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.

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.

Light energy and oxygen drive photo-oxidation of dissolved organic carbon (DOC). The longer the wavelength the greater its depth of penetration into water, changing the spectral environment with depth. We asked how oxygen concentration and light spectral composition might affect photo-oxidation processes in DOC. Outdoor experiments compared responses of fluorescence and absorbance indices to photo-oxidation of filtered (0.45 µm) Rio Negro water (Amazon Basin) under near-anoxia, normoxia and hyperoxia exposed to natural sunlight or reduced sunlight (≥340, reduced-UVR). Near-anoxia decreased all absorbance and fluorescence indices. Absorbance changed across the spectrum (≥250 nm) even under reduced-UVR provided that oxygen was present. This phenomenon maintains broader photo-oxidation and the release of CO2 at depth. Slope350-400 was responsive to changes in the irradiance field but not to oxygen concentration, while Slope275-295 responded to both. Thus, larger molecules are broken down near the water\'s surface and medium to smaller molecules continue to be processed at depth. The production of fulvic acid-like fluorescence required both UVB and oxygen, restricting its production to surface waters. The relatively small increase in R254/365 compared with the loss of SUVA254 under near-anoxia indicated a slower breakdown of larger DOC molecules as oxygen becomes limiting. Breakdown of larger molecules which absorb in the 350-400 nm range, appears to involve two steps - one by radiant energy and another involving oxygen. The study results reflect the dynamic gradients in photo-oxidation with depth.

Rare earth elements (REEs) are generally defined as a homogenous group of elements with similar physical-chemical properties, encompassing Y and Sc and the lanthanides elements series. Natural REEs contents in soils depend on the parent material, the soil genesis processes and can be gradually added to the soil by anthropogenic activities. The REEs have been considered emerging pollutants in several countries, so the establishment of regulatory guidelines is necessary to avoid environmental contamination. In Brazil, REE soils data are restricted to some regions, and knowledge about them in the Amazon soils is scarce, although this biome covers more than 40% of the Brazilian territory. Thus, the objectives of this study were to determine the REE content in soils of two hydrographic basins (Solimões and Rio Negro) of the Amazon biome, establish their Quality Reference Values (QRV) and to investigate the existence of enrichment of REEs in urban soils. The ΣREE(Y + Sc) content of Solimões surface samples was 109.28 mg kg-1 and the ΣREE(Y + Sc) content in the subsurface samples was 94.11 mg kg-1. In soils of Rio Negro basin, the ΣREE(Y + Sc) was 43.95 15 mg kg-1 surface samples and 38.40 mg kg-1 in subsurface samples. The ΣREE(Y + Sc) in urban topsoils samples was 38.62 mg kg-1. The REEs contents pattern in three studied areas are influenced in different amplitude by natural soil properties. The REEs content in urban topsoils were slightly higher than the Rio Negro pristine soils, but the ecological risk was low. QRVs recommend for Solimões soils ranged from 0.01 (Lu) to 145.6 mg kg-1 (Ce) and for Rio Negro soils ranged from 0.05 (Lu) to 15.8 mg kg-1 (Ce).

The aim of the present study was to characterize the effects of particles on metal aquatic toxicity in a tropical system. To this end, we investigated the effects of natural suspended solids on copper (Cu) geochemistry and acute toxicity to the cardinal tetra (Paracheirodon axelrodi), in 2 Amazonian rivers with different total suspended solids (TSS) levels: the Rio Negro (low TSS ~ 8 mg L-1 ) and the Rio Solimões (high TSS ~ 70 mg L-1 ). The effect of particles on Cu aqueous geochemistry was assessed by measuring total, dissolved, and free ionic Cu concentrations in filtered (<0.45 μm) and unfiltered waters. Furthermore, acute Cu toxicity to fish was assessed in both filtered and unfiltered waters, by measuring physiological net fluxes of Na+ , Cl- , K+ , and total ammonia (in both river waters) and 96-h fish mortality (in Rio Solimões only). The particles in the Rio Negro were not abundant enough to play a significant role in our study. On the other hand, the Rio Solimões particles bound approximately 70% of total aqueous Cu in our tests. In agreement with bioavailability-based models, this decrease in dissolved (and free ionic) Cu concentration decreased Cu lethality in the 96-h toxicity tests. In the physiological measurements, the best evidence of particle protection was the total alleviation of Cu-induced Cl- losses. These flux tests also suggested that the particles themselves may negatively affect Na+ balance in the fish. Overall, the present study supports the use of bioavailability concepts to account for the role of natural suspended solids on metal biological effects in the Amazon River basin. Environ Toxicol Chem 2019;38:2708-2718. © 2019 SETAC.

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.

The Amazon basin includes 1000s of bodies of water, that are sorted according to their color in three types: blackwater, clearwater, and whitewater, which significantly differ in terms of their physicochemical parameters. More than 3,000 species of fish live in the rivers of the Amazon, among them, the sardine, Triportheus albus, which is one of the few species that inhabit all three types of water. The purpose of our study was to analyze if the gene expression of T. albus is determined by the different types of water, that is, if the species presents phenotypic plasticity to live in blackwater, clearwater, and whitewater. Gills of T. albus were collected at well-characterized sites for each type of water. Nine cDNA libraries were constructed, three biological replicates of each condition and the RNA was sequenced (RNA-Seq) on the MiSeq® Platform (Illumina®). A total of 51.6 million of paired-end reads, and 285,456 transcripts were assembled. Considering the FDR ≤ 0.05 and fold change ≥ 2, 13,754 differentially expressed genes were detected in the three water types. Two mechanisms related to homeostasis were detected in T. albus that live in blackwater, when compared to the ones in clearwater and whitewater. The acidic blackwater is a challenging environment for many types of aquatic organisms. The first mechanism is related to the decrease in cellular permeability, highlighting the genes coding for claudin proteins, actn4, itgb3b, DSP, Gap junction protein, and Ca2+-ATPase. The second with ionic and acid-base regulation [rhcg1, slc9a6a (NHE), ATP6V0A2, Na+/K+-ATPase, slc26a4 (pedrin) and slc4a4b]. We suggest T. albus is a good species of fish for future studies involving the ionic and acid-base regulation of Amazonian species. We also concluded that, T. albus, shows well defined phenotypic plasticity for each water type in the Amazon basin.

We examined ionoregulatory characteristics of two species of characiform fish and two species of cichlids that are not native to the Rio Negro, an extremely ion-poor, acidic tributary of the Amazon River, in order to gain insight into the origin of the specializations possessed by Rio Negro fish. The two characiform species examined, Congo tetras (Phenacogrammus interruptus) and black neon tetras (Hyphessobrycon herbertaxelrodi), had high-affinity/high-capacity transporters that produce high rates of uptake in dilute water. Na+ uptake for both was pH insensitive (down to pH 3.5). Exposure to 100 μmol L-1 phenamil had no effect on Na+ uptake in either species, while exposure to 1 mmol L-1 NH4Cl- (high external ammonia [HEA]) slightly stimulated Na+ uptake in Congo tetras. Exposure to \"Na+-free\" water significantly inhibited Na+ uptake (by 65%-85%) but had no effect on net ammonia flux. The two cichlid species examined, convict cichlids (Amatitlania siquia) and red point cichlids (Archocentrus sp.), had high-affinity but low-capacity transporters that yield low rates of uptake in dilute media. Sodium transport was pH sensitive and completely inhibited at pH 3.5. Phenamil exposure inhibited Na+ uptake by 60% in convict cichlids but had no effect on red point cichlids, and exposure to HEA reduced Na+ uptake in both species by 70%-85%. Exposure to \"Na+-free\" water reduced Na+ uptake by 80%-85%, and in convict cichlids it also reduced net ammonia flux by about 50%. The ionoregulatory characteristics described for both groups are strikingly similar to those for Rio Negro species, and we suggest that they may be ancestral physiological traits for these two groups. Further, if this is the case, it seems likely that these traits existed before the Rio Negro and may explain the great success of these species-rich groups in colonizing the river despite its challenging chemistry.

Copper at high concentrations is an ionoregulatory toxicant in fish and its toxicity is known to be strongly modulated by the water chemistry. The toxicity of Cu to the tropical fish cardinal tetra (Paracheirodon axelrodi) was investigated in waters from two major rivers of the Amazon watershed: the Rio Negro (filtered <0.45μm, pH 5.6, DOC=8.4 mgL-1, Na=33μM, Ca=8μM) and the Rio Solimões (filtered <0.45μm, pH 6.7, DOC=2.8 mgL-1, Na=185μM, Ca=340μM), as well as in a natural \"reference water\" (groundwater) which was almost DOC-free (pH 6.0, DOC=0.34 mgL-1, Na=53μM, Ca=5μM). Acute 96-h mortality, Cu bioaccumulation and net flux rates of Na+, Cl-, K+ and total ammonia were determined in P. axelrodi exposed in each water. Copper speciation in each water was determined by two thermodynamic models and by potentiometry, and its toxicity was predicted based on the biotic ligand model (BLM) framework. Our results indicate that high Na+ loss is the main mode of toxic action of Cu in P. axelrodi, in accordance with general theory. Cardinal tetra showed a particularly high ability to tolerate Cu and to maintain Na+ balance, similar to the ability of this and other endemic Rio Negro species to tolerate low pH and ion-poor conditions. Cu toxicity was lower in Rio Negro than in the other two waters tested, and the free [Cu2+] at the LC50, as determined by any of the three speciation methods tested, was approximately 10-fold higher. This variation could not be captured by a realistic set of BLM parameters. At least in part, this observation may be due to gill physiological alterations induced by the abundant dissolved organic matter of the Rio Negro. The implication of this observation is that, for metals risk assessment in tropical waters, similar to the Rio Negro, care must be used in applying BLM models developed using temperate DOC and temperate species.

Blood-O2 affinities (P50 ) were measured over a physiologically relevant pH range at 31 (highest temperature average of Rio Negro over the last 8 years), 33 and 35° C for 10 species of the Rio Negro, aiming to test the acute effects of temperature foreseen by the IPCC (Intergovernmental Panel on Climate Change) for coming years. The animals were collected during an expedition to the Anavilhanas Islands of the Rio Negro, 110 km upstream from Manaus (2° 23\' 41″ S; 60° 55\' 14″ W). Hoplias malabaricus showed higher blood-O2 sensitivity to pH changes (Bohr effect, Φ = Δlog10  P50  ΔpH(-1) ) at both 31° C (Φ = -0·44) and 35° C (Φ = -0·26) compared to Osteoglossum bicirrhosum (Φ = -0·54 at 31° C and Φ = -0·58 at 35° C), but lower P50 under most conditions, and a greater sensitivity of P50 to temperature. Two out of the 10 analysed species had significant increases of P50 (lower blood-O2 affinity) at the highest temperature throughout the pH range tested. For all other species, a minor increase of P50 over the assay-tested temperatures was observed, although all presented a normal Bohr effect. Overall, a diversity of intensities of pH and temperature effects on blood-O2 affinities was observed, which seems to be connected to the biological characteristics of the analysed species. Thermal disturbances in their habitats, likely to occur due to the global warming, would impair blood-O2 binding and unloading in some of the analysed fish species. Copyright © 2016 John Wiley & Sons, Ltd.