Ecotoxicity of Malathion® 500 CE before and after UVC radiation and UV/H2O2 treatment

Since 2008, Brazil has been the largest consumer of agrochemicals in the world, using pesticides to combat pests and vectors, impacting both microbiota and human health. Aiming at the degradation of the contaminants present in the environment, treatments by advanced oxidative processes are based on the synthesis of free radicals that allow the degradation of the pollutant. Among these processes are included UV/H2O2. By evaluating the ecotoxicity and phytotoxicity of the commercial Malathion® 500 CE product, using Aedes aegypti larvae and Lactuca sativa seed, the high toxicity of this formulation was observed. EC50 values for A. aegypti being equal to 0.4 μg L-1 and for L. sativa equal to 550 μg L-1. The commercial agrochemical degradation was carried out by UVC radiation and UV/H2O2, and the toxicity was evaluated after 30 and 120 minutes. After both treatments, the percentage of immobility was zero for A. aegypti, inferring the efficiency of the processes. However, for L. sativa, no treatment was able to remove or reduce initial toxicity.


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][6][7][8][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 ABSTRACT: Since 2008, Brazil has been the largest consumer of agrochemicals in the world, using pesticides to combat pests and vectors, impacting both microbiota and human health. Aiming at the degradation of the contaminants present in the environment, treatments by advanced oxidative processes are based on the synthesis of free radicals that allow the degradation of the pollutant. Among these processes are included UV/H2O2. By evaluating the ecotoxicity and phytotoxicity of the commercial Malathion ® 500 CE product, using Aedes aegypti larvae and Lactuca sativa seed, the high toxicity of this formulation was observed. EC50 values for A. aegypti being equal to 0.4 μg L -1 and for L. sativa equal to 550 μg L -1 . The commercial agrochemical degradation was carried out by UVC radiation and UV/H2O2, and the toxicity was evaluated after 30 and 120 min. After both treatments, the percentage of immobility was zero for A. aegypti, inferring the efficiency of the processes. However, for L. sativa, no treatment was able to remove or reduce initial toxicity. 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][11][12] . Recent works have shown the efficiency of AOPs, as photo-Fenton process 13 , UVC/H2O2 14 and others 15 , 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][21][22] . The species Aedes aegypti was also chosen for toxicity and larvicide evaluation because it is the target organism of malathion 23 .

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).

Ecotoxicological tests with Aedes aegypti
Aedes aegypti tests were performed according to methodology described by the World Health Organization 24 . 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 water 25 .
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 -1 16 . 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.

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 Ronco 22 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 > 1.2 26 .

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.

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 metavanadate 27 .

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. aegypt were based on EC50 of Malathion 500 CE to Daphnia magna, which ranges between 0.36-3.8 ng L -1 28-30 .
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 .
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 ecosystems 19 .
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 work 33 .
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][29][30] .

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 organisms 17 . 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][35][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. Table 2. Evaluation of the phytotoxicity of Malathion500 CE (550 μg L -1 ) using Lactuca sativa seeds before and after treatment with UVC radiation and UV/H2O2 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 .

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.