Matix effects and method accuracy
The complexity of the vegetable matrix may have some effect on the analysis, and may inhibit or enhance the response, thus affecting the accuracy, selectivity and sensitivity of the method35, 36. If more than 20 % signal suppression or enhancement, the matix effect (ME) should to be addressed in calibration38. In this study, 11.4% of the thirty-five pesticides showed negligible ME (ME <20%), and 48.6% of them showed medium ME (20% < ME <50%), while 40.0% of them suffered strong signal suppression (ME > 50%) (Table 4). It has been reported that 98% of the total compounds analyzed by GC-MS/MS presented significant enhancement caused by the co-extraction of matrix components36. On the contrary, only 7% of pesticides showed signal suppression in complex herb matrices40. According to Krynitsky et al., even after comprehensive extensive sample extracts, there were still sufficient co-extraction compounds that could result in signal inhibition or signal enhancement, affecting quantity analysis adversely41. Therefore, to avoid the ME, the results were quantified by external standard method with matrix standard solution.
The results of method accuracy by external standard method with matrix standard solution are listed in Table 4. It showed that the average recoveries of 35 pesticides were 83.2%~116.5% when the spiked levels were 50, 100, and 250 µg/kg, with relative standard deviations (RSDs) in the range of 0.8%~9.8% (n=5). The calibration curves of 35 pesticides were from 2.5 µg/L to 500 µg/L, and the correlation coefficients were all greater than 0.9990. In addition, the LODs were ranged from 0.3 to 2.5 μg/kg, and the LOQs were ranged from 1.0 to 8.0 μg/kg, which were lower than the Chinese MRLs (Table 3). According to the Guidance SANTE/12682/201938, this method can meet the requirements for determination of the selected pesticides in cowpea sample.
Verification and analysis of cowpea samples
The validated analytical method was used to analyse 35 pesticide residues in 574 cowpea samples collected from market, super-market and planting bases from hainan province, China. As shown in Fig 1, 27 of the 35 pesticides were detected at least once. There were 8 pesticides with detection rate above 10%. The most frequently detected pesticide was the insecticide chlorfenapyr (46.3%), followed by the fungicide difenoconazole (39.9%), insecticide cypermethrin (36.8%), acaricide pyridaben (19.7%), then by insecticide profenofos (18.1%), chlorpyrifos-ethyl (14.5%), cyhalothrin (12.0%) and fenpropathrin (11.0%). According to the prohibited pesticides on legumes in China, twelve banned pesticides, acephate, chlordimeform, chlorpyrifos-ethyl, coumaphos, dicofol, fipronil, isazofos, isocarbophos, methamidophos, parathion-methyl, sulfotep, and triazophos, were found in 2.8%, 5.9%, 14.5%, 0.5%, 0.7%, 5.6%, 5.4%, 0.9%, 1.2%, 3.8%, 0.2% and 3.8% of the total samples, respectively. Furthermore, of the 27 detected pesticide residues, the maximum residue limits (MRLs) priority referenced in GB/T 2763-202142, followed by in Chinese regulations (the MRLs of pesticide in vegetable routine monitoring in 2015)43, the list of prohibited pesticides on legumes in China44, and the MRLs of pesticide in European Commission45, which shown in Table 3. It showed that the residues for ten pesticides in 17.1% of the samples exceeded their MRLs with the highest exceedance of 380%. In addition, the MRLs exceedance rates were found for cypermethrin (8.5%), difenoconazole (6.4%), parathion-methyl (3.8%), chlorfenapyr (3.1%), cyfluthrin (1.4%), cyhalothrin (1.4%), pyridaben (0.9%), fenvalerate (0.3%), fenitrothion (0.2%) and fenpropathrin (0.2%), respectivly. This implied that these frequently detected pesticides were used widely and extensively in the cultivation of cowpea in Hainan provice, China. For the production and safe supply of agricultural products, the goverment need to strengthen oversight over the agricultural supplies market, strictly control the sale and use of prohibited pesticides, and strengthen the training and management of sales staff in agricultural stores. It also suggested that the rational use of these pesticides should be regulated.
As shown in Fig 2, samples with multiple pesticide residues (two or more detected pesticide residues) accounted for 59.5% of the total number of samples, samples contained one pesticide for 21.3%, and residue-free samples for 19.2%. The overall rate of multiple residues samples was higher than the rate of samples with no residue and a single residue, and sample numbers decreased with the increase of pesticide residues. This finding is consistent with those previous studies in cowpea, green pepper, cucumber, peach and apple1, 3, 39, 46, 47. However, up to 10 different pesticides were detected in three samples of cowpea. Moreover, 99 of the 122 samples with one pesticide residue, 47 of the 109 samples with two pesticide residues, 32 of the 76 samples with three pesticide residues, 14 of the 42 samples with four pesticide residues, 6 of the 50 samples with five pesticide residues, exceeded their MRLs (Fig 2). This could be due to the overuse of mixture pesticides for plants protect, which can lead to major multiresidue regarding food safety39. Therefore, effective national food control systems, such as good Agricultural Practices (GAP), which establish a national pesticide monitoring program widely accepted in most countries, are essential to protect the health and safety of domestic consumers.
Comparison of different years
A total of 574 samples of cowpea (Vigna unguiculata L. Walp) were collected includes 61 samples from 2018, 152 samples from 2019, 199 samples from 2020, and 162 samples from 2021. The samples from 2018 are relatively small and unrepresentative, so they will not be included in the comparison. A total of 17 pesticides were detected in 2021, 24 pesticides in 2019 and in 2020 (Fig 3a). Moreover, it has a tendency that the detection rate of the same pesticide decreased year by year. In addition, Pesticide residues of azoxystrobin, chlorfenapyr, chlorpyrifos-ethyl, cyhalothrin, cypermethrin, difenoconazole, fenpropathrin, fenvalerate, fipronil, malathion, methamidophos, profenofos, pyridaben, pyrimethanil, and triazophos were detected in 2019 to 2021, indicating that these pesticides were all used in various years. Compared with 2019, 2 new pesticides were detected in 2020: acephate, coumaphos. In addition, compared with 2020, one new pesticide was detected in 2021: fenitrothion. However, Duan et al. reported that the most important residues of the 433 fresh cowpea samples from Hainan province in 2012 and 2013 were triazophos, carbofuran, isocarbophos, phoxim and omethoate3. It showed that the pesticide triazophos was still used now. This might be due to in addition to spraying conventional pesticides, there are also exploratory pesticides used in the annual use of cowpea farmers. As shown in Fig 3b, 10 banned pesticides were detected in 2019, 11 in 2020, and 5 in 2021. Therefore, there is a tendency to decrease the use of banned pesticides. Moreover, 4 pesticides (cyhalothrin, cypermethrin, difenoconazole, and pyridaben) which exceeded their MRLs, were detected in 2019 to 2021, indicating that there may be excessive dosage and times of pesticides used in cowpea.
Comparison by Region
From 2018 to 2021, a total of 166 samples from Ledong, 136 samples from Lingshui, 115 samples from Sanya, 52 samples from Chengmai, 29 samples from Wanning, 40 samples from Haikou, and 36 sample from Danzhou were collected. The samples from Ledong, Lingshui, and Sanya, which are more relatively and representative than other region, so they will be included in the comparison. A total of 26 pesticides were detected in Ledong, 24 pesticides in Lingshui and 21 pesticides in Sanya (Fig. 4a). In addition, pesticide residues of azoxystrobin, chlordimeform, chlorfenapyr, chlorpyrifos-ethyl, cyfluthrin, cyhalothrin, cypermethrin, dicofol, difenoconazole, fenpropathrin, fenvalerate, fipronil, isazophos, isocarbophos, parathion-methyl, profenofos, pyridaben, pyrimethanil, and triazophos were all detected in Ledong, Lingshui, and Sanya, indicating that these pesticides were all used in each production area. As shown in Fig 4b, 8 banned pesticides (chlordimeform, chlorpyrifos-ethyl, dicofol, fipronil, isazophos, isocarbophos, parathion-methyl, and triazophos) and 3 pesticides (chlorfenapyr, difenoconazole and pyridaben) which exceeded their MRLs, were detected in Ledong, Lingshui, and Sanya. It shows that farmers use prohibited pesticides in these areas, and the source needs to be traced. Therefore, there may be excessive dosage and times of pesticides used in cowpea.
Dietary exposure risk assessment
Dietary exposure was used to assess the possible exposure routes and dose levels and to clarify the actual and expected exposure levels and possible harm caused to sensitive groups. The chronic hazard quotients of different populations calculated based on average pesticide residues were listed in shown in Table 348. The chronic hazard quotient of all pesticides detected in cowpea was less than 100%, indicating that the pesticide residues in Hainan cowpea had little contribution to the risk of chronic dietary exposure. The magnitude of chronic hazard quotient in different groups of the same gender was consistent with (2~7 years old)>(8~12 years old)>(13~19 years old)≥(>65 years old)≥(20~50 years old)≥(51~65 years old), which was caused by the weight difference of different groups and cowpea intake. The magnitude of the chronic hazard quotient of the same population of different genders was consistent with that of female≥ male, because female's weight and daily intake was lower than that of male. The analysis revealed that dietary exposure gradually decreased with age, and children (2~7 years old) had the highest dietary exposure. In addition, the dietary exposure of female was slightly higher than that of male of the same age group. A similar phenomenon has also been observed in previous studies1, 46, 47. In fact, unlike foreigners, Chinese people are used to stir-frying vegetables before eating, which could reduce the risk3. Similarly, it also has been reported that blanching (5 min) followed by stir-frying (3 min) was recommended to the citizens as the safest household processing way to cowpea49. Therefore, we suggested that the cowpeas should be blanching or stir-frying before eating to reduce the risk.