Validation of qPCR reference genes in the endangered annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions.

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.

Organic contamination and multi-biomarker assessment in watersheds of the southern Brazil: an integrated approach using fish from the Astyanax genus.

We aimed to examine the responses of pollution biomarkers in feral fish from Astyanax genus collected at three hydrographic regions in southern Brazil and the capacity of these tools to differentiate between various levels of contamination. To achieve this, levels of organochlorine pesticides (liver), as well as the biomarkers AChE (muscle and brain), TBARS (liver), and EROD (liver) were assessed. Collections were conducted in four municipalities (Alegrete, Caraá, Lavras, and Santa Vitória) during 1 year, encompassing winter and summer. Fish from Alegrete were the most contaminated overall, but animals sampled in Caraá, and Lavras also displayed elevated levels of current-use pesticides. Elevated levels of endosulfans, DDTs, HCHs, and current-use pesticides were accompanied by elevated levels of TBARS in the liver. Conversely, fish from Santa Vitória exhibited the highest levels of PAHs, accompanied by elevated levels of EROD in the liver and reduced levels of AChE in muscle and brain. TBARS proved to be a reliable biomarker for assessing impacts arising from pesticide accumulation, while EROD and AChE served as valuable indicators of impacts resulting from PAHs accumulation. Ultimately, the results obtained in this study demonstrate the reliable use of the proposed biomarkers for tracking biological impacts stemming from aquatic pollution using feral Astyanax as biomonitoring species.

Chlorothalonil as a potential endocrine disruptor in male zebrafish (Danio rerio): Impact on the hypothalamus-pituitary-gonad axis and sperm quality.

Chlorothalonil is a broad-spectrum organochlorine fungicide widely employed in agriculture to control fungal foliar diseases. This fungicide enters aquatic environments through the leaching process, leading to toxicity in non-target organisms. Organic contaminants can impact organism reproduction as they have the potential to interact with the neuroendocrine system. Although there are reports of toxic effects of chlorothalonil, information regarding its impact on reproduction is limited. The aim of the present study was to evaluate the influence of chlorothalonil on male reproductive physiology using the zebrafish (Danio rerio) as ecotoxicological model. Zebrafish were exposed for 7 days to two concentrations of chlorothalonil (0.1 and 10 µg/L) along with a control group (with DMSO - 0.001%). Gene expression of hypothalamus-pituitary-gonad axis components (gnrh2, gnrh3, lhr, fshr, star, hsd17b1, hsd17b3, and cyp19a1), as well as hepatic vitellogenin concentration were assessed. In sperm cells, reactive oxygen species (ROS) content, lipid peroxidation (LPO), mitochondrial functionality, and membrane integrity and fluidity were evaluated. Results indicate that exposure to the higher concentration of chlorothalonil led to a reduction in brain gnr2 expression. In gonads, mRNA levels of lhr, star, and hsd17b1 were decreased at both chlorothalonil concentrations tested. Similarly, hepatic vitellogenin concentration was reduced. Regarding sperm cells, a decreased ROS level was observed, without significant difference in LPO level. Additionally, a higher mitochondrial potential and lower membrane fluidity were observed in zebrafish exposed to chlorothalonil. These findings demonstrate that chlorothalonil acts as an endocrine disruptor, influencing reproductive control mechanisms, as evidenced by changes in expression of genes HPG axis, as well as hepatic vitellogenin concentration. Furthermore, our findings reveal that exposure to this contaminant may compromise the reproductive success of the species, as it affected sperm quality parameters.

Temporal changes in metal and arsenic concentrations in blood and feathers of tropical seabirds after one of the largest environmental disasters associated with mining.

Monitoring of contaminant levels in wildlife over time is a tool for assessing the presence and persistence of environmental impacts at ecosystem, community and population levels. Tropical seabirds breeding in the Abrolhos Archipelago, 70 km off the Brazilian coast, forage in areas under the influence of the Doce River discharge. In 2015, the Fundão Dam collapsed and released ca 60 million tons of iron ore tailings into the ocean. In the present study, red-billed tropicbirds Phaethon aethereus and brown boobies Sula leucogaster breeding in Abrolhos were monitored over four years (2019-2022) for metal (Fe, Mn, Zn, Cu, Cr, Hg, Pb, Cd) and metalloid (As) concentrations in blood and feathers. Over six sampling events, metal (loid) concentrations showed strong temporal variation in both tissues. Overall, feathers showed greater element concentrations than blood, with stronger correlations between elements, especially Mn and the nonessential As, Cd, Hg and Pb. Mn is one of the major chemical markers of the Fundão Dam tailings. Metal (loid) concentrations in the tropical seabirds evaluated were above suggested threshold levels for most nonessential elements (As, Cd and Pb), especially in February 2021, when metal (loid) concentrations peaked in feathers. In this case, values were orders of magnitude higher than those observed in other sampling events. This occurred one year after a major rainy season in the Doce River basin, which increased river discharge of contaminated mud into the ocean, where contaminants are further remobilized by winds and currents, resulting in transference through the marine food web. This finding is consistent to what has been observed for other ecosystem compartments monitored in the region under the influence of the Doce River. Our findings highlight the utility of using tropical seabirds as sentinels of marine pollution, revealing strong temporal patterns in metal (loid) concentrations associated to bottom-up climatic processes.

Assessment of oxidative stress biomarkers in the threatened annual killifish Austrolebias charrua exposed to Roundup.

This study aimed to analyze the toxic effects of Roundup Transorb® on the endangered Neotropical annual killifish Austrolebias charrua through the assessment of molecular and biochemical biomarkers. The fish were collected in temporary ponds and exposed to environmentally realistic concentrations of the herbicide (5 mg.L-1 for 96 h). The production of ROS, lipid peroxidation, DNA damage, and membrane fluidity were evaluated in the blood cells by flow cytometry. The mRNA expression of the antioxidant-related genes sod2, cat, gstα, atp1a1, gclc, and ucp1 across the brain, liver, and gills was quantified. The acute exposure of annual killifish to Roundup significantly increased ROS production, lipid peroxidation, and DNA damage in their erythrocytes. Likewise, Roundup Transorb® decreased membrane fluidity in the blood cells of the exposed fish. Gene expression analysis revealed that Roundup exposure alters the relative expression of genes associated with oxidative stress and antioxidant defense. Our results give rise to new insights into adaptive mechanisms of A. charrua in response to Roundup. Since Brazilian annual killifishes strongly risk extinction, this study paves the way for developing novel biotechnologies applied to environmental monitoring and aquatic toxicology assessment.

Sex-biased response of pollution biomarkers in fish: Insights from the killifish Poecilia vivipara.

Copper ions (Cu) are one of the most frequent trace-contaminants found in Brazilian waters and, although considered as an essential element, in high concentrations can accumulate and induce toxicity. Biomarkers are important tools that can be used to assess these impacts, but to be considered trustworthy, they have to be previously tested in target organisms through laboratory studies under controlled conditions. However, many of these experiments are conducted using only males, as it is believed that the hormonal variation of females can bias the results, increasing data variability. Notwithstanding, few studies have actually tested this hypothesis, highlighting the importance of considering and measuring the role of sex in ecotoxicological studies. The aim this study was to evaluate the influence of sex on biomarkers classically used in environmental monitoring programs using the fish Poecilia vivipara as model. For this, females and males were exposed for 96 h to two Cu concentrations (9 and 20 µg/L) and a control group. In liver and gills, Cu accumulation, total antioxidant capacity (TAC) and lipid peroxidation (LPO) were evaluated. In addition, samples of peripheral blood were used for neutrophil to lymphocyte ratio determination, a measure of the onset of secondary stress. Results show that Cu hepatic accumulation did not differ between females and males, but higher levels of this metal were observed in exposed animals compared to control fish. Additionally, interactive effects were observed for hepatic LPO, as males showed elevated oxidative damage in comparison to females. Moreover, Cu exposure elevated hepatic LPO relative to control only in males, but this increase in oxidative damage was not accompanied by changes in liver TAC. On the other hand, differences in branchial Cu accumulation and LPO were not observed. Conversely, control females showed elevated TAC in comparison to control males, but Cu exposure eliminated this difference. Cu exposure also induced an increase in the N:L ratio, indicating the presence of a secondary stress response unrelated to sex. Ultimately, the findings of this study demonstrate that sex can influence the response of biomarkers that are typically used in ecotoxicological investigations in a multifaceted manner. As a result, using animals from a singular sex in such studies may result in consequential outcomes, potentially leading to underestimation or overestimation of results.

Oxidative Stress in a Wild Population of the Freshwater Fish Hyphessobrycon Luetkenii Chronically Exposed to a Copper Mining Area: New Insights into Copper Toxicology.

In this study, we examine markers of oxidative stress in the tetra Hyphessobrycon luetkenii collected from two locations in the copper contaminated João Dias creek (southern Brazil). Also, specimens were translocated from a clean reference section of the creek to a polluted stretch and vice-versa. Fish were held at in submerged cages for 96 h and then sacrificed. Nuclear abnormalities in erythrocytes and total antioxidant capacity, lipid peroxidation and protein carbonylation in gills, brain, liver and muscle displayed similar trends in both groups. Lipid peroxidation increased in all tissues of individuals translocated to the polluted site but only in liver and muscle of those translocated to the reference site. Increased protein carbonylation was also observed in gills of individuals translocated to the reference location. These results suggest similar oxidative stress among fish from the reference and polluted locations and that long-term metals exposure may require adaptations toward oxidative stress responses.

Biomineralization biomarkers to assess microplastics toxic effects in the freshwater snail Pomacea canaliculata.

Microplastics (MPs) pollution has increased the number of reports on the toxic effects on biota, especially aquatic organisms. Recently, studies highlighted changes in ion transport and concentration, especially Ca2+, in organisms exposed to MPs. For calcifying organisms, such as mollusks, Ca2+ homeostasis is critical for their shells construction. We investigated the effects of polyethylene (PE) MPs at 20 µg/L on biomineralization biomarkers (Ca2+ATPase, carbonic anhydrase, hemolymph [Ca2+], and shell regeneration) of the freshwater gastropod Pomacea canaliculata. Two experimental sets were performed: (1) animals in physiological condition and (2) animals with their shells excised. The results of the first set showed that within 24 h, the hemolymph [Ca2+] decreased, and the Ca2+ATPase activity increased in the mantle edge. For carbonic anhydrase (CA), the activity decreased in the gland and increased in the mantle. By 72 h, the hemolymph [Ca2+] had not changed, whereas both enzymes had increased in both tissues. In the second set, the hemolymph [Ca2+] increased after 72 h, whereas Ca2+ATPase activity decreased in both tissues. For AC, the opposite results were observed. At 120 h, calcium pumping was still reduced and CA values increased in the digestive gland. Additionally, MPs exposure increased the capacity of the gastropods to recover their shells. Based on this, our work provides novel data associating PE microplastic exposures (at 20 µg/L) and their potential to stimulate biomineralization enzymes of P. canaliculata, as well as increase shell regeneration in excised animal; a good prerogative for further investigations on both subjects that still lacks of more robust evidence.

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.

Distribution of PAHs and trace elements in Spartina densiflora and associated sediments from low to highly contaminated South American estuarine saltmarshes.

Estuarine saltmarshes from South America are exposed to several anthropogenic impacts due to diverse human activities that occur in both Atlantic/Pacific coastal environments. Primarily, chemical and petrochemical industries negatively impact saltmarshes generating inputs/deposition of non-essential trace elements (NTEs) and polycyclic aromatic hydrocarbons (PAHs) in sediments. The native cordgrass Spartina densiflora inhabits a wide range of environments, from non-impacted to highly impacted areas. It is important to know its performance towards pollution in different environmental settings in South America. The content of Cd, Hg, Pb, and PAHs was determined in the roots and leaves of S. densiflora, bulk sediments (Bs), and rhizosediments (Rs) of estuaries from Argentina, Brazil, and Chile. Differences in NTEs and PAHs levels were observed between Bs, Rs, and Spartina tissues from different saltmarsh areas. Differences in Rs/Bs (RHICF; rhizosediments concentration factors), roots/Bs (RCF; roots concentration factors) and leaves/roots (TF; translocation factors) factors were also found. In terms of NTEs, S. densiflora showed a high capability to increase levels in their Rs (RHICF>1) and bioconcentrate Cd in roots (RCF > 1), while no general translocation (TF < 1) was observed. Conversely, in cordgrass tissues, Bs and Rs, PAHs contents showed RCF and TF > 1, which was in line with lower levels in Rs related to Bs (RHICF<1) in most sites. These findings showed the S. densiflora capacity to retain, remove and/or translocate priority contaminants depending on intrinsic chemical characteristics and the level of contamination. The present study enables future considerations regarding the biomonitoring and phytoremediation/stabilization capabilities of Spartina in coastal environments.

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.

Health condition of Chelonia mydas from a foraging area affected by the tailings of a collapsed dam in southeast Brazil.

In 2015, the failure of the Fundão dam caused the release of 43 million m3 of tailings into the Doce River Basin, in southeast Brazil. It was considered the largest environmental disaster of the world mining industry. The tailings, composed mostly of heavy metals, caused massive destruction of the Doce River ecosystem endangering the organisms that live in the coastal zone where the mud reached the ocean. Among the exposed species are the sea turtles that use the region for food. The aim of this study was to evaluate the effect of contaminants on the health status of juvenile green sea turtles that feed in a coastal area exposed to ore mud (Santa Cruz) and to compare them with animals from an area not directly affected (Coroa Vermelha). A physical examination was performed to determine the health status. Blood samples were analyzed for hematological and biochemical parameters, and metal concentrations (As, Cd, Cr, Cu, Fe, Hg, Mn, Pb, and Zn). Santa Cruz sea turtles had more ectoparasites and a higher incidence of fibropapillomatosis. Statistically significant differences between sites were found for levels of calcium, phosphorus, glucose, protein, albumin, globulin, cholesterol, triglycerides, urea, CPK, ALT, and AST. The count of leukocytes, thrombocytes, and heterophils, as well as the concentrations of As and Cu were higher in Santa Cruz turtles. Together the results show a worse nutritional status and a greater degree of liver and kidney damage in animals affected by the tailings. The health status may indicate a physiological deficit that can affect their immune system and behavior, which is supported by the higher fibropapillomatosis tumor score and ectoparasite load in these animals. These results support the need for long-term monitoring of the exposed area to quantify the direct and indirect influence of the heavy metals levels on sea turtles and how this reflects the environmental health.

Metal accumulation induces oxidative stress and alters carbonic anhydrase activity in corals and symbionts from the largest reef complex in the South Atlantic ocean.

We evaluated the influence of metal accumulation on the oxidative status [lipid peroxidation (LPO) and total antioxidant capacity (TAC)] and carbonic anhydrase (CA) activity in host and symbionts of the coral Mussismilia harttii and the hydrocoral Millepora alcicornis collected in Abrolhos Reef Banks (Northeast Brazil), potentially impacted by a major mine dam rupture. Considering metal levels measured in reefs worldwide, Abrolhos corals had higher Fe and Mn levels than expected for preserved offshore reefs. Increasing concentrations of arsenic (As), chromium (Cr) and manganese (Mn) drove inhibition of CA and increased oxidative damage in the hydrocoral M. alcicornis. The impairment of enzymatic activity in the symbiotic algae of M. alcicornis may be related to the oxidative stress condition. The hydrocoral M. alcicornis was more affected by metals than the coral M. harttii, which did not show the expected CA inhibition after metal exposure. Our results suggest that CA activity can be applied as a complementary biomarker to evaluate the physiological impacts of environmental metal contamination in reefs. Also, the metal levels and biochemical biomarkers reported in the present study may provide reference data to monitor the health of reefs impacted by a relevant dam rupture.

Metal Accumulation and Ion Regulation in the Fish Hyphessobrycon luetkenii Living in a Site Chronically Contaminated by Copper: Insights from Translocation Experiments.

Fish living in the João Dias creek (southern Brazil) have to deal with trace-metal contamination in the long-term basis, as this aquatic environment has been historically impacted by copper mining activities. In order to survive in this harsh environment, the local biota had to develop adaptations related to pollution tolerance. The aim of this study was to test if biochemical mechanisms related to osmoregulation were among these adaptations, using translocation experiments. Water ionic and trace-metal compositions were measured in a nonmetal impacted site (NMIS) and in a metal impacted site (MIS) of this creek. Also, whole-body metal accumulation, ion concentration and branchial enzyme activity (Na,K-ATPase and carbonic anhydrase) were evaluated in Hyphessobrycon luetkenii. In both NMIS and MIS, fish were collected and immediately stored, kept caged or translocated from sites. The result shows that waterborne Cu was 3.4-fold higher at the MIS. Accordingly, animals that had contact with this site showed elevated whole-body Cu levels. Moreover, both translocated groups showed elevated Na,K-ATPase activity. Additionally, fish translocated from the NMIS to the MIS showed lower carbonic anhydrase activity. These findings indicate that H. luetkenii chronically or acutely exposed to naturally elevated waterborne Cu showed a rapid Cu bioaccumulation but was unable to readily excrete it. Moreover, classic Cu osmoregulatory toxicity related to Na,K-ATPase inhibition was not observed. Conversely, impacts in ammonia excretion related to carbonic anhydrase inhibition may have occurred.

Temporal and spatial variations in metals and arsenic contamination in water, sediment and biota of freshwater, marine and coastal environments after the Fundão dam failure.

Temporal and spatial variabilities in concentrations of metals (Cd, Cr, Cu, Fe, Hg, Mn, Pb and Zn) and metalloid (As) associated with the Fundão dam tailings were evaluated in water, sediment and biota from freshwater (tributary, river, lakes and lagoons), marine and coastal (mangroves and beaches) ecosystems affected by the Mariana dam disaster (southeastern Brazil). In freshwater shrimps and fishes, temporal increases in the concentrations of most elements analyzed were observed. This finding was clearly associated with temporal increases in the concentrations of As and metals observed in both water and sediment. In turn, freshwater plankton showed only a temporal increase in Hg concentration, which was also associated with an increased concentration of this metal in the abiotic matrices. In marine fishes, temporal increases were only observed for Cu, Fe and Pb concentrations. Also, temporal increase was observed for Fe concentration in marine plankton (phytoplankton and zooplankton) and shrimps. Marine phytoplankton also showed a temporal increase in Hg concentration. All these findings were clearly associated with temporal increases in the concentrations of these metals in marine sediments. Mangrove crabs showed temporal increases in Hg and Cd, which were associated with temporal increases in water Hg and sediment Cd concentrations, respectively. In turn, beach crabs displayed temporal increases in Mn and Zn, which were associated with temporal increases in the concentrations of these metals in sediments, especially for Mn. In summary, all environmental matrices evaluated in the present study were shown to be contaminated with metals and metalloid associated with the Fundão dam tailings. Additionally, findings reported in the present study relative to the spatial variabilities observed in the whole aquatic area affected by the Fundão dam failure clearly reinforce the need of incorporating biological diversity in monitoring programs aiming to assess environmental health of aquatic systems, considering that patterns of metals and metalloid contamination levels may vary among taxa.

Ecological trap for seabirds due to the contamination caused by the Fundão dam collapse, Brazil.

Human-induced rapid environmental changes can disrupt habitat quality in the short term. A decrease in quality of habitats associated with preference for these over other available higher quality is referred as ecological trap. In 2015, the Fundão dam containing iron mining tailings, eastern Brazil, collapsed and released about 50 million cubic meters of metal-rich mud composed by Fe, As, Cd, Hg, Pb in three rivers and the adjacent continental shelf. The area is a foraging site for dozens of seabird and shorebird species. In this study, we used a dataset from before and after Fundão dam collapse containing information on at-sea distribution during foraging activities (biologging), dietary aspects (stable isotopes), and trace elements concentration in feathers and blood from three seabird species known to use the area as foraging site: Phaethon aethereus, Sula leucogaster, and Pterodroma arminjoniana. In general, a substantial change in foraging strategies was not detected, as seabirds remain using areas and food resources similar to those used before the dam collapse. However, concentration of non-essential elements increased (e.g., Cd and As) while essential elements decreased (e.g., Mn and Zn), suggesting that the prey are contaminated by trace elements from tailings. This scenario represents evidence of an ecological trap as seabirds did not change habitat use, even though it had its quality reduced by contamination. The sinking-resuspension dynamics of tailings deposited on the continental shelf can temporally increase seabird exposure to contaminants, which can promote deleterious effects on populations using the region as foraging sites in medium and long terms.

Avian blood and feathers as biological tools to track impacts from trace-metals: Bioaccumulation data from the biggest environmental disaster in Brazilian history.

The Mariana's dam collapse was the worst environmental disaster in Brazilian history and one of the biggest worldwide. This perverse disaster resulted in the release of a contaminated mud tsunami that greatly impacted both aquatic and terrestrial biota. The aim of this study was to track environmental impacts resulting from Mariana's disaster using trace-element accumulation in avian blood and feathers as monitoring tool. For this, animals were collected at Doce River mouth (Regência), origin of the contaminated mud, and at southern (Aracruz) and northern (São Mateus) coastal areas. There were two sampling events (2018-2019), one during the winter period (first collection) and another during the summer period (second collection). Trace-element assessed were As, Cd, Cr, Cu, Fe, Pb, Hg, Mn and Zn. Findings show that inorganic contamination in birds followed a strong spatial and temporal behavior. In terms of time patterns, blood and feather contamination levels were markedly elevated in samples from the first collection event in comparison to the second. In terms of space, bioaccumulation was greater in Doce River mouth (Regência) and southern area (Aracruz). Additionally, levels found for Pb, Hg, As and Cd in birds from the first expedition were above proposed threshold levels, indicating possible health impacts. Finally, it is concluded that avian from areas impacted by Mariana's disaster still presents elevated levels of inorganic contamination even after 5 years following the event. Additionally, local climatic factors might pose as major drivers for bioaccumulation patterns in these animals, resulting in marked spatial and temporal fluctuations.

Response of biomarkers to metals, hydrocarbons and organochlorine pesticides contamination in crabs (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil).

Response of biomarkers to chemical contamination was evaluated in crabs of the Callinectes genus (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil). Biomarkers evaluated included hepatopancreatic metallothionein-like proteins (MTLP) and lipid peroxidation (LPO), as well as muscle acetylcholinesterase (AChE). Tissue concentrations of metals (pereiopod muscle and hepatopancreas), hydrocarbons (hepatopancreas) and organochlorine pesticides (hepatopancreas) were also evaluated. Crab samples were collected in three sites of each estuary (São Marcos Bay and São José Bay). Sampling was performed in August/2012 (dry season), January/2013 (rainy season), August/2013 (dry season), and January/2014 (rainy season). Concentrations of chemical contaminants and responses of biomarkers showed significant spatial (São Marcos Bay and São José Bay) and/or seasonal (dry and rainy seasons) and annual (2012-2014) variability. However, a general higher Zn concentration was observed in hepatopancreas of crabs from São José Bay. In turn, a general higher Cd concentration paralleled by oxidative damage (LPO) was observed in hepatopancreas of crabs from São Marcos Bay. As expected, these findings support the idea that this bay is more intensively or chronically impacted by industrial activities while the São José Bay is likely more affected by domestic activities. Interestingly, LPO level in crab hepatopancreas showed to be the most reliable and adequate biomarker to distinguish the two bays.

Combining elevated temperature with waterborne copper: Impacts on the energy metabolism of the killifish Poecilia vivipara.

We have previously demonstrated in a companion work that acclimation to 28 °C potentiated waterborne copper (Cu) toxic effects in Poecilia vivipara through oxidative stress-related processes. In the present study, we hypothesized that these results were related to kinetic metabolic adjustments in enzymes from aerobic and anaerobic pathways. To test this, P. vivipara was acclimated to two temperatures (22 °C or 28 °C) for three weeks and then exposed to Cu (control, 9 or 20 µg/L) for 96 h. The activity of enzymes from glycolysis (pyruvate kinase [PK] and lactate dehydrogenase [LDH]), Krebs cycle (citrate synthase [CS]) and the electron transport chain system (ETS) were assessed in gills, liver and muscle. Interactive effects were only seen for hepatic LDH activity, as both metal exposure and heat stress, combined or not, inhibited this enzyme, showing a suppression in anaerobic pathways. Conversely, a Cu main effect was present in the liver, expressed as an elevation in ETS activity, showing an enhancement in hepatic aerobic metabolism likely related with the very energy-demanding process of metal detoxification. Moreover, this study shows that P. vivipara has a remarkable ability to compensate heat stress in terms of energy metabolism, as we could not observe acclimation temperature effects for most of the cases. Nonetheless, a tissue-dependent effect of elevated temperature was observed, as we could observe an inhibition in muscular CS activity. Finally, it is concluded that kinetic adjustments in terms of the energy metabolism are not related with the temperature-dependent elevation of Cu toxicity in P. vivipara as we previously hypothesized.

Arsenic, lead and cadmium concentrations in caudal crests of the yacare caiman (Caiman yacare) from Brazilian Pantanal.

Wetlands are environments of extreme importance due to their high biodiversity and invaluable ecosystem services. Nevertheless, wetlands worldwide are under the increasing threat of the effects of contaminants, which put at risk the biota and the ecosystems. Herein the concentrations of non-essential and toxic elements arsenic (As), lead (Pb) and cadmium (Cd) in caudal crests of the yacare caiman (Caiman yacare) from the Brazilian Pantanal, one of the largest wetlands in the world, were investigated aiming to compare concentrations in individuals from a large pristine area (Southern Pantanal) to animals from an area close to potential sources of anthropogenic emissions (Northern Pantanal). Levels above the limit of detection were found for all the elements in the majority of the analysed samples. The highest mean concentration was detected for As in samples from both sites, followed by Pb and Cd in samples from Southern Pantanal, and Cd and Pb in samples from Northern Pantanal. Significant negative correlations were found between As and Cd concentrations and the size of the individuals from the Southern Pantanal. Surprisingly, concentrations of all three elements were higher in pristine Southern Pantanal, but with significant differences only for Cd. This result suggests that natural sources and processes may be acting for the mobilisation and availability of As, Pb and Cd for Southern Pantanal biota, which reflected in the contamination of C. yacare. Data from the literature showed higher levels of As, Pb and Cd in abiotic compartments in Southern Pantanal and adjacent plateaus in comparison to Northern Pantanal, corroborating the higher concentrations detected in biotic samples from the south. Thus, natural sources seem to play a relevant role in the contamination of biota by As, Pb and Cd in Southern Pantanal, characterising an intriguing pattern that could be tested for other species.