Introduction

Fluazinam (3-chloro-N-(3-chloro-5-trifluoromethyl-2-pyridyl)-α, α, α-trifluoro-2,6-dinitro-p-toluidine) is a 2,6-dinitrobenzene fungicide that was discovered by Ishihara Sangyo Kaisha, Ltd and first marketed in 1990 (Hu 2004). As a protective fungicide, fluazinam has long-lasting effects and is not readily removed by rain. It is used to control grey mold (Botrytis cinerea) and downy mildew (Peronospora parasitica) on vines; southern blight and white mold on peanuts; clubroot on brassicaceae; white and violet root rot on fruit trees; scab and mites in apples and Phytophthora infestans, tuber blight, scab, and powdery scab on potatoes (Tomlin 2011).

Potatoes (Solanum tuberosum) are an important crop in China, and the area devoted to their cultivation ranks behind only rice, wheat, and corn. The production of potatoes is increasing annually all over the world (Qu et al. 2005; Wu et al. 2012). However, potato crops suffer yield losses due to Phytophthora infestans during growth. Fluazinam is widely used worldwide to protect potatoes from Phytophthora infestans and it can increase potato yields (Dowley and Osullivan 1995; Leonard et al. 2001). However, residual fluazinam in the environment may cause dermatitis and occupational asthma (Draper et al. 2003). Therefore, it is important to determine the dissipation behavior of in edible fluazinam raw food and in the environment.

There have been a few reports of fluazinam extraction and analytical methods in grapes, must, wine (Cabras et al. 1998), pepper (Dong et al. 2008), potato, and soil (Zhang et al. 2012). The reported methods require large quantities of solvent and multiple steps. The Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method minimizes the number of sample-preparation steps and has been used mainly for the extraction of different classes of pesticides (Abd-Alrahman et al. 2011; Garrido Frenich et al. 2008; Lehotay et al. 2010; Paya et al. 2007). This is the first study of the dissipation behavior of fluazinam in two ecosystems, temperate monsoon and subtropical monsoon, in 2012.

In this study, we established a modified QuEChERS preparation approach coupled with gas chromatography-electron capture detector (GC-ECD) to analyze fluazinam in potatoes and soil. The dissipation dynamics of fluazinam in potato plants and soil were also investigated, as was the terminal residue in potato tubers and field soil.

Material and Methods

Chemical Material

HPLC-grade acetonitrile was purchased from Fisher Chemical (Fair Lawn, NJ, USA). Water was prepared using a Milli-Q water purification system (Millipore, USA). Analytical grade sodium chloride (99.5 %) was purchased from Sinopharm Chemical Reagent (Beijing, China). Cleanert PSA (Primary secondary amine) sorbent was purchased from Agela Technologies (Tianjin, China). Standard fluazinam (purity 98.0 %) and 500 g · L−1 fluazinam aqueous suspension concentrate (SC) were supplied by Jixi Agrochemicals Co., Ltd of Anhui Province, China. Stock standard solution of fluazinam (1000.0 mg · L−1) was prepared in acetonitrile and stored at -20 °C. The stock standard solution was diluted with acetonitrile as required.

Field Trials

Field experiments were carried out in Lulong (39°N, 118°E, Hebei Province, northern China, temperate monsoon) and Xiaoxian (34°N, 116°E, Anhui province, middle China, subtropical monsoon) in 2012 according to “The guideline for Pesticide Residue Field Experiment” issued by the Institute of the Control of Agrochemicals, Ministry and Agriculture, the People’s Republic of China.

The design of the field experiments for fluazinam residue and dissipation is shown in Table 1. There were seven treatments, including six fluazinam (500 g · L−1 SC formulation) treatments and a control treatment. Each experimental plot was 30 m2 and there were three replicate plots for each treatment. No pesticide was used at any time during the growth of potatoes in the control treatment. A 30 m2 buffer area is separated treatment plot.

Table 1 Design of the field experiments for fluazinam residue and dissipation in potato vine and field soil
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To investigate the dissipation dynamics of fluazinam in potato vines and soil, fluazinam was dissolved in 750 L water and applied to the surface of potato vines at the beginning of main stem elongation and bare soil using a JACTO-HD400 internal pump backpack sprayer at an active constituent dose of 375 g a.i. ha−1 (1.5 times the recommended high dosage). Representative potato vine and soil samples were collected randomly from several points in each plot at 2 h, and 1, 3, 7, 14, 28, and 42 days after treatment (DAT).

For the ultimate residue experiment, 500 g · L−1 SC fluazinam was applied at a low dosage of 250 g a.i. ha−1 (recommended high dosage) and a high dosage of 375 g a.i. ha−1 (1.5 times the recommended high dosage) for four and five times with sampling at 5, 7, and 10 DAT, respectively. Tubers and soil samples were collected 5, 7, and 10 DAT after the last application.

Sampling and Storage

Dissipation samples were collected randomly from each plot at different time intervals after treatment. Tuber and soil samples were collected at harvest for final determination. Potato vine (2 kg) and tuber (2 kg) samples were homogenized in a blender (Philips, China). Soil samples were collected randomly from each plot using a soil auger to a depth of 15 cm from the surface. Small stones, roots, stems and other unwanted materials were removed. The 2-month storage stability of fluazinam in potato vines and tubers, and soils at −18 °C were assessed in our lab. Fluazinam is freezer stable within 2 months, thus all samples were kept at −18 °C until analysis and were analyzed within 2 months.

Analytical Procedure

Portions (10.0 g) of homogenized potato, potato plant, and soil samples were weighed into 50-mL centrifuge tubes and 10 mL acetonitrile, 5 mL HPLC-grade water, and 4.0 g sodium chloride were added to each tube. Tubes were vortexed for 2 min and centrifuged at RCF 3802×g for 5 min. The acetonitrile supernatant layer (1.5 mL) was transferred into a 2-mL centrifuge tube containing 50 mg primary secondary amine (PSA) and 100 mg anhydrous magnesium sulfate (MgSO4). Samples were then vortexed for 1 min. The extract was centrifuged at RCF 9168×g for 5 min and then filtered through a 0.22-μm membrane filter (PTFE) into an autosampler vial and analyzed using GC-ECD.

Apparatus

An Agilent Technologies 6890 N network GC system equipped with an Agilent 7683 series auto injector, electron capture detector (ECD), and a 30 m × 0.25 mm AB-5 capillary column (0.25-μm film thickness) was used for quantification of fluazinam. The injector was held at 250 °C and the carrier gas was nitrogen at a constant flow rate of 1.0 mL · min−1. The oven temperature was maintained at 120 °C for 1 min, increased from 120 to 270 °C at a rate of 20 °C · min−1 and was held at 270 °C for 10 min. The detector was maintained at 280 °C. Samples were analyzed in splitless mode, and 1 μL of each sample was injected.

Statistical Analysis

The limit of detection (LOD) was defined as the concentration that resulted in a peak area that was three times the noise of the baseline at the retention time of the peak of target analyte. To determine LOQ, the LOD was multiplied by 3.3 (Clifton 1996). The dissipation of fluazinam is typically expressed in terms of DT50 i.e., time to disappearance of 50 % of the original pesticide concentration. DT50 values were obtained by fitting first-order kinetics to observe the degradation patterns as follows: C = Coe –kt, where C is the chemical concentration (mg · kg−1) at time t (d), Co is the initial concentration (mg · kg−1), and k is the first-order rate constant (d−1), which is independent of C and Co. The DT50 of fluazinam was calculated separately for each location using Hoskins’ formula DT50 = ln2 k−1 (Hoskins 1966).

Results and Discussions

Method Validation

Calibration, LOD and LOQ

A linear calibration curve was obtained for fluazinam by plotting the average peak area against concentration. The range of the six-point calibration curve was from 0.01 to 0.5 mg · L−1 in vines, tubers, and soil. The calibration curves showed good linearity with typical correlation coefficients (R2) between 0.9990 and 0.9994. The calibration curve was used to calculate the concentration of fluazinam residues in vines, tubers, and soil (Anagnostopoulos et al. 2006). The LOD value was 5.0 × 10−3 ng and the LOQ value was 0.01 mg · kg−1.

Accuracy and Precision

Acetonitrile or ethyl acetate are often used as the extraction solvent for multi-residue analysis that involves limited extraction (Anastassiades et al. 2003; Ferrer et al. 2005; Ferrer and Thurman 2007; Pan et al. 2008). In this study, acetonitrile was used as the extraction solvent and cleaned up with PSA sorbent and anhydrous magnesium sulfate.

Recovery is significant when attempting to quantify trace levels of analyte within a complex matrix. In this study, three concentrations of fluazinam were added to vines, tubers, and soil matrices. Five replicate measurements were performed for each concentration level (fortified level). In recovery experiment, ‘fortified level’ means the spiking concentration of fluazinam in three testing matrices, it indicated that when residues of fluazinam determined between these levels, results could be considered to be accurate. The LOQ of the target analyte in all matrices was 0.01 mg · kg−1 (signal-to-noise ratio of 10). The precision of the method, in terms of the relative standard deviations (RSD), ranged from 0.7 to 2.8 %. The recovery and precision results met the acceptability criteria of the Residues Analysis Quality Control Guide (General Administration of Quarantine of the People’s Republic of China 2002). Table 2 shows recoveries for three spiking levels. Average recoveries of fluazinam were between 85.1 and 99.7 % in vines, tubers, and soil samples.

Table 2 Recoveries and RSD of fluazinam in three matrices (n = 5)
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Dissipation of Fluazinam in Vines Under Field Conditions

Dissipation data for vines are shown in Fig. 1. The initial concentrations of fluazinam in plants were 13.28 and 23.33 mg · kg−1 in Hebei and Anhui, respectively. The dissipation dynamics of fluazinam were described by the following first-order kinetics equations: C = 13.5601e-0.2784t (Hebei) and C = 23.2665e-0.1935t (Anhui), with correlation coefficients (R2) of 0.9490 and 0.9928, respectively. The climate in Hebei was temperate monsoon, while it was subtropical monsoon in Anhui, but the calculated degradation half-life (DT50) of fluazinam in vines was 2.5 days in Hebei and 3.6 days in Anhui. This indicates that fluazinam dissipated rapidly in vines and was not affected by differences in weather conditions between sites.

Fig. 1
figure 1

Dissipation of fluazinam in vine in Hebei and Anhui

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Dissipation of Fluazinam in the Soil

Dissipation data for soil are shown in Fig. 2. The initial concentrations of fluazinam in soil were 7.89 and 1.11 mg · kg−1 in Hebei and Anhui, respectively. The dissipation dynamics of fluazinam were described by the following first-order kinetics equations: C = 6.83e-0.1475t (Hebei) and C = 0.95e-0.0519t (Anhui), with correlation coefficients (R2) of 0.88 and 0.93, respectively. The degradation half-life (DT50) of fluazinam in soil was 4.7 days in Hebei and 13 days in Anhui.

Fig. 2
figure 2

Dissipation of fluazinam in soil in Hebei and Anhui

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Both experimental locations contained soils characterized by clay loam. The organic matter content for the Hebei and Anhui soils were 21.0 and 27.0 g · kg−1, respectively, and the pHs were 7.4 and 6.4, respectively. Fluazinam degrade through hydrolysis faster in alkaline condition (Tomlin 2011). This indicates that the degradation of fluazinam in the soil was influenced to a greater degree by pH than by organic matter content. Alkaline conditions may promote the degradation of fluazinam.

Terminal Residues in Tuber and Soil

The terminal residue of fluazinam in tubers and soil were analyzed after application of fluazinam 500 g · L−1 SC at the recommended high dosage (250 g a.i. ha−1) and at a dosage 1.5-fold higher (375 g a.i. ha−1) at the two locations. Fluazinam was not detectable in tuber samples at either location harvested 5, 7, or 10 DAT. The residues in soil samples 5, 7, and 10 DAT were 0.116–0.700 mg · kg−1 in Hebei and 0.0925–0.949 mg · kg−1 in Anhui.

Conclusions

The methods used for extraction, clean-up and estimation of fluazinam residues in sample matrices were simple and quantitative. Fluazinam degraded quickly in both vines and soil in two experimental locations located in northern and central China. Alkaline soil conditions may promote the degradation of fluazinam. The residues of fluazinam in tuber samples were below the limit of detection (LOD) at five5, seven7, and 10 DAT at the tested dosages. Therefore, a safe pre-harvest interval (PHI) of 5 days is suggested before harvesting potatoes treated with fluazinam. The present study suggests that the fluazinam 500 g · L−1 SC can be safely used in potato fields at the recommended dosage. This study may also be helpful for establishment of the maximum residue level (MRL) of fluazinam in potatoes by the Chinese government to provide guidance as to the proper and safe use of this fungicide.