Combined physiological and behavioral approaches as tools to evaluate environmental risk assessment of the water accommodated-fraction of diesel oil.

There is an increasing concern related to the toxic effects of the soluble portion of diesel oil on aquatic ecosystems and the organisms living in them. In this context, the aim of this study was to analyze the effects of diesel water accommodated-fraction (WAF) on behavioral and biochemical responses of mussels Perna perna. Animals were exposed to 5 and 20% of WAF for 96 h. Prior to the beginning of the experiments, Hall effect sensors and magnets were attached to the valves of the mussels. Valve gaping behavior was continuously recorded for 12 h of exposure and tissues (gills and digestive gland) were separated after 96 h of exposure. Overall, both behavior and biochemical biomarkers were altered due to WAF exposure. Animals exposed to WAF reduced the average amplitude of the valves and the fraction of time opened, and presented greater transition frequency, demonstrating avoidance behavior over the 12 h period. Furthermore, the biochemical biomarkers (GSH, GST, SOD and CAT) were altered following the 96 h of exposure to WAF. Considering the results presented, this study demonstrates the toxic potential of WAF in both shorter and longer exposure periods.

Marine shrimps as biomonitors of the Fundão (Brazil) mine dam disaster: A multi-biomarker approach.

The disruption of the Fundão dam released 43 million m3 of mine tailings into the Doce River until it flowed into the ocean through the estuary. The mine tailing changed the composition of metals in water and sediment, creating a challenging scenario for the local biota. We used multivariate analyzes and the integrated biomarker response index (IBR) to assess the impact of mine tailings on the bioaccumulation profile (As, Cd, Cr, Cu, Fe, Mn, Pb and Zn) as well as the biomarkers response in gills, hepatopancreas and muscle of shrimps sampled from different sectors during two dry seasons (dry1 and dry2) (Sep/Oct 2018; 2019) and two wet seasons (wet1 and wet2) (Jan/feb 2019; 2020). There was seasonal and local effect under bioaccumulation and biomarker response revealing that the pattern responses seen in each sector sampled changed according to the season. The greater IBR added to the strong association among the most metals tissue content (Cd, Cr, Cu and Mn) and sectors sampled during dry 1 suggests greater bioavailability of these metals to the environment in this period. Estuarine sectors stand out for high Fe bioavailability, especially during wet1, which seems to be associated with greater metallothionein content in hepatopancreas of shrimps. Native species of marine shrimps proved to be successful indicators of sediment quality besides being sensitive to water contamination by metals. The multi-biomarkers approach added to multivariate analysis supports the temporal and seasonal effects, signalizing the importance of continuous monitoring of the estuarine region to better know about the bioavailability of these metals, mainly Fe, and their long-term effects on the local biota.

Ecotoxicological impacts of the Fundão dam failure in freshwater fish community: Metal bioaccumulation, biochemical, genetic and histopathological effects.

This study investigates the ecotoxicological impacts of the Fundão dam rupture, one of the major environmental disaster that occurred in Brazil and in the world mining industry history, through multi-biomarkers responses and metals bioaccumulation in the fish community of different trophic levels. Specimens of the fishes (omnivorous/herbivorous and carnivorous) were collected along the Doce River channel and its affluent Guandú River, and in different lakes and coastal lagoons adjacent to the river channel, in the Espirito Santo State, Southeast of Brazil. Four sampling collections were carried out over two years (2018 to 2020, during dry and rainy seasons). For both trophic groups the biomarkers responses indicated physiological alterations related to metals exposure and showed strong seasonal variations. The principal component analysis and integrated biomarker response index showed that DNA damage and lipid peroxidation were more associated with dry season 2 (Sep/Oct 2019) and the oxidative damage in proteins, metallothioneins concentration and the activity of superoxide dismutase in the gills showed a greater association with rainy season 2 (Jan/Feb 2020). On the other hand, the enzymes of energy metabolism, catalase and histological damage in the liver and the gills, were more associated with the dry and rainy campaigns of the first year of monitoring. The multivariate approach also suggested a temporal intensification in the bioaccumulation of metals and biological effects in the lacustrine environments. Thus, these results demonstrate that the release of mineral residues from the rupture of the Fundão mine dam affects the health status of the fish from the Doce River basin, provoking metals bioaccumulation, hepatic and branchial damage in the fish besides inducing of enzyme activity related to metal contamination, even four years after the rupture.

Impacts of tailings of Fundão dam (Brazil) rupture on marine fish: Metals bioaccumulation and physiological responses.

This study evaluated the impacts of the mining tailings after the rupture of the Fundão dam on fish communities on the Atlantic Ocean southeast coast. Four sample collections were carried out over two years (2018-2020), in seasonal periods. Omnivorous/herbivorous and carnivorous fish were collected for analysis of metal bioaccumulation, multibiomarkers of environmental contamination and histopathology. Metal bioaccumulation was stronger correlated in carnivorous fish in the dry-2018 collection, besides higher activity of antioxidant enzymes, energy metabolism and higher morphological damage; however, there was less oxidative damage and less metallothioneins induction, and these variables were strongly associated with the wet-2020 collection. In a temporal view, it was possible to observe a reduction in metal levels in fish, except in the mouth of the Doce River. These events can be explained by seasonal natural events, which tend the resuspension and boost metal levels, mainly in the mouth region during the rainy season.

Biochemical response and metals bioaccumulation in planktonic communities from marine areas impacted by the Fundão mine dam rupture (southeast Brazil).

The rupture of the Fundão dam (Mariana, MG, southeast Brazil) released a huge flood of mine tailings to Doce river basin and its adjacent coastal area, in November 2015. This catastrophic event exposed aquatic communities to metal contamination related to mine tailings, but its biological effects are still poorly understood. This study investigates how biochemical response related to metal exposure vary between locations and seasons during the years of 2018-2020, in planktonic communities (micro and mesoplankton). Marine microplankton collected in sectors in front and south of the Doce river mouth presented the highest lipid peroxidation (LPO) and induction of metallothioneins (MT). Mesoplankton collected in sectors in front and north of the Doce river mouth presented highest LPO, while MT in this size class did not respond to a clear spatial pattern. Our results showed that metals affected biomarkers in a non-linear pattern and highlighted the complex relationship between metals, biochemical parameters, and seasonality. The variation in biochemical biomarkers indicates physiological stress related to metals, once sectors contaminated by metals, especially Fe, Mn and Cd, presented stronger biochemical responses. Comparison of metal levels with bioaccumulation data collected before the impact indicates Fe, Cd, Cr and Cu more than 2-fold higher after disaster in sectors closer to the river. Literature showed that these sectors present zooplanktonic assemblages with lower biomass and biodiversity, suggesting that the opportunistic species that thrives in the area are also under biochemical stress, but possibly relies on repair or defense mechanisms. The physiological stress detected by this study is possibly related to the mine tailings, considering the metals that stood out and the proximity with the Doce river mouth. This suggests that the impacts related to the failure of Fundão dam are still affecting the marine planktonic community even three to four years after the environmental disaster.

The influence of the Doce River mouth on the microbiome of nearby coastal areas three years after the Fundão Dam failure, Brazil.

The failure of the Fundão Dam, considered the world's largest mining disaster, released more than 55 million m3 of ore tailings into the environment. The sediment plume formed by water and tailings spread along approximately 663 km of water bodies of the Doce River basin. It reached the Atlantic Ocean sixteen days after the dam failure. However, the effects of the dam failure in the marine coastal areas years after the disaster are still unknown. This study aims to evaluate water and sediment microbial communities of nearby coastal areas three years after the Fundão Dam failure, using 16S rRNA gene amplicon sequencing. A total of 441 samples from 25 locations were collected during two different seasons (dry and rainy). The results showed that the Doce River mouth seems to divide the microbial communities from the southern and northern stations into two groups. The plume of sediments from the Doce River seems to be impacting the marine microbiome even at the furthest sampling stations. Bacterial (Anaerolineaceae, Thermodesulfovibrionia and Rhodopirellula) and Archaeal (Bathyarchaeia and Woesearchaeia) taxa, found in high abundance in the sediment of the Doce River mouth, have been previously described in high abundance in heavy metal contaminated sediments, including the Doce River itself and in mine tailing sediments. Cyanobium, found in great abundance in the water samples from the Doce River mouth, was also reported as the most abundant in the water of the Doce River after the Fundão Dam failure. Overall, the farther from the Doce River mouth the sample was, the lower the relative abundances of these taxa were. These results provide strong evidence that the sediment plume released by the Fundão Dam failure is probably impacting the marine microbiome of nearby coastal areas, even three years after the dam failure.

Morphological characterization of hemocytes of the brown mussel Perna perna: An update.

Considering the importance of hemocyte characterization for immunological studies, this work aimed to characterize the hemocyte types of Perna perna mussels combining transmission electron microscopy and flow cytometry with the classical optical microscopy. The results indicated four type of hemocytes: hyalinocytes, semigranulocytes, granulocytes and blast-like cells.

DCOIT unbalances the antioxidant defense system in juvenile and adults of the marine bivalve Amarilladesma mactroides (Mollusca: Bivalvia).

DCOIT is a co-biocide that is part of the formulation of the commercial antifouling Sea-Nine 211® and although it is "safe to use", negative effects have been reported on the antioxidant defense system of non-target organisms. Therefore, the objective of this research was to verify and compare the response of antioxidant enzymes of juveniles and adults of Amarilladesma mactroides exposed to DCOIT. The animals were exposed to solvent control (DMSO 0.01%) and DCOIT (measured concentration 0.01 mg/L and 0.13 mg/L) for 96 h, then gills, digestive gland and mantle were collected for analysis of the enzymatic activity of glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). The results revealed that adults, in relation to juveniles, have low basal activity of GST and SOD enzymes in the gills and digestive gland and high basal activity of SOD and CAT in the mantle. DCOIT did not alter GST activity in the gills of any life stage, while both concentrations decreased SOD and CAT in adults. In the digestive gland, it was observed that DCOIT (0.13 mg/L) decreased the GST activity in adults and CAT in juveniles, and both concentrations of the co-biocide decreased the SOD and CAT in adults. In the mantle, DCOIT (0.13 mg/L) increased CAT in juveniles. We conclude that juveniles have greater basal activity of antioxidant enzymes than adults and, in addition, DCOIT negatively affected the adults of A. mactroides, mainly decreasing the activity of GST, SOD and CAT in the gills and digestive gland of these organisms.

Molecular and biochemical effects of the antifouling DCOIT in the mussel Perna perna.

Biological fouling is an unwanted phenomenon that results in economic losses to the shipping industry. To prevent fouling, antifouling paints are used. DCOIT (4,5- dichloro-2-n-octyl-4-isothiazolin-3-one) is a biocide present in many antifouling paint formulations, and is toxic to a wide range of organisms. The aim of the present study was to evaluate the effects of DCOIT on oxidative stress indicators of the brown mussel, Perna perna. Molecular (SOD-like, GSTO-like and MGST-like mRNA levels) and biochemical (activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH), reactive oxygen species (ROS) and protein carbonyls (PCO)) components were evaluated. Further, levels of biomarkers were assessed in the gills and digestive glands of mussels. Bivalves were exposed to DCOIT (control, 0.1 µg/L and 10 µg/L) for up to 96 h. DCOIT exposure decreased GSH content in gills. Moreover, exposure to DCOIT also decreased CAT activity in the gills and digestive glands of mussels. GST activity increased in digestive gland after exposure for 24 h to both concentrations of DCOIT tested. SOD activity, ROS levels and PCO content were not affected by exposure to the contaminant. Regarding the molecular biomarkers evaluated, DCOIT exposure altered mRNA levels of SOD-like in both tissues after 24 and 96 h of exposure, and decreased MGST-like mRNA levels in the digestive gland after 96 h of exposure to the chemical. These findings suggested that exposure to DCOIT may alter the biochemical and molecular functioning of P. perna, which may harm the species.

Antifouling biocide dichlofluanid modulates the antioxidant defense system of the brown mussel Perna perna.

Dichlofluanid is a fungicide employed as a booster biocide in antifouling paints, but information its toxicity to aquatic organisms is scarce. This study aims to evaluate biomarker responses in the mussel Perna perna exposed to dichlofluanid. Mussels were exposed to 0 (control), 0.1 µg/L (environmental concentration), 10, and 100 µg/L of dichlofluanid for 24 and 96 h. Byssus formation, oxygen consumption, and oxidative stress response were evaluated in gills and digestive glands. The results demonstrated that even the lowest dichlofluanid concentration causes a reduction in byssus biomass and water content. The higher concentrations caused an acute increase in oxygen consumption, which only returned to control levels after 96 h of exposure. ACAP levels and antioxidant enzyme activities were affected in both tissues with a larger effect observed in gill tissues as demonstrated by the IBR index. The overall results demonstrated that environmentally relevant concentrations of dichlofluanid would be deleterious to aquatic organisms.

First evidence of transcriptional modulation by chlorothalonil in mussels Perna perna.

Gills are considered a key player in organism defenses against environmental pollutants. Since it is the major site of uptake of waterborne chemicals, the modulation of important cellular defenses is expected in this tissue. Chlorothalonil, a fungicide presented in herbicides and antifouling paints, might be responsible for toxicity in marine biota. In this context, mussels were exposed to 0.1 µgL-1 and 10 µgL-1 of chlorothalonil for 24 h and 96 h. Genes from biotransformation and antioxidant defense pathways were investigated. Overall, we report, for the first time, an increase in the transcripts of the AhR-like, SULT1A1-like, CYP1A2-like, GSTO-like, MGST-like and SOD-like genes in the gills of the brown mussel Perna perna. This up-regulation was observed mostly after 96 h of exposure to chlorothalonil. Those results reinforce the important role of gills in xenobiotic metabolism and suggest the involvement of the mentioned genes in the detoxification of the compound. Throughout biotransformation and antioxidant defenses pathway, mussels exposed to chlorothalonil are activating mechanisms of defense against this contaminant.

Effects of DCOIT (4,5-dichloro-2-octyl-4-isothiazolin-3-one) to the haemocytes of mussels Perna perna.

Bivalve molluscs rely only on an innate immune system to execute cellular and humoral processes. Haemocytes, the haemolymph circulating cells, play a major role in this type of immunity, principally regarding cellular defences. Considering that environmental pollutants can affect the immune system of invertebrates, this work evaluated the effects of the antifouling biocide 4,5-dicloro-2-n-octil-4-isotiazolin-3-ona (DCOIT) on the haemocytes of mussels Perna perna. Individuals were exposed to 0 (control), 0.1 µg L-1 and 10 µg L-1 of DCOIT for up to 96 h. The analysed parameters included: total (THC) and differential (DHC) haemocyte count, cellular viability, adhesion capacity, phagocytic activity, levels of reactive oxygen species and DNA damage. Moreover, the stress on stress (SOS) response of mussels was analysed as a general stress index. The results show that DCOIT increased the haemocyte adhesion capacity and caused a decrease in THC and in the haemocyte viability after 24 h of exposure. After 96 h of exposure, DCOIT only affected the haemocyte adhesion capacity, which was decreased by biocide exposure. Moreover, exposure to DCOIT for 96 h did not affect the capacity for air survival of mussels. These results indicate that DCOIT interferes in important parameters associated with the innate immunity of P. perna, mainly after 24 h of exposure. It is suggested that the animals were able to develop some compensatory response strategy, making them more resistant to the biocide.

Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna.

Chlorothalonil is an effective fungicide used in agriculture and formulations of antifouling paints, which use and possible toxicity has been generating great concern. Thus, the present study investigated the effects of chlorothalonil on the antioxidant defense system (ADS) of the mussel Perna perna. The ADS was evaluated in gills and digestive gland after 24 h and 96 h of exposure to environmental relevant levels of chlorothalonil (0.1 and 10 µg/L). The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutamate cysteine-ligase (GCL) and glutathione S-transferase (GST), levels of non-enzymatic defenses, represented by glutathione (GSH), and lipoperoxidation (LPO) and protein carbonyls (PCO) were evaluated. Results indicated that exposure to chlorothalonil is affecting the ADS in both tissues. While the activity of SOD increased and GST and GSH were not altered in gills, they decreased in digestive gland after 24 h of exposure to 10 µg/L of chlorothalonil. The contrasting results indicate that gills and digestive gland presented different patterns of responses after exposure to chlorothalonil. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. It also should be highlighted that the fungicide reduced the glutathione metabolism in the digestive gland, which can lead to an imbalance of the redox state within the cells of animals.

Characterization of different DNA repair pathways in hepatic cells of Zebrafish (Danio rerio).

The concern about DNA damage has directed efforts toward evaluating the genotoxic potential of physical and chemical agents. Since the extent of DNA damage is also related to the capacity of the organism in repairing the DNA, the advance of toxicological studies on this area depends on the characterization of the DNA repair mechanisms in the available models. The cellular zebrafish models, for example, replace mammalian cells to answer ecologically relevant questions on aquatic toxicology. So, the aim of the present study was to characterize the nucleotide excision repair (NER) and photoreactivation (PER) in two cellular models of Danio rerio liver, primary hepatocytes and ZF-L (Zebrafish Liver) cell line. We performed kinetic studies of the DNA damage levels after exposure to 6.8 J/m2 UVC using the T4-PDG modified Comet Assay, and determined the expression levels of important genes involved in NER, PER and base excision repair using RT-qPCR. It was observed that both ZF-L cell line and primary hepatocytes exhibit similar NER and PER activity. Primary hepatocytes showed similarities in the gene expression of most of the evaluated repair genes with the original tissue. These results indicate that both primary hepatocytes and ZF-L cells are useful models for toxicological studies aiming to evaluate NER and PER in hepatic cells. Moreover, the similarities in gene expression between the cellular models suggest that the ZF-L cells retain the DNA repair characteristics of the primary hepatocytes and, thus, could serve as replacement to this primary culture, reducing the use of animals in research.

MXR response in sea anemones: Effect of temperature, salinity and copper.

Multixenobiotic resistance (MXR) phenotype is a cellular defense which can eliminate toxic substances from cells. Several studies describe the MXR activity after pollutant exposure, but little is known about the interference of abiotic factors in this mechanism. The present study aimed to evaluate MXR activity in sea anemones Bunodosoma cangicum after in vivo and in vitro exposures to different temperatures (15, 20 and 25C) and salinities (15, 30 and 45‰) associated or not with copper (0, 7.8 and 15.6 µg/L). Results showed that low temperature inhibited the MXR activity in vivo and in vitro, while salinity did not alter this activity. Copper could change the response, mainly at different temperatures (15 and 25 °C) - 7.8 µg/L Cu activated in vivo and in vitro and 15.6 µg/L Cu in vitro inhibited MXR activity in relation to same copper concentrations at 20 °C. Results for MXR activity found between in vivo and in vitro exposures were similar among temperature treatments and salinities; however, under hyperosmotic shock, in vivo exposure showed that animals has different response than isolated cells. The animals exposed to salinity 45‰ produced a mucus layer as a defense mechanism, because of this protection the response was different between in vivo and in vitro exposures. Concluding, temperature affects MXR activity independently of the presence of copper and each model of exposure contributes with different type of knowledge (cellular mechanism/systemic response).

Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (ZF-L).

Glyphosate is the active component of many commonly used herbicides; it can reach bodies of water through irrigated rice plantations. In the present study, we evaluated the effect of glyphosate and Roundup® (a glyphosate-based herbicide) in established culture of the zebrafish hepatocyte cell line ZF-L after 24 and 48 h of exposure to concentrations of 650 and 3250 µg/L. We observed a reduction in metabolic activity and lysosomal integrity, and an increase in cell number after 24 h of Roundup® exposure at the highest concentration. An increase in active mitochondria and apoptotic cells was observed following 24 h exposure to glyphosate and Roundup®, while only exposure to Roundup® induced an increase in necrotic cells. Rhodamine B accumulation decreased after 48 h exposure to 650 µg/L of Roundup®; this reduction is indicative of increased activity of ABC pumps. Overall, the present findings highlighted the hazard of glyphosate exposure not only in the commercial formulation but also glyphosate alone, since both can induce damage in the ZF-L cell line primarily through the induction of apoptosis.

Induction of oxidative stress by chlorothalonil in the estuarine polychaete Laeonereis acuta.

Chlorothalonil is an active biocide applied in antifouling paints, and also used as fungicide in agricultural activities with the purpose to protect plants from foliar and seed diseases. Thus, the aim of this study was to evaluate the effects of chlorothalonil exposure on biochemical biomarkers of oxidative metabolism as well as on cholinesterases in the estuarine polychaete Laeonereis acuta. Animals were exposed for 24 and 96 h to the following nominal concentrations of chlorothalonil: 0.1, 10.0 and 100.0 µg/L. The antioxidant capacity against peroxyl radicals (ACAP) and the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), glutamate cysteine ligase (GCL), acetylcholinesterase (AChE) and propionylcholinesterase (PChE) were evaluated in whole-body tissue. In addition, the levels of reduced glutathione (GSH), lipid peroxidation (LPO), glycogen and lactate levels were also analyzed. A reduction in ACAP levels was observed in animals exposed to the higher chlorothalonil concentration, concomitantly with an induction of GST activity as well as diminution in GSH content in these animals. This disturbance in the redox state of animal tissues leads to an oxidative stress situation, resulting in an induction in LPO levels. It was also demonstrated that chlorothalonil exposure causes alteration in AChE activity, possibly related to damage to membrane lipids. These results demonstrated that chlorothalonil possesses harmful effects to estuarine animals and its use as antifouling biocide has to be carefully reconsidered in risk analysis studies.

Comparison of the Base Excision Repair Activity in Liver Cell Models of Zebrafish (Danio rerio).

Fish cellular models are commonly used to study the toxic potential of environmentally relevant compounds. Several of these pollutants act on DNA and compromise its integrity. Little is known, however, about the DNA repair ability of these cellular models. Therefore, the aim of this study was to evaluate the DNA base excision repair (BER) of zebrafish Liver (ZF-L) cell line and primary hepatocytes. We performed kinetic studies of the DNA damage levels after exposure to hydrogen peroxide (H2O2, 20 µM for 10 min) using the Comet Assay. Ten minutes after H2O2 treatment, 16% and 50% of the initial damage, measured as comet tail length, were repaired in ZF-L cell line and primary hepatocytes, respectively. Primary hepatocytes repaired 50% of the damages twice as fast as ZF-L cell line and showed DNA damage levels similar to control 40 min after H2O2 treatment. The total recovery time for ZF-L model was of 180 min, which indicates the culture cells have a less efficient BER. In conclusion, both ZF-L cell line and primary hepatocytes exhibit BER activity; however, these cellular models have different repair capacity. In addition, we demonstrated that ZF-L cell line and primary hepatocytes are useful tools for ecotoxicological studies focusing on DNA single-strand breaks and BER.

Characterization of MXR activity in the sea anemone Bunodosoma cangicum exposed to copper.

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6µg/L) in presence and absence of MXR blocker (verapamil 50µM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6µg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8µgCu/L, but 15.6µgCu/L was similar to 0 and 7.8µg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves.