Fungicide dissipation kinetics and dietary exposure risk assessment in squash fruit and leaf

doi:10.21203/rs.3.rs-2006286/v1

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Fungicide dissipation kinetics and dietary exposure risk assessment in squash fruit and leaf

https://doi.org/10.21203/rs.3.rs-2006286/v1

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Dissipation behavior and dietary exposure risk assessment of four pesticides (dimethomorph, mandipropamid, myclobutanil, and metalaxyl) were studied in fruits and leaves of squash grown under greenhouse conditions. Squash fruit and leaf samples were collected randomly at 0, 3, 5, 7, and 14 days after the last pesticide application. The analytical method was set up using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). The quick, easy, cheap, effective, rugged, and safe method (QuEChERS) was performed for sample preparation. Recovery rates at two spiked levels (0.01 and 0.1 mg/kg) were in the range of 76.4–101.9% for the analyzed pesticides and their relative standard deviations were ≤ 4%.Pesticide half-lives were 2.1 and 4.9 days for dimethomorph, 4.6 and 8.1 days for mandipropamid, 4.7 and 8.2 days for myclobutanil, and 2.7 and 5 days for metalaxyl in squash fruit and leaf, respectively. Regarding total surveyors, hazard quotient values for squash fruit and leaf were ≤ 1.03 × 10^− 3and ≤ 2.39 × 10^− 3, respectively. These values in the case of true consumers were ≤ 3.14 × 10^− 3and ≤ 3.91 × 10^− 1, respectively.

Cucurbita pepo

squash leaf

dietary risk assessment

dissipation pattern

ultra-high-performance liquid chromatography–mass spectrometry

pesticide

Food safety is receiving more attention worldwide than ever before (Magkos et al., 2006) andpesticide residues are a major harmful factor affecting human health(Torres et al., 1996).Regarding human toxicity, an earlier study dealing with residual pesticides indicates that the exposure route primarily originates from food and the intake via this route is considerably higher than that via any other route, such as air and water(Margni et al., 2002).Even though pesticides have apotential risk to human health, they effectively protect crops against both pests and diseases, which results in an increase in crop yield(Aguilera et al., 2012).

Levels of residual pesticides in food are usually legislated to minimize unnecessary exposure of consumers (Na et al., 2012).In the Republic of Korea (ROK), a positive list system (PLS) was enforced since 2019(Chang et al., 2018). PLS representsa legislative rule that the tolerance limit of a pesticide residue (0.01mg/kg) must be applied to agricultural products that have no maximum residue limit (MRL).Consequently, consumers intake agricultural products that are less exposed to pesticides(Yang et al., 2017). Whilethis system is a strict standard for producers, setting the MRL is significant for both producers and consumers. Since major crops have a high economic value, pesticide manufacturers are focused on setting MRLsfor major cropsratherthan for minor crops(Walorczyk et al., 2015).

Being a minor crop from the Cucurbitaceae family, squash is mainly consumed as fruit, leaf, and seed in the ROK. In the 5th Korea National Health and Nutrition Examination Survey (5th KNHNES) (KCDC, 2019), squash fruit was reported to be a major source of potassium and thiamine intake.Squash leaf is very ofteneaten intheROK, and although frequency of consumption is lower than of fruit, it is known that its calcium content is about 6 to 45 times higher(RDA, 2011).

According to the national nutrition statistics of the Korea Health Industry Development Institute(KHIDI, 2019), squash fruit intake of children 1–2 years old, 3–5 years old, and 6–11 years old, adolescents 12–18 years old, and young adults 19–29 yearsold were 6.32, 4.72, 5.18, and 5.27g, respectively, implying that humans between 1 and 29 yearsold consume a similar amount of squash fruit. On the other hand, squash fruit intake of adults 30–49 years old, 50–64 years old, and those having 65 years or more were 8.93g, 12.02g, and 12.97g, respectively, indicating that squash fruit intake increased with the age. In the case of squash leaf, consumption of children having 1–2 years and 3–5 years were 0.04 and 0.01g, while intake of those 6–11 years old, 12–18 years old, and 19–29 years old were all zero. In addition, squash leaf intake of people aged 30 to 49, 50 to 64, and 65 years or older were 0.13, 0.13, and 0.23g, respectively. Similar to squash fruit, squash leaf intake increases after the age of 30. Comparing the averagesquash fruit and leaf intakeper capita by the type of settlement, it is 9.31g and 0.02 gfor large cities, 8.46and 0.12 g for small and medium-sized cities, and 10.32 and 0.33 g for suburbs, respectively. It was found that the population of small/medium-sized cities and suburbs consumed 6 and 16.5 times more squash leaf than the population living in large cities, respectively. Considering the average intake by the age and settlement type, it was found that itincreased when people lived in suburbs than in large cities and when they were at a higher age. Therefore,studies are needed to assess dietary risks in people who actually consume squash fruit and leaf.

At the present time, in the ROK,MRLsare set for only several pesticides for squash fruit and leaf(MFDS, 2019). However, in the European Union (EU) they are set for squash fruit only(EU Pesticides Database, 2021).The MRL valuesfor squash fruit in the EU and the ROK are 0.5 and 1.5 mg/kg for dimethomorph, 0.3 mg/kg and undetermined for mandipropamid, 0.3 and 0.1 mg/kg for myclobutanil, and 0.01 and 0.2 mg/kg for metalaxyl, respectively.In the case of squash fruit, previous studies reported on dissipation pattern and exposure risk assessment ofchlorfenapyr(Abdel Ghani and Abdallah, 2016),sulfoxaflor(O. Abdallah et al., 2021), tebuconazole, penconazole, myclobutnil, and triadimenol(O. I. Abdallah et al., 2021).However, there are no publications on dissipation pattern or human exposure risk assessment of any pesticide in squash leaf.

In this paper, experiments wereperformed in greenhouse conditions after which four pesticides present in harvested squash fruits and leaves were analyzed. This study was aimed at (1) developing and validatingan ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS/MS) method using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation procedure to determine residues of four pesticides in squash fruit and leaf,(2) calculating the half-lifeof four pesticides based on their dissipation patterns, and(3) estimating the health risk to a true consumer by assessing pesticides’ initial residues. The results will provide a reference towardproposing usage recommendations of four pesticides in squash fruit and leaf.

2.1 Chemicals, reagents, and materials

Dimethomorph, mandipropamid, myclobutanil, and metalaxyl were purchased from Kemidas Co. (Suwon, the ROK). Chemical structures and physicochemical properties of the four pesticides are presented in Supplementary material Table S1. Formulations of dimethomorph (wettable powder (WP), 25%, DongbangAgro Corporation), mandipropamid (suspension concentrate (SC), 22.59%, Syngenta Korea Ltd.), myclobutanil (WP, 6%, Kyung Nong Corporation), and metalaxyl (WP, 25%, Enbio Co., Ltd.,) used in greenhouse experiments were purchased at a local market (Seoul, the ROK). HPLC-grade acetonitrilewas provided by J.T.Baker (Phillipsburg, NJ, USA). Formic acid (> 98% purity) was obtained from Merck KGaA (Darmstadt, Germany). Membrane filters (0.2 µm) were purchased from Phenomenex (Torrance, CA, USA). An EN-QuEChERS kit was purchased from Chiral Technology Korea (Daejeon, the ROK).

2.2 Greenhouse experiments

Squash plants were grown under greenhouse conditions. The greenhousefacilitywas located in Cheongju-si,Chungcheongbuk-do, the ROK (36.675120°N, 127.317190°E). The experiments were carried out complying relevant institutional, national, and international Good Laboratory Practice guidelines and legislation(RDA, 2020).Each pesticide treatment took the surface of 45 m (length) × 1 m (width), divided into three replicates. A control plot was located in an area separated from the treated areas. Each experimental plot was treated with one of the four pesticides three times before harvest. The applications were performed weekly and the first applicationwas performed on April 6th .Dimethomorph,mandipropamid, myclobutanil, andmetalaxyl formulations were diluted in water in the following ratios: 1:1000, 1:2000, 1:1000,and 1:1500, respectively. The total spraying volume was 27.5 L per plot (45 m²). Squash fruit samples (> 2 kg) and squash leaf samples (> 1 kg) were collected 0, 3, 5, 7, and 14 days after the last pesticide application, randomly from each replicate.During cultivation, the greenhouse air temperature range was maintained at 13.7°C–20.9°C and humidity was 63.0–90.9% (Supplementary material Table S2) that was measured using an electric data logger (TandD, Japan).After harvesting, all plant samples were immediately transferred to the laboratory. Squash fruit and leaf samples were homogenized in dry ice and subsequently stored at − 20°C in polyethylene bags.

2.3 Standard solutions

Standard stock solutions of the four pesticides were prepared at a concentration of 1000 mg/kgin acetonitrile. Working solutions at concentrations of0.01, 0.02, 0.04, 0.1, and 0.2 mg/kg were prepared by serial dilution of the stock solutions with acetonitrile. Each of the standard solutions was mixed with the extract of squash fruit or squash leaf (1:1, v/v).

2.4 Sample preparation and extraction

An EN-QuEChERS kit(Anastassiades et al., 2003) was used to extract the pesticides. Homogenized squash fruits or squash leaves (10 g) were weighed into 50-mL falcon tubes. Ten milliliters of acetonitrile were added to each tube. The EN-QuEChERS kit (4 g MgSO₄, 1 g NaCl, 1 g sodium citrate, and 0.5 g disodium citrate sesquihydrate) was added into the falcon tubes. The tubes were shaken at 1,200 rpm for 1 min. After centrifugation at 4,000 rpm for 5 min, 1 mL of the supernatant was filtered using a 0.2-µm PTFE syringe filter.

2.5 LC–MS/MS analytical conditions

The analysis of all samples was performed using a Shimadzu LCMS-8045 device with a UHPLC Nexera X2 system (Kyoto, Japan), coupled with a Kinetex C18 column (2.1 × 150 mm; 2.6 µm particle size; Phenomenex, USA). The column oven temperature was maintained at 40°C and the injection volume was 5 µL (for metalaxyl) or 2 µL (for other pesticides). Mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The total mobile phase flow rate was 0.2 mL/min.The gradient conditions followedthe two different programs as follows (A:B): for dimethomorph—90:10 (0–0.5 min), 55:45 (1–5.5 min), 10:90 (6–6.5 min), and 90:10 (7–8 min); other pesticides—90:10 (0–1 min), 90:10 (2–5 min), and 90:10 (6–7 min).

The MS instrumental conditions with a positive ESI source were applied as follows: an interface temperature of 350°C, a heat block temperature of 400°C, a nebulizing gas flow of 3 L/min, and a drying gas flow of 10 L/min.The analysis of the four pesticides was performed in the multiple reaction monitoring modeusing a triple quadrupole (QqQ) mass spectrometer (Shimadzu, Japan) (Table 1).

Table 1

Multiple reaction monitoring conditions of the four analytes.
Analyte	Ionization	Precursor ion (m/z)	Product ion (m/z)		Retention time (min)
Analyte	Ionization	Precursor ion (m/z)	Quantitation (collision energy, eV)	Qualification (collision energy, eV)	Retention time (min)
Dimethomorph	[M + H]⁺	388.1	301.15(− 21)	165.1(− 33)	5.3; (E)-isomer
Dimethomorph	[M + H]⁺	388.1	301.15(− 21)	165.1(− 33)	5.6; (Z)-isomer
Mandipropamid	[M + H]⁺	412.1	328.2(− 15)	125.1(− 35)	3.5
Myclobutanil	[M + H]⁺	289.4	70.15(− 18)	125.05(− 31)	3.6
Metalaxyl	[M + H]⁺	280.1	220.1(− 14)	248.1(− 11)	3.5

2.6 Method performance

The method was validated in terms of linearity of calibration curvesand the recovery test throughlimits of quantitation (LOQs). Matrix-matched calibration was examined using pure standard solutionsat five concentrations: 5, 10, 20, 50, and 100 ng/mL and homogenized pesticide-free squash fruit and leaf. Among the chromatograms that produced a signal-to-noise ratio (S/N) > 10, the lowest concentration was defined as the LOQ.To estimate accuracy and precision though relative standard deviations (RSD), the recovery test was performed at two different concentrations: 0.01 and 0.1 mg/kg. Each test was performed in triplicate.

2.7 Statistical analysis

The dissipation patterns of the four pesticides in squash fruit and leafover time were expressed by the following first-order kinetics (Eq. (1)) and the half-lives were calculatedusing the Eq. (2)(Farha et al., 2018):

$${C}_{t}= {C}_{0}\times {e}^{-kt}$$

$${t}_{1/2}= \text{ln} \times 2/k, $$

whereC₀is the initial residue concentration of a pesticide (mg/kg), which represents the residue levels 2 h after the last treatment, C_tis the residue concentration (mg/kg), trepresents days after the last spraying, and kis the constant rate of dissipation.

2.8 Dietary risk assessment

Human health risk related to pesticide residues present in squash fruit and leaf was estimated though the dietary intake rate compared to acceptable daily intake (ADI). Hazard quotient (HQ) based on estimated daily intake (EDI) and ADI was calculated following the equations (3) and (4):

$$EDI = {C}_{0} \times IR/BW$$

$$HQ = EDI/ADI, $$

where EDI represents the estimated daily intake (mg/kg day), C₀is the initial residue concertation of a pesticide in squash fruit orleaf (mg/kg),and IR is the average squash fruit or leaf intake (kg/day) thatwas obtained from the Standardization Guidelines for Food Intake Calculation and Contamination Monitoring conducted in 2019 by the National Institute of Food and Drug Safety Evaluation (NIFDS) on 43,602 individuals. The average consumption of squash fruit and leaf was 10.42g and 0.18g per person, respectively. Among the total surveyors (n = 43,602), true consumers of squash fruit were 14,013 individuals (32.14%). The average squash fruit intake ofatrue consumer (n = 14,013) was 31.88g per day. Considering squash leaf, the true consumer percentage among the total surveyors was 0.73% andthe average squash leaf intake of a true consumer (n = 319) was 29.46g per day. Body weightwas obtained from 5th KNHNES(KCDC, 2019) and the average weight of total surveyorswas 59.44kg.The ADI valuesof dimethomorph, mandipropamid, myclobutanil, and metalaxylare0.2, 0.05, 0.03, and 0.08 mg/kg day, respectively. If a value of HQ is less than 1, it indicates that a population is at no risk of that exposure. All of statistical data was taken and used for the study from a publicly available database.

3.1 Method validation

Method validation was set in terms of linearity, accuracy, precision, and recovery.Calibration curves of all pesticides were linear from 5 to 100 ng/mLof standard solutions in both squash fruit and leaf (Table 2).Method limit of quantification was 0.01 mg/kg for all pesticides.The accuracy and precision were determined using recovery rate and RSDof recovery tests performed in triplicate at two different concentrations (0.01 and 0.1 mg/kg). The results presented in Table 3 show satisfactory recovery rates and RSD for two spiked levels in squash fruit and leaf for all pesticides. The range of recovery rates was76.4–101.9% at two different concentrations. The RSD values for all pesticides were less than 4%.

Table 2

Linear equation of the calibration curves for quantification of the four pesticide residues in squash fruit and leaf.
Pesticide	Linear range (ng/mL)	Squash fruit		Squash leaf
Pesticide	Linear range (ng/mL)	Linear equation	R²	Linear equation	R²
Dimethomorph	5–100	y = 31,386,895.5097 × +8,396.1323	0.9993	y = 27,666,075.9306 × +34,369.7811	0.9996
Mandipropamid		y = 23,414,787.2168 × +8,024.7460	0.9951	y = 15,504,048.6650 × +9,435.4988	0.9997
Myclobutanil		y = 8,773,295.1861 × −9,848.4438	0.9965	y = 7,088,559.9919 × −3,798.8394	0.9923
Metalaxyl		y = 145,612,794.4175 × +276,181.3131	0.9987	y = 61,474,433.9401 × +146,444.2884	0.9992

Table 3. Recovery tests in squash fruit and leaf.

Pesticide	Squash fruit				Squash leaf
	Fortification (mg/kg)				Fortification (mg/kg)
	0.01 mg/kg		0.1 mg/kg		0.01 mg/kg		0.1 mg/kg
	Recovery (%)	RSD	Recovery (%)	RSD	Recovery (%)	RSD	Recovery (%)	RSD
	Avg. ± SD	RSD	Avg. ± SD	RSD	Avg. ± SD	RSD	Avg. ± SD	RSD
Dimethomorph	98.0 ± 1.1	1.1	100.0 ± 1.7	1.7	92.3 ± 0.9	1.0	98.8 ± 0.3	0.3
Mandipropamid	85.7 ± 3.0	3.5	95.4 ± 0.8	0.8	87.9 ± 2.6	3.0	89.3 ± 3.2	3.6
Myclobutanil	99.9 ± 1.5	1.5	96.7 ± 1.7	1.7	88.5 ± 4.0	4.5	89.8 ± 1.5	1.7
Metalaxyl	87.6 ± 1.0	1.2	96.5 ± 0.7	0.7	76.6 ± 0.2	0.3	94.0 ± 1.3	1.4

SD, standard deviation; RSD, relative standard deviation.

3.2 MRL of pesticides in the EU and the ROK

3.2.1 MRL in squash fruit

MRL for squash fruit was set in both the EU (EU Pesticides Database, 2021)and the ROK(MFDS, 2019) for all pesticides, except for mandipropamid in the ROK (Table 4). Table 4also shows pesticide residues 0, 3, 5, 7, and 14 days after the last application.The initial residue of dimethomorph was 0.36 mg/kg. It was 24% of the domestic MRL (1.5 mg/kg) and 72% as compared to the MRL in the EU (0.5 mg/kg). Initial residue of mandipropamid (0.29 mg/kg) was 97% of the EU’sMRL. In the case of myclobutanil, the initial residue was 0.06 mg/kg, which is 60% of the domestic MRL (0.1 mg/kg) and 20% of the MRL set in the EU (0.3 mg/kg). The initial residue of metalaxyl (0.33 mg/kg) was 165% as compared to the domestic MRL (0.2 mg/kg), but it was 3300% in comparison to the MRL set in the EU (0.01 mg/kg). AMRL value of mandipropamid in squash was not set in the ROK. The residual amount data formandipropamid may be used as a basis for the establishment of a domestic MRL.There are differences between domestic and EU MRLs for dimethomorph, myclobutanil, and metalaxyl in squash fruit. It was shown that differences in pesticide residual amounts may occur owing to growing conditions (air temperature and humidity), climate, and crop species.

Table 4

(a). Average residues of four pesticides in squash fruit.
Pesticide	Days after the last treatment	Mean ± SD(mg/kg)	EU MRL of squash fruit (mg/kg)	Domestic MRL of squash fruit (mg/kg)
Dimethomorph	0	0.36 ± 0.00	0.5	1.5
	3	0.19 ± 0.01
	5	0.12 ± 0.02
	7	0.02 ± 0.00
	14	0.00 ± 0.00
Mandipropamid	0	0.29 ± 0.04	0.3	-
	3	0.13 ± 0.02
	5	0.10 ± 0.00
	7	0.08 ± 0.00
	14	0.03 ± 0.00
Myclobutanil	0	0.06 ± 0.00	0.3	0.1
	3	0.03 ± 0.00
	5	0.03 ± 0.00
	7	0.01 ± 0.00
	14	0.01 ± 0.00
Metalaxyl	0	0.33 ± 0.02	0.01	0.2
	3	0.16 ± 0.03
	5	0.05 ± 0.01
	7	0.05 ± 0.01
	14	0.01 ± 0.00

Table 4

(b). Average residues of four pesticides in squash leaf.
Pesticide	Days after the last treatment	Mean ± SD(mg/kg)	EU MRL of squash leaf (mg/kg)	Domestic MRL of squash leaf (mg/kg)
Dimethomorph	0	69.88 ± 6.46	-	-
	3	56.71 ± 3.06
	5	30.32 ± 1.57
	7	25.97 ± 1.55
	14	10.18 ± 3.23
Mandipropamid	0	39.41 ± 4.22	-	-
	3	27.49 ± 0.85
	5	20.92 ± 4.09
	7	16.86 ± 1.53
	14	11.69 ± 1.60
Myclobutanil	0	11.65 ± 1.52	-	20
	3	9.15 ± 0.65
	5	6.99 ± 0.38
	7	6.07 ± 0.93
	14	3.57 ± 0.32
Metalaxyl	0	31.71 ± 0.54	-	30
	3	15.59 ± 0.77
	5	11.64 ± 1.03
	7	8.09 ± 1.38
	14	4.22 ± 0.23
SD, standard deviation; EU, the European Union; MRL, maximum residue limit.

3.2.2 MRL in squash leaf

Currently, MRL for squash leaf was set in the ROK formyclobutanil and metalaxylonly (MFDS, 2019) and in the EU none of the pesticides has a defined MRL for squash leaf (EU Pesticides Database, 2021). Therefore, the initial residues (mg/kg) of myclobutanil and metalaxyl were compared to the domestic MRLs. The initial residue of myclobutanil (11.65 mg/kg) was 58% of the domestic MRL (20 mg/kg) and the initial residue of metalaxyl was 31.71 mg/kg, which is 106% of the domestic MRL (30 mg/kg). In the cases of dimethomorph and mandipropamid, the initial residues were69.88 and 39.41 mg/kg, respectively. The obtained residual amount data for these pesticides in squash leaf may be used as a basis for the establishment of their MRLs.

3.3 Residual characteristics of pesticides in squash fruit and leaf

The residue dissipation patterns of the four pesticides applied in agreenhouse are shown in Fig. 1.The data implythat the pesticide residue levels were closely related to the specific surface area and texture of the crop.Since a squash fruit has a smaller specific surface area and a smoother texture than a squash leaf, they show a large difference in initial residue levels, despite being sprayed in the same cultivation conditions. Specifically, the initial residue levels in squash fruit 2 h after the last treatment were 0.36, 0.29, 0.06, and 0.33 mg/kg for dimethomorph, mandipropamid, myclobutanil, and metalaxyl, respectively, while the residue levels in squash leaf were 69.88, 39.41, 11.65, and 31.71 mg/kg, respectively.By comparing the initial residual amounts between the two organs, these values in squash leaf were 193, 135, 180, and 95 times higher for dimethomorph, mandipropamid, myclobutanil, and metalaxyl, respectively.

All the pesticide residual amounts decreased as the harvest date after the last spraying was postponed and the correlation coefficients of dissipation kinetics were 0.94–0.98, as can be seen in Fig. 1. Figure 1a and 1b show the regression curves and dissipation equations of the studied four pesticides by first-order kinetics in squash fruit and leaf. Half-life of all pesticideswas shorter in fruit than in leaf.In this study, it might be hypothesized that the significant difference recorded between pesticides’ half-life in squash fruit and leaf could be due to the effect of dilution during plant growth(Fenoll et al., 2008).

In squash fruit, half-lives of dimethomorph, mandipropamid, myclobutanil, and metalaxylwere 2.1, 4.6, 4.7, and 2.7 days, respectively. In previous studies, half-lives of various pesticides were reported such aschlorfenapyr (3.05 days)(Abdel Ghani and Abdallah, 2016),sulfoxaflor (6.13 days)(O. Abdallah et al., 2021), tebuconazole (2.30 days),triadimenol (2.81 days), and myclobutanil (2.98 days)(O. I. Abdallah et al., 2021).In field experiments,temperature rising accelerates numerous processes associated with pesticide dissipation(Katagi, 2004; Stenersen, 2004; Willis and McDowell, 1987).Moreover, the higher the air humidity, the higher the pesticide adsorption affinity to the plant surface and the lower its volatilization(Karthika and Muraleedharan, 2009; Sundaram, 1997).In a previous study on myclobutanil residues in squash fruit(O. I. Abdallah et al., 2021), the average temperature and average humidity during field experiments were reported to be 13°C and 60%, respectively. In this study, it was expected that the average temperature of 18°Cand the average humidity of 71% were relatively high and contributed to a short half-life of the pesticide, but various factors involved in its half-life(e.g., microbial activity, rainfall, sunlight) seem to have been combined.The half-lives of dimethomorph, mandipropamid, myclobutanil, and metalaxyl in squash leafwere 4.9, 8.1, 8.2, and 5 days, respectively. Consequentially, their half-liveswere higher in squash leaf than in squash fruit.Unlike squash fruit, no studies have been reported on half-lives of these pesticides in squash leaf.

Previous studies have shown that dimethomorph tend to decompose quickly in iceberg lettuce (0.86 days) (Horská et al., 2020)and pepper (3.2–3.8 days)(Liang et al., 2011), whereas slowly in potato (9.4 days)(Chen et al., 2018), grape (9–9.8 days)(Liu et al., 2012a), and pak choi (6.2 days)(Tang et al., 2021).Mandipropamid has been reported to have a short or a similar half-life in grape (2.2 days)(Malhat and Mahmoud, 2012), Korean cabbage (3.9–4 days)(Choung et al., 2016), and sesame leaf (5.1–5.4 days)(Farha et al., 2016).A few studies on myclobutanil half-life showed similar trends, reporting 2.2–3.4 days for lychee(Y. Liu et al., 2012), 2.5–4.5 days for wheat(Liu et al., 2009), and 4.9–6.8 days for green tobacco leaf(Wang et al., 2012).Regarding chemical classes, dissipation half-lives of triazoles were reported to be in the range of 2–12.8 days (Fantke and Juraske, 2013), which is similar toour results and previous studies.The half-life of metalaxyl was reported to be 2.5 days in Chinese cabbage(Ripley et al., 2003), 3–3.5 days in cucumber(Rattan and Sharma, 2012), 4.9 days in grape(C. Liu et al., 2012), and 16.5 days in durian leaf(Phetkhajone et al., 2021).Decomposition behavior of pesticides isgenerally influenced by many factors such as their physicochemical characteristics, formulations, environmental conditions, crop species, and dilution factor based on plant growth(Edwards, 1975; Nash, 1983; Willis and McDowell, 1987; Zongmao and Haibin, 1988).

3.4 Dietary risk assessment

Dietary risk assessment was performed by calculating dietary intake rates compared to ADI. If aHQ value was greater than 1, it indicates that a population is at risk of that exposure.Considering total surveyors (n = 43,602), the HQ values of squash fruit were ≤ 1.03 × 10^− 3 (3.18 × 10^− 4, 1.03 × 10^− 3, 3.78 × 10^− 4, and 7.32 × 10^− 4 for dimethomorph, mandipropamid, myclobutanil, and metalaxyl, respectively)and thoseof squash leaf were ≤ 2.39 × 10^− 3(1.06 × 10^− 3, 2.39 × 10^− 3, 1.18 × 10^− 3, and 1.20 × 10^− 3for the same pesticides, respectively) (Table 5 (a)).Since there was a large difference in squash intake between actual eaters and non-eaters, additional risk assessments were conducted in true consumers. In this case the HQ values of squash fruit were ≤ 3.14 × 10^− 3 (9.73 × 10^− 4, 3.14 × 10^− 3, 1.16 × 10^− 3, and 2.24 × 10^− 3 for dimethomorph, mandipropamid, myclobutanil, and metalaxyl, respectively) and those of squash leaf were ≤ 3.91 × 10^− 1(1.73 × 10^− 1, 3.91 × 10^− 1, 1.93 × 10^− 1, and 1.96 × 10^− 1for the same pesticides, respectively) (Table 5 (b)). It implies that the exposure risk of true consumers of squash fruit and leaf to these pesticides wasthree times and 164 times higher, respectively,than that of all surveyors. Considering daily consumption of squash fruit and leaf, the exposure risk to pesticides is regarded as safe.

Table 5

(a). HQ of four pesticides in total surveyors related to squash fruit and leaf consumption.
Pesticide	Initial concentration (mg/kg)	ADI	Squash fruit		Squash leaf
Pesticide	Initial concentration (mg/kg)	ADI	EDI (mg/kg)	HQ	EDI (mg/kg)	HQ
Dimethomorph	0.36	0.20	0.004	3.18 × 10^− 4	0.012	1.06 × 10^− 3
Mandipropamid	0.29	0.05	0.003	1.03 × 10^− 3	0.009	2.39 × 10^− 3
Myclobutanil	0.06	0.04	0.001	3.78 × 10^− 4	0.002	1.18 × 10^− 3
Metalaxyl	0.33	0.07	0.003	7.32 × 10^− 4	0.011	1.20 × 10^− 3

Table 5

(b). HQ of four pesticides in true consumers related to squash fruit and leaf consumption.
Pesticide	Initial concentration (mg/kg)	ADI	Squash fruit		Squash leaf
Pesticide	Initial concentration (mg/kg)	ADI	EDI (mg/kg)	HQ	EDI (mg/kg)	HQ
Dimethomorph	69.88	0.20	0.004	9.73 × 10^− 4	0.012	1.73 × 10^− 1
Mandipropamid	39.41	0.05	0.003	3.14 × 10^− 3	0.009	3.91 × 10^− 1
Myclobutanil	11.65	0.04	0.001	1.16 × 10^− 3	0.002	1.93 × 10^− 1
Metalaxyl	37.71	0.07	0.003	2.24 × 10^− 3	0.011	1.96 × 10^− 1
ADI, acceptable daily intake; EDI, estimated daily intake; HQ, hazard quotient.

We studied residual amounts and dissipation patterns and performed dietary risk assessment of four fungicides: dimethomorph, mandipropamid, myclobutanil, and metalaxylin squash fruit and leaf duringgreenhouse cultivation.To determine pesticide residues, simple and rapid analytical methods based on a modified QuEChERS method were developed and validated using UHPLC-MS/MS.Half-lives of the studied pesticides in squash fruit and leaf were 2.1–4.7 d and 4.9–8.2 d, respectively.Among total surveyors, the HQ values of these compounds in squash fruit and leaf were ≤ 1.03 × 10^− 3 and ≤ 2.39 × 10^− 3, respectively. Their HQ values in true consumers were ≤ 3.14 × 10^− 3 and ≤ 3.91 × 10^− 1 in squash fruit and leaf, respectively.Based on the obtained HQ values, it can be concluded that the intake of pesticide residues from squash fruit and leaf does not pose a health riskto even a true consumer.Overall, it is expected that these results contribute in understanding the properties of dimethomorph, mandipropamid, myclobutanil, and metalaxyl residues as well as in establishing their domestic MRL values.

Acknowledgments

This study was supported by a grant (No. PJ01594905) from the Rural Development Administration in the Republic of Korea.

Author contributions

JL conceptualized and designed the study. RK, JK, JL, and KL collected samples. RK and DA performed chemical analysis. RK, DA, and JL compiled the data and prepared the graphs and maps. JL drafted the manuscript. DA and JL interpreted the data and revised and completed the manuscript. All authors have read and approved the final manuscript.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Fungicide dissipation kinetics and dietary exposure risk assessment in squash fruit and leaf

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Abstract

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1 Introduction

2 Material And Methods

2.1 Chemicals, reagents, and materials

2.2 Greenhouse experiments

2.3 Standard solutions

2.4 Sample preparation and extraction

2.5 LC–MS/MS analytical conditions

2.6 Method performance

2.7 Statistical analysis

2.8 Dietary risk assessment

3 Results And Discussion

3.2 MRL of pesticides in the EU and the ROK

3.2.1 MRL in squash fruit

3.2.2 MRL in squash leaf

3.4 Dietary risk assessment

4 Conclusions

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