Assessing the water quality in a World Heritage Site using biomarkers in top fish predators.

The use of biomarkers in fish for biomonitoring is a valuable approach to reveal effects of human impacts on biota health. Top predator fish are effective models for monitoring human activities' impacts on aquatic ecosystems. The Guaraguaçu River is the largest river-system on coastal region of South Brazil and a World Heritage site. The river receives contaminants from disorderly urban growth, including discharges of domestic sewage and small fishery boats, particularly during the tourist season. Our study aimed to assess impact of anthropogenic activities on water quality in the Guaraguaçu River by analyzing environmental contamination biomarkers in the top fish predator Hoplias malabaricus. Fish were collected using a fyke net trap across sectors representing a gradient of anthropic impact: sector 1 - pristine; sector 2 - impacted; and sector 3 - less impacted. Water samples were collected to analyze the presence of trace elements and pesticide. Biomarkers of the antioxidant system, histopathology, genotoxicity, neurotoxicity, and concentration of trace elements were analyzed in fish tissues. In water samples Al, Fe and Mn were detected, but no pesticides were found. In fish muscle, zinc and iron were detected. Brain acetylcholinesterase activity decreased in impacted sectors, indicating neurotoxic effects. The antioxidant system increased activity in gills and liver, and damage from lipoperoxidation was observed, particularly in sector 2 when compared to sector 1, suggesting oxidative stress. Histopathological biomarkers revealed lesions in the liver and gills of fish in impacted sectors. Micronuclei, a genotoxicity biomarker, were observed in organisms from all sectors. Our results demonstrate detrimental effects of poor water quality on biota health, even when contaminants are not detected in water.

Different response of females and males Neotropical catfish (Rhamdia quelen) upon short-term temperature increase.

Climate change has been one of the most discussed topics in the world. Global warming is characterized by an increase in global temperature, also in aquatic environments. The increased temperature can affect aquatic organisms with lethal and sublethal effects. Thus, it is necessary to understand how different species respond to temperature. This study aimed to evaluate how the Neotropical catfish species Rhamdia quelen responds to temperature increases. The fish were exposed to temperatures of 25 °C (control) and 30 °C after gradual temperature increase for 7 days. After 96 h in each temperature, the fish were anesthetized, blood was collected, and after euthanasia, brain, liver, posterior kidney, gills, muscle, and gonads were collected. The gonads were used for sexing, while other tissues were used for the hematological, biochemical, genotoxic, and histopathological biomarkers analysis. Hepatic proteomic analysis with a focus on energy production was also carried out. Blood parameter changes in both sexes, including an increase in glucose in males, leukopenia in females, and genotoxicity in both sexes. Hepatic proteins related to energy production were altered in both sexes, but mainly in males. Others biomarker alterations, such as histopathological, were not observed in other tissues; however, the antioxidant system was affected differently between sexes. These showed that R. quelen juveniles, at temperatures higher than its optimum temperature such as 30 °C, has several sublethal changes, such as hematological alterations, antioxidant system activation, and energetic metabolism alteration, especially in males. Thus, short-term temperature rise can affect females and males of R. quelen differently.

Effects of cadmium on the female reproductive axis of a Neotropical fish.

Metals are one of the contaminants released from the increase of anthropic activities. They can be classified as endocrine disruptors once they can affect the reproductive parameters of different organisms. The aim of the study was to evaluate the potential effects of cadmium on regulatory reproduction axis (Hypothalamic-Pituitary-Gonadal-Liver, the HPGL axis) in females of Rhamdia quelen exposed to nominal concentrations of 0.1; 1; 10 and 100 µg.L-1 of cadmium. After 15 days, tissues were collected for hormonal quantification, brain aromatase (cyp19a1b), hepatic vitellogenin (vtg) gene expression, and biomarkers analysis. Cadmium was quantified in water, gonad and liver samples. The plasma levels of estradiol, testosterone and gonad and hepatosomatic indexes did not changed after Cd exposure. The cyp19a1b was not different among the groups. Cadmium was detected at higher concentrations in the liver compared to the gonads. No genotoxicity was observed, only erythrocytes nuclear alterations. Metallothionein was reduced at 10 µg.L-1 in the liver and 10 and 100 µg.L-1 in the gonad. Hepatic superoxide dismutase activity increased and this can lead to a hydrogen peroxide increase, one of reactive oxygen species. This increase without a compensation of other enzymes of the antioxidant system can lead to lipoperoxidation, as occurred at 100 µg.L-1. Hepatic vitellogenin gene expression increased as well as the injury index at 0,1 and 100 µg.L-1. The tested cadmium concentrations have been found in the freshwater ecosystems and can affect the female reproductive regulation axis HPGL of the Neotropical species R. quelen.

Sublethal effects of microcystin-LR in the exposure and depuration time in a neotropical fish: Multibiomarker approach.

Eutrophication is an ecological process that results in cyanobacterial blooms. Microcystin-LR is the most toxic variant of microcystins and may cause toxic effects in the organisms, mainly in hepatic tissues. The aims of this study were to use multiple biomarkers in order to evaluate the sublethal effects of a low concentration of MC-LR (1 µg/L) in fish Geophagus brasiliensis by waterborne exposure; and evaluate the depuration of this toxin during 15 days. A group of 30 fish was exposed to 1 µg/L of MC-LR solution for 96 h in a static bioassay. After this time, blood, brain, muscle, liver, gonad and gills were collected from half of the exposed fish group in order to evaluate chemical, biochemical, histological and genotoxic biomarkers. The rest of the fish group was submitted to the depuration experiment with free MC-LR water for 15 days. After this time the same tissues were collected and evaluated using biomarkers analysis. Toxic effects were found mostly in the fish liver from depuration time as alterations on the antioxidant system and histopathologies. The results showed that even low concentrations can cause sublethal effects to aquatic organisms, and cyanotoxins monitoring and regulation tools are required.

Integrated biomarker response index to assess toxic effects of environmentally relevant concentrations of paracetamol in a neotropical catfish (Rhamdia quelen).

The nonsteroidal anti-inflammatory drugs (NSAIDs) are amongst the most commonly detected classes of pharmaceuticals in freshwater environments, with paracetamol being the most abundant. The aim of this study was to evaluate the possible toxic effects of environmentally relevant concentrations (0.25, 2.5 and 25 µg.L-1) of paracetamol in Rhamdia quelen fish exposed for 14 days using different biomarkers. The total count of leukocytes and thrombocytes was reduced at the highest concentration. In the gills, all concentrations of paracetamol reduced the glutathione S-transferase (GST) activity and the reduced glutathione (GSH) levels compared to the control group. The activity of catalase (CAT) was not altered and glutathione peroxidase (GPx) activity increased at the highest concentrations. The superoxide dismutase (SOD) activity decreased at 25 µg.L-1 and the LPO levels increased at 2.5 µg.L-1 when compared to the control group. The concentration of ROS was not different among the groups. In the posterior kidney the activities of GST (2.5 µg.L-1), CAT (2.5 µg.L-1 and at 25 µg. L-1) and GPx and GSH levels increased at all concentrations when compared to the control group. The SOD activity and LPO levels did not change. Paracetamol caused genotoxicity in the blood and gills at concentrations of 2.5 µg.L-1 and in the posterior kidney at 2.5 and 25 µg.L-1. An osmoregulatory imbalance in plasma ions and a reduction in the carbonic anhydrase activity in the gills at 0.25 µg.L-1 were observed. Histopathological alterations occurred in the gills of fish exposed to 25 µg.L-1 and in the posterior kidney at 0.25 and 25 µg.L-1 of paracetamol. The integrated biomarker index showed that the stress caused by the concentration of 25 µg.L-1 was the highest one. These results demonstrated toxic effects of paracetamol on the gills and posterior kidneys of fish, compromising their physiological functions and evidencing the need for monitoring the residues of pharmaceuticals released into aquatic environment.

Toxicological effects of anthropogenic activities in Geophagus brasiliensis from a coastal river of southern Brazil: A biomarker approach.

This study aimed to assess the water quality of the Perequê River, Porto Belo, Santa Catarina, Brazil, through a biomarker approach in the native fish species Geophagus brasiliensis, and the sediment chemical quantification of the herbicides glyphosate and bentazone used in irrigated rice plantations. This river is used for the public water supply of two municipalities. The first sampling (S1) was in November 2016, in the spring season and the second (S2), in March 2017, in the fall season. In each sampling, two points of the river were analyzed, one upstream of the accumulation dam and the water catchment point for water supply of the Porto Belo WTS (P1), and another downstream (P2) with water, sediment, and fish sampling. Biotransformation, oxidative stress, histopathological and genotoxic biomarkers were analyzed in different tissues. The presence of glyphosate was detected in the sediment (11.7 µg·kg-1) from the upstream point of the water catchment site (P1) in spring. The lower activity of the antioxidant enzyme superoxide dismutase (SOD), as well as the increased damage to renal DNA and hepatic tissue, coincided with the lower muscular and cerebral acetylcholinesterase activities (AChE) at P1, in relation to P2 in the spring season, with a lower rainfall index (81.8 mm3). A seasonal variation was also observed between the spring and fall seasons, in fish responses to biomarkers. Reduction of muscle AChE activity and biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione S-transferase and antioxidant enzymes such as, SOD and glutathione peroxidase, as well as increased brain DNA damage, coincided with the highest number of tissue lesions in the liver and gills in the spring, regardless of the sampling point. The results suggested that the Perequê River is contaminated by xenobiotics and probably herbicides from irrigated rice plantations, indicating damages to the biota and a potential public health risk.

Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish.

Nature-based tourism is gaining extensive popularity, increasing the intensity and frequency of human-wildlife contacts. As a consequence, behavioral and physiological alterations were observed in most exposed animals. However, while the majority of these studies investigated the effects of punctual exposure to tourists, the consequences of constant exposition to humans in the wild remains overlooked. This is an important gap considering the exponential interest for recreational outdoor activities. To infer long-term effects of intensive tourism, we capitalized on Odontostilbe pequira, a short-lived sedentary Tetra fish who spends its life close to humans, on which it feeds on dead skin. Hence, those fish are constantly exposed to tourists throughout their lifecycle. Here we provide an integrated picture of the whole phenomenon by investigating, for the first time, the expression of genes involved in stress response and neurogenesis, as well as behavioral and hormonal responses of animals consistently exposed to tourists. Gene expression of the mineralocorticoid (and cortisol) receptor (mr) and the neurogenic differentiation factor (NeuroD) were significantly higher in fish sampled in the touristic zone compared to those sampled in the control zone. Additionally, after a simulated stress in artificial and controlled conditions, those fish previously exposed to visitors produced more cortisol and presented increased behavioral signs of stress compared to their non-exposed conspecifics. Overall, nature-based tourism appeared to shift selection pressures, favoring a sensitive phenotype that does not thrive under natural conditions. The ecological implications of this change in coping style remain, nevertheless, an open question.

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.

Comparative study of photoreceptor and retinal ganglion cell topography and spatial resolving power in Dipsadidae snakes.

The diurnal Dipsadidae snakes Philodryas olfersii and P. patagoniensis are closely related in their phylogeny but inhabit different ecological niches. P. olfersii is arboreal, whereas P. patagoniensis is preferentially terrestrial. The goal of the present study was to compare the density and topography of neurons, photoreceptors, and cells in the ganglion cell layer in the retinas of these two species using immunohistochemistry and Nissl staining procedures and estimate the spatial resolving power of their eyes based on the ganglion cell peak density. Four morphologically distinct types of cones were observed by scanning electron microscopy, 3 of which were labeled with anti-opsin antibodies: large single cones and double cones labeled by the antibody JH492 and small single cones labeled by the antibody JH455. The average densities of photoreceptors and neurons in the ganglion cell layer were similar in both species (∼10,000 and 7,000 cells·mm(-2), respectively). The estimated spatial resolving power was also similar, ranging from 2.4 to 2.7 cycles·degree(-1). However, the distribution of neurons had different specializations. In the arboreal P. olfersii, the isodensity maps had a horizontal visual streak, with a peak density in the central region and a lower density in the dorsal retina. This organization might be relevant for locomotion and hunting behavior in the arboreal layer. In the terrestrial P. patagoniensis, a concentric pattern of decreasing cell density emanated from an area centralis located in the naso-ventral retina. Lower densities were observed in the dorsal region. The ventrally high density improves the resolution in the superior visual field and may be an important adaptation for terrestrial snakes to perceive the approach of predators from above.

Modulatory effect of nano TiO2on Pb in Hoplias malabaricus trophically exposed.

This study investigated the hepatic and neural effects of TiO2 nanoparticle and Pb in Hoplias malabaricus trophically exposed. The alanine transaminase activity was altered at the high dose of exposed group to Pb and at the lowest doses of co-exposed groups. It may reflect the hepatic effects of TiO2 on Pb toxicity, but the aspatate transaminase activity was not altered. The decreased injury index observed at the highest dose of co-exposed group compared to TiO2 may be related to the increased energy demand and can explain the more pronounced toxic effects observed in this group. The liver authomethallography revealed the metals presence at high dose groups. Serotonin concentration increased at the Pb lowest dose and at the highest dose of co-exposed group compare to control. Most importantly, when associated the contaminants were able to interact and altered some biomarkers. However, further studies, about action mechanisms of this co-exposure are needed.

Mercury distribution in target organs and biochemical responses after subchronic and trophic exposure to neotropical fish Hoplias malabaricus.

In the present study, we investigated the mercury distribution, mercury bioaccumulation, and oxidative parameters in the Neotropical fish Hoplias malabaricus after trophic exposure. Forty-three individuals were distributed into three groups (two exposed and one control) and trophically exposed to fourteen doses of methylmercury each 5 days, totalizing the doses of 1.05 µg g⁻¹ (M1.05) and 10.5 µg g⁻¹ (M10.5 group). Autometallography technique revealed the presence of mercury in the intestinal epithelia, hepatocytes, and renal tubule cells. Mercury distribution was dose-dependent in the three organs: intestine, liver, and kidney. Reduced glutathione concentration, glutathione peroxidase, catalase, and glutathione S-transferase significantly decreased in the liver of M1.05, but glutathione reductase increased and lipid peroxidation levels were not altered. In the M10.5, most biomarkers were not altered; only catalase activity decreased. Hepatic and muscle mercury bioaccumulation was dose-dependent, but was not influenced by fish sex. The mercury localization and bioaccumulation corroborates some histopathological findings in this fish species (previously verified by Mela et al. in Ecotoxicol Environ Saf 68:426-435, 2007). However, the results of redox biomarkers did not explain histopathological findings previously reported in M10.5. Thus, fish accommodation to the stressor may reestablish antioxidant status at the highest dose, but not avoid cell injury.

Hematologic and hepatic responses of the freshwater fish Hoplias malabaricus after saxitoxin exposure.

The bioaccumulation of saxitoxins (STX) in the trophic chain, mainly in freshwater, are not completely known. This work aimed to elucidate the effects of STX on Hoplias malabaricus through trophic bioassay. The fish were fed once every five days with Astyanax sp. before being subjected to an intraperitoneal inoculation with the lysate of Cylindrospermopsis raciborskii culture containing 97% STX and 3% by neosaxitoxin and gonyautoxin during 20 days. The animal's liver was assessed using biomarkers as activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx), and concentrations of reduced glutathione (GSH) and lipoperoxidation (LPO) and protein carbonylation (PCO). In the blood was analyzed the genotoxic and hematological parameters. The hepatosomatic index and the relative condition factor did not show a significant difference between the exposed and control groups. The values of mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin increased in the STX group. The hepatic tissue from both groups exhibited a typical pattern that have been already described for most teleost fish. The results suggested the generation of reactive oxygen species, with increased activity of GPx and concentrations of LPO and GSH; whereas the specific activity of SOD decreased. However, no changes were observed in the CAT, PCO, and DNA damage. Although the STX effects are known as neurotoxic, this cyanotoxin caused liver biochemical alterations that can be considered ecologically relevant.

Genotoxic evaluation of different doses of methylmercury (CH3Hg⁺) in Hoplias malabaricus.

The survey of the effects of toxic metals and its organic compounds are of critical importance because these compounds tend to accumulate in aquatic environments. In the present work, the genotoxic potential of methylmercury, an organomercurial compound with high toxicity and present in large amounts in fish of the Amazon due to the mining process, was evaluated using the piscine micronucleus test and comet assay in fish. Specimens of Hoplias malabaricus (popularly known as traira), a neotropical specie, was exposed to methylmercury via food web, over 70 days, in two different concentrations: two groups of fifteen fish were tested with 0.075 µg CH(3)Hg(+)/g and 0.75 µg CH(3)Hg(+)/g of methylmercury per gram of fish, at 5-day intervals and over 14 successive intervals whereas control group, composed by nine fish, was fed by uncontaminated prey fish (Astyanax sp). One of the aims of this study was to reproduce the contamination found in nature in an attempt to increase our biological knowledge on the neotropical species. Hoplias malabaricus specimens were then anesthetized for removal of blood samples and dissected, for cephalic kidney removal. As a result, it was observed that the piscine micronucleus test showed no significant differences between exposed groups and control group. The comet assay performed on erythrocyte system cells, showed a significant difference between controls and contaminated, but there was no difference between doses. In contrast, the kidney cells comet assay showed no significant difference between groups, probably due to the type of damage caused by xenobiotics to be related to the tissues of most contact with it, as well as its mode of action and the chain of bioaccumulation within bodies.

Morphological evidence of neurotoxicity in retina after methylmercury exposure.

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 µgg(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: a basis for neurotoxicology.

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure.

Enzymatic inhibition and morphological changes in Hoplias malabaricus from dietary exposure to lead(II) or methylmercury.

Neotropical fish traíra (Hoplias malabaricus) were used to investigate the effects of dietary doses of metals through individual exposures to either inorganic lead(II) or methylmercury, respectively, 21 microg Pb2+g(-1) w.w. and 75 ng H(3)C-Hg+g(-1) w.w., every 5 days, for 70 days (14 doses). The erythrocyte delta-aminolevulinic acid dehydratase (ALAd) activity was inhibited after 14 doses of Pb2+ and H(3)C-Hg+. The muscle cholinesterase (ChE) activity was inhibited after 14 doses of H(3)C-Hg+. Damage in cytoskeleton and nuclei were observed after exposure to inorganic lead. Individuals exposed to H(3)C-Hg+ showed the presence of atypical granules and vesicles, cytoplasm disorganization, and mitochondria damages in hepatocytes also after 14 doses. The present results demonstrate that erythrocyte ALAd and muscle ChE activities can be used as long-term biomarkers of sublethal, subchronic, and trophic exposures to Pb2+, and H(3)C-Hg+ in fish. Also the morphological aspects described in the present work confirm the toxicity of both studied metals.