Determination of Quizalofop-p-ethyl in onion spices: Weed control efficiency, residual profile, and food safety assessment under field conditions

doi:10.21203/rs.3.rs-1413281/v1

Monitoring for pesticide residues in onion (Allium cepa L.) is important to protect consumer health and to ensure food safety. A field experiment was conducted for two consecutive seasons with quizalofop ethyl (5% EC) @ 50 and 100 g a.i. ha^-1 to evaluate weed control efficiency and to determine terminal residues. Double the recommended dose (100 g a.i. ha^-1) produced higher weed control efficiency (88.53-95.13 %), lower weed density (4.15-7.73 m^-2), and weed dry weight. A rapid, sensitive, and analytical method was developed using high-performance liquid chromatography with excellent linearity (r²> 0.99). Accuracy of the method in terms of average recoveries ranged from 94.4 to 99.9 % at spiking levels of 0.04, 0.20 and 0.40 mg kg^-1with intra and inter-day precision expressed relative standard deviation RSD (%) between 0.1-2.3 and 1.7-3. The limit of quantification for quizalofop-p-ethyl was established at 0.04 mg kg^-1 with signal to noise (S/N) ratio ≥10. The method was successfully applied and initial quantified residues were in the range of 2.5-4.4 mg kg^-1irrespective of seasons and doses. Finally, the presence of targeted herbicide residues in harvested samples was confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS) under optimized operating conditions. The herbicide dissipated to the lowest validated level (0.04 mg kg^-1) between 10-15 days with half-life values (DT₅₀) of 1.5-1.6 days. Dietary risk assessment assured harvested onions were safe for consumption at the recommended dose.

Allium cepa

Herbicide residue

Chromatography

Accuracy and precision

Dietary risk assessment

Phytotoxicity

Weed management

LC-MS/MS

Uncontrolled weed growth reduces onion (Allium cepa L.) bulb yield by 40–80% depending upon the intensity and duration of weed competition (Souza et al. 2020). Herbicide formulations extensively applied for controlling a wide range of weed flora are required to be evaluated for their bio-efficacy, crop growth parameters, and effects on the yield of onion. Quizalofop p-ethyl [ethyl (R)-2-{4-(6-chloroquinoxanlin-2-yloxy)-phenoxyl}propionate], a very effective post-emergence herbicide, belongs to the aryloxy phenoxy propionate (fops) group according to the classification given by the Herbicide Resistance Action Committee of Argentina (ARG-HRAC) (Li et al. 2020). Application of this herbicide could lead to concerns of accumulation of residues in onion. As the consumption of onion green leaves, immature and mature onion bulbs (either raw or processed) are increasing, monitoring pesticide residue levels therefore becomes important to maintain quality and ensure food safety (Narenderan et al. 2020).

Analysis of pesticide residues in onion matrices is often challenging due to its high pigment and chlorophyll content (Lawal et al. 2018). It is necessary to develop a suitable method for analysis of this herbicide and test residues in field studies to ensure residue data which can be used in fixation of the maximum residue level (MRL) (Saha et al. 2014; Szpyrka et al. 2020). Chromatographic methods commonly used in the determination and separation of target pesticides including herbicides are gas chromatography (GC) and high performance liquid chromatography (HPLC) with precise detection systems (Toledano et al. 2010; Masiá et al. 2013; Wang et al. 2019; Majumder et al. 2022). The HPLC is preferred over GC methods due to the water solubility of phenoxy acids, which allows direct analysis of the phenoxy acids and their esters without derivatization (Mei et al. 2015).

The objective of this experiment was to establish a rapid, sensitive, and selective analytical method to determine the targeted quizalofop ethyl residues in onion and onion cropped soil. Dietary risk assessment was also performed to know whether the harvested onions were safe for consumption or not.

Chemicals and Reagents

The formulation of quizalofop ethyl (5% EC) was supplied by M/S Dhanuka Agritech Limited, New Delhi. The reference standard of quizalofop ethyl (purity 99.0%) (Fig. 1) was procured from Sigma Aldrich (Humburg, Germany). The HPLC grade solvents - acetonitrile and water were obtained from J.T. Baker (Phillipsburg, New Jersey). The N-propyl ethylene diamine-based primary secondary amine (PSA), graphitized carbon (C18), and florist sorbents were procured from Agilent Technology (Santa Clara, CA). The analytical reagent grade anhydrous magnesium sulfate (MgSO₄), and sodium chloride (NaCl) were purchased from Avantor Performance Materials India Limited (Gurugram, India).

Apparatus

The HPLC analysis was performed with an HPLC (1200 series, Agilent, Santa Clara, CA) coupled to a ultra-violet (UV) detector with an isocratic pump. Separation was with an Agilent Eclipse Plus chromatographic C18 column (4.6 mm × 250 mm × 5 µm). The degassed, filtered (0.45 µm Nylon filter), the mobile phase was composed of acetonitrile: water (70:30, v: v) with flow rate at 1 mL min^− 1 and column temperature of 20°C. Aliquots (10 µL) were injected directly to identify and quantify Quizalofop ethyl compared to retention time (RT) of external standard peak area.

Briefly, LC–MS/MS analysis was performed with Acquity ultra performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) and API-3200 triple quadrupole mass spectrometry (MS/MS) system (ABSciex, Vaughan, ON, Canada) coupled with an electrospray ionization (ESI) source operating in positive ion mode.

Preparation of Standard Solutions

A stock solution containing 1g L^− 1 of the target herbicide was prepared by dissolving 10 ± 0.1 mg reference standard in 10 mL of acetonitrile and stored at -4°C. All intermediate working solutions (0.04, 0.1, 0.20, 0.4 or 1.0 mg L^− 1 were prepared by serial dilution to determine the accuracy, precision, linearity, and sensitivity.

Sample Preparation

Onion samples were prepared following the QuEChERS method (Anastassiades et al. 2003) and validated as per guidelines of the European Commission (SANTE 2020) for residues determination and dissipation kinetics of quizalofop ethyl. A representative chopped homogenized sample of onion (leaf/bulb) (500g) was smeared thoroughly in a Robot Cuope Blixer 6 v.v (Ridgeland, MS) vertical mixer at 756 ×g speed for 5 min. An aliquot of 10 g was placed in a centrifuge tube (50 mL), to which 10 mL acetonitrile was added and the mix vortexed for 1 min. Afterward, 4 g MgSO₄ and 1 g NaCl were added and the mix was immediately vigorously hand shaken and centrifuged in Superspin, Plasto-Craft Centrifuge for 5 min at 8495×g. An aliquot from the upper layer (5 mL) was subjected to clean-up by dispersive solid-phase extraction (d-SPE) using primary secondary amine (PSA) sorbent (250 mg), anhydrous MgSO₄ (750 mg), and centrifugation at 8495 ×g speed for 5 min. The final supernatant (1 mL) was filtered through 0.22 µm nylon 66 membrane filter paper (SGE Analytical Science Pvt. Ltd, Mumbai, India) and subjected to HPLC chromatographic analysis at 235 nm absorbance. All experiments were performed in triplicate.

Method Validation

The method validation study was carried out on onion (leaf, immature and mature bulb) and soil samples (not treated with target pesticide) with linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, and precision. The performance of the analytical method was assessed as per the single laboratory validation approach of Kollipara et al. (2011).

Optimization of Chromatographic and Spectrometric Conditions

Optimization of chromatographic conditions of Quizalofop ethyl was done with separation using reverse-phase HPLC columns. Separation and peak shapes were determined with reverse-phase BDS Hypersil C18 column (4.6 mm×250 mm×5 µm) using water: acetonitrile (30:70, v/v) as the mobile phase. Since Quizalofop ethyl produced good absorption at 235 nm, this wavelength was used for detection and quantification.

The final determination of quizalofop-p-ethyl was performed by liquid chromatography tandem mass spectrometry (UPLC-MS/MS) by injecting 10 µL analytes to the system controlled by Analyst 1.5 software for data collection and analysis. Analytes were separated onto a Zorbax ultra aqueous reverse phase C-18 (150 × 3 mm i.d., 3.5 µm particle size) column (Agilent Technologies, USA) maintained at 35°C temperature. The mobile phase was prepared using (A) 5mM ammonium formate in methanol and (B) 5 mM ammonium formate in water. The gradient elution program was as follows: A (95%) B (5%) at the initial time (0 min), A (95%) B (5%) (at 0.96 min), A (50%) B (50%) (at 2.79 min), A (10%) B (90%) (at 5.64 min), A (10%) B (90%) (at 6.55 min), A (95%) B (5%) (at 7.54 min), A (95%) B (5%) (at 8.00 min). The mobile phase flow rate was 0.35 ml min^− 1 using binary pump with total run time of 14 min.

Calibration and Linearity

Calibration and linearity were assessed by internal standardization over the range 0.04-1.0 mg kg^-1. The matrix-matched standard calibration curves were created at 5 levels (and blank) and injected in duplicates.

Selectivity and Sensitivity

The selectivity and sensitivity limits of detection and quantification were estimated following the IUPAC approaches which consisted of analyzing the blank sample to establish noise levels and then testing experimentally estimated LOQs for signal/noise ≥ 10.

Accuracy and Precision

Accuracy and precision of the analytical method are defined as the similarity of results obtained by the analytical method to the true value. Accuracy of the method was determined as % recovery by spiking quizalofop ethyl at concentration levels of 0.04, 0.20, and 0.40 mg L^− 1 at specification level (1000 mg L^− 1). The precision of the method was estimated by repeatability (intra-day precision) and reproducibility (inter-day precision) at 3 concentrations. Repeatability is the intra-day variation obtained at 3 concentration levels and expressed in terms of relative standard deviation (RSD).

Field Experiment

The flora Cynodon dactylon (L.), Acrachne racemosa (Roemer & Schultes), Dactyloctenium aegyptium (L.), Boerhaavia diffusa (L.), Parthenium hysterophorus (L.) and Digeria arvensis (L.) were dominant weeds in the experimental field. Associated species of weeds were sampled and identities confirmed by a taxonomist. A field experiment was conducted in a randomized complete block design with red onion, expected bulb weight 30–35 g (variety: Sukhsagar), in 2009 and 2010 at Sonakhali village, Dist. Nadia, West Bengal, India (Bastiaans et al., 2000). The geographical location of the experimental site was 24.11''N latitude and 88.48''E longitude with an altitude of 44.70 m above mean sea level (MSL). The soil was an alluvial sandy loam with the texture of 38.2% clay, 31.8% silt, 21.3% fine sand, and 8.7% coarse sand, low in available nitrogen and medium in available phosphorus and potassium. A plot (50 m²) was used for individual treatment and quizalofop ethyl 5% EC (Targa Super) was applied 30 days after transplanting (DAT) @ 50 and 100 g a.i. ha^− 1 at 50% vegetative stage with a volume of 500 L ha^− 1. Data were recorded on predominant weed flora, weed density, and dry weight in onion.

Sample Collection

Two-kg of randomly selected immature onion leaves from treated plots and the untreated control were collected at 0, 1, 3, 5, 7, 10, and 15 days, immature onion bulbs at 30 days, and mature onion bulbs and soil samples at 55 days after the last treatment. Immediately after collection, samples were placed in polyethylene bags and transported to the laboratory. Samples were thoroughly mixed and 500 g of sub-samples obtained by quartering were stored at -20°C until analysis (Meng et al. 2020).

Observations on Weeds

Weed Density

Weed count was recorded by species from 0.5 × 0.5 m quadrat (a frame, traditionally square, used in ecology and geography to isolate a standard unit of area for the study of the distribution of an item over a large area) from 3 randomly selected places in each plot. The density of grasses, sedges, and broadleaf weeds, and total weeds were recorded at 20, 40, and 60 days after sowing.

Weed Dry Weight

Weeds within frames of the quadrate were shade dried and then dried in a hot-air oven at 80°C for 72 hrs. Dry weights were recorded.

Weed Control Efficiency

WCE = × 100%Weed control efficiency (WCR) was calculated as per the reported procedure (Zia-Ul-Haq et al. 2019).

Where,

WCE = Weed control efficiency (per cent);

WDC = Weed biomass (g m^− 2) in control plot;

WDT = Weed biomass (g m^− 2) in treated plot.

Weed Index

The weed index (WI) was calculated as per the method of Kaur et al. (2020).

Where,

X = Yield (kg ha^− 1) from minimum weed competition plot;

Y = Yield (kg ha^− 1) from the treatment plot for which WI is to be worked out.

Food Safety Evaluation

To evaluate the safety of Quizalofop ethyl in onion the theoretical maximum residue contribution (TMRC) of Quizalofop ethyl was compared to the maximum permissible intake (MPI) at the recommended dose. The TMRC was calculated with an assumption that the onion used for cooking would contain Quizalofop ethyl residue at the highest level applied at the recommended dose (Saha et al. 2014). Per capita consumption of onion (green and bulb, red onion or otherwise) is 35,606.48 mg day^− 1, of 1.5 kg of food consumption per day, i.e. 2.37% can be taken as food factor for onion (NHB 2018). No observed effect level (NOEL) of Quizalofop-p-ethyl in mice was < 0.9 mg kg^− 1 day^− 1. The TMRC was estimated by multiplying the initial residue level quantified at the recommended dose of application with food factor and total intake of food per day. After calculation, TMRC for season-I was 0.088 mg person^− 1 day^− 1; for the 2nd season, it was 0.071 mg person^− 1 day^− 1. The MPI was estimated at 0.58 mg person^− 1 day^− 1.

The estimated daily intake (EDI) of quizalofop ethyl residues was calculated by multiplying the highest residue concentration (mg kg^− 1) with the food consumption rate (kg day^− 1) divided by the mean body weight of an adult. The long-term risk assessment of intakes compared to pesticide toxicological data was made by calculating the risk quotient (RQ) obtained by dividing the EDI by the relevant acceptable daily intake (ADI). The acceptable risk for long-term human dietary intake of quizalofop ethyl is confirmed when RQ is < 1; if RQ is > 1, it is an unacceptable risk (Song et al. 2020).

Weed Control Efficiency

Weed Density

Post-emergence application of new formulation of quizalofop ethyl at 50 and 100g a.i. ha^− 1 followed by one hand weeding on 40 DAS resulted in significantly (p < 0.05) control of broad-leaved weeds, grasses, and to some extent sedge due to its broad-spectrum action (Table 1). Higher doses of 100 g a.i. ha^− 1 gave more impressive control of broad-leaved weeds like Boerhaavia diffusa, Parthenium hysterophorus, and Digeria arvensis due to the herbicidal effect over inhibition of ATP synthesis and ultimately death of weeds (Dayan et al. 2019). The post-emergence application of quizalofop ethyl at 100 g a.i. ha^− 1 reduced the density of broad-leaved weeds (70–90%) in onion compared with non-treated plots. Several research findings showed that herbicide has successfully controlled broad-leaved weeds in vegetable systems including broccoli, onion, chili pepper, cauliflower, and cabbage (Dhananivetha et al. 2017; Ghorai et al. 2013). Post-emergence application of quizalofop ethyl most effectively decreased the number of annual broad-leaved weeds in onion and cabbage.

Table 1

Effects of weed management practices on total weed density in onion.
Treatments	Total weed density (No. m^− 2)
	Season-I			Season-II
	20 DAT^a	40 DAT	60 DAT	20 DAT	40 DAT	60 DAT
T₁: 50 g a.i.ha^− 1	5.59 (28.93)^b	8.60 (70.20)	6.98 (47.54)	5.67 (30.14)	8.41 (68.94)	7.58 (60.13)
T₂: 100ga.i. ha^− 1	4.47 (18.11)	7.73 (57.52)	5.93 (33.92)	4.15 (15.25)	7.48 (54.12)	6.25 (37.11)
T₃: HW^c (twice)	14.53 (210.37)	9.39 (67.29)	9.15 (49.00)	13.08 (169.14)	9.24 (83.29)	9.79 (58.55)
T₄: Unweeded control	13.22 (200.13)	24.78 (611.50)	11.05 (120.12)	13.67 (184.80)	20.1 (448.63)	10.89 (112.24)
SE_d	0.50	1.30	0.82	0.43	0.80	0.67
CD (P < 0.05)	1.05	2.74	1.72	0.90	1.68	1.40
^a DAT = days after transplanting
^bParentheses are original values must need for differentiating the original and transformed values; HW^c = Hand weeding

Weed Dry Weight

Weed dry weight is the most important parameter to assess the weed competitiveness for crop growth and productivity (Pannacci et al. 2020). Sparse weeds with high biomass might be more competitive for crops than dense weeds with the lesser dry matter. Considerable reduction in weed dry weight was recorded with the application of Quizalofop ethyl at 100 g a.i. ha^− 1 at all the stages of observation (Table 2). Maximum dry weed biomass was noticed in the weedy check, where weeds were not controlled. Weed control efficiency, which indicates the comparative magnitude of reduction in weed dry matter, was highly influenced by different weed control treatments. The application of Quizalofop ethyl was evident even after 60 days after transplanting as shown by the minimum weed count, weed dry matter production, and the maximum weed control efficiency. Throughout the experimental period, weed control efficiency was higher with the post-emergence application of Quizalofop ethyl at 100 g a.i. ha^− 1 followed by Quizalofop ethyl spray at 50 g a.i. ha^− 1; since it registered lesser weed density and weed dry weight.

Table 2

Total weed dry weight and weed control efficiency as influenced by different weed management practices in onion.
Treatments	Season-I				Season-II
	Total weed dry weight (kg ha^− 1)		WCE^d (%)		Total weed dry weight (kg ha^− 1)		WCE (%)
	Total weed dry weight (kg ha^− 1)		40 DAT	60 DAT	Total weed dry weight (kg ha^− 1)		40 DAT	60 DAT
T₁: 50 g a.i. ha^− 1	5.60 (26.48)^b	10.69 (133.35)	88.44	83.04	5.98 (32.87)	12.10 (140.87)	85.47	83.10
T₂: 100 g a.i. ha^− 1	3.81 (12.49)	9.49 (87.93)	95.13	88.53	3.87 (11.94)	9.24 (83.13)	94.83	90.10
T₃: HW^c (twice)	15.24 (210.30)	12.94 (141.74)	-	84.25	14.44 (207.69)	13.58 (131.87)	-	84.15
T₄: Unweeded control	16.03 (253.65)	29.05 (841.23)	-	-	15.88 (248.96)	28.89 (831.34)	-	-
SE_d	0.43	0.78	-	-	0.59	0.77	-	-
CD (P < 0.05)	0.88	1.66	-	-	1.2	1.62	-	-
^a DAT = days after transplanting
^bParentheses are original values must need for differentiating the original and transformed values; HW^c = Hand weeding; WCE^d = Weed control efficiency

Phytotoxicity Effect

The new formulation showed no phytotoxicity symptoms like bleaching, leaf tip burn, leaf curling and also stunting of growth in onion.

Effect on Crop Yields

Weeds are one of the biggest problems in onions that not only reduce the yield and quality, but utilize essential nutrients also. During both the years of study, among the weed control treatments, post-emergence application of Quizalofop ethyl at 100 g ha^− 1 recorded a higher bulb yield of 13760 and 13,830 kg ha^− 1 due to better control of weeds at critical stages thus providing a favorable environment for better growth and development leading to enhanced bulb yield (Table 3). Onion productivity is mainly decided by the weed control efficiency of weed management methods. The post-emergence herbicides offer the most practical, effective, and economical method of weed control for increasing bulb yield of onion. Bulb yield reduction in onion is directly related to increasing weed density, dry weight, and intensity of weed interference throughout the crop period (Ikeda et al. 2019).

Table 3

Effects of weed management methods on bulb weight (g), yield kgha^− 1) and weed index (%) of onion
Treatment	Season-I			Season-II
Treatment	Bulb weight (g)	Bulb yield (kgha^− 1)	Weed index (%)	Bulb Weight (g)	Bulb yield (kgha^− 1)	Weed index (%)
T₁: 50 g a.i. ha^− 1	35.87	12540	22.34	35.19	12680	18.95
T₂: 100 g a.i. ha^− 1	38.68	13760	19.01	36.20	13890	11.44
T₃: HW^c (twice)	37.82	12870	10.15	39.44	14690	5.97
T₄: Unweeded control	13.84	6180	61.63	18.41	6841	56.19
SE_d	0.88	689	-	0.93	713	-
CD (P = 0.05)	1.89	1383	-	1.91	1434	-
^a DAT = days after transplanting
HW^b = Hand weeding

Method Validation

Quizalofop ethyl showed a linear response over the concentration range (0.04-1.0 mg L^-1) tested (Fig. 2). The selectivity of the method was evaluated by comparing the chromatograms obtained from fortified samples with blank samples. In the chromatogram obtained for the blank sample, no peaks were recorded at the retention times of the quizalofop ethyl. Moreover, the chromatograms of the fortified sample extracts presented a satisfactory chromatographic resolution (Fig. 3).

MS/MS method was applied to study the optimal two ion transitions (primary and secondary transitions of a precursor to product ions) from which the identification of target fungicide in samples was confirmed through optimization of instrument acquisition parameters (Table 4). LOQ values obtained were 0.04 mg L^− 1 for the fops' herbicide. Thus, MRL required by the European and international regulations for the selected compound could have complied without any difficulty.

Table 4

MS-MS transitions for Quizalofop ethyl analysis
Substance	RT (min)	Ion transitions (m/z), Positive mode		Energy and Potential (V)
Substance	RT (min)	Ion transitions (m/z), Positive mode		DP	EP	CEP	CE	CXP
Quizalofop-p-ethyl	4.3014	Primary (373 > 299)	Secondary (375 > 300.9)	54	5	19	26/49	7/4
Abbreviations: DP = Declustering potential; EP = Entrance potential; CEP = Cell entrance potential; CE = Collision energy; CXP = Cell exit potential

Once the extraction procedure had been optimized, accuracy-recovery and precision of the method were assessed. The lowest concentrations were selected at or below the MRLs. Recovery values of quizalofop ethyl range from 94.4–99.9% and RSD values range from 0.1–2.7% (Table 5). Results indicate that the developed method has good accuracy for the estimation of quizalofop ethyl.

Table 5

Accuracy data of the validated method for Quizalofop ethyl standard
Added quantity (µgmL^− 1)	Recovery (% ± SD^a)	RSD^b (%)
0.04	94.37 ± 0.0025	2.73
0.20	99.96 ± 0.6971	1.2
0.40	99.98 ± 0.1550	0.15
^aSD = Standard deviation
^bRSD = Relative standard deviation

The precision of the method was determined by repeatability and reproducibility studies, expressed by RSD. The RSD values at each level were below 3% for quizalofop ethyl indicating good intraday precision (Table 6). The intermediate precision is the inter-day variation at the same concentration levels determined on two different days. The RSD values at each level were < 3.5% for quizalofop ethyl indicating good intermediate precision. The method was found to be precise (RSDr and RSDR < 20%) at all the spiking levels (Table 7). The overall recovery percent in onion substrates was high, which proves the efficiency of the residue methodology.

Table 6

Precision data of validated method for Quizalofop ethyl standard
Added quantity (µg mL^− 1)	Intraday precision, RSD%		Inter-day precision, RSD % (n = 6)
Added quantity (µg mL^− 1)	Day 1	Day 2	Inter-day precision, RSD % (n = 6)
0.04	2.73	2.53	3.13
0.20	1.20	1.08	2.11
0.40	2.38	1.30	1.72

Table 7

Recovery indicating level and stage of fortification of Quizalofop Ethyl
Sample	Fortification level (µg g^− 1)	Mean recovery (%)^a	Standard deviation	Coefficient of variance (%)
Onion leaf	0.04	88.67	0.003	7.92
	0.20	89.33	0.015	6.84
	0.40	89.33	0.038	8.48
Onion immature bulb	0.04	88.69	0.002	5.68
	0.20	92.00	0.010	4.35
	0.40	88.00	0.026	6.01
Onion mature bulb	0.04	88. 76	0.003	7.25
	0.20	93.33	0.015	6.55
	0.40	90.00	0.026	5.88
Soil	0.04	87.33	0.002	4.77
	0.20	86. 75	0.016	7.05
	0.40	88.67	0.015	3.45
^aMean value of 3 replicates.

Persistence Pattern

In the experiment, two doses of the quizalofop ethyl were tested for their persistence in the soil, rhizome, and plant. The developed method was applied for the analysis of different onion samples collected from the experimental fields. The initial detected amount (IDA) (2 hr. after the last spray) was found to be 2.0-4.4 mg kg^− 1 irrespective of treatments and seasons. For quizalofop-p-ethyl residues, the transition at m/z 373.0→299.0 was monitored in positive mode for primary quantification; the transition at m/z 375.0→300.9 for confirmation (Table 4 & Fig. 4). Dissipation patterns of quizalofop ethyl in onion when applied at different rates on the onion field were presented in Fig. 5. The quizalofop ethyl exhibited a faster dissipation rate and reached below its quantification limit (0.04 mg kg^− 1) in onion, soil rhizome 10 days after treatment.

Different curves of fit were tested to predict the dissipation behavior of the herbicide. Among the models tested (linear, polynomial, logarithmic, and exponential), the exponential model was found to give a better fit for field dissipation of the quizalofop ethyl at 50 and 100 g a.i. ha^− 1 doses (Fig. 6). Mathematically, quizalofop ethyl dissipation followed a first-order (pseudo-first-order) decay process and residue found in green onion leaves below the quantifiable level of 0.04 mg kg^− 1 on the 10th and 15th day during the season I and II respectively.

Using the exponential equations, half-life (DT₅₀) i.e. time taken, in days, for 50% dissipation of initial residues) was worked out (Table 8). The field dissipation from onion green leaves at doses above the recommended rate of application (50 g a.i. ha^− 1) and double the recommended rate indicated that, the half-life (DT₅₀) of quizalofop ethyl in onion was 1.5 − 1.7 days, irrespective of seasons which is a similar half-life (0.6 and 2.9 days) of the herbicide with the earlier report (Mantzos et al. 2017).

Table 8

Residues of Quizalofop ethyl (µg∙g^-1) in onion collected at intervals after application of herbicides
Treatment	Season	^aResidues of Quizalofop ethyl at different days after application								Regression equation	Half- life (days)
Treatment	Season	0	1	3	5	7	10	15	Harvest	Regression equation	Half- life (days)
T₁: 50 g a.i. ha^− 1	I	2.46	1.35	0.69	0.29	0.08	BQL^b	BQL	BQL	Y = 3.39–0.18X	1.67
T₁: 50 g a.i. ha^− 1	II	2.00	0.91	0.45	0.16	0.07	BQL	BQL	BQL	Y = 3.24–0.20X	1.41
T₁: 100 g a.i. ha^− 1	I	4.41	2.64	1.01	0.43	0.16	0.06	BQL	BQL	Y = 3.24–0.20X	1.81
T₁: 100 g a.i. ha^− 1	II	3.37	1.69	0.50	0.26	0.12	0.06	BQL	BQL	Y = 3.39–0.18X	1.51
Control	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	-	-
Hand weeding	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	BQL	-	-
^aMean value of three replicates;
^b BQL = Below Quantifiable Limit (0.05 µg g^− 1).

Both immature and mature onion bulbs were analyzed for quizalofop residues and no residues were estimated at harvest irrespective of doses and seasons. No residue of quizalofop ethyl was detected in untreated control (T₀) samples also. Residues of the tested herbicide could not be detected in soil samples at the harvest stage of the crop in any of the doses applied. The quizalofop ethyl residues in soil were ranged from 0.012 to 0.038 mg kg^− 1 at harvest at the earlier study (Janaki et al. 2018). Zhou et. al. (2018) stated that micro-organisms, especially fungi, were a critical factor that influenced quizalofop depletion in soils and could accelerate greatly the degradation in soil.^[28]

Dietary Risk Exposure Assessment

The residue dissipation data are used for calculating the risk assessment of quizalofop ethyl applied in onion grown under open field conditions. The TMRC of quizalofop ethyl from onion from both Seasons I (0.088 mg person^− 1 day^− 1) and Season II (0.071 mg person^− 1 day^− 1) is much lower than Maximum Permissible Intake (MPI) of quizalofop ethyl (0.58 mg person^− 1 day^− 1), which indicates that the application of quizalofop ethyl at the recommended dose is safe for human consumption and the finding is complying with the guidelines of World Health Organization (WHO 2004). Thus, residue data generated from multi-seasonal supervised field trials coupled with safety risk assessment may be helpful in the fixation of MRL from a national context of the view. Moreover, the calculated dietary risk quotient (RQ) based on the highest residue concentration obtained in the treated dose reflected less than one from 0 (2 hr. after) days after the application of quizalofop ethyl, indicating onion collected from the field were safe for consumption. Since no residues were detected irrespective of doses in any harvested samples, the risk associated with dietary exposure of quizalofop ethyl in onion is considered safe for the human being.

From the present investigation, it could be concluded that the post-emergence application of quizalofop p-ethyl (5% EC) at 100 g a.i. ha^− 1 kept the weed density and dry weight reasonably at a lower level and enhanced the productivity of onion with higher economic returns. In this work, a simple QuEChERS based HPLC method has also been established and validated for the determination of terminal residues of quizalofop ethyl and its dissipation kinetics. It also can be stated that quizalofop ethyl did not adversely influence soil at recommended rates of application. Onion is safe for consumption at harvest upon the application of quizalofop ethyl 5% EC at the recommended dose (50 g a.i. ha^-1), considering an LOQ value of 0.04 mg kg^-1.

a.i.	Active Ingredient
ADI	Acceptable Daily Intake
CE CEP CXP	Collision Energy Cell Entrance Potential Cell Exit Potential
DP EP ESI	Declustering Potential Entrance potential Electrospray Ionization
GC-MS	Gas Chromatography – Mass Spectrometry
HPLC	High Performance Liquid Chromatography
LC-MS	Liquid Chromatography – Mass Spectrometry
LOD	Limit of Detection
LOQ	Limit of Quantification
MPI	Maximum Permissible Intake
MRL	Maximum Residue Limit
PSA	Primary Secondary Amine
RT	Retention Time
UPLC UV	Ultra-Performance Liquid Chromatography Ultraviolet

Ethical Approval

Not applicable.

Competing Interests

No potential conflict of interest was reported by the authors.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' Contributions

This work was carried out in collaboration among all authors. DKH, RK and PM designed the study and wrote the protocol. DKH, RK, SM and SB managed the analysis of the study. AP, SR, TB and HB made data interpretation. DKH and PM were major contributors in writing the manuscript. All authors thoroughly read and approved the final manuscript.

Funding

The authors thank the administrative authority of M/S Dhanuka Agritech Limited, New Delhi, for providing financial support to conduct the work on residue analysis.

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Determination of Quizalofop-p-ethyl in onion spices: Weed control efficiency, residual profile, and food safety assessment under field conditions

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Chemicals and Reagents

Apparatus

Preparation of Standard Solutions

Sample Preparation

Method Validation

Optimization of Chromatographic and Spectrometric Conditions

Calibration and Linearity

Selectivity and Sensitivity

Accuracy and Precision

Field Experiment

Sample Collection

Observations on Weeds

Weed Density

Weed Dry Weight

Weed Control Efficiency

Weed Index

Results And Discussions

Weed Control Efficiency

Weed Density

Weed Dry Weight

Phytotoxicity Effect

Effect on Crop Yields

Method Validation

Persistence Pattern

Dietary Risk Exposure Assessment

Conclusion

Abbreviations

Declarations

References

Status:

Version 1