Rafael M. Duarte

Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus): new angles to the osmorespiratory compromise

SUMMARY Earlier studies demonstrated that oscars, endemic to ion-poor Amazonian waters, are extremely hypoxia tolerant, and exhibit a marked reduction in active unidirectional Na+ uptake rate (measured directly) but unchanged net Na+ balance during acute exposure to low PO2, indicating a comparable reduction in whole body Na+ efflux rate. However, branchial O2 transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O2 permeability. Direct measurements of 22Na appearance in the water from bladder-catheterized fish confirmed a rapid 55% fall in unidirectional Na+ efflux rate across the gills upon acute exposure to hypoxia (PO2=10–20 torr; 1 torr=133.3 Pa), which was quickly reversed upon return to normoxia. An exchange diffusion mechanism for Na+ is not present, so the reduction in efflux was not directly linked to the reduction in Na+ influx. A quickly developing bradycardia occurred during hypoxia. Transepithelial potential, which was sensitive to water [Ca2+], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K+ loss rates, and 3H2O exchange rates (diffusive water efflux rates) across the gills fell by 55–75% during hypoxia, with recovery during normoxia. Osmotic permeability to water also declined, but the fall (30%) was less than that in diffusive water permeability (70%). In total, these observations indicate a reduction in gill transcellular permeability during hypoxia, a conclusion supported by unchanged branchial efflux rates of the paracellular marker [3H]PEG-4000 during hypoxia and normoxic recovery. At the kidney, glomerular filtration rate, urine flow rate, and tubular Na+ reabsorption rate fell in parallel by 70% during hypoxia, facilitating additional reductions in costs and in urinary Na+, K+ and ammonia excretion rates. Scanning electron microscopy of the gill epithelium revealed no remodelling at a macro-level, but pronounced changes in surface morphology. Under normoxia, mitochondria-rich cells were exposed only through small apical crypts, and these decreased in number by 47% and in individual area by 65% during 3 h hypoxia. We suggest that a rapid closure of transcellular channels, perhaps effected by pavement cell coverage of the crypts, allows conservation of ions and reduction of ionoregulatory costs without compromise of O2 exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

Acute effects of chemically dispersed crude oil on gill ion regulation, plasma ion levels and haematological parameters in tambaqui (Colossoma macropomum).

The main goal of this study was to investigate the toxicological effects of the chemical dispersant Corexit 9500, crude oil and the combination of the two components in the form of chemically dispersed crude oil (CO+DIS) on the ion regulation of the tropical fish tambaqui (Colossoma macropomum). Gill ion regulation was evaluated on the basis of unidirectional flux measurements (influx-J(in), efflux-J(out) and net flux-J(net)) of Na(+), Cl(-) and K(+). Plasma ion composition, haematocrit, haemoglobin and glucose concentrations in the blood of tambaqui were determined by classical methods. The exposure of fish to chemically dispersed crude oil promoted a significant increase in J(out) Na(+) across the gills, which, together with the inability of fish to stimulate Na(+) uptake to compensate for these losses resulted in significantly higher J(net) Na(+) outward, particularly within the first 3h of exposure. Increased outward J(net) Cl(-) was also seen in fish that were exposed to dispersed crude oil, whereas outward J(net) K(+) was only increased at crude oil dispersed in higher concentration of Corexit 9500. Plasma Na(+) and Cl(-) concentrations decreased between 6 and 12h of exposure, whereas Ca(2+) concentrations remained significantly lower than those of the control group over the entire experimental period. There were significant increases in plasma K(+) concentrations and in the haematocrit after 6 and 24h of exposure to dispersed crude oil, suggesting significant changes in the permeability of the erythrocytic membrane. Collectively, our results suggest that chemically dispersed crude oil promotes a more extensive impairment of gill ion regulation, in addition to changes in plasma ion levels and blood parameters, in tambaqui compared with exposure to Urucu crude oil or Corexit 9500 alone. Thus, in the event of an oil spill in Amazonian waters, the chemical dispersion of Urucu crude oil could represent a great risk to tambaqui, challenging their ability to maintain ionic and osmotic gradients in native ion-poor waters.

Roundup® exposure promotes gills and liver impairments, DNA damage and inhibition of brain cholinergic activity in the Amazon teleost fish Colossoma macropomum

Roundup Original® (RD) is a glyphosate-based herbicide used to control weeds in agriculture. Contamination of Amazon waters has increased as a consequence of anthropogenic pressure, including the use of herbicides as RD. The central goal of this study was to evaluate the toxic effects of RD on juveniles of tambaqui (Colossoma macropomum). Our findings show that biomarkers in tambaqui are organ specific and dependent on RD concentration. Alterations in gills structural and respiratory epithelium were followed by changes in hematological parameters such as concentration of hemoglobin, particularly in fish exposed to the higher concentration tested (75% of RD LC50 96 h). In addition, both RD concentrations affected the biotransformation process in gills of tambaqui negatively. Instead, liver responses suggest that a production of reactive oxygen species (ROS) occurred in fish exposed to RD, particularly in the animals exposed to 75% RD, as seen by imbalances in biotransformation and antioxidant systems. The increased DNA damage observed in red blood cells of tambaqui exposed to RD is in agreement with this hypothesis. Finally, both tested sub-lethal concentrations of RD markedly inhibited the cholinesterase activity in fish brain. Thus, we can suggest that RD is potentially toxic to tambaqui and possibly to other tropical fish species.

Dissolved organic carbon from the upper Rio Negro protects zebrafish ( Danio rerio ) against ionoregulatory disturbances caused by low pH exposure

The so-called “blackwaters” of the Amazonian Rio Negro are rich in highly coloured dissolved organic carbon (DOC), but ion-poor and very acidic, conditions that would cause fatal ionoregulatory failure in most fish. However these blackwaters support 8% of the world’s ichthyofauna. We tested the hypothesis that native DOC provides protection against ionoregulatory dysfunction in this extreme environment. DOCs were isolated by reverse-osmosis from two Rio Negro sites. Physico-chemical characterization clearly indicated a terrigenous origin, with a high proportion of hydroxyl and phenolic sites, high chemical reactivity to protons, and unusual proteinaceous fluorescence. When tested using zebrafish (a model organism), Rio Negro DOC provided almost perfect protection against ionoregulatory disturbances associated with acute exposure to pH 4.0 in ion-poor water. DOC reduced diffusive losses of Na+ and Cl−, and promoted a remarkable stimulation of Na+ uptake that otherwise would have been completely inhibited. Additionally, prior acclimation to DOC at neutral pH reduced rates of branchial Na+ turnover, and provided similar protection against acid-induced ionoregulatory disturbances, even if the DOC was no longer present. These results reinforce the important roles that DOC molecules can play in the regulation of gill functions in freshwater fish, particularly in ion-poor, acidic blackwaters.

Copper sensitivity of wild ornamental fish of the Amazon.

The copper sensitivity of 10 wild ornamental fish from Rio Negro, an ion-poor river in the Amazon, was analyzed in the absence of dissolved organic carbon (DOC). No mortality was observed at concentrations up to 700mug Cul(-1) for two species exposed to CuCl(2).2H(2)O dissolved in standard EPA-USA water. Copper sensitivity of fish was also tested in INPA groundwater, which is similar to that of the Rio Negro, but lacking typical DOC of Rio Negro water. In these experiments, all four characid fish species were more sensitive to copper than the catfish and the cichlids. LC(50) values ranged from 12.8mug Cul(-1) for the characid Hemigrammus rhodostomus to 74.1mug Cul(-1) for the callichtyid Dianema urostriatum. Copper sensitivity of Hyphessobrycon socolofi was 52-fold lower in EPA (1405.5mug Cul(-1)) than in INPA water (26.8mugCul(-1)). The differences in values of LC(50) may be related to species-specific physiological abilities to ion-regulate under the original conditions of the habitat of the analyzed species.

Recovery of fat snook, Centropomus parallelus (Teleostei: Perciformes) after subchronic exposure to copper

We studied the recovery of juvenile fat snook (Centropomus parallelus) after subchronic exposure to different concentrations of copper. Healthy juveniles (1.98 g) were exposed to 25 or 50 μg Cu/L for 30 days (12 replicates with 5 fish in each one), and recovery was observed at 0, 4, 10, and 30 days after exposure (3 replicates with 5 fish in each one). Copper genotoxicity in exposed individuals was observed using a micronucleus assay, and recovery was not observed even 30 days post-exposure. Copper accumulation was observed in fish exposed to 25 or 50 μg/L of copper in the gills (14.4 and 34.4 μg/g, respectively) and muscle (5.7 and 5.5 μg/g, respectively), and a return to normal copper levels (6.0 μg/g for gills and 2.5 μg/g for muscle) was observed 4 and 30 days post-exposure in the gills and muscle tissues, respectively. Glutathione S-transferase (GST) was 80% inhibited in individuals exposed to copper and returned to normal levels for fish exposed to basal concentrations within 10 days. Although copper accumulation in tissues dispersed 30 days post-exposure, no recovery from genotoxicity was observed during this time. Thirty days was not enough to recover juvenile fat snook following subchronic exposure to copper.

Influence of the natural Rio Negro water on the toxicological effects of a crude oil and its chemical dispersion to the Amazonian fish Colossoma macropomum.

The increment in crude oil exploitation over the last decades has considerably increased the risk of polycyclic aromatic hydrocarbon (PAH) contamination to Amazonian aquatic environments, especially for the black water environments such as the Rio Negro. The present work was designed to evaluate the acute toxicity of the Urucu crude oil (CO), the chemically dispersed Urucu crude oil (CO + D), and the dispersant alone (D) to the Amazonian fish Colossoma macropomum. Acute toxicity tests were performed, using a more realistic approach, where fish were acclimated to both groundwater (GW), used as internal control, and natural Rio Negro water (RNW) and exposed to CO, CO + D and D. Then, biomarkers such as ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), lipid peroxidation (LPO), serum sorbitol dehydrogenase (s-SDH) in liver, DNA damage in blood cells, and the presence of the benzo[a]pyrene-type, pyrene-type, and naphthalene-type metabolites in fish bile were assessed. Fish exposed to CO and CO + D, at both water types tested, presented increased biomarker responses and higher PAH-type metabolites in the bile. However, fish exposed to these treatments after the acclimation to RNW increased the levels of LPO, s-SDH (hepatotoxicity), DNA damage in blood cells (genotoxicity), and benzo[a]pyrene-type metabolites when compared to fish in GW. Our data suggests that some physicochemical properties of Rio Negro water (i.e., presence of natural organic matter (NOM)) might cause mild chemical stress responses in fish, which can make it more susceptible to oxidative stress following exposure to crude oil, particularly to those chemically dispersed.

Biomarker responses and PAH ratios in fish inhabiting an estuarine urban waterway

Many cities worldwide are established adjacent to estuaries and their catchments resulting in estuarine contamination due to intense anthropogenic activities. The aim of this study was to evaluate if fish living in an estuarine urban waterway were affected by contamination, via the measurement of a suite of biomarkers of fish health. Black bream (Acanthopagrus butcheri) were sampled in a small urban embayment and a suite of biomarkers of fish health measured. These were condition factor (CF), liver somatic index (LSI), gonadosomatic index (GSI), hepatic EROD activity, polycyclic aromatic hydrocarbon (PAH) biliary metabolites, serum sorbitol dehydrogenase (s‐SDH) and branchial enzymes cytochrome C oxidase (CCO), and lactate dehydrogenase (LDH) activities. The biomarkers of exposure EROD activity, and pyrene‐ and B(a)P‐type biliary metabolites confirmed current or recent exposure of the fish and that fish were metabolizing contaminants. Relative to a reference site, LSI was higher in fish collected in the urban inlet as was the metabolic enzyme LDH activity. CF, GSI, s‐SDH, CCO, and naphthalene‐type metabolites were at similar levels in the urban inlet relative to the reference site. PAH biliary metabolite ratios of high‐molecular‐weight to low‐molecular‐weight suggest that fish from the urban inlet were exposed to pyrogenic PAHs, likely from legacy contamination and road runoff entering the embayment. Similarly, the sediment PAH ratios and the freshness indices suggested legacy contamination of a pyrogenic source, likely originating from the adjacent historic gasworks site and a degree of contamination of petrogenic nature entering the inlet via storm water discharge. Biomarkers of exposure and effect confirmed that black bream collected in the Claisebrook Cove inlet, Western Australia, are currently exposed to contamination and are experiencing metabolic perturbations not observed in fish collected at a nearby reference site.

Mechanisms of toxic action of copper and copper nanoparticles in two Amazon fish species: Dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi)

Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints and are released into the environment, potentially inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper (Cu) on two ornamental Amazon fish species: dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi). Fish were exposed to 50% of the LC50 for nCuO (dwarf cichlid 58.31μgL-1 and cardinal tetra 69.6μgL-1) and Cu (dwarf cichlid 20μgL-1 and cardinal tetra 22.9μgL-1) for 24, 48, 72 and 96h. Following exposure, aerobic metabolic rate (ṀO2), gill osmoregulatory physiology and mitochondrial function, oxidative stress markers, and morphological damage were evaluated. Our results revealed species specificity in metabolic stress responses. An increase of ṀO2 was noted in cardinal tetra exposed to Cu, but not nCuO, whereas ṀO2 in dwarf cichlid showed little change with either treatment. In contrast, mitochondria from dwarf cichlid exhibited increased proton leak and a resulting decrease in respiratory control ratios in response to nCuO and Cu exposure. This uncoupling was directly related to an increase in reactive oxygen species (ROS) levels. Our findings reveal different metabolic responses between these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differential osmoregulatory strategies among species.

Physiological protective action of dissolved organic carbon on ion regulation and nitrogenous waste excretion of zebrafish (Danio rerio) exposed to low pH in ion-poor water

Dissolved organic carbon (DOC) represents a heterogeneous group of naturally-occurring molecules in aquatic environments, and recent studies have evidenced that optically dark DOCs can exert some positive effects on ionoregulatory homeostasis of aquatic organisms in acidic waters. We investigated the effects of Luther Marsh DOC, a dark allochthonous DOC, on ion regulation and N-waste excretion of zebrafish acutely exposed to either neutral or low pH in ion-poor water. In the first experiment, simultaneous exposure to pH 4.0 and DOC greatly attenuated the stimulation of Na+ diffusive losses (JoutNa), and prevented the blockade of Na+ uptake (JinNa) seen in zebrafish exposed to pH 4.0 alone, resulting in much smaller disturbances in Na+ net losses (JnetNa). DOC also attenuated the stimulation of net Cl− losses (JnetCl) and ammonia excretion (JnetAmm) during acidic challenge. In the second experiment, zebrafish acclimated to DOC displayed similar regulation of JinNa and JoutNa, and, therefore, reduced JnetNa at pH 4.0, effects which persisted even when DOC was no longer present. Protective effects of prior acclimation to DOC on JnetCl and JnetAmm at pH 4.0 also occurred, but were less marked than those on Na+ balance. Urea fluxes were unaffected by the experimental treatments. Overall, these effects were clearly beneficial to the ionoregulatory homeostasis of zebrafish at low pH, and were quite similar to those seen in a recent parallel study using darker DOC from the upper Rio Negro. This suggests that dark allochthonous DOCs share some chemical properties that render fish tolerant to ionoregulatory disturbances during acidic challenge.