and G6PDH activities indicated a liver oxidative stress status. The observed increases in kidney NADH cytochrome c reductase and in both cytochromes P-450 and b5 contents were not reflected in enhanced oxyradical production, suggesting a well coupled P-450 catalytic cycle in this tissue. However, the antioxidant enzymes had a similar pattern in liver. The decrease in GSH observed in kidney is probably associated with conjugation reactions for subsequent excretion. All these results furnish useful data for future investigations of polluted aquatic sites in order to provide a data base relating the presence of pollutants to biological effects at a molecular level.

Cholinesterase inhibition and behavioral toxicity of carbofuran on Oreochromis niloticus early life stages

Nile tilapia Oreochromis niloticus at 9 days post-hatch were exposed in semi-static experiments to the carbamate insecticide carbofuran, which is applied in agricultural systems in Brazil. Although the molecular mechanism of carbofuran toxicity is well known, a detailed understanding of the ecological mechanisms through which carbofuran effects can propagate towards higher levels of biological organization in fish is incomplete. Mortality rates were quantified for larvae exposed for 96 h to 8.3, 40.6, 69.9, 140, 297 and 397 μg/L carbofuran, and the LC(50) 96 h was 214.7 μg/L. In addition, the biochemical biomarker cholinesterase inhibition and behavioral biomarkers related to vision, swimming, prey capture and predator avoidance were quantified in individual larvae, as well as their growth in weight. The behavioral parameters were quantified by analysis of digitally recorded videos of individual larvae within appropriate experimental setups. The activity of the enzyme cholinesterase decreased after exposure to carbofuran with a lowest observed effects concentration (LOEC) of 69.9 μg/L. Visual acuity deficits were detected after carbofuran exposure with a LOEC of 40.6 μg/L. Swimming speed decreased with carbofuran exposure, with a LOEC of 397.6 μg/L. The number of attacks to prey (Daphnia magna nauplii) decreased in larvae exposed to carbofuran, with a LOEC of 397.6 μg/L. Growth in weight was significantly reduced in a dose dependent manner, and all carbofuran groups exhibited a statistically significant decrease in growth when compared to controls (p<0.05). The number of predator attacks necessary to capture larvae decreased after exposure to carbofuran, and the LOEC was 69.9 μg/L. These results show that exposure of sensitive early life stages of tilapia O. niloticus to sublethal concentrations of carbofuran can affect fundamental aspects of fish larval ecology that are relevant to recruitment of fish populations, and that can be better understood by the application of behavioral biomarkers.

Thiamine Deficiency Effects on the Vision and Foraging Ability of Lake Trout Fry

The exact causes of the historical recruitment failures of Great Lakes lake trout Salvelinus namaycush are unknown. Thiamine deficiency has been associated with neurological abnormalities in lake trout that lead to early mortality syndrome (EMS) in salmonine swim-up fry, and EMS-related mortality at the swim-up stage is a factor that contributes to the reproductive failure of lake trout populations in the Great Lakes. The potential for adverse effects of thiamine deficiency beyond the swim-up stage is unknown. We investigated the effects of low egg thiamine on behavioral functions in young, post-swim-up lake trout fry. The behavioral endpoints included visual acuity and prey capture rates in the same groups of lake trout fry from each family. Low-thiamine eggs were produced by feeding lake trout broodstock diets entailing thiaminase activity. The thiamine content of the spawned eggs ranged from 0.3 to 26.1 nmol/g. Both visual acuity and prey capture rates were affected by the thiamine content of the eggs. The visual acuity of lake trout was severely affected by low egg thiamine, mainly at thiamine concentrations below the threshold of 0.8 nmol/g but also at higher concentrations in field-collected eggs. Feeding was also reduced with low egg thiamine content. The reduction of prey capture rates was dramatic below 0.8 nmol/g and less dramatic, but still significant, in a portion of the families with egg thiamine concentrations of less than 5.0 nmol/g from both laboratory and field samples. Approximately one-third of the latter families had reduced feeding rates. Deficits in visual acuity may be part of the mechanism leading to decreased feeding rates in these fry. The effects of low egg thiamine on both of the behavioral endpoints studied increase the risk of low recruitment rates in Great Lakes lake trout populations.

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: glutathione reductase as a target.

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.

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.

Effects of naphthalene and phenanthrene on visual and prey capture endpoints during early stages of the dourado Salminus Brasiliensis

Naphthalene (NAP) and phenanthrene (PHE) effects on Salminus brasiliensis, a carnivorous freshwater fish, were investigated using behavioral tests. Larval stages of S. brasiliensis were exposed to water concentrations of 0, 0.04 mg/l, 0.20mg/l and 0.50mg/l for naphthalene and 0, 0.01 mg/l, 0.05 mg/l and 0.1mg/l for phenanthrene during two developmental phases. The prey fish Prochilodus lineatus were not exposed. Visual acuity was measured at the end of phase 2 in individual S. brasiliensis, which were also tested at the end of each phase for number of attacks on prey, number of prey captured, prey capture efficiency, and distance swam. Vision was impaired by PHE exposure, as acuity angles increased in exposed fish. At Stage I 2.3+/-1.2 prey were captured with 46% efficiency in controls compared to 0.4+/-0.3 prey captured with 13.4% efficiency in fish exposed to 0.05 mg PHEl(-1), the LOEL for both endpoints. At Stage II 4.0+/-1.1 preys were captured in controls compared to 2.5+/-0.9 preys captured in fish exposed to 0.01 mg PHEl(-1), the LOEL. Stage II control fish captured prey with 70% efficiency compared to 30% efficiency at Stage II fish exposed to 0.05 mg PHEl(-1), the LOEL. Distance swam was not affected by either NAP or PHE exposure. The exposure of larval stages of S. brasiliensis to realistic water concentrations of PHE impairs foraging skills and could affect recruitment of the species.

Petroleum hydrocarbons in water from a Brazilian tropical estuary facing industrial and port development.

A fast paced industrial and port development has occurred at Suape Estuary, Northeast Brazil, but no information about hydrocarbon concentrations in water is available to this area. Considering that, the contamination level of Suape was determined by UV-Fluorescence in terms of dissolved and/or dispersed petroleum hydrocarbons (DDPHs), during wet and dry seasons. DDPHs ranged between 0.05 and 4.59 μg L(-1) Carmópolis oil equivalents and 0.01-1.39 μg L(-1) chrysene equivalents, indicating DDPHs close to a baseline contamination level. Some relatively high concentrations (>1 μg L(-1)) were probably associated with shipyard operations (hull paintings and ship docking), pollutants remobilization by dredging operations, occasional industrial discharges and oil derivatives released by vessels. DDPHs concentrations were lower in the wet season suggesting that the increased dilution rates caused by rainfall dominated upon the wet deposition of atmospheric combustion-derived PAHs process. Results in this study may be used as baseline to further studies in this area.

Bioconcentration of phenanthrene and metabolites in bile and behavioral alterations in the tropical estuarine guppy Poecilia vivipara

Quantification of polycyclic aromatic hydrocarbon (PAH) metabolites in fish bile is widely used to evaluate levels of internal PAH contamination in fish, whereas behavioral effects are deemed important to address potential risks to fish populations. The estuarine guppy Poecilia vivipara was exposed for 96h to waterborne phenanthrene at concentrations of 10, 50, 200 and 500μgL(-1). Phenanthrene and metabolites in bile were analyzed by fixed fluorescence at 260/380nm (excitation/emission) wavelengths. Phenanthrene increased in the bile of exposed fish in a dose-dependent pattern, and log bile bioconcentration factors ranged from 4.3 to 3.9 at 10 and 500μgL(-1) phenanthrene, respectively, values that are similar to predicted bioconcentration factors based on phenanthrene Kow. Swimming resistance index was reduced to 81% of control values at 500μgL(-1). Alteration of swimming speed was non monotonic, with a significant speed increase relative to control fish in treatments 50 and 200μgL(-1) phenanthrene, respectively, followed by a speed decrease in fish exposed to 500μgL(-1). However, swimming trajectories of fish exposed to 50, 200 and 500μgL(-1) was altered by the development of a repetitive circular swimming behavior, in contrast to the controls that explored the entire experimental arena. This change in swimming patterns apparently explains the reduction in prey capture rates at 200μgL(-1) phenanthrene. This study provides important information enabling the use of the estuarine guppy P. vivipara to monitor PAH metabolites in bile and its bioconcentration, linking internal exposure with ecologically relevant behavioral effects in the species.

Contaminant concentrations, biochemical and hematological biomarkers in blood of West Indian manatees Trichechus manatus from Brazil.

The West Indian manatee Trichechus manatus is threatened with extinction in Brazil, and this study focused on nondestructive blood samples analyzed for metals, polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), as well as biochemical and hematological biomarkers. Studied manatees were kept at Projeto Peixe-Boi headquarters in Pernambuco State, and at two natural areas in estuaries where they are released to the wild. Manatees kept at the natural estuary in Paraiba State have blood concentrations of Al, Pb, Cd, Sn that are 11, 7, 8 and 23 times greater, respectively, than the concentrations found in blood of animals from the same species in Florida, USA. An inhibition of butyrylcholinesterase in manatees kept at the two reintroduction sites in Alagoas and Paraiba States indicated possible exposure of the animals to cholinesterase inhibitor insecticides. PCBs and OCPs were not detected. Results from this study will help delineate conservation efforts in the region.

Stereotypical behaviour in captive West Indian manatee ( Trichechus manatus )

There is great difficulty in maintaining aquatic mammals in captivity, since the attempt to replicate the environment they live in poses an enormous challenge. Poor captivity facilities without environmental enrichment can lead to different consequences for animal health, including the appearance of stereotypical movements. The aim of this study was to identify these behaviours in three groups of animals, one group of manatees inhabiting a reintroduction oceanarium in Pernambuco (PE) state, and two other groups confined in corrals constructed in natural areas (estuaries), one located in Paraiba state and the other in Alagoas state, all in north-eastern Brazil. Observations were conducted using the focal animal sampling method. It was found that the animals inhabiting the reintroduction oceanarium with no environmental enrichment showed stereotypical behaviour such as ‘Back-and-forth’ movements, ‘Hitting head against the limiting structure’ and ‘Hitting the muzzle’, while animals constrained within estuaries did not. The ‘circle swimming’ behaviour was present in a higher percentage of the animals captive in the reintroduction oceanarium, although no significant difference between the sites was found. The number and frequency of occurrence of stereotypical behaviours was significantly higher in animals kept in the reintroduction oceanarium in PE than in those of other locations. Based on these results we recommend the use of appropriate environmental enrichment and the reduction of time manatees stay confined in the reintroduction oceanarium.