Ecotoxicological effects of the emerging contaminant ivermectin on Rhinella arenarum: A comparative study of active ingredient and commercial formulation.

Ivermectin (IVM) is a broad-spectrum veterinary antiparasitic used worldwide in cattle breeding. The aim of this study was to evaluate the lethal effects of the active ingredient and a commercial formulation of IVM (1 % active ingredient) in the embryonic stage (S. 4-6) and larval stage (S. 25) of the South American amphibian Rhinella arenarum through chronic standardized bioassays. Also, behavior analysis and oxidative stress and cholinergic effects biomarkers were analyzed at 1, 10 and 100 µg IVM/L concentrations. For the embryonic stage, the active ingredient (96 h- LC50: 15900 µg/L) was more toxic than the commercial formulation (96 h-LC50: 51230 µg/L) during the acute period, while at chronic exposure the commercial formulation was more toxic (504 h-LC50: 10.25 µg/L), compared to the active ingredient (504 h-LC50: 312.80 µg/L). For the larval stage, in acute exposure, the active ingredient (96 h-LC50: 800 µg/L) was more toxic than the commercial formulation (96 h-LC50: 1550 µg/L). In the chronic exposure, the commercial formulation (504 h-LC50: 77.33 µg/L) was more toxic than the active ingredient (504 h-LC50: 195.25 µg/L). Overall, larvae exhibited greater sensitivity to both the active ingredient and the commercial formulation. However, during chronic exposure, embryos were more sensitive to the commercial formulation than larvae. The commercial formulation primarily induced oxidative stress, and both forms of the compound affected behavior and cholinergic effect biomarkers, even at low environmentally relevant concentrations (1 µg/L). These results highlight the potential impact of IVM on aquatic ecosystems.

A multi-approach analysis of the toxicity of a commercial formulation of monensin on Rhinella arenarum embryos and larvae.

Monensin, an antibacterial commonly used in animal fattening, can enter aquatic ecosystems and harm non-target organisms. Since there are no previous studies about the effects of monensin on amphibians, the aim of the present study was to evaluate the lethal and sublethal toxicity of a commercial formulation of monensin (CFM) through standardized bioassays with embryos and larvae of the amphibian Rhinella arenarum. Oxidative stress (catalase and glutathione S-transferase activities, and reduced glutathione and lipid peroxidation levels), cholinesterasic effect (acetylcholinesterase and butyrylcholinesterase activities) and mutagenicity (micronuclei frequency) biomarkers were evaluated. The CFM produced teratogenic effects, with a teratogenic index of 6.21. Embryos (504 h-LC50: 273.33 µg/L) were more sensitive than larvae, as no significant mortality was observed on larvae exposed up to 3000 µg/L for 504 h. However, oxidative stress, cholinesterasic effect and mutagenicity biomarkers were altered on larvae exposed for 96 h to environmentally relevant concentrations (4, 12 and 20 µg/L of monensin active ingredient). The CFM caused adverse effects on the exposed organisms, primarily on embryos, leading to lethal and sublethal effects, which could impact the wildlife when it reaches aquatic ecosystems.

Assessing the transformation products and fate of Oxytetracycline by simulated aerobic degradation tests.

Oxytetracycline (OTC) is a widely used broad-spectrum antibiotic, whose presence in water and sediments was reported in various regions of the world. The effects of OTC and other tetracyclines on the environment have been intensively studied although many of their transformation products (TPs) formed in the environment and their impact have not been yet fully characterized. Abiotic and biotic degradation tests under aerobic conditions at two pH values were carried out using OTC in artificial water/sediment systems to assess the effect of these variables on the environmental fate of the pollutant. HPLC-MSn was employed to detect and identify the main degradation products and pathways. Several transformations involved in the process were identified including alcohol oxidation, decarbonylation and hydroxylation. Differences in TPs and kinetics were found among degradation conditions, remarking a faster degradation of both OTC and TPs in the presence of microorganisms and at lower pH values. In summary, a total of 44 TPs were detected and structures were proposed for 20 of them, none of them having been previously reported. Furthermore, OTC degradation generated 24 TPs which remained in either solution or sediment, although none of them displayed higher algae toxicity than OTC. These results might be useful for planning future remediation and monitoring strategies.

Water and sediment quality of an agro-industrial area through different approaches: Physicochemical parameters and ecotoxicity with amphibians.

Amphibians are subject to several stressors in the aquatic and terrestrial environments, and human activities have profoundly impacted this vertebrate group. The aim of the present study was to analyze physicochemical parameters, metals and pesticide residues, and the toxicity of water and sediment samples from an environment with high agricultural activity (S1: Salto stream; S2: drainage channel downstream from S1) by means of bioassays using Rhinella arenarum (Amphibia: Anura) larvae. Metals and pesticides were analyzed in water and sediment samples by fluorescence spectrometer of X-ray by total reflection and ultra-high-performance liquid chromatography-MS/MS, respectively. For lethality bioassays, 10 larvae (in triplicate) were exposed for 504 h to water and sediment samples. Also, 50 larvae were exposed for 96 h (in triplicate) to water and sediment samples for the evaluation of biomarkers of neurotoxicity, oxidative stress, and genotoxicity. Twenty-six different pesticides (mainly herbicides) were detected in both sites, and Cu, Zn, and Pb exceeded the limit for protection of aquatic life. Lethality was observed in larvae exposed to water and sediment samples from both sites at chronic exposure. Oxidative stress was observed in larvae exposed to both sites. In larvae exposed to samples from S1, alterations in the neurotoxicity biomarkers were observed. These results alert about the degradation of the sites and highlight the need to monitor and control the use of pesticides. PRACTITIONER POINTS: Twenty-six pesticides were detected in water and sediment from Salto stream basin. Significant mortality was observed in larvae exposed to samples from all sites. Sublethal effects were observed mainly in larvae exposed to samples from Salto stream. The degraded quality can be associated with the agricultural activities of the area.

Metals, pesticides, and emerging contaminants on water bodies from agricultural areas and the effects on a native amphibian.

In the Paraná River lower basin, an important agro-productive area of Argentina, crop fields and cattle breeding activities are common and may affect water quality. So, the aim of this study was to analyze the impacts of cattle breeding and agricultural activities on a stream from Buenos Aires, through physicochemical parameters (metals, pesticides, and emerging contaminants) and ecotoxicological parameters with Rhinella arenarum larvae, a native amphibian species. Three sites were selected on an ordinary plain stream that goes through agricultural fields and a cattle breeding establishment (upstream -S1-, near -S2- and downstream -S3- the establishment). Physicochemical parameters were measured in situ (in water) and in laboratory (in water and sediment samples: metals, pesticides, ivermectin and oxytetracycline). A semi-static chronic toxicity bioassay (504 h) was performed with water samples, and neurotoxicity, oxidative stress and genotoxicity biomarkers were measured after acute exposure (96 h). According to the index, a degradation in the water quality was observed in all sites. Ivermectin (8.03 mg/kg) and oxytetracycline (1.9 mg/kg) were detected in sediment samples from S2. Pesticides were detected in all sites, mainly in water samples: S1 presented the highest variability (7 residues) and in S3 AMPA, glyphosate and acetochlor concentrations were higher (10.3, 22.4 and 23.8 µg/L). Also, all sites significantly produced lethality at chronic exposure. Lethality at 504h was 40% for S1, 56.66% for S2 and 93.33% for S3. At acute exposure, the oxidative stress biomarkers were altered on R. arenarum larvae exposed to all sites and the neurotoxicity biomarkers were altered on larvae exposed to S1 and S3. Water quality was severely degraded by the surrounding agricultural and cattle breeding activities, which may represent a threat to the ecosystems.

Environmental quality and ecotoxicity of sediments from the lower Salado River basin (Santa Fe, Argentina) on amphibian larvae.

The lower Salado River basin receive agricultural, industrial and domestic waste water. So, the aim was to evaluate the quality of three sampling sites that belong to the Salado River basin (S1: Cululú stream; S2: Salado River, at Esperanza City, S3: Salado River at Santo Tomé City) based on physicochemical parameters, metals and pesticides analyses and ecotoxicity on Rhinella arenarum larvae. R. arenarum larvae (Gosner Stage -GS- 25) were chronically exposed (504h) to complex matrixes of surface water and sediment samples of each site for the determination of the survival rate. Biomarkers of oxidative stress, neurotoxicity and genotoxicity were analyzed in R. arenarum larvae (GS. 25) after exposure (96h) to the complex matrix of water and sediment. The water quality index showed a marginal quality for all sites, influenced mainly by low dissolved oxygen, high total suspended solid, phosphate, nitrite, conductivity, Pb, Cr and Cu levels. Metal concentrations were higher in sediment than in water samples (˜34-35000 times). In total, thirty different pesticides were detected in all water and sediment samples, S1 presented the greatest variety (26). Glyphosate and AMPA were detected in sediments from all sites, being higher in S3. N,N-Diethyl-meta-toluamide (DEET) and atrazine were detected in all water samples. Greatest mortality was observed in larvae exposed to samples from S1 from 288h (43.3%), reaching a maximum value of 50% at 408h. Oxidative stress and genotoxicity were observed in larvae exposed to S1 and S3 matrix samples. Neurotoxicity was observed in larvae exposed to all matrix samples. The integrated biomarker response index showed that larvae exposed to S1 and S3 were the most affected. According to the physicochemical data and the ecotoxicity assessment, this important river basin is significantly degraded and may represent a risk to aquatic biota, especially for R. arenarum larvae.

Ecotoxicological assessment of complex environmental matrices from the lower Paraná River basin.

Sediments of aquatic ecosystems constitute the fate of most atmospheric and terrestrial pollutants. Since aquatic organisms, such as amphibians, interact with sediments, the presence of pollutants may affect their survival, growth and reproduction. So, the aim of this study was to evaluate, the sediment and water quality of five sites from the lower basin of the Paraná River (Buenos Aires, Argentina) with different anthropic impacts: Morejón stream (S1), de la Cruz stream upstream (S2) and downstream (S3), Arrecifes river (S4), tributary stream of Arrecifes river (S5). Physicochemical parameters were measured in situ (water) and in laboratory (water and sediment samples). Also, a screening of metals and pesticides was performed. Chronic (504 h) lethal toxicity bioassays were performed exposing Rhinella arenarum larvae to sediment and water samples. Oxidative stress (catalase, superoxide dismutase, glutathione S transferase, reduced glutathione and lipid peroxidation) and genotoxicity (micronuclei test) biomarkers were analyzed at acute (96 h) exposure. According to the calculated water quality index, S1 and S3 showed excellent quality, S2 good quality and, S4 and S5 poor quality. Dissolved oxygen was low in all sites (2.26-5.63 mg/L) and S5 had the highest organic matter content. Copper levels exceeded the limit for the protection of aquatic life in S2 and S4; arsenic levels exceeded its limit in S4; and selenium levels exceeded its limit in S4 and S5. Pesticides were mainly detected in water samples. Sediment from S5 showed higher sulfide and organic matter concentrations. At 504 h, no significant mortality was observed in the control group while S5 caused the greatest mortality (80%), followed by S2 (66.67%), S1 (63.33%), S3 (46.67%) and S4 (43.4%). All samples caused oxidative stress and lipid peroxidation, and samples from S4 also caused genotoxicity. The analysis of sediment and water samples was a suitable approach to assess the effects of water bodies on a native amphibian species.

Exposure to toxic Microcystis via intact cell ingestion and cell crude extract differently affects small-bodied cladocerans.

Cyanobacterial blooms are increasingly common in aquatic environments worldwide. These microorganisms cause concern due to their ability to produce cyanotoxins. Aquatic organisms, especially zooplankton, are exposed to cyanobacterial toxins by different routes, depending on the bloom phase. During cyanobacterial dominance, zooplankton is exposed to cyanotoxins through the ingestion of cyanobacterial cells, while at the bloom senescence, dissolved toxins are the most representative route. In this study, we assessed the effects of a microcystin-producing strain of Microcystis aeruginosa (NPLJ-4) on clones of the tropical small cladocerans Macrothrix spinosa (two clones) and Ceriodaphnia cornuta (one clone) exposed to intact cells and aqueous cell crude extracts. Short-term toxicity assays and life-table experiments were performed to assess the effects of the toxic M. aeruginosa on the survival and life history of the cladocerans. In the short-term toxicity assay, we found that cladocerans were more affected by intact cells. Both clones of M. spinosa were more affected when exposed to intact cells, while C. cornuta displayed about 5-fold more resistance. On the other hand, crude extracts had a low impact on cladocerans' survival. Also, we observed a significant decrease in survival, fecundity, and growth of animals exposed to sublethal and environmentally relevant concentrations of M. aeruginosa cellular biomass. However, even at high concentrations of dissolved microcystins, the crude extract did not have significant effects on the life history parameters of the cladocerans. Although they can be found during cyanobacterial bloom events, small-bodied cladocerans are still affected by toxic Cyanobacteria depending on the exposure route.

Comprehensive assessment of water quality through different approaches: Physicochemical and ecotoxicological parameters.

Traditionally, water quality was assessed by physicochemical parameters. However, a more comprehensive analysis is needed to study the effects of polluted water bodies on key species over time. So, the aim of this study was to monitor through physicochemical and ecotoxicological indicators the surface water quality of four study sites with different land uses from the lower Paraná river basin (Argentina) during spring and summer of two years: Morejón stream (S1), De la Cruz stream upstream (S2), downstream (S3) and Arrecifes river (S4). Physicochemical parameters were measured in situ and in laboratory, and a Water Quality Index (WQI) was calculated. Chronic toxicity bioassays were performed with surface water samples using Rhinella arenarum embryos and larvae. Also, oxidative stress (catalase, superoxide dismutase, glutathione S-transferase, reduced glutathione and lipid peroxidation), neurotoxicity (butyrylcholinesterase) and genotoxicity (micronuclei frequency) biomarkers were measured at acute exposure, and an Integrated Biomarkers Response (IBR) index was calculated. The water quality varied between excellent and bad in S1, good and bad in S2 and S3, and bad and marginal in S4. S1 presented the greatest variability of pesticides and S4 the highest number of metals exceeding the limits for the local protection of aquatic life. Mainly, S4 caused lethality in R. arenarum larvae, reaching a maximum mortality of 83.3% at 504 h of exposure. The lethal toxicity of S1 and S2 varied between periods. Water samples from all sites altered the oxidative stress, neurotoxicity and genotoxicity biomarkers, and the IBR was negatively correlated with the WQI. The IBR reflected the effects of the degraded water quality on the exposed organisms. So, the importance of evaluating both physicochemical and ecotoxicological parameters to analyze integrally the water quality of polluted areas is highlighted. A degradation of the studied water bodies and its negative impact to the native amphibian R. arenarum were observed.

Toxicity characterization and environmental risk assessment of Mancozeb on the South American common toad Rhinella arenarum.

Agricultural activity, especially the increasing use of pesticides, is considered one of the main reasons for the decline of amphibian populations. Mancozeb (MCZ) is one of the most used fungicides worldwide, despite its ancient use and toxicity demonstrated in different taxa. However, there is limited information about the effects of MCZ in amphibians, which are keystones of riparian ecosystems. For species conservation purposes, it is essential to identify the most sensitive developmental period(s) of a given species to a xenobiotic. We evaluated the toxicity of a commercial fungicide of mancozeb, (80% active ingredient) on the early development of the common South American toad Rhinella arenarum (Anura, Bufonidae). Embryos from early blastula (S.4) and larvae from complete operculum (S.25) stages were exposed to a wide range of MCZ concentrations during acute, subchronic and chronic exposure (up to 504 h) periods. The toxicity profiles for lethal and sublethal effects were performed. At all exposure times, MCZ was more toxic to embryos, for instance, NOEC 504 h were 0.01 and 0.05 mg MCZ/L for embryos and larvae, respectively. Thus, embryo sensitivity was 5-fold higher than larvae. A Teratogenic Index of 14 indicated the significant teratogenic potential of this fungicide. Among sublethal effects, embryos exhibited a wide range of abnormalities with high incidence. The ecological risk assessment demonstrated that the estimated Risk Quotient value for Rhinella arenarum embryos at chronic exposure was higher than the Level of Concern value, which warns about the potential risk of MCZ for this native species.

Developmental toxicity of bisphenol A diglycidyl ether (epoxide resin badge) during the early life cycle of a native amphibian species.

Bisphenol A diglycidyl ether (BADGE) is used in packaging materials, in epoxy adhesives, and as an additive for plastics, but it is also a potential industrial wastewater contaminant. The aim of the present study was to evaluate the adverse effects of BADGE on Rhinella arenarum by means of standardized bioassays at embryo-larval development. The results showed that BADGE was more toxic to embryos than to larvae at all exposure times. At acute exposure, lethality rates of embryos exposed to concentrations of 0.0005 mg/L BADGE and greater were significantly higher than rates in the vehicle control, whereas lethality rates of larvae were significantly higher in concentrations of 10 mg/L BADGE and greater. The toxicity then increased significantly, with 96-h median lethal concentrations (LC50s) of 0.13 mg/L and 6.9 mg/L BADGE for embryos and larvae, respectively. By the end of the chronic period, the 336-h LC50s were 0.04 mg/L and 2.2 mg/L BADGE for embryos and larvae, respectively. This differential sensitivity was also ascertained by the 24-h pulse exposure experiments, in which embryos showed a stage-dependent toxicity, with blastula being the most sensitive stage and S.23 the most resistant. The most important sublethal effects in embryos were cell dissociation and delayed development, whereas the main abnormalities observed in larvae related to neurotoxicity, as scare response to stimuli and narcotic effect. Environ Toxicol Chem 2016;35:3031-3038. © 2016 SETAC.