The biotic ligand model as a promising tool to predict Cu toxicity in amazon blackwaters.

The Rio Negro basin of Amazonia (Brazil) is a hotspot of fish biodiversity that is under threat from copper (Cu) pollution. The very ion-poor blackwaters have a high dissolved organic carbon (DOC) concentration. We investigated the Cu sensitivity of nine Amazonian fish species in their natural blackwaters (Rio Negro). The acute lethal concentration of Cu (96 h LC50) was determined at different dilutions of Rio Negro water (RNW) in ion-poor well water (IPW), ranging from 0 to 100%. The IPW was similar to RNW in pH and ionic composition but deficient in DOC, allowing this parameter to vary 20-fold from 0.4 to 8.3 mg/L in tests. The Biotic Ligand Model (BLM; Windward version 3.41.2.45) was used to model Cu speciation and toxicity over the range of tested water compositions, and to estimate lethal Cu accumulations on the gills (LA50). The modeling predicted a high relative abundance of Cu complexes with DOC in test waters. As these complexes became more abundant with increasing RNW content, a concomitant decrease in free Cu2+ was observed. In agreement with this modeling, acute Cu toxicity decreased (i.e. 96 h LC50 values increase) with increasing RNW content. The three most sensitive species (Hemigrammus rhodostomus, Carnegiella strigatta and Hyphessobrycon socolofi) were Characiformes, whereas Corydoras schwartzi (Siluriformes) and Apistogramma agassizii (Cichliformes) were the most tolerant. These sensitivity differences were reflected in the BLM-predicted lethal gill copper accumulation (LA50), which were generally lower in Characiformes than in Cichliformes. Using these newly estimated LA50 values in the BLM allowed for accurate prediction of acute Cu toxicity in the nine Amazonian fish. Our data emphasize that the BLM approach is a promising tool for assessing Cu risk to Amazonian fish species in blackwater conditions characterized by very low concentrations of major ions but high concentrations of DOC.

Investigating the mechanisms of dissolved organic matter protection against copper toxicity in fish of Amazon's black waters.

We investigated how natural dissolved organic matter (DOM) of the Rio Negro (Amazon) affects acute copper (Cu) toxicity to local fish: the cardinal tetra (Paracheirodon axelrodi) and the dwarf cichlid (Apistogramma agassizii). It is established that Cu2+ complexation with DOM decreases Cu bioavailability (and thus toxicity) to aquatic organisms, as conceptualized by the Biotic Ligand Model (BLM). However, we also know that Rio Negro's DOM can interact with fish gills and have a beneficial effect on Na+ homeostasis, the main target of acute Cu toxicity in freshwater animals. We aimed to tease apart these potential protective effects of DOM against Cu-induced Na+ imbalances in fish. In the laboratory, we acclimated fish to Rio Negro water (10 mg L-1 DOC) and to a low-DOM water (1.4 mg L-1 DOC) with similar ion composition and pH (5.9). We measured 3-h Cu uptake in gills and unidirectional and net Na+ physiological fluxes across a range of Cu concentrations in both waters. Various DOM pre-acclimation times (0, 1 and 5 days) were evaluated in experiments with P. axelrodi. Copper exposure led to similar levels of net Na+ loss in the two fish, but with distinct effects on Na+ influx and efflux rates reflecting their different ionoregulation strategies. Rio Negro DOM protected against Cu uptake and toxicity in the two fish species. Both Cu uptake in fish gills and Na+ regulation disturbances were relatively well predicted by the modelled aqueous free Cu2+ ion concentration. These findings suggest that protection by DOM occurs mainly from Cu complexation under the tested conditions. The prevalence of this geochemical-type protection over a physiological-type protection agrees with the BLM conceptual framework, supporting the use of the BLM to assess the risk of Cu in these Amazonian waters.

The effects of dissolved organic carbon on the reflex ventilatory responses of the neotropical teleost (Colossoma macropomum) to hypoxia or hypercapnia.

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O2 and/or CO2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l-1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l-1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro.

Boron Oxide Nanoparticles Exhibit Minor, Species-Specific Acute Toxicity to North-Temperate and Amazonian Freshwater Fishes.

Boron oxide nanoparticles (nB2O3) are manufactured for structural, propellant, and clinical applications and also form spontaneously through the degradation of bulk boron compounds. Bulk boron is not toxic to vertebrates but the distinctive properties of its nanostructured equivalent may alter its biocompatibility. Few studies have addressed this possibility, thus our goal was to gain an initial understanding of the potential acute toxicity of nB2O3 to freshwater fish and we used a variety of model systems to achieve this. Bioactivity was investigated in rainbow trout (Oncorhynchus mykiss) hepatocytes and at the whole animal level in three other North and South American fish species using indicators of aerobic metabolism, behavior, oxidative stress, neurotoxicity, and ionoregulation. nB2O3 reduced O. mykiss hepatocyte oxygen consumption (MO2) by 35% at high doses but whole animal MO2 was not affected in any species. Spontaneous activity was assessed using MO2 frequency distribution plots from live fish. nB2O3 increased the frequency of high MO2 events in the Amazonian fish Paracheirodon axelrodi, suggesting exposure enhanced spontaneous aerobic activity. MO2 frequency distributions were not affected in the other species examined. Liver lactate accumulation and significant changes in cardiac acetylcholinesterase and gill Na+/K+-ATPase activity were noted in the north-temperate Fundulus diaphanus exposed to nB2O3, but not in the Amazonian Apistogramma agassizii or P. axelrodi. nB2O3 did not induce oxidative stress in any of the species studied. Overall, nB2O3 exhibited modest, species-specific bioactivity but only at doses exceeding predicted environmental relevance. Chronic, low dose exposure studies are required for confirmation, but our data suggest that, like bulk boron, nB2O3 is relatively non-toxic to aquatic vertebrates and thus represents a promising formulation for further development.

Does dissolved organic carbon from Amazon black water (Brazil) help a native species, the tambaqui Colossoma macropomum to maintain ionic homeostasis in acidic water?

To assess how the quality and properties of the natural dissolved organic carbon (DOC) could drive different effects on gill physiology, we analysed the ionoregulatory responses of a native Amazonian fish species, the tambaqui Colossoma macropomum, to the presence of dissolved organic carbon (DOC; 10 mg l-1 ) at both pH 7.0 and pH 4.0 in ion-poor water. The DOC was isolated from black water from São Gabriel da Cachoeira (SGC) in the upper Rio Negro of the Amazon (Brazil) that earlier been shown to protect a non-native species, zebrafish Danio rerio against low pH under similar conditions. Transepithelial potential (TEP), net flux rates of Na+ , Cl- and ammonia and their concentrations in plasma and Na+ , K+ ATPase; v-type H+ ATPase and carbonic anhydrase activities in gills were measured. The presence of DOC had negligible effects at pH 7.0 apart from lowering the TEP, but it prevented the depolarization of TEP that occurred at pH 4.0 in the absence of DOC. However, contrary to our initial hypothesis, SGC DOC was not protective against the effects of low pH. Colossoma macropomum exposed to SGC DOC at pH 4.0 experienced greater net Na+ and Cl- losses, decreases of Na+ and Cl- concentrations in plasma and elevated plasma ammonia levels and excretion rates, relative to those exposed in the absence of DOC. Species-specific differences and changes in DOC properties during storage are discussed as possible factors influencing the effectiveness of SGC DOC in ameliorating the effects of the acid exposure.

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 (J outNa ), and prevented the blockade of Na+ uptake (J inNa ) seen in zebrafish exposed to pH 4.0 alone, resulting in much smaller disturbances in Na+ net losses (J netNa ). DOC also attenuated the stimulation of net Cl- losses (J netCl ) and ammonia excretion (J netAmm ) during acidic challenge. In the second experiment, zebrafish acclimated to DOC displayed similar regulation of J inNa and J outNa , and, therefore, reduced J netNa at pH 4.0, effects which persisted even when DOC was no longer present. Protective effects of prior acclimation to DOC on J netCl and J netAmm 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.

Physiological impacts and bioaccumulation of dietary Cu and Cd in a model teleost: The Amazonian tambaqui (Colossoma macropomum).

Increasing anthropogenic activities in the Amazon have led to elevated metals in the aquatic environment. Since fish are the main source of animal protein for the Amazonian population, understanding metal bioaccumulation patterns and physiological impacts is of critical importance. Juvenile tambaqui, a local model species, were exposed to chronic dietary Cu (essential, 500 µg Cu/g food) and Cd (non-essential, 500 µg Cd/g food). Fish were sampled at 10-14, 18-20 and 33-36 days of exposure and the following parameters were analyzed: growth, voluntary food consumption, conversion efficiency, tissue-specific metal bioaccumulation, ammonia and urea-N excretion, O2 consumption, Pcrit, hypoxia tolerance, nitrogen quotient, major blood plasma ions and metabolites, gill and gut enzyme activities, and in vitro gut fluid transport. The results indicate no ionoregulatory impacts of either of the metal-contaminated diets at gill, gut, or plasma levels, and no differences in plasma cortisol or lactate. The Cd diet appeared to have suppressed feeding, though overall tank growth was not affected. Bioaccumulation of both metals was observed. Distinct tissue-specific and time-specific patterns were seen. Metal burdens in the edible white muscle remained low. Overall, physiological impacts of the Cu diet were minimal. However dietary Cd increased hypoxia tolerance, as evidenced by decreased Pcrit, increased time to loss of equilibrium, a lack of plasma glucose elevation, decreased plasma ethanol, and decreased NQ during hypoxia. Blood O2 transport characteristics (P50, Bohr coefficient, hemoglobin, hematocrit) were unaffected, suggesting that tissue level changes in metabolism accounted for the greater hypoxia tolerance in tambaqui fed with a Cd-contaminated diet.

High waterborne Mg does not attenuate the toxic effects of Fe, Mn, and Ba on Na+ regulation of Amazonian armored catfish tamoatá (Hoplosternum litoralle).

Formation water (FoW) is a by-product from oil and gas production and usually has high concentrations of soluble salts and metals. Calcium (Ca) and magnesium (Mg) have been shown to reduce the toxicity of metals to aquatic animals, and previous study showed that high waterborne Ca exerts mild effect against disturbances on Na+ regulation in Amazonian armored catfish tamoatá (Hoplosternum littorale) acutely exposed to high Fe, Mn, and Ba levels. Here, we hypothesized that high Mg levels might also reduce the toxic effects of these metals on Na+ regulation of tamoatá. The exposure to 5% FoW promoted an increase in Na+ uptake and a rapid accumulation of Na+ in all tissues analyzed (kidney

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 (MO2), 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 MO2 was noted in cardinal tetra exposed to Cu, but not nCuO, whereas MO2 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.

Nitrogen metabolism in tambaqui (Colossoma macropomum), a neotropical model teleost: hypoxia, temperature, exercise, feeding, fasting, and high environmental ammonia.

The total rate of N-waste excretion (M N) in juvenile tambaqui living in ion-poor Amazonian water comprised 85 % ammonia-N (M Amm-N) and 15 % urea-N (M Urea-N). Both occurred mainly across the gills with only ~5 % of M Amm-N and ~39 % of M Urea-N via the urine. Tambaqui were not especially tolerant to high environmental ammonia (HEA), despite their great resistance to other environmental factors. Nevertheless, they were able to maintain a continued elevation of M Amm-N during and after 48-h exposure to 2.5 mmol L-1 HEA. The normally negative transepithelial potential (-18 mV) increased to -9 mV during the HEA period, which would help to reduce branchial NH4+ entry. During 3 h of acute environmental hypoxia (30 % saturation), M Amm-N declined, and recovered thereafter, similar to the response seen in other hypoxia-tolerant teleosts; M Urea-N did not change. However, during gradual hypoxia, M Amm-N remained constant, but M Urea-N eventually fell. The acute temperature sensitivities of M Amm-N and M N were low from 28 °C (acclimation) to 33 °C (Q10 ~1.5), but high (~3.8) from 33 to 38 °C, relative to [Formula: see text] (~1.9 throughout). In contrast, M Urea-N exhibited a different pattern over these temperature ranges (Q10 2.6 and 2.1, respectively). The nitrogen quotient (NQ = 0.16-0.23) was high at all temperatures, indicating a 60-85 % reliance on protein to fuel aerobic metabolism in these fasting animals. During steady-state aerobic exercise, [Formula: see text] and M Urea-N increased in parallel with velocity (up to 3.45 body lengths s-1), but M Amm (and thus M N) remained approximately constant. Therefore, the NQ fell progressively, indicating a decreasing reliance on protein-based fuels, as work load increased. In group feeding trials using 45 % protein commercial pellets, tambaqui excreted 82 % (range 39-170 %) of the dietary N within 24 h; N-retention efficiency was inversely related to the ration voluntarily consumed. M Amm-N peaked at 4-6 h, and M Urea-N at 6-9-h post-feeding, with an additional peak in M Amm-N only at 21 h. During subsequent fasting, M N stabilized at a high endogenous rate from 2 through 8 days post-feeding. Possible reasons for the high wasting of protein-N during both fasting and feeding are discussed.

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.

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.

Effect of low pH exposure on Na(+) regulation in two cichlid fish species of the Amazon.

We evaluated the effects of acute exposure to low pH on Na(+) regulation in two Amazon cichlids collected from natural ion-poor "blackwaters", angelfish (Pterophyllum scalare) and discus (Symphysodon discus). Na(+) uptake kinetic parameters, unidirectional Na(+) fluxes, and net Cl(-) fluxes were determined at pH6.0 and 3.6. At pH6.0, both species presented low unidirectional Na(+) flux rates, with kinetics showing a relatively low affinity for Na(+) (angelfish Km=79, discus Km=268µmolL(-1)), with similar maximum transport capacities (Jmax~535nmolg(-1)h(-1)). Overall, there appeared to be high sensitivity to inhibition by low pH, yet low intrinsic branchial permeability limiting diffusive ion effluxes, resulting in relatively low net loss rates of Na(+), the same strategy as seen previously in other blackwater cichlids, and very different from the strategy of blackwater characids. At low pH, Na(+) uptake in angelfish was inhibited competitively (increased Km=166µmolL(-1)) and non-competitively (decreased Jmax=106nmolg(-1)h(-1)), whereas in discus, only a decrease in Jmax (112nmolg(-1)h(-1)) was statistically significant. An acute reduction in H(+)-ATPase activity, but not in Na(+)/K(+)-ATPase activity, in the gills of angelfish suggests a possible mechanism for this non-competitive inhibition at low pH. Discus fish were more tolerant to low pH than angelfish, as seen by lesser effects of exposure to pH3.6 on unidirectional Na(+) uptake and efflux rates and net Na(+) and Cl(-) loss rates. Overall, discus are better than angelfish in maintaining ionic balance under acidic, ion-poor conditions.

Sodium fluxes in tamoatá, Hoplosternum litoralle, exposed to formation water from Urucu Reserve (Amazon, Brazil).

Formation water (produce water or oil field brine) from oil and gas production usually has high concentrations of soluble salts and metals. The objective of this study was to examine the effect of formation water from Urucu Reserve, Amazon, on whole-body uptake and internal distribution of newly accumulated Na+ in juvenile tamoatá, Hoplosternum litoralle. Groups of fish were submitted to nine treatments for 3 h in 400-ml chambers: control (well water), 5% formation water, and well water with respective concentrations of 5% formation water of Ca2+, Fe, Mn, Ba2+, Fe+Ca2+, Mn+Ca2+, and Ba+Ca2+ added. Specimens of tamoatá exposed to 5% formation water presented a very high Na+ influx, probably due to the high Na+ levels in this water. Waterborne Fe and Mn stimulated Na+ influx, but Fe increased Na+ efflux, causing Na+ loss. Waterborne Mn, on the other hand, decreased Na+ efflux, reducing Na+ loss by this species. Waterborne Ca2+ also affected Na+ influx but had no significant effect on net Na+ fluxes. These results demonstrated that spilling of formation water in ion-poor Amazon rivers would dramatically disrupt osmoregulatory balance of tamoatá and probably other Amazon fish species, impairing their survival and reduce biodiversity.