Analysis of Multi-Pesticide Residues and Dietary Risk Assessment in Fresh Tomatoes (Lycopersicum esculentum) from Local Supermarkets of the Metropolitan Region, Chile

In recent years, the official authorities in Chile have reported transgressions in the maximum residue levels of pesticides in fresh vegetables. There is no official information about traceability, pesticide levels, and potential health risks. The aim of this study was to analyse pesticide residues and their corresponding dietary risk assessments in tomatoes from supermarkets in the Metropolitan Region. Pesticides were extracted using the Quick, Easy, Cheap, Effective, Rugged and Safe, QuEChERS method, and their concentrations were determined by using chromatography with HPLC-FL/UV and GC-MS/ECD/NPD, following the Analytical Quality Control and Method Validation Procedures for Pesticides Residues Analysis in Food and Feed, SANTE guide and ISO 17025:2017 standard. In addition, a dietary risk assessment was carried out by comparing Chilean data to international references. The results reported that 9% of the samples had pesticide residue levels above the maximum residue levels permitted in Chile. All the scenarios evaluated revealed the highest estimated daily intake and hazard quotients for methamidophos and chlorpyrifos. Both the active substances used were acetylcholinesterase inhibitors and were neurotoxic under chronic risk assessment. The results showed the highest chronic hazard index in the Chilean scenario for all age groups and genders. The evidence obtained revealed that methamidophos, methomyl, and chlorpyrifos should be restricted for their use in Chilean agriculture.


Introduction
Pesticides have different physicochemical characteristics, structures, modes of action, and uses in agriculture [1,2]. Depending on their molecular structure, pesticides are classified into different groups, such as organochlorines, organophosphates, neonicotinoids, carbamates, triazine, urines, phenoxyacids, pyrethroids, and triazoles [3]. According to their use in agriculture, pesticides are classified as insecticides, fungicides, nematicides, acaricides, and herbicides [4]. Nowadays, pesticides are widely used in the agricultural industry to reduce the impact of pests, weeds, and diseases in different crops, leading to increases in productivity and a higher quality of crops [5]. However, the improper use of pesticides involves risks for human health, as pesticide residues remain in fresh vegetables [6][7][8], food [9], soil [10], and water bodies [11] after harvest. Hence, the overuse and misuse of pesticides can greatly impact the environment [12,13] and poses a serious risk for human health, since short-or long-term exposure to pesticide residues may cause acute or chronic toxicity [14]. Pesticide residues with different mechanisms of action on humans tomatoes wrapped in aluminium foil. The samples were processed using a grinder, and then stored in a flask and frozen at −20 • C, according to the protocol previously described elsewhere [22].

Pesticide Analysis and Quality Assurance
Pesticide extraction was performed using the QuEChERS method [34], as previously described elsewhere [21,22]. Briefly, 10 g of tomato sample were extracted with acetonitrile and extraction mix (4 g MgSO 4 ; 1 g NaCl and 1.5 g citrate), manually shaken and centrifugated. The supernatant was transferred and the clean-up step was performed using 900 mg MgSO 4 , 150 mg PSA and 150 mg C18, manually shaken and centrifugated, and the extract was transferred to a vial until analysis at −20 • C. Pesticides residues were analyzed by GC or HPLC according to their functional groups, volatility and derivatisation properties. The concentration of organophosphates was quantified using a GC-NPD Agilent 7890 with autosampler (Santa Clara, CA, USA). The concentration of halogenated proteins was quantified using a GC-electron capture detector (Thermo Scientific Trace-Ultra) with an autosampler (Waltham, MA, USA) and Perkin Elmer Auto-System XL (Waltham, MA, USA). The concentration of dithiocarbamates was determined using distillation and quantification with a Thermo 10VIS spectrophotometer (Thermo Scientific Inc., Madison, WI, USA). Results were expressed as mg of carbon disulphide (CS 2 ) per kg. The concentration of methyl-carbamates was quantified using HPLC with a Merck Hitachi LaChrom D-7000-autosampler (Dartford, United Kingdom) coupled to a fluorescence detector and a reaction pump (655A-B) from Merck Hitachi (Dartford, United Kingdom). To determine the concentration of imidacloprid and carbendazim, a HPLC system Merck Hitachi D-6000 with a UV detector was used (Burladingen, Germany) [21,22]. The concentration of dithiocarbamates was determined using distillation and quantification with a Thermo 10VIS spectrophotometer (Thermo Scientific Inc., Madison, WI, USA). Results were expressed as mg of carbon disulphide (CS2) per kg [21,22].
Quality assurance was carried out following the SANTE 12682/2019 guidelines. Standard ISO/IEC 17025:2017 of the Accredited Laboratory of Pesticide Residues was implemented. The λ-cyhalothrin, myclobutanil, buprofezin, indoxacarb, pyrimethanil, difenoconazole, azoxystrobin, boscalid, chlorfenapyr and chlorpyrifos were analysed by gas chromatography ECD detector. Additionally, methamidophos and acetamiprid were analysed by GC-NPD detector. The imidacloprid was analysed by liquid chromatography HPLC-DAD and methomyl by HPLC-FL detector. The accuracy was expressed as percentage of recovery, and the precision as repeatability both were used for the validation process [21,22]. Tomato blank samples (free of pesticides) were used to perform the quality assurance. The recovery was studied at a concentration of 20 µg/kg and the precision was determined as the relative standard deviation (RSD). The Limit of detection (LOD) was determined as the signal-to-noise ratio and Limit of Quantification (LOQ) as the lowest concentration quantifiable with acceptable recoveries. Calibration curves were evaluated in blank tomato samples between 10 and 320 µg/kg for the detected pesticides residues.

Compliance of Chilean Maximum Residue Levels
The results were verified under the current regulation of MRLs of Chilean RES 762/2011, which was mandatory for all fresh food commercialised in supermarkets under the national regulation RSA Nº 977/1996. In addition, we checked that the pesticide residues detected in the tomato samples were authorised by the Agricultural and Livestock Service.

Screening of Pesticide Residues and Their Compliance of MRL
In our study, pesticide residues were analysed in 57 samples of tomatoes to assess their compliance with national regulations. The first screening detected the presence of different residues of λ-cyhalothrin, buprofezin, indoxacarb, chlorfenapyr, chlorpyrifos, methamidophos, acetamiprid, imidacloprid, methomyl, pyrimethanil, difenoconazole, azoxystrobin, boscalid, and myclobutanil. Of the total samples evaluated, 39% of samples were free of pesticide residues, 35% contained one residue, 17% contained two residues, and 39% contained three or more residues. The residue concentration in 9% of the contaminated samples was above the MRL. Interestingly, all samples containing methamidophos had concentrations above the corresponding MRL.

Multi Pesticide Residues Analysis
All the pesticides detected in this study were authorised by the Chilean authorities for their use in tomato production. More than 60% of pesticide residues detected in the samples were insecticides: λ-cyhalothrin, buprofezin, indoxacarb, chlorfenapyr, chlorpyrifos, methamidophos, acetamiprid, imidacloprid, and methomyl ( Table 1). The main insecticide detected was acetamiprid, which was present in 21% of the total samples. In addition, the main fungicide detected was difenoconazole, present in 11.5% of the samples. Pyrimethanil had the highest mean concentration: 0.23 mg/kg. The mean concentration of methamidophos was 0.12 mg/kg, which was 12 times higher than the Chilean MRL. Ac-cording to these results, methamidophos represented the highest transgression in this study. This result was in accordance with previous studies.

Discussion
The limits of detection in our work ranged from 5-10 µg/kg and the limit of quantification was between 10-20 µg/kg. Supporting our findings, in a validation study the LOD ranged from 2.35 µg/kg for benthiavalicarb to 6.49 µg/kg for allethrin in fresh tomatoes [51]. On another hand, the recovery rates of our study ranged from 89.7-107.3% for all the pesticides quantified according to the range proposed by the SANTE guide from 0.1 to 19.6%. Supporting our results, similar results reported a range from 80.1 and 112% for 24 pesticide residues in tomatoes marketed in Colombia [52]. In addition, results from 72-116% were obtained in multiclass pesticide residues in tomato samples collected from different markets of Iran [53]. The RSD values were as follows: λ-cyhalothrin 2.7%; buprofezin 3.6%; indoxacarb 3.3%; chlorfenapyr 2.9%; chlorpyrifos 4.3%; methamidophos 4.5%; acetamiprid 2.3%; imidacloprid 10.2%; methomyl 9.5%; pyrimethanil 6.1%; difenoconazole 4.5%; azoxystrobin 3.9%; boscalid 3.1%; and myclobutanil 5.8%. These results showed that the results and their accuracy were positive and the laboratory competences were confirmed. Comparing the results obtained in this work to other studies, the RSD values from 2.1-17.9% were obtained for pesticide residues in tomatoes in Turkey [54].
Previous surveillance studies reported the existence of pesticide residues in local markets worldwide. A recent study analysed tomato samples (n = 20) from the local markets of Majmaah Province, Saudi Arabia, and determined that 27% of the analysed samples contained pesticide residues, cypermethrin in most of the cases [55]. Moreover, a surveillance study of organophosphates in Northern Thailand was performed using 160 samples of vegetables, including tomatoes from local markets. They described a rate of chlorpyrifos residues of 33.8% in all the samples detected [56]. Finally, a monitoring study in tomatoes marketed in Bogota, Colombia (n = 400) discovered at least one pesticide in 70.5% of the total samples evaluated. In this study, the most frequently detected active substances were pyrimethanil, carbendazim, dimethomorph, and acephate [52].
Based on our results and the previously reported literature, we considered that methamidophos residues posed a potential risk to human health [21,22]. A previous study conducted on the residues in tomatoes from Iranian markets (n = 150) reported the presence of both chlorpyrifos and diazinon residues above the MRL [53]. Similar results were obtained in tomatoes from Ghana (n = 20), in which the residues of methamidophos, malathion, and dimethoate exceeded the corresponding MRL [57]. A surveillance study in vegetables in Saudi Arabia, including tomatoes (n = 26), reported a high frequency of methomyl, imidacloprid, metalaxyl, and cyproconazole residues [58]. In Argentina, pesticide residues were evaluated in several vegetables, including tomatoes from domestic markets (n = 10), and found that 65% of the total samples were contaminated and that 20% were above the MRL [59]. A surveillance study in Kuwait also analysing tomatoes (n = 16), determined that 21% of samples had a pesticide residue concentration above the MRL. The pesticides more frequently detected in this study were imidacloprid, deltamethrin, cypermethrin, malathion, acetamiprid, monocrotophos, chlorpyrifos, and diazinon, all of which exceeded their corresponding MRLs [60]. In a study including tomato samples (n = 17) from the Burkina Faso market, 36% of the pesticide-containing samples exceeded the MRL. The main residues detected were acetamiprid, carbofuran, chlorpyrifos, λ-cyhalothrin, dieldrin, imidacloprid, and profenofos. According to a health risk assessment included in this study, chlorpyrifos and λ-cyhalothrin posed a threat to human health [61]. A study conducted in Canada on 133 samples of vegetables, including 17 samples of tomatoes, reported that 47% of samples were above the limit of detection for at least one pesticide. Among all the pesticide residues detected, the active substances imidacloprid, acetamiprid, and clothianidin were the most recurrent [62]. Furthermore, similar results were reported in tomatoes obtained from the Jordan Valley, with transgressions of chlorothalonil and daminozide [63]. Finally, another surveillance study determined that 61% of the tomatoes cultivated in greenhouses in the Mediterranean region of Turkey contained the active substances: chlorpyrifos methyl, cyfluthrin, deltamethrin, and acetamiprid [54].
Several previous studies reported that the EDI of pesticide residues in tomatoes were higher than our results. A study conducted on tomatoes from Northeast China (n = 36) reported that the EDIs of methamidophos, dichlorvos, diazinon and omethoate were 4.2-fold, 1.7-fold, 1.2-fold, and 4.1-fold higher than the respective AIDs for adults. The maximum EDIS for children reported in this study for methamidophos, dichlorvos, diazinon and omethoate were 3.2-fold, 1.3-fold, 0.96-fold and 3.17-fold higher than their corresponding ADI [70]. A study conducted on tomatoes from Zhejiang, China (n = 237), reported that the EDI of chlorpyrifos and cypermethrin in a group of children (2 to 6 years old) was 48.9% and 31.8% of ADI, respectively [71]. In contrast with these results, another study assessing the presence of pesticide residues in several vegetables in Zambia, including tomatoes (n = 9), reported an EDI below the ADI estimated by the World Health Organization and the Food and Agriculture Organization (FAO) [72]. Another study assessed the pesticide concen-trations in tomato samples from Tanzania (n = 50), and reported that the EDIs for chlorpyrifos, permethrin, and ridomil were higher than the values permitted, indicating that consumption of fresh tomatoes could pose health risks to the consumer [73]. Furthermore, a study of pesticide residues in tomatoes from Kazakhstan (n = 44) reported EDI values ranging from 0.01% of the ADI established for pyrimethanil, to 12.05% of the ADI established for λ-cyhalothrin. The most critical pesticides were triazophos and flusilazole, contributing 70.8% and 42.5% to the cHI [74].
The HQs for all scenarios are described in Figure 1. Methamidophos had the highest value. Methamidophos showed the highest value in the Chilean model, age group 15-24 (23.8 in the WOMAN model and 20.8 in the MEN model). As shown in Figure 1a, the HQ for MEN decreased in the following order: methamidophos > chlorpyrifos > buprofezin > difenoconazole > myclobutanil > λ-cyhalothrin. Moreover, as shown in Figure 1b, the HQs for the WOMAN model decreased in the same order as that described in the model, MEN. A study conducted on Nepalese tomatoes assessed the HQ and cHI in adolescents and adults, and reported similar finding to our study [65]. 9, x FOR PEER REVIEW to 12.05% of the ADI established for λ-cyhalothrin. The most critical pesticides w azophos and flusilazole, contributing 70.8% and 42.5% to the cHI [74].

21,
The HQs for all scenarios are described in Figure 1. Methamidophos had the value. Methamidophos showed the highest value in the Chilean model, age grou (23.8 in the WOMAN model and 20.8 in the MEN model). As shown in Figure 1a, for MEN decreased in the following order: methamidophos > chlorpyrifos > bupr difenoconazole > myclobutanil > λ-cyhalothrin. Moreover, as shown in Figure 1b, t for the WOMAN model decreased in the same order as that described in the mode A study conducted on Nepalese tomatoes assessed the HQ and cHI in adolesce adults, and reported similar finding to our study [65]. A previous study carried out on 19 tomato samples reported a high expo chlorpyrifos and ethion, supporting our findings. In this study, the HI was approx 100% of the ADI, and chlorpyrifos was reported as a risk for adults [54]. HQ > 1 served for the active substances profenofos, triazophos, dimethoate, omethoate, ch ifos, and carbendazim with high HQs [68]. Contrasting results were reported in to A previous study carried out on 19 tomato samples reported a high exposure to chlorpyrifos and ethion, supporting our findings. In this study, the HI was approximately 100% of the ADI, and chlorpyrifos was reported as a risk for adults [54]. HQ > 1 was observed for the active substances profenofos, triazophos, dimethoate, omethoate, chlorpyrifos, and carbendazim with high HQs [68]. Contrasting results were reported in tomatoes cultivated in greenhouses from the Turkish Mediterranean region, with 61% of samples containing chlorpyrifos methyl, cyfluthrin, deltamethrin, or acetamiprid. All the pesticides mentioned showed an HI of 9.5% for adults and 11.02 for children (3 to >10 years), mainly owing to the presence of chlorpyrifos [66]. Moreover, a study, assessing the health risk of tomatoes from Kazakhstan, reported triazophos and flusilazole residues, indicating that pesticide residues could be considered a public health issue [74]. In addition, some samples of tomato with an HI higher than 1 for chlorothalonil were reported and could pose a threat to children's health [75].

Conclusions
Chilean supermarkets conduct internal testing programmes on pesticide residues. The aim of this private surveillance is to identify non-compliances with the Maximum Residue Levels set by the Chilean government. If the pesticide residues levels in fresh tomatoes are above the MRL, the supermarkets force the farmers to reduce the number of pesticide applications and to improve the use of good agricultural practices. Therefore, supermarkets should implement a larger sampling test to cover a higher number of tomato samples and identify hazardous pesticides such as methamidophos, chlorpyriphos and methomyl. In addition, the Chilean authorities should increase the effort for testing fresher tomato samples for national consumption in supermarkets of the Metropolitan Region.
In our study, 9% of the total samples evaluated showed concentrations of pesticide residues above the Maximum Residue Levels of pesticides permitted in Chile. Based on the results obtained, methamidophos, chlorpyriphos and methomyl, which are internationally considered to be highly hazardous pesticides by the Food and Agriculture Organization and the World Health Organization of the United Nations, should be restricted in their use on tomatoes marketed in Chile. However, the main limitations of our study are the lack of consumption frequency and body weight data for children. Further dietary consumption studies are necessary for conducting a health risk assessment in Chile.