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Introduction
Nowadays there is a trend from the phytosanitary authorities to require food safety, in order to guarantee consumers’ health. From the different chemical risks associated with food primary production, pesticides and specific residues deriving from their use represent the main group of attention, since residues analyses of pesticides in food determine the trade of these products (Codex Alimentarius, 2016). International Organizations such as Food and Agriculture Organization (FAO), European Commission, Food and Drug Administration of the United States (FDA) and the Federal Commission of Health Risks Protection in Mexico (COFEPRIS) establish the socalled Maximum Residue Limits (MRL), which refer to the lowest concentration of pesticide residues found in food that has previously been demonstrated to be safe for consumers (CICOPLAFEST, 2016; Codex Alimentarius, 2016; EPA, 2016a, b; European Commission, 2016; FDA, 2016). It is important to mention that MRL which are emitted and published by the Codex Alimentarus is just a guide and each country is free to adopt or establish its own MRLs, adjusting them on the basis of their own characteristics regarding food consuming habits and modality of use of pesticides in agriculture, ensuring that they consider environmental and health aspects (Codex Alimentarius, 2016).
In this sense, the presence and/or absence of agrochemical contaminants (phytosanitary) in food must to be demonstrated, therefore it is necessary to rely on increasingly sensitive and selective analytical techniques, allowing warning on the presence of these potentially harmful contaminants for consumers’ health. To do so, laboratories for pesticide residues analysis have used multi-residues methods which have been modified throughout the years, mainly adjusting to the technological development of the measurement tools (FDA, 1999). These methods were firstly semi-quantitative and were performed using paper chromatography, then the development of gas chromatography allowed for more precise measurements, in addition to the use of specific and selective detectors (Mills et al. 1963; Luke et al. 1975, 1981, 1983; Lee et al., 1991). However, these traditional methods generally include procedures of sample preparation which require large periods of time, high quantities of toxic organic solvents, various steps of extraction of analytes and elimination of interferences (Ramos, 2012; Puri, 2014; Nollet & Rathore, 2016).
An alternative to these methods is the called “QuEChERS method” (acronym of Quick, Easy, Cheap, Effective, Rugged and Safe), developed by Anastassiades et al. (2003) in order to analyze veterinary drugs, is used nowadays due to its high potential to adapt to analysis of pesticide residues in both agricultural and animal products and has been widely accepted by the international community for the analysis of pesticide residues in different foods. Some of the food matrixes in which it has been demonstrated to be successful for pesticide extraction are: avocado (Benavides & Echeverría, 2014), rice (Hou et al., 2013), shrimp (Omar et al., 2013), peach (Pinho Costa et al., 2014), cocoa grains (Dankyi et al., 2015), orange juice (Rizzetti et al., 2016), olive oil (García-Reyes et al., 2007), tamarind (Paz et al., 2015), corn and soy (Marchis et al., 2012).
For the above mentioned, the aim of the present study was to perform a comparative study between the QuEChERS method and the conventional PAM 302 method (liquid-liquid extraction) for determining organophosphorus pesticides residues in agricultural products of high (> 80 %) and low (< 10 %) moisture content. To that end, 4 pesticides, representative of the organophosphorus group (acephate, methamidophos, dichlorvos and dimethoate) were analyzed, using Ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) and gas chromatography with flame photometric detector (GCFPD); the comparison of the methods was performed using different parameters of validation like: recuperation, linearity, precision (like repeatability), accuracy and detection and quantification limits.
Material and methods
Analytical standards
An individual stock solution was prepared for each one of the analytical standards of high purity organophosphorus pesticides (> 97 %), in acetone, from which mixtures were prepared at intermediate concentrations of 10 ng/µL and 50 ng/µL that were used to fortify control samples and prepare working solutions in the same solvent (Table 1).
Material of analysis
Control samples of fresh tomato (as a product with high moisture content) were obtained from a producer of organic fruits while wheat flour samples (as product with low moisture content) were acquired in the local market of the city of Culiacan, Sinaloa, both were previously analyzed in duplicate to demonstrate that they were free of analytes under scope.
Sample preparation
Products with high (tomato) and low (flour) moisture content were prepared according to that reported in the Pesticide Analytical Manual (PAM), specifically in the 102-A section, which refers to sample preparation for pesticide analysis. In the case of tomato, the edible part was blended and homogenized in an industrial blender (Brand: Waring, Model: 7010G), where a portion was taken as a representative sample for the analysis; while in the case of flour, a subsample required for the analysis was taken (FDA, 1999).
Determination of pesticides by PAM 302 method
In the case of samples with high moisture content, 100 ± 1.0 g of the prepared sample were weighted in a blander glass and 200 mL of acetone were added; in the case of samples with low moisture content, between 10 and 50 ± 0.5 g were weighted in a blender glass and 350 mL of a mixture of water-acetone at 35 % were added, and they were blended at high speed for 2 min. In both cases, the extract was vacuum filtered using a pomp (Brand: Welch, Model: 2567B-50B), and an aliquot of 40 mL was placed in a 1,000 mL separating funnel containing 7.0 g of sodium chloride, then 50 mL of methylene chloride and 50 mL of acetone (all the solvents were HPLC grade), it was shaken for 1 minute and the phases were let separated, the aqueous phase (lower phase) was transferred to a second 500 mL separating funnel, while the organic phase was filtered through anhydrous sodium sulphate and received in a 500 mL Kuderna-Danish concentrator. The aqueous layer contained in the 500 mL separating funnel was re-extracted again twice with 50 mL of methylene chloride, each reextraction was passed through anhydrous sodium sulphate and was received in the Kuderna-Danish concentrator, at the end sodium sulphate was rinsed with 30 mL of methylene chloride. Some heating pearls were added to the heater and a Snyder column was placed inside, initiating the evaporation, in a concentric rings steam bath (Brand: Therm Scientific, Model: 2898), at low temperature (to avoid abrupt reaction of solvents), which was progressively increased. It was evaporated almost until dryness and then 100 mL of petroleum ether were added, it was evaporated almost until dryness and 50 mL more of petroleum ether were added, the evaporation was pursued almost until dryness and 30 mL of acetone were added, doing it on the walls of the column to drag possible rest of samples, it was evaporated to obtain approximately 1.0 mL of the extract. Then it was let cooling and after that the intern standard of triphenyl phosphate (TPP) was added and were diluted with 4 mL of acetone, until subsequent determination of the extract by means of gas chromatography with flame photometric detector (CG-FPD) (FDA, 1999).
Instrumentation and chromatographic conditions
Standards and samples were automatically injected by means of an autosampler 7693 of Agilent with split/ splitless injection ports with a 30 m x 0.25 mm x 0.25 μm capillary column Brand Agilent VF-5 Pesticides, under the following conditions (Table 2).
Table 2 Program of temperature of the oven used in the chromatographic separation
| Stage | Temperature (ºC) | Rate (ºC/min) | Duration (min) | Total (min) |
|---|---|---|---|---|
| Inicial | 100 | -- | 2 | 2 |
| 1 | 170 | 20 | 1.25 | 6.75 |
| 2 | 275 | 4 | 12 | 45 |
Temperature of the Injector: 250ºC
Gas: Helium 1.5 mL/min
Make up: Nitrogen 30 mL/min
Mode: Split - Rate 10:1
Volume of injection: 2.0 µL
Temperature of the detector: FPD-250 oC
Calculations
To calculate the concentration of analyte in the sample, an external standard was used by means of the use of the following formula:
Where:
Determination of pesticides by QuEChERS method
Extraction and partition
15 ±0.1 g of sample were weighted in the case of products with high moisture content (fresh fruits) and 1-5 ± 0.05 g of dry samples (flour) in a 50 mL centrifuge tube. In the case of dry samples, 10 g of water were added and it was let resting for 10 minutes to ensure moisturizing, later 15 mL of acidified acetonitrile at 1 % with acetic acid were added and shaken in an ultrasound bath (Brand: Bransonic, Model: CPX8800H) for 10 minutes, posteriorly 6.0 g of magnesium sulphate and 1.5 g of sodium acetate were added, mixed manually and vigorously for one minute and centrifuged (centrifuge Brand: Hettich, Model: EBA21) at 4,000 r.p.m. for 5 minutes.
Clean-up (dispersion in solid phase)
From 1 to 8 mL of supernatant were placed into a centrifuge tube containing 50 mg of primary/secondary amine (PSA) and 150 mg of anhydrous magnesium sulphate for each millimeter of extract, it was mixed with vortex for one minute and then centrifuged at 4,000 r.p.m. for 5 minutes.
Dilution
100 μL of dispersed supernatant were taken and diluted with 890 μL of mobile phase of ammonium formate 5 mM pH 3.0 + 10 μL of the internal standard solution (d5 atrazine at 0.4900 ng/μL) for its determination by means of Ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS).
Calculations
The calculation of results (concentration in mg/kg) was realized by means of the method of internal standard as expressed below:
Where:
Instrumentation and chromatographic conditions
Each sample was automatically injected through a Sample-Manager system - FTN Acquity of Waters to an equipment of Ultra Performance Liquid Chromatography (UPLC Acquity serie H) equipped with a column Brand Waters Acquity UPLC BEH C18 1.7µm, 2.1 x 50mm, in a volume of 5.0 µL. Conditions employed were established by the laboratory during the development of the chromatographic method, with mobile phase A (ammonium formate 5 mM, pH 3.0) and mobile phase B (methanol + ammonium formate 5 mM + 0.1 % of formic acid), with the following gradient (Table 3).
Table 3 Gradient of the chromatographic method UPLC / MS-MS
| Gradient | Time (min) | Flow (mL/ min) | % A | %B |
|---|---|---|---|---|
| 0 | Inicial | 0.35 | 83 | 17 |
| 1 | 5 | 0.35 | 10 | 90 |
| 2 | 5.1 | 0.35 | 10 | 90 |
| 3 | 7.5 | 0.35 | 83 | 17 |
With a total running time of 9.0 minutes. The identification and quantification were performed by means of ESI+ mode in a Mass Spectrometer Xevo TQ-S of Waters and workstation MassLynx. Ions were monitored using MRM (Multiple Reaction Monitoring) for at least two transitions under the following conditions of tandem mass (Table 4).
Table 4 MS tandem conditions for the analytes under the scope
| Analite | Parent m/z | Daughter m/z | Dwell (s) | Cone (V) | Collision (eV) |
|---|---|---|---|---|---|
| Acephate | 184.1 | 125.1 | 0.003 | 8 | 18 |
| 143 | 0.003 | 8 | 8 | ||
| Methamidophos | 142 | 93.9 | 0.003 | 17 | 13 |
| 124.9 | 0.003 | 17 | 13 | ||
| Dichlorvos | 221 | 79 | 0.003 | 23 | 34 |
| 109 | 0.003 | 23 | 22 | ||
| Dimethoate | 230.1 | 125 | 0.003 | 12 | 20 |
| 199 | 0.003 | 12 | 10 | ||
| Atrazine d5 | 221.3 | 78.89 | 0.003 | 40 | 24 |
| 100.98 | 0.003 | 40 | 22 | ||
| 136.99 | 0.003 | 40 | 22 |
Experiments for verification
Fortified control samples (in distinct days) for fresh product
Level 1. Control sample (fresh tomato) fortified with 200 µL of mixture at the concentration of 10 ng/µL (low level).
Level 2. Control sample (fresh tomato) fortified with 1 mL of mixture at the concentration of 10 ng/µL (medium level).
Level 3. Control sample (fresh tomato) fortified with 2.5 mL de mixture at the concentration of 50 ng/µL (high level).
Fortified control samples (in distinct day) for dry product
Level 1. Control sample (grain wheat) fortified with 200 µL of mixture at the concentration of 10 ng/µL (low level).
Level 2. Control sample (grain wheat) fortified with 1 mL of mixture at the concentration of 10 ng/µL (medium level).
Level 3. Control sample (grain wheat) fortified with 2.5 mL of mixture at the concentration of 50 ng/µL (high level).
Parameters of verification evaluated in the methodological comparison
In both methods, the parameters limit of detection (LOD), limit of quantification limit (LOQ), linearity of the equipment (working interval), linearity of the method, accuracy and precision of the method (in conditions of repeatability) were assessed according to EURACHEM validation guide and posteriorly a comparison of these parameters were performed between both methods.
Linearity of the method and working linear interval (measuring equipment)
To establish the linearity of the system, a calibration curve was constructed for each analyte with six levels of concentration (range 0.05 to 1.0 ng/µL) for PAM 302 method and seven levels of concentration (range 1.0 to 40 µg/L) for QuEChERS method. For this, standards were used in mixture, each level of concentration of the mixture of standards was analyzed under operating conditions and graphs of area were obtained under the curve of theoretical concentration against the response of the detectors (counts of area), using Excel 2010 and confirming with the graph generated by MS Workstation 7.01 and OpenLab Chemstation software for the PAM 302 method and by MassLynx software for QuEChERS method.
The linearity of the method was established with the fortification of control sample in three levels of concentration, high, medium and low, previously mentioned within the working linear range and drawing a graph with the average quantity of recovering against the quantity of added analyte, in both cases (linearity of the system and of the method), the criterion of acceptation according to EURACHEM (1998) is that the coefficient of determination (R2) is higher or equal to 0.98.
Precision (Repeatability)
It was determined by fortifying control samples in three levels of concentration (low, medium and high), seven replicates of each level of concentration and each level in different days were performed and in the determining step they were ran in simple replicate, percentages of recovering were calculated (% R) and the precision in repeatability conditions was assessed as the coefficient of variation (% CV) of the repetitions of the medium level.
Where:
The criterion of acceptation is that percentage of CV is ≤ 20 % (EURACHEM, 1998).
Accuracy (% of recovering)
To evaluate this parameter, data of the percentage of recovering obtained in each level were taken and the average recuperation of the three levels was obtained for each analyte.
The criterion of acceptation was that the percentage of recovering is between 70 and 120 % (EURACHEM, 1998).
Limit of Detection (LOD) and Limit of Quantification (LOQ)
These parameters were estimated from the data of repeatability of first level of fortification calculating the recuperated concentration and obtaining the standard deviation (SD) which was used to estimate detection limits (LODs) and quantification limits (LOQs) of the method, as described in the following formula (USEPA, 2000):
LOD= t0.99*SD (t0.99 = 3.1427; value from tables of one-tail t-Student with 6 degrees of freedom (n = 7) and 99 % of confidence level).
Results and Discussion
Detection Limits (LODs) and Quantification Limits (LOQs)
Detection limits obtained for each analyte by means of analytical method for the matrix of high moisture content are presented in Table 5, and in Table 6 for the matrix of low moisture content. As observed, PAM 302 method presented lower limits, both detection and quantification than the QuEChERS method. However, detection and quantification limits of the latest were lesser than those reported by Ahumada and Zamudio (2011) for acephate (LOD = 0.01 mg/kg and LOQ = 0.04 mg/kg) and dimethoate (LOD = 0.005 mg/kg and LOQ = 0.02 mg/kg) using the QuEChERSUPLC/MS-MS method in the same matrix (tomato). Meanwhile in the case of the whole wheat flour, LOQs of 0.01 mg/kg have been reported for dichlorvos and methamidophos using the QuEChERS-UPLC/MS-MS method, which were lower than those reported in the present study (Bordin et al., 2016).
Table 5 Detection limits by analytical method for the high humidity matrix
| Analites | Limit of detection (LOD) (mg/kg) PAM 302 Method |
Limit of detection (LOD) (mg/ kg) QuEChERS Method |
|---|---|---|
| Methamidophos | 0.006 | 0.009 |
| Acephate | 0.005 | 0.006 |
| Dichlorvos | 0.008 | 0.006 |
| Dimethoate | 0.009 | 0.006 |
Table 6 Detection limits by analytical method for the low humidity matrix
| Analites | Limit of detection (LOD) (mg/kg) PAM 302 Method |
Limit of detection (LOD) (mg/ kg) QuEChERS Method |
|---|---|---|
| Methamidophos | 0.006 | 0.04 |
| Acephate | 0.011 | 0.039 |
| Dichlorvos | 0.007 | 0.033 |
| Dimethoate | 0.009 | 0.026 |
Other methods employed in determining organophosphorus compounds in tomato as the QuEChERS method and determining by means of gas chromatography coupled to a detector of simple mass spectrophotometry (GC-MS) reported LODs and LOQs higher for dimethoate (LOD = 0.1 and LOQ = 0.4 mg/kg) (Domínguez et al., 2014) and dichlorvos (LOD = 9.72 and LOQ = 32.4 mg/kg) (Jahanmard et al., 2015).
On the other hand, inferior limits have been reported as well for some compounds as diazinon (LOD = 0.0001 and LOQ = 0.0002 mg/kg) (Fenoll et al., 2007), dimethoate and dichlorvos (LOD = 0.003 and LOQ = 0.009 mg/kg) (Trivedi et al., 2014) in tomato using the same quantification technic employed here for PAM 302 method.
Table 7 Quantification limits by analytical method for the high humidity matrix
| Analites | Limit of quantification (LOQ) (mg/kg) PAM 302 Method |
Limit of quantification (LOQ) (mg/kg) QuEChERS Method |
|---|---|---|
| Methamidophos | 0.017 | 0.027 |
| Acephate | 0.014 | 0.018 |
| Dichlorvos | 0.023 | 0.018 |
| Dimethoate | 0.026 | 0.018 |
working linear interval
All calibration curves of the analytes under this study, regardless of the method, achieved the criterion specified in EURACHEM. Figure 1 shows an example of calibration curves of acephate and methamidophos in matrix of high moisture content.
Linearity of the method
Both methods showed a linear response for the evaluated analytes which achieved the established criterion, results obtained as for both matrixes of high and low moisture content were summarized in Table 9.
Table 9 Linearity of the methods in matrices of high and low moisture content
| Analites | High moisture content matrix | Low moisture content matrix | ||
|---|---|---|---|---|
| PAM 3021 | QuEChERS2 | PAM 3021 | QuEChERS2 | |
| r2 | r2 | |||
| Methamidophos | 0.997 | 0.997 | 0.999 | 0.9919 |
| Acephate | 0.999 | 0.999 | 0.999 | 0.9958 |
| Dichlorvos | 0.999 | 0.997 | 0.999 | 0.9967 |
| Dimethoate | 0.995 | 0.999 | 1.000 | 0.9995 |
| Criterion | r2 > 0.99 | |||
1GC-FPD= Gas Chromatography with Flame Photometric Detector; 2UPLC-MS/MS= Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry Detector.
Precision in repeatability conditions
Precision was estimated as relative deviation standard or % CV in the low level of concentration. Results of precision for the assessed methods are presented in Table 10, which indicate that both methods are precise, since in all cases they achieved the evaluation criterion.
Table 10 Precision of the methods in matrices of high and low moisture content
| Analites | Coefficient of variation in matrix of high moisture content (%) |
Coefficient of variation in matrix of low moisture content (%) |
||
|---|---|---|---|---|
| PAM 3021 | QuEChERS2 | PAM 3021 | QuEChERS2 | |
| Methamidophos | 10.07 | 8.38 | 4.8 | 13.54 |
| Acephate | 7.54 | 7.08 | 9.08 | 14.66 |
| Dichlorvos | 12.23 | 5.69 | 5.68 | 10.46 |
| Dimethoate | 13.71 | 5.82 | 7.43 | 8.95 |
| Criterion | %CV ≤ 20 | |||
1GC-FPD= Gas Chromatography with Flame Photometric Detector; 2UPLC-MS/MS= Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry Detector.
Accuracy of the methods
The percentage of recovery or recovering is a parameter demonstrating the efficiency of the extraction method for each analyte in intentionally fortified samples. Both methods demonstrated to be exact for the set objective, percentages of recovering for the QuEChERS method were between 85 and 95 % for the matrix of low moisture content and between 91 and 101 % for the one of high moisture content; while for the PAM 302 method, recovering were observed between 93 and 99 % for the matrix with low moisture content and between 91 and 110 % for the one of high moisture content, in all cases they achieved the established criterion (Table 11).
Table 11 Accuracy of the methods in matrices of high and low moisture content
| Percentage of average recovery (%R) in a high moisture content matrix | Percentage of average recovery (%R) in a low moisture content matrix | |||
|---|---|---|---|---|
| Analites | PAM 3021 | QuEChERS2 | PAM 3021 | QuEChERS2 |
| Methamidophos | 91.64 ± 9.23 | 100.50 ± 9.26 | 99.94 ± 4.79 | 99.03 ± 6.91 |
| Acephate | 94.61 ± 7.14 | 91.44 ± 5.54 | 97.65 ± 8.87 | 84.89 ±4.46 |
| Dichlorvos | 97.24 ± 11.90 | 100.04 ± 4.18 | 92.77 ± 5.27 | 95.40 ±5.29 |
| Dimethoate | 110.16 ± 13.73 | 101.10 ± 1.68 | 95.72 ± 7.11 | 98.59 ± 2.74 |
| Criterion | 70<%R <120 | |||
1GC-FPD= Gas Chromatography with Flame Photometric Detector; 2UPLC-MS/MS= Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry Detector.
Regarding the extraction methods evaluated in the present study, PAM 302 is based on a liquid-liquid extraction (LLE) consisting in the distribution of the analytes in different organic solvents due to the coefficient of partition of each analyte. It is a technic of extraction commonly used in standardized methods for pesticide analysis (Farajzadeh et al., 2014; Puri, 2014). However, it presented more disadvantages than advantages, such as: use of high volumes of toxic organic volumes, formation of emulsions, low sensitivity, it is a delayed process that normally requires an extra step for washing the extract, it is expensive and tedious (Ramos, 2012; Farajzadeh et al., 2014).
While QuEChERS method is based on a dispersive solid phase extraction (d-SPE) consisting in dispersing the simple with anhydrous magnesium sulphate to achieve the separation of the water present in the organic solvent used to extract the analytes. Posteriorly, the obtained extract was mixed with PSA sorbent which is used to eliminate polar compounds co-extracted from the matrix (fatty acids, some sugars, polar pigments, etc.) and in some cases other sorbents were added as well such as C18 or carbon-graphite to eliminate other interferents (sterols, pigments as chlorophyll, etc.) (Anastassiades et al., 2003). Among the advantages of this process are its velocity, robustness, high percentage of recovering, ease of handling, minimum requirement of solvent and low cost. One of its disadvantages is that evaporation and reconstitution of the final extract is required to improve the sensitivity of the studied pesticides (Anastassiades et al., 2003; Ramos, 2012; Bruzzoniti et al., 2014; Puri, 2014).
Regarding pesticide quantification in food samples, gas and liquid chromatography coupled to mass spectrophotometry are currently the most used instrumental technics in residues analysis, since they allow the unequivocal identification of the analytes, while the use of selective detectors normally requires realizing a confirmation by means of a different column and/or detector (Di Stefano et al., 2012; Gómez-Ramos et al., 2013; Bruzzoniti et al., 2014; Hird et al., 2014; Puri, 2014; Saraji & Boroujeni, 2014).
Monitoring the method applied to real samples
In order to demonstrate the performance of the assessed methods, real samples were obtained from markets and supermarkets in Culiacan, Sinaloa. A total of four samples were analyzed (three representative samples for matrixes with high moisture content and one representative sample for matrixes with low moisture content).
In one of the tomato samples, two of the analytes under scope were detected (methamidophos and dimethoate). Residues were detected in concentrations of 0.056 and 0.038 mg/kg, respectively by means of the QuEChERS methods and 0.031 and 0.023 mg/kg, respectively for the PAM 302 method.
Conclusions
Results of the verification of performance in both proposed methods were satisfactorily in accordance to what was indicated in the validation guide of EURACHEM methods for parameters of linearity, accuracy, precision in repeatability conditions, detection and quantification limits.
Percentages of recovering varied in the range of 91.6 to 110.1 % with CV in the range of 4.8 to 13.7 %. LOD of the different analytes was established in the range of 0.006 to 0.0009 mg/kg in matrixes of high moisture content and of 0.006 to 0.040 mg/kg in matrixes of low moisture content, while LOQ was established in the range of 0.014 to 0.027 mg/kg in matrixes of high moisture content and of 0.018 to 0.121 mg/kg in matrixes of low moisture content.
The proposed methods were successfully applied in the analysis of real samples, detecting pesticide residues in a tomato sample.
For the notorious advantages presented by the QuEChERS methods with respect to the PAM 302 method and in addition to the use of detector of tandem mass spectrometry, using or implementing of this method was recommended for matrixes of both low and moisture content, even increasing the scope of the here proposed method to other pesticides of the same or different chemical class.
