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Risk assessment of aclonifen use in Ukraine
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Abstracts / Toxicology Letters 259S (2016) S73–S247
toxicology studies we are conducting to assess the joint action of low levels of NNTs and other modern pesticides, here we show preliminary results of a DT assessment of the NNT compound acetamiprid (ACP) in Wistar rats. Methods: We selected two exposure levels: 1 and 6 mg/kg/day. These daily doses were both below the reported NOAELs for maternal and developmental toxicity of ACP in rats, i.e., 16 mg/kg/day (USEPA), but quite above its ADI of 0.07 mg/kg/day. ACP was dissolved in the water offered ad libitum to the pregnant dams from gestational day (GD) 5 to 20. The study was divided into blocks, and 4–6 l per treatment have been so far evaluated. Dams and their male and female offspring were monitored for body weight gain, developmental landmarks such as eye opening, fur appearance, and anogenital distance. Last, experimental pups were evaluated using a 7-session long, associative-motor test (Circling Training Test, CTT) at late adolescence to explore potential enduring effects of gestational treatments on fine nervous system function. Results: No evident alteration was observed in the dams or the offspring in any case. Nevertheless, ACP treated pups showed trends for a dose-related decrease in body weight and a poorer performance through the first sessions of the associative-spatial conditioning in the CTT test. Animals were sacrificed soon after the CTT assays, and various target tissues (brain, kidney and gonads) were dissected out for determining organ/body weight ratios (work in progress). http://dx.doi.org/10.1016/j.toxlet.2016.07.517 PP23.1 Clinical extrapolation of chlorfenapyr poisoning on delayed neurotoxicity in rats M.Y. Liu 1 , S. Periasamy 1 , J.F. Deng 2 1 Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan 2 Division of Clinical Toxicology, Department of Medicine, Veterans General Hospital, Shih-Pai, Taipei, Taiwan
Introduction: Chlorfenapyr [4-bromo-2-(4-chlorophenyl)-1(ethoxymethyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile] is a widely used insecticide. It uncouples oxidative phosphorylation in the mitochondria, resulting in disrupted ATP production, cellular death, and ultimately, death of the organism. Recently, several cases of suicidal attempt using chlorfenapyr have been reported in Asian countries. Case studies revealed delayed neurotoxic symptom and fatalities after 7–10 days. Objective: We investigated clinical extrapolation of chlorfenapyr poisoning in a rat model. Materials and methods: Commercial chlorfenapyr contained 10% chlorfenapyr and 90% detergent, it was gavaged to male Wistar rats. Chlorfenapyr solution (0.125 mL, 0.25 mL, and 0.5 mL) was given to male Wistar rats. Rats were killed at 18 days, and serum and organs were harvested. Biochemical parameters and brain damage markers were examined. Results: After 2 h, chlorfenapyr-treated rats were lethargic and weak. Mortality rates were 14.28%, 28.57%, and 57.14%, respectively, in three treated doses at 24 h. Motor activity was abnormal in 0.25 mL and 0.5 mL groups. Dead animal’s feet were blue in color. After 18 days, we found that body weights were decreased, but no changes in the organ weight. AMYL, GOT and NO were significantly increased. CPK and BUN were unaltered. MDA was significantly decreased. In addition, no significant alternation was found in the brain GABAA Ra1 receptor. Brain HSP60 and Cyt c expression were significantly decreased.
Conclusion: Chlorfenapyr ingestion after 18 days might result in delayed neurotoxicity in the brain in rats. http://dx.doi.org/10.1016/j.toxlet.2016.07.518 PP23.2 Glufosinate ammonium alters quality and DNA in mouse spermatozoa E. Guinto-Ruiz 1 , J.J. Sánchez-Carlos 1 , M.E. Moreno-Godínez 2 , C. González-Calixto 3 , M. Calixto-Gálvez 1 , B. Quintanilla-Vega 4 , I. Hernández-Ochoa 4 , M. Urióstegui-Acosta 1 1
Unidad Académica de Ciencias Naturales, Ciudad de México, Mexico Unidad Académica de Ciencias Químico Biológicas, Ciudad de México, Mexico 3 Unidad Académica de Enfermería, Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, Mexico 4 Departamento de Toxicología, CINVESTAV-IPN, Ciudad de México, Mexico 2
Introduction: Glufosinate ammonium (GLA) is a non-selective organophosphorus herbicide commonly used in domestic gardens and agriculture, and it is classified as a persistent contaminant. In Guerrero, a state of Mexico, GLA is highly used in agriculture. Previous studies have reported that GLA induces preimplantation and implantation losses, altering male reproduction. Objective: We evaluated the effect of GLA on sperm quality and DNA damage exploring the sensitive stage(s) of spermatogenesis, particularly mature spermatozoa and spermatids. Materials and Methods: Adult male mice were administered GLA [5 or 10 mg/kg bw/v.o./day/5–15 days of treatment (dt)], and euthanized 24 h post-treatment. Spermatozoa were obtained from cauda epididymis-vas deferens, and then evaluated for sperm quality and DNA damage (Alkaline Comet assay, pH 13). Results: GLA at 5 mg/kg/d/5-dt altered all sperm quality parameters that decreased in a dose-dependent manner. Cells collected from the group of 5 mg/kg/d/15-dt (low dose) showed decreased viability (22%), motility (17%), and normal morphology (3%). GLA increased %DNA (1.4- and 1.8-fold) and OTM (7.4- and 8.4fold) at both assessed times in a dose-dependent manner. At 10 mg/kg/d/5- and 15-dt all sperm quality parameters: viability (27–30%), motility (27–31%), and morphology (4–10%) were altered at both times and increases in %DNA (2.3- and 4.7-fold) and OTM (8.7- and 10.7-fold) were observed compared to the control group. Conclusions: These data suggest that spermatids and mature spermatozoa are targets of GLA exposure. Financial support: Study supported by UAGro and the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775). http://dx.doi.org/10.1016/j.toxlet.2016.07.519 PP23.3 Risk assessment of aclonifen use in Ukraine M. Prodanchuk, A. Kravchuk, I. Leposhkin, N. Nedopytanska, P. Zhminko, A. Grynko L.I.Medved’s Research Center of Preventive Toxicology, Ministry of health, Kyiv, Ukraine Introduction: Aclonifen is systemic, selective herbicide for the pre-emergence control of grass and broad- leaved weeds in sunflower, carrots and other crops. The mode of action of aclonifen is the inhibition of carotenoid biosynthesis.
Abstracts / Toxicology Letters 259S (2016) S73–S247
Materials and methods: The toxicological assessment of formulations and their active ingredients have been conducted in Ukraine during State trial. The level of aclonifen residues and safety of grown products have been investigated in different agroclimatic zones of Ukraine. Residual quantities of aclonifen in the sunflower and carrots have been studied by high-performance liquid chromatography (HPLC). Results: Aclonifen is not acutely toxic via different routes of exposure, and is not a skin irritant. Active ingredient caused delayed contact hypersensitivity in guinea pigs. Aclonifen is not genotoxic, neurotoxic, or developmentally toxic. On the basis of toxicological assessment of active substances the ADI – 0,01 mg/kg were recommended and approved in Ukraine. As a result of conducted dissipation experiments it was establish that the rate of degradation of active ingredients is quite slow. The aclonifen residues disappeared mono exponentially. Herbicide residues in seeds, edible root and forage were not detectable at harvest time. Conclusions: On the basis of conducted experiments, an MRL has been recommended, providing safe use of new herbicides in Ukraine. Theoretical maximum daily intake of new herbicides with food products did not exceed 2% of acceptable daily intake. Based on conducted researches we consider that a dietary intake of aclonifen residues is unlikely to present public health concern. http://dx.doi.org/10.1016/j.toxlet.2016.07.520 PP23.4 Persistent organic pollutants (POPs) in geoduck clam Panopea globosa from the northwestern Mexican Pacific D.R. Murrieta 1 , H.J. García 1 , L.G. Cortez 1 , P. Bastidas 2 , M.D. Aguilera 1 1 Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Guaymas, Guaymas, Sonora, Mexico 2 Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Culiacán, Sinaloa, Mexico
Introduction: Persistent organic pollutants (POPs) are toxic substances that when available on the environment produce biological alterations on organisms through the food chain. Coastal areas are highly susceptible to this contamination because of their proximity to human settlements. Objective: To determine the persistent organic pollutants concentrations on geoduck clams Panopea globosa of different sizes and ages of Northwest Mexico Materials and methods: The study was conducted on the North of Sonora State and West of Baja California Sur, Mexico. Sampling of the geoduck clam was conducted on the spring of 2015 in coordination with cooperative fisheries of the specified sites. The determination and quantification of the analytes in tissue of geoduck clams were made following the USEPA methods for organochlorine pesticides and polychlorinated biphenyls; an Agilent Technologies Gas Chromatographer model 7890B with an electron capture detector (CG-DCE) was used for the analysis. Results: The obtained results revealed that the concentrations of total POPs ranged from 0.10 ng g−1 to 44.25 ng g−1 . The organochlorine pesticide (OC) concentration in collected molluscs followed the order: total cyclodienes> ␣-BHC> p,p -DDD. The highest concentration was aldrin (44.25 ng g−1 ). Meanwhile, for PCB’s only congeners 28, 52 and 153 were detectable at low concentrations. The highest concentration was PCB-28 (0.33 ng g−1 ). The concentrations of PCB’s followed the order: PCB-52>PCB-28>PCB-153. Conclusions: These compounds are of particular environmental interest because they have a long half-life and easily bioaccumulate
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along the trophic chain. According to the world health authorities (FDA and NOM-242), the concentrations of POPs in geoduck Panopea globosa of the studied area did not pose a health risk for consumers. Financial support: This study was partially supported by the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775). http://dx.doi.org/10.1016/j.toxlet.2016.07.521 PP23.5 Cardiorespiratory impairment of chemoreflex responses after acute exposure to the organophosphorus insecticide (OP) chlorpyrifos I.S.A. Felippe, A.A. Siqueira, V. Beijamini, K.N. Sampaio Department of Pharmaceutical Sciences, Federal University of Espírito Santo, Vitória, Espirito Santo, Brazil Introduction: Although it is well known that in severe poisoning by OP the cholinergic overstimulation leads to a cardiorespiratory arrest, the effects of sublethal exposure to these compounds on the cardiorespiratory regulation are poorly explored. Objective: To evaluate the effects of acute sublethal exposure to CPF on chemoreflex, which exerts an integrative function upon the cardiorespiratory system. Materials and methods: Under sodium pentobarbital anesthesia (50 mg/kg, ip), femoral artery and vein catheterization procedure was conducted in adult male Wistar rats for pressure recordings and chemoreflex activation, respectively. After recovery, animals were injected with a single dose of CPF (30 mg/Kg, ip, n = 10) or saline (0.9% w/v, ip, n = 9). 24 hours later, the respiratory frequency, mean arterial pressure and heart rate recordings were performed in awake rats placed inside a plethysmography chamber. KCN doses (10, 20, 40 and 80 g, iv) were used to induce the hypertensive, bradycardic and tachypnea chemoreflex responses. Blood samples were taken for measurements of plasma cholinesterase activity, while the acetylcholinesterase activity was measured in the brainstem. Data was analyzed by two-way ANOVA for repeated measures followed by Bonferroni post hoc test or by Student’s t test as appropriate. Project was approved under the Ethics Committee in Animal Experimentation (CEUA-UFES n◦ 058/2014). Results: The chemoreflex induced hypertensive (F = 13.97, P = 0.0016), bradycardic (F = 6.570, P = 0.0202), as well as the tachypnea responses (F = 4.901, P = 0.0408) were reduced in CPF treated animals when compared with control animals. Plasma cholinesterase (F = 5.662, P = 0.041) as well as brainstem acetylcholinesterase activity (P = 0.0001) was significantly reduced in CPF treated animals. Conclusions: Our results showed that acute exposure to a sublethal dose of CPF induces impairment in all chemoreflex cardiorespiratory responses. These effects are associated with a marked inhibition of the acetylcholinesterase activity within the brainstem which holds the main nuclei controlling cardiorespiratory function. Financial support: Bioclin, CAPES, FAPES. http://dx.doi.org/10.1016/j.toxlet.2016.07.522
Abstracts / Toxicology Letters 259S (2016) S73–S247
toxicology studies we are conducting to assess the joint action of low levels of NNTs and other modern pesticides, here we show preliminary results of a DT assessment of the NNT compound acetamiprid (ACP) in Wistar rats. Methods: We selected two exposure levels: 1 and 6 mg/kg/day. These daily doses were both below the reported NOAELs for maternal and developmental toxicity of ACP in rats, i.e., 16 mg/kg/day (USEPA), but quite above its ADI of 0.07 mg/kg/day. ACP was dissolved in the water offered ad libitum to the pregnant dams from gestational day (GD) 5 to 20. The study was divided into blocks, and 4–6 l per treatment have been so far evaluated. Dams and their male and female offspring were monitored for body weight gain, developmental landmarks such as eye opening, fur appearance, and anogenital distance. Last, experimental pups were evaluated using a 7-session long, associative-motor test (Circling Training Test, CTT) at late adolescence to explore potential enduring effects of gestational treatments on fine nervous system function. Results: No evident alteration was observed in the dams or the offspring in any case. Nevertheless, ACP treated pups showed trends for a dose-related decrease in body weight and a poorer performance through the first sessions of the associative-spatial conditioning in the CTT test. Animals were sacrificed soon after the CTT assays, and various target tissues (brain, kidney and gonads) were dissected out for determining organ/body weight ratios (work in progress). http://dx.doi.org/10.1016/j.toxlet.2016.07.517 PP23.1 Clinical extrapolation of chlorfenapyr poisoning on delayed neurotoxicity in rats M.Y. Liu 1 , S. Periasamy 1 , J.F. Deng 2 1 Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan 2 Division of Clinical Toxicology, Department of Medicine, Veterans General Hospital, Shih-Pai, Taipei, Taiwan
Introduction: Chlorfenapyr [4-bromo-2-(4-chlorophenyl)-1(ethoxymethyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile] is a widely used insecticide. It uncouples oxidative phosphorylation in the mitochondria, resulting in disrupted ATP production, cellular death, and ultimately, death of the organism. Recently, several cases of suicidal attempt using chlorfenapyr have been reported in Asian countries. Case studies revealed delayed neurotoxic symptom and fatalities after 7–10 days. Objective: We investigated clinical extrapolation of chlorfenapyr poisoning in a rat model. Materials and methods: Commercial chlorfenapyr contained 10% chlorfenapyr and 90% detergent, it was gavaged to male Wistar rats. Chlorfenapyr solution (0.125 mL, 0.25 mL, and 0.5 mL) was given to male Wistar rats. Rats were killed at 18 days, and serum and organs were harvested. Biochemical parameters and brain damage markers were examined. Results: After 2 h, chlorfenapyr-treated rats were lethargic and weak. Mortality rates were 14.28%, 28.57%, and 57.14%, respectively, in three treated doses at 24 h. Motor activity was abnormal in 0.25 mL and 0.5 mL groups. Dead animal’s feet were blue in color. After 18 days, we found that body weights were decreased, but no changes in the organ weight. AMYL, GOT and NO were significantly increased. CPK and BUN were unaltered. MDA was significantly decreased. In addition, no significant alternation was found in the brain GABAA Ra1 receptor. Brain HSP60 and Cyt c expression were significantly decreased.
Conclusion: Chlorfenapyr ingestion after 18 days might result in delayed neurotoxicity in the brain in rats. http://dx.doi.org/10.1016/j.toxlet.2016.07.518 PP23.2 Glufosinate ammonium alters quality and DNA in mouse spermatozoa E. Guinto-Ruiz 1 , J.J. Sánchez-Carlos 1 , M.E. Moreno-Godínez 2 , C. González-Calixto 3 , M. Calixto-Gálvez 1 , B. Quintanilla-Vega 4 , I. Hernández-Ochoa 4 , M. Urióstegui-Acosta 1 1
Unidad Académica de Ciencias Naturales, Ciudad de México, Mexico Unidad Académica de Ciencias Químico Biológicas, Ciudad de México, Mexico 3 Unidad Académica de Enfermería, Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, Mexico 4 Departamento de Toxicología, CINVESTAV-IPN, Ciudad de México, Mexico 2
Introduction: Glufosinate ammonium (GLA) is a non-selective organophosphorus herbicide commonly used in domestic gardens and agriculture, and it is classified as a persistent contaminant. In Guerrero, a state of Mexico, GLA is highly used in agriculture. Previous studies have reported that GLA induces preimplantation and implantation losses, altering male reproduction. Objective: We evaluated the effect of GLA on sperm quality and DNA damage exploring the sensitive stage(s) of spermatogenesis, particularly mature spermatozoa and spermatids. Materials and Methods: Adult male mice were administered GLA [5 or 10 mg/kg bw/v.o./day/5–15 days of treatment (dt)], and euthanized 24 h post-treatment. Spermatozoa were obtained from cauda epididymis-vas deferens, and then evaluated for sperm quality and DNA damage (Alkaline Comet assay, pH 13). Results: GLA at 5 mg/kg/d/5-dt altered all sperm quality parameters that decreased in a dose-dependent manner. Cells collected from the group of 5 mg/kg/d/15-dt (low dose) showed decreased viability (22%), motility (17%), and normal morphology (3%). GLA increased %DNA (1.4- and 1.8-fold) and OTM (7.4- and 8.4fold) at both assessed times in a dose-dependent manner. At 10 mg/kg/d/5- and 15-dt all sperm quality parameters: viability (27–30%), motility (27–31%), and morphology (4–10%) were altered at both times and increases in %DNA (2.3- and 4.7-fold) and OTM (8.7- and 10.7-fold) were observed compared to the control group. Conclusions: These data suggest that spermatids and mature spermatozoa are targets of GLA exposure. Financial support: Study supported by UAGro and the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775). http://dx.doi.org/10.1016/j.toxlet.2016.07.519 PP23.3 Risk assessment of aclonifen use in Ukraine M. Prodanchuk, A. Kravchuk, I. Leposhkin, N. Nedopytanska, P. Zhminko, A. Grynko L.I.Medved’s Research Center of Preventive Toxicology, Ministry of health, Kyiv, Ukraine Introduction: Aclonifen is systemic, selective herbicide for the pre-emergence control of grass and broad- leaved weeds in sunflower, carrots and other crops. The mode of action of aclonifen is the inhibition of carotenoid biosynthesis.
Abstracts / Toxicology Letters 259S (2016) S73–S247
Materials and methods: The toxicological assessment of formulations and their active ingredients have been conducted in Ukraine during State trial. The level of aclonifen residues and safety of grown products have been investigated in different agroclimatic zones of Ukraine. Residual quantities of aclonifen in the sunflower and carrots have been studied by high-performance liquid chromatography (HPLC). Results: Aclonifen is not acutely toxic via different routes of exposure, and is not a skin irritant. Active ingredient caused delayed contact hypersensitivity in guinea pigs. Aclonifen is not genotoxic, neurotoxic, or developmentally toxic. On the basis of toxicological assessment of active substances the ADI – 0,01 mg/kg were recommended and approved in Ukraine. As a result of conducted dissipation experiments it was establish that the rate of degradation of active ingredients is quite slow. The aclonifen residues disappeared mono exponentially. Herbicide residues in seeds, edible root and forage were not detectable at harvest time. Conclusions: On the basis of conducted experiments, an MRL has been recommended, providing safe use of new herbicides in Ukraine. Theoretical maximum daily intake of new herbicides with food products did not exceed 2% of acceptable daily intake. Based on conducted researches we consider that a dietary intake of aclonifen residues is unlikely to present public health concern. http://dx.doi.org/10.1016/j.toxlet.2016.07.520 PP23.4 Persistent organic pollutants (POPs) in geoduck clam Panopea globosa from the northwestern Mexican Pacific D.R. Murrieta 1 , H.J. García 1 , L.G. Cortez 1 , P. Bastidas 2 , M.D. Aguilera 1 1 Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Guaymas, Guaymas, Sonora, Mexico 2 Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Culiacán, Sinaloa, Mexico
Introduction: Persistent organic pollutants (POPs) are toxic substances that when available on the environment produce biological alterations on organisms through the food chain. Coastal areas are highly susceptible to this contamination because of their proximity to human settlements. Objective: To determine the persistent organic pollutants concentrations on geoduck clams Panopea globosa of different sizes and ages of Northwest Mexico Materials and methods: The study was conducted on the North of Sonora State and West of Baja California Sur, Mexico. Sampling of the geoduck clam was conducted on the spring of 2015 in coordination with cooperative fisheries of the specified sites. The determination and quantification of the analytes in tissue of geoduck clams were made following the USEPA methods for organochlorine pesticides and polychlorinated biphenyls; an Agilent Technologies Gas Chromatographer model 7890B with an electron capture detector (CG-DCE) was used for the analysis. Results: The obtained results revealed that the concentrations of total POPs ranged from 0.10 ng g−1 to 44.25 ng g−1 . The organochlorine pesticide (OC) concentration in collected molluscs followed the order: total cyclodienes> ␣-BHC> p,p -DDD. The highest concentration was aldrin (44.25 ng g−1 ). Meanwhile, for PCB’s only congeners 28, 52 and 153 were detectable at low concentrations. The highest concentration was PCB-28 (0.33 ng g−1 ). The concentrations of PCB’s followed the order: PCB-52>PCB-28>PCB-153. Conclusions: These compounds are of particular environmental interest because they have a long half-life and easily bioaccumulate
S217
along the trophic chain. According to the world health authorities (FDA and NOM-242), the concentrations of POPs in geoduck Panopea globosa of the studied area did not pose a health risk for consumers. Financial support: This study was partially supported by the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775). http://dx.doi.org/10.1016/j.toxlet.2016.07.521 PP23.5 Cardiorespiratory impairment of chemoreflex responses after acute exposure to the organophosphorus insecticide (OP) chlorpyrifos I.S.A. Felippe, A.A. Siqueira, V. Beijamini, K.N. Sampaio Department of Pharmaceutical Sciences, Federal University of Espírito Santo, Vitória, Espirito Santo, Brazil Introduction: Although it is well known that in severe poisoning by OP the cholinergic overstimulation leads to a cardiorespiratory arrest, the effects of sublethal exposure to these compounds on the cardiorespiratory regulation are poorly explored. Objective: To evaluate the effects of acute sublethal exposure to CPF on chemoreflex, which exerts an integrative function upon the cardiorespiratory system. Materials and methods: Under sodium pentobarbital anesthesia (50 mg/kg, ip), femoral artery and vein catheterization procedure was conducted in adult male Wistar rats for pressure recordings and chemoreflex activation, respectively. After recovery, animals were injected with a single dose of CPF (30 mg/Kg, ip, n = 10) or saline (0.9% w/v, ip, n = 9). 24 hours later, the respiratory frequency, mean arterial pressure and heart rate recordings were performed in awake rats placed inside a plethysmography chamber. KCN doses (10, 20, 40 and 80 g, iv) were used to induce the hypertensive, bradycardic and tachypnea chemoreflex responses. Blood samples were taken for measurements of plasma cholinesterase activity, while the acetylcholinesterase activity was measured in the brainstem. Data was analyzed by two-way ANOVA for repeated measures followed by Bonferroni post hoc test or by Student’s t test as appropriate. Project was approved under the Ethics Committee in Animal Experimentation (CEUA-UFES n◦ 058/2014). Results: The chemoreflex induced hypertensive (F = 13.97, P = 0.0016), bradycardic (F = 6.570, P = 0.0202), as well as the tachypnea responses (F = 4.901, P = 0.0408) were reduced in CPF treated animals when compared with control animals. Plasma cholinesterase (F = 5.662, P = 0.041) as well as brainstem acetylcholinesterase activity (P = 0.0001) was significantly reduced in CPF treated animals. Conclusions: Our results showed that acute exposure to a sublethal dose of CPF induces impairment in all chemoreflex cardiorespiratory responses. These effects are associated with a marked inhibition of the acetylcholinesterase activity within the brainstem which holds the main nuclei controlling cardiorespiratory function. Financial support: Bioclin, CAPES, FAPES. http://dx.doi.org/10.1016/j.toxlet.2016.07.522