Effect of acute exposure to settleable atmospheric particulate matter emitted by the steel industry on hematology and innate immunity of fat snook (Centropomus parallelus).

The steel industry is a significant worldwide source of atmospheric particulate matter (PM). Part of PM may settle (SePM) and deposit metal/metalloid and metallic nanoparticles in aquatic ecosystems. However, such an air-to-water cross-contamination is not observed by most monitoring agencies. The region of Vitoria City is the main location of iron processing for exports in Brazil, and it has rivers, estuaries, and coastal areas affected by SePM. We have evaluated the effects of SePM on a local representative fish species, the fat snook, Centropomus parallelus. After acclimation, 48 fishes (61.67 ± 27.83 g) were individually exposed for 96 h to diverse levels of SePM (0.0, 0.01, 0.1 and 1 g/L-1). The presence of metals in the blood and several blood biomarkers were analyzed to evaluate the impact of SePM on stress signaling, blood oxygen transport capacity, and innate immune activity. Metal bioaccumulation was measured from blood in two separately analyzed compartments: intracellular (erythrocytes plus white blood cells) and extracellular (plasma). The major metals present at all contamination levels in both compartments were Fe and Zn, followed by Al and Cu, plus traces of 'Emerging metals': Ba, Ce, La, Rb, Se, Sr, and Ti. Emerging metals refer to those that have recently been identified in water as contaminants, encompassing rare earth elements and critical technology elements, as documented in previous studies (See REEs and TCEs in Cobelo-García et al., 2015; Batley et al., 2022). Multivariate analysis revealed that SePM had strong, dose-dependent correlations with all biomarker groups and indicated that blood oxygen-carrying capacity had the highest contamination responsiveness. Metal contamination also increased cortisol and blood glucose levels, attesting to increased stress signaling, and had a negative effect on innate immune activity. Knowledge of the risks related to SePM contamination remains rudimentary. However, the fact that there was metal bioaccumulation, causing impairment of fundamental physiological and cellular processes in this ecologically relevant fish species, consumed by the local human population, highlights the pressing need for further monitoring and eventual control of SePM contamination.

Oxidative stress and neurotoxicity induced by exposure to settleable atmospheric particulate matter in bullfrog tadpoles, Aquarana catesbeiana, (Shaw, 1802).

Bullfrog tadpoles, Aquarana catesbeiana, were exposed to settleable particulate matter (SePM), (1 g L-1, 96 h) and their organs were collected for analysis of metal/metalloid, oxidative stress and neurotoxicity in liver, muscle, kidney and brain. The SePM water of the exposed groups contained 18 of the 28 metals/metalloids detected in ambient particulate matter (APM). Fe56 and Al were those that presented the highest concentrations, Cr, Mn, Pb and Cu increased from 10 to 20 times and Ti, V, Sr, Rb, Cd, Sn and Ni increased from 1 to 3 times compared to the control. Bioaccumulation of metals/metalloids in the exposure water varied significantly between organs, with the muscle and liver showing the highest concentrations of metals, followed by the brain. Lipoperoxidation and malondialdehyde increased only in muscle, while carbonyl proteins increased only in the liver and brain. Regarding nitric oxide synthase, there was an increase in the liver and brain in the group exposed to SePM. Catalase activity decreased in the liver and muscle, while the activity of glutathione peroxidase, increased in the liver and kidney and decreased in muscle. Glutathione S-transferase, which is mainly responsible for detoxification, increased in the liver and decreased in muscle and the kidney. Cholinesterase activity increased only in the muscle. The results indicate oxidative stress, due to oxidation catalyzed by metals, components of SePM. Thus, the results contribute to the understanding that SePM has a deleterious effect on the aquatic environment, negatively affecting bullfrog tadpoles, in different ways and levels in relation to the analyzed organs.

Toxic risk evaluation of effluents from a swine biodigester in the plant models Lactuca sativa and Allium cepa.

Pig farming is recognized as an activity with great polluting potential. The aim was to investigate possible environmental risks of effluents from the stabilization pond (SP) and the raw effluent (RE) from the biodigestion process of swine residues, in different concentrations in the models Lactuca sativa and Allium cepa. Seeds were germinated in different dilutions, 100% (C1), 50% (C2), 25% (C3), 12.5% (C4), 6.25% (C5), 3.12% (C6), 0.78% (C7), and 0.39% (C8). Distilled water was used as the negative control (CN) and trifluralin (0.84 g/L-1) as the positive control. Germination (GR), root growth (RG), cell cycle, and oxidative stress (OS) were analyzed. To assess OS, the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) and the quantification of glutathione (GSH) and lipid peroxidation (LPO) were analyzed. Data were submitted to ANOVA (one way), followed by the Kruskal-Wallis mean test (P ≤ 0.05). Chemical analysis showed high values of Cu, Fe, Mn, and Zn. Dilutions (C1, C2, C3 RE) and (C1 and C2 SP) inhibited GR and RG of L. sativa and A. cepa than other concentrations. The mitotic index showed a reduction in C5 (RE), C6, and C7 (SP) of L. sativa and C3 and C4 (SP) of A. cepa in relation to CN and higher frequencies of chromosomal alterations. Regarding the OS, only the concentrations of SP treatment showed statistical difference in relation to the NC: in L. sativa model, GSH at (C5 and C8) concentrations and LPO (C7); in A. cepa model, SOD (C3 and C4), GST (C4, C5 and C6), GSH (C5 and C8), and CAT (C3 and C7). The alterations in metabolism are possibly related to the metals, such as zinc and copper, observed in high amounts in the raw waste. The results allowed us to conclude that the raw and stabilization pond effluents offer environmental risks, requiring caution and monitoring in the use of these effluents.

Toxicogenetic, biochemical, and anatomical effects of the herbicide Clethodim on Allium cepa L.

Pesticides are compounds with several chemical or biological agents developed to potentiate the biocide action. Their use is associated with increased economic and agricultural productivity worldwide but can harm health and the environment, damaging existing biota. Clethodim is a systemic post-emergent herbicide for grasses, highly selective for cotton, coffee, onions, carrots, soybeans, etc. Therefore, this work aimed to evaluate the harmful effect of the herbicide Clethodim with the model plant Allium cepa. A series of tests were conducted to evaluate the effects of the herbicide under study. Germination tests, root growth, cell, and nucleolar cycle analysis, as well as oxidative stress assessment and histological analysis of the roots, were performed. The results indicated that the herbicide demonstrated phytotoxicity, inhibiting germination at C1 (1.92 g/L) and C3 (0.84 g/L), and root growth at all concentrations, presenting mutagenicity at C1 (1.92 g/L) and C4 (0.24 g/L), evidenced by the increased frequency of micronuclei. In addition, changes were observed in the enzymatic activity of the enzymes catalase at concentrations C1 (1.92 g/L) and C2 (0.96 g/L) and ascorbate peroxidase at concentrations C1 (1.92 g/L), C2 (0. 96 g/L), and C3 (0.48 g/L) and in cell elongation at concentrations C1 (1.92 g/L) and C3 (0.48 g/L), demonstrated in histological analyses of the root apex.

Multi-level biological responses of Daphnia magna exposed to settleable atmospheric particulate matter from metallurgical industries.

Metallurgical industries are a continuous source of air pollution due to the amount of settleable particulate matter (SePM) they release. This SePM is a complex mixture formed by metallic nanoparticles and metals, which reach terrestrial and aquatic ecosystems and can be a significant source of contamination. The aim of this study was to evaluate the adverse effects of SePM at different levels of biological organization in order to estimate its ecological impacts on aquatic ecosystems. For this purpose, the crustacean Daphnia magna was exposed to different concentrations of SePM (0.01, 0.1, 1, 5, 10 g/L) using a multi-level response approach. The endpoints studied were: avoidance throughout 24 h in a non-forced exposure system, reproduction (total number of neonates per female after 21 days of exposure), acetylcholinesterase activity (AChE) after 48 h, and finally, the feeding rates during a short-term exposure (48 h) and a long-term exposure (21 day + 48 h). There was a negative effect of SePM on all responses measured at high concentrations. The avoidance was concentration-dependent and represented 88 % and 100 % at the two highest concentrations. The AChE activity was significantly inhibited at 5 and 10 g/L. The total number of neonates increased from 1 g/L of SePM and the first brood occurred earlier as of 5 g/L compared to control. The post-exposure feeding rates were lower during long-term exposure at the highest concentration. Chemical analyses were performed to characterize the metals present in this SePM, but this study did not report any direct relationship with toxicity, due to the chemical heterogeneity of the particles. The emission of compounds caused by anthropogenic activity may have significant ecological consequences, so it is important to consider these possible effects on aquatic biota generated by the mixture of metals present in SePM originated from metallurgical activities. Environmental and sectorial regulations are needed to prevent contamination and ecological disturbances.

Metal/metalloid bioconcentration dynamics in fish and the risk to human health due to water contamination with atmospheric particulate matter from a metallurgical industrial area.

Settleable atmospheric particulate matter (SeAPM) containing a mixture of metals, including metallic nanoparticles, has increased throughout the world, and caused environmental and biota contamination. The metal bioconcentration pattern in Nile tilapia (Oreochromis niloticus) was evaluated during a 30-day exposure to 1 g L-1 SeAPM and assessed the human health risk from consuming fish fillets (muscle) based on the estimated daily intake (EDI). SeAPM was collected surrounding an iron ore processing and steel industrial complex in Vitória city (Espírito Santo, Brazil) area. Water samples were collected daily for physicochemical analyses, and every 3 days for multi-elemental analyses. Metal bioconcentrations were determined in the viscera and fillet of fish every 3 days. The elements B, Al, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Rb, Sr, Ag, Cd, Pb, Hg, Ba, Bi, W, Ti, Zr, Y, La, Nb, and Ce were analyzed in SeAPM, water, and fish using inductively coupled plasma mass spectrometry. The metal concentration in SeAPM-contaminated water was higher than in control water. Most metals bioconcentrated preferentially in the fish viscera, except for the Hg and Rb, which bioconcentrated mostly in the fillet. The bioconcentration pattern was Fe > Al > Mn > Pb > V > La > Ce > Y > Ni > Se > As > W > Bi in the viscera; it was higher than the controls throughout the 30-day exposure. Ti, Zr, Nb, Rb, Cd, Hg, B, and Cr showed different bioconcentration patterns. The Zn, Cu, Sr, Sn, Ag, and Ta did not differ from controls. The differences in metal bioconcentration were attributed to diverse metal bioavailability in water and the dissimilar ways fish can cope with each metal, including inefficient excretion mechanisms. The EDI calculation indicated that the consumption of the studied fish is not safe for children, because the concentrations of As, La, Zr, and Hg exceed the World Health Organization's acceptable daily intake for these elements.

rGO outperforms GO in generating oxidative stress and DNA strand breaks in zebrafish liver cells.

Graphene oxide (GO) and reduced graphene oxide (rGO) are both widely applicable and there is a massive production throughout the world which imply in inevitable contamination in the aquatic environment by their wastes. Nevertheless, information about their interaction at the cellular level in fish is still scarce. We investigated the metabolic activity, reactive oxygen species (ROS) production, responses of antioxidant defenses, and total antioxidant capacity (TAC) as well as oxidative stress and DNA integrity in zebrafish liver cells (ZFL) exposed to (0.001, 0.01, 0.1 and 1 µg mL-1) of GO and rGO after two exposure period (24 and 72 h). Higher ROS production and no significant changes in the antioxidant defenses resulted in lipid peroxidation in cells exposed to rGO. Cells exposed to GO increased the activity of antioxidant defenses sustaining the TAC and avoiding lipid peroxidation. Comet assay showed that both, GO and rGO, caused DNA strand breaks after 24 h of exposure; however, only rGO caused DNA damage after 72 h of exposure. The exposure to rGO was significantly more harmful to ZFL cells than GO, even at very low concentrations. The cells showed a high capacity to neutralize ROS induced by GO preventing genotoxic effects and metabolic activity, thus sustaining cell viability. The time of exposure had different impacts for both nanomaterials, GO caused more changes in 24 h showing recovery after 72 h, while cells exposed to rGO were jeopardized at both exposure times. These results indicate that the reduction of GO by removal of the oxygen functional groups (rGO) increased toxicity leading to adverse effects in the cells, even at very low concentrations.

Integrating chemical and biological data by chemometrics to evaluate detoxification responses of a neotropical bivalve to metal and metalloid contamination.

Mangroves represent a challenge in monitoring studies due to their physical and chemical conditions under constant marine and anthropogenic influences. This study investigated metals/metalloids whole-body bioaccumulation (soft tissues) and the risk associated with their uptake, biochemical and morphological detoxification processes in gills and metals/metalloids immobilisation in shells of the neotropical sentinel oyster Crassostrea rhizophorae from two Brazilian estuarine sites. Biochemical and morphological responses indicated three main mechanisms: (1) catalase, superoxide dismutase and glutathione played important roles as the first defence against reactive oxygen species; (2) antioxidant capacity against peroxyl radicals, glutathione S-transferase, metallothionein prevent protein damage and (3) metals/metalloids sequestration into oyster shells as a mechanism of oyster detoxification. However, the estimated daily intake, target hazard quotient, and hazard index showed that the human consumption of oysters would not represent a human health risk. Among 14 analysed metals/metalloids, chemometrics indicate that Mn, As, Pb, Zn and Fe overload the antioxidant system leading to morphological alterations in gills. Overall, results indicated cellular vacuolization and increases in mucous cell density as defence mechanisms to prevent metals/metalloids accumulation and the reduction in gill cilia; these have long-term implications in respiration and feeding and, consequently, for growth and development. The integration of data from different sites and environmental conditions using chemometrics highlights the main biological patterns of detoxification from a neotropical estuarine bivalve, indicating the way in which species can cope with metals/metalloids contamination and its ecological consequences.

Settleable atmospheric particulate matter harms a marine invertebrate: Integrating chemical and biological damage in a bivalve model.

Some atmospheric pollutants may affect aquatic ecosystems after settling, generating contamination, bioaccumulation, and threats to aquatic species. Metallurgical processes result in the emission of settleable atmospheric particulate matter (SePM), including metals and metalloids, along with rare earth elements (REE) that are considered emerging contaminants. We report the 30-day exposure of brown mussels (Perna perna) to SePM collected in a metallurgical area of southeast Brazil close to estuarine ecosystems, followed by a 30-day clearance period, to evaluate the toxic potential of SePM to this model mollusk. The bioaccumulation of 28 elements identified in SePM and the sublethal effects were evaluated. REEs were found in SePM (Ce, Y, and La). Significant bioaccumulation of eight metals (Fe, Ni, Cu, Zn, Rb, Sr, Cd, and Ba) was found in the bivalves and correlates with the cytotoxicity and genotoxicity, showing a dose-dependent mode and suggesting a pre-pathological condition that could lead to ecological disturbances over time. Conversely, the unchanged lipid-peroxidation level after SePM exposure could indicate the effectiveness of the antioxidant system in protecting gills and digestive glands. The clearance period was not enough to successfully reverse the negative effects observed. So far, the current results enhance the comprehension of the negative role of SePM on metal bioaccumulation and metal-induced toxicity to aquatic biota. Thus, this report adds innovative findings on the role of SePM in aquatic pollution in coastal areas affected by atmospheric pollution, which should be relevant for future public policies to verify and control the environmental pollution.

Metalliferous atmospheric settleable particulate matter action on the fat snook fish (Centropomus parallelus): Metal bioaccumulation, antioxidant responses and histological changes in gills, hepatopancreas and kidneys.

Metallic smoke released by steel industries is constitute by a mixture of fine and gross particles containing metals, including the emerging ones, which sedimentation contaminates soil and aquatic ecosystems and put in risk the resident biota. This study determined the metal/metalloids in the atmospheric settleable particulate matter (SePM, particles >10 µm) from a metallurgical industrial area and evaluated metal bioconcentration, antioxidant responses, oxidative stress, and the histopathology in the gills, hepatopancreas and kidneys of fat snook fish (Centropomus parallelus) exposed to different concentrations of SePM (0.0, 0.01, 0.1 and 1.0 g L-1), for 96 h. From the 27 metals (Al, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Rb, Sr, Y, Zr, Nb, Mo, Ag, Cd, Sn, Ba, La, Ce, W, Hg, Pb, Bi) analyzed, 18 were quantified in SePM and dissolved in seawater. Metal bioconcentrations differed among organs; Fe and Zn were the metals most bioconcentrated in all organs, Fe was higher in hepatopancreas and Zn > Fe > Sr > Al was higher in kidneys. The activity of superoxide dismutase (SOD) decreased in the gills; SOD, catalase (CAT) decreased, and glutathione peroxidase (GPx) increased in hepatopancreas and, CAT, glutathione-S-transferase (GST) and the level of glutathione (GSH) increased in kidneys. The unchanged levels of lipid peroxidation and oxidized protein in any organ indicate that the antioxidant responses were efficient to avoid oxidative stress. Organ lesion indices were higher in the gills > kidneys > hepatopancreas, being higher in fish exposed to 0.01 g L-1 SePM. All changes indicate a tissue-specific metal/metalloids bioconcentration, antioxidant and morphological responses that all together compromise fish health. Regulatory normative are needed to control the emission of these metalliferous PM to preserve the environment and biota.

Changes in blood parameters and metabolism in bullfrog tadpoles, Lithobates catesbeianus, (Shaw, 1802) after exposure to the Sorocaba River (São Paulo, Brazil) water.

This study evaluated the genetic damage, oxidative stress, neurotoxicity, and energy metabolism in bullfrog tadpoles (Lithobates catesbeianus) exposed to water from two sites of the Sorocaba River, Ibiúna (PI), and Itupararanga reservoir (PIR), in summer and winter. After 96-h exposure, the erythrocyte number decreased in PI and increase in PIR in summer. Bullfrogs show oxidative unbalance (liver, kidney, and muscle), with alterations in the nitric oxide synthase and glucose 6-phosphate dehydrogenase. Cholinesterase increased in the brain in PI and PIR in the summer and decreased in PI in the winter. It also increased in the muscle in both PI and PIR in the winter. Tadpoles show alterations in the activity of the metabolic enzymes (liver, kidney, and muscle), such as phosphofructokinase, pyruvatokinase, malate dehydrogenase, and lactate dehydrogenase; and in the amount of glucose and triglycerides metabolites. Exposure to the Sorocaba River reflected a stressful situation for L. catesbeianus as the changes caused to their metabolism associated with oxidative stress and neurotoxicity may have effects on the development of tadpoles.

A multi-biomarker approach to assess the sublethal effects of settleable atmospheric particulate matter from an industrial area on Nile tilapia (Oreochromis niloticus).

Iron and steel industries discharge a large amount of atmospheric particulate matter (PM) containing metals and metallic nanoparticles (NPs) that contaminate not only the air, but also settle into the aquatic environments. However, the effects of settleable atmospheric particulate matter (SePM) on aquatic fauna are still poorly understood. This study aimed to evaluate the sublethal effects of a short-term exposure to a realistic concentration of SePM on Nile tilapia (Oreochromis niloticus) using a multi-biomarker approach: relative ventricular mass (RVM) and heart function, blood oxidative stress, stress indicators, hemoglobin concentration, metallic NPs internalization, and metal bioaccumulation. Exposed fish exhibited reduced hemoglobin content and elevated plasma cortisol and glucose levels, reflecting stressed states. Furthermore, SePM caused blood oxidative stress increasing lipid and protein oxidation, decreasing glutathione levels, and inhibiting superoxide and glutathione reductase activities. SePM exposure also increased RVM and improved cardiac performance, increasing myocardial contractile force and rates of contraction and relaxation. In the heart tissue there was a significant accumulation of Fe > Zn > > Cr > Cu > Rb > Ni > V > Mn > Se > Mo > As. On the other hand, in the erythrocytes there was significant accumulation of Sn > Zn > > Cr > Ti > Mn = Ni > Nb > As > Bi. The highest bioaccumulation factors were found for Cr, Zn and Ni in both tissues. NPs (Ti, Sn, Al, Fe, Cu, Si, Zn) were also detected in ventricular myocardium of fish exposed and nanocrystallographic analysis revealed a predominance of anatase phase of TiO2-NP, which is regarded to be more cytotoxic. The association between blood oxidative stress and energy expenditure to sustain increased cardiac pumping capacity under stress condition suggests that SePM has negative impacts on fish physiological performance, threatening their survival, growth rate and/or population establishment.

Liver dysfunction and energy storage mobilization in traíra, Hoplias malabaricus (Teleostei, Erythrinidae) induced by subchronic exposure to toxic cyanobacterial crude extract.

Microcystins (MC) are hepatotoxic for organisms. Liver MC accumulation and structural change are intensely studied, but the functional hepatic enzymes and energy metabolism have received little attention. This study investigated the liver and hepatocyte structures and the activity of key hepatic functional enzymes with emphasis on energetic metabolism changes after subchronic fish exposure to cyanobacterial crude extract (CE) containing MC. The Neotropical erythrinid fish, Hoplias malabaricus, were exposed intraperitoneally to CE containing 100 µg MC-LR eq kg-1 for 30 days and, thereafter, the plasma, liver, and white muscle was sampled for analyses. Liver tissue lost cellular structure organization showing round hepatocytes, hyperemia, and biliary duct obstruction. At the ultrastructural level, the mitochondria and the endoplasmic reticulum exhibited disorganization. Direct and total bilirubin increased in plasma. In the liver, the activity of acid phosphatase (ACP) increased, and the aspartate aminotransferase (AST) decreased; AST increased in plasma. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were unchanged in the liver, muscle, and plasma. Glycogen stores and the energetic metabolites as glucose, lactate, and pyruvate decrease in the liver; pyruvate decreased in plasma and lactate decreased in muscle. Ammonia levels increased and protein concentration decreased in plasma. CE alters liver morphology by causing hepatocyte intracellular disorder, obstructive cholestasis, and dysfunction in the activity of key liver enzymes. The increasing energy demand implies glucose mobilization and metabolic adjustments maintaining protein preservation and lipid recruitment to supply the needs for detoxification allowing fish survival.

Baroreflex responses of decerebrate rattlesnakes (Crotalus durissus) are comparable to awake animals.

A decerebrate rattlesnake, Crotalus durissus, has previously been used as a model Squamate for cardiovascular studies. It enabled instrumentation for concomitant recordings of diverse variables that showed autonomic responses. However, to validate the preparation and its scope for use, it is necessary to assess how close its cardiovascular variables are to non-decerebrate snakes and the effectiveness of its autonomic responses. Similarly, it is important to analyze its recovery profile after instrumentation and observe if it maintains stability throughout the duration of experimental protocol. Here we have objectively assessed these points by comparing decerebrate preparations and non-decerebrate snakes, after the occlusive cannulation of the vertebral artery. We have assessed cardiovascular variables and the baroreflex to analyze the presence, magnitude and stability of complex autonomic-controlled parameters as indicators of autonomic nervous system (ANS) functionality. After instrumentation, mean heart rates were high but recovered to stable values within 24 h. Mean arterial pressure stabilized within 24 h in control snakes and 48 h in decerebrate preparations. After that, both parameters remained stable. The operational gain and effectiveness index of the baroreflex recovered within the first 6 h after instrumentation in both experimental groups. In addition, the baroreflex capacities and its limits were also equivalent between the groups. These experiments demonstrated that decerebrate preparations and inactive, non-decerebrate snakes showed comparable recovery profiles following anesthesia and cannulation, maintained similar values of cardiovascular variables during experimental manipulation and exhibited functional, ANS modulated reflexes. Accordingly, the present results attest the relevance of this decerebrate preparation for studies on cardiovascular modulation.

Distribution and behavior of lipid droplets in hepatic cells analyzed by variations of citochemical technique and scanning electron microscopy.

Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.

Validation Testing of the European Portuguese Critical-Care Pain Observation Tool.

Aim. The study aim was to validate the Portuguese version of the Critical-Care Pain Observation Tool (CPOT) in the critically ill adult population of Portugal. Methods. A prospective, observational cohort study was conducted to evaluate the CPOT in mechanically ventilated patients who were admitted to an intensive care unit. A consecutive sample of 110 patients was observed at rest pre-procedure, during a nociceptive procedure (NP) which includes turning/positioning and endotracheal or tracheal suctioning and 20 min post-procedure. Two raters participated in the data collection. The discriminative validity, criterion validity, convergent validity and inter-rater reliability of the CPOT were examined. Results. The inter-rater reliability was excellent (0.93 ≤ α ≤ 1.00) at rest and fair to moderate (0.39 ≤ α ≤ 0.60) during the NP. The CPOT could discriminate between conditions with higher scores during the NP when compared to CPOT scores at rest (p < 0.001). The optimal CPOT cut-off score was >2, with a sensitivity of 71% and a specificity of 80%, and self-reported pain was the gold standard criterion. Significant correlations (<0.40) were found between CPOT scores, the heart rate and the respiratory rate during the nociceptive procedure. Conclusions. The CPOT appears to be a valid alternative for both ventilated and non-ventilated patients who are unable to communicate.

Concentration- and time-dependence toxicity of graphene oxide (GO) and reduced graphene oxide (rGO) nanosheets upon zebrafish liver cell line.

Graphene oxide (GO) and reduced graphene oxide (rGO) are carbon-based nanomaterials that have a wide range of applicability. Therefore, it is expected that their residual traces reach the aquatic environment, accumulate, and interact with its different compartments and the biota living in them. The concentration- and time-dependency response to GO and rGO in aquatic organisms are still poorly known. In the present study, the effects of GO and rGO on zebrafish hepatocytes were investigated using in vitro assays performed with established liver cell lines from zebrafish (ZFL). GO and rGO nanosheets were applied on ZFL cells at a concentration range of 1-100 µg mL-1 for 24 and 72 h. The internalization of GO and rGO nanosheets, reactive oxygen species (ROS) production, cell viability, and cell death were evaluated. The internalization of GO increased as the concentrations of GO increased. The rGO nanosheets were smaller than GO nanosheets, and their hydrophobic characteristic favors their interaction with the cell membrane. However, the rGO nanosheets were not observed in the uptake assay. Exposure for 72 h was found to cause harmful effects in ZFL cells, causing higher ROS production in cells exposed to rGO and stopping cell replication. Nevertheless, GO did not stop cell replication, but exposed cells had higher levels of apoptosis and necrosis. After 72 h, both GO and rGO were toxic, but with different mechanisms of toxicity.

Settleable atmospheric particulate matter induces stress and affects the oxygen-carrying capacity and innate immunity in Nile tilapia (Oreochromis niloticus).

Steel industry emissions of atmospheric particulate matter are responsible for air to water cross-contamination, which deposits metal/metalloid contaminants in aquatic ecosystems. This source of contamination has not been considered in most of the environmental monitoring protocols. Settleable atmospheric particulate matter (SePM) collected in an area of steel industry influence was used to analyze the sublethal effects on the hematological and innate immunological variables in Nile tilapia (Oreochromis niloticus) after short-term exposure (96 h). Blood samples were analyzed to evaluate the oxygen-carrying transport capacity, innate immune activity and stress biomarkers after exposure to ecologically relevant concentration of SePM. The exposure reduced blood oxygen-carrying capacity by lessening hematocrit, hemoglobin, erythrocyte, and mean corpuscular hemoglobin concentration. Compensatory increments in mean corpuscular volume and mean corpuscular hemoglobin have also been observed. The contaminant impacted the immune system by reducing the number of leukocytes, thrombocytes, and monocytes, total plasma protein, leukocyte respiratory activity, and by increasing lysozyme concentration. Furthermore, the contaminant caused endocrine stress response, raising plasma cortisol and glucose. Therefore, the alterations caused by SePM threatened the capacity of sustaining aerobic metabolism, impaired the immune system, and changed the energy allocation due to both stress response and immune effect. This may have important implications for the impact of SePM on aquatic ecosystems. Future investigations should assess SePM impact on general physiology and aerobic performance, especially to face common ecological challenges such as hypoxia and sustained swimming. These results point out the need to develop proper protocols to address the air-to-water cross-contamination risks by iron ore processing industries.

Metallic nanoparticle contamination from environmental atmospheric particulate matter in the last slab of the trophic chain: Nanocrystallography, subcellular localization and toxicity effects.

Atmospheric particulate material (PM) from mining and steel industries comprises several metallic contaminants. PM10 samples collected in a Brazilian region with a recognized influence of the steel and iron pelletizing industries were used to investigate metallic nanoparticle incorporation into human fibroblast cells (MRC-5). MRC-5 cells were exposed to 0 (control, ultrapure water), 2.5, 5, 10, 20 and 40 µg PM10 mL-1, for 24 h. Cytotoxic and genotoxic dose-response effects were observed on lysosome and DNA structure, and concentrations high as 20 and 40 µg PM10 mL-1 induced elevated cell death. Ultrastructure analyses showed aluminosilicate, iron, and the emerging metallic contaminants titanium, bismuth, and cerium nanoparticles were incorporated into lung cells, in which the nanocrystallography analysis indicated the bismuth as Bi2O3. All internalized metallic nanoparticles were free and unbound in the cytoplasm and nucleus thereby indicating bioavailability and potential interaction to biological processes and cellular structures. Pearson's correlation analysis showed Fe, Ni, Al, Cr, Pb and Hg as the main cytotoxic elements which are associated with the stainless steel production. The presence of internalized nanoparticles in human lung cells exposed to environmental atmospheric matter highlights the need for a greater effort by regulatory agencies to understand their potential damage and hence the need for future regulation, especially of emerging metallic contaminants.

Bioconcentration and toxicological impacts of fipronil and 2,4-D commercial formulations (single and in mixture) in the tropical fish, Danio rerio.

The insecticide fipronil and the herbicide 2,4-D are the most applied pesticides in sugarcane crops leading to aquatic contamination. The whole-body bioconcentration of fipronil and 2,4-D, single and in mixture, was evaluated in Danio rerio after 96-h exposure. The activities of catalase (CAT) and glutathione S-transferase(GST) in whole body and in the gills and the acetylcholinesterase (AChE) in muscle were determined. The gill histopathology and the morphology of the pavement (PVC) and the mitochondria-rich(MRC) cells at gill surface were analyzed. Bioconcentration occurred after exposure to fipronil (2.69 L kg-1) and 2,4-D (1.73 L kg-1) single and in mixture of fipronil (3.10 L kg-1) and 2,4-D (1.27 L kg-1). Whole-body CAT activity was unchanged, and its activity decreased in the gills after exposure to fipronil and increased after exposure to 2,4-D and mixture. GST and AChE increased after single exposure to each pesticide and mixture of both. Fish exposed to mixture increased the MRC fractional area (MRCFA) which suggested possible ionic regulation disturbance and reduced the microridge of the PVC surface. Synergistic interactions occurred in the CAT activity and MRCFA after exposure to mixture of pesticides. The results indicate that the recommended application dose of fipronil and 2,4-D, single or in mixture, for sugarcane crops affects this fish species altering its homeostasis.