Surveillance of fungicidal dithiocarbamate residues in fruits and vegetables

Food Chemistry 134 (2012) 366–374

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Surveillance of fungicidal dithiocarbamate residues in fruits and vegetables O. López-Fernández, R. Rial-Otero, C. González-Barreiro, J. Simal-Gándara ⇑ Nutrition and Bromatology Group, Analytical and Food Chemistry Department. Faculty of Food Science and Technology, University of Vigo, Ourense Campus, E-32004 Ourense, Spain

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Article history: Received 18 January 2011 Received in revised form 25 October 2011 Accepted 28 February 2012 Available online 6 March 2012 Keywords: Dithiocarbamates (DTCs) Fungicides Fruits Vegetables Surveillance Violations of MRL (maximum residue limits)

a b s t r a c t Derivatisation, solid–liquid extraction, solid-phase purification and HPLC–DAD separation is a simple, fast, and accurate method developed for the determination of dithiocarbamate (DTC) fungicides in fruits and vegetables. Quantification is based on external standard calibration curves made with DTCs-spiked blank-matrices. Limits of detection (LODs) and limits of quantitation (LOQs) are in the range of 0.01– 0.3 and 0.02–0.5 mg/kg, respectively. Recoveries higher than 78% were obtained for the target DTCs in all the commodities studied. The aim of this research was to evaluate the quality and determine the concentrations of DTC fungicide residues in plant material harvested from September to November of 2010. Several fruits and vegetables produced in Spain were collected at small shops and large food chains distributed by the four provinces of Galicia (North-Western Spain). In total, 150 samples were studied (32 apples, 12 wine grapes, 32 lettuces, 32 peppers, 32 tomatoes, and 10 strawberries). Residues of DTCs were found in all the products analysed with the exception of strawberries, being ethylenebis(dithiocarbamates) (EBDCs) vs. propylenebis (dithiocarbamates) (PBDCs) the main contributors to the total pesticide load. Moreover, fungicide residues exceeding maximum residue limits (MRLs) were identified in 6% of the samples analysed, specifically on lettuces and peppers. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Pesticides are chemicals used world-wide to destroy or control weeds, insects, fungi and other pests. When pesticides are applied improperly, the resulting residues in the produce (Fernández González, Rial-Otero, Cancho Grande, & Simal Gándara, 2003; González-Rodríguez, Cancho-Grande, & Simal-Gándara 2009; González-Rodríguez, Cancho-Grande, & Simal-Gándara 2011; González-Rodríguez, Cancho-Grande, Torrado-Agrasar, Simal-Gándara, & Mazaira-Pérez 2009; González-Rodríguez, Rial-Otero, CanchoGrande, González-Barreiro, & Simal-Gándara, 2011; GonzálezRodríguez, Rial-Otero, Cancho-Grande, & Simal-Gándara, 2008a; González-Rodríguez, Rial-Otero, Cancho-Grande, & Simal-Gándara, 2008b; López-Pérez et al., 2006; Pose-Juan, Cancho Grande, RialOtero, & Simal Gándara, 2006; Rial Otero, Cancho Grande, & Simal Gándara, 2003; Rial-Otero, Arias-Estévez, López-Periago, CanchoGrande, & Simal-Gándara, 2005) can pose significant health risks to consumers, who are increasingly aware of the potential for contamination of food and drinking water. To protect consumer health, national programmes have been established in many countries to monitor levels of pesticide residues in domestic and imported foods and to prevent the marketing of food containing residues that either exceeds specific tolerances set by regulations ⇑ Corresponding author. Tel.: +34 988 387000; fax: +34 988 387001. E-mail address: [email protected] (J. Simal-Gándara). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.02.178

or for which no tolerances have been established for that food (Hotchkiss, 1992). Dithiocarbamates are a group of organosulphur fungicides characterised by a broad spectrum of activity against various plant pathogens, low acute mammal toxicity, and low production costs (Crnogorac & Schwack, 2009). Thus, DTCs are widely used in agricultural especially in combination with modern systemic fungicides to control resistances and to expand the spectrum of activity (Crnogorac & Schwack, 2009; Szolar, 2007). As a consequence, DTCs are some of the most frequently detected pesticides in the European Union and this group also showed the highest frequency in exceeding maximum residue limits (MRLs) in the monitoring program of pesticide residues in products of plant origin in the European Union developed in 2005 (EC, 2007). DTCs can be categorised into three subclasses depending upon their carbon skeleton: (i) dimethyldithiocarbamates (DMDCs) such as ziram, thiram and ferbam; (ii) ethylenebis(dithiocarbamates) (EBDCs) such as mancozeb, maneb, zineb, and metiram; and (iii) propylenebis(dithiocarbamates) (PBDCs) such as propineb. This distinction is important because many countries are restricting the use of EBDCs because of their toxicity. In fact, today zineb is not considered as active substance approved for the manufacture of pesticides in Spain (MARM, 2010). Traditional methods for measuring DTCs consists of acid hydrolysis of the DTCs to CS2 and its posterior determination either by gas chromatography (Kazos, Stalikas, Nanos, & Konidari, 2007;

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Royer, Ménand, Grimault, & Communal, 2001; USEPA Method 630.1 (2007); Vryzas, Papadakis, & Papadopoulou-Mourkidou, 2002) or by absorption spectrophotometry (Banerjee et al., 2010; Cabras et al., 2001; Caldas, Conceição, Miranda, De Souza, & Lima, 2001; Singh et al., 2009; USEPA Method 630, 2007). However, these methods do not discriminate between the subclasses of DTCs (Van Lishaut & Schwack, 2000). Distinguishing between these subclasses is required due to their toxicological differences (Tomlin, 1994) and for the monitoring of unauthorised fields of application of different DTC pesticides. Also, legal limits, which until now have restricted only the amount of CS2 that can be released by hot hydrolysis of the sample, have recently required differentiation between CS2 from polymeric and non polymeric DTCs (Van Lishaut & Schwack, 2000). In addition, incidences of phytogenic CS2 blinds in various crops of the Brassica family may result in false positive results (Crnogorac, Schmauder, & Schwack, 2008; Perz, Van Lishaut, & Schwack, 2000). In this way, approaches based on high performance liquid chromatography (HPLC) (Crnogorac & Schwack, 2007; Garcinuño, Fernández-Hernando, & Cámara, 2004; Gustafsson & Thompson, 1981; Hanada, Tanizaki, Koga, Shiraishi, & Soma, 2002; Hayama & Takada, 2008; Hayama et al., 2007; Håkan Gustafsson & Fahlgren, 1983; Ozhan & Alpertunga, 2008) or ion pair chromatography (IPC) (Nakazawa et al., 2004; Van Lishaut & Schwack, 2000; Weissmahr, Houghton, & Sedlak, 1998) are most selective. In addition, other alternative analytical methods based on capillary electrophoresis, biosensors, spectrophotometry or gas chromatography can be found in the recent literature (Nakamura et al., 2010; Szolar, 2007). In this work a new method for the determination of residues of mancozeb, maneb and propineb in fruits and vegetables by means of HPLC–DAD is presented. Finally, the proposed method was applied to the determination of these compounds in commercial samples of tomatoes, lettuces, peppers, apples, wine grapes and strawberries in order to evaluate their compliance with the MRLs established on these products (EU, 2010). Ripe produce was collected from the market-shelves of small and large stores located in Galician (NW Spain).

2. Experimental 2.1. Chemical, solvents and disposables Mancozeb [CAS No. 8018-01-7] (purity 96.8%), Maneb [CAS No. 12427-38-2] (purity 91.6%) and Propineb [CAS No. 12071-83-9] (purity 103.1%) were purchased from Fluka (Steinheim, Germany). Water and acetonitrile for liquid chromatography were purchased from Sigma–Aldrich (Steinheim, Germany). L-Cystein [CAS No. 5290-4] (purity P97%) was from Sigma–Aldrich. Other reagents used were: ethylenediaminetetraacetic acid disodium salt 2-hydrate PAACS (EDTA, purity P99%), sodium chloride PA-ACS-ISO (purity 99.5%), magnesium sulphate anhydrous QP (purity 96%), di-sodium hydrogen phosphate anhydrous (USP) purissimum-CODEX (purity >98%), sodium di-hydrogen phosphate 1-hydrate (USP, BP) PRSCODEX (purity >98%), anhydrous sodium carbonate (purity 99.8%) and sodium polyphosphate (purity >65%), all of them from Panreac (Barcelona, Spain). For solid–liquid extraction (SLE) and derivatisation, samples were placed in 50 mL polypropylene centrifuge tubes from Sterilin (Newport, UK). Polypropylene tubes were centrifuged in a Rotina 35 R centrifuge from Hettich Lab Technology (Tuttlingen, Germany). Strata SI-1 silica cartridges (55 lm, 70 Å, 500 mg, 6 mL) from Phenomenex (Utrecht, The Netherlands); florisil Plus Sep-Pak cartridges (500 mg, 6 mL) from Waters (Milford, CT, USA); dual layer Envi-Carb II/PSA (500 mg, 6 mL) SPE tube and dispersive SPE PSA

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clean-up Tube-1 from Supelco (Bellefonte, PA, USA) were used as solid-phase extraction (SPE) minicolumns for purification. Organic extracts were concentrated to 1 mL in a TurboVap LV concentration workstation (Caliper Life Sciences, Barcelona, Spain) using nitrogen C-50 purchased from Carburos Metálicos (Vigo, Spain). Final organic extracts were filtered through a Chromafil Xtra PET-20/ 25 (0.20 lm) filters from Macherey–Nagel (Düren, Germany) and placed in 2 mL amber vials from Supelco prior to chromatographic analysis. 2.2. Standard solutions A stock standard solution (ca. 100 mg/L) of each fungicide was separately prepared as a turbid liquid in acetonitrile by weighing approximately 1 mg of the analyte into a 10 mL volumetric flask and diluting to volume. Stock standard solutions were stored in the dark at 4 °C and were prepared each 2 weeks due to the instability of the standards. Working solutions were prepared daily in distilled water or buffer solution by appropriate dilution. For recovery studies, some samples were spiked with the appropriate volume of each stock standard solution and homogenised by shaking. These spiked samples were maintained at room temperature for 30 min before extraction to allow the solution to penetrate the test material. 2.3. Sampling Tomato samples used for the optimisation and characterisation of the proposed method were purchased at local markets in Ourense, NW Spain. The surveillance program to screen for the presence of these fungicides was performed with fruit (strawberries, apples, and grapes) and vegetable (tomatoes, lettuces, and peppers) samples collected at small fruit shops and large food chains from the four Galician’s provinces (Ourense, Lugo, Pontevedra and A Coruña) in NW Spain. It is important to remark that the sampling by Galician’s province is only attending at selling place and not at production origin. This study was conducted during the 3 months from September to November of 2010. In total, 150 samples (32 apples, 12 wine grapes, 32 lettuces, 32 peppers, 32 tomatoes, and 10 strawberries) were collected and frozen at 20 °C until usage. In general sampling was uniformly distributed as follows: 4 units for type of sample  2 kinds of markets  4 provinces. A lower amount of strawberry samples was obtained due to their availability in the selected sampling time. Also, samples of wine grape were obtained only in the provinces of Ourense and Pontevedra, the main winemaking provinces in Galicia. 2.4. Extraction, derivatisation and purification of samples and standard-spiked blank samples After peel and cut the plant food sample, an aliquot (about 10 g) was further homogenised with a pH 7.8 aqueous buffer (7.5 mL), Lcystein (0.1 g), EDTA-2Na (0.5 g) and 0.05 M dimethyl sulphate solution in acetonitrile (10 mL). The whole mixture was orbital shaken for 15 min; then, 4 g of anhydrous magnesium sulphate (Mg2SO4) and 1 g of sodium chloride (NaCl) were added, and it was shaken with a vortex for 1 min. After centrifugation for 5 min at 4000 rpm and 10 °C, the organic phase was removed. An aliquot of the organic extract (3 mL) was collected for cleaningup with the silica cartridge. After conditioning the silica cartridge with 5 mL of acetonitrile, the organic extract (3 mL) was loaded onto the cartridge and 3 mL of acetonitrile were then passed through; both fractions, load and wash, were collected together and further concentrated to a final volume of 3 mL. Final extract was filtered through a Chromafil Xtra PET-20/25 (0.20 lm) filters

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prior to HPLC analysis. Standard-spiked blank samples were processed in the same way for quantification of the fungicidal DTCs. 2.5. HPLC–DAD system and operating conditions High-performance liquid chromatography (HPLC) analyses were carried out on a Thermo Surveyor HPLC system equipped with a LC Pump Plus, an Autosampler Plus Lite, a Gecko 2000 column heater from Cil Cluzeau Info Labo (France) and a PDA Plus detector linked to a PC computer running the ChromQuest 5.0 v. software programme (TermoQuest, Madrid, Spain). The analytical column was a C18 Kinetex 2.6 lm/100 Å (50  4.6 mm I.D.) from Phenomenex (Utrecht, The Netherlands). The guard column (50  4.6 mm I.D.) was packed with dry 40 lm Pelliguard LC-18

from Supelco (Madrid, Spain). For HPLC analysis, an extract aliquot (25 lL) was injected into the column thermostatised at 40 °C with a constant flow-rate of 1.1 mL/min at the following gradient conditions: water/acetonitrile (95:5, v/v) was changed for 9 min to 10:90 (v/v), before recovering initial conditions in just 1 min and keeping them for 5 min for column stabilisation. Detection was carried out at the wavelength of 270 nm. 3. Results and discussion 3.1. Optimisation of liquid chromatographic separation Chromatographic conditions were optimised for the simultaneous determination of the target pesticides. After assaying dif-

Fig. 1. HPLC–DAD chromatogram obtained for the analysis of a 2 mg DTC/mL standard mix of mancozeb and propineb (up) and for a commercial lettuce sample (down) at the optimised and validated conditions. Peaks: 1, mancozeb; and 2, propineb.

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ferent mobile phase systems and gradients, the mobile phase acetonitrile/HPLC water was selected. In addition, a Symmetry C18 (150  4.6 mm; 2.6 lm) and a Kinetex C18 100 Å (50  4.6 mm; 2.6 lm) columns were assayed. Although the individual elution of the target compounds was possible with both columns, the Kinetex column was finally selected. With this column a better resolution, throughput, and sensitivity can be achieved and also a decrease in solvent consumption. This can be related with the properties of the KinetexÒ core–shell particles; particles not fully porous and an extremely narrow particle size distribution. The optimised conditions described above allowed the elution of pesticides between 5 and 7 min (Fig. 1). It is important to note that this method cannot distinguish between residues of mancozeb and maneb as both results in the same compound after derivatisation.

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3.2. Sample extraction performance DTCs are not systemic pesticides and therefore it is likely to be found only on the food surface. For this reason, sample homogenisation is not essential and surface extraction can be used, avoiding in this manner loss of analytes due to the rapid breakdown of the DTCs in the presence of plant material (Crnogorac & Schwack, 2009; Gustafsson & Thompson, 1981; Håkan Gustafsson & Fahlgren, 1983; Van Lishaut & Schwack, 2000). Moreover, sample homogenisation give rises to more coextractives which interfere in the determination. In addition, DTCs are insoluble in general solvents, and they are readily transformed into soluble salts in water by the addition of alkaline EDTA solution. In previous studies, DTCs are transformed into DTC-dimethyl from their sodium salts by S-methylation with

A

B

C

Fig. 2. (A) Effect of the extraction time and shaking mode on the extraction efficiency of mancozeb and propineb; (B) influence of pH of the aqueous extractant on fungicide recovery; and (C) optimisation of the purification step. Columns show mean recovery from three replicate tomato samples and standards; error bars show standard deviations.

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methyl iodide through the ion-pair alkylation (Gustafsson & Thompson, 1981; Hanada et al., 2002; Hayama et al., 2007; Håkan Gustafsson & Fahlgren, 1983). However, in later works the use of dimethyl sulphate as methylation reagent was preferred because the methyl derivative yield obtained was >15% higher than with methyl iodide (Hayama & Takada, 2008). On the basis of this, dimethyl sulphate was selected as methylation reagent in this work. Uncontaminated fresh peel of tomato samples were chopped using a knife in small pieces (1 cm2 and 5 mm of thickness) immediately before use, and spiked with the target pesticides (mancozeb and propineb) at levels of ca. 2 mg DTC/kg of peel for each compound. These spiked samples were maintained at room temperature for 30 min before extraction to allow the solution to penetrate the test material. After equilibration, samples were initially processed according to the QuEChERS method developed by Hayama and Takada (2008) for the determination of EBDCs in fruits and vegetables. In brief, 10 g of the food sample was weighed in a 50 mL centrifuge tube and a volume of water (7.5 mL), L-cysteine (0.1 g), EDTA-2Na (0.5 g) and 0.05 M dimethyl sulphate in acetonitrile (10 mL) were added. The mixture was orbital shaken for 15 min and then Mg2SO4 (4 g) and NaCl (1 g) were added. Afterwards, the mixture was vigorously shaken for 1 min and centrifuged for further 5 min at 5000 rpm. Then, 6 mL of the upper layer (acetonitrile), was vortexed for another minute with the content of a dispersive SPE PSA clean-up Tube 1 (900 mg of anhydrous Mg2SO4 and 150 mg of dispersive PSA), centrifuged for 5 min at 3000 rpm and injected in the HPLC equipment. Analyses were performed by triplicate. L-Cysteine has been added to the sample to prevent the degradation of DTCs during the analytical procedure (Hayama et al., 2007). With this method, good results were obtained for mancozeb according with the data obtained for Hayama and Takada (2008). However, as it can be seen in Fig. 2A, lower recoveries were obtained for propineb from tomato samples (ca. 40%). These results did not improve by increasing the extraction time to 30 min (Fig. 2A) or by doing two consecutive extractions (data not shown). Several studies have reported the use of high intensity ultrasound to accelerate chemical and enzymatic reactions (Galesio et al., 2010). In this sense, ultrasound-assisted shaking using an ultrasonic bath during 5 and 10 min was assayed in order to improve the extraction process and also accelerate the derivatisation step. Triplicate analyses were performed for each time. As it can be seen in Fig. 2A, similar results to those obtained with orbital shak-

ing were obtained with ultrasonication for standards in water. On the contrary, the use of ultrasonication with spiked samples promotes a decrease in the extraction efficiency of propineb. This can be explained because disruption of plant cells may lead to analyte loss (Van Lishaut & Schwack, 2000). Therefore, orbital shaking for 15 min was selected as optimum. Derivatisation efficiency could also be influenced by the pH of the media (Hayama & Takada, 2008). In order to optimise this parameter, assays were performed by triplicate in distilled water, in a buffer solution with pH 7.8 (0.2 M NaH2PO4 + 0.2 M Na2HPO4) and in a buffer solution with pH 10.6 (3.6% w/v anhydrous sodium carbonate +0.8% w/v sodium polyphosphate). As it can be seen in Fig. 2B, alkaline buffers can help to improve the derivatisation yield for dithiocarbamates, especially for propineb in vegetal samples. Nevertheless, recoveries higher than 100% were obtained for both compounds, what is indicative of a matrix effect and of the need for a further purification of the extracts. 3.3. Extract purification performance Organic solvent extracts from samples have many interfering compounds from the sample matrix. To remove matrix interferences, the purification efficiency of PSA dispersive SPE (Supelco) as proposed by Hayama and Takada (2008) was tested on organic extracts from spiked samples. In addition, florisil Plus (Waters), Strata silica-1 (Phenomenex) and ENVI-CarbII/PSA (Supelco) cartridges were tested. In the case of PSA dispersive SPE, 6 mL of the organic extract was mixed with the content of a dispersive SPE PSA clean-up Tube 1 (900 mg of anhydrous Mg2SO4 and 150 mg of dispersive PSA by vortex agitation for 1 min and then, the mix was centrifuged, filtered and injected into the HPLC. For florisil Plus and Strata silica-1 cartridges, 3 mL of the organic extract were loaded through the cartridge, previously conditioned with 5 mL of acetonitrile. Three more mL of acetonitrile was passed through the cartridge, and then the collected eluates were concentrated to 3 mL, filtered and injected for HPLC analysis. Finally, for ENVICarbII/PSA cartridges 3 mL of the organic extract were loaded through the cartridge, previously conditioned with 5 mL of acetonitrile/toluene mixture (3/1, v/v), and the retained pesticides eluted with 3 mL of the same mixture, filtered and injected into the HPLC. It is important to comment that target fungicides are not retained in the cartridges except when ENVI-CarbII/PSA cartridges

Table 1 Quality parameters of the proposed method. Fungicide

Repeatabilitya RSD (%) (n = 5)

Reproducibilitya RSD (%) (n = 9)

(A) Precision, linearity and limits of detection and quantification Mancozeb 9.8 17.1 Maneb 9.8 11.8 Propineb 7.6 12.9 Sample

Calibration Points

(B) Recovery data for various DTCs spiked samples Apples 7 Grapes 7 Strawberries 7 Lettuces 7 Peppers 7 Tomatoes 7 a

Linear rangea (mg DTC/kg peel) (n = 7)

Determination coefficienta (r2)

0.5–9.3 0.4–9.3 0.2–9.3

0.9995 0.9989 0.9996

Fruitsb (mg DTC/ kg peel) (n = 5)

Vegetablesb (mg DTC/kg peel) (n = 5)

LOD

LOQ

LOD

LOQ

0.31 0.22 0.11

0.54 0.38 0.16

0.04 0.04 0.01

0.11 0.12 0.02

Mean recoveryc (%) Mancozeb

Maneb

Propineb

95 74 115 139 99 111

90 115 90 82 78 96

128 106 99 125 111 114

These parameters were evaluated using fresh peel of strawberries. These values should be corrected taking into account the ratio between weight of peel/total weight for each commodity (0.19 for apples, 0.20 for wine grapes, 1 for lettuces, 0.71 for peppers, 0.20 for tomatoes and 0.56 for strawberries). c Mean recovery evaluated in the concentration range between 0.3 and 7 mg DTC/kg of peel sample. b

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were used. In addition, a minimal reduction of the colour of the organic extract after purification was observed with the exception of ENVI-CarbII/PSA cartridges where colourless extracts were obtained. However, as it can be seen in Fig. 2C, lower recoveries were obtained for the ENVI-CarbII/PSA cartridges for both, standards and samples. Probably due to the compounds are strongly retained on active carbon. On the contrary, PSA dispersive and florisil Plus cartridges gave recoveries higher than 130%; it could be explained with the presence of not-removed interferences. High recoveries were corrected with the use of Strata silica-1 cartridges. In order to increase the sensibility of the method, purified extracts could be concentrated prior to injection in the HPLC. In this way, it is important to remark that evaporation to dryness should be avoided as they can cause significant losses of compounds. 3.4. Method performance Method performance was assessed by evaluating quality parameters such as recovery values, repeatability, reproducibility, linearity and limits of detection and quantitation. All values obtained are summarised in Table 1A and B. For this purpose, uncontaminated samples of fresh peel of strawberries were previously fortified with the target fungicides and treated following the opti-

mised experimental conditions described above. Strawberry samples were selected for this purpose because major interferences of the matrix were observed on the chromatogram. The repeatability and reproducibility of the method were assessed by analysing 5 spiked uncontaminated samples in the same day and a total of nine spiked uncontaminated samples along 3 days in two different weeks, respectively. All samples were spiked at levels of ca. 0.5 mg DTC/kg of peel sample for each fungicide. The relative standard deviations (RSD%) obtained for repeatability and reproducibility were lower than 10% and 17%, respectively, as it can be seen in Table 1A. These values show the good precision of the proposed method. Calibration curves for the fungicides were prepared by plotting the area of the fungicide vs. the analyte concentration using a total of seven spiked uncontaminated strawberry samples (0.2–10 mg DTC/kg of peel). Analysis of an unspiked strawberry sample did not give any response at the retention time of the studied fungicides. Linear ranges and determination coefficients (r2) corresponding to each fungicide are shown in Table 1A. Limits of detection and quantitation were evaluated following the recommendations of the American Chemical Society (American Chemical Society & ACS Subcommittee on Environmental Analytical Chemistry, 1980). As tested experimentally, quantitation limits

Table 2 Levels of DTC fungicide residues on fruits and vegetables. MRLsa (mg/kg)

Commodity

N° samples analysed

Positive samples

Samples above MRLs

Concentration rangeb in mg/kg (rank)

(A) Concentrations ranges found in samples from NW Spain, together with number of samples with levels above the maximum residue limits. Apples (1.43 cm2/g) 5 32 5 (15.6%) 0 0.03–0.19 (4) Grapes (1.9 cm2/g) 5 12 4 (33.3%) 0 0.11–0.61 (2) Strawberries (2.30 cm2/g) 10 10 0 (0%) 0
Commodity

(B) Concentrations ranges found in samples Belgium Tomatoes Brazil Apples Bananas Beans Carrots Dry beans Dry beans Grapes Lettuce Oranges Papaya Potatoes Rice Strawberries Tomatoes India Apples Italy Apples Morocco Tomatoes Neatherlands Pears Senegal Cherry tomatoes Spain Broccoli Cocktail tomatoes Tomatoes Thailand Tamarillos Turkey Apple juices Cucumbers Grapes Grape juice Tomato juice

Levels of DTC (mg CS2 kg1)

Concentration rangeb in ng/cm2(rank) 21–132 (4) 58–321 (2)
References

from different countries according to the literature n.d. Crnogorac et al. (2008) 0.309–0.450 Caldas, Miranda, Conceição, and De Souza (2004) and Caldas, Tressou, and Boon (2006) 0.099–0.220 Caldas et al. (2004) and Caldas et al. (2006) 0.052 Caldas et al. (2006) 0.040 Caldas et al. (2006) <0.100 Caldas et al. (2004) <0.10 Caldas et al. (2004) 0.012 Crnogorac et al. (2008) 0.361 Caldas et al., 2006 0.065–0.18 Caldas et al. (2004) and Caldas et al. (2006) 0.003–0.350 Caldas et al. (2004, 2006) and Crnogorac et al. (2008) 0.052–0.110 Caldas et al. (2004) and Caldas et al. (2006) <0.100–0.050 Caldas et al. (2004) and Caldas et al. (2006) 0.154–0.180 Caldas et al. (2004) and Caldas et al. (2006) 0.202–0.220 Caldas et al. (2004) and Caldas et al. (2006) n.d. Singh et al. (2009) 0.010 Crnogorac et al. (2008) 0.023 Crnogorac et al. (2008) 0.038 Crnogorac et al. (2008) 0.077 Crnogorac et al. (2008) 0.040 Crnogorac et al. (2008) 0.185 Crnogorac et al. (2008) * 2.9 Blasco and Font (2004) 0.065 Crnogorac et al. (2008) n.d. Ozhan and Alpertunga (2008) n.d. Crnogorac et al. (2008) n.d. Crnogorac et al. (2008) n.d. Ozhan and Alpertunga (2008) ** 0.450 Ozhan and Alpertunga (2008)

n.d.: not detected. a Dithiocarbamates expressed as CS2, including maneb, mancozeb, metiram, propineb, thiram and ziram. b Sum of mancozeb, maneb and propineb expressed as CS2, based on the whole fruit weight. * This value is expressed as mg of Thiram per kg of commodity. ** This value is expressed as mg Maneb per L.

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were between 30 and 90 times lower than MRLs established by European legislation for each commodity. In this case, a distinction between the LODs and LOQs for fruits and vegetables was done, due to the increased complexity of the fruit’s matrix (dirty chromatograms) compared to vegetables. LODs and LOQs values included in the table should be corrected for the corresponding sample factor (ratio between weight of peel/total weight). Recoveries and the matrix-induced suppression/enhancement effects were evaluated by comparing the response for DTCsdimethyl in acetonitrile solution with that obtained for the same amount of DTCs-dimethyl in the blank extracts of various crops at the same concentration level (0.3, 0.5, 0.75, 1, 2, 5 and 7 mg DTC/kg of peel). Table 1B shows the means recoveries obtained from the ratio of slopes. As it can be seen in this table, recoveries between 74% and 139% were obtained, indicating the existence of matrix-induced effects. To overcome these effects, the matrixmatched calibrations recommended in the EU guidelines (EC, 2006) were used, and they provided stable results for determination of DTCs-dimethyl. 3.5. Surveillance of DTCs residues in fruits and vegetables from NW Spain in summer–autumn 2010 The degradation of the food quality by pesticides pollution is a cause of concern. As a result of the great sensibility of pest attack and the necessity to launch them quickly in the market, the fruits and vegetables need special attention regarding pesticide use in order to evaluate food risk. Maximum residue limits (MRLs) in the European Union for dithiocarbamates in fruits and vegetables are expressed as mg/kg of CS2, including the sum of maneb, mancozeb, metiram, propineb, thiram and ziram (EU, 2010). MRLs for each crop are also showed in Table 2A. The occurrence and distribution of DTCs in fresh fruits and vegetables collected in different markets from Galicia (Spain) was monitored. In total, 150 samples were analysed (32 apples, 12 wine grapes, 32 lettuces, 32 peppers, 32 tomatoes and 10 strawberries). Samples were analysed directly by duplicate, without wash. Results obtained are summarised in the Table 2A. As it can be seen, residues of DTCs were found in all the products analysed with

the exception of strawberries. Peppers was the commodity with more positive samples (96.9%) followed by tomatoes (87.5%), lettuces (71.9%), grapes (33.3%) and apples (15.6%); being EBDCs (vs. PBDCs) the main contributors to the total pesticide load (Fig. 3). Plant foodstuffs ranking from higher to lower residual concentrations in mg/kg is as follows: lettuces (1) > grapes (2) > peppers = tomatoes (3) > apples (4) > strawberries (5); instead, expressing the results in ng/cm2, the ranking is as follows: lettuces = tomatoes (1) > grapes (2) > peppers (3) > apples (4) > strawberries (5). In the latter ranking, lettuces and tomatoes become the food products with the largest residual concentration per surface, even though their ratio surface/mass is very different (for lettuces is the maximum, 38.5 cm2/g, whereas for tomatoes is the minimum, 0.55 cm2/g); in this way, this is showing the dilution effect with growing mass in tomatoes. If we normalise the concentration values for each commodity with respect to their MRL (Fig. 3), it is possible to observe that, in general, residues of DTCs were lower than the MRLs established in the European legislation. Only for lettuces (12.5%) and peppers (15.6%) residues above the MRLs were found, surpassing some samples more than three times the legal limit. However, the degree of accumulated pesticide pollution is 35 times higher in lettuces than in peppers, and no differences were observed on this degree of pollution between large stores or small shops (Fig. 4A). The higher pesticide concentrations in lettuces compared to peppers can be explained because lettuces are highly sensitive to pests and need for successive applications of pesticide treatments, leaving in consequence higher level of residues that are tolerated. Thus, MRLs for lettuces are fifty times higher than for peppers. If the data are considered by provinces, we can even observe that the CS2 accumulated concentration in lettuces is higher for the provinces of A Coruña and Ourense (Fig. 4B). However, while in A Coruña the degree of pollution is four times higher for small fruit shops than for large stores; in the province of Ourense the degree of pollution is 25 times higher in large food chains. In the case of peppers, the high pesticide pollution corresponds to the provinces of Lugo and Ourense, especially in large stores (Fig. 4C). Italy and Spain are by far the largest producer countries of fresh vegetables within the EU-27 (EUROSTAT, 2010). Currently, the

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Fig. 3. Residues above and below MRLs after normalisation of their concentrations by referring to the foodstuff MRL, and distribution of the residues in the foodstuff between EBDCs and PBDCs.

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Fig. 4. (A) Pareto chart of CS2 accumulated concentration in vegetables according to factors: (1) type of commodity plus (2) type of seller (small specialised shop or large food chain); (B) Pareto chart of CS2 accumulated concentration in lettuces according to factors: (1) province plus (2) type of seller; and (C) Pareto chart of CS2 accumulated concentration in peppers according to factors: (1) province plus (2) type of seller.

gross consumption of lettuce per capita in Spain is 5.02 kg/capita/ year, representing a consumption of about 14 g per day (Ministerio de Agricultura, Pesca y Alimentación, 2010). Taking into account that the acceptable daily intake (ADI) for the group of EBDCs is 0.03 mg/kg/day, in the case of a reference person weighing 70 kg, the maximum allowable intake would be 2.1 mg/day. Thus, consumption of 14 g of the lettuce sample most contaminated found in this study (22.5 mg CS2/kg corresponding to 39.7 EBDCs mg/kg) represents an intake of 0.56 mg EBDCs/day or what is the same, 26.7% of the ADI. Overall, DTC fungicides found in this study were similar to those found in other studies at different countries as it can be seen in Table 2B. For instance, residues of dimethyldithiocarbamates (DMDs) and EBDs ranging between 0.003 and 0.185 mg kg1 were detected in several fruits and vegetables from different countries of origin (apples, pears, grapes, cherry tomatoes, cocktail tomatoes, cucumbers, tomatoes, tamarillos, papaya, and broccoli) (Crnogorac et al., 2008). In addition, residues of dithiocarbamates were detected in tomato, cabbage and cucumber samples from the market of the Republic of Serbia during 2007 (Lazic´, Bursic´, Vukovic´, Šunjka, & Pucarevic´, 2009). In 2009, Singh et al. studied the presence of mancozeb and other pesticide residues in apple of integrated pest management (IPM) and non-IPM samples collected from the IPM and non-IPM fields of Shimla (India) and residues of mancozeb were below its detectable limits (0.35 mg/kg) (Singh et al., 2009). 4. Conclusions The proposed method has been demonstrated to be suitable for the control of mancozeb, maneb and propineb in several fruits and vegetables at concentrations lower than their MRLs established. Both mancozeb and maneb give place to the same derivatisation product, what makes no possible to distinguish between them. Anyway, the distinction is of none interest since from a legal point of view their limits are established for the addition of DTCs and not for individual DTCs. To completely remove any matrix effect, their quantification should be performed with calibration curves made with spiked blank matrices. When the proposed method was applied to 150 samples of fruits and vegetables collected in Galicia in 2010, residues of DTCs were found in all the products analysed with the exception of strawberries. Peppers was the commodity with more positive samples (96.9%) followed by tomatoes (87.5%), lettuces (71.9%), grapes

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