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.

Effects of the emulsifiable herbicide Dicamba on amphibian tadpoles: an underestimated toxicity risk?

The effects of exposure to the herbicide Dicamba (DIC) on tadpoles of two amphibian species, Scinax nasicus and Elachistocleis bicolor, were assessed. Mortality and biochemical sublethal effects were evaluated using acetylcholinesterase (AChE), glutathione S-transferase (GST), glutathione reductase (GR), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities and thyroid hormone (T4) levels. The LC50 value at 48h was 0.859 mg L-1 for S. nasicus and 0.221 mg L-1 for E. bicolor tadpoles. After exposure to sublethal DIC concentrations for 48 h, GST activity increased in S. nasicus but significantly decreased in E. bicolor with respect to controls. GR activity decreased only in S. nasicus at all the tested DIC concentrations. AChE activity was significantly inhibited in both S. nasicus and E. bicolor tadpoles at 48 h. DIC also caused significant changes in transamination, as evidenced by an increase in AST and ALT activities in both amphibian species. T4 levels were higher in DIC-treated tadpoles of both species than in controls. The DIC-induced biochemical alterations in glutathione system enzymes and transaminases indicate lesions in liver tissues and cellular function. Moreover, the observed AChE inhibition could lead to the accumulation of acetylcholine, excessively stimulating postsynaptic receptors, and the increase in T4 levels in both species may indicate an overactive thyroid. The commercial DIC formulation showed a high biotoxicity in the two amphibian native species after short-term exposure, controversially differing from the toxicity level indicated in the official fact sheet data. This fact highlights the need for an urgent re-categorization and reevaluation of DIC toxicity in native species.

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.

Biotoxicity of diclofenac on two larval amphibians: Assessment of development, growth, cardiac function and rhythm, behavior and antioxidant system.

The non-steroidal anti-inflammatory drug diclofenac (DCF) threatens the health of aquatic animals and ecosystems. In the present study, different biological endpoints (mortality, development and growth, abnormalities, cardiotoxicity, neurotoxicity and antioxidant system) were used to characterize the acute and chronic effects of DCF (at concentrations ranging between 125 and 4000 µg L-1) on two amphibian species from Argentina (Trachycephalus typhonius and Physalaemus albonotatus). Results showed that the larval developmental, growth rates, and body condition of DCF-exposed individuals of both species were significantly reduced. DCF-exposed individuals also showed several morphological abnormalities, including significantly altered body axis, chondrocranium and hyobranchial skeleton, and organ and visceral abnormalities including cardiac hypoplasia, malrotated guts, asymmetrically inverted guts, and cholecystitis. DCF also had a significant effect on the swimming performance of both species: at low concentrations (125 and 250 µg L-1), swimming distance, velocity and global activity decreased, whereas, at high concentrations (1000 and 2000 µg L-1), these behavioral responses increased. Regarding cardiac function and rhythm, at DCF concentrations higher than 1000 µg L-1, the heart frequency and ventricular systole interval of both species were significantly reduced. Regarding the antioxidant system, the activity of acetylcholinesterase indicated that DCF is neurotoxic and thus related to the changes in behavioral performance. The DCF concentrations studied produced a biochemical imbalance between radical oxygen species production and antioxidant systems. The sensitivities to sublethal and chronic DCF exposure in both anuran species were similar, thus indicating the inherent complexity involved in understanding the biotoxic effects of DCF.

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.

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae.

The ecological risks posed by two ß-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000µgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10µgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000µgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10µgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.