High toxicity of agro-industrial wastewater on aquatic fauna of a South American stream: Mortality of aquatic turtles and amphibian tadpoles as bioindicators of environmental health.

The aim of this study was to characterize an aquatic system of Santa Fe province (Argentina) receiving wastewater from agro-industrial activities (mainly dairy) by in situ assessment (fauna mortality, physicochemical, microbiological, and pesticide residues measurement), and ecotoxicity bioassays on amphibian tadpoles. Water and sediment samples were obtained from the Los Troncos Stream (LTS), previous to the confluence with the "San Carlos" drainage channel (SCC), and from the SCC. Biological parameters (mortality and sublethal biomarkers) were used to evaluate ecotoxicity during 10-day exposure of Rhinella arenarum tadpoles to LTS and SCC samples. Nine pesticides were detected in both LTS and SCC. Chemical and biochemical oxygen demand, ammonia, and coliform count recorded in SCC greatly exceeded limits for aquatic life protection. At SCC and LTS after the confluence with SCC, numerous dying and dead aquatic turtles (Phrynops hilarii) were recorded. In the ecotoxicity assessment, no mortality of tadpoles was observed in LTS treatment, whereas total mortality (100%) was observed in SCC treatments in dilution higher than 50% of water and sediment. For SCC, median lethal concentration and the 95% confidence limits was 18.30% (14.71-22.77) at 24 h; lowest-observed and no-observed effect concentrations were 12.5% and 6.25%, respectively. Oxidative stress and neurotoxicity were observed in tadpoles exposed to 25% SCC dilution treatment. In addition, there was a large genotoxic effect (micronuclei test) in all sublethal SCC dilution treatments (6.25%, 12.5%, and 25%). These results alert about the high environmental quality deterioration and high ecotoxicity for aquatic fauna of aquatic ecosystems affected by agro-industrial wastewater. PRACTITIONER POINTS: Great mortality of turtles was observed in a basin with a high load of agro-industrial wastewater. San Carlos Channel (SCC), where effluents are spilled, is environmentally deteriorated. The water-sediment matrix of SCC caused 100% lethality in tadpoles. SCC dilutions caused neurotoxicity, oxidative stress, and genotoxicity on tadpoles.

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.