Introduction

Pesticides play an inevitable role in modern agriculture. Rising concern about food safety and environmental impact has led to increasing number of studies on the impact of pesticide residues in agricultural products consumed by humans (Cun-Zheng Zhang et al. 2010; Mestres 1988; Rozemeijer and Broers 2007).

Tomatoes are cultivated over a large area in Egypt. This important dietary component is used fresh or processed and canned for later use. (El Nabarawy et al. 1992).The cultivation of tomatoes demands frequent application of a large number of pesticides, and >100 pesticides have been recommended for use in Egypt to control a variety of pests and diseases (APC 2010).

Agricultural committees are required by law to safeguard the interests of consumers, and hence, regulations have been formed and stringently implemented to eliminate harmful pesticide residues in foods. The management of pesticide residues in tomatoes is challenging because of direct application of pesticides over fruits, pesticides accumulated in the soil and absorbed through the roots, and drifting pesticides from adjoining fields of other agricultural crops. The intensive use of pesticides on tomato crop may cause accumulation of pesticide residues more than the permitted levels and hence needs frequent field evaluations. Accurate measurements of dissipation or degradation rates of various pesticides under field conditions will be helpful in their optimal application (Fenoll et al. 2009; Omirou et al. 2009)

The dissipation/degradation of various pesticide compounds after their application depends on various factors, including plant species, chemical formulation, and mode of application (Cabras et al. 1989; Ebert et al. 1999; Womac et al. 1994), climatic conditions, physical phenomena (mainly volatilization), and photodegradation, in which sunlight plays a prominent role (Garau et al. 2002; Minelli et al. 1996; Papadopoulos et al. 1995). Therefore, dissipation studies for a given crop under the open field conditions of each growing area are necessary to test if the pesticide residue levels soon after the preharvest interval (PHI) are below the maximum residue limit (MRL). PHI, which is defined as the period between the last pesticide application and harvesting the crop, after which the pesticide residue level is expected to be below the established maximum residue level (MRL), is one of the important pesticide registration requirements in Egypt.

The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method is well known for its applicability in simultaneous analysis of a large number of pesticides in a variety of food matrices (Anastassiades et al. 2003; Lehotay et al. 2005). The method has received worldwide acceptance because of its simplicity and high throughput, enabling a laboratory to process significantly larger number of samples in a given time as compared to the earlier methods (AOAC 2000; Luke et al. 1975). Recently, the QuEChERS method has received the distinction as an AOAC official method for measuring multiple pesticide residues in fruits and vegetables (Lehotay 2007).

The objectives of this study were to: (a) determine the residue levels of six most commonly used pesticides in tomato cultivation in Egypt, (b) define the individual pesticide residue behavior in tomatoes using dissipation curves, and (c) determine the preharvest interval for each of these pesticides which may be suggested as a registration requirement. For this purpose, the QuEChERS method has been applied prior to high-performance chromatography (HPLC connected with photodiode array detector (DAD)).

Material and Methods

Plant Material

Pesticide residues of metalaxyl, abamectin, and propamocarb hydrochloride were studied in 200 m2 of open field, on tomato (Solanum lycopersicum) planted in January 2008, and pymetrozine, chlorothalonil, and metalaxyl-m were studied on tomato planted in July 2008, in certain governorates of Egypt.

Chemicals

Reference standards of all pesticides were of >98% purity and obtained from Central Agricultural Pesticides Laboratory (Egypt). Stock solutions of pesticides were prepared in acetonitrile and stored at −18 °C. All HPLC grade organic solvents, methanol, and acetonitrile were purchased from Sigma (Sigma GmbH, Germany). Primary secondary amine (PSA, 40 μm Bondesil) sorbent was purchased from Supelco (Supelco, Bellefonte, USA). Sodium acetate and anhydrous magnesium sulfate were of analytical reagent grade and purchased from Merck Ltd. These were activated by heating at 150 °C overnight and kept in desiccators.

Field Trial

For the field experiment, a random block scheme was used with three replications for each test. Pesticide treatments were carried out with a backpack motorized sprayer with an adjustable nozzle size of 1 mm. The following commercial formulations were used: Chess (50% of pymetrozine), Folio Gold (50% of chlorothalonil and 37.5% of metalaxyl-M), Ridomil Gold (537.5 metalaxyl-M), Previcure-N (72.2% of propamocarb hydrochloride), Vacomil plus (20% of metalaxyl and 50% of copper), and Vapcomic (1.8% abamectin); characteristics and details about the commercial formulations, the doses employed, a.i., and MRL are summarized in Table 1. The applications were carried out on January 9th and July 4th, 2008, respectively, at doses prescribed by the manufacturers. The relative humidity during the application period of January and July was 67.1% and 58.5%, and temperatures were 35.5 °C and 20.1 °C, respectively. Before application, samples of tomato of similar ripening stage, size, and shape were located and tagged. Samples, 1 kg, were collected 1 h after application at intervals of 1, 2, 3, 5, 7, 9, 12, and 15 days. During the experiment, a control sample was also collected after each sampling time interval. Immediately after collecting the tomatoes, all samples were put into plastic bags, transported to the laboratory, and homogenized using a food processor (Thermomix, Vorwerk). The homogenate of each sample was then placed into 50-ml polypropylene centrifuge tubes and frozen at −20 °C until further analysis.

Table 1 General characteristics of investigated pesticides
Full size table

Sample Preparation

The entire sample (1 kg) of tomatoes was chopped and homogenized for 5 min at high speed in a laboratory homogenizer and extracted according to the procedure described and modified by Lehotay (Lehotay et al. 2010). Briefly, 10 g of the homogenized sample was weighed into a 50-ml centrifuge tube. Ten milliliters of 1.0% acidified acetonitrile with acetic acid was added; the screw cap was closed and vigorously shaken for 1 min using a vortex mixer at maximum speed. Afterwards, 4 g of anhydrous MgSO4, 1 g of NaCl, 1 g sodium citrate dihydrate, and 0.5 g disodium hydrogen citrate sesquihydrate were added, then extract by shaking vigorously on vortex for 2 min and centrifuged for 10 min at 5,000 rpm. An aliquot of 3 ml was transferred from the supernatant to a new clean 5-ml centrifuge tube and cleaned by dispersive solid-phase extraction with 75 mg of PSA and 500 mg of magnesium sulfate. Afterwards, centrifugation was carried out at 6,000 rpm for 5 min. An aliquot (2 ml) from the supernatant was filtered through a 0.2-μm PTFE filter (Millipore, USA) and then analyzed by Agilent 1100 HPLC-DAD.

Apparatus and Chromatographic Analysis

Pesticide residue analysis was performed with Agilent technologies HP-1100 series high-performance liquid chromatographic system (Agilent Technologies, USA) equipped with a diode array detector and quaternary pump. The separation was performed on a C18 column (150 × 4.6 mm, 5 μm). The mobile phase, flow rate, and detection wavelength of each pesticide are mentioned in Table 2. Data analysis was performed using Chemistation software.

Table 2 Typical HPLC analysis of conditions used for investigated pesticides
Full size table

Statistical Analysis

Data were statistically evaluated by one-way analysis of variance. Determination of the differences among means was carried out by using the least significant differences test. All statistical analyses were done using the Statistical Package for Social Sciences (SPSS 16.0) program.

Results and Discussion

Analytical Performance

The analytical method was validated as per the single laboratory validation approach (Thompson et al. 2002). The performance of the method was evaluated considering different validation parameters that include the following items: the calibration curves of all of the compounds in pure solvent, and matrix were obtained by plotting the peak area against the concentration of the corresponding calibration standards at seven calibration levels ranging between 10 and 200 μg kg−1.

The limits of detection (LOD) were set to a signal-to-noise ratio of 3 with reference to the background noise obtained from blank sample, whereas the limits of quantification (LOQ) were set to a signal-to-noise ratio of 10. The LODs and LOQs obtained from six studied pesticides are presented in Table 3. The accuracy and precision of method were evaluated via recovery experiments with fortified samples at two fortification levels, 10 and 200 μg kg−1, each replicated thrice. The limits of quantification were, in all cases, lower than MRLs established by Codex Committee and Switzerland (FAO/WHO 2006a).

Accuracy and Recovery Experiments

Tomatoes obtained from the untreated area (which did not receive any treatment of the test pesticides) were used as blanks. Recovery experiments were carried out on fresh untreated tomatoes by fortifying the samples in three replicates of each pesticide separately at two concentration levels, 10 and 200 μg kg−1. The results are reported in Table 3.

Table 3 Dissipation rate of pesticides residues, mean ± SD (mg kg−1) detected in tomatoes under open field condition
Full size table

Residual Behavior of Pesticides

Degradation rate of six pesticides (chlorothalonil, pymetrozine, metalaxyl, metalaxyl-M, abamectin, and propamocarb) was studied after one application of these pesticides on tomatoes in open field trials, at the recommended doses of 300 ml/100 L, 240 g/Fed., 150 g/100 L, 200 g/100 L, 40 ml/100 L, and 250 ml/100 L for chlorothalonil, pymetrozine, metalaxyl, metalaxyl-M, abamectin, and propamocarb hydrochloride, respectively. Table 3 shows the values of the residues of all studied pesticides, maximum residues limit (MRL), half-life (t 1/2), and preharvest interval of each pesticide.

In this study, curve fitting for various pesticides was achieved by the modified coefficient of determination (r 2), and the correlation between residues and time was determined by the equation as proposed elsewhere (Timme and Frehse 1980; Timme et al. 1986). The correlation coefficient was calculated as follows:

$$ {r^2} = 1 - \frac{{\sum {_{{i = 1}}^n{{\left( {Ci - Ci,\bmod } \right)}^2}} }}{{\sum {_{{i = 1}}^n{{\left( {Ci - \overline C } \right)}^2}} }} $$

where C is the mean of the residue values and Ci, mod are the modified residue values calculated starting from the equation of the adjusted decline curve. By definition, r 2 ≤ 1; and the higher this coefficient, the better the decline curve fits the data in the sense of the least squares. Briefly, the following steps were used to evaluate and describe the dissipation process in tomatoes: the linear, quadratic, and cubic curves were obtained by plotting transformed residue values versus time; the coefficient of determination (r 2), the intercept, and the slope of each one line were also determined.

Fungicides

Tomato samples were collected during a 2-week period after the application of fungicides chlorothalonil, metalaxyl, metalaxyl-M, and propamocarb hydrochloride. The residue level of these fungicides tended to decrease with time (Fig. 1). Metalaxyl and chlorothalonil were the most stable fungicides showing first-order kinetics (r 2 = 0.889 and 0.954) with half-lives (t 1/2) of 4.88 and 2.06 days for metalaxyl and chlorothalonil, respectively. Residue level of these fungicides decreased with time from the initial concentration of 22.08 and 4.12 mg kg−1to 0.09 and 0.03 mg kg−1 in 15 days. Residues of metalaxyl and chlorothalonil decreased gradually from initial concentration of 22.08 and 4.12 mg kg−1 to 0.09 and 0.03 mg kg−1 during the experiment period of 15 days. Metalaxyl-M and propamocarb hydrochloride had shorter half-lives than the other fungicides of1.98 and 1.29 days, respectively, and showed first-order kinetics (r 2 = 0.991 and 0.903). Residues of these two fungicides decreased from the initial 2.13 and 3.07 mg kg−1 to 0.03 and 0.02 mg kg−1 in 12 and 9 days, respectively (Fig. 1 and Table 3). The preharvest interval (PHI), during which the residue level was below the maximum permitted residues level (MRL), of these fungicides was 6, 9, 4 and 4 days for chlorothalonil, metalaxyl, metalaxyl-M, and propamocarb hydrochloride, respectively. Different dissipation rates and half-lives (t 1/2 = 6.5 days) for chlorothalonil were obtained by Gabacorta et al. (2005), Gil Garcia et al. (1997), and Zhang et al. (2007).

Fig. 1
figure 1

Dissipation rate of chlorothalonil (a), pymetrozine (b), metalaxyl-m (c), metalaxyl (d), abamectin (e), and propamocarb hydrochloride (f) after application on tomato using different curves

Full size image

Insecticides

This study included one insecticide, pymetrozine. Figure 1 shows the residual behavior and degradation rate of this compound. Pymetrozine had a short t 1/2 of 3 days. The coefficient of regression of degradation curve was r 2 = 0.92. The insecticide pymetrozine had an initial deposit of 1.36 mg kg−1 after 1 h from the application and reached 0.07 mg kg−1 after 5 days. It required about 3 days for pymetrozine residue to be less than the established MRLof 0.5 mg kg−1. Same trend of pymetrozine dissipation rate was reported, and that the initial residues and half-life were 1.11 mg kg−1 and 0.89 day, respectively (Fenoll et al. 2009; Li et al. 2011; Shen et al. 2009; White et al. 2010).

Acaricide

One acaricide, abamectin, was included in this study. The results are presented in Table 2 and show that the initial deposit of abamectin on tomatoes of S. lycopersicum variety was 2.77 mg kg−1 which gradually decreased with time until the residue reached 0.009 mg kg−1 7 days after application. This is less than the upper limit of the maximum allowed residue set by the Codex Committee on Pesticide Residues under the Joint FAO/WHO Food Standards Program at 0.02 mg kg−1for fruits (FAO/WHO 2006b). Half-life (t 1/2) of abamectin was 1.06 day and the PHI was 6 days to have a residue less than MRL 0.02 mg kg−1. Dissipation rate of abamectin (Fig. 1) had a coefficient of regression of r 2 = 0.904. A different dissipation rate was reported in cases where the initial deposit of abamectin and residues after 7 days of application were 0.09 and 0.03 mg kg−1, respectively (Kamel 2007).

Conclusion

In this study, the dissipation rates of six pesticides after a single application at recommended doses on tomatoes were evaluated. We used an improved method (QuEChERS) for sample preparation. The half-life and PHI were determined for all pesticides. Different dissipation rates, ranging from 3–9 days, were seen for these pesticides. The long PHIs might lead to a higher risk of exposure to pesticides, especially that of metalaxyl, chlorothalonil, and abamectin. Further studies are required to assess the residual behavior, exposure risk, and the environmental fate of these pesticides, especially chlorothalonil, as a new fungicide.