The Assessment of Organophosphate Pesticide Exposure among School Children in Four Regions of Thailand: Analysis of Dialkyl Phosphate Metabolites in Students’ Urine and Organophosphate Pesticide Residues in Vegetables for School Lunch

In Thailand, pesticides containing organophosphates (OP) are frequently applied to crops to suppress insects. School children can be exposed to OPs on a daily basis, from food consumption to breathing and touching pesticides drifted near classrooms. Living in an agricultural area can also be one of the causes. As a result, it is important to monitor OPs residues in the food chain and biomarkers of exposure. The Gas Chromatography–Flame Photometric Detector method was employed to examine the relationship between OPs residue and DAPs (Diakly phosphate) in four targeted locations in Thailand, as well as to examine the residues of OPs in vegetable samples and DAPs in 395 school children’s urine samples. Vegetables were found to contain at least one OP, with chlorpyrifos being the most prevalent. The OPs detected frequencies for Sakon Nakhon, Chiang Mai, Phang Nga, and Pathum Thani are 96.1%, 94%, 91.7%, and 83.3%, respectively. The overall centration level of OPs showed 0.3261 mg/kg, 0.0636 mg/kg, 0.0023 mg/kg, 0.0150 mg/kg, 0.2003 mg/kg, 0.0295 mg/kg, and 0.0034 mg/kg for diazinon, dimethoate, pirimiphos-methyl, chlorpyrifos, profenofos, ethion, and triazophos, respectively. Nearly 98% of school children were detected with at least one DAP. The overall level of dimethyl phosphate metabolites (5.258 µmole/mole creatinine) in urine samples is higher than diethyl phosphate metabolites (2.884 µmole/mole creatinine), especially in the case of Pathum Thani. Our findings show a consistent relationship between OPs in vegetables from wet markets and DAPs in urine samples of school children in various parts of Thailand.


Introduction
Thai farmers frequently employ organophosphate (OP) pesticides to manage insects on their crops. Children exposed to pesticides are more likely to have cancer, respiratory issues, reproductive, endocrine, skin, and neurological disorders [1][2][3][4][5][6]. Since most of their organs are still developing, school children are more susceptible to pesticide exposure than adults are, as pesticides can affect the growth of different organs. They are also more vulnerable because of their distinct daily activity patterns and physiological traits [7][8][9]. They are more sensitive to the potential neurotoxic effects of pesticides because of their organ systems, such as the brain and central nervous system. These organs develop rapidly

Sample Collection
The OPs residues in vegetables were used as the contaminating markers, and DAP metabolites in urine were used as biomarkers of exposure among school children: (1) Vegetable samples: Two hundred and twenty-two vegetable samples were purchased from the same local wet markets where the cooker usually buys their ingredients, i.e., The vegetable and urine samples were transferred to the laboratory of Environmental and Occupational Health Sciences, Research Institute for Health Sciences (RIHES), Chiang Mai University, Chiang Mai, Thailand. The vegetable samples were finely chopped and stored at −20 • C in a freezer using a zip-lock plastic bag with individual code labeling. The urine samples were aliquoted in three tubes (10 mL in 15 mL-cryo-tube) for DAPs and creatinine analysis, then stored at −20 • C in a freezer prior to analysis.

Organophosphate Pesticide Analysis of Vegetables
Sample extraction and analysis were modified from the method of Koesukwiwat et al. [24] and Sapbamrer and Hongsibsong [15]. Briefly, 5 g of vegetable samples were added into a 50 mL centrifuge tube containing 10 mL acetonitrile (high-performance liquid chromatography [HPLC] grade; JT Baker, USA). The volume of 250 µL of 5 µg/mL triphenylphosphate (internal standard [IS]) was added, and centrifuging was performed at 2500 rpm for 5 min. The supernatant was transferred to a new 50 mL centrifuge tube that contained 6 g of MgSO 4 and 3 g of NaCl and was subsequently centrifuged at 2500 rpm for 5 min. The evaporation was performed to complete the dryness of the extracted solution. A vacuum rotary evaporator (Buchi, Switzerland) with a water bath at 30 to 35 • C was used. The pellet was then resuspended with 5 mL of ethyl acetate (HPLC grade; JT Baker, Phillipsburg, NJ, USA). The volume of 1 mL of ethyl acetate phase was pipetted to a dispersive solid-phase extraction tube (carbon black, Vertical Chromatography Co., LTD, Nonthaburi, Thailand) and centrifuged at 2000 rpm for 3 min. The extract was evaporated with a gentle stream of nitrogen at room temperature and resuspended by 0.5 mL of ethyl acetate for gas chromatography (GC) analysis. The GC analysis consisted of a Hewlett-Packard model 6890 equipped with a flame photometric detector, a capillary column (DB-5 MS, 0.25 mm × I.D. × 30 m length × 0.25 µm film thickness (Agilent J and W column; Agilent Technologies, Santa Clara, CA, USA)), and a computerized data handling system (GC Chemstation A.10.02; Agilent Technologies, Santa Clara, CA, USA). The injection port had a temperature of 220 • C (splitless mode). The oven temperature was programmed as follows: initial temperature of 100 • C for 10 min, the first ramp of 15 • C/min to 180 • C (5 min), the second ramp of 5 • C/min to 250 • C (3 min), and final temperature of 290 • C for 4 min. The carrier gas was 99.999 % helium.
For DAPs, each set of three samples was subjected to spike sample recovery for low (1-25 µg/L), medium (4-75 µg/L), and high (16-125 µg/L) concentrations of OPs standard and urine blank analysis. Pooled urine from a healthy volunteer who had not been given any medicines or directly exposed to chemicals was utilized as a blank for spike recoveries. The mean recovery of spiked DAPs standard ranges from 104.08% to 120.97%, 82.24% to 116.79%, and 80.34% to 102.34% for low, medium, and high spiked concentration, respectively. The within-series imprecision (CV%) ranges from 7.80% to 9.18%. The limit of detection (LOD) and quantitative limit (LOQ) were 2.25 and 20.0 µg/L for DMP, 0.20 and 5.50 µg/L for DMTP, 0.10 µg/L and 3.00 µg/L for DMDTP, 0.05 µg/L and 2.00 µg/L for DEP, 0.10 µg/L and 2.00 µg/L for DETP, 0.10 µg/L and 3.00 µg/L for DEDTP.

Data Analysis
The collected data were analyzed using SPSS Statistics Base 17.0, Thailand. All data were tested for normality before appropriate statistical analyses were performed. Mean, standard deviation (SD), and frequency was reported for variables associated with participant demographics and characteristics. An Independent T-Test was used to assess the difference between the demographics. Chi-square tests (χ 2 ) were used for the comparison of categorical data between participant groups. The OPs residues and urinary metabolites were reported as means, SD, median and interquartile range (IQR). The Kruskal-Wallis test was used to compare the urinary OP metabolites in urine samples of school children between the provinces and for OPs residue in vegetable samples between the provinces at the significance level of 0.05.

Results
The demographic characteristics of the participants are displayed below. There are 395 participants, including 231 males and 164 females, both of whom are the same age (9.73 ± 2.74 and 9.50 ± 2.63, respectively). The BMI, height, and weight all fall within the same range as that displayed in Table 1.

Results
The demographic characteristics of the participants are displayed below. There are 395 participants, including 231 males and 164 females, both of whom are the same age (9.73 ± 2.74 and 9.50 ± 2.63, respectively). The BMI, height, and weight all fall within the same range as that displayed in Table 1.
The correlation of detection and concentration of residues based on vegetable type revealed that chlorpyrifos had a high detection percentage in almost all vegetables. (81.8-100%) and showed low concentration except for yard long bean and kale (0.0035 and 0.0034 mg/kg) (Figure 1b). While dimethoate was found in Chinese cabbage (80.6%, 0.0308 mg/kg) (Figure 1c), high detection and concentration for diazinon was also found in just cabbage (64.4%, 0.3761 mg/kg) and kale (81.2%, 0.1229 mg/kg) (Figure 1d). The province's review of OPs residue results revealed a similar pattern to the overall detection result (Table 2). Briefly, the most detected is chlorpyrifos in any province, followed by diazinon and dimethoate. However, there is a difference between the kind of detected vegetables and the type of residue in each. The most detected OPs residues were found in Sakon Nakhon (96.1%), followed by Chiang Mai (94%), Phang Nga (91.7), and Pathum Thani (83.3%).
According to the results of the detected vegetable tests, the vegetables with at least The correlation of detection and concentration of residues based on vegetable type revealed that chlorpyrifos had a high detection percentage in almost all vegetables. (81.8-100%) and showed low concentration except for yard long bean and kale (0.0035 Toxics 2022, 10, 434 6 of 13 and 0.0034 mg/kg) (Figure 1b). While dimethoate was found in Chinese cabbage (80.6%, 0.0308 mg/kg) (Figure 1c), high detection and concentration for diazinon was also found in just cabbage (64.4%, 0.3761 mg/kg) and kale (81.2%, 0.1229 mg/kg) (Figure 1d).
The province's review of OPs residue results revealed a similar pattern to the overall detection result (Table 2). Briefly, the most detected is chlorpyrifos in any province, followed by diazinon and dimethoate. However, there is a difference between the kind of detected vegetables and the type of residue in each. The most detected OPs residues were found in Sakon Nakhon (96.1%), followed by Chiang Mai (94%), Phang Nga (91.7), and Pathum Thani (83.3%).
According to the results of the detected vegetable tests, the vegetables with at least one residue were found in Sukhothai to be cabbage, Chinese cabbage, morning glory, and Thai eggplant; in Chiang Mai, it was yard long bean, Chinese cabbage, kale, and carrot; in Phang Nga, it was Chinese cabbage, Thai eggplant, and cucumber; and in Pathum Thani, it was morning glory and pak choi.
According to each province, the frequency of detection and the concentration of OP residues were determined for all vegetables in each province, including 50 samples for CM, 60 samples for PT, 51 samples for SK, and 60 samples for PNG, and the results were displayed in Table 3. The detection showed a similar pattern of vegetable results in each province. Chlorpyrifos was the most detected in all targeted areas at 78%, 61%, 86.3%, and 80% for Chiang Mai (CM), Sakon Nakhon (SK), Pathum Thani (PT), and Phang Nga (PNG) province, respectively. Diazinon was found to be the second in PT and SK (40% and 39.2%), while CM and PNG were triazophos and dimethoate (32% and 21.7%). However, some OPs residues could not be detected in each target area, including pirimiphos-methyl in CM and PT, ethion in PT, profenofos, and triazophos in PNG. The differences in the median of OPs residues between the province were observed; dimethoate was found to be significantly higher in PT (0.1637 mg/kg) than in CM and PNG (0.0114 and 0.0193 mg/kg). In contrast, chlorpyrifos in PT (0.0021 mg/kg) was found significantly lower than in CM, PNG and SK (0.0031, 0.0029 and 0.0025 mg/kg).
In order to understand the relation and consequences of OPs contamination and exposure to children in Thailand, the children in government schools were chosen as the school has the same lunch-supported policy. Additionally, almost all the schools buy the food materials from local wet markets, especially vegetables, which were reported of high pesticide residues. DAP metabolites can be produced from several intact forms of OPs, and they can be categorized into DM and DE forms. However, some OP does not produce any DAP metabolites. The detected OPs and DAPs degradation products in the urine are shown in Table 5.     The relationship between OP residues in vegetables in local wet markets and DAPs in urine samples of school children was observed in this present study. The detection frequency of OP residues and DAPs were constructed in a bar chart to observe the relationship between OP residues and DAPs in each target area, as shown in Figure 2. The result confirmed the high detected DE metabolites consequences from high detected ethyl OP residues and low detected DM metabolites resulting from low detected methyl OP residues. While the overall concentration level of DM metabolites in urine samples is higher than DE metabolites even though the methyl OP residues levels are lower than ethyl OP residues, especially for PT, as shown in Figure 3.
The relationship between OP residues in vegetables in local wet markets and DAPs in urine samples of school children was observed in this present study. The detection frequency of OP residues and DAPs were constructed in a bar chart to observe the relationship between OP residues and DAPs in each target area, as shown in Figure 2. The result confirmed the high detected DE metabolites consequences from high detected ethyl OP residues and low detected DM metabolites resulting from low detected methyl OP residues. While the overall concentration level of DM metabolites in urine samples is higher than DE metabolites even though the methyl OP residues levels are lower than ethyl OP residues, especially for PT, as shown in Figure 3.

Discussion
In Thailand, during the past ten years, OPs have been the most widely used pesticides, particularly chlorpyrifos, until it was outlawed in 2020 [22]. However, it appears that today, over all of Thailand, chlorpyrifos and other OP group pesticides are the most often utilized. Several past studies reported OP residues in various types of vegetables, but most of them were studied just in one target area. The report on the finding of OP residues in vegetables from different sources such as farms, markets, and a supermarket in Phayao province, northern Thailand, showed that 59.3 % of farms and 13.2% of markets vegetables contained OP residues at or above the maximum residue limits established by

Discussion
In Thailand, during the past ten years, OPs have been the most widely used pesticides, particularly chlorpyrifos, until it was outlawed in 2020 [22]. However, it appears that today, over all of Thailand, chlorpyrifos and other OP group pesticides are the most often utilized.
Several past studies reported OP residues in various types of vegetables, but most of them were studied just in one target area. The report on the finding of OP residues in vegetables from different sources such as farms, markets, and a supermarket in Phayao province, northern Thailand, showed that 59.3% of farms and 13.2% of markets vegetables contained OP residues at or above the maximum residue limits established by the European Union. The most common OP residues detected in farms, markets, and supermarket samples were chlorpyrifos at 50.0%, 33.9%, and 33.3%, respectively [15]. Moreover, in the study by Silipaunyo et al., they reported OP residues were detected at 21.43% in fruit samples and 31.50% in vegetable samples collected from wet markets and supermarkets in Ching Mai province, northern Thailand [27]. The study in Trang province, southern Thailand, reported that 41.58% of 190 samples were contaminated with OPs and carbamates such as cilantro, kale, Chinese cabbage, cabbage, cauliflower, chili, celery, spring onion, yard long bean, cucumber, tomato, Thai eggplant, white radish, and lemon [28].
The present study is the first study that collected vegetable samples from four provinces that represent four regions of Thailand, including Chiang Mai, Sakon Nakhon, Pathum Thani, and Krabi provinces, which represent the northern, north-eastern, middle, and southern parts, respectively. The results of this investigation on OP residues in vegetables show that all samples contained at least one OP residue and that the earlier studies had also revealed chlorpyrifos, diazinon, and dimethoate as the first (76 percent), second (29.9 percent), and third (16.7%) OP residues. The highest concentration of chlorpyrifos found in this study was 0.4936 mg/kg in cabbage from a wet market, which is lower than the levels found in studies by Sapbamrer and Hongsibsong that found 2.423 mg/kg in lemon balm from a farm and 7.785 mg/kg in market-purchased garlic but higher than 0.027 mg/kg in parsley from a grocery store. Most OPs residues were discovered in the province of SK (96.1%), followed by CM (94%), PNG (91.7%), and PT (83.3%). Additionally, 86.3%, 78%, 80%, and 61% for SK, CM, PNG, and PT, respectively, of all target regions were found to have chlorpyrifos. The results did not distinguish across the provinces in terms of total OP residue detection. The quantities of each residue do vary, though, and dimethoate was discovered to be substantially higher in PT than in CM and PNG. As opposed to CM, PNG, and SK, chlorpyrifos levels in PT were found to be substantially lower. That may confirm the explanation that the level of exposure to OP pesticides in consumers depends on the vegetable source, vegetable type, and treatment process [17,29].
The DAPs were used as urinary biomarkers of OP exposure in several previous studies [12,15,17,[20][21][22]. In this study, at least one DAP was observed in almost all school children, and there were no differences in the detection between four target areas. However, a difference in concentration levels between the target areas was observed. The overall results found that the DAPs level of school children in SK province (median = 1.596 µmole/mole creatinine) is lower than the others province and significantly lower than those in CM province (median = 2.609 µmole/mole creatinine), especially for DEP metabolite. The DAPs levels in CM province were also reported to be higher levels than in other areas in the previous study [21], and the DAPs levels in SK were also reported to be lower than in children who lived in central Thailand [30,31].
According to a previous study conducted on American residents aged 6-59, DM levels were almost two times greater than DEs levels overall, and they noted that DAP concentrations in children aged 6 to 11 were significantly higher than in adults and adolescents [32]. The province of PT in central Thailand has the largest concentration of DMs. In line with a prior study in the same province that saw school-aged kids residing in a rice and aquaculture farming community [30]. The fact that this location is suburban and close to the capital, as opposed to other areas, where people live in rural settings, may be the source of the higher amount. It would be a good idea to look into this matter more in the future.
Few research discusses the connections between DAPs, particularly in children, and OP residues in local markets. The majority of them investigated contamination, exposure, or the danger of exposure and its repercussions. Several investigations [17,31,32] revealed residues in diverse target areas. In the study by Muoz-Quezada et al., which was conducted in Talca Province, Chile, more than 70% of children consume vegetables from the local market, which is primarily supplied by a large market in the city of Talca; it was reported that there is a relationship between chlorpyrifos residue and DAPs in school children. Similar to Thailand, free meals are provided to students in low-income public schools in Chile, and kids generally eat fresh fruit at school [33].

Conclusions
The correlation between OP residues in vegetables from local wet markets and DAPs in urine samples from school children in four major regions of Thailand is first reported in the present study. The findings demonstrated that OPs residue can be detected in veggies and that children were equally at risk of exposure in four parts of Thailand.