1. Introduction

Recently, epidemics of dengue, chikungunya, yellow fever and zika caused by the vector Aedes aegypti affected a large part of the Brazilian population. From this, government campaigns and control measures were adopted, providing large quantities of malathion insecticide to the environment^1-3. Malathion is an organophosphate type C widely used in rural and urban areas to control of aphids, flies. It is formulated as household sprays and being sprayed in areas with high proliferation rate of Aedes aegypti^{2, 4}. It presents the advantage of not being persistent in the environment and does not bioaccumulate. However, it presents high toxicity in animals due to its action mechanism, which inhibits the enzyme acetylcholinesterase, forming a complex with the esterase center of the enzyme. This results in accumulation of the neurotransmitter acetylcholine, interrupting the breakage of this substance in choline and acetic acid, causing several symptoms, such as nervous hyperactivity^5-9.

Aiming at the degradation of this pesticide as an alternative to make it less available in the environment, it is possible resort to simple and effective treatments, which includes advanced oxidative processes (AOPs). These processes are based on the generation of highly reactive and oxidizing species such as hydroxyl radicals, it can be obtained in various forms, including heterogeneous and homogeneous systems, with or without radiation. The main objective is to generate at the end of the process species less toxic such as water and carbon dioxide^10-12. Recent works have shown the efficiency of AOPs, as photo-Fenton process¹³, UVC/H₂O₂¹⁴ and others¹⁵, in the degradation of malathion in aqueous solutions. However, for the degradation of malathion, it is possible to obtain several by-products of the oxidation reaction, including substances like phorate sulfoxide, diethyl phosphate, pharatoxon sulfone and malaoxon, the latter being very toxic, which inhibition power under the enzyme acetylcholinesterase is 40 times higher than the parent compound^{16, 17}.

Like pesticides, their treatment products can also cause effects on the environment, which highlights the need for ecotoxicological assessments that can demonstrate and quantify possible adverse effects to organisms exposed to these substances^{18, 19}. In this sense, the present work evaluated the acute ecotoxicity of malathion before and after degradation by UVC radiation and UV/H₂O₂ using Lactuca sativa seeds and Aedes aegypti larve as bioindicators. Lettuce seed (Lactuca sativa) was chosen as the indicator organism, using the observation of interferences in seed germination and root growth^20-22. The species Aedes aegypti was also chosen for toxicity and larvicide evaluation because it is the target organism of malathion²³.

2. Experimental

For ecotoxicity evaluation, samples were collected at initial (0 min), intermediate (30 min), and final treatment times (120 min) in previously washed glass flasks. The pH was adjusted to 6∼7, and when necessary, bovine catalase (Sigma-Aldrich) was added for residual H₂O₂ removal. Samples were analyzed immediately or, when this was not possible, they were kept frozen at −20 °C for a maximum of 20 days until the tests were performed. Two tests were selected: Aedes aegypti (acute toxicity) and Lactuca sativa seeds (phytotoxicity).

2.1 Ecotoxicological tests with Aedes aegypti

Aedes aegypti tests were performed according to methodology described by the World Health Organization²⁴. Healthy eggs (Rockfeller variety) were used, provided by the Laboratory of Physiology and Control of Arthropod Vectors of the Oswaldo Cruz Foundation – RJ. Egg hatching was performed in a 1 L Becker containing 500 mL of mineral water with the addition of a small amount of fish feed. Packing was carried out in a B.O.D. incubator, with photoperiod of 16 h clear and 8 h dark, maintained at a temperature of 28 °C.

Commercial product Malathion^® 500 CE were used for the test solutions preparation, which were diluted with ultrapure water. In all of them, there were three preliminary tests (data not show). The concentration in focus refers to that recommended by the manufacturer, which indicates the dilution of 30 mL of the pesticide in 10 L of water²⁵.

Preliminary tests were performed in order to determine the concentration range necessary to determine the effective concentration at 50% of the organisms (EC₅₀), being the range of 0.05; 0.1; 0.2; 0.4; 0.8 and 1 μg L^-116. Tests were performed in triplicate, the definitive ones and the treated samples were done in quadruplicate, in each replicate, containing 25 mL of solution and 20 organisms, whose life stage is between the 3^rd and 4^th larval stage, approximately 96 h of life. The assays were maintained in B.O.D. at 23 °C, without photoperiod, for 24 h. After that, the number of immobile organisms was counted, thus obtaining the percentage of immobility.

2.2 Phytotoxicity with Lactuca sativa

In order to obtain the EC₅₀, solutions of the commercial product Malathion^® 500 CE were prepared for the realization of the bioassay with L. sativa. Preliminary tests were performed with concentrations ranging from 1 μg L^-1 to 400 μg L^-1, and the concentration range selected for the final test was 250, 350, 450, 550, 600 and 700 μg L^-1, where adverse effects were observed.

The seed germination/root elongation phytotoxicity assays were performed according to the methodology described by Sobrero and Ronco²² and Young et al.²⁶. Tests were carried out in Petri dishes lined with filter paper (Unifil, weight 80 g m^-2) and with 15 seeds each (cv. Boston), containing 4 mL of sample dilution or negative/positive control, with osmosis water and commercial glyphosate 3% (Dipil), respectively. The assay was done in triplicate. Seeds were incubated at 22 ± 2 °C, in the dark, for 120 h. At the end of the test, the number of germinated seeds and the root elongation data were used to calculate the germination index (GI)²⁰ and the relative growth index (RGI)²⁶. The RGI values were divided into three categories according to the observed toxicity effects: (a) inhibition of the root elongation: 0 < RGI < 0.8; (b) no significant effects: 0.8 ≤ RGI ≤ 1.2; and (c) stimulation of the root elongation: RGI > 1.2²⁶.

2.3 Statistical analysis

Statistical data evaluation was performed with the BioEstat 5.3 software (BioEstat Software, Belém, Brazil). The effective concentrations 50% (EC₅₀) were calculated using the Probit method. When the EC₅₀ was not reached by the tested effluent samples, toxicity was expressed as percentage of toxic effect. For L. sativa tests, data were subject to Kolmogorov-Smirnov normality test. As data were normally distributed, they were submitted to one-way analysis of variance (ANOVA) and Tukey test (p < 0.05). Significance values are indicated as follows:

*p < 0.05 and **p < 0.01.

2.4 Treatment of Malathion^® 500 CE by UVC radiation and UV/H₂O₂

Photolysis (UVC radiation) and UV/H₂O₂ treatments of the commercial product Malathion^® 500 CE were performed using the concentrations that gave rise to 50% of test organisms (EC₅₀), which were 550 μg L^-1 for L. sativa and 0.4 μg L^-1 for A. aegypti. The experiments were conducted in a borosilicate bench photoreactor with 300 mL capacity, equipped with a water-cooled system and a magnetic stirrer. Artificial radiation was provided by a high-pressure mercury vapor lamp (125 W, Philips) placed in the solution through a quartz jacket. The initial concentration of H₂O₂ was 1000 mg L^-1 and the residual hydrogen peroxide was determined by UV-Vis spectroscopy, using method based on ammonium metavanadate²⁷.

3. Results and discussion

3.1 Determination of EC₅₀ of Malathion^® 500 CE to A. aegypti and L. sativa

When it is intended to determine EC₅₀ of one (or more than one) substance to organisms that have not been reported yet, preliminary tests are of great value to explore and determine it correctly. Preliminary concentrations to A. aegypti were based on EC₅₀ of Malathion 500 CE to Daphnia magna, which ranges between 0.36-3.8 ng L^-128-30.

After preliminary results, new limits of concentration were settled down and this new range of values (0.05; 0.1; 0.2; 0.4; 0.8 and 1 μg L^-1) allowed determine a reliable EC₅₀ to A. aegypti larvae. In the definitive assays, EC₅₀ value obtained was 0.4 μg L^-1.

From data obtained at definitive assay (250, 350, 450, 550, 600 and 700 μg L^-1) with Lactuca sativa, it was possible to calculate the EC₅₀ (550 μg L^-1).

Comparing both organisms, it is possible to recognize their sensibility difference. L.sativa is about one hundred times more resistant than A.aegypti. This result was already expected once A. aegypti is a target organism, but comparing with literature, species like D.magna are two times more sensible than the target larvae showing the importance to treat this compound before achieve aquatic ecosystems¹⁹.

Brazilian and American legislation indicates maximum permissible values of pesticides in drinking water, but malathion is not contemplated^{31, 32}. In Brazil, resolution nº. 357/2005 stipulates the maximum concentration range of organophosphates in water, which ranging from 0.1 µg L^-1 (freshwater, saline and brackish class 1) to 100 µg L^-1 (freshwater class 3). This means that the higher values would bring acute toxicity effects, according to the value of EC₅₀ determined in this work³³.

Preliminary tests are of great value when it is intended to determine the EC₅₀ of organisms when exposed to one or more substances, therefore, preliminary tests with A. aegypti have been carried out using the EC₅₀ value of known organisms, such as Daphnia magna, which effective concentration varies between 0.36-3.8 ng L^-1 for Malathion 500 CE^28-30.

3.2 Ecotoxicity of Malathion^®500 CE to A. aegypti and L. sativa after treatment by photolysis and UV/H₂O₂

It was observed in both treatments that there was no immobility for A. aegypti (Table 1), inferring that the degradation of Malathion 500 CE was efficient, losing its larvicidal property, besides that; the byproducts generated were not toxic to the organisms¹⁷.

Comparing the results obtained before and after treatment, the toxicity of the commercial product reduced 100% when tested on A. aegypti, demonstrating that it is a viable alternative on the treatment of this organophosphate. This process has the advantage of no needing high pressures, temperatures, elevated times of exposure and the oxidizing agent (hydrogen peroxide) can be removed through catalase abatement^34-36.

The values of p (probability of significance) for ANOVA followed by Tukey's test, obtained from L. sativa bioassays (Table 2), indicate that in all samples there was statistical significance and RGI (Relative Growth Index) less than 0.80, it means that all treatments inhibited root growth. The Germination Index (GI) was greater than 90% in all samples, indicating that there was no inhibition of germination.

Three replicates with 15 seeds. Abbreviations: GI%, germination index; I, inhibition; RGI, relative growth index. Mean values with different letters (a, b, c, d) are significantly different (Tukey’s test, p < 0.05). *p < 0.05; **p < 0.01.

It can be verified that the treatments showed an efficiency in reduction of effects on root growth of lettuce seedlings in comparison to pre-treatment sample (T0 = 550 μg L^-1, with RGI = 0.50), since after UVC and UV/H₂O₂ processes all samples had RGI higher than 0.50.

However, UVC 120 min photolysis likely generated toxic degradation byproducts for lettuce, once in 30 min of radiation exposure there was 21% of inhibition in root growth (RGI = 0.79), while at 120 min the percentage of inhibition growth increased to 36% (RGI = 0.64). Even though there were no significant differences between them, this increase of the toxicity can be associated to the formation of inorganic anions such as sulfates and phosphates due to cleavage of the P-S bonds^{17, 18}.

For UV/H₂O₂, rootlets growth had high inhibition at 30 min of treatment (RGI = 0.55) and in 120 min was observed decrease of toxic effects (RGI = 0.76). It is possible that at the initial 30 min were formed intermediates by sulfur oxidation and generation of molecules from the combination of phosphorus, sulfur, carbon, hydrogen and oxygen, for example, phorate sulfoxide and phoratoxon sulfone^{17, 18}.

The influence of concentration of treated compound on processes efficiency is evident when comparing treatments carried out based on ecotoxicity of Malathion to A. aegypti, since concentration used was ten times lower in relation to L. sativa and after both treatments no toxic effects were obtained, it shows that, despite the objective of the treatment was to degrade the pesticide and eliminate sources of toxicity, the concentration of treated compound may interfere in the efficiency of the process and even increase toxic effects^{17, 36}.

4. Conclusions

As an insecticide, Malathion^® 500 CE was shown to be more toxic to Aedes aegypti larva, evidenced by EC₅₀, which is much lower than that obtained for L. sativa. As regards phytotoxicity, it was found that the L. sativa seed presented moderate resistance to the pesticide studied, with an EC₅₀ value of 536.11 μg L^-1, however; it was a low concentration, inferring that lower concentrations of Malathion^® 500 CE can cause damage to terrestrial plants.

Degradation of the commercial product in both treatments was efficient and the byproducts generated were not harmful to A. aegypti larvae. However, in the phytotoxicity assays, it was evidenced that there was inhibition of root growth.

This compound is present in agriculture for biological control and in urban areas being used in the fight against vectors of several diseases like dengue requiring several ecotoxicological and toxicological studies of this insecticide. In the present work, it is possible to see that low concentrations can cause harmful effects to target and non-target organisms.

5. References

[1] Diretoria de Vigilância Epidemiológica, Orientações para o uso de Malathion Emulsão Aquosa - EA 44% para o controle de Aedes aegypti em aplicações espaciais a Ultra Baixo Volume (UBV). Estado de Santa Catarina, Secretaria de Estado da Saúde, Superintendência de Vigilância em Saúde. Florianópolis, 2016.

[2] Instituto Oswaldo Cruz, Considerações técnicas sobre a aplicação aérea de inseticidas em área urbana, Nota Técnica nº 4/2016/IOC-FIOCRUZ/DIRETORIA, Rio de Janeiro, 2016.

[3] Brasil. Ministério da Saúde. Dispõe sobre a adoção de medidas de vigilância em saúde quando verificada situação de iminente perigo à saúde pública pela presença do mosquito transmissor do vírus da dengue, do vírus Chikungunya e do vírus da Zika, Lei nº 13.301, de 27 de junho de 2016.

[4] World Health Organization (WHO), Report of the fifteenth whopes working group meeting. Review of: Olyset®Plus, Interceptor®LN, Malathion 440 EW, Vectobac® GR. World Health Organization, Geneva, Switzerland, 2012.

[5] Barata, C., Solayan, A., Porte, C., Role of B-esterases in assessing toxicity of organophosphorus (chlorpyrifos, malathion) and carbamate (carbofuran) pesticides to Daphnia magna, Aquatic Toxicology 66 (2) (2004) 125-139. https://doi.org/10.1016/j.aquatox.2003.07.004.

[6] Saler, S., Saglan, N., Acute toxicity of Malathion on Daphnia magna Straus,1820, Journal of Biological Sciences 5 (3) (2005) 297-299. https://doi.org/10.3923/jbs.2005.297.299.

[7] Damásio, J., Guilhermino, L, Soares, A. M., Riva, M. C., Barata, C., Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion. Chemosphere. 70 (1) (2007) 74-82. https://doi.org/10.1016/j.chemosphere.2007.07.026.

[8] Oga, S., Camargo, M. A., Batistuzzo, J. A. O., Fundamentos de toxicologia, Atheneu, São Paulo, 3rd ed., 2008.

[9] Rebechi, D., Efeitos ecotoxicológicos em Chironomus sancticaroli Strixino & Strixino, 1981 (Diptera: Chironomidae) expostos ao Malathion. 2012. 85f Dissertação (Mestrado em Entomologia) – Universidade Federal do Paraná, Curitiba, 2012.

[10] Teixeira, C. P. A. B., Jardim, W. F., Advanced Oxidative Processes: theoretical concepts. Thematic Notebook, v. 3, State University of Campinas (UNICAMP), Institute of Chemistry - IQ, Laboratory of Environmental Chemistry - LQA. Campinas, 2004 (Português).

[11] Badawy, M. I., Ghaly, M. Y., Gad-Allah, T. A., Advanced oxidation processes for the removal or organophosphorus pesticides from waste water, Desalination. 194 (1-3) (2005) 166-175. https://doi.org/10.1016/j.desal.2005.09.027.

[12] Loures, C. C. A., Alcântara, M. A. K., Izário Filho, H. J., Teixeira, A. C. S. C., Silva, F. T., Paiva, T. C. B., Samanamud, G. R. L., Advanced oxidative degradation processes: Fundamentals and Applications, International Review of Chemical Engineering 5 (2) (2013) 102-120.

[13] Zhang, Y, Pagilla, K., Treatment of malathion pesticide wastewater with nanofiltration and photo-Fenton oxidation, Desalination 263 (1-30) (2010) 36-44. https://doi.org/10.1016/j.desal.2010.06.031.

[14] Chenna M., Messaoudi, K., Drouche, N., Lounici, H., Study and modeling of the organophosphorus compound degradation by photolysis of hydrogen peroxide in aqueous media by using experimental response surface design, Journal of Industrial and Engineering Chemistry 33 (2016) 307-315. https://doi.org/10.1016/j.jiec.2015.10.016.

[15] Clark, K. K., Mezyk, S. P., Abbott, A., Kiddle, J. J., Kinetic Studies of the AOP Radical-Based Oxidative and Reductive Destruction of Pesticides and Model Compounds in Water, Chemosphere 197 (2018) 193-199. https://doi.org/10.1016/j.chemosphere.2017.12.190.

[16] Brenner, L., Malathion Fact Sheet, Journal of Pesticide Reform 12 (4) (1992) 29-37.

[17] Konstantinou, I. K., Antonopoulou, M., Lambropoulou, D. A., Transformation products of emerging contaminants formed during advanced oxidation processes. In Transformation products of emerging contaminants in the environment: Analisys, processes, ocurrence, effects and risks, Lambropoulou, D. A, Nollet, L. M. L., ed., John Wiley & Sons: Chinchester, United Kingdom, 2014, Ch. 6.

[18] Kralj, M. B., Černigoj, U., Franko, M., Trebše, P., Comparison of photocatalysis and photolysis of malathion, isomalathion, malaoxon, and commercial malathion - Products and toxicity studies, Water Research 41 (19) (2007) 4504-4514. https://doi.org/10.1016/j.watres.2007.06.016.

[19] Zagatto, P. A., Bertoletti, E., Ecotoxicologia aquática: princípios e aplicações, RiMa, São Carlos, Brasil, 2nd ed., 2008.

[20] Garcia, J. C., Simionato, J. I., Cinque Almeida, V. de, Palácio, Z., Rossi, F. L., Schneider, M. V., Souza, N. E. de, Evolutive follow-up of the photocatalytic degradation of real textile effluents in TiO₂ and TiO₂/H₂O₂ systems and their toxic effects on Lactuca sativa seedlings, Journal of the Brazilian Chemical Society 20 (9) (2009) 1678-4790. https://doi.org/10.1590/S0103-50532009000900005.

[21] United States Environmental Protection Agency (USEPA), Protocols for short term toxicity screening of hazardous waste sites – 600/3-88/029, Washington, United States, 1989.

[22] Sobrero, M. C., Ronco, A., Ensayo de toxicidad aguda con semillas de lechuga Lactuca sativa L., In Ensayos toxicológicos para la evaluación de substancias químicas em água y suelo - La experiencia en México, Romero, P. R., Cantú, A. M., ed., Secretaria de Medio Ambiente y Recursos Naturales, Ciudad de México, México, 2004, 56-67.

[23] Muturi, E. J., Larval rearing temperature influences the effect of malathion on Aedes aegypti life history traits and immune responses, Chemosphere 92 (9) (2013) 1111-1116. https://doi.org/10.1016/j.chemosphere.2013.01.055.

[24] World Health Organization (WHO), Instructions for Determining the Susceptibility or Resistance on Mosquito Larvae to Insecticides, World Health Organization, Geneva, Switzerland, 1981.

[25] Dipil., Ficha técnica do produto: Malathion 500 CE Inseticida Líquido, Dipil Indústria Química.

[26] Young, B. J., Riera, N. I., Beily, M. E., Bres, P. A., Crespo, D. C., Ronco, A. E., Toxicity of the effluent from an anaerobic bioreactor treating cereal residues on Lactuca sativa, Ecotoxicology and Environmental Safety 76 (2) (2012) 182-186. https://doi.org/10.1016/j.ecoenv.2011.09.019.

[27] Nogueira, R. F. P., Oliveira, M. C., Paterlini, W. C., Simple and fast spectrophotometric determination of H₂O₂ in photo-Fenton reactions using metavanadate, Talanta 66 (2005) 86. https://doi.org/10.1016/j.talanta.2004.10.001.

[28] U. S. Environmental Protection Agency (USEPA), Reregistration Eligibility Decision (RED) for Malathion Revised, U. S. Office of Prevention, Pesticides and Toxic Substances, Washington, United States, 2009.

[29] Toumi, H., Boumaiza, M., Millet, M., Radetski, C. M., Camara, B. I., Felten, V., Ferard, J. F., Investigation of differences in sensitivity between 3 strains of Daphnia magna (Crustacean: Cladocera) exposed to malathion (organophosphorus pesticide), Journal of Environmental Science and Health 50 (1) (2014) 34-44. https://doi.org/10.1080/03601234.2015.965617.

[30] Nagato, E. G., Simpson, A. J., Simpson, M. J., Metabolomics reveals energetic impairments in Daphnia magna exposed to diazinon, malathion and bisphenol-A, Aquatic Toxicology 170 (1) (2016) 175-186. https://doi.org/10.1016/j.aquatox.2015.11.023.

[31] Brasil, Ministério da Saúde, Portaria nº 2914, de 12 de dezembro de 2011, “Dispõe sobre os procedimentos de controle e de vigilância da qualidade da água para consumo humano e seu padrão de potabilidade”, 2011.

[32] U. S. Environmental Protection Agency (USEPA), 2018 Edition of the Drinking Water Standards and Health Advisories, U. S. Office of Water, Washington, United States, 2018.

[33] Brasil, Ministério do Meio Ambiente, Conselho Nacional do Meio Ambiente, Resolução CONAMA no 357, de 17 de março de 2005, “Dispõe sobre a classificação dos corpos de água e diretrizes ambientais para o seu enquadramento, bem como estabelece as condições e padrões de lançamento de efluentes, e dá outras providências”, 2005.

[34] Lofrano, G., Pedrazzani, R., Libralato, G., Carotenuto, M., Advanced Oxidation Processes for Antibiotics Removal: A Review, Current Organic Chemistry 21 (12) (2017) 1054-1067. https://doi.org/10.2174/1385272821666170103162813.

[35] Santos, K. A., Antonelli, R., Gaydeczka, B., Granato, A. C., Pointer Malpass, G. R., Processos oxidativos avançados: uma revisão de fundamentos e aplicações no tratamento de águas residuais urbanas e efluentes industriais, Ambiente & Água-An Interdisciplinary Journal of Applied Science 11 (2) (2016) 387-401. https://doi.org/10.4136/ambi-agua.1862.

[36] Pignatello, J. J., Oliveros, S. E., Mackay, A., Advanced oxidation processes of organic contaminant destruction based of the Fenton reaction and related chemistry, Critical Reviews in Environmental Science and Technology 36 (1) (2006) 1-84. https://doi.org/10.1080/10643380500326564.