A biomarker approach to study the effects of polluted Brazilian urban reservoirs in a native fish.

Two of the largest water reservoirs in the Metropolitan Region of São Paulo, Brazil (MRSP), named Billings and Guarapiranga, are facing high levels of anthropic impact. This is evidenced by the presence of contaminants and pollutants, which are deteriorating their water quality. Therefore, this study evaluated antioxidant defense enzymes, lipoperoxidation and genotoxicity, in adult females of a native species, Astyanax altiparanae from the Guarapiranga and Billings reservoirs. The study also aimed to evaluate these biomarkers during two different periods of the year, the rainy (summer) and dry (winter) seasons. The oxidative stress was evaluated by the activity of enzymes such as glutathione peroxidase, glutathione S-transferases, superoxide dismutase, and catalase in the gills and liver, and the occurrence of lipoperoxidation was also evaluated in both organs. The genotoxicity was assessed by performing comet assay, micronucleus, and nuclear abnormality tests on blood samples. The results showed that fish from both reservoirs are subjected to oxidative stress and genotoxic damage, mainly during winter, but fish living in Billings showed greater alterations than fish from Guarapiranga. Likewise, the results of the principal component analysis suggested that caffeine, nitrogenous compounds, and some metals might be triggering these toxic effects in fish.

Neurotoxic, biotransformation, oxidative stress and genotoxic effects in Astyanax altiparanae (Teleostei, Characiformes) males exposed to environmentally relevant concentrations of diclofenac and/or caffeine.

The present study evaluated neurotoxic, biotransformation, genotoxic and antioxidant responses to relevant environmental concentrations of diclofenac (0.4 µg L-1) and caffeine (27.5 µg L-1), separate and combined, in adult males of the freshwater fish Astyanax altiparanae after a subchronic exposure (14 days). Fish exposed to diclofenac and caffeine, both separate and combined, revealed a neurotoxic effect through the inhibition of acetylcholinesterase activity in the muscle, while diclofenac alone and in combination caused cyclooxygenase inhibition. Caffeine alone produces genotoxicity on this species but, when combined with diclofenac, it potentiates hepatic lipoperoxidation and the inhibition of oxidative stress enzymes, while diclofenac alone or in combination produces a general inhibition of important enzymes. This study suggests that aquatic contamination produced by these pharmaceuticals has the potential to affect homeostasis and locomotion in A. altiparanae and compromise their immune system and general health.

Eutrophication effects on fatty acid profiles of seston and omnivorous fish in tropical reservoirs.

It has been postulated that eutrophication causes replacement of n3 highly unsaturated fatty acids (n3 HUFA) rich taxa, such as Bacillariophyta, Cryptophyta and Dinophyta, with taxa poor in these fatty acids (FA), such as Chlorophyta and Cyanobacteria. Such a change in community composition at the basis of the food web may alter the FA composition of consumer tissues. Here, we investigated the effects of eutrophication on phytoplankton composition and FA profiles of seston and muscle of two omnivorous fish species (Astyanax fasciatus and Astyanax altiparanae) from reservoirs of different trophic status in Southeast Brazil. The phytoplankton composition and seston FA profiles reflected the degree of eutrophication at most of the studied sites. Three of the five most eutrophic sites were dominated by cyanobacteria and had the highest saturated fatty acid (SFA) and lowest polyunsaturated fatty acid (PUFA) relative contents among all sites. In contrast, the remaining two sites presented a higher phytoplankton diversity and higher relative contribution of sestonic PUFAs with 18 carbons (C18) and HUFAs than less eutrophic systems. However, there were no clear effects of sestonic FA profiles on the FA profiles of muscle of both fish species. A higher percentage of n3 HUFAs was found in the fish samples from a hypereutrophic and cyanobacteria dominated reservoir than in those from sites with a more diverse phytoplankton community in which fish mainly showed higher percentages of C18 PUFA. These results suggest a lack of a direct relationship between the degree of eutrophication and the percentage of n3 HUFAs in both fish species, which can be caused by specific characteristics of the reservoirs that may modulate eutrophication effects. Therefore, consumer FA biochemistry seemed to be dictated by their ability to select, accumulate, and modify dietary FAs, rather than by the eutrophication degree of the studied tropical reservoirs.

Nonsteroidal anti-inflammatory drugs act as endocrine disruptors in Astyanax lacustris (Teleostei: Characidae) reproduction: An ex vivo approach.

Pharmaceutical products can act as endocrine-disrupting compounds (EDCs), affecting the physiological processes of animals, such as development or reproduction. This study aimed to investigate the influence of different concentrations of nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac (DCF) and ibuprofen (IBU) alone and mixed (MIX) on gonadotropin gene expression and gonadal steroid release using Astyanax lacustris pituitary and testes explant systems, respectively. The explant organs were maintained for 12 h in Leibovitz (L-15) medium supplemented with 0, 0.1, 1, 10, 100, and 1000 ng L-1 of DCF, IBU, and MIX (ratio 1:1 of the same concentrations of DCF and IBU alone) and gonadotropin releasing-hormone (cGnRH2) stimulation in pituitary explants and human chorionic gonadotropin (hCG) stimulation in testes explants. The pituitary glands and the media from the testicular explants were collected for gene expression analysis including the ß subunit of the follicle-stimulating hormone (fshß) and luteinizing hormone (lhß) and secreted gonadal steroid concentration analysis, respectively. Both DCF and IBU (alone and mixed) decreased pituitary gene expression of fshß and lhß and this inhibitory effect was evident even at low concentrations. In the testes, DCF and IBU did not change the levels of estradiol, and both pharmaceuticals increased the release of 11-ketotestosterone at low doses, while only IBU decreased the levels of testosterone in all concentrations. IBU's inhibitory effect in the testes was not triggered by the mixture of the two drugs. These results suggest that NSAIDs, may interfere in fish reproduction by acting as EDCs, thereby negatively affecting A. lacustris spermatogenesis and maturation.

Diclofenac and caffeine inhibit hepatic antioxidant enzymes in the freshwater fish Astyanax altiparanae (Teleostei: Characiformes).

Although concentrations of pharmaceutical compounds in aquatic ecosystems are low, they can cause toxic effects on organisms. The aim of this study was to evaluate the effects of diclofenac (DCF), a non-steroidal anti-inflammatory drug, and caffeine (CAF), a central nervous system stimulant, both alone or combined, in Astyanax altiparanae males under acute exposure (96 h), measuring neurotoxicity biomarkers, antioxidant response and damage at biochemical and cellular levels. DCF concentration in water, separated and combined, was 3.08 mg L-1 and that of CAF was 9.59 mg L-1. To assess neurotoxicity, brain and muscle acetylcholinesterase (AChE) activities were measured. To evaluate oxidative stress, the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST), as well as lipoperoxidation (LPO), were analyzed in liver and gills. Activity of hepatic cyclooxygenase (COX) was also evaluated. Genotoxicity was assessed in blood using comet assay and micronucleus test, as well as nuclear abnormalities. DCF and CAF, alone or combined, had neither effect on AChE activity, nor in the activity of SOD, CAT, GPx and GST in gills. In liver, DCF inhibited SOD and GPx activity, CAF inhibited CAT activity, the mixture inhibited SOD and GST activity; although only fish exposed to CAF showed increased hepatic LPO. Under these experimental conditions, no effect on COX activity was observed, nor cytotoxic and genotoxic damage. The most pronounced effects were caused by the drugs separately, since both compounds altered the enzymes, but only CAF triggered LPO, showing more harmful effects.

Integrated use of biomarkers to evaluate the reproductive physiology of Astyanax fasciatus and Hoplias malabaricus males (Teleostei: Characiformes) in polluted reservoirs.

The reproductive physiology of fish can be changed by the presence of pollutants in the water, which act as endocrine disrupting compounds (EDC). We evaluated the impacts of water contaminants in polluted reservoirs acting as possible EDC on the reproductive physiology of Astyanax fasciatus and Hoplias malabaricus males. We used biomarkers with different levels of biological organization. Hoplias malabaricus adult males were collected in the summer and winter at five different sites in the Tietê River Basin: the Ponte Nova reservoir (PN), considered a reference site due to the low anthropogenic influence; the Billings reservoir (BIL) at two different branches; and the Guarapiranga reservoir (GUA) at two different branches. Astyanax fasciatus adult males were collected at PN and BIL. BIL and GUA are subjected to great anthropogenic action. We analyzed gonadal histomorphology, testosterone (T), 11-ketotestosterone (11-KT), estradiol (E2) plasma levels, and gene expression of hepatic vitellogenin (vtgA) and pituitary follicle stimulating hormone (fshß). In the PN reservoir (reference), the biomarkers analyzed in both species did not differ between the periods analyzed. This is an evidence that the animals keep the same reproductive activity during both seasons. The changes in the plasma concentration of gonadal steroids in both species in polluted reservoirs suggest the presence of EDC compounds in the water and/or adjusts of the physiological setpoint to allow the reproduction in such adverse conditions. The use of vtgA as biomarker suggests the presence of estrogenic compounds, mainly in BIL, but with a more evident response of H. malabaricus. However, even considering physiological changes, both species present testes during the maturation phase that allow the reproduction in an environment with a high degree of pollution.

Endocrine disruptive action of diclofenac and caffeine on Astyanax altiparanae males (Teleostei: Characiformes: Characidae).

Diclofenac (DCF) and caffeine (CAF) are persistent pharmaceuticals that occur in mixtures in the aquatic ecosystems causing effects in the reproductive physiology of aquatic organisms. This study evaluated the physiological reproductive responses of Astyanax altiparanae males exposed to nominal concentrations of DCF (3.08 mg L-1) and CAF (9.59 mg L-1) separately and combined, for 96 h. The steroids profile, estrogenic biomarker vitellogenin (vtgA), testes and liver morphology, and also mortality of males were assessed. DCF and CAF degradation was 5% of the initial concentration for 24 h. The LC50 of the DCF and CAF were 30.8 mg L-1 and 95.9 mg L-1, respectively. Males exposed to DCF and CAF exhibited a reduction of 17ß-Estradiol (E2) concentration compared to control (CTL). Similarly, testosterone (T) was also reduced in the DCF treatment, but this response was not observed in 11-Ketotestosterone (11-KT). Males exposed to DCF + CAF combined did not exhibit differences in T, E2 and 11-KT steroids. The vtgA gene expression and the sperm concentration did not change among the treatments. Moreover, acute exposure revealed a hypertrophy of hepatocytes cells in the DCF and DCF + CAF treatments. In conclusion, DCF and CAF, isolated, exhibit an endocrine disruptive activity in A. altiparanae male, an opposite response observed with the mixture of both compounds that abolishes the endocrine disruptive effects. DCF seems to be more toxic for this species, altering also hepatocytes morphology.

Water temperature and acid pH influence the cytotoxic and genotoxic effects of aluminum in the freshwater teleost Astyanax altiparanae (Teleostei: Characidae).

The toxicity of metals, including aluminum (Al), can be potentiated by temperature and acid pH, a concern in view of the current global warming scenario. The aim of this study was to evaluate the bioconcentration of Al in the testes and semen of Astyanax altiparanae and the potential of this metal, at different environmental temperatures and acid pH, to cause cytotoxicity and genotocixity in erythrocytes and spermatozoa. A. altiparanae males were divided into nine experimental groups: at each of three different water temperatures (20, 25 and 30 °C), the fish were exposed to a neutral pH, an acid pH and acidic water containing Al (0.5 mg.L-1). The fish were subjected to subacute, semi-static exposure and sampled at 24 and 96 h. After each exposure period the comet assay (blood and semen) and micronucleus test (blood) were performed. Bioconcentration of Al was evaluated in the testes and semen. Exposure time and temperature influenced the Al bioconcentration pattern in the testes. Al concentration in the semen was higher in fish exposed at 20 and 25 °C (24 h). The DNA fragmentation score for the semen and blood was higher in fish exposed to Al at 20 (24 h) and 30 °C (96 h). The frequency of nuclear abnormalities in erythrocytes was higher in the group exposed to Al at 30 °C (96 h). It was concluded that Al bioconcentrates in the testes and semen of A. altiparanae at different temperatures and is potentially cytotoxic and genotoxic to erythrocytes and spermatozoa in this species.

Effect of culture medium on growth and protein content of Chlorella vulgaris / Efecto del medio de cultivo sobre el crecimiento y el contenido proteico de Chlorella vulgaris / Efeito do diferentes meios de cultura sobre o crescimento e o conteúdo proteico em Chlorella vulgaris

The use of live food in larviculture and fingerling stages has become an excellent option in pisciculture to reduce mortality and production costs. Live food is cost-effective, nutritious, and it has enzymes necessary for fish development. Objective: this study evaluated the effect of several culture media (chu10 , NPK complex fertilizer, worm humus, and equine manure) on growth and protein content of Chlorella vulgaris. Methods: 3 L volumes with constant light and aeration were used for algae culture. Six replicates were used per each treatment. One mL of C. vulgaris was added to each experimental unit to obtain 8.3 x 10(6) cel/mL as initial density. Algae counts were conducted every two days using the Neubauer chamber to determine cell density, parameters such as temperature, dissolved oxygen, pH, nitrates, and phosphates were measured. Crude protein was determined with the Kjeldahl method. The assay lasted 82 days. Results: microalgae reached the highest density with complex fertilizer NPK (10.9 ± 1.6 x 10(6) cel/mL) on day 22, followed by worm humus (5.3 ± 1.1 x 10(6) cel/mL) on day 48, equine manure (4.9 ± 0.9 x 10(6) cel/mL) on day 18, and the last was chu10 (2.2 ± 0.6 x 10(6) cel/mL) on day 12. The highest protein content was found in algae grown in worm humus (56.8%), and the equine manure (32.5%), in contrast, complex fertilizer NPK had the lowest value (16.8%). Conclusion: this study showed that organic media are a good choice for the culture of C. vulgaris, providing proper growth and high protein content of microalgae.

En piscicultura, el uso de alimento vivo durante las fases de larvicultura y alevinaje se ha convertido en una opción para reducir la tasa de mortalidad y los costos de producción, debido a que es económico, nutritivo y posee enzimas necesarias para el desarrollo de las larvas de peces. Objetivo: el presente trabajo evaluó el efecto de diferentes medios de cultivo (chu10, fertilizante complejo NPK, humus de lombriz y equinaza) sobre el crecimiento y el contenido proteico en Chlorella vulgaris. Métodos: para su cultivo se utilizaron volúmenes de 3 L, con luz y aireación constante. De cada tratamiento se realizaron seis réplicas y en cada unidad experimental se adicionó 1 mL de C. vulgaris con una densidad de 8.3 x 10(6) cel/mL. Para determinar la densidad celular se realizaron conteos cada dos días haciendo uso de cámara de Neubauer. Se midió temperatura, oxígeno disuelto, pH, nitratos y fosfatos. La proteína cruda se determinó por el método de Kjeldahl. La duración total del ensayo fue de 82 días. Resultados: la microalga alcanzó su mayor densidad con fertilizante complejo NPK (10.9 ± 1.6 x 10(6) cel/mL) en el día 22, seguido del humus de lombriz (5.3 x 106 ± 1.1 x 10(6) cel/mL) en el día 48, equinaza (4.9 ± 0.9 x 10(6) cel/mL) en el día 18, y por último con el chu10 (2.2 ± 0.6 x 10(6) cel/mL) en el día 12. Se encontró mayor contenido proteico en las células cultivadas en humus de lombriz (56.8%) y equinaza (32.5%); a diferencia, el del fertilizante complejo NPK fue muy bajo (16.8%). Conclusiones: este estudio permite concluir que los medios orgánicos son una buena opción para cultivar C. vulgaris, otorgando un adecuado crecimiento y un alto contenido proteico de la microalga.

Na piscicultura, o uso de alimento vivo durante as fases da cultura de larvas e juvenis têm sido convertidos em uma opção para reduzir a taxa de mortalidade e os custos de produção, porque é econômico, nutritivo e possui as enzimas necessárias para o desenvolvimento das larvas de peixe. Objetivo: o objetivo desta pesquisa foi avaliar a efeito de diferentes meios de cultura (chu10, fertilizante mineral complexo NPK, vermicomposto e esterco equino) sobre o crescimento e o conteúdo proteico em Chlorella vulgaris. Métodos: para a cultura, foram utilizados volumes de 3 L, com luz e aeração permanente. De cada tratamento, realizaram-se seis repetições e em cada unidade experimental, adicionou-se 1 mL de C. vulgaris com uma densidade de 8.3 x 10(6) cel/mL. Para determinar a densidade celular, realizaram-se contagens a cada dois dias usando a câmara de Neubauer. Aferiu-se a temperatura, oxigênio dissolvido, pH, nitratos e fosfatos. A proteína bruta foi determinada por o método de Kjeldahl. A duração total do ensaio foi de 82 dias. Resultados: os resultados mostraram que a microalga alcançou sua maior densidade com o fertilizante complexo NPK (10.9 ± 1.6 x 10(6) cel/mL) no dia 22, seguido do vermicomposto (5.3 ± 1.1 x 10(6) cel/mL) no dia 48, o esterco equino (4.9 ± 0.9 x 10(6) cel/mL) no dia 18 e por último com o chu10 (2.2 ± 0.6 x 10(6) cel/mL) no dia 12. Foi encontrado maior conteúdo proteico nas células cultivadas em vermicomposto (56.8%) e esterco equino (32.5%); a diferença, do fertilizante complexo NPK que foi muito baixo (16.8%). Conclusões: este estudo permitiu concluir que os meios orgânicos são uma boa opção para cultivar C. vulgaris, obtendo um crescimento adequado e um alto conteúdo proteico da microalga.