Epilithic biofilms, POCIS, and water samples as complementary sources of information for a more comprehensive view of aquatic contamination by pesticides and pharmaceuticals in southern Brazil.

Spatial-temporal monitoring of the presence of pesticides and pharmaceuticals in water requires rigor in the choice of matrix to be analyzed. The use of matrices, isolated or combined, may better represent the real state of contamination. In this sense, the present work contrasted the effectiveness of using epilithic biofilms with active water sampling and with a passive sampler-POCIS. A watershed representative of South American agriculture was monitored. Nine sites with different rural anthropic pressures (natural forest, intensive use of pesticides, and animal waste), and urban areas without sewage treatment, were monitored. Water and epilithic biofilms were collected during periods of intensive pesticide and animal waste application. After the harvest of the spring/summer crop, a period of low agrochemical input, the presence of pesticides and pharmaceuticals was monitored using the POCIS and epilithic biofilms. The spot water sampling leads to underestimation of the level of contamination of water resources as it does not allow discrimination of different anthropic pressures in rural areas. The use of endogenous epilithic biofilms as a matrix for the analysis of pesticides and pharmaceuticals is a viable and highly recommended alternative to diagnose the health of water sources, especially if associated with the use of POCIS.

Influence of adjuvants added to teflubenzuron spray on resistant and susceptible strains of the soybean looper Chrysodeixis includens (Lepidoptera: Noctuidae) / Influência da adição de adjuvantes à calda de pulverização de teflubenzuron em linhagens resistentes e suscetíveis da lagarta falsa-medideira Chrysodeixis includens em soja (Lepidoptera: Noctuidae)

The soybean looper (SBL), Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), is a soybean and cotton pest in South America countries. Field-evolved resistance of SBL to inhibitors of chitin biosynthesis has been reported in Brazil; however, this mode of action is still widely used against SBL. On this basis, we conducted laboratory bioassays to investigate if adjuvants (Nimbus®, TA 35®, Break-Thru® S 240, and Rizospray Extremo®) added to the teflubenzuron spray increase the mortality of SBL strains (resistant, heterozygous, and susceptible to chitin biosynthesis inhibitors). Using chromatography analysis, we also evaluated the amount of teflubenzuron on soybean leaves when applied alone or in combination with adjuvants. In laboratory bioassays, the biological activity of teflubenzuron increased against the susceptible SBL strain when adjuvants were added. In contrast, no relevant effects of adjuvants added to the teflubenzuron spray against heterozygous and resistant SBL larvae were detected. In leaf bioassays, even leaves from the upper third part of the plants containing a significantly higher amount of teflubenzuron (3.4 mg/kg vs 1.7 and 0.6 mg/kg); the mortality of SBL strains was similar when teflubenzuron was applied alone or in mixture with adjuvants. Our findings indicated that adjuvants added to teflubenzuron spray do not provide a substantial increase in the mortality of SBL strains resistant to chitin biosynthesis inhibitors. Therefore, it is necessary to reduce the use of this mode-ofaction insecticide against SBL and to give preference to other insecticides or control tactic.

A lagarta falsa-medideira, Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), é uma praga da soja e do algodão nos países da América do Sul. A resistência de C. includens a inibidores da biossíntese de quitina tem sido relatada no Brasil. Entretanto, esse modo de ação ainda é amplamente utilizado para controle de C. includens. Com base nisso, conduzimos bioensaios em laboratório para investigar se adjuvantes (Nimbus®, TA 35®, Break-Thru® S 240 e Rizospray Extremo®) adicionados à calda inseticida de teflubenzuron aumentam a mortalidade de linhagens de C. includens (resistentes, heterozigotos e suscetíveis a inibidores da biossíntese de quitina). Usando análise cromatográfica, também avaliamos a quantidade de teflubenzuron em folhas de soja quando aplicado isolado ou em combinação com adjuvantes. Em bioensaios de laboratório, a atividade biológica do teflubenzuron aumentou para a linhagem suscetível quando os adjuvantes foram adicionados à calda inseticida. Em contraste, nenhum efeito relevante de adjuvantes adicionados ao teflubenzuron foi detectado para os heterozigotos e resistentes. Em bioensaios de folhas, mesmo naquelas do terço superior das plantas, as quais apresentaram uma maior deposição de teflubenzuron (3,4 mg/kg vs 1,7 e 0,6 mg/kg); a mortalidade das linhagens de C. includens foi semelhante quando o teflubenzuron foi aplicado isolado ou com adjuvantes. Nossos resultados indicam que os adjuvantes adicionados ao teflubenzuron não fornecem um aumento substancial na mortalidade de linhagens de C. includens resistentes aos inibidores da biossíntese de quitina. Portanto, é necessário reduzir o uso desse modo de ação para o manejo de C. includens e dar preferência a outros inseticidas ou tática de controle.

Environmentally relevant pesticides induce biochemical changes in Nile tilapia (Oreochromis niloticus).

The high demand for food consequently increases the entry of agricultural residues into water resources, and this phenomenon can affect non-target organisms in different ways. Environmentally relevant pesticide effects (per se or in combinations) are scarce in the scientific literature. Therefore, the aim of this study was to investigate: (1) the presence of pesticide residues in an important Brazilian source of water supply and power generation (Jacuí river), during 1 year of monitoring. (2) in a laboratory study verify the effects of the most frequently, herbicide, fungicide, and insecticide found in Jacuí river (individualized or in a mixture) on biochemical parameters in different tissues of Oreochromis niloticus. Twenty pesticide residues were detected in superficial water samples, and two of them are banned in Brazilian territory. Atrazine (0.56 µg L-1), azoxystrobin (0.024 µg L-1), and imidacloprid (0.11 µg L-1) were the most frequently herbicide, fungicide, and insecticide, respectively, found in the river and were used in the laboratory assay. O. niloticus exposed to the pesticide mixture exhibited more biochemical effects than individualized exposure groups. This response can be a result of the combined pesticide effects, culminating in an additive or synergistic effect, depending on the biomarker. In individual exposure groups, atrazine presented the most pronounced alterations, followed by azoxystrobin and imidacloprid. Overall, pesticide exposure increased levels of oxidative stress parameters, reduced antioxidant enzyme activities, and induced acetylcholinesterase activity. These findings highlight the threat to aquatic organisms which may be exposed to a miscellaneous of toxic compounds in the environment.

Mobilization and transport of pesticides with runoff and suspended sediment during flooding events in an agricultural catchment of Southern Brazil.

Brazil is one of the largest consumers of pesticides in the world, and these chemicals present a high contamination risk for the country's water bodies. The mechanisms of mobilization and transport of pesticides from cropland to river systems are controlled by runoff and erosion processes occurring at the catchment scale. In addition to the excessive use of pesticides, the transport processes of these substances are also accelerated by inadequate soil management and the absence of soil conservation measures at the catchment scale. The current research relied on hydrological monitoring to investigate the transport and persistence of pesticides in response to hydrological dynamics. The study was conducted in the Conceição River watershed where runoff and suspended sediment fluxes are continuously monitored at the outlet. This study area is representative of the grain production system in southern Brazil including the application of large amounts of pesticides combined with extensive runoff and erosion problems. Sample collection in the river for pesticide analysis included the analysis of both water and suspended sediment. The sediment deposit analysis was performed in a single location at 4 depths. Results demonstrate the occurrence of pesticides including simazine, 2,4-D, carbendazim, imidacloprid, tebuconazole, propiconazole, tetraconazole, and trifloxystrobin in water, while glyphosate and AMPA were detected in suspended sediments, and AMPA and carbendazim were found in sediment deposits. The study demonstrated the strong dependence of the mechanisms of pesticide mobilization and transport in the catchment with the intra- and interevent variability of hydro-sedimentary processes. Pesticide detections can be related to several factors, including the magnitude of the rainfall event, the period of pesticide application, or the transport of suspended sediment. All these factors are correlated, and the mechanisms of transportation play an important role in the connections between sink and sources. The results suggest that pesticide monitoring should take into account the runoff and erosion pathways in each particular catchment, and it should especially include the monitoring of major rainfall events for identifying and quantifying the occurrence of pesticides in the environment. The transport of pesticides indicates to be potentiated by intensive pesticide use, the magnitude of rainfall-runoff events, and the absence of runoff control measures (e.g., terracing). These results demonstrate that water and soil conservation techniques should be planned and coordinated at the watershed scale to reduce the connectivity of water and sediment flows from agricultural areas to river systems with the implementation of effective runoff control practices. This will control the mobilization agents (runoff), as well as limit the connection between the sources and the water bodies.

A mixture of pesticides at environmental concentrations induces oxidative stress and cholinergic effects in the neotropical fish Rhamdia quelen.

The insecticides imidacloprid (IMI), a neonicotinoid, and propoxur (PRO), an N-methylcarbamate compound, are pesticides widely used throughout the world. Although they are not used together to combat pests, both are often found in freshwater near agricultural areas. Thereby, the goal of this study was to evaluate the additive effects of IMI and PRO mixtures at environmental concentrations in relation to isolated compounds on Rhamdia quelen, a neotropical fish. The fish was exposed to IMI (0.11 µg/L), PRO (0.039 µg/L), or Mix (0.11 µg/L IMI plus 0.039 µg/L PRO) during 96 h. Glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE) activities were determined. To verify oxidative damage thiobarbituric acid reactive substances (TBARS), protein carbonyl (PC), reactive oxygen species contents (ROS), antioxidant capacity against peroxides (ACAP) were determined in gills, liver, brain and muscle. The results shows that a mixture of these pesticides at environmental concentrations inhibited acetylcholinesterase activity in the brain and induced oxidative damage in all analyzed tissues. These results reinforce the hypothesis that mixture of contaminants present in environment could induce additive or synergistic effects on fish species.

Ecological impacts of pesticides on Astyanax jacuhiensis (Characiformes: Characidae) from the Uruguay river, Brazil.

Brazilian freshwater ecosystems are continuously exposed to pesticides and domestic sewage. The Uruguay River was chosen for this study because of its international importance, as it flows through Brazil, Argentina, and Uruguay. It receives contaminants such as pesticides and domestic residues. Thus, the aim of this study to assess the accumulation of pesticides in muscle of the fish Astyanax jacuhiensis, its biochemical responses, and the presence of pesticides in water. In total, seven pesticides were registered in water from both river sites. Eight pesticides were detected in fish muscle. The biochemical responses showed that brain lipid peroxidation (LPO) and protein carbonyl (PC) in A. jacuhiensis were higher in the summer. Muscle showed the highest LPO levels in the spring and the highest PC in the summer. Liver LPO and PC levels were higher in the spring and summer. In the gills, the PC was higher in the spring and the LPO in the spring and winter. In the brain and in the gills, glutathione-S-transferase activity was high in the summer and autumn. Catalase activity was lower during the winter and spring. Non-protein thiol (NPSH) levels were lower in the brain in the winter and spring. Muscle tissue showed lower NPSH in the winter (site 1). Liver NPSH showed increased levels in liver in the spring and winter (site 2). The biochemical results clearly is related to pesticides and/or to the presence of other contaminants in the water such as metals or domestic sewage. The accumulation of pesticides in fish muscle added evidence that pesticides have been used in the area surrounding the Uruguay River. In conclusion, the biomarkers assayed in the present study could be used in future investigations considering other sampling sites along Uruguay River.

Influence of pesticides and abiotic conditions on biochemical biomarkers in Aegla aff. longirostri (crustacea, anomura): Implications for conservation.

Freshwater ecosystems are constantly threatened by the advance of agricultural activities. Abiotic variables (such as temperature, ammonia, and nitrite) and contaminants (e.g. pesticides) can potentially interact, increasing metabolism and the absorption of toxic substances, which can alter the ability of organisms to establish adequate stress responses. This study aimed to verify which pesticides were most frequently found and in the greatest quantities in low-order streams, and whether the combination of these pesticides with the abiotic variables altered the biological metabolism of aeglids. These freshwater crustaceans are important shredders that inhabit low-order streams and are sensitive to disturbances and/or abrupt environmental variations. The animals were exposed in situ in four streams (reference site and sites 1, 2, and 3). The reference site is a preserved stream with no apparent anthropogenic interference where aeglids still occur, while the other sites no longer exhibit populations of these animals and are influenced by agricultural activities. The exposure was performed bimonthly from November 2017 to September 2018 and lasted 96 h. Measured abiotic data and water samples were collected through all days of exposure. The analyzed biochemical parameters were acetylcholinesterase activity in muscle; and glutathione S-transferase, lipid peroxidation, protein carbonylation, non-protein thiols, antioxidant capacity against peroxides, and reactive oxygen species (ROS) in muscle, gills, and hepatopancreas. We found 24 active principles of pesticides, the most frequently being clomazone, atrazine, and propoxur. Bentazone was present at the highest amounts. The parameters evaluated in this study, including biochemical biomarkers and abiotic factors measured from the water, provided a separation of the months as a function of environmental conditions. There was a difference in activity and biomarker levels throughout the year within the same site and in some months between sites. The greater concentration or variety of pesticides associated with extreme abiotic (very high temperatures) data generated increased oxidative stress, with high levels of protein damage and considerable lipid damage in all tissues, as well as elevation in ROS, even with high levels of antioxidant capacity and non-protein thiols. With these data, we intend to warn about the risks of exposure to these environmental conditions by trying to contribute to the preservation of limnic fauna, especially aeglid crabs, because most species are under some degree of threat.

Protective effects of diet containing rutin against trichlorfon-induced muscle bioenergetics disruption and impairment on fatty acid profile of silver catfish Rhamdia quelen.

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage.

Toxicological response of silver catfish (Rhamdia quelen) after acute exposure to a commercial insecticide containing thiamethoxam.

This study assessed the hematological, enzymatic and osmoregulatory responses of silver catfish (Rhamdia quelen) exposed to sublethal concentrations (1.125 and 3.750 µg/L) of a commercial thiamethoxam-containing insecticide used on rice crops. Groups of 6 fish per tank (in triplicate, n = 3, total 54 fish) were exposed for up to 96 h to different concentrations of the compound. After this period, fish were placed in clean water for 48 h. Two fish from each tank (6 per treatment) that had been exposed to the insecticide for 24 h were anesthetized with eugenol and blood was collected to evaluate hematological and biochemical parameters. Blood, liver and muscle were collected for determination of metabolic parameters, plasma cortisol, Cl-, Na+ and K+ levels and H+-ATPase and Na+/K+-ATPase activity in the gill. H+-ATPase activity was higher in fish exposed to 1.125 µg/L insecticide at 24 h compared to control (0.0 µg/L). Differences in cortisol levels were evidenced throughout the experimental period. These results indicated that exposure to the insecticide changed the hematological, biochemical and metabolic profile of the animals, suggesting concern about environmental safety. Therefore, we discourage the use of this pesticide in areas that come into contact with water bodies inhabited by fish.

"Modern agriculture" transfers many pesticides to watercourses: a case study of a representative rural catchment of southern Brazil.

The total cultivated area in Brazil reached to 62 million ha in 2018, with the predominance of genetically modified soybean and corn (36 and 17 million ha, respectively) in no-tillage systems. In 2018, 5.3 × 105 Mg of active ingredient of pesticides was applied in cropfields, representing about 7.3 L of commercial product by habitant. However, the monitoring of water courses contamination by pesticides remains scarce and is based on traditional grab sampling systems. In this study, we used the grab (water) and passive sampling (Polar Organic Chemical Integrative Sampler-POCIS) to monitor pesticide contamination in the river network of a representative agricultural catchment of southern Brazil. We selected 18 sampling sites located in tributaries and in the main course of the Guaporé River, in Rio Grande do Sul State, with different land use predominance including forest, urban, and agricultural areas. Altogether, 79 and 23 pesticides were, respectively, analyzed in water and POCIS samples. The water of Guaporé River and its tributaries were highly contaminated by many pesticides, especially by four herbicides (2,4-D, atrazine, deethyl-atrazine, and simazine), three fungicides (carbendazim, tebuconazole, and epoxiconazole), and one insecticide (imidacloprid). The amount, type, and concentration of pesticides detected were completely different depending on the sampling technic used. POCIS was effective to discriminate the contamination according to the main land use of each sampling site. The monitored areas with the predominance of soybean cultivation under no-tillage tended to have higher concentrations of fungicide, while in the more diversified region, the herbicides showed higher values. The presence of five herbicides used in corn and grassland forage production was correlated with areas of integrated crop-livestock systems, in contrast to higher contamination by 2,4-D in areas of intensive production of soybean and winter cereals.

Balls-in-tube matrix solid phase dispersion (BiT-MSPD): An innovative and simplified technique for multiresidue determination of pesticides in fruit samples.

Sample preparation of complex matrices, like food samples, continues to be a challenge demanding great effort for improvements. In this study, a new technique named balls-in-tube matrix solid-phase dispersion (BiT-MSPD) is proposed based on a simplification of the conventional MSPD technique being all sample preparation performed directly in a closed extraction tube with the assistance of steel balls. An innovative method using BiT-MSPD was successfully established for the determination of 133 pesticide residues in apple, peach, pear and plum by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). Several sorbents were evaluated as solid support in different proportions with the sample. The homogenization step using mortar, glass rod or steel balls, with methanol and acetonitrile as extraction solvent, was evaluated. Vortex and ultrasound assisted extractions were also tested. Best results were obtained with C18, homogenization with steel balls, acetonitrile as solvent and ultrasound assisted extraction. Validation presented adequate trueness and precision results for the evaluated pesticides with recovery results ranging from 72 to 113% and RSD ≤ 17%. Practical limit of detection (LOD) and quantification (LOQ) for the compounds were 3 and 10 µg kg-1, respectively. The developed method proved to be easier and faster to perform than the MSPD, considering that extraction and clean-up are performed in the same tube without the need to transfer to cartridges, recipients or to use a separate clean-up step. The proposed BiT-MSPD technique was successfully applied to fruit samples and has great potential to be applied in other matrices, like cereals and meat, since the steel balls promote an efficient sample dispersion and extraction of pesticides. The BiT-MSPD permit a fully automation of the entire sample preparation step.

Development and validation of a method for the analysis of pyrethroid residues in fish using GC-MS.

The presence of the pyrethroids residues in different samples and the impact on human health is an increasing concern due to their widespread use. So, a method to determine eighteen pyrethroids in fish samples using a modified QuEChERS was developed. The clean-up procedure was performed by freezing samples overnight followed by dispersive solid phase extraction. The combination of C18, PSA and Z-Sep+ was optimized using a mixture design approach and samples were analysed using gas chromatography mass spectrometry. The method was validated using trahira samples and a scope extension was performed for tilapia and tainha. Recoveries were within 63-129%, relative standard deviation was ≤21.5% and the quantification limits were 5 µg kg-1 or 10 µg kg-1. The applicability of the method was assessed by analysis of fourteen real samples, in which residues were detected in four samples. Such detections demonstrate the importance of this sensitive method.

Experimental reproduction of congenital anomalies in the progeny of cows fed apple pomace during pregnancy / Reprodução experimental de anomalias congênitas na progênie de vacas alimentadas com bagaço de maçã durante a gestação

This study aimed to describe and discuss the results of an experiment carried out in two stages with pregnant cows fed 25kg/apple pomace/day. The first stage involved 16 pregnant Holstein Friesian cows divided into four groups: Group 0 - Control (5 cows); Group I - 1 month-gestation (4 cows); Group II - 3 month-gestation (4 cows); Group III - 6 month-gestation (3 cows) and was performed from September to December 2015. The second stage comprised 12 pregnant Holstein Friesian cows divided into three groups: Group 0 - Control (6 cows), Group I - 1 month-gestation (3 cows), and Group II - 3 month-gestation (3 cows) and was conducted from April 2016 to February 2017. All study animals received apple pomace at a dose of 25kg/day. As for the first experiment stage, a cow in Group III bred a calf with complete absence of the coccygeal vertebrae and tail, slight bending of the hind limbs, scoliosis in the thoracic spine, and limited mobility. At 30 days, it presented with diarrhea and underdevelopment, and was euthanized for necropsy. At gross examination, malformations were observed in the thoracic spine, coxofemural joint, and genitourinary tract. Regarding the second experiment stage, a cow in Group I gave birth to a calf with curved pelvic and thoracic limbs with thick joints and flattening hooves. Microscopic examination of the femur showed disorganized, irregular hypertrophic zone and scarce growth zone, in addition to primary spongy zone with short, slightly mineralized trabeculae. Samples of the apple pomace used in this study were frozen and sent for laboratory evaluation of pesticide residues, which showed a positive result for the fungicide carbendazim.(AU)

O presente trabalho tem por finalidade descrever e discutir os resultados do experimento realizado em vacas prenhes que foram alimentadas com 25kg/dia de bagaço de maçã. Experimentos foram conduzidos em duas etapas, a primeira no ano de 2015, de setembro a dezembro onde foram utilizadas 16 vacas prenhes da raça holandês. Estas foram divididas em quatro grupos: Grupo 0, Controle (5 vacas); Grupo I, 1 mês gestação (4 vacas); Grupo II, 3 meses gestação (4 vacas); Grupo III, 6 meses gestação (3 vacas). A segunda etapa foi realizada em abril de 2016 a fevereiro de 2017. Foram utilizadas 12 vacas prenhes da raça holandês, divididas em três grupos: Grupo 0, Controle (6 vacas); Grupo I, 1 mês gestação (3 vacas); Grupo II, 3 meses gestação (3 vacas). Todas as vacas receberam bagaço de maçã na dose de 25kg/dia/vaca. Para o primeiro experimento, uma vaca do Grupo III pariu uma bezerra, com ausência completa das vértebras coccígeas e cauda, encurvamento leve dos membros posteriores, escoliose na coluna torácica e dificuldade de locomoção. Decorridos 30 dias do nascimento, manifestou diarreia e pouco desenvolvimento, sendo eutanasiada para necropsia. Na macroscopia, havia malformações na coluna torácica, articulação coxofemoral e no aparelho urogenital. Em relação ao segundo experimento uma vaca do Grupo I pariu uma bezerra com membros pélvicos e torácicos, curvos e com articulações consideravelmente grossas e "achinelamento" de cascos. Na microscopia do fêmur foi observado na placa epifisária, zona hipertrófica desorganizada, irregular e zona de crescimento escassa. Na zona esponjosa primária observou-se trabéculas curtas e pouco mineralizadas. Amostras do bagaço de maçã utilizado na experimentação foram congeladas e enviadas para avaliação de resíduos agrotóxicos, onde foi encontrado resultado positivo para o fungicida carbendazim.(AU)

Fish exposed to water contaminated with eprinomectin show inhibition of the activities of AChE and Na+/K+-ATPase in the brain, and changes in natural behavior.

The objective of this study was to evaluate whether antiparasitic eprinomectin may be an environmental contaminant in water compartment in low concentrations, negatively affecting neurotransmission and, consequently, the natural behavior of the jundiá (Rhamdia quelen). Fish were randomly allocated in tanks and exposed for 24 and 48 h to eprinomectin concentrations in water [0.0 (Control), 1.124 (T1), 1.809 (T2) and 3.976 (T3) µg L-1], followed by 48 h of recovery in eprinomectin-free water, in order to evaluate the behavioral parameters, levels of reactive oxygen species (ROS) in the brain, as well as cerebral enzymatic activities of acetylcholinesterase (AChE) and of the sodium-potassium ATPase pump (Na+/K+-ATPase). Especially at the two highest concentrations of eprinomectin (T2 and T3), the fish showed alterations in natural behavior, particularly hyperlocomotion and longer time on the surface. Furthermore, at these same concentrations, cerebral ROS levels increased and cerebral AChE activity decreased. At the highest concentration (T3) cerebral Na+/K+-ATPase activity was reduced. Increased ROS and impairment of AChE and Na+/K+-ATPase enzymes in the brain may have contributed directly to behavioral changes, due to neuronal damage and synapse impairment. Even after 48 h in water without eprinomectin, behavioral changes and neurotoxic effects were observed in fish, suggesting residual effects of the antiparasitic. In conclusion, eprinomectin even in low concentrations may be a hazardous environmental contaminant for aquatic organisms, as it causes brain damage and affects the natural behavior of fish.

Simultaneous determination of the quaternary ammonium pesticides paraquat, diquat, chlormequat, and mepiquat in barley and wheat using a modified quick polar pesticides method, diluted standard addition calibration and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry.

In this study, a modified Quick Polar Pesticides (QuPPe) method, optimized by a central composite design, was developed to determine quaternary ammonium pesticides (QUATs) residues in barley and wheat by ultra-high-performance liquid chromatographic tandem mass spectrometry (UHPLC-MS/MS) using a hydrophilic interaction chromatography (HILIC) column. Considering the high polarity of these compounds, special conditions of sample preparation and analysis are required. Different mobile phases, extraction procedure and clean-up were evaluated. An isocratic elution with aqueous solution of ammonium formate 60 mmol L-1 (pH 3.7) and acetonitrile, 40:60 (v/v), was selected. Water and acidified methanol as extraction solvent, without heating, and a clean-up with dichloromethane, chitosan and acetonitrile presented good results. The validated method presented satisfactory selectivity, linearity, matrix effect, trueness and precision, providing recoveries from 93 to 110% with RSD < 13% for barley, and 70 to 115% with RSD < 18% for wheat. The complexity of these matrices requires the calibration in matrix and the diluted standard addition calibration (DSAC) procedure has been shown to be an excellent option to compensate for the matrix effect and the losses of the analytes in the extraction. Real samples of barley and wheat were analyzed and 60% presented concentrations of paraquat above the maximum limits allowed by the European Union. The modified QuPPe method combined with DSAC and HILIC-UHPLC-MS/MS demonstrated to be an effective approach to determine QUATs in barley and wheat, and is a good alternative for routine analysis. The use of the biosorbent chitosan is effective, low cost and more ecological when compared to others conventional sorbents.

Disturbance of energetic homeostasis and oxidative damage provoked by trichlorfon as relevant toxicological mechanisms using silver catfish as experimental model.

Recent evidences have suggested the involvement of phosphoryl transfer, catalyzed by creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK), to metabolic alterations and impairment of bioenergetics homeostasis linked to adenosine triphosphate (ATP) production, and utilization during exposure to pesticides. It is recognized that sublethal concentrations of trichlorfon alter hepatic and branchial metabolism, but the pathways involved in this process remains unknown. Thus, the aim of this study was to evaluate whether phosphoryl transfer network can be a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon using silver catfish Rhamdia quelen as experimental model. Hepatic and branchial CK (cytosolic and mitochondrial isoforms) and PK activities were inhibited after 48 h of exposure to 11 and 22 mg/L trichlorfon compared to control group, while AK activity did not differ between groups. In addition, sodium-potassium pump (Na+, K+-ATPase) activity was lower after 48 h of exposure to 22 mg/L trichlorfon compared to control group. Thiobarbituric acid reactive substances (TBARS) were higher in liver samples after 24 h of exposure to 22 mg/L trichlorfon compared to control group, as well as after 48 h of exposure to 11 and 22 mg/L trichlorfon in liver and gills. Finally, hepatic and branchial non-protein thiol (NPSH) levels were lower after 48 h of exposure to 11 and 22 mg/L trichlorfon. All evaluated parameters did not recover after 48 h in clean water. Based on these evidence, the impairment of phosphoryl transfer network can be considered a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon. Moreover, alterations on CK and PK activities provoke an impairment on Na+, K+-ATPase activity, which can be mediated by lipid oxidative damage and reduction of NPSH content.

Organophosphate pesticide trichlorfon induced neurotoxic effects in freshwater silver catfish Rhamdia quelen via disruption of blood-brain barrier: Implications on oxidative status, cell viability and brain neurotransmitters.

The aim of this study was to evaluate whether rupture on blood-brain barrier (BBB) can be a pathway for trichlorfon-induced neurotoxic effects, and to investigate its implications on oxidative status, cell viability and brain neurotransmitters in silver catfish (Rhamdia quelen). The BBB permeability was increased in fish exposed for 24 h to 22 mg/L of trichlorfon compared to the control group, as well as in those exposed to 11 and 22 mg/L of trichlorfon for 48 h. Compared to the control group, brain reactive oxygen species and lipid peroxide levels were higher when exposed to 22 mg/L of trichlorfon and 11 and 22 mg/L of trichlorfon after 24 h and 48 h, respectively, while the antioxidant capacity against peroxyl radical levels was lower. Exposure to 22 mg/L of trichlorfon for 24 h reduced brain cell viability compared to the control group, together with 11 and 22 mg/L of trichlorfon for 48 h. Also, brain AChE, Na+ and K+-ATPase activities were reduced in those fish exposed to trichlorfon compared to the control group. Thus, the rupture of BBB can be considered an important pathway involved in trichlorfon-induced neurotoxic effects, which contributes to brain oxidative damage and important changes on brain neurotransmitters.

Prenatal Androgenization of Ewes as a Model of Hirsutism in Polycystic Ovary Syndrome.

The main clinical feature associated with hyperandrogenism in polycystic ovary syndrome (PCOS) in humans is hirsutism, where hair increases its length, pigmentation, and particularly its diameter. Currently, it is not known whether PCOS animal models also exhibit changes in the hair. Therefore, the aim of this study was to explore the wool characteristics in sheep prenatally androgenized (PA) with testosterone propionate. After 4 and 13 months of life, wool was collected from the top of the shoulder of both females and males (both androgenized and controls). The offspring sheep were followed for up to 19 months of life to evaluate testosterone and androstenedione serum levels by ultra-high-performance liquid chromatography-tandem mass spectrometry, determine insulin and glucose response to intravenous glucose tolerance test, and address estrus cyclicity during the second breeding season. PA male animals showed a reduction in wool fiber diameter at 4 months of age compared with controls (P = 0.02) but not at 13 months, whereas PA females showed increased hair diameter at 13 months (P = 0.002), with no difference at 4 months. No substantial changes in other hair parameters (length, color, and medullation) were identified. In addition, increased levels of serum testosterone were observed in PA female sheep compared with controls at 12 months (P = 0.03). Our results indicate for the first time, to our knowledge, that changes in wool fiber diameter observed in PA ewes replicate, at the translational level, the increase in hair diameter in hirsute women with PCOS.

Gill bioenergetics dysfunction and oxidative damage induced by thiamethoxam exposure as relevant toxicological mechanisms in freshwater silver catfish Rhamdia quelen.

Thiamethoxam is a neonicotinoid pesticide utilized on a worldwide scale, it has been reported in freshwater ecosystems, and detected in fishery products. Nevertheless, there is a lack of information about thiamethoxam sublethal effects on the gills of freshwater fish, principally linked to energetic metabolism. In this context, creatine kinase (CK) is an enzyme of the phosphoryl transfer network that provides a temporal and spatial energy buffer to maintain cellular energy homeostasis in tissues with high energy requirements, such as gills. Based on this evidence, the aim of this study was to evaluate whether exposure to thiamethoxam impairs the cytosolic and mitochondrial CK activities in gills of Rhamdia quelen, and the involvement of oxidative stress in the energetic imbalance. Branchial CK (cytosolic and mitochondrial) activity and sodium­potassium pump (Na+, K+-ATPase) were inhibited, and adenosine triphosphate (ATP) levels decreased after 96 h exposure to 1.125 and 3.75 µg/L thiamethoxam compared to the control group. Moreover, levels of branchial thiobarbituric acid reactive substances (TBARS) and protein carbonylation increased at 3.75 µg/L thiamethoxam after 96 h of exposure compared to the control group, while the non-protein thiol (NPSH) content did not differ between groups. It is important to emphasize that all evaluated parameters did not recover after 48 h in clean water. To summarize, the data presented here clearly demonstrated that thiamethoxan exposure severely impairs cytosolic and mitochondrial CK activities, a key enzyme for gill energy buffering to maintain cellular energy homeostasis, and this effect appears to be mediated by oxidation of lipid and protein molecules, which consequently thereby induces oxidative stress.

Thiamethoxam induced hepatic energy changes in silver catfish via impairment of the phosphoryl transfer network pathway: Toxicological effects on energetics homeostasis.

Precise coupling of spatially separated intracellular adenosine triphosphate (ATP)-producing and ATP-consuming processes exerts a pivotal role in bioenergetic homeostasis of living organisms, and the phosphotransfer network pathway, catalyzed by adenylate kinase (AK) and pyruvate kinase (PK), is fundamental in cellular and tissue energetic homeostasis. Measurement of the phosphotransfer network can provide new information for understanding the alterations in hepatic energetic metabolism during exposition to insecticides, such as thiamethoxam. Therefore, the aim of this study was to evaluate whether exposition to thiamethoxam negatively affects the hepatic enzymes of the phosphotransfer network in silver catfish (Rhamdia quelen). Hepatic AK and PK activities were inhibited at 3.75 µg L-1 after 24 h of exposure and at 1.125 and 3.75 µg L-1 after 96 h of exposure compared with the control group. The hepatic ATP levels were decreased following 3.75 µg L-1 thiamethoxam treatment after 24 h of exposure and at 1.125 and 3.75 µg L-1 after 96 h of exposure compared with the control group. The enzymatic activity of the phosphotransfer network and ATP levels did not recover after 48 h of recovery in clean water. Thus, the inhibition of hepatic AK and PK activities by thiamethoxam caused impairment of energy homeostasis in liver tissue, decreasing hepatic ATP availability. Moreover, the absence of a mutual compensatory mechanism between these enzymes directly contributes to ATP depletion and to a severe energetic dysregulation, which may contribute to toxic effects caused by thiamethoxam.