Standards of pesticides were used for calibration curves (0.001–5.00 μg/mL) and injected into the GC-NPD/ECD system under the conditions stated in the GC analysis section. The linearity of the method was evaluated within the range of 0.01–5.00 μg/mL, with correlation coefficients (R
2 > 0.995). The limit of quantification (LOQ) was determined with a signal-to-noise ratio (S/N) = 10, and the limit of detection (LOD) (S/N) = 3. LODs were below 0.003 mg/kg for all 16 analysed pesticides, and LOQs were at 0.01 mg/kg. The validation was performed for five replications at three different spiking levels (i.e. 0.01, 0.30 and 3.00 mg/kg). In all cases, the results of recovery tests were acceptable according to the validation and quality control criteria for pesticide residue analysis established by the European Commission (2014 SANCO/12571/2013) and average recoveries ranged from 78.5 to 101.0 %, with a maximum relative standard deviation (RSD) of 12.0 % (Table 1).
The unprocessed, raw strawberry samples were used to calculate the PFs, and these values describe the efficiency of reducing the pesticide residue level in food processing. Concentrations of the pesticides analyses in unprocessed samples are summarized in Table 2.
Physicochemical properties and mode of action of studied pesticides
The main physical and chemical properties of the studied pesticides, including octanol-water partition coefficient (logP), solubility in water (Sw), boiling point and molecular mass (M) and the mode of action, are presented in Table 3 and discussed individually.
Effect of processing
The processing conditions corresponded as closely as possible to actual conditions that are common in household and industrial practices. All processing methods were conducted over 1, 2 and 5 min, and the change of concentration level over time was analysed. A gradual reduction in the level of nearly all pesticides was noted when the time was increased to 5 min. The concentration changes of the fungicide and insecticide residue levels during processing (Fig. 3a, b) are discussed below for each individual treatment.
To evaluate the effects of processing on pesticide residues in strawberries, processing factors (PFs) related to each process were determined. Processing factors were generally below 1 for most of the studied pesticides, and only after the boiling process did three insecticides exhibit PFs above 1. The behaviour of fungicide and insecticide residue levels during processing is shown in Table 2.
Effects of washing with tap water (A)
Washing is the most common form of processing and is a preliminary step in both household and commercial preparations. In this study, washing was done in tap water. The effects of 1, 2 and 5 min of washing with tap water on pesticide residues in strawberries are shown in Table 2. Concentration changes for 16 pesticide residues were observed after 1, 2, and 5 min of treatment. The effectiveness of this process resulted in a 19.8 % reduction for bupirimate and 68.1 % for chlorpyrifos, with PF = 0.80 and PF = 0.32 (for 5 min), respectively. Washing with tap water significantly reduced (over 50 %) the concentrations of three insecticides and two fungicides. A gradual reduction was noted when the time was increased to 5 min for acetamiprid, alpha-cypermethrin, chlorpyrifos, cyprodinil and fenhexamid by 56.5, 53.4, 68.1, 54.1, and 57.2 %, respectively. These data are consistent with other studies conducted on raw cucumbers (Liang et al. 2012), where increasing the time of the washing process yielded a lower PF.
Our results can be explained through the analysis of the relationship between the physicochemical properties of the studied pesticides, including their solubility in water, octanol-water partition coefficient and PF values. Polar, water-soluble pesticides are more readily removed than low-polarity materials (Holland et al. 1994). A number of studies have reported that pesticides with a lower octanol-water partition coefficient are more easily removed by washing (Kong et al. 2012; Zhao et al. 2014). In our study, acetamiprid, with a low logP = 0.8 and high solubility in water 2950 mg/L, exhibited a low PF = 0.43, in contrast with deltamethrin (logP = 4.6, S
w = 0.0002 mg/L) PF = 0.73 (Table 2). As discussed above, the logP and solubility were the key factors affecting the reduction.
We can presume that a high solubility in water does not have an influence on the effectiveness of washing in every case, but the removal of residues also depends on the location of this substance in the plant material during transpiration. Thus, pesticides such as bupirimate and pirimicarb (with a systemic mode of action) are less likely to be transported into the internal parts of strawberries, despite their high water solubility, and thus, they exhibited PF = 0.80 and PF = 0.79, respectively.
Effects of washing with ozonated water (B)
Ozone (O3) is one of the most potent sanitizers against a wide spectrum of microorganisms (Khadre et al. 2001) and is considered to be the most suitable for removing pesticide residues from fruits and vegetables and for controlling microbes of food safety concern (Gabler et al. 2010). In our study, after 5 min of washing in ozone water, pesticide residues were reduced by between 36.1 % (PF = 0.64) for tetraconazole and 75.1 % (PF = 0.25) for chlorpyrifos (Table 2).
As in the case of washing with chlorinated water, the highest reduction was also observed for chlorpyrifos (Table 2). Chlorpyrifos is a non-systemic insecticide, acting only when it comes into direct contact with plant tissues, and is not transported to other plant parts; therefore, its residues were amenable to simple processing operations, and a larger decrease was expected. In contrast, systemic agents such as tetraconazole or bupirimate, which penetrate to deeper tissue layers of the strawberries, were more difficult to remove (PFs ≥ 0.56).
Comparing the above results, ozonated water was more effective than tap water in pesticide removal. Chen et al. (2013) concluded that removal efficiency increased when vegetables were treated with ozone, and our results confirmed this hypothesis.
Such a large reduction is possible because the dissolved ozone generates hydroxyl radicals that are highly effective at decomposing organic molecules such as pesticide residues (Sumikura et al. 2007; Takahashi et al. 2003).
Moreover, we can conclude that the molecular weight of each compound could affect the percentage of reduction. Washing with ozone water was more effective in the removal of pesticides with a lower molecular mass, such as boscalid or acetamiprid (M ≤ 343.21 g/mol) (both have PF = 0.37), compared with tetraconazole (M = 372.15 g/mol) and trifloxystrobin (M = 408.37 g/mol) (with PF = 0.64 and PF = 0.56, respectively (Table 2)).
Effects of ultrasonic cleaning (C)
Pesticide residues were eliminated most effectively by ultrasonic cleaning. The concentration levels of pesticide residues were strongly changed in this process. As shown in Table 2, pesticide reduction increased with time. The efficiency of ultrasonic cleaning after 5 min ranged between 45.1 % (fenhexamid, PF = 0.55) and 91.2 % (alpha-cypermethrin, PF = 0.09), and PFs were below 0.55 for all active substances.
High reductions, above 70 %, were also found for pyraclostrobin (89.4 %), tetraconazole (84.5 %) and chlorpyrifos (79.1 %) with PFs 0.11, 0.15 and 0.21, respectively (Table 2). Ultrasonic cleaning reduced those pesticide residues to a greater extent than did tap water soaking, because cavitation bubbles created many small air bubbles in the liquid. These bubbles grew, expanded and regularly broke out violently, which generated mechanical energy in the form of shockwaves and caused distribution within the very small pores on the asymmetric surfaces of strawberries. Thus, pesticide residue reduction was more efficient than cleaning without the ultrasonic cleaner.
We can assume that the effectiveness of ultrasonic cleaning also depended on the mode of action of the studied compounds. As reported in Table 2, the non-systemic pesticides (alpha-cypermethrin, chlorpyrifos, deltamethrin, folpet and iprodione) were easily removed by ultrasonication (PF ≤ 0.35), compared with bupirimate, acetamiprid, trifloxystrobin and cyprodinil (0.43 ≤ PF ≤ 0.48), which have systemic modes of action. Ultrasonic cleaning has rarely been studied, but our results confirmed that it is an attractive technique for the removal of pesticide residues in industry.
Effects of boiling (D)
Boiling is a method of cooking food using a high water temperature and is often used to make strawberry preserves. The concentrations of the studied pesticides were highly reduced using this process. Boiling for 5 min caused a greater reduction for most pesticides than did both types of washing. Pyraclostrobin showed the highest reduction during this process (92.9 %, PF = 0.07), while cyprodinil showed the lowest (42.8 %, PF = 0.57) (Table 2).
However, the results obtained from the thermal process indicated that the concentrations of three insecticides from the pyrethroid class (alpha-cypermethrin (PF = 1.02, 1.66 and 1.76), deltamethrin (PF = 1.03, 1.16 and 1.32) and lambda-cyhalothrin (PF = 1.19, 1.38 and 1.70)) increased, and processing factors above 1 were noted. These results could be because these pesticides were concentrated as the water evaporated from the strawberries during boiling (Amvrazi 2011). Notably, similar findings were obtained by Rasmusssen et al. (2003)), who found that boiling did not reduce pesticide residues in apples.
The decreases could also be explained by the fact that water-soluble pesticides such as pirimicarb (S
w = 3100 mg/L) and acetamiprid (S
w = 2950 mg/L) were significantly eliminated (PFs ≤ 0.33), in contrast with alpha-cypermethrin, deltamethrin and lambda-cyhalothrin (PFs > 1) with low solubilities of 0.004, 0.0002 and 0.005 mg/L, respectively. These results may also be interpreted based on the boiling point of each pesticide (Table 3). As shown in Table 3, compounds with high boiling points, such alpha-cypermethrin, lambda-cyhalothrin or deltamethrin, were barely reduced in comparison with acetamiprid or pirimicarb, which have low boiling points.
The disappearance of pesticide residues during thermal processing could be due to decomposition by the effect of heat, the stronger adsorption of pesticides onto plant tissues and/or the solubility of pesticides in water (Table 3). Processes involving heating can increase volatilization, hydrolysis or other forms of degradation and thus reduce residue levels (Holland et al. 1994).
In addition, each compound has different metabolites, and they are different in different matrices or may be produced under different processing conditions (European Commission 1997a, b). Thus, thermally unstable compounds, such as folpet, were significantly reduced (71.7 %), most likely by the formation of degradation products during boiling; however, these products were not investigated in this study.
The potential short-term consumer risk, before and after processing of strawberries, for alpha-cypermethrin was performed for two populations, adults and children. Alpha-cypermethrin, with an MRL = 0.07 mg/kg (EU Pesticide Database), showed more than twice the concentration of the safety limit in raw strawberries when we used a double dose of PPP during our experiments. Alpha-cypermethrin is a non-systemic, broad-spectrum, insecticidal pyrethroid. It acts through digestion in the target organism’s gut and affects the central and peripheral nervous system through sodium channel modulation. Thus, among the analysed pesticides, alpha-cypermethrin has a relatively low value of ARfD (0.04 mg/kg).
Table 4 presents the results of the short-term risk assessment where the ARfD in processed strawberries was below 4 % for adults and 12 % for children (without the correction for PF). Assessment of short-term risk calculated with the correction for PF was lower for both populations than when the PF values were excluded from the calculations with one exception. In boiling, value of PF was above 1 and after correction, IESTI and %ARfD were higher than those obtained without including PF. However, estimated short-term intakes for children and adults, in both cases (without and with correction for PFs), did not exceed the safety limit (100 % ARfD) in all treatments.
Both adults and children have similar values of LP (highest large portion is 333.0 and 251.8 g/person, respectively). However, the calculated IESTI was three times higher for children than that for adults without the correction for PF, and almost ten times higher when the PFs were used (IESTI*). This could be because children are a vulnerable group of consumers, who are, due to their lower body weight, exposed to relatively higher pesticide residue levels.
Correlation of selected parameters of pesticides and removal effectiveness using PCA
In order to better understand the correlation between the selected parameters of the pesticides and removal effectiveness (expressed as processing factors), a principal component analysis (PCA) was carried out. Figure 3 presents the correlation between the processing factors of pesticides and their physicochemical parameters for each treatment. The relationship between the solubility (S
w), polarity (logP), and the mode of action (systemic and non-systemic) of the studied pesticides and the effectiveness of technology (PFs) is discussed below, with respect to each water process.
The scree plots obtained in the PCA for each process are shown in Fig. 4a–d. According to the Kaiser’s eigenvalue-greater-than-one rule, the first two principal components (PC) fulfilled this criteria. Thus, the first PC1 and the second principal component PC2 were further analysed.
The PCA analysis revealed a correlation score (Fig. 4
a''–d'') and loading (Fig. 4
a'''–d''') plots that described more than 78 % of the variation in the first two principal components. The two significant PCs were extracted by covering 80.1 % of the variance in washing with tap water (PC1 51.62 % and PC2 28.48 %) (Fig. 4a), 80.3 % of the variance in washing with ozone water (PC1 51.71 % and PC2 28.58 %) (Fig. 4b), 78.1 % of the variance in ultrasonic cleaning (PC1 51.28 % and PC2 26.65 %) (Fig. 4c) and 82.5 % of the variance in boiling (PC1 65.21 % and PC2 17.30 %) (Fig. 4d).
Interpreting the scores and loadings, the pesticides were categorized, as shown in Fig. 4. Analysed pesticides were grouped into four clusters (Fig. 4a''–b''). The same two groups of compounds were noted in the cases of chlorine, ozone washing and ultrasonic cleaning. The first group was water-soluble pesticides (acetamiprid and pyraclostrobin) and the second was non-polar substances (alpha-cypermethrin, chlorpyrifos, deltamethrin and lambda-cyhalothrin). The dominant variables were solubility and polarity (Fig. 4a'''–d'''). In thermal processing (boiling), the variable with a high contribution was the effectiveness of the treatment and the concentration factors for alpha-cypermethrin, deltamethrin and lambda-cyhalothrin were observed. The results from the PCA confirmed our previous assumptions.