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Residues, dissipation kinetics, and dietary intake risk assessment of two fungicides in grape and soil
Accepted Manuscript Residues, dissipation kinetics, and dietary intake risk assessment of two fungicides in grape and soil Shouyi Wang, Qingtao Zhang, Yurong Yu, Ya Chen, Song Zeng, Ping Lu, Deyu Hu PII:
S0273-2300(18)30280-0
DOI:
https://doi.org/10.1016/j.yrtph.2018.10.015
Reference:
YRTPH 4245
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 22 May 2018 Revised Date:
3 October 2018
Accepted Date: 20 October 2018
Please cite this article as: Wang, S., Zhang, Q., Yu, Y., Chen, Y., Zeng, S., Lu, P., Hu, D., Residues, dissipation kinetics, and dietary intake risk assessment of two fungicides in grape and soil, Regulatory Toxicology and Pharmacology (2018), doi: https://doi.org/10.1016/j.yrtph.2018.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Residues, dissipation kinetics, and dietary intake risk assessment of
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two fungicides in grape and soil
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Shouyi Wang a, Qingtao Zhang b, Yurong Yu b, Ya Chen b, Song Zeng b, Ping Lu
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Deyu Hu a *
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a
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Education
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b
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Guiyang 550025, P.R. China
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*Address correspondence to Deyu Hu and Ping Lu, Key Laboratory of Green
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Pesticide and Agricultural Bioengineering, Ministry of Education, Center for
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Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025,
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P.R. China. Tel.: +86 851 88292170; Fax: +86 851 88292170; E-mail:
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[email protected]; [email protected].
a*
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Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
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Center for Research and Development of Fine Chemicals, Guizhou University,
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ABSTRACT The residue behavior and dietary intake risk of two fungicides (dimethomorph
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and pyraclostrobin) in grape (Vitis vinifera L.) were investigated from field trials. A
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modified quick, easy, cheap, effective, rugged, and safe method for simultaneously
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determining dimethomorph and pyraclostrobin residues in grape and soil was
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established
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spectrometry. The average recoveries of dimethomorph and pyraclostrobin in the
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grape and soil matrices varied from 76.88% to 97.05%, with relative standard
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deviations of 1.73% to 10.38%. The degradation half-lives of dimethomorph and
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pyraclostrobin were 7.3–12.0 days and 3.6–7.0 days in grape and soil, respectively.
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The terminal residues of dimethomorph and pyraclostrobin in the two matrices were
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0.05–0.87 mg/kg. For dietary exposure risk assessments, all of the hazard quotient
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and hazard quotient index values were below 100%, which indicated that the
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suspending agents of dimethomorph and pyraclostrobin were sprayed on grape at the
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recommended dosages with no significant potential risks for Chinese consumers.
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This study provides a reference for analytically evaluating residual degradation
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behavior and dietary intake risk of two fungicides under field conditions.
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Keywords: dimethomorph; pyraclostrobin; residue dynamics;
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assessment; grape.
high
performance
liquid
chromatography–tandem
mass
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2
dietary risk
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1 Introduction Grape (Vitis vinifera L.), belonging to the Vitaceae family, is one of the most
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commonly consumed fruits and an economically important crop globally. (Hassan,
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2012; Saad, 2017). It is highly nutritious, with its fruit containing components such
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as sugar, minerals, vitamins, amino acids, and organic acids (Conde et al., 2007).
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However, grape is prone to fungal diseases, such as downy mildew, gray mold, and
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powdery mildew during growth. The use of fungicides is necessary to control these
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diseases for improving grape quality and increasing its output (Zhang et al., 2017).
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Dimethomorph is a highly effective and low-toxicity systemic fungicide, which has
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demonstrated good curative and antisporulant activity, particularly against downy
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mildew (Cohen et al., 1995; Hengel and Shibamoto, 2000; Kim et al., 2015).
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Dimethomorph has been widely used to prevent many diseases of vegetables and
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fruits in China, such as downy mildew, late blight, crown, blue mold, and root rot
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(Yang et al., 2017). Pyraclostrobin is a broad-spectrum fungicide of the strobilurin
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family, which can effectively control diseases of blight, anthracnose, and fusarium
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head blight in cereals, vegetables, fruits, and oil seeds (Reddy et al., 2013).
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Pyraclostrobin is produced by BASF Corporation (Joshi et al., 2014); it transfers
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electrons between cytochromes b and c1 to inhibit mitochondrial respiration in the
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target fungus (Zhang et al., 2017). Mixed formulations of fungicides can more
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effectively control diseases and protect crop growth, decrease the application dose
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required, and reduce the risk of fungal resistance compared with a single fungicide
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(Wu et al., 2018). In this context, 45% suspending agent is most commonly used for
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has remarkable curative effects on bacterial and fungal diseases in grape. However,
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the use of these fungicides may have negative effects on the environment and human
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health. Consequently, it is important to monitor the residues of dimethomorph and
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pyraclostrobin in grape.
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In recent years, several methods for residue analysis of these two compounds
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from variety of matrixes are reported. Regarding some analytical methods of
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dimethomorph in tomato, cucumber, onion, apple, kiwi, orange, pear, pepper,
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Chinese cabbage, cauliflower and Swiss chard were introduced, most of which use
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gas chromatography with tandem mass spectrometry (GC-MS/MS), high
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performance liquid chromatography with ultraviolet visible detector (HPLC-UV),
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and liquid chromatography with tandem mass spectrometry (LC-MS/MS)
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(Walorczyk, 2013; Qi et al., 2015; Kim et al., 2015; Kocourek et al., 2017; Kabir et
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al., 2018a). Some studies for determination of Pyraclostrobin in different crops
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(green bean, spring onion, blueberry, sugarcane, winter jujube, banana, paddy, and
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apple) have been conducted, which include the use of GC-ECD/NPD (gas
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chromatography equipped with electron capture detector/nitrogen and phosphorus
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detector) (Munitz et al., 2014; Sadło et al., 2017), HPLC-UV (Fulcher et al., 2014;
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Fu et al., 2016), HPLC-ESI-MS (high performance liquid chromatography
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electrospray ionization source-mass spectrometry) (Hanafi et al., 2010), LC-MS/MS
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(Peng et al., 2014), UPLC-MS/MS (ultra- performance liquid chromatography with
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tandem mass spectrometry) (Guo et al., 2016). In China, the maximum residue limits
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(GB 2763-2016: MRLs for pesticides in food), whereas values of 3 and 1 mg/kg
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were set by the European Union (EU Pesticide Database, 2017), respectively.
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Chinese regulatory authorities and researchers have realized the necessity of such
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studies, not only for monitoring pesticide residues, but also for assessing the dietary
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exposure risk among consumers (Fang et al., 2015). Although many previous studies
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have reported dimethomorph and pyraclostrobin residues in different crops, to the
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best of our knowledge, there have been no systematic studies on the dissipation,
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residues, and dietary exposure risk assessment of dimethomorph and pyraclostrobin
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in grape.
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In the present study, a modified quick, easy, cheap, effective, rugged, and safe
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(QuEChERS) method was established to simultaneously determine dimethomorph
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and pyraclostrobin residues in grape and soil by LC-MS/MS. Field trials were
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conducted to investigate the dissipation rate and terminal residues of the mixed
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formulation (dimethomorph and pyraclostrobin) in grape, then, the dietary intake
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risk was evaluated through dietary exposure assessment based on the residue, food
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consumption, and toxicology data. Our study aimed to ensure the scientific
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application of this mixed formulation in grape and to provide residue data useful for
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the risk assessment of its presence in the diet on human health, offering guidance for
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the rational and safe use of this mixed fungicide.
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2 Materials and methods
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2.1 Materials and reagents
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Dimethomorph standards (99.0%) and pyraclostrobin standards (99.0%) were 5
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containing 30% dimethomorph and 15% pyraclostrobin was supplied by Hangzhou
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Yulong Chemical Co., Ltd (Hangzhou, China). LC-grade methanol (99.9%) was
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purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Analytical grade
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acetonitrile, dichloromethane, petroleum ether, acetone, sodium chloride, and
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anhydrous magnesium sulfate were obtained from Chengdu Jinshan Chemical
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Reagent Co. (Chengdu, China). C18 (50 µm) and primary secondary amine (PSA)
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(40-60 µm) sorbents were purchased from Agela Technologies Inc. (Tianjin, China).
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Distilled water was purchased from Watsons Corporation (Dongguan, China).
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Individual stock standard solutions of dimethomorph (200 µg/mL) and
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pyraclostrobin (200 µg/mL) were prepared in methanol and stored in volumetric
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flasks at −20°C. Mixed standard solutions of dimethomorph and pyraclostrobin were
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prepared by diluting stock solutions with methanol to the concentration range of
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0.01–2 µg/mL. A matrix-matched standard calibration method was prepared during
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the analytical procedure by adding the appropriate volumes of mixed standard
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solution in blank grape and soil extracts to eliminate matrix effects. All standard
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solutions were kept in the fridge at 4°C before use.
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2.2 Field experiment design
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According to the pesticide registration information and Guidelines on Pesticide
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Residue Trials (NY/T 788-2004) issued by the Ministry of Agriculture of the
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People’s Republic of China, field trials were conducted in Huishui (Guizhou
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Province) and Xiaoxian (Anhui Province) during the 2016 agricultural season (June 6
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Guizhou were 17°C (Anhui) and 14.4°C (Guizhou), the average annual durations of
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sunshine were 1400 h (Anhui) and 2350 (Guizhou) h, and the mean annual
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precipitation were 1200 mm (Anhui) and 1300 mm (Guizhou), respectively. The soil
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type in Huisui is loess with 6%–10% organic matter, pH 5.0–6.5, and the soil type in
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Xiaoxain is silt loam with 1.14% organic matter, pH 7.0–7.4. The treatments
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comprised grape dynamic test treatment, four terminal residual test treatments, and
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one control plot. The plot of each experimental treatment was 30 m2, with plots
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established in triplicate. To avoid cross-contamination, each plot was separated by a
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buffer zone.
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In the degradation dynamics experiment, the mixed formulation of
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dimethomorph and pyraclostrobin was dissolved in water and sprayed once when
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the grape fruits are about half the size of mature fruit, the dosage was 800 grams of
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active ingredient per hectare (g.a.i./ha). Grape samples of approximately 1 kg and
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soil samples of 1 kg (0–10 cm in depth) were collected randomly from each plot at
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2 h and 1, 2, 3, 5, 7, 10, 14, 21, and 28 days after spraying treatment. For the
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terminal residue experiment, the mixed formulation of dimethomorph and
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pyraclostrobin was sprayed on grapes at two dosage levels: 800 g.a.i./ha (low
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dosage, recommended dosage) and 1000 g.a.i./ha (high dosage). Both low and high
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dosages were sprayed three and four times, respectively. Samples were collected
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randomly after 7, 14, and 21 days of spraying treatment. All samples were placed in
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polyethylene bags and transported back to the laboratory.
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2.3 Analytical procedures
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2.3.1 Sample preparation The samples from the field trials were homogenized and divided into four equal
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portions by quartering technique; two portions (200 g, each) were used for the
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subsequent experiments. All samples were stored at −20°C before analysis.
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2.3.2 Sample extraction and purification
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A total of 5.0 g of grape samples or 10.0 g of soil samples was weighed and
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placed into a 50-mL polypropylene centrifuge tube, to which 20 mL of acetonitrile
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was added. The mixture was ultrasonically extracted for 15 min, followed by the
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addition of 3.0 g of anhydrous MgSO4 and 2.0 g of anhydrous Na2SO4. Next, the
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samples were mixed for 2 min at a speed of 2500 rpm and then centrifuged at a
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speed of 6000 rpm for 5 min. Subsequently, 1.0 mL of supernatant solution was
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accurately extracted to the pipette and transferred into a 2-mL centrifuge tube
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containing 50 mg of C18 and 50 mg of PSA. The tube was centrifuged at 1200 rpm
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for 3 min after vortexing for 1 min. Finally, the supernatant was filtered through a
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0.22-µm nylon syringe filter before analysis by LC-MS/MS.
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2.3.3 LC-MS/MS analysis
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Dimethomorph and pyraclostrobin were separated on a 20 AD-XR liquid
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chromatography (LC) system, with a Phenomenex Luna C18 column (150 mm × 2
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mm id, film thinness 5 µm; Phenomenex, CA, USA). The mobile phases contained
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methanol (A) and 0.1% formic acid aqueous solution (B). The gradient elution
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procedure was as follows: 95% B (0–0.4 min), 95% B (0.4–2.0 min), 40% B (2.0– 8
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min). The flow rate of the mobile phase was set at 0.3 mL/min, and the injection
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volume was 1 µL. The chromatographic column temperature was set at 40°C. An
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applied biosystems sciex API 4000Q trap quadrupole mass spectrometer equipped
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with an iron source turbo spray unit was used for the monitoring and quantitative
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analysis of dimethomorph and pyraclostrobin. The analysis of the two fungicides
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was performed in positive mode using multiple reaction monitoring (MRM).
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Nitrogen (99.999% purity) was used as the curtain, nebulizer, and collision gases;
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the pressures of the ion source gas (gases 1 and 2) and curtain gas were 413.68 kPa
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and 206.84 kPa, respectively; ion spray voltage was 5.5 kV; and ion source
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temperature was 600°C. The ion pair parameters of the two fungicides are presented
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in Table 1.
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Table 1 near here
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2.4 Calculations
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2.4.1 Method validation
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The performance of method was validated using certain parameters, including
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linearity, accuracy, precision, matrix effects, limit of detection (LOD) and limit of
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quantitation (LOQ) (Li et al., 2012; Kabir et al., 2018b). Linearity was evaluated
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using the correlation coefficient (R2), derived mainly from a six-point calibration
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curve; the calibration curves were obtained by plotting the peak area against the
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corresponding concentration of target analytes in pure solvent and matrix (Rahman
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et al., 2015). The matrix effect (ME) was calculated using the following formula:
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ME =
where m1 and m2 are the slopes of the calibration curves obtained in matrix and pure
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solvent. ME = 0 represents no ME, ME > 0 represents signal enhancement, whereas
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ME < 0 represents signal suppression. LOD and LOQ are defined by signal-to-noise
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ratios of 3 and 10, respectively. In recovery experiments, different concentrations of
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spiked samples for dimethomorph and pyraclostrobin (0.04, 0.4 and 4 mg/kg in
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grape and 0.02, 0.2, and 2 mg/kg in soil) were investigated. The precision and
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accuracy of the analytical method were evaluated by calculating recovery and
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relative standard deviation (RSD) for intra-day (five replicates) and inter-day (fifteen
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replicates in consecutive three days) (Li et al., 2015; Wang and Zhang, 2017).
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2.4.2 Degradation kinetics
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First-order kinetics equation was used to evaluate degradation of dimethomorph
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and pyraclostrobin over time in grape and soil, the specific calculation formula is as
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follows:
Ct = C0 e-kt t1/2 =
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ln 2 k
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Where C0 (mg/kg) denotes the initial concentration of the compound, k (day−1)
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denotes the rate constant of degradation, Ct (mg/kg) denotes the concentration of the
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compound at time t (day), and t1/2 denotes the half-life of compound degradation
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(Zhu et al., 2016; Song et al., 2018).
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2.4.3 Assessment of dietary exposure
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The chronic and acute dietary exposure risk quotients of dimethomorph and 10
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pyraclostrobin were calculated to comprehensively assess the risk of intake of the
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mixed formulation sprayed on grapes. The chronic assessment was performed using
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the following formulas: STMR × Fi bw EDI HQ = × 100% ADI
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Where STRM (mg/kg) denotes the supervised trials median residue, Fi denotes the
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food consumption data (g/day), bw denotes mean body weight (kg), EDI denotes the
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estimated daily intake (mg/kg·bw), ADI denotes the acceptable daily intake
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(mg/kg·bw), and HQ denotes the hazard quotient, (Lozowicka et al., 2014; Gad Alla
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et al., 2015)
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The acute assessment was performed using the following formulas: LP×HR bw ESTI αHI = × 100% ARfD
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Where, according to the Principles and Methods for the Risk Assessment of
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Chemicals
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(FAO) and the World Health Organization (WHO), LP denotes the large portion
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consumption for the commodity,
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percentile of food consumption derived from records of individual consumer days
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(i.e. survey days on which the food or foods of interest were consumed); HR is the
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highest residue in composite sample of edible portion found in the supervised trials
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used for estimating the maximum residue level (FAO/WHO, 2009). ESTI denotes
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the estimated short-term intake (mg/kg·bw), ARfD denotes the acute reference dose
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and the values have been based on the 97.5th
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indicates an acceptable risk for consumers, whereas a situation where HQ or αHI is >
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100% indicates that an unacceptable risk for consumers is posed (Fang, et al., 2015;
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Lin et al., 2017).
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3 Results and discussions
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3.1 Method validation Representative
LC-MS/MS
chromatograms
of
dimethomorph
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and
pyraclostrobin in standard solutions and spiked grape and soil samples are shown in
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Figure 1. The linearity of the method was evaluated using the standard calibration
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curves of dimethomorph and pyraclostrobin with a concentration range of 0.01–2
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µg/mL. As shown in Table 2, superior linearity was observed for two compounds (R2
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values of 0.9994–0.9999), and signal suppression for dimethomorph was found in
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grape and soil due to ME (−16.67) values < 0. Hence, calibration was implemented
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using external matrix-matched standards to eliminate the matrix effect in this study.
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The LOQ of dimethomorph and pyraclostrobin in grape were 0.04 mg/kg while the
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LOD were 0.012 mg/kg; the LOQ of the two fungicides in soil were 0.02 mg/kg
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while the LOD were 0.006 mg/kg. The recoveries and RSD of target analytes are
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rendered in Table 3. Average recoveries of dimethomorph in grape and soil were
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77.96%–97.05% (intra-day) and 82.93%–96.51% (inter-day), whereas intra-day and
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inter-day RSD were in the range of 1.73% to 10.38%. For pyraclostrobin, the
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recoveries in the two matrices were 76.88%–92.71% (intra-day) and 80.52%–89.22%
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(inter-day), with corresponding RSD between 2.57% and 8.36%. The satisfactory
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precision, so it is appropriate for analysis to detect dimethomorph and pyraclostrobin
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in grape and soil.
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Figure 1 near here
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Table 2 near here
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Table 3 near here
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3.2 Degradation of dimethomorph and pyraclostrobin in grape and soil
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According to the results of the monitoring analysis, the degradation curves of
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dimethomorph and pyraclostrobin in grape and soil from Anhui and Guizhou were
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plotted and are shown in Figure 2. The original residues of dimethomorph in grape at
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Anhui and Guizhou were 2.44 and 2.39 mg/kg after 2 h of spraying. Meanwhile, the
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equivalent values of residues were 0.41 and 0.68 mg/kg in soil, respectively. The
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degradation dynamics equations of dimethomorph were Ct = 1.7772 e−0.083t (Anhui)
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and Ct = 2.0592 e−0.095t (Guizhou) in grape, with half-lives (t1/2) of 8.4 and 7.3 days;
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the values were close to those obtained in previous studies (9.4 days; Liu et al.,
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2012). The degradation dynamics equations of dimethomorph in soil were Ct = 0.376
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e−0.06t (Anhui) and Ct = 0.619 e−0.058t (Guizhou), with t1/2 of 11.6 and 12.0 days. For
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pyraclostrobin, the original concentrations were 0.44 and 0.62 mg/kg in grape and
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soil from Anhui, and 0.47 and 0.41 mg/kg for Guizhou, respectively. The
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degradation dynamics equations of pyraclostrobin were Ct = 0.3868 e−0.099t (Anhui)
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and Ct = 0.3539 e−0.192t (Guizhou) in soil, and t1/2 values were 7.0 and 3.6 days,
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respectively. In contrast, the degradation dynamics equations of pyraclostrobin in
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values of 4.7 and 4.9 days. These t1/2 values were less than some presented in other
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reports, where the t1/2 values of pyraclostrobin were 5.5–8.0 days in blueberries
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(Munitz et al., 2014), 8.3–9.1 days in bananas (Fu et al., 2016), and 6.4–9.3 days in
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pepper (Wu et al., 2018). The degradation rate of dimethomorph in grape at 14 days
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after spraying was > 78% and that at 28 days after spraying was > 80% in soil.
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Meanwhile, the degradation rate of pyraclostrobin at 10 days after spraying was > 85%
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in the two matrices. The degradation rate and t1/2 values illustrate that pyraclostrobin
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dissipates faster than dimethomorph in grape and soil.
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Figure 2 near here
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3.3 Terminal residues of dimethomorph and pyraclostrobin in grape and soil
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The terminal residue results of the two fungicides in grapes and soil are
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presented in Table 4. The terminal residues of dimethomorph were 0.09–0.87 and
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0.05–0.19 mg/kg in grape and soil, respectively, at 7, 14, and 21 days after spraying
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for the two dosage levels. Meanwhile, the terminal residues of pyraclostrobin were
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0.05–0.32 and 0.05–0.87 mg/kg in grape and soil, respectively. It turned out that the
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residual levels of dimethomorph and pyraclostrobin in grape were below 3 and 1
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mg/kg (MRLs set by the EU), respectively.
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Table 4 near here
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3.4 Exposure risk assessment
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In this study, acute and chronic dietary exposure risk assessments for
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dimethomorph and pyraclostrobin in grape were performed on the basis of 14
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monitoring of pesticide residues and toxicity data for these two fungicides as well as
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data on grape consumption in China. The results of acute dietary exposure risk assessments are summarized in Table
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5. The HR values of dimethomorph and pyraclostrobin in grapes were 0.87 and 0.32
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mg/kg from the terminal residue experiments. The Chinese adult mean body weight
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is 63 kg (Lin et al., 2017). According to the World Health Organization, LP of grape
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is 570.3 g/d for Chinese residents (WHO, 2015). The ARfD values for
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dimethomorph and pyraclostrobin are 0.6 and 0.05 mg/kg·bw, respectively, as
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derived from the Joint Meeting on Pesticide Residues (JMPR) reports (FAO, 2007;
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FAO, 2003). All of the calculated ESTI values of dimethomorph and pyraclostrobin
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were far less than the corresponding ARfD values; the αHI values were < 10%,
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which means that the acute dietary exposure risk of dimethomorph and
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pyraclostrobin among humans is acceptable.
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Table 5 near here
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The chronic dietary exposure risk assessments for dimethomorph and
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pyraclostrobin in grape were performed on the basis of the consumption of fruit
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among Chinese from different age groups. The intakes of fruit and mean weight of
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different age groups are shown in Table 6 (Jin, 2008). The ADI values for
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dimethomorph and pyraclostrobin are 0.2 and 0.03 mg/kg·bw, as derived from the
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JMPR reports (FAO, 2007; FAO, 2003). The STRM values of dimethomorph and
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pyraclostrobin in grape were 0.34 and 0.10 mg/kg, respectively. As seen in Table 6,
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dimethomorph and pyraclostrobin have a higher chronic dietary exposure risk in
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adults. In addition, females were more sensitive to dimethomorph and pyraclostrobin
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than males within the same age group because the HQ values of females were higher
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than those of males. However, all of the EDI values were far below the ADI values,
317
and the HQ values ranged from 0.07% to 1.20% (< 10%), which implies that the
318
chronic dietary exposure risk of dimethomorph and pyraclostrobin among humans is
319
also acceptable. For dimethomorph and pyraclostrobin in grape, smaller HQ and αHI
320
values demonstrated that there is no significant potential risk for Chinese residents
321
according to the recommended application guide.
322
Table 6 near here
323
4 Conclusions
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In this study, the validated QuEChERS and LC-MS/MS analytical method was
325
developed and applied to simultaneously determine the levels of dimethomorph and
326
pyraclostrobin residues in grape and soil, achieved satisfactory precision and
327
accuracy. Subsequently, the degradation dynamics and terminal residues of
328
dimethomorph and pyraclostrobin in grape and soil were studied under field
329
conditions. The results show that the t1/2 values of dimethomorph and pyraclostrobin
330
were 7.3–12.0 days and 3.6–7.0 days in the two matrices, and pyraclostrobin
331
degraded more rapidly than dimethomorph. The levels of terminal residues for
332
dimethomorph and pyraclostrobin in all matrices were below the MRLs values set by
333
the EU (3 and 1 mg/kg, respectively). Based on the terminal residue results, dietary
334
exposure risk assessments for dimethomorph and pyraclostrobin in grape were
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ACCEPTED MANUSCRIPT performed. All of the HQ and αHI values were < 100%, which indicates no
336
significant potential risks of dimethomorph and pyraclostrobin in grape at the
337
recommended dosages. Our study could provide a valuable reference for the safe and
338
reasonable use of these two fungicides on grapes.
339
Acknowledgments
340
This work was supported by the National Key Research and Development Program
341
of China under grant numbers 2016YFD0201305 and 2016YFD0201306 and the
342
Science and Technology Programs of Guizhou Province under grant number
343
20136024.
344
Conflicts of interest
345
No potential conflict of interest was reported by the authors.
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Table 1. Mass spectrometric parameters of dimethomorph and pyraclostrobin.
509
Table 2. Calibration equation, determination coefficient (R2), LOD and LOQ, matrix
510
effect (ME) of dimethomorph and pyraclostrobin in different matrices.
511
Table 3. Average recoveries and RSD for dimethomorph and pyraclostrobin under
512
two matrices and spiking levels.
513
Table 4. Terminal residues levels of dimethomorph and pyraclostrobin in grape and
514
soil.
515
Table 5. Acute dietary risk assessment of dimethomorph and pyraclostrobin in grape.
516
Table 6. Chronic dietary risk assessment of dimethomorph and pyraclostrobin in
517
grape.
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Table 1. Mass spectrometric parameters of dimethomorph and pyraclostrobin. Analyte
Dimethomorph Pyraclostrobin
Precursor ion (m/z)
Product ion (m/z) Quantitation
388.2 388.3
301.10 194.20
Confirmation
Collision energy (ev)
Declustering potential (v)
165.10 164.20
30.05/43.18 13.05/24.81
82.62 82.62
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Table 2. Calibration equation, determination coefficient (R2), LOQ and LOD, matrix
521
effect (ME) of dimethomorph and pyraclostrobin in different matrices.
Dimethomorph
Pyraclostrobin
a
523
b
Regression equationa
Methanol Grape Soil Methanol Grape Soil
6
y = 6 × 10 x + 37197 y = 5 × 106 x + 320072 y = 5 × 106 x + 44349 y = 2 × 106 x + 6379.5 y = 2 × 106 x + 26514 y = 2 × 106 x + 24304
y, the Peak area value; x, concentration value.
R2
LOQ (mg/kg)
0.9999 0.9994 0.9997 0.9999 0.9998 0.9998
LOD (mg/kg)
ME (%)
—
—
—
0.04 0.02
0.012 0.006
−16.67 −16.67
—
—
—
0.04 0.02
0.012 0.006
0 0
b
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Matrix
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—, blank test with no matrix effect.
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Table 3. Average recoveries and RSD for dimethomorph and pyraclostrobin under two
526
matrices and spiking levels. intra-day (n = 5)
Inter-day (n = 15) a
Matrix
Spiked level (mg/kg)
Average recovery (%)
RSD (%)
Dimethomorph
Grape
0.04 0.4 4 0.02 0.2 2 0.04 0.4 4 0.02 0.2 2
79.37 92.35 97.05 77.96 91.01 90.02 80.30 82.93 89.17 76.88 92.71 89.22
4.23 7.87 1.73 7.04 2.86 2.57 4.19 6.12 2.57 5.72 3.92 5.54
Grape
Soil
527
a
RSD, relative standard deviation.
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Average recovery (%)
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Analyte
88.02 89.43 96.51 82.93 91.51 91.02 89.09 85.86 89.15 80.52 87.85 87.48
RSD (%) 9.08 8.59 2.81 10.38 5.48 6.87 8.36 4.95 3.74 7.64 7.38 5.37
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Table 4. Terminal residues levels of dimethomorph and pyraclostrobin in grape and
530
soil. Dosage(g.a.i./ha)a
Site
Spray times
Interval (days)
Average residual levels (n = 3) (mg/kg) Dimethomorph Grape Soil
4
1000
3
4
Anhui
800
3
1000
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a.i, active ingredient; ha, he.
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7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21
0.38±0.02 0.28±0.02 0.20±0.05 0.37±0.06 0.33±0.03 0.24±0.01 0.62±0.04 0.47±0.04 0.21±0.03 0.87±0.12 0.48±0.13 0.10±0.03 0.34±0.02 0.24±0.02 0.17±0.04 0.34±0.06 0.29±0.03 0.20±0.00 0.54±0.03 0.39±0.03 0.17±0.02 0.76±0.11 0.41±0.11 0.09±0.02
0.11±0.01 0.08±0.00 0.08±0.00 0.19±0.05 0.16±0.00 0.10±0.00 0.08±0.02 0.14±0.00 0.13±0.06 0.06±0.01 0.12±0.00 0.08±0.00 0.09±0.01 0.07±0.00 0.07±0.00 0.15±0.04 0.13±0.00 0.08±0.00 0.07±0.02 0.12±0.00 0.11±0.04 0.05±0.01 0.10±0.00 0.06±0.00
30
0.16±0.01 0.08±0.00 0.08±0.02 0.09±0.02 0.10±0.02 0.07±0.01 0.32±0.03 0.15±0.01 0.09±0.01 0.27±0.05 0.31±0.03 0.06±0.01 0.16±0.02 0.07±0.01 0.06±0.02 0.10±0.02 0.08±0.03 0.09±0.06 0.16±0.02 0.15±0.02 0.05±0.00 0.23±0.03 0.14±0.03 0.06±0.00
0.10±0.00 0.09±0.02 0.12±0.01 0.10±0.00 0.21±0.01 0.21±0.02 0.87±0.17 0.27±0.00 0.19±0.02 0.29±0.04 0.35±0.03 0.25±0.03 0.05±0.00 0.10±0.01 0.12±0.01 0.10±0.01 0.22±0.00 0.19±0.02 0.16±0.00 0.27±0.01 0.19±0.03 0.26±0.03 0.42±0.03 0.32±0.04
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Table 5. Acute dietary risk assessment of dimethomorph and pyraclostrobin in grape. Pesticides
HRa (mg/kg)
LPb (kg/d)
ESTIc (mg/kg bw day)
ARfDd (mg/kg bw)
αHIe (%)
Dimethomorph Pyraclostrobin
0.87 0.32
0.5703
0.0079 0.0029
0.6 0.05
1.32% 5.80%
a
“HR” is the highest residue.
535
b
“LP” values represent the estimated daily intake.
536
c
“ESTI” values represent the estimated short term intake.
537
d
“ARfD” is the acute reference dose, which is derived from the Joint Meeting on
538
Pesticide Residues (JMPR) reports.
539
e
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Table 6. Chronic dietary risk assessment of dimethomorph and pyraclostrobin in
541
grape.
Age
Sexa
14-17 18-29 30-44 45-59 60-69 ≥ 70
1.1256 1.2273 0.9552 1.0788 0.6993 0.7348 0.4686 0.4560 0.3551 0.4370 0.2434 0.3452 0.1881 0.2771 0.1730 0.2225 0.1869 0.2179 0.1569 0.1447
0.3311 0.3610 0.2810 0.3173 0.2057 0.2161 0.1378 0.1341 0.1039 0.1285 0.0716 0.1015 0.0553 0.0815 0.0509 0.0654 0.0550 0.0641 0.0462 0.0425
a
“M” and “F” represent “male” and “female”, respectively.
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b
“EDI” values represent the estimated daily intake.
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c
“HQ” values represent the hazard quotient.
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Dimethomorph
Pyraclostrobin
0.56% 0.61% 0.48% 0.54% 0.35% 0.37% 0.23% 0.23% 0.18% 0.22% 0.12% 0.17% 0.09% 0.14% 0.09% 0.11% 0.09% 0.11% 0.08% 0.07%
1.10% 1.20% 0.94% 1.06% 0.69% 0.72% 0.46% 0.45% 0.35% 0.43% 0.24% 0.34% 0.18% 0.27% 0.17% 0.22% 0.18% 0.21% 0.15% 0.14%
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43.7 44.4 47.2 51.4 47.1 46.9 47.0 45.6 48.5 58.1 41.8 52.9 35.9 45.4 32.1 37.3 33.8 34.8 27.0 21.7
HQc (%)
SC
7-10
13.2 12.3 16.8 16.2 22.9 21.7 34.1 34 46.7 45.2 58.4 52.1 64.9 55.7 63.1 57 61.5 54.3 58.5 51
EDIb (µg/kg, b.w.) Dimethomorph Pyraclostrobin
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M F M F M F M F M F M F M F M F M F M F
Fruit intake (g/d)
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Figure 1. LC-MS/MS chromatograms of dimethomorph and pyraclostrobin in
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standard solution (0.1 µg/mL), spiked grape (0.4 mg/kg) and soil (0.2 mg/kg) samples.
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Figure 2. Degradation of dimethomorph (A, B) and pyraclostrobin (C, D) in grape and
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soil.
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ACCEPTED MANUSCRIPT Highlights •
A validated method was established to simultaneously determine dimethomorph and pyraclostrobin in the grape and soil. Dissipation kinetics and terminal residue of two fungicides in grape and soil were
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investigated.
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The dietary intake risk of fungicides in grape was assessed.
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S0273-2300(18)30280-0
DOI:
https://doi.org/10.1016/j.yrtph.2018.10.015
Reference:
YRTPH 4245
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 22 May 2018 Revised Date:
3 October 2018
Accepted Date: 20 October 2018
Please cite this article as: Wang, S., Zhang, Q., Yu, Y., Chen, Y., Zeng, S., Lu, P., Hu, D., Residues, dissipation kinetics, and dietary intake risk assessment of two fungicides in grape and soil, Regulatory Toxicology and Pharmacology (2018), doi: https://doi.org/10.1016/j.yrtph.2018.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT 1
Residues, dissipation kinetics, and dietary intake risk assessment of
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two fungicides in grape and soil
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Shouyi Wang a, Qingtao Zhang b, Yurong Yu b, Ya Chen b, Song Zeng b, Ping Lu
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Deyu Hu a *
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a
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Education
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b
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Guiyang 550025, P.R. China
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*Address correspondence to Deyu Hu and Ping Lu, Key Laboratory of Green
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Pesticide and Agricultural Bioengineering, Ministry of Education, Center for
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Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025,
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P.R. China. Tel.: +86 851 88292170; Fax: +86 851 88292170; E-mail:
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[email protected]; [email protected].
a*
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Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
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Center for Research and Development of Fine Chemicals, Guizhou University,
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ACCEPTED MANUSCRIPT 14
ABSTRACT The residue behavior and dietary intake risk of two fungicides (dimethomorph
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and pyraclostrobin) in grape (Vitis vinifera L.) were investigated from field trials. A
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modified quick, easy, cheap, effective, rugged, and safe method for simultaneously
18
determining dimethomorph and pyraclostrobin residues in grape and soil was
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established
20
spectrometry. The average recoveries of dimethomorph and pyraclostrobin in the
21
grape and soil matrices varied from 76.88% to 97.05%, with relative standard
22
deviations of 1.73% to 10.38%. The degradation half-lives of dimethomorph and
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pyraclostrobin were 7.3–12.0 days and 3.6–7.0 days in grape and soil, respectively.
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The terminal residues of dimethomorph and pyraclostrobin in the two matrices were
25
0.05–0.87 mg/kg. For dietary exposure risk assessments, all of the hazard quotient
26
and hazard quotient index values were below 100%, which indicated that the
27
suspending agents of dimethomorph and pyraclostrobin were sprayed on grape at the
28
recommended dosages with no significant potential risks for Chinese consumers.
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This study provides a reference for analytically evaluating residual degradation
30
behavior and dietary intake risk of two fungicides under field conditions.
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Keywords: dimethomorph; pyraclostrobin; residue dynamics;
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assessment; grape.
high
performance
liquid
chromatography–tandem
mass
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using
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dietary risk
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1 Introduction Grape (Vitis vinifera L.), belonging to the Vitaceae family, is one of the most
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commonly consumed fruits and an economically important crop globally. (Hassan,
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2012; Saad, 2017). It is highly nutritious, with its fruit containing components such
37
as sugar, minerals, vitamins, amino acids, and organic acids (Conde et al., 2007).
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However, grape is prone to fungal diseases, such as downy mildew, gray mold, and
39
powdery mildew during growth. The use of fungicides is necessary to control these
40
diseases for improving grape quality and increasing its output (Zhang et al., 2017).
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Dimethomorph is a highly effective and low-toxicity systemic fungicide, which has
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demonstrated good curative and antisporulant activity, particularly against downy
43
mildew (Cohen et al., 1995; Hengel and Shibamoto, 2000; Kim et al., 2015).
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Dimethomorph has been widely used to prevent many diseases of vegetables and
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fruits in China, such as downy mildew, late blight, crown, blue mold, and root rot
46
(Yang et al., 2017). Pyraclostrobin is a broad-spectrum fungicide of the strobilurin
47
family, which can effectively control diseases of blight, anthracnose, and fusarium
48
head blight in cereals, vegetables, fruits, and oil seeds (Reddy et al., 2013).
49
Pyraclostrobin is produced by BASF Corporation (Joshi et al., 2014); it transfers
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electrons between cytochromes b and c1 to inhibit mitochondrial respiration in the
51
target fungus (Zhang et al., 2017). Mixed formulations of fungicides can more
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effectively control diseases and protect crop growth, decrease the application dose
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required, and reduce the risk of fungal resistance compared with a single fungicide
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(Wu et al., 2018). In this context, 45% suspending agent is most commonly used for
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ACCEPTED MANUSCRIPT mixed formulations of dimethomorph and pyraclostrobin registered in China, which
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has remarkable curative effects on bacterial and fungal diseases in grape. However,
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the use of these fungicides may have negative effects on the environment and human
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health. Consequently, it is important to monitor the residues of dimethomorph and
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pyraclostrobin in grape.
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In recent years, several methods for residue analysis of these two compounds
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from variety of matrixes are reported. Regarding some analytical methods of
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dimethomorph in tomato, cucumber, onion, apple, kiwi, orange, pear, pepper,
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Chinese cabbage, cauliflower and Swiss chard were introduced, most of which use
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gas chromatography with tandem mass spectrometry (GC-MS/MS), high
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performance liquid chromatography with ultraviolet visible detector (HPLC-UV),
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and liquid chromatography with tandem mass spectrometry (LC-MS/MS)
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(Walorczyk, 2013; Qi et al., 2015; Kim et al., 2015; Kocourek et al., 2017; Kabir et
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al., 2018a). Some studies for determination of Pyraclostrobin in different crops
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(green bean, spring onion, blueberry, sugarcane, winter jujube, banana, paddy, and
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apple) have been conducted, which include the use of GC-ECD/NPD (gas
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chromatography equipped with electron capture detector/nitrogen and phosphorus
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detector) (Munitz et al., 2014; Sadło et al., 2017), HPLC-UV (Fulcher et al., 2014;
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Fu et al., 2016), HPLC-ESI-MS (high performance liquid chromatography
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electrospray ionization source-mass spectrometry) (Hanafi et al., 2010), LC-MS/MS
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(Peng et al., 2014), UPLC-MS/MS (ultra- performance liquid chromatography with
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tandem mass spectrometry) (Guo et al., 2016). In China, the maximum residue limits
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(GB 2763-2016: MRLs for pesticides in food), whereas values of 3 and 1 mg/kg
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were set by the European Union (EU Pesticide Database, 2017), respectively.
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Chinese regulatory authorities and researchers have realized the necessity of such
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studies, not only for monitoring pesticide residues, but also for assessing the dietary
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exposure risk among consumers (Fang et al., 2015). Although many previous studies
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have reported dimethomorph and pyraclostrobin residues in different crops, to the
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best of our knowledge, there have been no systematic studies on the dissipation,
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residues, and dietary exposure risk assessment of dimethomorph and pyraclostrobin
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in grape.
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In the present study, a modified quick, easy, cheap, effective, rugged, and safe
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(QuEChERS) method was established to simultaneously determine dimethomorph
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and pyraclostrobin residues in grape and soil by LC-MS/MS. Field trials were
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conducted to investigate the dissipation rate and terminal residues of the mixed
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formulation (dimethomorph and pyraclostrobin) in grape, then, the dietary intake
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risk was evaluated through dietary exposure assessment based on the residue, food
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consumption, and toxicology data. Our study aimed to ensure the scientific
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application of this mixed formulation in grape and to provide residue data useful for
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the risk assessment of its presence in the diet on human health, offering guidance for
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the rational and safe use of this mixed fungicide.
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2 Materials and methods
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2.1 Materials and reagents
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Dimethomorph standards (99.0%) and pyraclostrobin standards (99.0%) were 5
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containing 30% dimethomorph and 15% pyraclostrobin was supplied by Hangzhou
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Yulong Chemical Co., Ltd (Hangzhou, China). LC-grade methanol (99.9%) was
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purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Analytical grade
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acetonitrile, dichloromethane, petroleum ether, acetone, sodium chloride, and
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anhydrous magnesium sulfate were obtained from Chengdu Jinshan Chemical
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Reagent Co. (Chengdu, China). C18 (50 µm) and primary secondary amine (PSA)
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(40-60 µm) sorbents were purchased from Agela Technologies Inc. (Tianjin, China).
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Distilled water was purchased from Watsons Corporation (Dongguan, China).
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Individual stock standard solutions of dimethomorph (200 µg/mL) and
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pyraclostrobin (200 µg/mL) were prepared in methanol and stored in volumetric
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flasks at −20°C. Mixed standard solutions of dimethomorph and pyraclostrobin were
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prepared by diluting stock solutions with methanol to the concentration range of
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0.01–2 µg/mL. A matrix-matched standard calibration method was prepared during
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the analytical procedure by adding the appropriate volumes of mixed standard
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solution in blank grape and soil extracts to eliminate matrix effects. All standard
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solutions were kept in the fridge at 4°C before use.
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2.2 Field experiment design
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According to the pesticide registration information and Guidelines on Pesticide
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Residue Trials (NY/T 788-2004) issued by the Ministry of Agriculture of the
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People’s Republic of China, field trials were conducted in Huishui (Guizhou
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Province) and Xiaoxian (Anhui Province) during the 2016 agricultural season (June 6
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Guizhou were 17°C (Anhui) and 14.4°C (Guizhou), the average annual durations of
124
sunshine were 1400 h (Anhui) and 2350 (Guizhou) h, and the mean annual
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precipitation were 1200 mm (Anhui) and 1300 mm (Guizhou), respectively. The soil
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type in Huisui is loess with 6%–10% organic matter, pH 5.0–6.5, and the soil type in
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Xiaoxain is silt loam with 1.14% organic matter, pH 7.0–7.4. The treatments
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comprised grape dynamic test treatment, four terminal residual test treatments, and
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one control plot. The plot of each experimental treatment was 30 m2, with plots
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established in triplicate. To avoid cross-contamination, each plot was separated by a
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buffer zone.
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In the degradation dynamics experiment, the mixed formulation of
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dimethomorph and pyraclostrobin was dissolved in water and sprayed once when
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the grape fruits are about half the size of mature fruit, the dosage was 800 grams of
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active ingredient per hectare (g.a.i./ha). Grape samples of approximately 1 kg and
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soil samples of 1 kg (0–10 cm in depth) were collected randomly from each plot at
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2 h and 1, 2, 3, 5, 7, 10, 14, 21, and 28 days after spraying treatment. For the
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terminal residue experiment, the mixed formulation of dimethomorph and
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pyraclostrobin was sprayed on grapes at two dosage levels: 800 g.a.i./ha (low
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dosage, recommended dosage) and 1000 g.a.i./ha (high dosage). Both low and high
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dosages were sprayed three and four times, respectively. Samples were collected
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randomly after 7, 14, and 21 days of spraying treatment. All samples were placed in
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polyethylene bags and transported back to the laboratory.
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2.3 Analytical procedures
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2.3.1 Sample preparation The samples from the field trials were homogenized and divided into four equal
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portions by quartering technique; two portions (200 g, each) were used for the
148
subsequent experiments. All samples were stored at −20°C before analysis.
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2.3.2 Sample extraction and purification
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A total of 5.0 g of grape samples or 10.0 g of soil samples was weighed and
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placed into a 50-mL polypropylene centrifuge tube, to which 20 mL of acetonitrile
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was added. The mixture was ultrasonically extracted for 15 min, followed by the
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addition of 3.0 g of anhydrous MgSO4 and 2.0 g of anhydrous Na2SO4. Next, the
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samples were mixed for 2 min at a speed of 2500 rpm and then centrifuged at a
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speed of 6000 rpm for 5 min. Subsequently, 1.0 mL of supernatant solution was
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accurately extracted to the pipette and transferred into a 2-mL centrifuge tube
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containing 50 mg of C18 and 50 mg of PSA. The tube was centrifuged at 1200 rpm
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for 3 min after vortexing for 1 min. Finally, the supernatant was filtered through a
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0.22-µm nylon syringe filter before analysis by LC-MS/MS.
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2.3.3 LC-MS/MS analysis
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Dimethomorph and pyraclostrobin were separated on a 20 AD-XR liquid
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chromatography (LC) system, with a Phenomenex Luna C18 column (150 mm × 2
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mm id, film thinness 5 µm; Phenomenex, CA, USA). The mobile phases contained
164
methanol (A) and 0.1% formic acid aqueous solution (B). The gradient elution
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procedure was as follows: 95% B (0–0.4 min), 95% B (0.4–2.0 min), 40% B (2.0– 8
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167
min). The flow rate of the mobile phase was set at 0.3 mL/min, and the injection
168
volume was 1 µL. The chromatographic column temperature was set at 40°C. An
169
applied biosystems sciex API 4000Q trap quadrupole mass spectrometer equipped
170
with an iron source turbo spray unit was used for the monitoring and quantitative
171
analysis of dimethomorph and pyraclostrobin. The analysis of the two fungicides
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was performed in positive mode using multiple reaction monitoring (MRM).
173
Nitrogen (99.999% purity) was used as the curtain, nebulizer, and collision gases;
174
the pressures of the ion source gas (gases 1 and 2) and curtain gas were 413.68 kPa
175
and 206.84 kPa, respectively; ion spray voltage was 5.5 kV; and ion source
176
temperature was 600°C. The ion pair parameters of the two fungicides are presented
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in Table 1.
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Table 1 near here
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2.4 Calculations
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2.4.1 Method validation
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The performance of method was validated using certain parameters, including
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linearity, accuracy, precision, matrix effects, limit of detection (LOD) and limit of
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quantitation (LOQ) (Li et al., 2012; Kabir et al., 2018b). Linearity was evaluated
184
using the correlation coefficient (R2), derived mainly from a six-point calibration
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curve; the calibration curves were obtained by plotting the peak area against the
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corresponding concentration of target analytes in pure solvent and matrix (Rahman
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et al., 2015). The matrix effect (ME) was calculated using the following formula:
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ME =
where m1 and m2 are the slopes of the calibration curves obtained in matrix and pure
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solvent. ME = 0 represents no ME, ME > 0 represents signal enhancement, whereas
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ME < 0 represents signal suppression. LOD and LOQ are defined by signal-to-noise
191
ratios of 3 and 10, respectively. In recovery experiments, different concentrations of
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spiked samples for dimethomorph and pyraclostrobin (0.04, 0.4 and 4 mg/kg in
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grape and 0.02, 0.2, and 2 mg/kg in soil) were investigated. The precision and
194
accuracy of the analytical method were evaluated by calculating recovery and
195
relative standard deviation (RSD) for intra-day (five replicates) and inter-day (fifteen
196
replicates in consecutive three days) (Li et al., 2015; Wang and Zhang, 2017).
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2.4.2 Degradation kinetics
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First-order kinetics equation was used to evaluate degradation of dimethomorph
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and pyraclostrobin over time in grape and soil, the specific calculation formula is as
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follows:
Ct = C0 e-kt t1/2 =
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ln 2 k
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Where C0 (mg/kg) denotes the initial concentration of the compound, k (day−1)
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denotes the rate constant of degradation, Ct (mg/kg) denotes the concentration of the
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compound at time t (day), and t1/2 denotes the half-life of compound degradation
204
(Zhu et al., 2016; Song et al., 2018).
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2.4.3 Assessment of dietary exposure
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The chronic and acute dietary exposure risk quotients of dimethomorph and 10
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pyraclostrobin were calculated to comprehensively assess the risk of intake of the
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mixed formulation sprayed on grapes. The chronic assessment was performed using
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the following formulas: STMR × Fi bw EDI HQ = × 100% ADI
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Where STRM (mg/kg) denotes the supervised trials median residue, Fi denotes the
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food consumption data (g/day), bw denotes mean body weight (kg), EDI denotes the
212
estimated daily intake (mg/kg·bw), ADI denotes the acceptable daily intake
213
(mg/kg·bw), and HQ denotes the hazard quotient, (Lozowicka et al., 2014; Gad Alla
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et al., 2015)
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The acute assessment was performed using the following formulas: LP×HR bw ESTI αHI = × 100% ARfD
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Where, according to the Principles and Methods for the Risk Assessment of
217
Chemicals
218
(FAO) and the World Health Organization (WHO), LP denotes the large portion
219
consumption for the commodity,
220
percentile of food consumption derived from records of individual consumer days
221
(i.e. survey days on which the food or foods of interest were consumed); HR is the
222
highest residue in composite sample of edible portion found in the supervised trials
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used for estimating the maximum residue level (FAO/WHO, 2009). ESTI denotes
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the estimated short-term intake (mg/kg·bw), ARfD denotes the acute reference dose
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indicates an acceptable risk for consumers, whereas a situation where HQ or αHI is >
227
100% indicates that an unacceptable risk for consumers is posed (Fang, et al., 2015;
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Lin et al., 2017).
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3 Results and discussions
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3.1 Method validation Representative
LC-MS/MS
chromatograms
of
dimethomorph
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and
pyraclostrobin in standard solutions and spiked grape and soil samples are shown in
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Figure 1. The linearity of the method was evaluated using the standard calibration
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curves of dimethomorph and pyraclostrobin with a concentration range of 0.01–2
235
µg/mL. As shown in Table 2, superior linearity was observed for two compounds (R2
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values of 0.9994–0.9999), and signal suppression for dimethomorph was found in
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grape and soil due to ME (−16.67) values < 0. Hence, calibration was implemented
238
using external matrix-matched standards to eliminate the matrix effect in this study.
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The LOQ of dimethomorph and pyraclostrobin in grape were 0.04 mg/kg while the
240
LOD were 0.012 mg/kg; the LOQ of the two fungicides in soil were 0.02 mg/kg
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while the LOD were 0.006 mg/kg. The recoveries and RSD of target analytes are
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rendered in Table 3. Average recoveries of dimethomorph in grape and soil were
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77.96%–97.05% (intra-day) and 82.93%–96.51% (inter-day), whereas intra-day and
244
inter-day RSD were in the range of 1.73% to 10.38%. For pyraclostrobin, the
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recoveries in the two matrices were 76.88%–92.71% (intra-day) and 80.52%–89.22%
246
(inter-day), with corresponding RSD between 2.57% and 8.36%. The satisfactory
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248
precision, so it is appropriate for analysis to detect dimethomorph and pyraclostrobin
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in grape and soil.
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Figure 1 near here
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Table 2 near here
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Table 3 near here
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3.2 Degradation of dimethomorph and pyraclostrobin in grape and soil
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According to the results of the monitoring analysis, the degradation curves of
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dimethomorph and pyraclostrobin in grape and soil from Anhui and Guizhou were
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plotted and are shown in Figure 2. The original residues of dimethomorph in grape at
257
Anhui and Guizhou were 2.44 and 2.39 mg/kg after 2 h of spraying. Meanwhile, the
258
equivalent values of residues were 0.41 and 0.68 mg/kg in soil, respectively. The
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degradation dynamics equations of dimethomorph were Ct = 1.7772 e−0.083t (Anhui)
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and Ct = 2.0592 e−0.095t (Guizhou) in grape, with half-lives (t1/2) of 8.4 and 7.3 days;
261
the values were close to those obtained in previous studies (9.4 days; Liu et al.,
262
2012). The degradation dynamics equations of dimethomorph in soil were Ct = 0.376
263
e−0.06t (Anhui) and Ct = 0.619 e−0.058t (Guizhou), with t1/2 of 11.6 and 12.0 days. For
264
pyraclostrobin, the original concentrations were 0.44 and 0.62 mg/kg in grape and
265
soil from Anhui, and 0.47 and 0.41 mg/kg for Guizhou, respectively. The
266
degradation dynamics equations of pyraclostrobin were Ct = 0.3868 e−0.099t (Anhui)
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and Ct = 0.3539 e−0.192t (Guizhou) in soil, and t1/2 values were 7.0 and 3.6 days,
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respectively. In contrast, the degradation dynamics equations of pyraclostrobin in
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270
values of 4.7 and 4.9 days. These t1/2 values were less than some presented in other
271
reports, where the t1/2 values of pyraclostrobin were 5.5–8.0 days in blueberries
272
(Munitz et al., 2014), 8.3–9.1 days in bananas (Fu et al., 2016), and 6.4–9.3 days in
273
pepper (Wu et al., 2018). The degradation rate of dimethomorph in grape at 14 days
274
after spraying was > 78% and that at 28 days after spraying was > 80% in soil.
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Meanwhile, the degradation rate of pyraclostrobin at 10 days after spraying was > 85%
276
in the two matrices. The degradation rate and t1/2 values illustrate that pyraclostrobin
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dissipates faster than dimethomorph in grape and soil.
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Figure 2 near here
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3.3 Terminal residues of dimethomorph and pyraclostrobin in grape and soil
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The terminal residue results of the two fungicides in grapes and soil are
281
presented in Table 4. The terminal residues of dimethomorph were 0.09–0.87 and
282
0.05–0.19 mg/kg in grape and soil, respectively, at 7, 14, and 21 days after spraying
283
for the two dosage levels. Meanwhile, the terminal residues of pyraclostrobin were
284
0.05–0.32 and 0.05–0.87 mg/kg in grape and soil, respectively. It turned out that the
285
residual levels of dimethomorph and pyraclostrobin in grape were below 3 and 1
286
mg/kg (MRLs set by the EU), respectively.
287
Table 4 near here
288
3.4 Exposure risk assessment
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In this study, acute and chronic dietary exposure risk assessments for
290
dimethomorph and pyraclostrobin in grape were performed on the basis of 14
ACCEPTED MANUSCRIPT 291
monitoring of pesticide residues and toxicity data for these two fungicides as well as
292
data on grape consumption in China. The results of acute dietary exposure risk assessments are summarized in Table
294
5. The HR values of dimethomorph and pyraclostrobin in grapes were 0.87 and 0.32
295
mg/kg from the terminal residue experiments. The Chinese adult mean body weight
296
is 63 kg (Lin et al., 2017). According to the World Health Organization, LP of grape
297
is 570.3 g/d for Chinese residents (WHO, 2015). The ARfD values for
298
dimethomorph and pyraclostrobin are 0.6 and 0.05 mg/kg·bw, respectively, as
299
derived from the Joint Meeting on Pesticide Residues (JMPR) reports (FAO, 2007;
300
FAO, 2003). All of the calculated ESTI values of dimethomorph and pyraclostrobin
301
were far less than the corresponding ARfD values; the αHI values were < 10%,
302
which means that the acute dietary exposure risk of dimethomorph and
303
pyraclostrobin among humans is acceptable.
304
Table 5 near here
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The chronic dietary exposure risk assessments for dimethomorph and
306
pyraclostrobin in grape were performed on the basis of the consumption of fruit
307
among Chinese from different age groups. The intakes of fruit and mean weight of
308
different age groups are shown in Table 6 (Jin, 2008). The ADI values for
309
dimethomorph and pyraclostrobin are 0.2 and 0.03 mg/kg·bw, as derived from the
310
JMPR reports (FAO, 2007; FAO, 2003). The STRM values of dimethomorph and
311
pyraclostrobin in grape were 0.34 and 0.10 mg/kg, respectively. As seen in Table 6,
312
dimethomorph and pyraclostrobin have a higher chronic dietary exposure risk in
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314
adults. In addition, females were more sensitive to dimethomorph and pyraclostrobin
315
than males within the same age group because the HQ values of females were higher
316
than those of males. However, all of the EDI values were far below the ADI values,
317
and the HQ values ranged from 0.07% to 1.20% (< 10%), which implies that the
318
chronic dietary exposure risk of dimethomorph and pyraclostrobin among humans is
319
also acceptable. For dimethomorph and pyraclostrobin in grape, smaller HQ and αHI
320
values demonstrated that there is no significant potential risk for Chinese residents
321
according to the recommended application guide.
322
Table 6 near here
323
4 Conclusions
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In this study, the validated QuEChERS and LC-MS/MS analytical method was
325
developed and applied to simultaneously determine the levels of dimethomorph and
326
pyraclostrobin residues in grape and soil, achieved satisfactory precision and
327
accuracy. Subsequently, the degradation dynamics and terminal residues of
328
dimethomorph and pyraclostrobin in grape and soil were studied under field
329
conditions. The results show that the t1/2 values of dimethomorph and pyraclostrobin
330
were 7.3–12.0 days and 3.6–7.0 days in the two matrices, and pyraclostrobin
331
degraded more rapidly than dimethomorph. The levels of terminal residues for
332
dimethomorph and pyraclostrobin in all matrices were below the MRLs values set by
333
the EU (3 and 1 mg/kg, respectively). Based on the terminal residue results, dietary
334
exposure risk assessments for dimethomorph and pyraclostrobin in grape were
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336
significant potential risks of dimethomorph and pyraclostrobin in grape at the
337
recommended dosages. Our study could provide a valuable reference for the safe and
338
reasonable use of these two fungicides on grapes.
339
Acknowledgments
340
This work was supported by the National Key Research and Development Program
341
of China under grant numbers 2016YFD0201305 and 2016YFD0201306 and the
342
Science and Technology Programs of Guizhou Province under grant number
343
20136024.
344
Conflicts of interest
345
No potential conflict of interest was reported by the authors.
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508
Table 1. Mass spectrometric parameters of dimethomorph and pyraclostrobin.
509
Table 2. Calibration equation, determination coefficient (R2), LOD and LOQ, matrix
510
effect (ME) of dimethomorph and pyraclostrobin in different matrices.
511
Table 3. Average recoveries and RSD for dimethomorph and pyraclostrobin under
512
two matrices and spiking levels.
513
Table 4. Terminal residues levels of dimethomorph and pyraclostrobin in grape and
514
soil.
515
Table 5. Acute dietary risk assessment of dimethomorph and pyraclostrobin in grape.
516
Table 6. Chronic dietary risk assessment of dimethomorph and pyraclostrobin in
517
grape.
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Table 1. Mass spectrometric parameters of dimethomorph and pyraclostrobin. Analyte
Dimethomorph Pyraclostrobin
Precursor ion (m/z)
Product ion (m/z) Quantitation
388.2 388.3
301.10 194.20
Confirmation
Collision energy (ev)
Declustering potential (v)
165.10 164.20
30.05/43.18 13.05/24.81
82.62 82.62
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Table 2. Calibration equation, determination coefficient (R2), LOQ and LOD, matrix
521
effect (ME) of dimethomorph and pyraclostrobin in different matrices.
Dimethomorph
Pyraclostrobin
a
523
b
Regression equationa
Methanol Grape Soil Methanol Grape Soil
6
y = 6 × 10 x + 37197 y = 5 × 106 x + 320072 y = 5 × 106 x + 44349 y = 2 × 106 x + 6379.5 y = 2 × 106 x + 26514 y = 2 × 106 x + 24304
y, the Peak area value; x, concentration value.
R2
LOQ (mg/kg)
0.9999 0.9994 0.9997 0.9999 0.9998 0.9998
LOD (mg/kg)
ME (%)
—
—
—
0.04 0.02
0.012 0.006
−16.67 −16.67
—
—
—
0.04 0.02
0.012 0.006
0 0
b
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522
Matrix
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—, blank test with no matrix effect.
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Table 3. Average recoveries and RSD for dimethomorph and pyraclostrobin under two
526
matrices and spiking levels. intra-day (n = 5)
Inter-day (n = 15) a
Matrix
Spiked level (mg/kg)
Average recovery (%)
RSD (%)
Dimethomorph
Grape
0.04 0.4 4 0.02 0.2 2 0.04 0.4 4 0.02 0.2 2
79.37 92.35 97.05 77.96 91.01 90.02 80.30 82.93 89.17 76.88 92.71 89.22
4.23 7.87 1.73 7.04 2.86 2.57 4.19 6.12 2.57 5.72 3.92 5.54
Grape
Soil
527
a
RSD, relative standard deviation.
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Pyraclostrobin
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Average recovery (%)
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Analyte
88.02 89.43 96.51 82.93 91.51 91.02 89.09 85.86 89.15 80.52 87.85 87.48
RSD (%) 9.08 8.59 2.81 10.38 5.48 6.87 8.36 4.95 3.74 7.64 7.38 5.37
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Table 4. Terminal residues levels of dimethomorph and pyraclostrobin in grape and
530
soil. Dosage(g.a.i./ha)a
Site
Spray times
Interval (days)
Average residual levels (n = 3) (mg/kg) Dimethomorph Grape Soil
4
1000
3
4
Anhui
800
3
1000
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a.i, active ingredient; ha, he.
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7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21 7 14 21
0.38±0.02 0.28±0.02 0.20±0.05 0.37±0.06 0.33±0.03 0.24±0.01 0.62±0.04 0.47±0.04 0.21±0.03 0.87±0.12 0.48±0.13 0.10±0.03 0.34±0.02 0.24±0.02 0.17±0.04 0.34±0.06 0.29±0.03 0.20±0.00 0.54±0.03 0.39±0.03 0.17±0.02 0.76±0.11 0.41±0.11 0.09±0.02
0.11±0.01 0.08±0.00 0.08±0.00 0.19±0.05 0.16±0.00 0.10±0.00 0.08±0.02 0.14±0.00 0.13±0.06 0.06±0.01 0.12±0.00 0.08±0.00 0.09±0.01 0.07±0.00 0.07±0.00 0.15±0.04 0.13±0.00 0.08±0.00 0.07±0.02 0.12±0.00 0.11±0.04 0.05±0.01 0.10±0.00 0.06±0.00
30
0.16±0.01 0.08±0.00 0.08±0.02 0.09±0.02 0.10±0.02 0.07±0.01 0.32±0.03 0.15±0.01 0.09±0.01 0.27±0.05 0.31±0.03 0.06±0.01 0.16±0.02 0.07±0.01 0.06±0.02 0.10±0.02 0.08±0.03 0.09±0.06 0.16±0.02 0.15±0.02 0.05±0.00 0.23±0.03 0.14±0.03 0.06±0.00
0.10±0.00 0.09±0.02 0.12±0.01 0.10±0.00 0.21±0.01 0.21±0.02 0.87±0.17 0.27±0.00 0.19±0.02 0.29±0.04 0.35±0.03 0.25±0.03 0.05±0.00 0.10±0.01 0.12±0.01 0.10±0.01 0.22±0.00 0.19±0.02 0.16±0.00 0.27±0.01 0.19±0.03 0.26±0.03 0.42±0.03 0.32±0.04
RI PT
3
SC
800
M AN U
Guizhou
Pyraclostrobin Grape Soil
ACCEPTED MANUSCRIPT 533
Table 5. Acute dietary risk assessment of dimethomorph and pyraclostrobin in grape. Pesticides
HRa (mg/kg)
LPb (kg/d)
ESTIc (mg/kg bw day)
ARfDd (mg/kg bw)
αHIe (%)
Dimethomorph Pyraclostrobin
0.87 0.32
0.5703
0.0079 0.0029
0.6 0.05
1.32% 5.80%
a
“HR” is the highest residue.
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b
“LP” values represent the estimated daily intake.
536
c
“ESTI” values represent the estimated short term intake.
537
d
“ARfD” is the acute reference dose, which is derived from the Joint Meeting on
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Pesticide Residues (JMPR) reports.
539
e
AC C
EP
TE D
SC
M AN U
“αHI” values represent hazard quotient index.
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534
31
ACCEPTED MANUSCRIPT 540
Table 6. Chronic dietary risk assessment of dimethomorph and pyraclostrobin in
541
grape.
Age
Sexa
14-17 18-29 30-44 45-59 60-69 ≥ 70
1.1256 1.2273 0.9552 1.0788 0.6993 0.7348 0.4686 0.4560 0.3551 0.4370 0.2434 0.3452 0.1881 0.2771 0.1730 0.2225 0.1869 0.2179 0.1569 0.1447
0.3311 0.3610 0.2810 0.3173 0.2057 0.2161 0.1378 0.1341 0.1039 0.1285 0.0716 0.1015 0.0553 0.0815 0.0509 0.0654 0.0550 0.0641 0.0462 0.0425
a
“M” and “F” represent “male” and “female”, respectively.
543
b
“EDI” values represent the estimated daily intake.
544
c
“HQ” values represent the hazard quotient.
AC C
EP
542
545
32
Dimethomorph
Pyraclostrobin
0.56% 0.61% 0.48% 0.54% 0.35% 0.37% 0.23% 0.23% 0.18% 0.22% 0.12% 0.17% 0.09% 0.14% 0.09% 0.11% 0.09% 0.11% 0.08% 0.07%
1.10% 1.20% 0.94% 1.06% 0.69% 0.72% 0.46% 0.45% 0.35% 0.43% 0.24% 0.34% 0.18% 0.27% 0.17% 0.22% 0.18% 0.21% 0.15% 0.14%
RI PT
11-13
43.7 44.4 47.2 51.4 47.1 46.9 47.0 45.6 48.5 58.1 41.8 52.9 35.9 45.4 32.1 37.3 33.8 34.8 27.0 21.7
HQc (%)
SC
7-10
13.2 12.3 16.8 16.2 22.9 21.7 34.1 34 46.7 45.2 58.4 52.1 64.9 55.7 63.1 57 61.5 54.3 58.5 51
EDIb (µg/kg, b.w.) Dimethomorph Pyraclostrobin
M AN U
4-6
M F M F M F M F M F M F M F M F M F M F
Fruit intake (g/d)
TE D
2-3
Body weight (kg)
ACCEPTED MANUSCRIPT Figure captions
547
Figure 1. LC-MS/MS chromatograms of dimethomorph and pyraclostrobin in
548
standard solution (0.1 µg/mL), spiked grape (0.4 mg/kg) and soil (0.2 mg/kg) samples.
549
Figure 2. Degradation of dimethomorph (A, B) and pyraclostrobin (C, D) in grape and
550
soil.
AC C
EP
TE D
M AN U
SC
RI PT
546
33
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights •
A validated method was established to simultaneously determine dimethomorph and pyraclostrobin in the grape and soil. Dissipation kinetics and terminal residue of two fungicides in grape and soil were
RI PT
•
investigated.
EP
TE D
M AN U
SC
The dietary intake risk of fungicides in grape was assessed.
AC C
•