Chiang Mai Journal of Science

Determination of Organophosphate, Carbamate, and Pyrethroid Pesticide Residues in Vegetables from Hotpot and Shabu Sectors and Local Markets within Bangkok

1. INTRODUCTION

Pesticides are chemical compounds applied mainly in the agricultural sector to control pest populations, protect plants from harmful diseases or organisms, and enhance crop productivity throughout the plant growth cycle [1]. Although the usefulness of pesticides is apparent, the widespread use of these compounds has exposed humans to adverse health outcomes, ranging from acute toxicity, including nausea, skin irritation, eye pain, and respiratory distress [2], to chronic toxicity from prolonged exposure, such as disruption of thyroid function or cancer [3]. Moreover, the integrity of the relationship between abiotic and biotic components in ecosystems is impaired by the persistence of pesticides, which affects soil fertility, contaminates water sources, and leads to adverse consequences for the microbiome and wildlife [4].

More than 40% of Thailand’s land area is dedicated to agriculture, making the country a leading global supplier and exporter of rice, durian, and processed products like pet food. Consequently, pesticides are extensively utilized, with the Thai market supporting a vast diversity of types and quantities. Based on their chemical composition, these pesticides are primarily classified into four groups: organochlorines, organophosphates, carbamates, and pyrethroids [5]. Imports of agricultural substances containing these chemicals remained stable between 2019 and 2023; herbicides accounted for the largest share of these imports in Thailand, totaling approximately 90,000 metric tons [6]. Previous studies have shown that numerous pesticides were found contaminating various crops and vegetables, such as chlorpyrifos in school lunch boxes [7] and a group of pyrethroids (cyfluthrin, flumethrin, and cypermethrin) in coffee beans [8], which is consistent with the large quantity of pesticide use. Dietary exposure to these pesticide residues via vegetables could adversely affect human health, ranging from acute symptoms (such as headaches, nausea, and blurred vision) to chronic, severe diseases like cancer [26–27]. A prominent trend in the Thai food service sector is the prevalence of hotpot and suki-shabu restaurants, characterized by the tabletop boiling of raw ingredients by the consumer. With a market valuation of approximately 740 million USD (23 billion THB), this sector represents a significant pathway for potential daily exposure to pesticide residues via contaminated produce [9]. Despite its popularity, documentation concerning the pesticide contamination profiles of the ingredients specific to these restaurants is still unavailable.

Therefore, the aim of this study is to determine the level of pesticide contamination (organophosphates, carbamates, and pyrethroids) in raw vegetables from the hotpot and suki-shabu food sector in Bangkok, Thailand, using a screening process (certified test kits) and confirmatory testing (LC-MS/MS). The vegetables were also sampled from local markets for comparison. Additionally, the effect of boiling vegetables via the tabletop pot was simulated to assess whether pesticide residues could eventually lead to human oral exposure.

2. MATERIALS AND METHODS

2.1 Sample Collection and Screening Test Preparation
Five vegetables, including Chinese cabbage (Brassica rapa subsp. pekinensis), water spinach (Ipomoea aquatica), cabbage (Brassica oleracea var. capitata), baby corn (Zea mays L.), and carrot (Daucus carota var. sativa), were selected for analysis due to the high frequency of reported residues in Thai literature [22-23]. A total of 85 samples were obtained from 12 hotpot and suki-shabu restaurants (S01–S12) and 5 Type 1 (structured) markets (M01–M05) within the Ratchathewi district, Bangkok Metropolitan Area. Samples were selectively bought from the restaurants and markets that include all five vegetables. All samples underwent preliminary screening using two distinct colorimetric assays. The GT-test kit (30-minute version) was utilized to determine organophosphates (dicrotophos and EPN) and carbamates (carbofuran and methomyl), while the PY-test kit was used to detect pyrethroids (cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, esfenvalerate, fenvalerate, and fenpropathrin). Both test kits were obtained from GT Trading, the developer and sole manufacturer of the GT and PY test kits. The sampling period spanned from December 2023 to April 2025. The vegetables were not subjected to washing prior to the screening test to determine the actual contamination levels present at the point of source.

The GT-test kit utilizes a cholinesterase inhibition technique to detect organophosphate and carbamate residues. This method is based on the principle that these pesticides act as cholinesterase inhibitors, effectively suppressing the activity of the cholinesterase (ChE) enzyme. Briefly, the edible portions of the samples were finely chopped and homogenized. A 5 g portion of each sample was transferred into a 30 mL polypropylene (PP) bottle, followed by the addition of 5 mL of Solvent-1. The mixture was shaken for 1 minute and allowed to stand for 10 minutes to facilitate residue extraction. One milliliter of the extract was then transferred to a glass test tube, and 1 mL of Solvent-2 was added to separate the residues. The tube was placed in a water bath (37^oC) and purged with air until the solvent had completely evaporated. Next, positive and negative controls were prepared by adding 0.25 mL of Solvent-2 into new glass test tubes and placing them in the water bath to ensure accuracy. A series of reagents were then added to each tube and mixed sequentially: GT-1 (0.5 mL, incubated for 10 min), GT-2 (0.25 mL for samples and negative control; 0.375 mL for positive control, incubated for 30 min), GT-3 (1 mL), GT-4 (0.5 mL), and GT-5 (0.5 mL). Finally, the color intensity of the reaction was visually inspected. Pesticide residues inhibit the cholinesterase enzyme (GT-1), preventing it from hydrolyzing the acetylcholine (GT-2). The concentration of unhydrolyzed acetylcholine determines the color depth, which reflects the levels of organophosphates and carbamates present in the vegetable samples (Figure 1). The test kit was validated for vegetables and fruits, demonstrating a sensitivity, specificity, and accuracy of 92.3%, 85.1%, and 87.1%, respectively. The detection limit for the assay was 0.05 mg/kg, calculated based on the insecticide trichlorfon.

For the PY-test kit analysis, 20 g of the vegetable sample was placed on a PP plastic sheet. One milliliter of Extract-1 reagent was transferred into a glass tube, from which 0.25 mL was pipetted onto a cotton ball (1 cm in diameter). Forceps were used to handle the saturated cotton ball, which was thoroughly wiped across the sample surface to recover pesticide residues. The cotton ball was then returned to the original tube, vortex-mixed, and subsequently removed. For the analytical phase, a 0.25 mL aliquot of the sample extract was transferred into a new glass tube. A separate tube was prepared as a blank control, and a series of reagents was added as follows: PY-1 (0.25 mL), followed by incubation in a water bath (37^oC for 10 min), PY-2 (0.75 mL), PY-3 (0.15 mL), PY-4 (0.25 mL), PY-5 (0.5 mL), and PY-6 (0.5 mL). Finally, the resulting color change in the solution was observed and recorded according to the interpretation criteria illustrated in Figure 1. The detection limit for this assay was 0.2 mg/kg, calculated as cypermethrin.

2.2 Simulation of Hotpot Consumption and Thermal Processing
To simulate the conditions of hotpot consumption, where vegetables are blanched in a large communal pot immediately before eating, a boiling simulation was conducted to assess potential pesticide residue exposure. Samples that had previously tested positive for residues from test kits (at both "safe" and "unsafe" levels) were selected. Type I ultrapure water was subjected to boiling (100^oC) in an electric cooking pot. The vegetable samples were then boiled for 10 minutes. Following thermal processing, the samples were re-analyzed using the specific test kit that originally detected the contamination, allowing for a direct comparison of the heat's effect on residual pesticide levels.

2.3 Determination of Pesticide Concentration
Samples exhibiting contamination during the preliminary screening were also subjected to confirmatory analytical testing to verify the specific pesticide residues. Positive samples from prior screening tests were prepared and submitted to the Food and Biological Safety Assessment Laboratory, Faculty of Medical Technology, Mahidol University. The laboratory utilized an in-house method based on WI-PC-025-10-02 AOAC (2023) 2007.01, employing high-specificity Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). This method is capable of detecting three major pesticide groups: organophosphates, carbamates, and pyrethroids. The facility is an ISO/IEC 17025 accredited laboratory, ensuring international standards of technical competence and data reliability. Calibration curves with R² ≥ 0.995 were used to validate linearity. Injections of a 20-ppb pesticide reference standard at the start, middle, and end of each analytical batch (%RSD < 5%), together with duplicate sample injections every ten runs (%RPD < 10%), were used to monitor system precision. Method accuracy was verified using spiked samples (5 and 10 ppb), with the recoveries between 70% and 120%. Reagent blanks were examined to make sure there was no contamination or carryover. The target pesticides' limits of detection (LOD) and quantification (LOQ) were determined to be 0.005 mg/kg and 0.01 mg/kg, respectively. The residues were classified as not detected if they fell below the LOQ.

3. RESULTS AND DISCUSSION

The screening test results for pesticide residues in vegetables acquired from the hotpot and suki-shabu and Type 1 market sectors are summarized in Table 1. Analysis using the GT-test kit revealed that organophosphate and carbamate residues were detected in only 6 out of 85 samples (7.06%), comprising 4 samples from the hotpot and suki-shabu sector and 2 samples from the Type 1 markets. All detected residues were within the safe level for consumption, with no samples from either source exceeding the safety threshold for these two pesticide groups. Regarding the PY-test kit analysis for pyrethroids, samples from hotpot and suki-shabu restaurants exhibited a higher frequency of contamination, with 8 samples (13.33%) at a safe level and 4 samples (6.67%) at an unsafe level. In contrast, only one sample from the Type 1 markets was detected with safe-level contamination, and no unsafe levels were observed.

The Department of Agricultural Extension, Ministry of Agriculture and Cooperatives [13], suggests using both test kits as preliminary screening tools for pesticide contamination among farmers and consumers. The limits of detection (LOD) for the GT-test kit and PY-test kit were 0.05 mg/kg (as trichlorfon) and 0.2 mg/kg (as cypermethrin), respectively. Although these tests are rapid and user-friendly, they can be unreliable; they tend to underestimate the frequency of vegetables exceeding the maximum residue limit (MRL) or, in some cases, produce false-positive results. The monitoring by the Thai Food and Drug Administration using the GT-test kit detected organophosphate residues in the unsafe range in only 4–5% of samples, which was significantly lower than the 35–71% detected via GC-MS/MS analysis [14]. Conversely, a food safety monitoring program in Indonesia utilized the G-9 test kit to screen for carbamates (LOD = 2 ppm) and organophosphates (LOD = 0.5 ppm) in various fresh plant-based foods. The test kits overestimated contamination levels, reporting residues in 11.45% of samples compared to only 1.42% confirmed by laboratory GC-MS analysis [15]. Due to the qualitative nature of these kits and the limited number of residues they can detect, confirmatory testing using a standard method or the development of advanced test kits should be considered to better ensure food safety. Numerous newly developed rapid test kits are currently in the research phase; for instance, some utilize magnetic particles coated with esterase enzymes to achieve enhanced sensitivity and specificity when screening for organophosphates (LOD = 0.001 mg/L for dichlorvos) and carbamates (LOD = 0.004 mg/L for carbaryl) [16]. Moreover, the integration of a colorimetric paper-chip biosensor with a smartphone to detect carbofuran, a highly toxic carbamate, represents a promising development for future large-scale monitoring applications [24].

To validate the preliminary screening results of the pesticide residues, the selected positive samples were submitted for laboratory analysis. The analysis covered a total of 73 pesticide compounds across five chemical groups, including organophosphates, carbamates, and pyrethroids. The laboratory results showed no detectable pesticide residues in any of the five vegetable types that had initially screened positive using the two test kits (Appendix A). All analytical values were below the limit of detection (LOD), reported as non-detected (ND) at a threshold of 0.005 mg/kg. Using confirmatory testing with advanced detection techniques such as LC-MS/MS helped validate that the vegetables served in the restaurants were safe for consumption. The discrepancy between the screening test and confirmatory test results could be due to the vast operational differences between broad-spectrum colorimetric assays and quantitative mass spectrometry. The LC-MS/MS negative results suggest the occurrence of false-positive results. Therefore, the positive screening outcomes of test kits should be interpreted as early indicators of potential pesticide residue. This safety level could be attributed to the strict surveillance program implemented by the Food Sanitation Division, Department of Health, Bangkok Metropolitan Administration. Under the “Bangkok Food Safety City” project, the administration not only samples food for chemical and biological contaminants using test kits and laboratory analysis but also promotes hygiene practices among food handlers across all food sectors annually [17]. These good practices, especially thoroughly washing vegetables before serving, have contributed to the low detection rates of pesticide residues reported in multiple studies [18–19]. For example, washing vegetables under running water for 5 minutes effectively reduces residues by up to 77.0%. Furthermore, adding substances such as vinegar (0.1% acetic acid) or 0.001% KMnO₄ to the wash water helps degrade specific organophosphates, such as chlorpyrifos and malathion. These practices significantly reduce dietary exposure to pesticide contaminants during the pre-cooking stage in a hotpot and suki-shabu setting. Novel techniques, such as cold plasma technology and ultrasonic irradiation, should also be considered promising alternatives for effectively reducing pesticide residues in food, especially in the leafy and root vegetables found to have high contamination levels in this study [25].

The vegetable samples that initially tested positive, at either safe or unsafe levels, using the test kits were identified from five hotpot and suki-shabu restaurants and two Type 1 markets. Interestingly, samples from three restaurants were contaminated with both organophosphate, carbamate and pyrethroid residues. A total of 16 contaminated samples were subjected to boiling in water and reexamined with test kits to assess the effect of thermal processing on their contamination profiles (Table 3). These indicated that no detectable residues were found in any boiled sample, representing a 100% reduction rate across all samples. The result is consistent with other studies on the effect of different thermal processing applications. The effect of boiling on pesticide contamination in Chinese kale and yard-long beans was determined, with results demonstrating reduction rates of up to 71% and 100%, respectively, after a 10-minute cooking duration [20]. Notably, the study also observed a decrease in pesticide residues using a "blanching" method, which reduced the thermal processing time to 2 minutes, though the average reduction achieved was just slightly lower for organophosphate and pyrethroid residues. Another study observed a reduction of organophosphate residues in tomato samples (100% for dimethoate and 60% for chlorpyrifos) after boiling for 5 minutes [21]. The decrease in pesticide levels during heat treatment is driven by evaporation, hydrolysis, or co-distillation, which varies upon different chemical structures. Given the short duration of dietary exposure within hotpot and suki-shabu restaurants, the consumption of large quantities of blanched vegetables in these settings remains safe for consumers on a daily basis. However, the pesticide contamination in the hotpot broth was not evaluated and should be further explored in a future study.

4. CONCLUSIONS

This study found that while rapid screening kits (GT and PY) detected low-level pesticide residues in a small percentage of samples from both hotpot and suki-shabu restaurants and local markets, subsequent LC-MS/MS confirmatory analysis revealed no detectable contamination (<LOD). Notably, Chinese cabbage and carrots exhibited the highest initial detection rates. Furthermore, the thermal process of boiling, which simulates the typical hotpot and suki-shabu dining experience, successfully reduced all previously detected residues to non-detectable levels. These results likely reflect the success of the Bangkok Metropolitan Administration’s food sanitation policies, particularly regarding the thorough washing of vegetables by food handlers. The utilization of rapid screening kits for routine monitoring remains necessary; however, users must account for potential misinterpretation stemming from the limited sensitivity and specificity of these tools. Therefore, confirmatory testing by certified laboratories should be conducted concurrently to validate results. A larger sample size, a wider variety of vegetables, and seasonal sampling should be considered in future research or long-term monitoring programs. Additionally, to more accurately reflect diverse hotpot and suki-shabu dining behaviors, future studies should explore the effects of the blanching method across varied time intervals. Ultimately, while consumers can be assured of the safety of food in hotpot and suki-shabu settings, the requirement for regular, systematic surveillance persists as a vital measure in safeguarding public health.

ACKNOWLEDGEMENTS

This study was financially supported by Faculty of Public Health, Mahidol University, Thailand.

AUTHOR CONTRIBUTIONS

Teerapong Lertassavakorn: Writing - Original Draft, Methodology, Funding acquisition; Pimpannin Phodaeng: Writing - Original Draft, Investigation; Tanutcha Pramnanun: Investigation, Visualization; Teerapat Arawan: Investigation, Formal analysis; Anuphan Siangyai: Resources, Methodology; Duangrat Inthorn: Conceptualization, Resources, Writing - Review & Editing

CONFLICT OF INTEREST STATEMENT

The authors declare that they hold no competing interests.

DECLARATION OF GENERATIVE AI IN PREPARATION OF MANUSCRIPT

During the preparation of this work, the author used “Google Gemini 3” and “Quillbot” Generative AI to improve the readability and language of the manuscript. After using this tool, the author reviewed and edited the content as necessary and takes full responsibility for the publication's content.

FUNDING

This study was financially supported by Faculty of Public Health, Mahidol University, Thailand (PH-RIU/RF 1/2567).

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