1. Introduction
Monitoring pesticide residue is especially important for fresh leafy vegetables, as they are widely consumed food items. A variety of vegetable crops are being used as greenhouse cultivation crops to meet the demand imposed by the growing human population [
1]. In Korea, lettuce is cultivated as a typical rotational plant consumed throughout the year. The National Agricultural Products Quality Management Service of Korea has demonstrated that, among all crops on the market, lettuce ranks as the top crop in terms of the content of unregistered pesticides [
2]. The unregistered pesticides in lettuce are transported to the plant from the soil after being used for the primary crop. Thus, managing pesticides remaining in soil is necessary to prevent pesticide contamination of secondary crops with unregistered pesticides. To ensure the safety of rotational crops, the positive list system (PLS), a pesticide safety system, was implemented in Korea for all agricultural products. Under PLS regulations, agricultural products containing unregistered pesticides for which the maximum residue limit (MRL) has not been established can be sold only when pesticides levels are <0.01 mg/kg. However, amounts in excess of this threshold may enter rotational crops through an unintentional route, i.e., even if the pesticides are not sprayed directly onto the secondary crops. In Korea, a cropping system in which different crops with short growing periods are rotationally cultivated in a greenhouse is commonly used; as such, pesticides used for primary crops could remain in the soil, thus contaminating secondary crops. For soil-residual pesticides used for primary crops that are not registered for use in secondary crops and remain at levels >0.01 mg/kg, products derived from the secondary crops cannot be sold under the PLS system. Thus, methods are required to prevent unintentional contamination with unregistered pesticides when cultivating secondary crops. To address this issue, the Rural Development Administration (RDA) of Korea requires the registration of pesticides for use with secondary crops, to decrease the possibility that soil-residual pesticides may contaminate secondary crops and violate the PLS. The RDA also applies the plant-back interval (PBI) as an alternative method for preventing unregistered pesticide contamination. However, in rotational cultivation systems, this requires information on the uptake and residue patterns of pesticides remaining in soil that have been used for primary crops, to avoid violation of the PLS.
Diazinon is an organophosphorus insecticide widely used for pest control in Korea. The World Health Organization (WHO) has categorized diazinon as a moderately hazardous pollutant of Class II [
3]. The number of studies have demonstrated the toxicity of diazinon in aquatic organisms, including potential acute toxicity [
4,
5], body length shortening and endothelial cell changes [
6], enzyme reaction and gene expression changes [
7], lipid and protein oxidative damage [
8] and adaptation adverse effects [
9]. The reduction of intestinal microorganism, involving in the production of fatty acids, and the induction of bile acid disorder, by the exposure of diazinon have been reported in mice [
10,
11]. Moreover, the toxicological effects of diazinon on histophysiological and biochemical parameters in mammalian animals, particularly in the liver and kidney, suggest that diazinon residue cannot be ignored in life systems [
12]. Taken from these studies, monitoring diazinon residue in food products has become increasingly important to ensure the safety of human health. According to the Institute of Public Health and Environment of Korea, diazinon ranks first among the top pesticides found in agricultural products on the market in the last 3 years [
13]. Their report states that diazinon residue must be monitored continuously in leafy vegetable products, as it is mainly detected in vegetables in which diazinon is not registered for use. In this study, we aimed to investigate the magnitude of soil-residual diazinon translocated into lettuce as a representative rotational crop in Korea. The acceptable soil residue of diazinon when growing lettuce (<0.01 mg/kg) was estimated on the basis of the uptake ratio between the soil and crop. In turn, the PBI of diazinon was evaluated based on this threshold and the dissipation kinetics of diazinon in soil.
2. Materials and Methods
2.1. Chemicals and Reagents
Diazinon standard (98.2%) was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) and its analytical standard (1000 mg/L) was obtained from Kemidas Corp. (Gunpo, Gyeonggi-do, Korea). Organic solvents used in this study were of HPLC grade, obtained from J. T. Baker (Phillipsburg, NJ, USA). Other chemicals were of analytical grade, obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA), unless otherwise stated. Sample extraction and purification were performed using Agilent QuEChERS kits (San Francisco, CA, USA). A granular diazinon (3%) was kindly provided by Sungbo Chemicals Co., Ltd. (Seoul, Korea).
2.2. Diazionon Application
A granular formulation of diazinon (3%) was mixed thoroughly with soil (1:4 (
g/g) in a stainless serum bottle and applied evenly onto each plot of soil at a rate of 50 kg/10a. The applied rate was determined based on the diazinon level used in greenhouse soil consecutively for five years. The soils of each plot were mixed by using a land management machine (Dongyang Tech., Daegu, Korea), as previously described [
14].
2.3. Greenhouse Conditions and Experiments
The experiments were performed at an agricultural greenhouse (Damyang, Jeonnam, Korea). The soil composition was found as sand (50.4%), silt (37.6%) and clay (12.0%), 95.08 g kg
−1 organic matter, 31.63 cmolc kg
−1 cation exchange capacity and pH 6.8 by the method described previously [
15]. The soil was investigated as a loam texture based on its composition data. The experimental plots were designed as prepared previously [
5]. The plot size was 30 m
2 with triplicates of each 10 m
2. Lettuce was planted in the soil with the distance of 20 × 20 cm, 7 days after diazinon application. The plots were prepared to have a buffer zone (1.0 m) between each plot in order to prevent sample contamination. The greenhouse temperature and the humidity were monitored daily by a digital thermo-hydrometer during the experiment. The average temperature was 24.8 °C with the maximum 30.0 °C and the minimum 16.4 °C. The relative humidity was from about 48 to 88% during the experiments. The light was natural conditions during the experiment.
2.4. Sample Preparation
The soils were sampled by a stainless auger (Shinill Sci., INC., Paju, Korea) at a depth of 0–20 cm from eight points in each plot after diazinon treatment. The soil samples were dried overnight under dark shadow and subjected to pass through a 2 mm sieve. For the plant samples, lettuces (1 kg per plot) were collected from each plot 32, 36, 40, 44, 48, 51 and 56 days after treatment (DAT) with diazinon. The samples were put in polyethylene bags with ice and immediately transported to the laboratory. The plant root and leaf samples were prepared into small cakes after washed the debris with running water. The samples were then blended using a homogenizer and stored at −20 °C until used for the experiments.
For the determination of diazinon, the samples were prepared by methods modified from QuEChERS [
16,
17]. The methods were optimized to meet the requirements of the Organization for Economic Cooperation and Development (OECD) that has guided pesticide residue analysis. A 10 g soil sample was mixed with 10 mL distilled water in a 50 mL centrifuge tube for 15 min. The sample was then extracted with 10 mL of acetonitrile for 2 min and 4 g anhydrous MgSO
4 and 1 g MgCl
2 was added, followed by mixing thoroughly (2 min) and centrifugation at 3500 rpm (5 min). The supernatant (1 mL aliquot) was added with 150 mg MgSO
4, 25 mg C18 and 25 mg PSA in a centrifuge tube and mixed for 2 min before centrifugation at 8000 rpm for 3 min. The supernatant was then subjected to passing through a 0.2 µm syringe membrane filter (PTFE-H) and used for liquid chromatography/tandem mass spectroscopy analysis (LC/MS/MS). For clean-up of the plant samples, the samples were prepared as described above, replacing C18 by graphitized carbon black (GCB, 2.5 mg).
2.5. Instrumentals
A Waters model Xevo TQ-XS triple quadrupole LC/MS/MS equipped with a Waters model ACQUITY
TM UPLC system was used for the sample analysis. The analytical column was a C18 stainless column (Osaka Soda CAPCELL CORE, 150 × 2.1 mm length, 2.7 μm particle size, 90 Å pore size). A solvent mixture of acetonitrile and water with 0.1% (
v/v) formic acid was used as the mobile phase and flowed as: isocratic flow with 60% acetonitrile for 0.5 min, flow rate 0.3 mL min
−1, gradient flow with 95% acetonitrile for 2.5 min, isocratic flow with 95% acetonitrile for 2 min. The collision energy values were 23 eV and 38 eV for quantitative and qualitative ions of diazinon, respectively. The electron spray ionization (ESI) method at positive ion mode was used for acquiring LC/MS/MS spectra with optimized conditions as follows: capillary voltage 3 KV, de-solvation N
2 flow 650 L h
−1, ion source temperature 150 °C, cone gas flow 50 L h
−1, de-solvation temperature 350 °C and de-clustering potential value 31 eV. For optimizing LC/MS/MS, instrumental validation was conducted as guided by SANTE/11312/2021 [
18]. The tolerances of ion ratio was permitted absolutely within ±30% by considering the relativity to the ratio of standard calibration. The diazinon MS ions were
m/z 305.2 and
m/z 169.1 for quantitative detection and
m/z 305.2 and
m/z 153.2 for qualitative detection, respectively. For quantitative analysis of diazinon in the samples, the matrix-matched calibration curve was applied in the range of 5 to 250 µg L
−1 in the working solutions that had been diluted with the control extracts from the stock solutions (100 mg L
−1). The limit of quantification (LOQ) at the signal-to-noise (S/N) ratio of 10:1 was calculated. The recovery tests at levels of LOQ and 10× the LOQ were conducted in triplicate.
4. Discussion
Lettuce is usually rotated into cultivation within a few weeks after primary crop cultivation in greenhouses in Korea. Lettuce is an important income crop for farmers because it is consumed throughout the year. However, unregistered pesticide levels in lettuce are among the highest for all crops. Unregistered pesticides may violate the PLS threshold for agricultural products; only crops with levels <0.01 mg/kg can be brought to market. Diazinon is one of the top 10 unregistered pesticides in crops on the market. It is mostly found in vegetables, such as lettuce and spinach. Thus, managing the residual diazinon in soil after its application to primary crops is required for safe rotational cultivation of vegetables in greenhouses. We studied the uptake of soil-residual diazinon in lettuce, as a typical rotational crop. LC-MS/MS methods were validated and developed for this purpose, in accordance with OECD guidelines. The methods are capable of quantitatively and qualitatively detecting diazinon without interfering with the samples, as evidenced by the matrix effects and ion ratio tolerance ratios.
Diazinon dissipated rapidly in soil after treatment, exhibiting a half-life of 22 days. Previous studies demonstrated rapid dissipation of diazinon in soil after treatment by microbial oxidation and hydrolysis [
20,
21,
22,
23]. Adsorption and volatilization are also important for the rapid dissipation of diazinon in agricultural soil [
24,
25]. Based on our data and the results of previous studies, the accumulation of diazinon in soil is not significant.
Diazinon residues in lettuce fluctuated during the harvest period, different from the residue patterns in soil. Diazinon uptake is likely greatest in the initial period after treatment, where the amounts in soil were low during the harvest. Diazinon uptake by carrot was high in the early growth stage [
26], which is related to the high residue in soil in that stage. In this study, diazinon levels differed significantly between leaf and root, such that transport of diazinon from root to leaf may not be concentration-dependent. The peak UTR of diazinon in leaf was 0.028, while that of root was 0.389. These results suggest that the root took up diazinon continuously as the plant grew during the experiments. Moreover, diazinon dilution appeared to occur during plant growth, as in other studies [
27,
28]. The dilution effect was higher in leaf than root, probably due to leaf growth. Diazinon has a water solubility of 40 mg L
−1 and octanol–water (log K
ow) partition coefficient of 3.81 [
29], indicating high sorption to soil [
30]. There is a positive relationship between K
ow and the UTR [
31]. High sorption of diazinon to soil could contribute to its rapid dissipation therein. Meanwhile, lettuce root hair would contact soil organics containing diazinon and affect the UTR. This hypothesis could explain approximately 32–42%, of the diazinon in soil, accumulated in lettuce root, relatively higher than the levels in lettuce leaf. Diazinon uptake by carrot was found higher on the root outer layer than the inside root body, meaning that direct contact of root with soil residue may contribute to the uptake [
32]. The translocation of
14C-ring-labeled diazinon by bean plants documented the presence of diazinon only in the primary leaves by two days but not thereafter, persisting mostly in the roots [
33]. Leaf vegetables such as lettuce spinach have been known to be a typical plant that takes pesticide moderately [
34,
35], but dissipation of pesticide is generally faster in lettuce than in other plants [
36]. The residue behavior of methoxyfenozide and pymetrozine in Chinese cabbage was conducted for their risk assessment, demonstrating temperature-dependent dissipation, leading to low health risk [
37]. The uptake of total soil-residual dinotefuran by lettuce has recently demonstrated that about a 24–28% level of initial concentration was translocated into leaves 30 and 60 days after treatment [
38]. This high uptake ratio was explained by high water solubility and lower sorption to soil organics [
39,
40]. Thus, the evaluation of soil-residual pesticide in leaf vegetables is important for pesticide safety in agricultural products. In this study, diazinon in soil was taken up continuously by lettuce during the experiment, and the uptake was dependent on the soil-residual amount. Thus, managing diazinon residue in treated soil is necessary for safe rotation of lettuce in the greenhouse setting.
We aimed to identify an MSR resulting in lettuce diazinon uptake amounts <0.01 mg/kg, as stipulated by the PLS; the MSR satisfying this threshold was estimated as 0.357 mg kg
−1 for lettuce leaf and 0.026 mg kg
−1 for lettuce root. Considering the dissipation kinetics, a diazinon residence time in soil of 93.9 days for leaf and 177.7 days for root would be needed to achieve this MSR in soil; thus, a PBI of ≥3 months is needed to comply with the PLS. Efforts should also be made to reduce diazinon residue in soil to below the MSR before cultivating lettuce. An MRL for diazinon is required for lettuce. According to the OECD calculator [
41], the MRL of diazinon for edible leaf parts was 0.3 mg kg
−1. However, governmental input is needed for setting the MRL. According to the OECD guidelines [
42], the PBI should be studied over a 7–30 day period for lettuce, given that it is a rotational crop. Following OECD guidelines, the RDA of Korea recommends conducting PBI studies over 30 and 60 days for lettuce and spinach, respectively [
43,
44]. Based on soil dissipation and UTR values for diazinon, we estimated a PBI of diazinon for lettuce complying with the PLS threshold.
The PLS was implemented in Korea in 2020 to promote safe pesticide use for all agricultural products intended for the market. However, the threshold of 0.01 mg kg
−1 may be exceeded in rotational crops due to the uptake of residual pesticides in soil used for treating primary crops, as stated above. This may be especially common in Korea, where farmers cultivate a variety of rotational crops in greenhouses. Thus, studies on the uptake patterns of pesticides by secondary crops are needed to satisfy the PLS threshold. Here, we examined plant uptake of diazinon, as a typical unregistered pesticide that can accumulate in lettuce. Diazinon dissipated rapidly in the soil after treatment, and was taken up by lettuce over the growth period. To comply with the PLS threshold, a PBI of ≥3 months is needed for lettuce grown in greenhouse soil containing diazinon in an amount equivalent to the MSR. An MRL for diazinon should be set for lettuce grown in greenhouses, as an alternative to application of the PBI. Furthermore, a strategy for accelerating the dissipation of diazinon in soil [
45] or suppressing diazinon uptake by the plant [
46] would be helpful to satisfy the PLS threshold.