Glyphosate and glufosinate ammonium, herbicides commonly used on genetically modified crops, and their interaction with microplastics: Ecotoxicity in anuran tadpoles.

The effects of glyphosate (GLY)-based and glufosinate ammonium (GA)-based herbicides (GBH and GABH, respectively) and polyethylene microplastic particles (PEMPs) on Scinax squalirostris tadpoles were assessed. Tadpoles were exposed to nominal concentrations of both herbicides (from 1.56 to 100 mg L-1) and PEMPs (60 mg L-1), either alone or in combination, and toxicity evaluated at 48 h. Acetylcholinesterase (AChE), carboxylesterase (CbE), and glutathione-S-transferase (GST) activities were analyzed at the three lowest concentrations (1.56, 3.12 and 6.25 mg L-1, survival rates >85%) of both herbicides alone and with PEMPs. Additionally, the thermochemistry of the interactions between the herbicides and polyethylene (PE) was analyzed by Density Functional Theory (DFT). The median-lethal concentration (LC50) was 43.53 mg L-1 for GBH, 38.56 mg L-1 for GBH + PEMPs, 7.69 for GABH, and 6.25 mg L-1 for GABH+PEMPs. The PEMP treatment increased GST but decreased CbE activity, whereas GBH and GABH treatments increased GST but decreased AChE activity. In general, the mixture of herbicides with PEMPs increased the effect observed in the individual treatments: the highest concentration of GBH + PEMPs increased GST activity, whereas GABH+PEMP treatments decreased both AChE and CbE activities. DFT analysis revealed spontaneous interactions between the herbicides and PE, leading to the formation of bonds at the herbicide-PE interface, significantly stronger for GA than for GLY. The experimental and theoretical findings of our study indicate that these interactions may lead to an increase in toxicity when pollutants are together, meaning potential environmental risk of these combinations, especially in the case of GA.

Toxicity assessment at different experimental scenarios with glyphosate, chlorpyrifos and antibiotics in Rhinella arenarum (Anura: Bufonidae) tadpoles.

The presence of pesticides as well as that of several antibiotics provided at a great scale to poultry, cattle, and swine in aquatic environments within agroecosystems is a matter of growing concern. The objective of the present study was to characterize the sublethal effects of four environmental toxic compounds at two experimental pollution scenarios on the morphology, development and thyroid (T4), acetylcholinesterase (AChE) and glutathione S-transferase (GST) levels in Rhinella arenarum tadpoles. The first experimental pollution scenario aimed to evaluate the individual and mixed toxicity (50:50% v/v) of a glyphosate-based herbicide (GBH) and the antibiotic ciprofloxacin (CIP) on earlier developmental stages. The second experimental pollution scenario aimed to evaluate the effects of other toxic compounds (the insecticide chlorpyrifos (CP) and the antibiotic amoxicillin (AMX)) added to the ones from the first scenario on previously exposed premetamorphic tadpoles. In all the treatments of the first pollution scenario, the most conspicuous effect observed in early-stage tadpoles was a high prevalence of morphological abnormalities. Exposure to GBH and to its mixture with CIP also led to a significant decrease in T4 levels and lower development. Both pollutant combinations from the second experimental scenario significantly increased T4 levels, inhibited AChE activities, and led to lower development, whereas the quaternary mixture led to a significant decrease in GST levels. The alterations here revealed by our approaches in several morphological and biochemical endpoints allow characterizing the ecotoxicological risk for anurans exposed to complex mixtures of pollutants that frequently occur in aquatic systems.

Morphological and histological abnormalities of the neotropical toad, Rhinella arenarum (Anura: Bufonidae) larvae exposed to dexamethasone.

Dexamethasone (DEX) is a glucocorticoid highly effective as an anti-inflammatory, immunosuppressant and decongestant drug. In the present study, a preliminary acute toxicity test was assayed in order to determinate DEX median-lethal, lowest-observed-effect and the no-observed-effect concentrations (LC50, LOEC and NOEC, respectively) on the common toad embryos (Rhinella arenarum). Also, morphological and histological abnormalities from five body larval regions, liver melanomacrophages (MM) and glutathione S-transferase (GST) activity were evaluated in the toad larvae to characterize the chronic sublethal effects of DEX (1-1,000 µg L-L). Results of the acute test showed that the LC50 of DEX at 96 h of exposure for the toad embryos (GS 18-20) was 10.720 mg L-g, and the LOEC was 1 µg L-g. In the chronic assay, the larval development and body length were significantly affected. DEX exposition also induced teratogenic effects. Most frequent external abnormalities observed in DEX-treated larvae included abdominal edema and swollen body, abnormal gut coiling and visceral congestion. Intestinal dysplasia was recurrent in cross-section of all DEX-treated larvae. Neural, conjunctive and renal epithelial cells were also affected. Significant increase in liver MM number and size, and GST activity levels were also registered in DEX treatments with respect to controls. The evaluation of a variety of biomarkers provided clear evidence of toad larvae sensitivity to DEX, and the ecotoxicological risk of these pharmaceuticals, commonly found in different water bodies worldwide on aquatic animals.

Comparative Toxicity of Two Different Dimethoate Formulations in the Common Toad (Rhinella arenarum) Tadpoles.

Dimethoate (D) are among the most commonly used organophosphates insecticides in the world. To evaluate the toxicity of two D formulations were selected as test organisms tadpoles of Rhinella arenarum. This toad species has an extensive neotropical distribution and is easy to handle and acclimate to laboratory conditions. The tadpoles were exposed in an acute assay for 48 h to D soluble concentrates (DSC) and emulsifiable concentrates (DEC). The 48 h-LC50 (95% confidence limits) value of DSC was 57.46 mg L-1 (40.52-81.43) and to DEC was 12.76 mg L-1 (10.39-15.68). These differences in toxicity were statistically significant (p < 0.05). In both formulations, acetylcholinesterase), carboxylesterase, and glutathione-S-transferases enzyme activities varied significantly respect to those of control group (p < 0.05). The DEC formulation was the most toxic. These results would allow the assessment and characterization of potential ecological risks following the application of those formulations.

First evaluation of novel potential synergistic effects of glyphosate and arsenic mixture on Rhinella arenarum (Anura: Bufonidae) tadpoles.

The toxicity of glyphosate-based herbicide (GBH) and arsenite (As(III)) as individual toxicants and in mixture (50:50 v/v, GBH-As(III)) was determined in Rhinella arenarum tadpoles during acute (48 h) and chronic assays (22 days). In both types of assays, the levels of enzymatic activity [Acetylcholinesterase (AChE), Carboxylesterase (CbE), and Glutathione S-transferase (GST)] and the levels of thyroid hormones (triiodothyronine; T3 and thyroxine; T4) were examined. Additionally, the mitotic index (MI) of red blood cells (RBCs) and DNA damage index were calculated for the chronic assay. The results showed that the LC50 values at 48 h were 45.95 mg/L for GBH, 37.32 mg/L for As(III), and 30.31 mg/L for GBH-As(III) (with similar NOEC = 10 mg/L and LOEC = 20 mg/L between the three treatments). In the acute assay, Marking's additive index (S = 2.72) indicated synergistic toxicity for GBH-As(III). In larvae treated with GBH and As(III) at the NOEC-48h (10 mg/L), AChE activity increased by 36.25% and 33.05% respectively, CbE activity increased by 22.25% and 39.05 % respectively, and GST activity increased by 46.75% with the individual treatment with GBH and by 131.65 % with the GBH-As(III) mixture. Larvae exposed to the GBH-As(III) mixture also showed increased levels of T4 (25.67 %). In the chronic assay at NOEC-48h/8 (1.25 mg/L), As(III) and GBH-As(III) inhibited AChE activity (by 39.46 % and 35.65%, respectively), but did not alter CbE activity. In addition, As(III) highly increased (93.7 %) GST activity. GBH-As(III) increased T3 (97.34%) and T4 (540.93%) levels. Finally, GBH-As(III) increased the MI of RBCs and DNA damage. This study demonstrated strong synergistic toxicity of the GBH-As(III) mixture, negatively altering antioxidant systems and thyroid hormone levels, with consequences on RBC proliferation and DNA damage in treated R. arenarum tadpoles.

Insecticide pyriproxyfen (Dragón®) damage biotransformation, thyroid hormones, heart rate, and swimming performance of Odontophrynus americanus tadpoles.

Odontoprynus americanus tadpoles were used to determine the safety concentration of pyriproxyfen (PPF) insecticide by acute and sublethal toxicity tests (nominal range tested 0.01 to 10 [± 15%] PPF mg/L). Median lethal concentration (LC50) and no, and lowest-observed-effect concentrations (NOEC and LOEC, respectively) were calculated. We also assessed the effect on the activities of glutathione S-transferse (GST), acetylcholinesterase (AChE), and carboxylesterase (CbE) and compared to control (CO) tadpoles. Based on the 48-h NOEC value, two sublethal concentrations of PPF (0.01 and 0.1 mg/L) were assayed to detect effects on enzymes activities (GST and CbE), thyroid hormone's levels (triiodothyronine; T3 and thyroxine; T4), heart function, and tadpoles swimming behaviour. The results showed that the LC50 values of O. americanus tadpoles were 3.73 PPF mg/L and 2.51 PPF mg/L at 24-h and 48-h, respectively (NOEC = 0.1 mg/L; LOEC = 1 mg/L, for both times). PPF concentrations at 48 h, induced enzymatic activities such as GST (212.98%-242.94%), AChE (142.15%-165.08%), and CbE (141.86%-87.14%) significantly respect to COs. During the 22 days of chronic PPF exposure, GST (0.01 mg/L 88%-153% NOEC), AChE (177.82% NOEC), and T4 (70% NOEC) also significantly increased respect to COs. Similarly, heart rate (fH) and ventricular cycle length (VV interval) in CO tadpoles were significantly higher than PPF treated. Finally, at NOEC tadpoles exhibited significant effects on the behavioral endpoint (swimming distance, mean speed, and global activity; P < 0.05).

Blood biomarkers of common toad Rhinella arenarum following chlorpyrifos dermal exposure.

Chlorpyrifos (CPF) is a broad spectrum pesticide commonly used for insect control, has great affinity for lipids and is thus a potential for bioaccumulation in aquatic organisms. The aim of this study was to evaluate the toxicity of CPF using the common toad Rhinella arenarum via dermal uptake in plastic bucket to simulate their natural exposition in ponds. R. arenarum toads were exposed individually to solutions containing a nominal concentration of a commercial formulation of CPF insecticide (5 and 10 mg/L). Different enzyme biomarkers (BChE: butyrylcholinesterase, CbE: carboxylesterase, and CAT: catalase) were measured in blood tissue after exposition. The capacity of pyridine-2-aldoxime methochloride (2-PAM) to reverse OP-inhibited plasma BChE and the ratio of heterophils and lymphocytes (H/L) as hematological indicators of stress were also determined. The normal values of plasma B-sterases (BChE and CbE) were highly inhibited (until ≈ 70%) in toads 48 h after exposure to CPF. The results indicate that 2-PAM produced BChE reactivation as well. The activity of CAT was also inducted for dermal exposure at more than double of that in the control toads (CPF; 5 mg/L). H/L ratios did not reveal a significantly increased stress. The study suggests that CPF via dermal uptake induced neurotoxicity and oxidative stress in the common toad R. areanum. Thus, some blood biomarkers employed in our study (i.e. BChE, CbE, 2-PAM, and CAT) might be used as predictors in health and ecological risk assessment of amphibian populations exposed to CPF.