Microchemical Journal 96 (2010) 139–145
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Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
Development, validation and application of a method based on DI-SPME and GC–MS for determination of pesticides of different chemical groups in surface and groundwater samples Adalberto Menezes Filho a,b, Fábio Neves dos Santos a, Pedro Afonso de P. Pereira a,c,d,⁎ a
Instituto de Química, Universidade Federal da Bahia, Campus Universitário de Ondina, 40170-290 Salvador, BA, Brazil Instituto Federal de Educação, Ciência e Tecnologia (IF), 49055-260 Aracaju, SE, Brazil c CIEnAm – Centro Interdisciplinar de Energia e Ambiente – Salvador, BA, Brazil d INCT – Instituto Nacional de Ciência e Tecnologia de Energia e Ambiente – Salvador, BA, Brazil b
a r t i c l e
i n f o
Article history: Received 20 November 2009 Received in revised form 23 February 2010 Accepted 24 February 2010 Available online 10 March 2010 Keywords: Water Pesticides SPME GC–MS
a b s t r a c t A simple and rapid method based on solid-phase micro extraction (SPME) technique followed by gas chromatography–mass spectrometry with selected ion monitoring (GC–MS, SIM) was developed by the simultaneous determination of 16 pesticides of seven different chemical groups [Six organophosphorus (trichlorfon, diazinon, methyl parathion, malathion, fenthion and ethyon), three pyrethroids (bifenhin, permethrin, cypermethrin), two imidazoles (imazalil and prochloraz), two strobilurins (azoxystrobin and pyraclostrobin), one carbamate (carbofuran), one tetrazine (clofentezine), and one triazole (difenoconazole)] in water. The pesticides extraction was done with direct immersion mode (DI-SPME) of the polyacrilate fiber (PA 85 µm). The extraction temperature was adjusted to 50 °C during 30 min, while stirring at 250 rpm was applied. After extraction, the fiber was introduced in the GC injector for thermal desorption for 5 min. at 280 °C. The method was validated using ultra pure water samples fortified with pesticides at different concentration levels and shows good linearity in the concentrations between 0.05 and 250.00 ng mL− 1. The LOD and LOQ ranged, from 0.02 to 0.30 ng mL− 1 and 0.05 to 1.00 ng mL− 1, respectively. Intra-day and inter-day precisions were determined in two concentration levels (5.00 and 50.00 ng mL− 1). Intra-day relative standard deviation (%R.S.D.) ranged between 3.6 and 13.6%, and inter-day (%R.S.D.) ranged between 6.3 and 18.5%. Relative recovery tests were carried out spiking the ultra pure sample with standards in three different concentration levels 0.20, 5.00 and 50.00 ng mL− 1. The recovery at 0.20 ng mL− 1 level varied from 86.4 ± 9.4% to 108.5 ± 10.5%, at 5.00 ng mL− 1 level varied from 77.5 ± 10.8% to 104.6 ± 9.6% and at 50.00 ng mL− 1 level varied from 70.2 ± 4.6% to 98.4 ± 8.5%. The proposed SPME method was applied in twenty-six water samples collected in the “Platô de Neópolis”, State of Sergipe, Brazil. Methyl parathion was detected in five samples with an average concentration of 0.17 ng mL− 1 and bifenthrin, pyraclostrobin and azoxystrobin residues were found in three samples with average concentrations of 2.28, 3.12 and 0.15 ng mL− 1, respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Pesticides are widely used in pest control in agriculture with the objective of increasing agricultural productivity. It is one of the sources of water contamination with serious risks to human and animal health. Additionally, it causes changes in the ecosystem with harmful consequences for the environment and agriculture, due to the emergence and spread of new pests and disease with consequent increasing in the need for using more pesticides [1]. Due to the intensive use of pesticides and the persistence of these compounds, ⁎ Corresponding author. Instituto de Química, Universidade Federal da Bahia, Campus Universitário de Ondina, 40170-290 Salvador, BA, Brazil. E-mail address:
[email protected] (P.A.P. Pereira). 0026-265X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2010.02.018
residues can be found in different environmental compartments, including groundwater and surface water [2]. Water resources are the most affected due to agriculture requires a water supply, leading the development of this activity to areas close to rivers and lakes [3]. For the control of level concentration of pesticide residues in water, the Ministry of Health in Brazil established the Value Maximum Allowable by decree no. 518 on March 25, 2004 [4]. For environmental and drinking water, the maximum admissible concentration of a single compound established by the European Union (EU) is 0.1 µg L− 1, and 0.5 µg L− 1 is the maximum allowed for the total concentration of all pesticides [5]. Because of the pesticides toxicity and their harmful effects to the environment, especially in water, the development of efficient analytical methods to detect the presence of pesticides in water is
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in crescent interest and significant importance to environmental protection. Thus, continuous monitoring of pesticides residues in environmental samples has great importance. However, it has paramount importance to develop faster and more selective analytical methodologies, with higher cost-benefit ratios, that are less harmful to the environment and more sensitive to trace levels of pesticide residues in natural and drinking waters [6]. Multi-residue analytical methods have been proposed for the simultaneous determination of pesticides in water. However, these methods are generally difficult to develop, since the targeted compounds present different degrees of polarity, solubility, and volatility, as well as different values of pKa, making their extraction and analysis difficult [7], as shown in Table 1. Traditionally, conventional multi-residue analytical methods have been developed for the analysis of pesticides in water based on liquid– liquid extraction [8] and solid-phase extraction [9,10]. Recently, singledrop micro extraction [6,11] and solid-phase micro extraction [12,13] were applied to determine pesticides in water as an alternative to such traditional analytical methods establishing certain quality compromises [14]. SPME is an important method of sample preparation; it is solvent free and incorporates sample extraction, concentration and sample introduction into a single procedure [15]. The use of techniques such as solid-phase micro extraction can be considered such as a selective and useful tool for automatic sampling and sample treatment, avoiding the systematic errors [14]. Analytical methods have been proposed to determine compound belonging to different pesticide families by SPME, as organochlorine [12,16–18], pyrethroids [13], anilide and triazine [1] and organophosphorus [19,20]. However, most of these studies do not include analytical validation data for strobilurin, imidazole, triazole and tetrazine or these combined with organophosphates, pyrethroids or carbamates in water samples. Gas chromatography is the determination technique usually used with different detectors. Organophosphorus compounds are detected with higher sensitivity when using gas chromatography/nitrogenphosphorous detection (GC-NPD) [8,21] and gas chromatography– mass spectrometry [14,22]. Pyrethroids and imidazoles compouds are detected from gas chromatography/electron capture detection (GC-ECD) with excellent sensitivity [8,13,16,23,24]. Strobilurins fungicides were detected in low concentrations in food by gas chromatography–mass spectrometry (GC–MS) [14,25]. This detection system is most suitable for multi-residue analysis of pesticides belonging to different chemical groups as organophosphorus, carbamate, tetrazine, triazole, imidazoles, pyrethroids and strobilurins. The GC–MS provide high selectivity and mass resolution to reduce potential interference and enable the method to routinely achieve low levels of chemical detection. Recognizing the negative effects of water contamination by pesticides, the present work reports a simple methodology for simultaneous
Table 1 Chemical groups, log kow and solubility in water of the pesticides selected for the study. Pesticides
Chemical groups
log kow
Trichlorfon Diazinon Methyl parathion Malathion Fenthion Ethion Clofentezine Carbofuran Difenoconazole Imazalil Prochloraz Bifenthrin Permethrin Cypermethrin Pyraclostrobin Azoxystrobin
Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Organophosphate Tetrazine Carbamate Triazole Imidazole Imidazole Pyrethroid Pyrethroid Pyrethroid Strobilurin Strobilurin
0.43 3.69 3.00 2.75 4.84 5.07 3.10 1.70 4.20 2.56 3.53 7.30 6.10 5.50 3.99 2.50
Solubility in water (mg L− 1) 1.20 · 105 6.00 · 10 5.50 · 10 1.45 · 102 4.20 2.00 2.00 · 10− 3 3.19 · 102 1.50 · 10 14.20 · 10 3.44 · 10 2.50 · 10− 3 2.00 · 10− 1 4.00 · 10− 3 1.90 6.70
determination of sixteen pesticides from seven different chemical groups in water for DI-SPME and GC–MS. The method developed and validated was applied to samples of water surface and groundwater collected in Plateau of Neópolis, region with projects of irrigated fruit culture, State of Sergipe, Brazil. The pesticides selected for the study belong to the following chemical groups: organophosphate (trichlorfon, diazinon, methyl parathion, malathion, fenthion and ethyon), carbamate (carbofuran), tetrazine (clofentezine), triazole (difenoconazole), imidazoles (imazalil and prochloraz), pyrethroids (bifenhin, permethrin, cypermethrin) and strobilurins (azoxystrobin and pyraclostrobin). Table 1 shows the pesticides selected for the study. 2. Experimental 2.1. Standards, reagents and materials Certified standards of clofentezine, carbofuran, fenthion, ethion, trichlorfon, imazalil, difenoconazole, bifenthrin, permethrin, cypermethrin, prochloraz, pyraclostrobin and azoxystrobin were acquired from AccuStandard (New Haven, CT, USA); methyl parathion (1000 µg ml− 1), malathion (1000 µg ml− 1) and diazinon (1000 µg ml− 1) were obtained from Absolute Standards (Hamden, Connecticut, USA). All standards had purities greater than 97.0%. Methanol and acetonitrile HPLC grade were acquired from J. T. Baker (USA); isopropanol was obtained from Merck (Dalmstadt, Germany); and sodium chloride (99.0%) was obtained from Nuclear (São Paulo, Brazil). Individual stock solutions of each standard were prepared in methanol in the 1000 µg mL− 1 concentration and stored at −18 °C. The exception was clofentezine, which was prepared in acetonitrile. The work standard containing the 16 pesticides was prepared by dilution in methanol of the stock solution up to a concentration of 10.0 µg mL− 1. This standard was used both for matrix spike, in order to optimize the extraction conditions (10.0 ng mL− 1) and in the validation study in different concentration levels (0.05 to 250 ng mL− 1). Calibration standards were prepared in the 0.05, 0.1, 0.2, 1.0, 5.0, 10.0, 50.0, 100.0 and 250.0 ng mL− 1 concentrations, by diluting the work standard directly into the matrix. Deionized water with conductivity of 18.2 MΩs was generated from a Nanopure Diamond water purification systems, Barnsted Model D11911 (Dubuque, Iowa, USA). 2.2. Instrumentation Extraction and routine analysis of pesticides were done with an auto-sampler CTC Combi-PAL (Zwinger, Sweden) coupled to a GC–MS Shimadzu QP2010 Plus (Kyoto, Japan) equipped with a split/split less injector in the split less mode and at 280 °C during the chromatographic run. A Restek Rtx®-1 MS (Crossbond® 100% polydimethylsiloxane) fused silica capillary column 30 m × 0.25 mm ID × 0.25 µm (film thickness) manufactured by Supelco (Bellefonte, PA, USA) was employed in the separation of pesticides, using helium 99.99% as carrier gas at a 1.0 mL min− 1 flow rate. The oven temperature was as followed: 60 °C (1 min); then 170 °C at 25 °C min− 1; then 290 °C at 6 °C min− 1 and hold at 290 °C for 1 min. The mass detector conditions were: transfer line temperature – 250 °C; ion source temperature – 230 °C; ionization mode – electron impact at 70 eV. The optimization of the retention times and chromatographic resolution were done in the SCAN mode and with a 1 µg mL− 1 standard. In order to quantify the pesticides in water, SIM (Selected ion monitoring) mode was then chosen and three specific ions were selected for each analysis. The following ions were then monitored: 137, 102 and 109 (clofentezine); 109, 79 and 185 (trichlorfon); 164, 149 and 131 (carbofuran); 137, 179 and 152 (diazinon); 109, 125 and 263 (methyl parathion); 127, 173 and 93 (malathion); 278, 125 and 109 (fenthion); 41, 215 and 173 (imazalil); 231, 97 and 125 (ethion);
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181, 165 and 166 (bifenthrin); 183, 163 and 165 (permethrin); 43, 70 and 180 (prochloraz); 181, 163 and 165 (cypermethrin); 132, 164 and 325 (pyraclostrobin); 265, 323 and 267 (difenoconazole) and 344 , 388 and 329 (azoxystrobin), being the first for measurement and the other two for confirmation. Fig. 1(a) shows the chromatogram of standard solution of the pesticides at 1.0 µg mL− 1 obtained by GC–MS in the SIM mode and Fig. 1(b) shows the chromatogram obtained to a spiked ultra pure water sample at 10 µg L− 1. The resolution was considered satisfactory. For the pesticides permethrin, cypermethrin and difenoconazole, which present stereoisomerism, two peaks were detected for each one, corresponding to the cis(Z) and trans (E) isomers. The SPME was done with a holder designed for auto sampler use model 23GA which was acquired from Supelco (Bellafonte, PA, USA). Silica SPME fiber polyacrilate (85 µm), polydimethylsiloxane (100 µm), polydimethylsiloxane-divinylbenzene (65 µm), divinylbenzene- carboxen-polydimethylsiloxane (50 µm) and carboxen-polydimethylsiloxane (85 µm) and amber glass screw cap vials (20 ml) for SPME with polytetrafluoroethylene (PTFE)/silicone septa were obtained from Supelco (Bellafonte, PA, USA). Since the selected pesticides belong to different chemical groups, with significant variations in their polarity, these fiber phases were chosen for testing in order to ensure the best extraction efficiency.
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2.3. The study area and pesticides selection The irrigation project Plateau of Neópolis, situated in the municipality of Neópolis (State of Sergipe, Brazil: 10°19′12″S, 36°34′ 46″W), the right margin of the São Francisco river (Fig. 2) has a total area of 104.32 km2, with 72.3 km2 of irrigated area and 32.0 km2 of environmental reserve [26]. The irrigated area is divided into 27 modules destined for fruit production for the internal and external markets. The main crops are citrus fruits, mango, pineapple, banana, papaya, passion and coconut. The coconut occupies an area of approximately 34.0% of the irrigated area. The expansion of fruit crops in the region was favored by the prevalence of large plane areas and proximity to a rich source of water for irrigation, allow intensify crops and increase the productivity of plants to maintain the rate cost/ benefice in competitive levels [27]. The compounds studied were selected after a visit to fruit producers in the irrigated project Plateau of Neópolis (area of study). 2.4. Sample collection Water samples (250 ml) were collected in new polyethylene bottles in October 2009. Twenty samples were collected at eight points in the Plateau of Neópolis, four samples were collected in the
Fig. 1. (a) — GC/MS (SIM) chromatogram of a standard solution of the pesticides at 1.0 mg L− 1. Peak identification: 1, clofentezine; 2, trichlorfon; 3, carbofuran; 4, diazinon; 5, methyl parathion; 6, malathion; 7, fenthion; 8, imazalil; 9, ethion; 10, bifenthrin; 11, permethrin; 12, prochloraz; 13, cypermethrin; 14, pyraclostrobin; 15, difenoconazole and 16, azoxystrobin. (b) — DI-SPME-GC/MS (SIM) chromatogram corresponding to a spiked ultra pure water sample (10 µg L− 1). Peak identification: 1, clofentezine; 2, trichlorfon; 3, carbofuran; 4, diazinon; 5, methyl parathion; 6, malathion; 7, fenthion; 8, imazalil; 9, ethion; 10, bifenthrin; 11, permethrin; 12, prochloraz; 13, cypermethrin; 14, pyraclostrobin; 15, difenoconazole and 16, azoxystrobin.
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Fig. 2. Map of the Plateau of Neópolis showing sampling points.
community Tenório, located 1 km from the irrigated area and used good water in their day-to-day. Two samples were collected in an agribusiness located 8 km from the irrigated area (see Fig. 2). The bottles were sealed immediately after sampling and stored in ice until arrival in the laboratory. After arriving at the laboratory, samples were filtered through 0.45 μm membrane to remove particulate matter and maintained under refrigeration at 4 °C until analysis.
followed by 10 s stopped and then followed by 30 s counterclockwise, in order to avoid any settlement in the vial's wall. After extraction, the fiber was introduced in the GC injector for thermal desorption for 5 min at 280 °C. After each extraction, the fiber was cleaned at 280 °C for 1 min in a helium atmosphere.
2.5. SPME procedure
3.1. SPME optimization
For extraction, samples of distilled and deionized water was spiked with 10 µL of working solution and the volume was adjusted to 10 ml and let for resting for 30 min. The resulting solution was then transferred to a 10.0 ml sample vial, capped with a silicone septum interfaced with Teflon. The vial was full up to the top with the sample solution in all cases. The SPME fiber utilized (85 µm polyacrilate) was firstly conditioned 2 h in the injection port as recommended by the manufacturer. A blank injection was performed to confirm removal of impurities from GC system and other blank experiments also carried out were those of fiber blanks. The pesticides extraction was done through the direct immersion mode (DI-SPME) by exposing 2.4 cm length of the polymer-coated fiber to the sample solution. The extraction temperature was adjusted to 50 °C during 30 min, while stirring at 250 rpm was applied in successive cycles of 30 s clockwise,
Fig. 3 shows that the 85 μm PA fiber gave the best results, followed by the 65 μm PDMS/DVB and finally the 100 μm PDMS fiber. A possible explaining for the fact that pyrethoids (bifenthrin, permethrin and cypermethrin) were better extracted onto 100 µm PDMS is their more pronounced lipophilic characteristic when compared to the other pesticides (Table 1). Thus, the PA fiber was selected for the method development throughout the study. Conventionally, DI-SPME is more sensitive than HS-SPME and it is thus the method of choice for the analysis of clean aqueous samples. These two extraction modes were evaluated and, despite the less sensitivity than HS-SPME in the case of the more volatile compounds, DI-SPME mode successfully extracted all the 16 pesticides while HS-SPME was able to extract only 12 compounds (Fig. 4). The first mode was then elected as the best choice for the development of the method. The influence of temperature was
3. Results and discussion
Fig. 3. Evaluation of five SPME fibers: PA (85 μm); PDMS (100 μm); PDMS/DVB (65 μm); CARB/PDMS (85 μm) and DVB/CARB/PDMS (50 μm). Pesticides identification: 1 = Trichlorfon; 2 = Diazinon; 3 = Methyl parathion; 4 = Malathion; 5 = Fenthion; 6 = Ethion; 7 = Carbofuran; 8 = Difenoconazole; 9 = Clofentezine; 10 = Imazalil; 11 = Prochloraz; 12= Bifenthrin; 13= Permethrin; 14 = Cypermethrin; 15 = Pyraclostrobin and 15= Azoxytrobin.
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Fig. 4. Evaluation of extraction mode (ID and HS).
evaluated starting from 30 to 60 °C. The majority of pesticides showed a signal enhancement at temperatures up to 50 °C. A temperature of 50 °C was chosen, since it was found to yield the best analytical signal for most of the compounds. The extraction time was evaluated through direct immersion extractions at 50 °C from 10 to 40 min. It was observed that the analytical signal for most of the compounds was enhanced for times up to 30 min, and thus this extraction time was chosen. The extraction was carried out at 250, 400 and 600 rpm and it was observed that, except for clofentezine, the signal decreased for all pesticides as the stirring velocity increased. Since the autosampler has a different way for stirring the sample (the fiber immersed in the sample remains immobilized while the vial rotates), this stirring mode causes an effect of washing the fiber and thus reduces the efficiency of extraction of the analytes as the stirring velocities increase. This phenomenon has been observed by Menezes Filho, et al (2010) [28]. The velocity of 250 rpm was thus chosen. The influence of ionic strength was evaluated by adding different amounts of NaCl. The 5% concentration yielded the best results for majority pesticides. However, this concentration impaired the repeatability of the results, therefore chose not add salt. The complete desorption of analytes from the fiber was evaluated with the injector temperature varied from 250 to 300 °C from 3 to 6 min. The conditions chosen were therefore 280 °C and 5 min. 3.2. Analytical parameters Figures of merit including the linear range (LR), limit of detection (LOD), limit of quantification (LOQ), repeatability (RSD, %) and recovery, in different levels of fortification, were evaluated for the validation of the SPME method proposed. The external standard calibration curve was constructed with eight standard solutions, sequentially analyzed in order of crescent concentrations. Each concentration was analyzed in triplicate. The linearity of the SPME method was studied by extracting aqueous solutions of the mixture of pesticides in the concentrations between 0.05 and 250.00 ng mL− 1. The correlation coefficients (R2) obtained varied from 0.9912 to 0.9998 as shown in Table 2. These are comparable to results obtained by other workers [12,17]. The LOD and LOQ were established using the signal to noise ratio for each compound, obtained through a standard for which a minimum signal was still measurable for each analyte. A 3:1 ratio was used as the limit of detection while a 10:1 ratio was used
as the limit of quantification. The LOD and LOQ ranged, respectively, from 0.02 to 0.30 ng mL− 1 and 0.05 to 1.00 ng mL− 1 as shown in Table 2. Intra-day and inter-day precisions of the method were determined from mixed aqueous solutions in two concentration levels (5.00 and 50.00 ng mL− 1) of each pesticide. As shown in Table 3, the intra-day coefficient of variation (RSD %) in the small level varied from 3.6% for azoxystrobin to 12.2% for imazalil and in the major level varied from 4.6% to methyl parathion to 13.6% to cypermethrin, while the inter-day (RSD %) in the small level varied from 6.4% for carbofuran to 18.5% for prochloraz and in the major level varied from 6.3% to azoxystrobin to 16.9% to prochloraz. Due to an extension at the base of the peak of prochloraz, causing variations in the integration, this compound showed values (RSD %) greater than 15% in the study of the inter-day precision. Since the SPME is a non-exhaustive extraction procedure, the relative recoveries were determined for each compound studied. The relative recoveries were based in the comparison of the average (n = 3) detector signals produced when directly spiking a known amount of each pesticide in surface or groundwater sample extracts, with the average (n = 3) detector signals produced when directly spiking the same amount of the pesticide in ultra pure water extracts.
Table 2 Analytical figures of merit obtained using proposed SPME method. Pesticides
R2
Linearity (ng mL− 1)
LOD (ng mL− 1)
LOQ (ng mL− 1)
Clofentezine Trichlorfon Carbofuran Diazinon Methyl parathion Malathion Fenthion Imazalil Ethion Bifenthrin Permethrin Prochloraz Cypermethrin Pyraclostrobin Difenoconazole Azoxystrobin
0.9998 0.9944 0.9966 0.9990 0.9997 0.9981 0.9920 0.9976 0.9912 0.9968 0.9967 0.9996 0.9987 0.9934 0.9980 0.9952
0.05–250.0 0.20–100.0 0.10–100.0 0.05–250.0 0.05–250.0 0.10–250.0 0.05–250.0 0.10–250.0 0.05–100.0 0.10–250.0 1.00–100.0 0.10–250.0 0.10–50.0 1.00–100.0 0.05–100.0 0.05–250.0
0.02 0.07 0.03 0.02 0.02 0.03 0.02 0.03 0.02 0.03 0.30 0.03 0.03 0.30 0.02 0.02
0.05 0.20 0.10 0.05 0.05 0.10 0.05 0.10 0.05 0.10 1.00 0.10 0.10 1.00 0.05 0.05
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Table 3 Intra-day and inter-day precisions (RSD %) of the proposed SPME method in two concentration levels. Pesticides
Clofentezine Trichlorfon Carbofuran Diazinon Methyl parathion Malathion Fenthion Imazalil Ethion Bifenthrin Permethrin Prochloraz Cypermethrin Pyraclostrobin Difenoconazole Azoxystrobin
Table 5 Evaluation of the pH effect on the extraction recoveries. Recovery and R.S.D (%) (5.0 ng mL− 1) (n = 3)
Pesticides
RSD % Intra-day (n = 5)
Inter-day (n = 22)
5.0 ng mL− 1
50.0 ng mL− 1
5.0 ng mL− 1
50.0 ng mL− 1
4.0 8.6 3.7 5.7 7.2 8.7 7.3 12.2 6.3 6.9 8.6 11.3 9.4 7.3 6.3 3.6
6.2 9.6 10.3 7.2 4.6 6.9 4.7 10.6 8.0 4.9 7.4 9.5 13.6 9.0 7.8 6.7
7.2 9.8 6.4 9.8 13.4 6.8 9.8 14.6 7.1 8.0 6.6 18.5 13.9 12.6 11.7 8.4
8.4 11.9 10.4 10.4 14.7 12.4 7.4 11.5 9.2 9.9 7.8 16.9 14.6 13.7 7.8 6.3
Clofentezine Trichlorfon Carbofuran Diazinon Methyl parathion Malathion Fenthion Imazalil Ethion Bifenthrin Permethrin Prochloraz Cypermethrin Pyraclostrobin Difenoconazole Azoxystrobin
pH 3.0
pH 7.0
pH 8.0
94.6 ± 9.4 86.0 ± 6.8 84.7 ± 7.6 93.7 ± 12.6 93.9 ± 12.4 86.2 ± 9.7 89.1 ± 8.7 86.7 ± 12.4 97.6 ± 14.0 72.3 ± 8.4 83.8 ± 12.3 94.8 ± 7.9 74.4 ± 12.0 97.6 ± 13.8 86.5 ± 7.9 89.0 ± 10.7
91.9 ± 6.8 83.9 ± 8.6 89.4 ± 4.8 87.0 ± 9.4 95.3 ± 14.3 82.3 ± 8.4 86.0 ± 11.4 98.4 ± 6.3 104.6 ± 9.6 77.5 ± 10.8 92.2 ± 7.2 90.7 ± 6.4 84.2 ± 8.4 93.4 ± 8.3 82.3 ± 9.5 92.6 ± 5.0
85.6 ± 4.9 79.4 ± 12.4 76.8 ± 11.7 77.0 ± 7.6 88.3 ± 8.7 78.8 ± 9.2 81.4 ± 6.9 93.4 ± 9.6 86.2 ± 7.4 84.1 ± 8.4 98.6 ± 10.6 88.3 ± 9.8 93.9 ± 10.5 84.6 ± 11.4 79.8 ± 5.7 87.0 ± 7.8
decomposition in alkaline medium, while the pyrethroids undergo hydrolysis in acidic medium [29]. No pesticides were detected in this ultra pure water sample using MS detection. Relative recovery tests were carried out at three different concentration levels, namely 0.20, 5.00 and 50.00 ng mL− 1, as shown in Table 4. The recovery at 0.20 ng mL− 1 level varied from 86.4 ± 9.4% for imazalil to 108.5 ± 10.5% for carbofuran, at 5.00 ng mL− 1 level varied from 77.5 ± 10.8% for bifenthrin to 104.6 ± 9.6% for ethion and 50.00 ng mL− 1 level varied from 70.2 ± 4.6% for azoxystrobin to 98.4 ± 8.5% for fenthion.
3.3. Evaluation of the pH effect Since other critical parameters of the method have been already evaluated and optimized, the effect of the pH on the sample extraction was also evaluated. Extractions were performed on samples of ultra pure water, spiked at a concentration of 5.0 ng mL− 1 and at pH values of 3.0, 7.0 and 8.0. Table 5 shows the mean recoveries and the respective R.S.D values. Briefly, the lowest recovery attained were 72.3 ± 8.4 for bifenthrin at pH 3.0, while the highest was 104.6 ± 9.6 for ethion at pH 7.0, thus showing that pH had little or no effect on the sample extraction, despite organophosphorous pesticides, in general, are more stable in acidic pH between 3.0 and 6.0 and undergo
Table 4 Average recovery (%) and R.S.D (%) of the proposed SPME method at three concentration levels. Pesticides
Clofentezine Trichlorfon Carbofuran Diazinon Methyl parathion Malathion Fenthion Imazalil Ethion Bifenthrin Permethrin Prochloraz Cypermethrin Pyraclostrobin Difenoconazole Azoxystrobin
3.4. Application of optimized SPME method to the water samples To apply the proposed SPME method in the analyzes of real samples, twenty-six water samples were collected in the Plateau of Neópolis (area of study), in accordance with item 2.4. In a first step, a fast screening was applied on each sample using the optimized conditions, in order to identify those ones containing pesticides. In a second step, the samples identified as containing pesticide residues were re-analyzed with three replicates, in order to confirm and quantify the pesticides detected in the previous screening. The results for these analyses are summarized in Table 6. Methyl parathion was detected in five samples, either from Plateau of Neópolis and Tenório, with an average concentration of 0.17 ng mL− 1. Besides it, in three samples from Neópolis, bifenthrin, pyraclostrobin and azoxystrobin residues were found, with average concentrations of 2.28, 3.12 and 0.15 ng mL− 1, respectively. The contamination levels of methyl parathion in the groundwater samples from the Tenório community was quite significant, compared to the maximum limits established by the Ministry of Health in Brazil (0.04 ng mL− 1) [4], since the local population uses the groundwater for personal consumption. Regarding the Plateau of Neópolis areas, it is expected that they become seriously damaged in the future, considering not only the results found in this work but specially the intense agricultural activity which is carried out in the region presently. 4. Conclusions
Average recovery (%) (R.S.D %) (n = 3) 0.2 ng mL− 1
5.0 ng mL− 1
50.0 ng mL− 1
94.3 ± 9.7 89.3 ± 8.8 108.5 ± 10.5 101.3 ± 6.7 93.8 ± 7.4 89.6 ± 6.8 93.7 ± 8.3 86.4 ± 9.4 105.3 ± 9.4 91.5 ± 6.8 – 87.3 ± 6.2 98.4 ± 8.3 – 104.4 ± 7.3 93.8 ± 4.6
91.9 ± 6.8 83.9 ± 8.6 89.4 ± 4.8 87.0 ± 9.4 95.3 ± 14.3 82.3 ± 8.4 86.0 ± 11.4 98.4 ± 6.3 104.6 ± 9.6 77.5 ± 10.8 92.2 ± 7.2 90.7 ± 6.4 84.2 ± 8.4 93.4 ± 8.3 82.3 ± 9.5 92.6 ± 5.0
82.3 ± 5.1 72.4 ± 3.8 97.4 ± 9.8 79.3 ± 4.9 82.8 ± 12.4 76.6 ± 6.4 98.4 ± 8.5 88.4 ± 8.3 79.0 ± 7.5 79.4 ± 9.8 84.9 ± 4.5 92.5 ± 6.5 82.0 ± 6.5 87.4 ± 4.6 91.3 ± 9.3 70.2 ± 4.6
Pesticides residues were determined in water, employing a developed method based on DI-SPME and GC–MS analysis. The method has proved to be selective, sensitive, precise and robust for the simultaneous determination of residues of sixteen pesticides, Table 6 Mean pesticides concentrations in 26 field-collected water samples from Plateau of Neópolis. Pesticides
Samples analyzed
Samples where pesticides were detected
Range and average concentration (ng mL− 1)
% RSD
Methyl parathion Bifenthrin Pyraclostrobin Azoxystrobin
26 26 26 26
5 3 3 3
0.13–0.23 1.89–2.57 2.48–3.65 0.13–0.19
8.6 9.3 8.2 11 . 6
(0.17) (2.28) (3.12) (0.15)
A.M. Filho et al. / Microchemical Journal 96 (2010) 139–145
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