Evaluation of pesticide residues in vegetables from Mekong, Delta, Vietnam using LC-MS/MS

Purpose: To assess the levels of pesticide residues in commonly used vegetables in Vietnam as a reference for future monitoring. Methods: A total of 180 samples of six different fresh vegetables including watercress, mustard green, choy sum, daikon, okra, and yam were analyzed from Mekong Delta, Vietnam. Ten popular pesticides were evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after extraction with a multi-residue method (QuEChERS method). Results: The results were assessed according to the maximum residue limit (MRL) provided by Codex for each pesticide in each commodity. Pesticide residues above the MRL were detected in 107 samples (59 %) and 63 samples (47 %) contained residues below the MRL. Multiple residues were present in 38.3% of the samples with two to five pesticides, and 0.6 % of samples were contaminated with more than five pesticide residues. Of the pesticides investigated, eight pesticides were detected, of which abamectin, alpha-cypermethrin, acetamiprid, chlorpyrifosethyl, chlorantraniliprole, fenobucarb, fipronil, and trichlorfon exceeded their MRLs. Pesticide residues were detected above MRLs in samples of watercress (14 samples), mustard green (24 samples), choy sum (25 samples), daikon (26 samples), and yam (18 samples). Chlorpyrifos-ethyl, and fipronil were detected in most of the vegetable samples (100% and 89.44%, respectively). Conclusion: The results indicate the occurrence of pesticide residues in commonly consumed vegetables in Vietnam. The need for the regular monitoring of pesticide residues and the sensitization of farmers to better pesticide safety practices, especially the need to adhere to recommended pre-harvest intervals is recommended.


INTRODUCTION
Chemical pesticides have continually proven their usefulness by enhancing worldwide agricultural output, lowering insect-borne and endemic diseases, and safeguarding plantations, forests, and harvested areas [1]. Pesticides are currently more valuable in developing nations, particularly those in tropical regions aiming to enter the global economy by supplying offseason fresh fruits and vegetables to nations with more moderate climates, such as Vietnam. Integrated Pest Management (IPM) programs were implemented by Food and Agriculture Organization (FAO) and other organizations in the 1990s [2]. In Vietnam, these IPM programs significantly improved the knowledge of local farmers about pesticide use, resulting in the reduction of pesticide application rates and a plunge in the total consumption of pesticides in this country [3].
In order to ensure food safety for consumers and protect human health, many organizations and countries around the world have established maximum residue limits (MRLs) for pesticides in food commodities. The maximum residue limits (MRLs), expressed as the highest concentration of pesticide residues in vegetables and fruits (mg/kg), are used to determine the maximum intake legally permitted by regulations introduced by different countries and internationally by the Codex Alimentarius Commission [4]. The human intake of pesticide residues in vegetables can be much higher than the intake related to water consumption and air inhalation. It is very important to monitor such chemicals in vegetables and to assess their potential risks to human health [5]. Some indicators can estimate the number of chemicals in food, which is represented as an acceptable daily intake (ADI). The ADI can be used to predict the daily intake during a lifetime that would prevent health risks to the consumer [6]. The aim of this study was to assess the levels of pesticide residues in commonly used vegetables in Vietnam for use as a reference for future monitoring. The information is also critically important for developing measures that aim at reducing or preventing human health risks from harmful pesticide residues in primary and derived agricultural products. Ten kinds of pesticides included in this study, were selected on the basis of their wide use in vegetable production in Mekong Delta, Vietnam.

Sample collection
On the basis of information received during the survey, it was concluded that 10 pesticides were most widely used by the farmers ( Table 1). As was expected, all the farmers interviewed in this study reported using various kinds of chemical pesticides. A total of 180 samples of 6 different kinds of vegetable samples including watercress, mustard green, choy sum, daikon, okra and yam was collected after harvesting from farmers' fields in Mekong delta for pesticide residue analysis. The sampling was performed in accordance with the general principles and methods of the European Commission (EC) directive 2002/63/EC for establishing MRLs in food commodities. Each representative vegetable was collected at preparing harvest for sale and avoiding the time of intense sun or rain. All the samples (1.5 -2.1 kg each) were placed in sterile polythene bags, stored 4 o C, to avoid contamination and deterioration, labeled, and transported to the laboratory for processing and analyzed within 12 h. The working standard solutions were used to prepare matrix-matched calibration and solvent calibration standards for the validation study. The ISs were prepared as described above. The working standard solutions and ISs were stored at −20 °C until they were used for the analysis.

Extraction procedure
A modified QuEChERS method was utilized to extract pesticides from the vegetable samples. After homogenization, a 10 g portion of each sample was placed in a 50 mL centrifuge tube. The ISs (TPP and CPR-d10) were added to the centrifuge tube, yielding a sample concentration of 20 μg/kg. Then, 10 mL of 1 % acetic acid in acetonitrile was added, and the samples were vortexed for 1 min. Then, 6.0 g of anhydrous MgSO4 and 1.5 g of CH3COONa were gradually added to the sample tubes. The centrifuge tubes were then tightly capped, vortexed for 1 min, and centrifuged at 6,000 rpm for 5 min. For the cleanup procedure, 6 mL of the supernatant was transferred to a 15 mL centrifuge tube containing 900 mg anhydrous MgSO4, 150 mg PSA, and 45 mg GCB. The centrifuge tube was vortexed for 1 min and centrifuged at 12,000 rpm for 1 min. A 4 mL aliquot of the supernatant was dried under a nitrogen stream at 40 °C. The residue was then reconstituted with 1 mL of ACN:H2O (40:60), and the extract was filtered through a 0.22 μm PTFE filter. Finally, the filtrate was transferred into a vial.

UPLC-MS/MS parameters
The analysis was performed using an LC-MS/MS system consisting of an Acquity UPLC I-Class (Waters, USA) coupled with a Xevo TQ-S micro triple quadrupole mass spectrometer (Waters, USA) equipped with an electrospray ionization (ESI) source working simultaneously in the positive (ESI + ) mode for nine analytes and in the negative (ESI⁻) mode for fipronil. The source settings were as follows: capillary voltage, 3.0 kV in the positive mode or −1.0 kV in the negative mode; source temperature, 150°C; desolvation temperature, 400°C; desolvation gas flow rate, 900 L/h. Argon was used as the collision gas. For the chromatographic conditions, a Waters Acquity BHE C18 column (1.7 μm; 50 × 2.1 mm) was used and maintained at 25°C. The flow rate and injection volumes were 0.25 mL/min and 10 μL, respectively. The mobile phases consisted of (A) 5 mM ammonium acetate/0.1 % formic acid in methanol and (B) 5 mM ammonium acetate/0.1% formic acid in water. The gradient elution program was as follows: 5 % A (0 -1 min), 100 % A (2 -3 min), 100% A (4 -6 min), and 5 % A (until 7 min). The total chromatography run time was 7 min. The data acquisition in the multiple reaction monitoring (MRM) mode was optimized after direct infusion into the detector. Two ion transitions were selected for each compound; the quantifier and qualifier MRM and the analytical method were validated according to Association of Official Analytical Chemists (AOAC) guidelines [8].

Monitoring results
A total of 180 samples were analyzed for pesticide residues. Table 2 shows the seven chemical groups of pesticides analyzed in each commodity. Surprisingly, all of the samples were positive for at least one pesticide (≥LOD). Residual of OPs pesticide and pyrethroid pesticide was found in 6 kinds of vegetables with high ratio. All of the samples were pesticides detectable OPs in watercress, daikon and okra; whereas 100 and 96.7 % of the samples were detected pyrethroid pesticide in choy sum and mustard green, respectively. In contrast, no residual neonicotinoid was in the majority of samples. Obviously, seven chemical groups of

Pesticides residues in analyzed samples
After validation, the proposed method was applied to determine pesticide residue in 180 vegetable samples from farmers' fields in Mekong delta. Surprisingly, the result in Table 3 showed that all of the samples contained at least one detectable pesticide residue, whereas 107/180 samples (59%) contained pesticide residue above the MRLs established by Codex and those percentages were extremely higher than of Thailand's vegetables which had only 77.7 % detectable and 22.3 % exceeding samples [7]. Except for okra, all of the vegetables had high percentage of samples exceeding MRLs called exceeding samples (> 40 %). In 107 exceeding samples, the leaf vegetables (watercress, mustard green, choy sum) had higher percentages (59 %) than the root vegetables (daikon and yam). All of okra samples contained pesticide residue below MRLs, whereas daikon (87 %) had the highest number of detected samples.  Figure 1 shows that alpha-cypermethrin, fipronil and abamectin also had the highest number of exceeding samples with 82 (37.3 %), 52 (23.6 %) and 27 (12.3 %) samples respectively. Whereas a residue of trichlorfon and chlorantraniliprole was found to exceed the MRLs in one choysum and watercress sample, respectively.

Samples with multiple residues
In 107 exceeding MRL samples, the results showed that the percentages of samples which contained more than one exceeding MRLs pesticide was 64.5 % (69/107 samples). In addition, the samples containing two exceeding pesticides had 35.51 % (38 samples), three exceeding pesticides had 17.76 % (19 samples), four and five exceeding pesticides had 10.28 % (11 samples) and 0.93 % (1 daikon sample), respectively. The most frequent combination of two pesticides was alpha-cypermethrin and fipronil in mustard green and choysum samples and alpha-cypermethrin and chlorpyriphos ethyl in daikon. Besides, the frequent combination of three pesticides was abamectin, fenobucarb and fipronil found in daikon (Figure 2).

Acceptable Daily Intake (ADI) results
Intake (ADI) is the estimate of the amount of a pesticide in food or drinking water that can be ingested daily over a lifetime without appreciable health risk to the consumer, on the basis of all known facts at the time of the evaluation. ADI in Figure 3 and Table 5 is calculated by using the sum of ADI of each pesticide detected sample followed the Ministry of Health and the Ministry of Agriculture regulation. The high ADI level (>200 µg/kg) accounted for 33.9 % of the total 180 samples, whereas, leafy vegetables had ADI >200 µg/kg higher than root vegetables. On average, ADI between the two groups of leafy vegetables and root vegetables had a statistically significant difference (95% significance level), in particular, leafy vegetables had an average ADI of 614.87 µg/kg higher than root vegetables 275.69 µg/kg (Figure 3).  The maximum number of pesticides detected in choysum were 6 pesticides (  [11]. There are their extreme harmful effects on human health and their long-term persistence in the environment, therefore, chlorpyrifos-ethyl and fipronil pesticides are banned or restricted in many countries.
On February 12, 2019, the Deputy Minister of Agriculture and Rural Development signed Decision No. 501/QD-BNN-BVTV on the removal of pesticides containing chlorpyrifos ethyl and fipronil chemicals from the list of real protection drugs objects allowed to be used in Vietnam, together with 228 commercial drugs containing ethyl chlorpyrifos and 152 commercial drugs containing Fipronil active ingredients [12]. However, these pesticides are still being used clandestinely in some developing countries. Previous studies have detected chlorpyrifos-ethyl and fipronil pesticide residues in different commodities, including vegetables from Ghana [13], Thailand [7] but most residues were below the MRL allowed. Nevertheless, continuous consumption of food products even with moderate pesticide contamination may have negative consequences on human health in the long term [14]. Pesticides can accumulate in the tissues of organisms as they are not easily soluble [15].
The detection of multiple pesticides in this study is a cause of concern. In 180 samples, the results showed that the percentages of samples that contained more than one exceeding MRLs pesticide were 38.34 % (69 samples). Multiple residues may be expected in some crops because the application strategy is usually to alternate pesticide classes to prevent the buildup of pest resistance. According to a survey on Vietnamese Farmers' Knowledge of Nguyen et al [16], a high number of farmworkers received training on integrated approaches to pest management but still relied heavily on pesticides. Perhaps the farmers believe that using different types of pesticides is the most effective solution to control pests and diseases [17].
Most farmers apply chemicals at high frequency, resulting in use in excess of label specifications. Excessive application of pesticides may lead to high residue levels on plants, which may be dangerous to farmers and to vegetable consumers [16]. Almost farmers mix pesticides inappropriately and do not consider that this could reduce pesticide effectiveness and cause damage to their health or the environment. Farmers explained that mixing chemicals saves time and labor cost and that they anticipated higher pest control efficacy. However, in addition to health and environmental concerns, inappropriate mixtures of various pesticides can increase pest resistance and reduce effectiveness [18].
Greater priorities must be given to develop strategies for pesticide reduction in agriculture through farmer training pesticide use and promote alternatives to chemical pest control. Training farmers should be mandatory, yearly, and include assessment of knowledge and understanding gained as well as intent to adopt improved practices. Current training events reach the majority of farmers, but there is no assessment of the knowledge gained or level of understanding of the impact of their actions. Moreover, intervention strategies by regulatory agencies to strengthen the enforcement mechanisms of current pesticide laws at the farm and retail level are a necessity in promoting safe pesticide use.
Pesticide residues should be monitored on agricultural products and soil and water resources. A baseline of food residues and soil and water pesticide levels is the first step in implementing a monitoring program [16]. It is also critical to raise awareness among the general public who may be directly or indirectly exposed to pesticides, about the risk of these chemicals and how to reduce this risk. Consumers should be aware of practical measures to reduce the contamination of pesticides in fresh agricultural produce, especially vegetables that may be consumed raw. Finally, due to the increasing trend in pesticide use in Vietnam, routine monitoring of pesticide residues in agricultural produce is a necessity to ensure the safety of consumers.