Biochemical changes and bioaccumulation of manganese in Astyanax lacustris (Teleostei: Characidae).

Major tailings dam failures have occurred recently around the world and resulted in severe environmental impacts, such as metal contamination. Manganese is a metal highly associated with mining activities, largely detected in mining dam collapses. This metal is considered necessary for different organisms, but it can be toxic and cause oxidative stress and genetic damage in fishes. In this study, we investigated the toxic effects of manganese on Astyanax lacustris, by exposing the fish individually to different concentrations of this metal (2.11, 5.00, and 10.43 mg/L) for 96 h. To assess the effects of manganese, we used biochemical biomarkers (glutathione S-transferase, catalase, and acetylcholinesterase enzyme activity) and the manganese bioaccumulation in different tissues (liver and gills). The obtained data showed that only at concentrations of 5.00 mg/L and 10.43 mg/L the activity of glutathione S-transferase differed significantly. Additionally, the acetylcholinesterase activity in the brain tissue was inhibited. The highest level of manganese bioaccumulation was observed in the liver and branchial tissue. Overall, we concluded that high concentrations of manganese may cause physiological changes in Astyanax lacustris.

Cyanotoxins and water quality parameters as risk assessment indicators for aquatic life in reservoirs.

We assessed the extent of pollution in an essential public water supply reservoir (southeastern Brazil). An environmental monitoring study was performed at the Billings Reservoir (at the water catchment site) to assess the water quality in 2017, 2018, and 2019. Physicochemical parameters were analyzed, quantifying the total cyanobacteria and the cyanotoxins microcystins (MCs) and saxitoxins (SXTs), as well as their possible ecological risk to the aquatic environment. We also determined metals and metalloids (As, Ba, Cd, Pb, Cu, Cr, Fe, Mn, Ni, Zn, and Sb) and fecal bacteria (Escherichia coli). Monthly samplings were performed for 2017, 2018, and 2019 (totaling 36 sampling campaigns). Metals, metalloids, and E. coli values were below the maximum limit allowed by the Brazilian legislation. High concentrations of total cyanobacteria (3.07 × 104 - 3.23 × 105 cells/mL), microcystin variants MC-LR (0.67-23.63 µg/L), MC-LA (0.03-8.66 µg/L), MC-RR (0.56-7.92 µg/L), and MC-YR (0.04-1.24 µg/L), as well as the saxitoxins GTX2 (0.18-5.37 µg/L), GTX3 (0.13-4.40 µg/L), and STX (0.12-2.92 µg/L) were detected. From an ecotoxicological point of view, the estimated values for the risk quotient (RQ) for microcystins and saxitoxins were largely greater than 1, indicating a high risk to aquatic life. Therefore, further efforts need to be made to delay the eutrophication of the reservoir.

Do Manganese and Iron in Association Cause Biochemical and Genotoxic Changes in Oreochromis Niloticus (Teleostei: Cichlidae)?

The present study aimed to evaluate the toxicity of the association between Fe and Mn in Oreochromis niloticus through genotoxic (micronucleus test and comet assay) and biochemical (CAT and GST enzymes) assays. The tested treatments were T1 = control group (without metal addition), T2 = 2.60 mg L-1 of Fe + 0.2 mg L-1 of Mn, and T3 = 4.40 mg L-1 of Fe + 3.49 mg L-1 of Mn, during 96-h bioassays. All animals exposed to the metals showed a significant increase in erythrocyte micronucleus frequency and DNA damage. The hepatic GST activity increased two times in animals exposed to T3 compared to the control group. The results indicate that Fe + Mn caused genotoxic and biochemical changes in exposed fish. Therefore, excess metals in ecosystems, even those essential for organisms, can be dangerous for the local biota due to the risk associated with high concentrations of these metals.

Effects of the water-soluble fraction of diesel oil (WSD) on the fertilization and development of a sea urchin (Echinometra lucunter).

Considering the high number of accidents with diesel oil spills occurring in the marine ecosystem, toxicity tests aimed at assessing the effects of this pollutant on biota are necessary and urgent. Thus, the present study aimed to evaluate the toxicity of the soluble fraction of diesel oil (WSD) in the fertilization success of gametes and pluteu larvae of the sea urchin Echinometra lucunter. To do this, gametes and embryos were exposed to concentrations of 0% (control group), 0.5%, 1.5% and 2.5% of WSD. The fertilization success of exposed gametes and embryos were reduced significantly when compared to the control group in all tested concentrations. With this finding, it is evident that diesel oil can be significantly promoted in the early and adult life stages of a particular organism, and a better way of evaluating this toxicity is through the analysis of contaminant effects throughout the reproductive cycle of a species.

Genotoxic, biochemical and bioconcentration effects of manganese on Oreochromis niloticus (Cichlidae).

Manganese and iron were found at high concentrations (3.61 mg/L and 19.8 mg/L, respectively) in the water of the Rio Doce after the dams of Fundão and Santarém broke in Mariana/MG (Brazil). These same metals were found in fish and crustacean muscle (15 mg/kg and 8 mg/kg wet weight, respectively) in the specimens collected near the Rio Doce's outfall. Due to the variation in Mn concentration found in the lower Rio Doce, this study aimed to determine the effects of Mn in Oreochromis niloticus, at the concentrations allowed by CONAMA, and in concentrations found in the Rio Doce after the dams broke. The animals were exposed to the following dissolved concentrations: control group (0.0 mg/L), 0.2; 1.5 and 2.9 mg/L manganese for 96 h. In addition, a positive control was conducted, injecting intraperitoneally with cyclophosphamide (at 25 mg/kg). These exposures caused significant erythrocyte micronucleus formation in the organisms exposed to the highest concentration, as well a significant increase in the DNA damage index of erythrocytes from organisms exposed to 1.5 mg/L and 2.9 mg/L treatments. The glutathione S-transferase enzyme activity also showed a significant increase in the liver of the organisms exposed to 2.9 mg/L. However, catalase activity increased significantly in the gills of the animals exposed to all concentrations of manganese that were tested. Manganese bioconcentrated in greater quantities in the liver than the gills. Thus, manganese causes significant damage to genetic material, generates nuclear abnormalities, activates the body's detoxification system and can accumulate in animal tissue.

A rapid LC-MS/MS method for multi-class identification and quantification of cyanotoxins.

Cyanobacteria can form harmful blooms in specific environmental conditions due to certain species producing toxic metabolites known as cyanotoxins. These toxins pose significant risks to public health and the environment, making it critical to identify and quantify them in food and water sources to avoid contamination. However, current screening methods only focus on a single class of cyanotoxins, limiting their effectiveness. Thus, fast and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed to analyze eighteen cyanotoxins simultaneously. A simplified extraction procedure using lyophilized samples of cyanobacterial biomass was also used, eliminating the need for traditional solid-phase extraction methods. This method uses multiple reaction monitoring and allows accurate determination and quantification of eighteen cyanotoxins, including anatoxin-a, homoanatoxin-a, cylindrospermopsin, deoxy-cylindrospermopsin, nodularin, guanitoxin, seven microcystins (RR, [D-Asp3] RR, LA, LR, LY, LW, and YR), and five saxitoxins (gonyautoxins - GTX-1&4, GTX-2&3, GTX-5), decarbamoylgonyautoxin (dcGTX-2&3), and N-Sulfocarbamoylgonyautoxin (C1&C2), all in a short acquisition time of 8 min. Therefore, this method provides a simple and efficient approach to identify and quantify harmful compounds produced by cyanobacteria. Hence, this represents the first method to detecting guanitoxin among cyanotoxins. By expanding the range of toxins analyzed, this method can help ensure high-quality food and drinking water and protect recreational users from exposure to cyanotoxins.

Multiple biomarkers response of Astyanax lacustris (Teleostei: Characidae) exposed to manganese and temperature increase.

The present study aimed to evaluate the toxicity of Mn (6.65 mg/L) at different exposure times (96 h, 7, 14, and 21 days) and evaluate its possible toxic effects on the fish Astyanax lacustris through multi-biomarkers and the maximum critical temperature (CT Max). The results show an increase in the Mn accumulation (liver and gills) with increasing exposure time. The glutathione S-transferase (GST) activity showed differences in the group exposed to Mn for 96 h compared to the group exposed for 21 days. The acetylcholinesterase (AChE) activity increased in the fish exposed for 7 days compared to the control group. On the other hand, no genotoxic changes were observed. The CT Max showed that the loss of equilibrium of 50% of the fish occurs at a temperature of 39ºC, with and without the Mn presence. Furthermore, the catalase gene expression (oxidative stress) did not show alterations.

Ecotoxicological assessment of guanitoxin-producing cyanobacteria in Danio rerio and Daphnia similis.

Anthropogenic activity has dramatically deteriorated aquatic ecosystems in recent years. Such environmental alterations could change the primary producers' composition, exacerbating the proliferation of harmful microorganisms such as cyanobacteria. Cyanobacteria can produce several secondary metabolites, including guanitoxin, a potent neurotoxin and the only naturally occurring anticholinesterase organophosphate ever reported in the literature. Therefore, this study investigated the acute toxicity of guanitoxin-producing cyanobacteria Sphaerospermopsis torques-reginae (ITEP-024 strain) aqueous and 50% methanolic extracts in zebrafish (Danio rerio) hepatocytes (ZF-L cell line), zebrafish embryos (fish embryo toxicity - FET) and specimens of the microcrustacean Daphnia similis. For this, hepatocytes were exposed to 1-500 mg/L of the ITEP-024 extracts for 24 h, the embryos to 31.25-500 mg/L for 96 h, and D. similis to 10-3000 mg/L for 48 h. Non-target metabolomics was also performed to analyze secondary metabolites produced by the ITEP-024 using LC-MS/MS. Metabolomics indicated the guanitoxin presence just in the aqueous extract of the ITEP-024 and the presence of the cyanopeptides namalides, spumigins, and anabaenopeptins in the methanolic extract. The aqueous extract decreased the viability of zebrafish hepatocytes (EC(I)50(24h) = 366.46 mg/L), and the methanolic extract was not toxic. FET showed that the aqueous extract (LC50(96) = 353.55 mg/L) was more toxic than the methanolic extract (LC50(96) = 617.91 mg/L). However, the methanolic extract had more sublethal effects, such as abdominal and cardiac (cardiotoxicity) edema and deformation (spinal curvature of the larvae). Both extracts immobilized daphnids at the highest concentration analyzed. However, the aqueous extract was nine times more lethal (EC(I)50(48h) = 108.2 mg/L) than the methanolic extract (EC(I)50(48h) = 980.65 mg/L). Our results showed an imminent biological risk for aquatic fauna living in an ecosystem surrounded by ITEP-024 metabolites. Our findings thus highlight the urgency of understanding the effects of guanitoxin and cyanopeptides in aquatic animals.

Gene expression changes in Epinephelus marginatus (Teleostei, Serranidae) liver reveals candidate molecular biomarker of iron ore contamination.

Wastes from iron ore mining activities are potentially damaging to adjacent aquatic ecosystems. We aimed to determine biomarkers of environmental exposure to this xenobiotic in the dusky grouper Epinephelus marginatus by differential gene expression analysis. For this, fish were exposed to iron ore (15.2 mg/L) and gene expression in liver was assessed by RNA-Seq and compared to the control group. A total of 124 differentially expressed genes were identified, from which 52 were upregulated and 72 were downregulated in response to iron ore. From these, ferritin (medium subunit), cytochrome b reductase and epoxide hydrolase genes were selected for validation by RT-qPCR that confirmed the upregulation of epoxide hydrolase in fish exposed to iron ore.

Response of Oreochromis niloticus (Teleostei: Cichlidae) exposed to a guanitoxin-producing cyanobacterial strain using multiple biomarkers.

Changes in environmental conditions in aquatic ecosystems caused by anthropic actions can modify the composition of primary producers, promoting the excessive proliferation of cyanobacteria. These organisms can form cyanobacterial blooms, which directly affect aquatic life. The present study investigated the mutagenicity of the cyanobacterium Sphaerospermopsis torques-reginae (strain ITEP-024), guanitoxin-producing (natural organophosphate), and sublethal effects on fish in relevant environment concentrations. For this, the Ames test (Salmonella/microsome) was performed as a mutagenic assay for extracts of the ITEP-024 strain. Specimens of Oreochromis niloticus (Teleostei: Cichlidae) were subjected to acute 96 h exposure to different concentrations of aqueous extract of the strain: C = control group; T1 = 31.25 mg/L; T2 = 62.5 mg/L; T3 = 125 mg/L; and T4 = 250 mg/L. Genotoxic, biochemical, osmoregulatory, and physiologic biomarkers were analyzed. Our results showed that the cyanobacterium had a weak mutagenic response for the TA102 strain of Salmonella with and without metabolic activation by S9. Strains TA98 and TA100 were not affected. Fish from treatments T3 and T4 showed changes in oxidative stress (CAT, SOD, and GST enzymes), inhibition of the enzyme acetylcholinesterase activity, micronucleus formation, and osmoregulatory disorders. No guanitoxin accumulation was detected in the different tissues of O. niloticus by LC-MS/MS. Our results showed unprecedented mutagenicity data of the guanitoxin-producing cyanobacteria by the Ames test and biochemical, osmoregulatory, and genotoxic disorders in fish, providing efficient aquatic contamination biomarkers. Despite the great concern related to the presence of guanitoxin in blooms in freshwater ecosystems, its concentration is not yet regulated, and thus there is no monitoring agenda in current legislation.

Can the insecticide Imidacloprid affect the health of the Neotropical freshwater fish Astyanax altiparanae (Teleostei: Characidae)?

Female juveniles of the Neotropical fish Astyanax altiparanae were exposed for 96 h to four treatments containing the active ingredient from Imidacloprid® commercial formulation (IMI 1, IMI 2, IMI 3, and IMI 4) and to a control treatment (only dechlorinated tap water). Glutathione content, glutathione S-transferase activity, lipid peroxidation (LPO) and protein carbonylation levels, acetylcholinesterase (AChE) activity, and frequency of micronuclei and erythrocyte nuclear abnormalities (ENA) were measured in the fish. The muscle and gills were the most affected organs; their antioxidant defense was not enough to prevent oxidative damage (LPO) in the IMI 2 and IMI 4 treatment fish. IMI also inhibited AChE activity in the muscle (IMI 3 and IMI 4) and increased ENA frequency (IMI 4). IMI can affect the health of A. altiparanae in environmentally relevant concentrations, causing oxidative damage in different organs, neurotoxic effects in the muscle, and genotoxicity.

Is the Doce River elutriate or its water toxic to Astyanax lacustris (Teleostei: Characidae) three years after the Samarco mining dam collapse?

In 2015, after the Fundão dam failure, in Minas Gerais State, Brazil, around 50 million cubic meters of sludge from iron mining tailings were discharged into the Doce River. After the dam collapse, surpassing concentrations of metals were observed in the river sediment, which could be harmful to aquatic organisms, including the fishes. The present study aimed to evaluate the toxic effects of both elutriate and water, collected from the Doce River, on Astyanax lacustris three years after the dam failure. A bioassay was carried out through subchronic exposure to Doce River water (E0) and three elutriate concentrations (10, 50 and 100%). Biochemical analyses (CAT, GST, AChE), metal bioaccumulation assays and calculation of the integrated biomarker response index, version 2 (IBRv2) were performed. The outcomes uncovered deleterious consequences on organisms exposed to E0, with AChE inhibition and bioaccumulation of Fe and Mn in both liver and gills. IBRv2 values were more elevated in fishes exposed to E0 for all tissues. Thus, the elutriate was not harmful for the assessed fishes, since complexing agents presented in the sediment, such as goethite and hematite, may have triggered metals' chelation. In this scenario, the elutriate may have acted as a protective agent for the subjected organisms, unlike the Doce River waters, in which contaminants were proven to be hazardous for the aquatic biota.

The success of the fertilization and early larval development of the tropical sea urchin Echinometra lucunter (Echinodermata: Echinoidea) is affected by the pH decrease and temperature increase.

The decrease in the pH of oceans and the increase in their temperature are the two main problems observed in the marine ecosystems due to the increasing emission of CO2 in the atmosphere. Both conditions can affect the ecological processes of reproduction, recruitment and survival of the marine biota. Thus, the objective of the present study was to evaluate the effects of pH decrease and temperature increase of seawater on the fertilization success and embryo-larval development of a species of tropical sea urchin. For this purpose, fertilization success (gametes) and embryo-larval development rate were determined by exposing gametes and embryos to decreasing pH values (8.0 (control), 7.7, and 7.4) and increasing temperatures (26 (control), 28, 30, 34, and 38 °C). These conditions were tested associated with each other (in synergy). The gamete test was sensitive to all investigated scenarios, the fertilization success was significantly reduced in the conditions of increased temperature (28, 30, 34, and 38 °C) associated with the ideal pH (pH 8.0) and the conditions of reduced pH (pH 7.7, and 7.4), remaining unchanged only in the ideal condition (pH 8.0 + 26 °C). However, the embryo test displayed enhanced sensitivity in the scenarios of temperature increase (28, 30, 34, and 38 °C) associated with pH decrease conditions. A significantly reduction of 29%, 23% and 10% was observed in all tested pH values at 38 °C, when compared to the control group (80%, 79.5% and 63%, respectively). Therefore, the present study suggests that the occurrence of both scenarios may have a significant impact, in the coming years, on the population of Echinometra lucunter.

Metals, arsenic, pesticides, and microcystins in tilapia (Oreochromis niloticus) from aquaculture parks in Brazil.

The production of Nile tilapia (Oreochromis niloticus) in Brazil exhibits the highest growth rate in the world and represents approximately 45% of the total fish production. The objective of the present study was to assess the risk for human health due the consumption of tilapia farmed in net cages in eight aquaculture parks in Brazil. The concentrations of pesticides (40 compounds), metals (Mn, Ni, Zn, Cd, Pb, and Sn), arsenic, and cyanotoxins (microcystins) were evaluated in 16 fish from each park. Among analyzed pesticides, pyraclostrobin (0.18-0.32 mg/kg) and fenthion (0.0026-0.0037 mg/kg) exhibited values above the limit of quantification in the tilapia from Aracoiaba, Castanhão, and Ilha Solteira. The highest concentrations of As (0.44 µg/g) in fish tissues were found in Juara, Mn (0.21 µg/g) in Castanhão, and Zi (11.5 µg/g) were found in Três Marias. Furnas and Linhares exhibited the lowest metal concentrations. The estimated daily intake of muscle by the average Brazilian with 70 kg body weight is below the reference dose for all studied metals in all parks. Total free microcystins showed an accumulation pattern (muscle < gill < liver). The highest concentration in muscle was found in Castanhão (1043 µg/kg) samples. The results showed that fish exhibited metal, As, and pesticide tolerable daily intake (TDI) below the limit and pose low risk for human consumption. Otherwise, TDI for microcystins in fish of all studied parks was above the maximum level recommended by the World Health Organization, indicating that there exists a toxicity risk of fish consumption.

Temperature affects the toxicity of lead-contaminated food in Geophagus brasiliensis (QUOY & GAIMARD, 1824).

Lead is toxic to fish, and its toxicity can be aggravated by the water temperature. Geophagus brasiliensis populations are geographically widespread and thus live in areas with different temperatures. The objective of this work was to evaluate the effects of lead-contaminated feed in fish (Geophagus brasiliensis) exposed to different temperatures. A factorial experiment was performed with two temperatures (25 and 28°C), and two feeds (control and lead contaminated - 60 mg/kg) for a sum of four treatments (25°C, Control = 25/C; 25°C, 60 mg/kg = 25/60; 28°C, Control = 28/C and 28°C, 60 mg/kg = 28/60). Analyses of the lead accumulation, oxidative stress and genotoxic damage were performed. The gills and liver showed increased lead concentrations in fish receiving lead-contaminated food at both temperatures. The lead concentrations in the intestines and muscles of fish exposed to the 25/60 treatment was greater than it was in fish exposed to the 25/C treatment. The enzyme response in the gills and the micronuclei count increased in fish exposed to the 25/60 treatment. Higher temperatures can be a beneficial factor for Geophagus brasiliensis because they can hinder the absorption of lead, thereby reducing the damage caused to the organism.