Evaluation of cytotoxicity, genotoxicity and ecotoxicity of nanoemulsions containing Mancozeb and Eugenol.

Mancozeb is a fungicide widely used in agriculture, mostly against the pathogen Glomerella cingulata responsible for the rot of ripe grape, but presents high toxicity. Strategies are sought to reduce the toxicity of this fungicide and alternative treatments are welcome. An alternative could be the use of clove oil, which has Eugenol as its major compound, and has antifungal potential against G. cingulata, however, Eugenol is susceptible to degradation processes which may compromise its efficacy. The nanoencapsulation of Mancozeb and Eugenol is a possible strategy to overcome the limitations of toxicity, solubility and instability of these compounds. Therefore, the objective of this study is to develop nanoemulsions containing Mancozeb (0.1 mg/mL) and Eugenol (33 mg/mL), isolated or associated, and evaluate the safety of these formulations through cytotoxicity, genotoxicity and ecotoxicity tests. Nanoemulsions were developed by the spontaneous emulsification method, cytotoxicity and genotoxicity were evaluated in healthy human cells through MTT, Dichlorofluorescein diacetate and Picogreen tests, and ecotoxicity assessment was carried out using the chronic toxicity test in springtails. After preparation, the physicochemical characterization of the nanoemulsions were performed which presented mean particle size between 200 and 300 nm, polydispersity index less than 0.3, negative zeta potential and acid pH. The nanoencapsulation was able to avoid the reduction of the cell viability caused by Mancozeb, while Eugenol was shown to be safe for cell use in both free and nanostructured forms, however the association of the two active compounds showed toxicity in the higher doses of Mancozeb. In the ecotoxicity tests, both free Mancozeb and Eugenol forms presented high toxic potential for soil, whereas the nanoencapsulation of these compounds did not cause a reduction in number of springtails. Therefore, from the tests performed, it was possible to observe that nanoencapsulation of Mancozeb and Eugenol is a safe alternative for the application of these compounds mainly in agriculture.

Ecotoxicological effects of fipronil, neem cake and neem extract in edaphic organisms from tropical soil.

Veterinary medicines are widely applied for the treatment and prevention of animal diseases. Consequently, animal manure contains significant amounts of environmental pollutants that are potential sources of environmental pollution when inappropriately applied in soils. This work aimed to evaluate ecotoxicological effects of doses of commercial fipronil, neem cake and neem extract in the survival and reproduction of earthworms (Eisenia andrei), enchytraeidae (Enchytraeus crypticus) and springtails (Folsomia candida) in Oxisol and tropical artificial soil (TAS). Applications of fipronil, neem cake and extract in soil were carried out according to standardized ISO methodologies by using a random experimental design with five replicates. Toxic effects of fipronil for springtails in Oxisol and TAS were observed with LC50 of 0.26 mg kg-1 (0.18-0.35 mg kg-1) and 0.29 mg kg-1 (0.22-0.37 mg kg-1), respectively. It was not observed significant toxic effects of fipronil for earthworms and enchytraeidae in both soils. However, significant amounts of juvenile earthworm and adult enchytraeidae decreased in fipronil doses higher than 10 mg kg-1. Neem cake and extract were not toxic for earthworms and enchytraeidae but, significant amounts of juvenile springtails decreased in neem cake doses from 500 to 1000 mg kg-1. It can be concluded that the use of veterinary medicines containing synthetic compounds for preventing diseases in animals needs to be controlled to avoid environmental pollution after applying manure in soil. Veterinary medicines containing natural compounds as neem cake and extract are eco-friendly and could be efficiently applied in soil in a sustainable way.

Ecotoxicological effect of fipronil and its metabolites on Folsomia candida in tropical soils.

The aim of this study is to evaluate the toxicity of the fipronil and its metabolites, fipronil sulfone and fipronil desulfinyl on Folsomia candida. Three types of soils were used to perform the ecotoxicological tests, two natural soils (Oxisol and Entisol), and an artificial substrate (Tropical Artificial Soil). The treatments consisted of increasing doses of a veterinary medicinal product with active ingredient based on fipronil, and of its two main metabolites, fipronil sulfone and fipronil desulfinyl. The doses of fipronil were 0.00, 0.02, 0.04, 0.08, 0.15, 0.30, 0.60, and 1.00 mg kg-1 of the active ingredient. For fipronil sulfone and fipronil desulfinyl the doses tested were: 0.00, 0.01, 0.02, 0.04, 0.06, 0.08, 0.15, 0.30, 0.60, 1.00, and 5.00 mg kg-1. The effects of toxicity on F. candida were evaluated by survival and reproduction tests, based on the methods described by ISO 11267. There was a completely randomized design, with six replicates for the fipronil tests and eight for the metabolites. We show that fipronil and its metabolites, even at very low concentrations, are toxic to F. candida. Reduction of reproduction can be observed at concentrations of 0.15 mg kg-1. There was little difference in toxicity (LC50 and EC50) between fipronil and its metabolites. Rather, differences were related more to soil type, where the artificial soil was more sensitive than the two natural soils (Oxisol and Entisol). We conclude that it is important to perform ecotoxicological tests in natural soils, in order to generate more realistic representations of veterinary drug toxicity on the environment.