Using mesocosms to evaluate the impacts of pasture intensification and pasture-sugarcane conversion on tadpoles in Brazil.

This study evaluated the effects of environmental contamination caused by pasture intensification and pasture-sugarcane conversion on oxidative stress, biotransformation, esterase enzymes, and development of Scinax fuscovarious and Physalaemus nattereri. Tadpoles were exposed in mesocosms allocated in three treatments: (1) untreated extensive pasture (EP); (2) intensive-pasture conversion (IP) (2,4-D herbicide + fertilizers); and (3) pasture-sugarcane conversion (SC) (fipronil + 2,4-D + fertilizers). After 7 days of exposure, IP reduced catalase (CAT) and increased malondialdehyde (MDA) levels in P. nattereri, while this treatment decreased glucose-6-phosphate dehydrogenase (G6PDH) and CAT activities in S. fuscovarious. SC decreased CAT, G6PDH, and glutathione S-transferase (GST) activities in P. nattereri. In S. fuscovarius, SC reduced G6PDH, acetylcholinesterase (AChE), and carboxylesterase (CbE) activities. MDA was raised in both tadpole species exposed to SC, evidencing oxidative stress. Integrated biomarker responses showed higher scores in both species exposed to SC. Our results warn that management practices currently applied to sugarcane cultivation in Brazil can negatively impact the functional responses of amphibians at natural systems.

Irrigation with Water Contaminated by Sugarcane Pesticides and Vinasse Can Inhibit Seed Germination and Crops Initial Growth.

Sugarcane crops are dependent on chemicals for maintaining plantations. Therefore, environmental consequences concern adjacent areas that can be affected by contaminants in common use, including pesticides and vinasse (i.e., a by-product from the ethanol industry). This study aimed to evaluate phytotoxicity through two plant bioassays with water from mesocosms contaminated with the herbicide 2,4-D (447.0 µg L-1), the insecticide fipronil (63.5 µg L-1), and sugarcane vinasse (1.3%). First, the germination test (4 d) with Eruca sativa L. assessed water samples collected three times after the contamination (2 h, 14 d, and 30 d), considering germination, shoot, and root growth. The results from this bioassay indicated higher phytotoxicity for 2,4-D as it fully inhibited the shoot and root growth even in low concentrations (0.2 µg L-1). However, no significant effect was reported for fipronil and vinasse. Also, the 2,4-D effects drastically decreased due to an expressive concentration reduction (99.4% after 30 d in mixture with vinasse). Second, the irrigation test with Phaseolus vulgaris L. and Zea mays L. considered shoot and root growth and biomass under 21 days after plants emergence. The herbicide 2,4-D inhibited the initial growth of tested species, especially the roots (up to 45% inhibition). Furthermore, sugarcane vinasse caused harmful effects on plant growth (up to 31% inhibition). Therefore, our data showed that these contaminants could inhibit plant germination and initial growth under our tested conditions. These evaluations can endorse risk assessments and water management in sugarcane crops surrounding areas.

Functional responses of Hyalella meinerti after exposure to environmentally realistic concentrations of 2,4-D, fipronil, and vinasse (individually and in mixture).

Sugarcane crops management in Brazil includes the use of pesticides, as well as alternative organic fertilizers such as vinasse obtained from waste of the ethanol industry. In order to assess the effects of the environmental contamination generated by such sugarcane practices, this study was aimed to investigate the effects of the pesticides 2,4-Dichlorophenoxyacetic acid (2,4-D) and fipronil, as well as vinasse, on the survival, behavior, and reproduction of the native epibenthic macroinvertebrate Hyalella meinerti through in situ and laboratory experiments. In situ assays were conducted in mesocosms with six treatments, i.e. untreated control, 2,4-D, fipronil, and vinasse, the mixture of the two pesticides, and both pesticides mixed with vinasse. Survival, swimming behavior, and reproduction were evaluated over time post contamination, from 0-96 h (T1) and 7-14 days (T2) through in situ experiments and 30-44 days (T3) and 75-89 days (T4) post contamination by laboratory bioassays with mesocosm water. In the T1 period, survival of H. meinerti was registered only in controls and mesocosms treated with 2,4-D. In the T2 period, treatments containing fipronil and vinasse (isolated or in both mixture treatments) still caused 100 % of mortality. Survival was recorded only in 2,4-D and control treatments, whereas reproduction only occurred in the control. In the T3 period, no survival occurred to fipronil and both mixture treatments. Vinasse and 2,4-D decreased total reproduction in comparison to control. In the T4 period, amphipods survival was detected when exposed to fipronil and its mixture with 2,4-D. However, these same treatments decreased the amplexus rates and total reproduction, with synergism denoted for the pesticide mixture. The swimming activity of males, females, and couples was decreased in surviving organisms exposed to 2,4-D, fipronil, vinasse, and the mixture of pesticides along all experimental periods. Our study showed that the application of fipronil, 2,4-D, and vinasse isolated or mixed at realistic concentrations of actual sugarcane management practices may negatively impact functional responses of indigenous amphipods in natural aquatic systems.

Oxidative stress, biotransformation enzymes and histopathological alterations in Nile tilapia (Oreochromis niloticus) exposed to new and used automotive lubricant oil.

Lubricant oils are among oil-based products that are not fully consumed during its use, thereby producing non-biodegradable residues which can cause contamination of natural systems. This study evaluated the toxicity of new and used lubricating oil (0.01 and 0.1 mL L-1) in adult Nile tilapia (Oreochromis niloticus), by assessing the effects on oxidative stress, biotransformation enzymes (liver and gills), and histopathological alterations on hepatic and pancreatic tissues after 3 and 7 days of exposure. Results showed that 3-days exposure to 0.1 mL L-1 of used and new lubricating oil increased the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) levels in liver of O. niloticus, respectively. In gills, catalase (CAT) was decreased in fish exposed to 0.1 mL L-1 of non-used oil after 3 days, but pronounced increases in CAT was detected after 7 days-exposure to both new and used oil. Shorter exposure to both concentrations of new and used oil also raised glutathione-S-transferase activity (GST) in gills. Ethoxyresorufin-O-deethylase (EROD) was induced in liver of fish exposed to 0.1 mL L-1of used oil after 3 and 7 days, however a reduced response of this enzyme was detected in gills of animals from both oil treatments. In vitro analysis showed that hepatic EROD was inhibited by lubricating oil exposures, with more pronounced responses in treatments containing used oil. Hepatic lesions, such as cytoplasmic vacuolization, nuclei abnormally, changes in hepatocytes shape, steatosis, cholestasis, eosinophilic inclusions and necrosis were mainly increased by 7 days exposure to used lubricating oil at higher concentration.

Effects of 2,4-D-based herbicide (DMA® 806) on sensitivity, respiration rates, energy reserves and behavior of tadpoles.

Increased use of sugarcane pesticides and their destination to non-target environments in Brazil has generated concerns related to the conservation of more vulnerable groups, such as amphibians. Besides the high skin permeability, tadpoles are constantly restricted to small and ephemeral ponds, where exposure to high concentrations of pesticides in agricultural areas is inevitable. This study evaluated chronic effects caused by sub-lethal concentrations of 2,4-dichlorophenoxyacetic acid herbicide on energy storage, development, respiration rates, swimming performance and avoidance behavior of bullfrog tadpoles (Lithobates catesbeianus). Firstly, we conducted acute toxicity test (96 h) to estipulate sub-lethal concentrations of 2,4-D and evaluate the sensitivity of three tadpoles' species to this herbicide. Results showed that Leptodactylus fuscus presented the lowest LC50 96 h, 28.81 mg/L, followed by Physalaemus nattereri (143.08 mg/L) and L. catesbeianus (574.52 mg/L). Chronic exposure to 2,4-D (125, 250 and 500 µg/L) delayed metamorphosis and inhibited the growth of tadpoles at concentrations of 125 µg/L. Effects on biochemical reserves showed that 2,4-D increased total hepatic lipids in tadpoles, although some individual lipid classes (e.g. free fatty acids and triglycerides) were reduced. Protein and carbohydrates contents were also impaired by 2,4-D, suggesting a disruption on energy metabolism of amphibians by the herbicide. In addition to biochemical changes, respiration rates and swimming speed were also decreased after chronic exposure to 2,4-D, and these responses appeared to be correlated with the changes detected in the basic energy content. Avoidance test indicated that tadpoles of L. catesbeinus avoided the presence of 2,4-D, however they were unable to detect increasing gradients of the contaminant. Our data showed that chronic exposure to 2,4-D impaired biochemical, physiological and behavioral aspects of tadpoles, which may compromise their health and make them more vulnerable to environmental stressors in natural systems.

Isolated and mixed effects of diuron and its metabolites on biotransformation enzymes and oxidative stress response of Nile tilapia (Oreochromis niloticus).

Diuron is one of the most used herbicide in the world, and its field application has been particularly increased in Brazil due to the expansion of sugarcane crops. Diuron has often been detected in freshwater ecosystems and it can be biodegraded into three main metabolites in the environment, the 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU). Negative effects under aquatic biota are still not well established for diuron, especially when considering its presence in mixture with its different metabolites. In this study, we evaluated the effects of diuron alone or in combination with its metabolites, DCPMU, DCPU and 3,4-DCA on biochemical stress responses and biotransformation activity of the fish Oreochromis niloticus. Results showed that diuron and its metabolites caused significant but dispersed alterations in oxidative stress markers and biotransformation enzymes, except for ethoxyresorufin-O-deethylase (EROD) activity, that presented a dose-dependent increase after exposure to either diuron or its metabolites. Glutathione S-transferase (GST) activity was significant lower in gills after exposure to diuron metabolites, but not diuron. Diuron, DCPMU and DCA also decreased the multixenobiotic resistance (MXR) activity. Lipid peroxidation levels were increased in gill after exposure to all compounds, indicating that the original compound and diuron metabolites can induce oxidative stress in fish. The integration of all biochemical responses by the Integrated Biomarker Response (IBR) model indicated that all compounds caused significant alterations in O. niloticus, but DCPMU caused the higher alterations in both liver and gill. Our findings imply that diuron and its metabolites may impair the physiological response related to biotransformation and antioxidant activity in fish at field concentrations. Such alterations could interfere with the ability of aquatic animals to adapt to environments contaminated by agriculture.

Combined effects of temperature and clomazone (Gamit®) on oxidative stress responses and B-esterase activity of Physalaemus nattereri (Leiuperidae) and Rhinella schneideri (Bufonidae) tadpoles.

Temperature is an important factor influencing the toxicity of chemicals in aquatic environments. Neotropical tadpoles experience large temperature fluctuations in their habitats and many species are distributed in areas impacted by agriculture. This study evaluated the effects caused by the exposure to clomazone (Gamit®) at different temperatures (28, 32 and 36 °C) on biochemical stress responses and esterase activities in Physalaemus nattereri and Rhinella schneideri tadpoles. Results evidenced that temperature modulates the effects of clomazone on biochemical response of tadpoles. Antioxidant enzymes, including catalase, superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase had their activities increased by clomazone in P. nattereri treated at higher temperatures. The biotransformation enzyme glutathione-S-transferase (GST) was also induced by clomazone at 32 and 36 °C. In R. schneideri, clomazone failed to alter antioxidant enzymes at 28 °C, but SOD and GST were increased by clomazone at higher temperatures after three days. All enzymes had their activities returned to the control levels after eight days in R. schneideri. Lipid peroxidation was induced in both species exposed to clomazone at 32 and 36 °C, but not at 28 °C. Acetylcholinesterase was not sensitive to clomazone and temperature, while most treatments impaired carboxylesterase activity. Integrated biomarker response (IBR) was notably induced by temperature in both species, and a synergic effect of temperature and clomazone was mostly observed after three days of exposure. These findings imply that tadpoles from tropical areas may present differential responses in their physiological mechanism linked to antioxidant defense to deal with temperature fluctuations and agrochemicals presence in their habitats.

Influence of temperature on the antioxidant responses and lipid peroxidation of two species of tadpoles (Rhinella schneideri and Physalaemus nattereri) exposed to the herbicide sulfentrazone (Boral 500SC®).

Amphibians can experience large temperature fluctuations in their habitats, especially during the larval stage, when tadpoles are restricted to small and ephemeral ponds. Changes in water temperature can alter development, metabolism and behaviour of cold-blooded animals but also the toxicokinetics of chemicals in the environment. In Brazil, pesticides application is intensified during the rainy season, which is the period of reproduction for many amphibian species. We evaluated here the influence of temperature (28, 32, and 36°C) on the toxicity of the herbicide sulfentrazone (Boral®SC) in tadpoles of Physalaemus nattereri and Rhinella schneideri, by analysis of oxidative stress biomarkers. Exposure of tadpoles to sulfentrazone altered the antioxidant enzymes activities and induced lipid peroxidation with temperature-associated responses. Catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (G6PDH) were impaired by combined effect of temperature and sulfentrazone in both species. G6PDH was increased in most groups exposed to 36°C. Biotransformation enzyme glutathione-S-transferase had more evident alterations in P. nattereri at higher temperatures and changes in tGSH contents presented different patterns between the species. Lipid peroxidation was particularly induced in tadpoles of P. nattereri. Integrated biomarker response (IBR) index indicated a synergic effect of temperature and sulfentrazone for tadpoles of P. nattereri, while the IBR was mainly influenced by temperature in R. schneideri. Our study showed that temperature modulates biochemical responses in tadpoles exposed to sulfentrazone with a species-specific pattern. These findings imply that the effects of abiotic factors should be taken into account to evaluate the real risks of exposure of amphibians to commonly used pesticides.