Special issue paper Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3094
Optimization of supercritical fluid extraction method for detection of fluquinconazole and tetraconazole in soil using gas chromatography and confirmation using GC-MS: application to dissipation kinetics Sang Im Parka†, Jong-Hyouk Parka†, Ah-Young Koa, A. M. Abd El-Atya,b*, Ayman Goudahb, Jin Janga, Md. Musfiqur Rahmana, Mi Ra Kimc and Jae-Han Shima* ABSTRACT: The aim of this study was to establish an analytical method to detect fluquinconazole and tetraconazole in soil using supercritical fluid extraction (SFE) and gas chromatography (GC). The optimal extraction conditions for SFE were: temperature, 60 °C; pressure, 280 kg/cm2; extraction time, 50 min; and a 10% modifier ratio. The linearity of the calibration curves was good and yielded a determination coefficient (R2) ≥ 0.995. The soil samples were fortified with known quantities of the analytes at three different concentrations (0.01, 0.02 and 0.1 μg/g for fluquinconazole; 0.05, 0.1 and 0.5 μg/g for tetraconazole), and the recoveries ranged between 83.7 and 94.1%. The intra- and inter-day relative standard deviations were 1.3–10.6 and 2.2–11.9% for fluquinconazole and tetraconazole, respectively. The limit of detection and limit of quantitation were 0.002 and 0.01 μg/g for fluquinconazole and 0.01 and 0.05 for tetraconazole, respectively. The method was successfully applied to the analysis of soil residues collected from an onion field. The results show that a combination of SFE and GC can be used as an environmentally friendly technique to detect fungicides in soil. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fluquinconazole; tetraconazole; soil; supercritical fluid extraction; gas chromatography
Introduction
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Many environmental pollutants, pesticides in particular, are used widely to protect crops and seeds; however, their presence in food and soil poses a potential human health threat (Jin et al., 2004). Soil is an important agricultural resource that has the ability to retain agrochemicals, including pesticides. Soil is exposed to pesticides through direct application, accidental spillage, run-off from plant surfaces or incorporation of pesticidecontaminated plant material. Once in the soil, a pesticide may undergo adsorption, leaching and/or degradation (Fernandes et al., 2013). The possibility of groundwater pollution also exists, as soil leachate may contain pesticide residues. Thus, the persistence of fungicides in soil needs to be evaluated for their safe and judicious use (Alam et al., 2013). Fluquinconazole [3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2, 4-triazol-1-yl)quinazolin-4(3H)-one, Fig. 1] is a quinazoline-based sterol demethylation inhibitor fungicide that is active against Ascomycetes, Basidiomycetes, Deuteromycetes, and Mycosphaerella graminicola (anamorph Septoria tritici; Metcalfe et al., 2000; Mavroeidi and Shaw, 2005). Tetraconazole [(RS)-2-(2,4-dichlorophenyl) -3-(1H-1,2,4-triazol-1-yl)propyl 1,1,2,2-tetrafluoroethyl ether, Fig. 1] is a triazole fungicide that exerts its antifungal activity by inhibiting sterol biosynthesis (Stenersen, 2004). Tetraconazole has a broad spectrum of bioefficacy against powdery
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mildew and rust diseases of various agricultural crops (Banerjee et al., 2008). The half-life of fluquinconazole in apple is 15–32 days (Szpyrka and Walorczyks, 2013), and that of tetraconazole is 7 days in cucumber (Khalfallah et al., 1998), 5 days in sugar beet (Menkissoglu-Spiroudi et al., 1998), 4–5 days in mango (Alam et al., 2011) and 66.9–86 days in soil under different pH conditions (Alam et al., 2013).
* Correspondence to: J. H. Shim and A. M. Abd El-Aty, Biotechnology Research Institute, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500–757, Republic of Korea. Email: [email protected]; [email protected] †
The first two authors contributed equally to this article.
a
Biotechnology Research Institute, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea
b
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt
c
Regional FDA Hazardous Substances Analysis Division, Daejeon, Republic of Korea Abbreviations used: μ-ECD, micro-electron-capture detection; MeOH, methanol; NPD, nitrogen–phosphorus detection; SC-CO2, supercritical carbon dioxide
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Detecting fungicides in soil
Figure 1. Structures of (A) fluquinconazole (structural formula C16H8Cl2FN5O, molecular weight 376.172) and (B) tetraconazole (structural formula C13H11Cl2F4N3O, molecular weight 372.146).
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Experimental Chemicals and reagents Fluquinconazole (purity 98.5%) and tetraconazole (purity 98.0%) were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Acetone and methanol were supplied by Merck (Darmstadt, Germany). Air, nitrogen (purity 99.999%), helium (purity 99.999%) and hydrogen (purity 99.85%) used for gas chromatography were supplied by Gaswell Co. Ltd (Gwangju, Republic of Korea). All other chemicals were of analytical and/ or high-performance liquid chromatography grade.
Preparation of the standard solutions Standard compounds (10.2 mg of fluquinconazole and tetraconazole) were separately dissolved in acetone, and their volume was brought up to 100 mL to obtain a 100 μg/mL stock solution. A 20 μg/mL standard mixture of the two fungicides was prepared via volumetric dilutions of each stock solution with acetone.
SFE system A Jasco (Tokyo, Japan) PU980 system with two pumps was used for SFE. The master pump was fitted with a cooling jacket on the pump head for CO2 delivery, and a second pump was used to add the modifier. The pumps were connected to a 10 mL stainless SFE vessel (inside diameter, 10 mm; length, 15 cm) in a column oven chamber (Jasco, model CO-965) that was directly connected to a Jasco 880–81 backpressure regulator, used to control the pressures of the supercritical fluid CO2 and methanol in the system.
Sample preparation and extraction procedure Soil was air-dried in the shade and powdered by passing it through a 2 mm sieve before extraction. Exactly 2 g (Sartorius, BSA2202S-CW, d
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The efficient extraction of pesticides from soil samples is crucial owing to the complexity of the matrix and the diverse physicochemical properties of the tested analytes (Rashid et al., 2010), generally requiring a technique capable of extracting the bound residues at low concentrations. Therefore, methods that are simple, rapid, accurate, environmentally friendly and provide clean extracts are needed for the proper risk assessment of pesticide residues in water, soil and crops (Yang et al., 2010). Conventional liquid–liquid/solid extraction techniques such as Soxhlet extraction, sonication and mechanical shaking have been applied to extract pesticides from soil (Rissato et al., 2005). However, concerns about the hazards associated with most of the solvents used and the costs and environmental dangers of waste solvent disposal have led to the development of more efficient environmentally friendly techniques such as supercritical fluid extraction (SFE) (Dean, 1996). SFE has gained increased attention due to properties of supercritical fluids such as higher diffusivity and low viscosity (Smith, 1999). These properties are generally obtained by optimizing the pressure of the supercritical CO2 used for extraction and the temperature of the extraction cell (Dean, 1996). The experimental SFE parameters have to be varied continuously during the extraction process to maximize selectivity and recovery for pesticide residue analysis (Rissato et al., 2005). SFE provides selective extraction of different chemicals without an additional cleanup step, and also allows the use of a smaller sample size (Pace et al., 1999). Despite these advantages, SFE has not been widely adopted in official methods or analytical protocols for soil pesticide analyses that rely on liquid–liquid/solid extraction (Alam et al., 2013). Forero-Mendieta et al. (2012) extracted a mixture of 31 pesticides in soil by SFE, using supercritical carbon dioxide
(SC-CO2) with methanol (MeOH) added as a co-solvent with simultaneous micro-electron-capture detection (μ-ECD) and nitrogen–phosphorus detection (NPD). It should be noted that none of the analytes tested in the current study was studied in their work. In addition, these authors did not report any details regarding the application of the method or analyte confirmation by GC-MS. No studies on the extraction of fluquinconazole and/or tetraconazole from soil using SFE have been reported, although Alam et al. (2013) did report the use of liquid–liquid extraction to evaluate the persistence of tetraconazole in soil and water. According to the Rural Developmental Administration (Republic of Korea), data concerning the efficacy and residues of a new pesticide product should be generated for each particular crop in order to establish the maximum residue limit. Thus, the aim of this study was to develop an analytical method to detect fluquinconazole and tetraconazole residues in soil using SFE and gas chromatography (GC). Development of an SFE method included the optimization of various parameters such as the inclusion of modifiers, temperature, pressure, and time. The method developed was then used to determine pesticide residues in soil collected from an onion field, and the dissipation pattern was investigated from the data. Both analytes were confirmed by GC-MS. This study was a part of an effort to use clean sample preparation techniques as a replacement for Soxhlet extraction and to reduce the occupational exposure of the analysts to toxic solvents and to prevent the disposal of these solvents to the environment by laboratories whose principal role should be to ensure environmental safety (Gonçalves et al., 2006).
S. I. Park et al. 0.01 g, China) of soil was inserted into a 10 mL stainless extraction vessel. The texture and physiochemical properties of the soil samples are shown in Table 1. The samples were extracted by SFE procedure using SC-CO2 containing methanol. The dynamic extraction time, extraction temperature,
pressure and modifier ratios were optimized for SFE. The optimal extraction conditions were achieved by varying the experimental parameters sequentially, one at a time, while all other parameters remained fixed (Abd El-Aty et al., 2009). The extraction conditions were changed as
Table 1. Soil texture and physiochemical properties Particle size (%) Clay Silt 13.2
Sand Soil texture
33.6 53.2
Cation exchange capacity (cmol/kg)
Sandy loam
pH
6.4
6.3
Organic Total nitrogen matter (g/kg) (g/kg) 11.4
0.6
NH4 +
NO3 -1
(mg N kg ) 51.8
111.7
Available phosphate (mg P/kg) 51.0
Figure 2. Extraction profile of fluquinconazole and tetraconazole from fortified soil at various temperatures, modifier ratios, pressures and extraction times.
Table 2. Calibration curve and determination coefficients in the linear range, limit of detection (LOD) and limit of quantification (LOQ) for fluquinconazole and tetraconazole in soil using solid-phase extraction Compound Fluquinconazole Tetraconazole
Linear range (μg/mL)
Linear regression equation
R2
0.002–0.2 0.01–1
y = 31,608x +213 y = 2,700,174x + 23,572
0.998 0.995
LOD (μg/g) 0.002 0.01
LOQ (μg/g) 0.01 0.05
Table 3. Recoveries and repeatability (intra-day and inter-day) for fluquinconazole and tetraconazole in soil using the solid phase extraction method Compound
Fortification levels (μg/g) (n = 3)
Fluquinconazole
Tetraconazole
0.01 0.02 0.1 0.05 0.1 0.5
Mean recovery (%) 92.4 89.4 94.1 83.7 89.2 92.7
Intra-daya RSD (%) 2.3 10.6 7.2 2.3 5.2 1.3
Inter-dayb RSD (%) 4.0 11.9 7.1 5.2 6.2 2.2
Intra-day repeatability was calculated by analyzing triplicate samples at three fluquinconazole and tetraconazole concentrations on the same day (n = 9). b Inter-day repeatability was calculated by analyzing of triplicate samples at three fluquinconazole and tetraconazole concentrations on three consecutive days (n = 27). a
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Detecting fungicides in soil follows: temperature, 50, 60 and 80 °C; pressure, 250, 280, 300 and 2 320 kg/cm ; and modifier ratio, 7.5, 10 and 15% methanol. The prelimi2 nary conditions were: oven temperature, 80 °C; pressure, 300 kg/cm ; extraction time, 30 min; modifier ratio, 10%; and total flow rate, 3 mL/min. After SFE, the extract was placed in a round-bottomed flask and evaporated at 40 °C in a rotary vacuum evaporator (Büchi Rotavapor R-114, Essen, Germany). The residue was then reconstituted in 2 mL of acetone for GC analysis.
Gas chromatography GC-μECD analysis of fluquinconazole. A 7890 A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a 63Ni microelectron capture detector and an Agilent Technologies 7683 B auto-injector, was utilized to detect fluquinconazole residues in soil. Separations were
conducted on an HP-Ultra 2 column (50 m × 0.32 mm i.d. × 0.17 μm film thickness; J&W Scientific Products, Folsom, CA, USA) with the following operating conditions: oven temperature 120 °C increased to 280 °C at 10 C/min, and held for 4 min. The temperatures in the injection port and detector were 270 and 300 °C, respectively. Nitrogen was utilized as the carrier and make-up gas at a flow rate of 2 and 60 mL/min, respectively. All injections were made in a split ratio of 10:1 with a 2 μL injection volume. The target analyte was eluted at 15.586 min. GC-NPD analysis of tetraconazole. An Agilent Technologies 6890 N, Network GC system, equipped with a nitrogen–phophorus detector and an Agilent Technologies 7683 B auto-injector, was used to detect tetraconazole in soil residues. The gas chromatograph was fitted with a DB-5 fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific Products). Helium was used as a carrier and
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Figure 3. Gas chromatography–electron capture detection chromatograms of blank uncontaminated soil (A), standard fluquinconazole (B), fortified soil sample at 0.1 μg/g (C), and a field sample collected on day 56 post-application (D).
S. I. Park et al. make-up gas with a flow rate of 1 and 4 mL/min, respectively. The air and hydrogen detector flow rates were set at 60 and 3 mL/min, respectively. The following temperature program was employed: initial temperature of 120 °C, increased at 10 °C/min to 280 °C, and held for 4 min. The detector and injector temperatures were 250 and 280 °C, respectively. All injections were made in splitless mode with a 2 μL injection volume. The target analyte was eluted at 11.190 min. GC-MS confirmation. An Agilent Technologies 6890 gas chromatograph was used to confirm the identity of fluquinconazole and tetraconazole. The system was fitted with an Agilent 5973 N mass-selective quadrupole detector and an Agilent 7683B auto-sampler. Compounds were separated on an HP-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific Products). The initial oven temperature was 120 °C and then increased to 280 °C at 15 °C/min and held for 9 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The temperatures of the ion source and quadrupole were 230 and 150 °C, respectively. The injector temperature was 250 °C, and a 2 μL sample was injected in splitless mode. The mass spectrometer was operated in electron ionization mode (70 eV), and the analysis was carried out in selected ion monitoring mode in which three characteristic ions (m/z 298, 340, and 342 for fluquinconazole; and m/z 171, 336, and 338 for tetraconazole) were selected (Húšková et al., 2008). The preferred (target) ions are in bold. Under the above-mentioned conditions, tetraconazole and fluquinconazole were confirmed at retention times of 13.360 and 21.929 min, respectively. Field experiment. A field experiment was conducted at an onion farm located at Chonnam National University, Gwangju, Republic of Korea. Three plots were used for each treatment along with one plot as a control. No fungicides were applied to the control plot prior to or during the experiment. The pesticide product applied was a suspoemulsion (Donbu Farm Hannong, Seoul, Republic of Korea) that contained 14% fluquinconazole and 7% tetraconazole. The pesticide product was diluted 1000 times to reach the manufacturer’s recommended dose. The commercial formulations were sprayed onto the soil before planting the onions, and the field was covered with mulching film. Samples were collected randomly at 0 (2 h), 7, 14, 28, 56, 105 and 133 days post-application.
SFE optimization A standard mixture of the two fungicides was added to 2 g of soil sample to reach a concentration of 1 μg/g, and the soil sample was then extracted as described above. The fortified soil samples were extracted by SFE using the preliminary conditions with the temperature being increased from 50 to 80 °C to achieve a high extraction yield. As shown in Fig. 2, the efficiency of extraction of both analytes increased when the temperature was increased from 50 to 60 °C, and decreased thereafter. Therefore, the optimal extraction temperature of 60 °C was used throughout the study. Selecting a suitable modifier for SFE is important. Supercritical CO2 is a nonpolar solvent that extracts nonpolar compounds efficiently but it is not suitable for the extraction of polar pesticides and/or metabolites (Gonçalves et al., 2006; Choi et al., 2009). Adding a modifier to the CO2 makes it possible to quantitatively extract such polar compounds. As methanol has been shown to behave as a universal modifier for pesticide analysis (Rissato et al., 2005; Gonçalves et al., 2006; ForeroMendieta et al., 2012), various ratios of methanol (7.5, 10 and 15%) were investigated for their extraction efficiencies at a constant temperature in soil. A modifier ratio of 10% methanol was found to be optimal for the maximum extraction efficiency of the tested analytes in soil (Fig. 2).
Method performance. The quantification was conducted using calibration curves based on matrix-matched standard solutions. The 2 linearity was estimated by the determination coefficient (R ) of the calibration standards at concentrations of 0.002, 0.01, 0.02, 0.05, 0.1 and 0.2 μg/mL for fluquinconazole, and 0.01, 0.05, 0.1, 0.2, 0.5 and 1 μg/mL for tetraconazole. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as three and ten times the signalto-noise ratio, respectively. Recovery experiments for the SFE method were carried out at three different levels (0.01 (LOQ), 0.02 (2 × LOQ) and 0.1 (10 × LOQ) μg/g for fluquinconazole; and 0.05 (LOQ), 0.1 (2 × LOQ) and 0.5 (2 × LOQ) μg/g for tetraconazole) spiked in soil samples, in triplicate. The precision was calculated in terms of intra- and inter-day repeatability using the relative standard deviation.
Results and discussion
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We initially tried to analyze both compounds by GC-ECD; however, the sensitivity for tetraconazole was poor, and peak tailing was observed (data not shown). Thereafter, we improved the sensitivity and peak sharpness by using a pepper leaf matrix as an analyte protectant (Rahman et al., 2013). Although the sensitivity improved, overlapping peaks were noticed near the tetraconazole retention time when using the pepper leaf components (data not shown). Finally, the analyte was successfully identified using GC-NPD with good sensitivity and a sharp peak without interference.
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Figure 4. Gas chromatography–nitrogen–phosphorus detection chromatograms of blank uncontaminated soil (A), standard tetraconazole (B), fortified soil sample at 0.5 μg/g (C), and a field sample collected on day 7 post-application (D).
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Detecting fungicides in soil To determine the optimal extraction pressure, the fortified soil samples were extracted by SFE at 60 °C and a 10% modifier ratio with the pressure being varied from 250 to 320 kg/cm2. A higher extraction pressure at a fixed temperature leads to a higher SC-CO2 density, which results in an increase in its dissolving ability and selectivity. A pressure of 280 kg/cm2 was selected as optimal for the extraction of the target analytes from soil (Fig. 2). The fortified soil samples were extracted by SFE under the optimal conditions of temperature, pressure and modifier ratio, and extracts were recovered every 10 min up to 60 min. Both fungicides were fully extracted (>80%) within 50 min (Fig. 2). The recovery of both analytes from the soil samples was independent of the SFE flow rate. The extraction process appeared to have been controlled by desorption and not by analyte solubility (Hawthorne et al., 1995). Therefore, the rate-limiting step in the extraction process was the initial desorption of the analyte from the bound matrix sites.
Method validation Quantification was conducted via a matrix-matched standard solution, and the calibration curves were assessed at a concentration range of 0.002–0.2 μg/mL for fluquinconazole and 0.01–1 μg/mL for tetraconazole. The two compounds showed good linearity with r2 ≥ 0.995 (Table 2). The sensitivity of the method was provided as the LOD, which was calculated as three times the signal-to-noise ratio (Forero-Mendieta et al., 2012): 0.002 μg/g for fluquinconazole, and 0.01 μg/g for tetraconazole. The LOQ was evaluated as 10 times the signal-to-noise ratio (Forero-Mendieta et al., 2012): 0.01 μg/g for fluquinconazole and 0.05 μg/g for tetraconazole. Accuracy data were obtained from the recovery test conducted in triplicate using the fortified blank soil samples at three different fortification levels. The recovery rates were 89.4–94.1% for fluquinconazole and 83.7–92.7% for tetraconazole (Table 3). The
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Figure 5. Gas chromatography–mass spectrometry (MS) chromatogram (selected ion monitoring mode) of (A) standard mixture of fluquinconazole and tetraconazole (1 μg/g) and MS spectra of (B) a field sample treated after 7 days with tetraconazole and (C) fluquinconazole.
S. I. Park et al. precision was provided from the intra- and inter-day repeatability of target compound recovery at the same concentration levels, and the results were 1.3–10.6% for fluquinconazole and 2.2–11.9% for tetraconazole, respectively. As shown in Table 3, this technique afforded high extraction efficiency and accuracy. Sample chromatograms of the pesticides from blank uncontaminated soil, matrix-matched standards, fortified soil and field soil samples are shown in Figs 3 and 4. The soil blank did not interfere with the analyte peak. The GC-MS chromatogram (selected ion monitoring mode) is shown in Fig. 5. It should be noted that low recovery rates are more related to the clay content than to the organic matter of the soil (Clausen et al., 2001). The clays may have strong interaction with pesticides owing to their specific area and surface reactivity (Dean, 1996; Clausen et al., 2001; Li et al., 2003).
The rate of disappearance of fluquinconazole and tetraconazole was described with first-order kinetics, and the calculated formulations were y = 0.4669e0.0.0325t for fluquinconazole and y = 0.2052e0.0345t for tetraconazole. In addition, the half-lives of fluquinconazole and tetraconazole were estimated to be 21.3 and 20.1 days, respectively. Both half-lives were shorter than those reported by Alam et al. (2013) for tetraconazole in soil (66.9–86 days). This difference could be attributed to variations in the organic carbon content of the tested soils, which was somewhat higher in our samples (1.5%) than in those of Alam (0.5–1.21%). Our findings and those of Alam et al. (2013) were shorter than those reported for propiconazole (200 days; Thorstensen and Lode, 2001) and for flutriafol, epoxiconazole and triadimenol (more than 2 years; Singh, 2005).
Conclusion Dissipation of fungicides in soil under field conditions The initial fluquinconazole and tetraconazole concentrations in soil were 0.547 and 0.328 μg/g, respectively. A steady decrease in the residue levels was observed, to 0.24 and 0.079 μg/g by day 14, respectively. No fluquinconazole residue was found 133 days post-application and no tetraconazole was found 105 days post-application. The higher the soil organic carbon content, the lower the fungicide persistence in the soil. Fluquinconazole is strongly adsorbed to soil, and its degradation rate depends on temperature, soil moisture, soil pH and organic contents (MacBean, 2012). The fluquinconazole dissipation pattern is mainly a hydrolytic process; however, further degradation and mineralization may occur, possibly involving microbial action. The metabolites are finally transformed to soil-bound residues and CO2 (MacBean, 2012). Tetraconazole degraded slightly more rapidly in soil than fluquinconazole. Figure 6 shows the dissipation curves for fluquinconazole and tetraconazole in soil under field conditions. The residue data of both analytes in soil were analyzed using the following mathematical expression: C t ¼ C 0 ekt where Ct is the residual concentration at time t, C0 is the residual concentration at time t = 0, and k is the dissipation constant reflecting the degradation potential of the pesticides. The halflife (DT50) in soil was evaluated using the following equation: DT50 ¼ lnð2Þ k 1 :
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Figure 6. Dissipation of fluquinconazole and tetraconazole residues in soil under field conditions.
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An extraction and analytical method using SFE and GC-ECD/NPD was successfully established to detect fluquinconazole and tetraconazole in soil. SFE was an attractive technique for the determination of soil pesticide residues. The analytical performance parameters reflected good detection and precision of the SFE technique coupled to GC. Only small volumes of organic solvents (9 mL of methanol and 2 mL of acetone) were needed for SFE compared with other extraction methods. Thus, SFE, as an environmentally friendly technique, can be used as an extraction method to detect fluquinconazole and tetraconazole in soil.
Acknowledgement This study was supported by the MSIP (Ministry of Science, Ict & future Planning).
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(wileyonlinelibrary.com) DOI 10.1002/bmc.3094
Optimization of supercritical fluid extraction method for detection of fluquinconazole and tetraconazole in soil using gas chromatography and confirmation using GC-MS: application to dissipation kinetics Sang Im Parka†, Jong-Hyouk Parka†, Ah-Young Koa, A. M. Abd El-Atya,b*, Ayman Goudahb, Jin Janga, Md. Musfiqur Rahmana, Mi Ra Kimc and Jae-Han Shima* ABSTRACT: The aim of this study was to establish an analytical method to detect fluquinconazole and tetraconazole in soil using supercritical fluid extraction (SFE) and gas chromatography (GC). The optimal extraction conditions for SFE were: temperature, 60 °C; pressure, 280 kg/cm2; extraction time, 50 min; and a 10% modifier ratio. The linearity of the calibration curves was good and yielded a determination coefficient (R2) ≥ 0.995. The soil samples were fortified with known quantities of the analytes at three different concentrations (0.01, 0.02 and 0.1 μg/g for fluquinconazole; 0.05, 0.1 and 0.5 μg/g for tetraconazole), and the recoveries ranged between 83.7 and 94.1%. The intra- and inter-day relative standard deviations were 1.3–10.6 and 2.2–11.9% for fluquinconazole and tetraconazole, respectively. The limit of detection and limit of quantitation were 0.002 and 0.01 μg/g for fluquinconazole and 0.01 and 0.05 for tetraconazole, respectively. The method was successfully applied to the analysis of soil residues collected from an onion field. The results show that a combination of SFE and GC can be used as an environmentally friendly technique to detect fungicides in soil. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: fluquinconazole; tetraconazole; soil; supercritical fluid extraction; gas chromatography
Introduction
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Many environmental pollutants, pesticides in particular, are used widely to protect crops and seeds; however, their presence in food and soil poses a potential human health threat (Jin et al., 2004). Soil is an important agricultural resource that has the ability to retain agrochemicals, including pesticides. Soil is exposed to pesticides through direct application, accidental spillage, run-off from plant surfaces or incorporation of pesticidecontaminated plant material. Once in the soil, a pesticide may undergo adsorption, leaching and/or degradation (Fernandes et al., 2013). The possibility of groundwater pollution also exists, as soil leachate may contain pesticide residues. Thus, the persistence of fungicides in soil needs to be evaluated for their safe and judicious use (Alam et al., 2013). Fluquinconazole [3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2, 4-triazol-1-yl)quinazolin-4(3H)-one, Fig. 1] is a quinazoline-based sterol demethylation inhibitor fungicide that is active against Ascomycetes, Basidiomycetes, Deuteromycetes, and Mycosphaerella graminicola (anamorph Septoria tritici; Metcalfe et al., 2000; Mavroeidi and Shaw, 2005). Tetraconazole [(RS)-2-(2,4-dichlorophenyl) -3-(1H-1,2,4-triazol-1-yl)propyl 1,1,2,2-tetrafluoroethyl ether, Fig. 1] is a triazole fungicide that exerts its antifungal activity by inhibiting sterol biosynthesis (Stenersen, 2004). Tetraconazole has a broad spectrum of bioefficacy against powdery
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mildew and rust diseases of various agricultural crops (Banerjee et al., 2008). The half-life of fluquinconazole in apple is 15–32 days (Szpyrka and Walorczyks, 2013), and that of tetraconazole is 7 days in cucumber (Khalfallah et al., 1998), 5 days in sugar beet (Menkissoglu-Spiroudi et al., 1998), 4–5 days in mango (Alam et al., 2011) and 66.9–86 days in soil under different pH conditions (Alam et al., 2013).
* Correspondence to: J. H. Shim and A. M. Abd El-Aty, Biotechnology Research Institute, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500–757, Republic of Korea. Email: [email protected]; [email protected] †
The first two authors contributed equally to this article.
a
Biotechnology Research Institute, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea
b
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt
c
Regional FDA Hazardous Substances Analysis Division, Daejeon, Republic of Korea Abbreviations used: μ-ECD, micro-electron-capture detection; MeOH, methanol; NPD, nitrogen–phosphorus detection; SC-CO2, supercritical carbon dioxide
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Detecting fungicides in soil
Figure 1. Structures of (A) fluquinconazole (structural formula C16H8Cl2FN5O, molecular weight 376.172) and (B) tetraconazole (structural formula C13H11Cl2F4N3O, molecular weight 372.146).
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Experimental Chemicals and reagents Fluquinconazole (purity 98.5%) and tetraconazole (purity 98.0%) were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Acetone and methanol were supplied by Merck (Darmstadt, Germany). Air, nitrogen (purity 99.999%), helium (purity 99.999%) and hydrogen (purity 99.85%) used for gas chromatography were supplied by Gaswell Co. Ltd (Gwangju, Republic of Korea). All other chemicals were of analytical and/ or high-performance liquid chromatography grade.
Preparation of the standard solutions Standard compounds (10.2 mg of fluquinconazole and tetraconazole) were separately dissolved in acetone, and their volume was brought up to 100 mL to obtain a 100 μg/mL stock solution. A 20 μg/mL standard mixture of the two fungicides was prepared via volumetric dilutions of each stock solution with acetone.
SFE system A Jasco (Tokyo, Japan) PU980 system with two pumps was used for SFE. The master pump was fitted with a cooling jacket on the pump head for CO2 delivery, and a second pump was used to add the modifier. The pumps were connected to a 10 mL stainless SFE vessel (inside diameter, 10 mm; length, 15 cm) in a column oven chamber (Jasco, model CO-965) that was directly connected to a Jasco 880–81 backpressure regulator, used to control the pressures of the supercritical fluid CO2 and methanol in the system.
Sample preparation and extraction procedure Soil was air-dried in the shade and powdered by passing it through a 2 mm sieve before extraction. Exactly 2 g (Sartorius, BSA2202S-CW, d
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The efficient extraction of pesticides from soil samples is crucial owing to the complexity of the matrix and the diverse physicochemical properties of the tested analytes (Rashid et al., 2010), generally requiring a technique capable of extracting the bound residues at low concentrations. Therefore, methods that are simple, rapid, accurate, environmentally friendly and provide clean extracts are needed for the proper risk assessment of pesticide residues in water, soil and crops (Yang et al., 2010). Conventional liquid–liquid/solid extraction techniques such as Soxhlet extraction, sonication and mechanical shaking have been applied to extract pesticides from soil (Rissato et al., 2005). However, concerns about the hazards associated with most of the solvents used and the costs and environmental dangers of waste solvent disposal have led to the development of more efficient environmentally friendly techniques such as supercritical fluid extraction (SFE) (Dean, 1996). SFE has gained increased attention due to properties of supercritical fluids such as higher diffusivity and low viscosity (Smith, 1999). These properties are generally obtained by optimizing the pressure of the supercritical CO2 used for extraction and the temperature of the extraction cell (Dean, 1996). The experimental SFE parameters have to be varied continuously during the extraction process to maximize selectivity and recovery for pesticide residue analysis (Rissato et al., 2005). SFE provides selective extraction of different chemicals without an additional cleanup step, and also allows the use of a smaller sample size (Pace et al., 1999). Despite these advantages, SFE has not been widely adopted in official methods or analytical protocols for soil pesticide analyses that rely on liquid–liquid/solid extraction (Alam et al., 2013). Forero-Mendieta et al. (2012) extracted a mixture of 31 pesticides in soil by SFE, using supercritical carbon dioxide
(SC-CO2) with methanol (MeOH) added as a co-solvent with simultaneous micro-electron-capture detection (μ-ECD) and nitrogen–phosphorus detection (NPD). It should be noted that none of the analytes tested in the current study was studied in their work. In addition, these authors did not report any details regarding the application of the method or analyte confirmation by GC-MS. No studies on the extraction of fluquinconazole and/or tetraconazole from soil using SFE have been reported, although Alam et al. (2013) did report the use of liquid–liquid extraction to evaluate the persistence of tetraconazole in soil and water. According to the Rural Developmental Administration (Republic of Korea), data concerning the efficacy and residues of a new pesticide product should be generated for each particular crop in order to establish the maximum residue limit. Thus, the aim of this study was to develop an analytical method to detect fluquinconazole and tetraconazole residues in soil using SFE and gas chromatography (GC). Development of an SFE method included the optimization of various parameters such as the inclusion of modifiers, temperature, pressure, and time. The method developed was then used to determine pesticide residues in soil collected from an onion field, and the dissipation pattern was investigated from the data. Both analytes were confirmed by GC-MS. This study was a part of an effort to use clean sample preparation techniques as a replacement for Soxhlet extraction and to reduce the occupational exposure of the analysts to toxic solvents and to prevent the disposal of these solvents to the environment by laboratories whose principal role should be to ensure environmental safety (Gonçalves et al., 2006).
S. I. Park et al. 0.01 g, China) of soil was inserted into a 10 mL stainless extraction vessel. The texture and physiochemical properties of the soil samples are shown in Table 1. The samples were extracted by SFE procedure using SC-CO2 containing methanol. The dynamic extraction time, extraction temperature,
pressure and modifier ratios were optimized for SFE. The optimal extraction conditions were achieved by varying the experimental parameters sequentially, one at a time, while all other parameters remained fixed (Abd El-Aty et al., 2009). The extraction conditions were changed as
Table 1. Soil texture and physiochemical properties Particle size (%) Clay Silt 13.2
Sand Soil texture
33.6 53.2
Cation exchange capacity (cmol/kg)
Sandy loam
pH
6.4
6.3
Organic Total nitrogen matter (g/kg) (g/kg) 11.4
0.6
NH4 +
NO3 -1
(mg N kg ) 51.8
111.7
Available phosphate (mg P/kg) 51.0
Figure 2. Extraction profile of fluquinconazole and tetraconazole from fortified soil at various temperatures, modifier ratios, pressures and extraction times.
Table 2. Calibration curve and determination coefficients in the linear range, limit of detection (LOD) and limit of quantification (LOQ) for fluquinconazole and tetraconazole in soil using solid-phase extraction Compound Fluquinconazole Tetraconazole
Linear range (μg/mL)
Linear regression equation
R2
0.002–0.2 0.01–1
y = 31,608x +213 y = 2,700,174x + 23,572
0.998 0.995
LOD (μg/g) 0.002 0.01
LOQ (μg/g) 0.01 0.05
Table 3. Recoveries and repeatability (intra-day and inter-day) for fluquinconazole and tetraconazole in soil using the solid phase extraction method Compound
Fortification levels (μg/g) (n = 3)
Fluquinconazole
Tetraconazole
0.01 0.02 0.1 0.05 0.1 0.5
Mean recovery (%) 92.4 89.4 94.1 83.7 89.2 92.7
Intra-daya RSD (%) 2.3 10.6 7.2 2.3 5.2 1.3
Inter-dayb RSD (%) 4.0 11.9 7.1 5.2 6.2 2.2
Intra-day repeatability was calculated by analyzing triplicate samples at three fluquinconazole and tetraconazole concentrations on the same day (n = 9). b Inter-day repeatability was calculated by analyzing of triplicate samples at three fluquinconazole and tetraconazole concentrations on three consecutive days (n = 27). a
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Detecting fungicides in soil follows: temperature, 50, 60 and 80 °C; pressure, 250, 280, 300 and 2 320 kg/cm ; and modifier ratio, 7.5, 10 and 15% methanol. The prelimi2 nary conditions were: oven temperature, 80 °C; pressure, 300 kg/cm ; extraction time, 30 min; modifier ratio, 10%; and total flow rate, 3 mL/min. After SFE, the extract was placed in a round-bottomed flask and evaporated at 40 °C in a rotary vacuum evaporator (Büchi Rotavapor R-114, Essen, Germany). The residue was then reconstituted in 2 mL of acetone for GC analysis.
Gas chromatography GC-μECD analysis of fluquinconazole. A 7890 A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a 63Ni microelectron capture detector and an Agilent Technologies 7683 B auto-injector, was utilized to detect fluquinconazole residues in soil. Separations were
conducted on an HP-Ultra 2 column (50 m × 0.32 mm i.d. × 0.17 μm film thickness; J&W Scientific Products, Folsom, CA, USA) with the following operating conditions: oven temperature 120 °C increased to 280 °C at 10 C/min, and held for 4 min. The temperatures in the injection port and detector were 270 and 300 °C, respectively. Nitrogen was utilized as the carrier and make-up gas at a flow rate of 2 and 60 mL/min, respectively. All injections were made in a split ratio of 10:1 with a 2 μL injection volume. The target analyte was eluted at 15.586 min. GC-NPD analysis of tetraconazole. An Agilent Technologies 6890 N, Network GC system, equipped with a nitrogen–phophorus detector and an Agilent Technologies 7683 B auto-injector, was used to detect tetraconazole in soil residues. The gas chromatograph was fitted with a DB-5 fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific Products). Helium was used as a carrier and
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Figure 3. Gas chromatography–electron capture detection chromatograms of blank uncontaminated soil (A), standard fluquinconazole (B), fortified soil sample at 0.1 μg/g (C), and a field sample collected on day 56 post-application (D).
S. I. Park et al. make-up gas with a flow rate of 1 and 4 mL/min, respectively. The air and hydrogen detector flow rates were set at 60 and 3 mL/min, respectively. The following temperature program was employed: initial temperature of 120 °C, increased at 10 °C/min to 280 °C, and held for 4 min. The detector and injector temperatures were 250 and 280 °C, respectively. All injections were made in splitless mode with a 2 μL injection volume. The target analyte was eluted at 11.190 min. GC-MS confirmation. An Agilent Technologies 6890 gas chromatograph was used to confirm the identity of fluquinconazole and tetraconazole. The system was fitted with an Agilent 5973 N mass-selective quadrupole detector and an Agilent 7683B auto-sampler. Compounds were separated on an HP-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific Products). The initial oven temperature was 120 °C and then increased to 280 °C at 15 °C/min and held for 9 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The temperatures of the ion source and quadrupole were 230 and 150 °C, respectively. The injector temperature was 250 °C, and a 2 μL sample was injected in splitless mode. The mass spectrometer was operated in electron ionization mode (70 eV), and the analysis was carried out in selected ion monitoring mode in which three characteristic ions (m/z 298, 340, and 342 for fluquinconazole; and m/z 171, 336, and 338 for tetraconazole) were selected (Húšková et al., 2008). The preferred (target) ions are in bold. Under the above-mentioned conditions, tetraconazole and fluquinconazole were confirmed at retention times of 13.360 and 21.929 min, respectively. Field experiment. A field experiment was conducted at an onion farm located at Chonnam National University, Gwangju, Republic of Korea. Three plots were used for each treatment along with one plot as a control. No fungicides were applied to the control plot prior to or during the experiment. The pesticide product applied was a suspoemulsion (Donbu Farm Hannong, Seoul, Republic of Korea) that contained 14% fluquinconazole and 7% tetraconazole. The pesticide product was diluted 1000 times to reach the manufacturer’s recommended dose. The commercial formulations were sprayed onto the soil before planting the onions, and the field was covered with mulching film. Samples were collected randomly at 0 (2 h), 7, 14, 28, 56, 105 and 133 days post-application.
SFE optimization A standard mixture of the two fungicides was added to 2 g of soil sample to reach a concentration of 1 μg/g, and the soil sample was then extracted as described above. The fortified soil samples were extracted by SFE using the preliminary conditions with the temperature being increased from 50 to 80 °C to achieve a high extraction yield. As shown in Fig. 2, the efficiency of extraction of both analytes increased when the temperature was increased from 50 to 60 °C, and decreased thereafter. Therefore, the optimal extraction temperature of 60 °C was used throughout the study. Selecting a suitable modifier for SFE is important. Supercritical CO2 is a nonpolar solvent that extracts nonpolar compounds efficiently but it is not suitable for the extraction of polar pesticides and/or metabolites (Gonçalves et al., 2006; Choi et al., 2009). Adding a modifier to the CO2 makes it possible to quantitatively extract such polar compounds. As methanol has been shown to behave as a universal modifier for pesticide analysis (Rissato et al., 2005; Gonçalves et al., 2006; ForeroMendieta et al., 2012), various ratios of methanol (7.5, 10 and 15%) were investigated for their extraction efficiencies at a constant temperature in soil. A modifier ratio of 10% methanol was found to be optimal for the maximum extraction efficiency of the tested analytes in soil (Fig. 2).
Method performance. The quantification was conducted using calibration curves based on matrix-matched standard solutions. The 2 linearity was estimated by the determination coefficient (R ) of the calibration standards at concentrations of 0.002, 0.01, 0.02, 0.05, 0.1 and 0.2 μg/mL for fluquinconazole, and 0.01, 0.05, 0.1, 0.2, 0.5 and 1 μg/mL for tetraconazole. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated as three and ten times the signalto-noise ratio, respectively. Recovery experiments for the SFE method were carried out at three different levels (0.01 (LOQ), 0.02 (2 × LOQ) and 0.1 (10 × LOQ) μg/g for fluquinconazole; and 0.05 (LOQ), 0.1 (2 × LOQ) and 0.5 (2 × LOQ) μg/g for tetraconazole) spiked in soil samples, in triplicate. The precision was calculated in terms of intra- and inter-day repeatability using the relative standard deviation.
Results and discussion
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We initially tried to analyze both compounds by GC-ECD; however, the sensitivity for tetraconazole was poor, and peak tailing was observed (data not shown). Thereafter, we improved the sensitivity and peak sharpness by using a pepper leaf matrix as an analyte protectant (Rahman et al., 2013). Although the sensitivity improved, overlapping peaks were noticed near the tetraconazole retention time when using the pepper leaf components (data not shown). Finally, the analyte was successfully identified using GC-NPD with good sensitivity and a sharp peak without interference.
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Figure 4. Gas chromatography–nitrogen–phosphorus detection chromatograms of blank uncontaminated soil (A), standard tetraconazole (B), fortified soil sample at 0.5 μg/g (C), and a field sample collected on day 7 post-application (D).
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Detecting fungicides in soil To determine the optimal extraction pressure, the fortified soil samples were extracted by SFE at 60 °C and a 10% modifier ratio with the pressure being varied from 250 to 320 kg/cm2. A higher extraction pressure at a fixed temperature leads to a higher SC-CO2 density, which results in an increase in its dissolving ability and selectivity. A pressure of 280 kg/cm2 was selected as optimal for the extraction of the target analytes from soil (Fig. 2). The fortified soil samples were extracted by SFE under the optimal conditions of temperature, pressure and modifier ratio, and extracts were recovered every 10 min up to 60 min. Both fungicides were fully extracted (>80%) within 50 min (Fig. 2). The recovery of both analytes from the soil samples was independent of the SFE flow rate. The extraction process appeared to have been controlled by desorption and not by analyte solubility (Hawthorne et al., 1995). Therefore, the rate-limiting step in the extraction process was the initial desorption of the analyte from the bound matrix sites.
Method validation Quantification was conducted via a matrix-matched standard solution, and the calibration curves were assessed at a concentration range of 0.002–0.2 μg/mL for fluquinconazole and 0.01–1 μg/mL for tetraconazole. The two compounds showed good linearity with r2 ≥ 0.995 (Table 2). The sensitivity of the method was provided as the LOD, which was calculated as three times the signal-to-noise ratio (Forero-Mendieta et al., 2012): 0.002 μg/g for fluquinconazole, and 0.01 μg/g for tetraconazole. The LOQ was evaluated as 10 times the signal-to-noise ratio (Forero-Mendieta et al., 2012): 0.01 μg/g for fluquinconazole and 0.05 μg/g for tetraconazole. Accuracy data were obtained from the recovery test conducted in triplicate using the fortified blank soil samples at three different fortification levels. The recovery rates were 89.4–94.1% for fluquinconazole and 83.7–92.7% for tetraconazole (Table 3). The
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Figure 5. Gas chromatography–mass spectrometry (MS) chromatogram (selected ion monitoring mode) of (A) standard mixture of fluquinconazole and tetraconazole (1 μg/g) and MS spectra of (B) a field sample treated after 7 days with tetraconazole and (C) fluquinconazole.
S. I. Park et al. precision was provided from the intra- and inter-day repeatability of target compound recovery at the same concentration levels, and the results were 1.3–10.6% for fluquinconazole and 2.2–11.9% for tetraconazole, respectively. As shown in Table 3, this technique afforded high extraction efficiency and accuracy. Sample chromatograms of the pesticides from blank uncontaminated soil, matrix-matched standards, fortified soil and field soil samples are shown in Figs 3 and 4. The soil blank did not interfere with the analyte peak. The GC-MS chromatogram (selected ion monitoring mode) is shown in Fig. 5. It should be noted that low recovery rates are more related to the clay content than to the organic matter of the soil (Clausen et al., 2001). The clays may have strong interaction with pesticides owing to their specific area and surface reactivity (Dean, 1996; Clausen et al., 2001; Li et al., 2003).
The rate of disappearance of fluquinconazole and tetraconazole was described with first-order kinetics, and the calculated formulations were y = 0.4669e0.0.0325t for fluquinconazole and y = 0.2052e0.0345t for tetraconazole. In addition, the half-lives of fluquinconazole and tetraconazole were estimated to be 21.3 and 20.1 days, respectively. Both half-lives were shorter than those reported by Alam et al. (2013) for tetraconazole in soil (66.9–86 days). This difference could be attributed to variations in the organic carbon content of the tested soils, which was somewhat higher in our samples (1.5%) than in those of Alam (0.5–1.21%). Our findings and those of Alam et al. (2013) were shorter than those reported for propiconazole (200 days; Thorstensen and Lode, 2001) and for flutriafol, epoxiconazole and triadimenol (more than 2 years; Singh, 2005).
Conclusion Dissipation of fungicides in soil under field conditions The initial fluquinconazole and tetraconazole concentrations in soil were 0.547 and 0.328 μg/g, respectively. A steady decrease in the residue levels was observed, to 0.24 and 0.079 μg/g by day 14, respectively. No fluquinconazole residue was found 133 days post-application and no tetraconazole was found 105 days post-application. The higher the soil organic carbon content, the lower the fungicide persistence in the soil. Fluquinconazole is strongly adsorbed to soil, and its degradation rate depends on temperature, soil moisture, soil pH and organic contents (MacBean, 2012). The fluquinconazole dissipation pattern is mainly a hydrolytic process; however, further degradation and mineralization may occur, possibly involving microbial action. The metabolites are finally transformed to soil-bound residues and CO2 (MacBean, 2012). Tetraconazole degraded slightly more rapidly in soil than fluquinconazole. Figure 6 shows the dissipation curves for fluquinconazole and tetraconazole in soil under field conditions. The residue data of both analytes in soil were analyzed using the following mathematical expression: C t ¼ C 0 ekt where Ct is the residual concentration at time t, C0 is the residual concentration at time t = 0, and k is the dissipation constant reflecting the degradation potential of the pesticides. The halflife (DT50) in soil was evaluated using the following equation: DT50 ¼ lnð2Þ k 1 :
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Figure 6. Dissipation of fluquinconazole and tetraconazole residues in soil under field conditions.
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An extraction and analytical method using SFE and GC-ECD/NPD was successfully established to detect fluquinconazole and tetraconazole in soil. SFE was an attractive technique for the determination of soil pesticide residues. The analytical performance parameters reflected good detection and precision of the SFE technique coupled to GC. Only small volumes of organic solvents (9 mL of methanol and 2 mL of acetone) were needed for SFE compared with other extraction methods. Thus, SFE, as an environmentally friendly technique, can be used as an extraction method to detect fluquinconazole and tetraconazole in soil.
Acknowledgement This study was supported by the MSIP (Ministry of Science, Ict & future Planning).
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