Abstract
Residues of the fungicides difenoconazole, propiconazole, cyflufenamid, and mandipropamid were determined in tomato fruit using acetonitrile for extraction and LC-MS/MS for quantification. Validation criteria include linearity range, the limit of detection (LOD) and limit of quantitation (LOQ), accuracy in terms of precision and trueness, and matrix effect were studied. The recovery rates of the method ranged from 91.8 to 106.3%. The precision of the method in terms of repeatability at one day (RSDr) and between three days (RSDR) ranged from 2.8 to 6.4% and from 4.3 to 7.6%, respectively, with good trueness from 92.2 to 96.4%. Matrix effects (suppression effects) ranged from 3.8% to 11.1%. The validated method was used to evaluate the dissipation kinetics of three different premix formulations: 30% EC (15% difenoconazole + 15% propiconazole), 14% DC (12.5% difenoconazole + 1.5% cyflufenamid), and 50% SC (25% difenoconazole + 25% mandipropamid) used on field tomatoes in Egypt. A first-order kinetic equation best describes residue dissipation. The calculated half-lives of difenoconazole, propiconazole, cyflufenamid, and mandipropamid were 2.01–2.27, 1.89, 1.97, and 1.71 days, respectively. The dissipation rate of difenoconazole did not differ significantly in the three premix formulations. Mandipropamid also dissipated faster compared to the other fungicides tested. The chronic dietary risk assessment results showed a minimal risk to adult Egyptian consumers. Waiting periods were advised for the safe consumption of tomatoes treated with the tested premix formulations.
Introduction
Tomato fruits (Lycopersicon esculentum Mill.) are one of the most important vegetable crops in the world, which contains vital elements such as antioxidants as lycopene [1]. Egypt produces over 6.7 million tons of tomatoes, ranking sixth among global producers [2]. Fungal diseases are among the most common and widespread pests in tomato crops, and many fungicides are used to control fungal diseases in the field [3]. Difenoconazole (Fig. 1A) and propiconazole (Fig. 1B) are broad-spectrum systemic 1,2,4-triazole fungicides that are popular for their preventive and therapeutic properties because they can inhibit fungal development and multiplication by inhibiting the synthesis of fungal cell wall sterols [4, 5].
The chemical structure of difenoconazole (A), propiconazole (B), mandipropamid (C) and cyflufenamid (D)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
Cyflufenamid (Fig. 1C) and mandipropamid (Fig. 1D) are new amide fungicides. Cyflufenamid acts as a preventive and curative fungicide against powdery mildew on vegetables via a mechanism that is likely different from that of known fungicides by inhibiting phospholipid biosynthesis at several stages of the fungal life cycle. Mandipropamid is effective against oomycete plant pathogens in several crops, including Phytophthora infestations on potatoes and tomatoes, downy mildew (Bremialactucae) and blue mold (Peronospora effuse) on leafy vegetables [6, 7].
Direct application of pesticides in ready-to-use premixes is becoming more common in Egypt; for example, difenoconazole with propiconazole and difenoconazole with mandipropamid are registered for late blight control, difenoconazole with cyflufenamid for powdery mildew control in many crops.
This combination can minimize the number of application doses required and the associated costs while reducing the likelihood of fungal resistance. However, residues above the maximum residue limit can accumulate and pose a risk to consumers as an increased risk of cardiovascular disease, developmental abnormalities, thyroid endocrine disorders, immune system damage, metabolic disorders and reproductive toxicity [8]. Therefore, measuring pesticide residues in crops to ensure food safety is taken seriously by the public.
The dissipation behavior of a single fungicide, such as difenoconazole, propiconazole, mandipropamid, or cyflufenamid, has been extensively studied [4, 9–14], and their residues have been determined mainly by HPLC-UV [13], GC-ECD [9], GC-FID [14], GC-MS [14–17] and LC-MS/MS [12, 18, 19]. In contrast, there are few reports examining the dissipation behavior of pesticides in ready-to-use, premixed formulations [20, 21]. Due to its lower limit of quantitation (LOQ), and high accuracy, QuEChERS “Quick-Easy-Cheap-Effective-Rugged-Safe” protocol is often performed in conjunction with the HPLC-MS/MS technique [22].
A valuable tool for analyzing residue behavior and determining the time required to reduce residue levels below maximum residue limits is the rate at which a pesticide dissipates after application. Therefore, this study aims to: (1) optimize and verify a rapid, and effective analytical procedure for the simultaneous quantitation of difenoconazole, propiconazole, cyflufenamid, and mandipropamid residues in tomato; (2) evaluate the safety of the premixed fungicides tested based on their residues in tomatoes compared to maximum residue limits established by international authorities; (3) assess the dissipation kinetics of difenoconazole in combination with propiconazole, cyflufenamid, and mandipropamid in tomatoes grown in the field; and (4) include these residue data in the risk assessment, which could provide guidance to authorities on the appropriate and safe use of the fungicides tested.
Experimental
Chemicals and reagents
The reference standards difenoconazole, propiconazole, cyflufenamid, and mandipropamid with purities of more than 98% were brought from Chem Service Inc (West Chester, PA, USA). A commercial ready-to-use mixture containing the following ingredients was purchased at the local market: 30% EC (emulsifiable concentrate) (Montoro 30% EC®, Zhejiang Sega Science and Technology Co., Ltd. – China) consist of difenoconazole 15% + propiconazole 15%, 50% SC (suspension concentrate) (Revus top 50% SC®, Syngenta Agro, Egypt) consist of difenoconazole 25% + mandipropamid 25%, and 14% DC (dispersible concentrate) (Cidely top 14% DC®, Syngenta Agro, Egypt) consist ofdifenoconazole 12.5% + cyflufenamid 1.5%. Fisher Scientific provided HPLC grade acetonitrile and methanol, anhydrous magnesium sulfate (99%), analytical grade sodium chloride, LC-MS grade formic acid (Loughborough, UK). Agilent Technologies Inc. supplied the primary secondary amine (PSA) (DE, USA). The BarnsteadTM Micro Purification System produced ultra-pure deionized water (Thermo Fisher Scientific, Hungary).
Field experiments
The field trial was conducted in Menof, Menofia Governorate, Egypt. The pre-ready mixes of 30% EC, 50% SC, and 14% DCwere diluted in water (1000 L. ha−1) and applied once at rates of 150 ga.i.ha−1, 240 ga.i.ha−1 and 84 ga.i.ha−1, respectively, at the time of plant fruiting using a knapsack sprayer. Tomato samples (approximately 2 kg) were collected at 0 (2 h), 1, 3, 7, 10, 15, and 21 days after application. Three replicate plots of 50 m2 were sprayed for each formulation type, with buffer zones between each treatment. The daily average temperature during the field trial ranged from 24 to 36 °C, and there was no rainfall. Samples were brought to the laboratory under strict control, then cut into small pieces (2–3 cm) and stored overnight in the freezer. The samples were crushed and homogenized using a HOBART mill and kept at −20 °C until analysis.
Extraction procedure
10 ± 0.1 g of the homogenized frozen sample was weighed into a 50-mL centrifuge tube. Then 10 mL of acetonitrile was added and shaken by hand for two minutes after adding a ceramic homogenizer. For phase separation, a salt mixture of 4 g magnesium sulfate and 1 g sodium chloride was added, shaken for 60 s, and then centrifuged at 5,000 rpm for 3 min. The upper phase (extract) was collected. The extract was filtered after 6-fold dilution (0.2 mL of extract plus 1 mL of pure acetonitrile) using a nylon syringe filter with a filter fineness of 0.22 μm and then shaken well for chromatographic analysis.
LC-MS/MS
The Thermo Scientific Dionex Ultimate 3000 RS UHPLC instrument was coupled to a TSQ Altis triple quadrupole mass spectrometer (MS/MS) (Thermo Fisher Scientific, Austin, TX, USA). An Accucore RP-MS C18 column from Thermo Scientific (Lithuania, 100 × 2.1 mm, 2.5 µm particle size) set at 40 °C was used for chromatographic separation. Analytes were eluted at a flow rate of 0.3 mL min−1 with mobile phases of water (A) and acetonitrile (B) with a gradient of 40% of B for 1 min, then increasing to 95% over 3 min (1–4) and holding for 5 min (4–9), then returning to 40% of B at 9.1 and equilibrating for 6 min (9.1–15). The injection volume was 5 μl. Positive ion mode (H-ESI+) was selected for ionization in multiple reaction monitoring (MRM). The sheath and auxiliary gas pressures were 40 and 10 Arb, respectively, and the desolvation and ion source temperatures were 350 °C and 325 °C, respectively. The spray voltage was set at 3.8 KV. System management and data acquisition were performed using the Trace Finder software package (version 4.1).
Preparation of calibration standards
A reference standard solution and intermediate solutions of 1,000 mg L−1 and 100 mg L−1 were prepared individually in acetonitrile and stored at −20 °C. A standard solution mixture of 10 mg L−1 was prepared by further dilution in acetonitrile. A tomato blank extract prepared by the proposed method was mixed with the standard mixture in the appropriate volumes to prepare matrix-matched standards.
Method validation
The procedure's effectiveness was evaluated using the criteria of the SANTE/12682/2019 guideline [22]. The linearity range was determined by constructing calibration curves in the range of 0.5–500 μg kg−1 in the matrix, then the correlation coefficient (R2) and residuals were calculated. The matrix effect (ME) was evaluated by comparing the slopes of the solvent-based standard curve and the matrix-matched calibration curve. Accuracy was assessed regarding precision and trueness at a spiking concentration of LOQ for each analyte tested. Method precision was evaluated in one day (RSDr, n = 6) and three different days, 7 days' interval (RSDR, n = 18), while trueness was assessed by percent recovery. Method recovery was determined at four spiking levels of 0.01, 0.5, 1, and 5 mg kg−1. The limit of detection (LOD) was defined as the concentration at which a signal-to-noise ratio of S/N = 3. The limit of quantitation (LOQ) was defined as the lowest analyte concentration in the sample at which satisfactory recovery (70–120%) was achieved with a relative standard deviation of less than 20%.
Calculations
To calculate field findings and assess significance between groups, the one-way test ANOVA and Fisher's least significant difference (LSD) test were performed using Microsoft Excel 2021 software, and a probability value of P < 0.05 was considered significant. The dissipation rates and other kinetics was described using first-order kinetic model Ct = C0e−kt, where Ct is the concentration (mg kg−1) at time t (days), C0 is the initial concentration (mg kg−1), and k is the corresponding rate constant. The half-life was calculated using equation t1/2 = ln2/k [24]. The pre-harvest interval (PHI) was estimated using Equation (1) [12].
Results and discussion
LC-MS/MS optimization
The LC-MS/MS parameters were optimized for quantifying the studied analytes based on the determination of the retention times and the relative abundance of the two selected ion transitions [23]. The direct infusion mode using a Harvard infusion pump (South Natick, MA, USA) was chosen for optimization. The system was operated in positive electrospray ionization (ESI+) mode. Cone voltage was automatically optimized for the selected precursor ions [M + H] + of difenoconazole (m/z, 406.1), propiconazole (m/z, 342.08), cyflufenamid (m/z, 413.1) and mandipropamid (m/z, 412.08), which were selected based on their higher responses, and then the precursor ion was used to generate the product ions. The final MS/MS parameters are shown in Table 1.
LC-MS/MS parameters
| Pesticide | tR (min) | Precursor ion (m/z) | Product ion (m/z) | Collision energy (v) | RF lens (v) | Dwell time (ms) |
| Difenoconazole | 7.52 | 406.1 | 251 | 25 | 70.4 | 7.54 |
| 337.1 | 17 | 70.4 | ||||
| Propiconazole | 7.39 | 342.08 | 158.9 | 30 | 65.6 | 7.38 |
| 204.9 | 19 | 65.6 | ||||
| Cyflufenamid | 7.45 | 413.1 | 295.1 | 15 | 63 | 7.45 |
| 241 | 23 | 63 | ||||
| Mandipropamid | 6.76 | 412.08 | 328.2 | 14 | 63 | 6.76 |
| 356.2 | 10 | 63 |
The underlined ions used as a quantifier ion.
Experiments were performed with methanol/water or acetonitrile/water with or without formic acid (0.1%) to achieve optimal separation and peak shape. The results showed that acetonitrile/water or methanol/water at a ratio of 40/60 as the mobile phase for the initial gradient resulted in acceptable separation, symmetric peak shape, and rapid elution. Nevertheless, we decided to use acetonitrile/water as a mobile phase component for further analysis because the instrument's sensitivity for propiconazole and difenoconazole was significantly increased (P < 0.05), 280% and 319%, respectively. In contrast, the sensitivity of cyflufenamid was significantly (P < 0.05) decreased by 50%, and in the case of mandipropamid, a non-significant (P > 0.05) difference was observed compared to the use of methanol/water as the mobile phase.
Adding 0.1% formic acid to the mobile phase leads to a significant (P < 0.05) reduction in the signal sensitivity of the tested analytes by more than 70%. The target analytes eluted after 6.76 min (mandipropamid), 7.38 min (propiconazole), 7.45 min (cyflufenamid), and 7.52 min (difenoconazole) (Fig. 2).
LC/MS/MS representative chromatograms of blank sample extract (A, B, C), fortified sample extract of difenoconazole (a), propiconazole (b), mandipropamid (c), and cyflufenamid (d) at 2 μg kg−1
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
Effect of dilution on the matrix effect
The weak anion exchanger PSA is the most commonly used adsorbent to remove various fatty acids and sugars from sample extract [28]. The evaluation of the efficiency of PSA (25 mg mL−1 extract) compared to the dilution effect was performed by determining the matrix effect. The ME (suppression effect) resulted from direct analysis of spiked raw extract of tomatoes without cleanup was ranged from 32.5 to 58.7%. The using of PSA adsorbent in cleanup of spiked tomato extract leads to significant (P > 0.05) decrease in signal sensitivity of cyflufenamid and mandipropamid by 21.2–47.3% compared to standard in pure acetonitrile, and non-significant (P > 0.05) decrease in case of propiconazole and difenoconazole. To minimize the laboratory equipment used, reduce material costs, and shorten the time required to perform the cleanup step, a different dilution factor of 2–10× of the raw extract was tested. Figure 3 shows that, non-significant differences were observed in the ME when using PSA adsorbent compared to the diluted extract in case of difenoconazole, whereas the difference was significant for other tested fungicides. The minimum significant ME was Observed at a dilution factor of 5×, and a signal suppression was ranged from 6.3% to 14.6%. So, it is possible to dilute the raw extract at 5-fold using acetonitrile instead of using PSA as adsorbent for cleanup, which saves tools, cost and analysis time.
The effect of using PSA (25 mg mL−1) adsorbent comparing to matrix dilution on the matrix effect at concentration of 10 μg kg−1
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
Method validation
The linearity ranges of the targeted analytes were examined in the range of 0.5–500 μg kg−1. Good linearity and a strong correlation coefficient were obtained in the range of 2–100 μg kg−1 for difenoconazole and propiconazole, and 1–100 μg kg−1 for cyflufenamid and mandipropamid, respectively, with a correlation coefficient (R2) and relative residuals of ≥ 0.996 and ≤ 11.7%, respectively. The matrix effect (ME) is one of the challenges that require an appropriate strategy to eliminate possible interference during the analysis processes using LC-ESI-MS/MS. The signal response was weakly suppressed with a percentage of 7.4, 4.1, 3.8, and 11.1%, for difenoconazole, propiconazole, cyflufenamid, and mandipropamid, respectively, overall, the matrix effect considered negligible [29–31]. Even though the matrix-matched standard calibration was used to compensate for possible interferences in the measurement of the target analytes [32, 33]. The recovery experiment was conducted by spiking tomato samples with the tested analytes at four concentrations of 10, 500, 1,000, and 5,000 μg kg−1 on tomato blank samples. The achieved average percentage recoveries were ranged from 91.8 to 106.3%, with a relative standard deviation (RSD) of less than 9.8% (Table 3). Accuracy of the method was evaluated in terms of precision and trueness. Precision was evaluated in one day (RSDr, n = 6) (intra-day repeatability, RSDr) and within three different days (n = 18) (Inter-days repeatability, RSDR) by analyzing samples spiked at concentration level of 5 μg kg−1. Results in Table 2 show that, The RSDr and RSDR were lower than 6.4% and 7.6%, respectively, and the trueness (% recovery) were more than 96.4%. According quality control guideline for pesticide residue analysis, recoveries of 70–120% and an RSD of <20% are considered acceptable [23]. LOQ was tested at a concentration of 5 μg kg−1 as the minimum concentration at which satisfactory recovery and precision were obtained. The values obtained from LOQ showed that the optimized method was able to detect pesticide residues at concentrations approximately 8-fold, 6-fold, 40-fold, and 60-fold below the MRLs set by the EU Commission [34] for cyflufenamid, mandipropamid, difenoconazole, and propiconazole, respectively. As shown in Tables 2 and 3, the proposed method was suitable and sufficient for simultaneously determine the tested analytes in real tomato samples.
Calibration parameters, LOD, LOQ, ME (%), and precision of the four fungicides in tomato fruits
| Analyte | Linear range (µg kg−1) | R2 | Residual (%) | LOD (µg kg−1) | LOQ (µg kg−1) | ME (%) | Precision | |||
| Intra-day (n = 6) | Inter-day (n = 18) | |||||||||
| R (%) | RSDr (%) | R (%) | RSDR (%) | |||||||
| Difenoconazole | 2–200 | 0.997 | 11.7 | 1.22 | 5 | −7.4 | 93.7 | 3.7 | 96.1 | 5.5 |
| Propiconazole | 2–200 | 0.996 | 6.4 | 0.93 | 5 | −4.1 | 92.2 | 6.4 | 94.4 | 7.3 |
| Cyflufenamid | 1–200 | 0.998 | 4.8 | 0.53 | 5 | −3.8 | 96.4 | 2.8 | 91.7 | 4.3 |
| Mandipropamid | 1–200 | 0.999 | 9.3 | 0.42 | 5 | −11.1 | 94.3 | 5.8 | 93.8 | 7.6 |
Recovery and RSD of the four fungicides in tomato fruits
| Analyte | Recovery ± RSD (%), (n = 6) | |||
| 0.01 mg kg−1 | 0.5 mg kg−1 | 1 mg kg−1 | 5 mg kg−1 | |
| Difenoconazole | 92.2 (4.7) | 97.1 (8.1) | 94.3 (3.1) | 101.2 (2.8) |
| Propiconazole | 91.8 (6.5) | 96.3 (5.2) | 95.1 (7.7) | 97.2 (3.3) |
| Cyflufenamid | 93.1 (8.2) | 98.4 (3.8) | 101.8 (5.9) | 96.7 (8.8) |
| Mandipropamid | 106.3 (9.8) | 95.1 (4.1) | 96.7 (6.9) | 98.3 (5.7) |
Dissipation study
Dissipation of difenoconazole and propiconazole in ready premix formulation
The initial residues of difenoconazole and propiconazole were 1.77 and 1.34 mg kg−1, after applying the recommended dose of 75 g a.i.ha−1 and 75 g a.i.ha−1, respectively. On day 7, after spraying, the residues were relatively rapidly dissipated at 77.9% and 91.8%, respectively. By day 15, more than 98% of the residues were dissipated (Fig. 4). The higher initial deposition of difenoconazole compared to propiconazole may be attributed to the high vapor pressure value of 1 × 10−6 mmHg for propiconazole compared to 2 × 10−10 mmHg for difenoconazole. The first-order kinetics described the dissipation behavior of difenoconazole: (Ct = 1.8232e−0.269t) and propiconazole (Ct = 1.0116e−0.299t) with correlation coefficients of 0.985 and 0.981, respectively (Table 4). The half-lives (t1/2) of difenoconazole and propiconazole residues in tomatoes were 2.01 and 1.89 days, respectively. The high rate of dissipation of propiconazole (k = 0.299) compared to difenoconazole residues (k = 0.269) can be explained by the differences in the octanol/water partition coefficient (Log Kow) of 4.4 and 3.7 for difenoconazole and propiconazole, respectively, suggesting that difenoconazole is relatively more lipophilic than propiconazole and has a higher penetration effect into the outer layer of tomato, so that the remaining propiconazole residues on the outer surface may be higher than those in the cuticle layer, exposing them to photodegradation and exceeding the rate of dissipation. The present t1/2 results differ from those that are 6.66–10.35 days [35] in ginseng, 6.3–10.2 days [36] in apples, for propiconazole, and 4.68–8.09 days [10] for difenoconazole in chili fruits. Few studies have been conducted on the dissipation dynamic of the combination of difenoconazole and propiconazole in the ready-mix. Zhang et al. (2015) reported that the half-lives of difenoconazole and propiconazole in wheat straws were 3.6–5.5 days and 5.1–6.9 days, respectively [19].
Dissipation rate of difenoconazole and propiconazole residues in ready-mix formulation of 30% EC in tomato fruits
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
Kinetics of the tested ready-mix formulations dissipation in tomato fruits
| Ready mix formulation (30%, EC) | Ready mix formulation (14%, DC) | Ready mix formulation (50%, SC) | ||||
| Difenoconazole | Propiconazole | Difenoconazole | Cyflufenamid | Difenoconazole | Mandipropamid | |
| Application rate (g.ai/ha) | 75 | 75 | 75 | 9 | 120 | 120 |
| Regression equation | 1.8232e−0.269× | 1.0116e−0.299× | 2.1779e−0.207× | 0.1012e−0.279× | 2.5598e−0.232× | 2.8728e−0.362× |
| Correlation coefficient (r2) | 0.985 | 0.981 | 0.972 | 0.961 | 0.982 | 0.970 |
| K (days−1) | 0.269 | 0.299 | 0.207 | 0.279 | 0.232 | 0.362 |
| MRLs (mg kg−1)a | 2 | 3 | 2 | 0.04 | 2 | 0.3 |
| t0.5 (days) | 2.01 | 1.89 | 2.27 | 1.97 | 2.15 | 1.71 |
| PHI (days)b | 0.34 | 3.63 | 0.41 | 3.33 | 1.06 | 6.24 |
aMaximum residue limits.
bPre-harvest interval.
Dissipation of difenoconazole and mandipropamid in ready premix formulation
The initial deposits of difenoconazole and mandipropamid in tomatoes were 3.11 and 3.72 mg kg−1, after applying the recommended dose of 120 g a.i.ha−1 and 120 g a.i.ha−1, respectively. Mandipropamid exhibited a faster dissipation rate of >91% than difenoconazole of >82% on day 7 after spraying. Residues of mandipropamid were below the limit of quantitation of 5 μg kg−1, while those of difenoconazole were 0.089 mg kg−1 on day 15 (Fig. 5). The initial deposition of mandipropamid was higher than that of difenoconazole despite the same spray dose of 120 g a.i.ha−1 each. The dissipation kinetics of difenoconazole and mandipropamid could be described by the first-order kinetic equations: Ct = 2.5598e−0.232t and Ct = 2.8728e−0.362t, with correlation coefficients of 0.982 and 0.970, respectively (Table 4). The half-lives of difenoconazole and mandipropamid were 2.15 and 1.71 days, respectively. The dissipation rate of mandipropamid was higher than that of difenoconazole; due to the high vapor pressure of 7.1 × 10−9 mmHg and the low log Kow value of 3.2 compared to 2 × 10−10 mmHg and 4.4 for mandipropamid and difenoconazole, respectively. The present results on the rapid dissipation of mandipropamid residues agree with those obtained in grapes with a half-life of 2.20 days [13].
Dissipation rate of difenoconazole and mandipropamid residues in ready-mix formulation of 50% SC in tomato fruit
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
Dissipation of difenoconazole and cyflufenamid in ready premix formulation
The initial deposits of difenoconazole and cyflufenamid in tomatoes were 2.46 and 0.12 mg kg−1, after applying the recommended dose of 75 g a.i.ha−1and 9 g a.i.ha−1, respectively (Fig. 6). The initial deposition was higher for difenoconazole than for cyflufenamid due to the higher application dose of difenoconazole compared to cyflufenamid. Difenoconazole and cyflufenamid showed higher dissipation rates on day 3 and day 10, with dissipation rates of 65% and 87%, and 71% and 93%, respectively. Residues of cyflufenamid were below the limit of quantitation of 0.01 mg kg−1 after 15 days, while the residue concentration of difenoconazole was 0.085 mg kg−1. The dynamic equations of difenoconazole and cyflufenamid were fitted by the first-order kinetic model of Ct = 2.1779e−0.207t and Ct = 0.1012e−0.279t with correlation coefficients of 0.971 and 0.961, respectively (Table 4). The dissipation rate of cyflufenamid was higher than that of difenoconazole due to the high vapor pressure of cyflufenamid (2.65 × 10−7 mmHg), while the log Kow value of cyflufenamid was 4.7, which was almost equal to that of difenoconazole. The calculated half-lives of difenoconazole and cyflufenamid were 2.27 and 1.97 days, respectively. As far as we know, few papers have addressed the dissipation of cyflufenamid alone or in combination. The half-life of difenoconazole and cyflufenamid determined in our work is consistent with a previous study that found a half-life of 2.35–2.37 and 1.67 days in pea pods and whole pods, respectively, for difenoconazole and cyflufenamid when both were applied in a ready premix [37].
Dissipation rate of difenoconazole and cyflufenamid residues in ready-mix formulation of 14% DC in tomato fruits
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01134
The previous results show that the difenoconazole dissipation rate was high when 30% EC was applied compared to 50% SC and 14% DC premix formulations; this could be due to the different formulation types, which could affect residue deposition proportionally to the change in application rates (kg active ingredient/ha). Increasing the application rate also leads to a higher concentration of surfactants and adjuvants in the spray solution, which could result in longer residue residence time in the plant [38]. Several factors, such as formulation types, temperature, humidity, plant composition (lipids, sugars, proteins), and pH, and their interactions may play a crucial role in the dissipation process, which is complex and highly variable [39–42].
Risk assessment and pre-harvest intervals
The consumer risk assessment of the tested fungicides in tomatoes was based on the estimated daily intake at harvest of 0 and 1 days. It was determined that it is logical to use the selected harvest dates for consumer risk assessment because tomatoes are harvested multiple times and are harvested each day after the last application. The four fungicide residue concentrations, EDI, and RQ values were calculated and are shown in Table 5. The EDI values of the four fungicides are relatively low. These results indicate that the potential health risks of the tested fungicides are insignificant. Generally, an RQ value greater than 100% suggests high pesticide residues and a significant impact on consumer health. The RQ values of the fungicides tested were less than 100%, except for difenoconazole in the 50% SC formulation at 0 days, indicating that the application of the three mixture formulations to tomatoes poses a low, acceptable risk to human health. The results showed that residues on tomatoes are unlikely to pose a risk to consumers after the tested ready-to-use mixtures are applied.
Residues (mg kg−1) (PHI = 0 and 1 days), acceptable daily intake (ADI), estimated daily intake (EDI) and risk quotient (RQ) of the tested fungicide residues in tomatoes
| Formulation type | Pesticides | Dosage (g.ai/ha) | Interval (days) | Residues (mg kg−1) | ADI (mg kg−1, bw) | EDI (mg kg−1, bw) | RQ (%) |
| 50% SC | Difenoconazole | 125 | 0 | 3.11 | 0.01 | 1.2 × 10−2 | 120.3 |
| 1 | 2.12 | 8.2 × 10−3 | 82.3 | ||||
| Mandipropamid | 125 | 0 | 3.72 | 0.15 | 1.4 × 10−2 | 9.5 | |
| 1 | 1.67 | 6.5 × 10−3 | 4.3 | ||||
| 14% DC | Difenoconazole | 78 | 0 | 2.46 | 0.01 | 9.6 × 10−3 | 95.5 |
| 1 | 1.69 | 6.6 × 10−3 | 65.7 | ||||
| Cyflufenamid | 9.4 | 0 | 0.12 | 0.04 | 0.5 × 10−3 | 1.1 | |
| 1 | 0.08 | 0.3 × 10−3 | 0.7 | ||||
| 30% EC | Difenoconazole | 75 | 0 | 1.77 | 0.01 | 6.9 × 10−3 | 68.7 |
| 1 | 1.16 | 4.5 × 10−3 | 45.1 | ||||
| Propiconazole | 75 | 0 | 1.16 | 0.04 | 5.2 × 10−3 | 13 | |
| 1 | 0.58 | 2.3 × 10−3 | 5.6 |
Since there is no legal maximum residue limit in Egypt for the analytes tested, the maximum residue limit established by the European Union was considered in the pre-harvest calculation (PHI). The estimated PHI of difenoconazole and propiconazole in a ready-mix of 30% EC were 0.34 and 3.63 days and 1.06 and 6.24 days, respectively, for difenoconazole and mandipropamid in a ready-mix of 50% SC, and 0.41 and 3.33 days for difenoconazole and cyflufenamid in a ready-mix of 14% DC. A shorter PHI was required for difenoconazole, while a longer PHI was required for mandipropamid than for other fungicides tested. Because the ready-to-use mixtures tested contain two different fungicides with different PHI, the recommended PHI for the ready-to-use mixtures tested depends on the choice of the longer of the two fungicides in the mix. Thus, for safe consumption of tomatoes treated with 30% EC and 14% DC or 50% SC ready-to-use mixtures, the recommended PHI is 4, 4, and 7 days, respectively.
Conclusion
A rapid and accurate method for simultaneous quantifying difenoconazole, propiconazole, mandipropamid, and cyflufenamid in tomato based on acetonitrile extraction combined with LC-MS/MS spectrometry was optimized. The proposed method was verified according to SANTE/12682/2019 guidelines. The criteria related to linearity, LOQs, recoveries, accuracy, and matrix effect of the developed method showed satisfactory validation results. Propiconazole, mandipropamid, and cyflufenamid are used in a ready premix formulation with difenoconazole on tomato. Data obtained during field trials showed that residues of difenoconazole, propiconazole, mandipropamid, and cyflufenamid were below the maximum residue level of 2, 3, 0.3, and 0.04 mg kg−1 (EU-MRL database), respectively, 4, 4 and 7 days after application of the recommended dosage of the tested formulations. Despite the faster dissipation of mandipropamid and slower dissipation of difenoconazole compared to the tested fungicides, difenoconazole on tomato required a shorter PHI. In contrast, mandipropamid required a longer PHI. The premixes of the fungicides tested don't expose customers to concentrations higher than toxicologically safe levels, so no adverse health effects are expected. The current results could make PHI safer, and the tested fungicides can be safely used in a premix for the tomato to control fungal diseases.
Compliance with ethical requirements
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgement
The researchers would like to thank the Deanship of Scientific Research, Qassim University for funding the publication of this project.
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