Ozone as a Safety Post-Harvest Treatment for Chlorpyrifos Removal from Vegetables and its Effects on Vegetable Quality

The removal of chlorpyrifos pesticide residues from vegetables was achieved by using low level of ozonated water (OZW) for 15 60 min as contact times at 25 and 35oC. Recovered amount of chlorpyrifos was extracted using solid phase extraction (SPE) and then analyzed by gas chromatography-mass spectrometry (GC-MS). The initial levels of residual chlorpyrifos varied with the kind of vegetables, where arugula had the highest level followed by parsley, leek, tomato, carrot, cucumber, cabbage, and then bell pepper. When vegetables washed in OZW was at 25 oC, the percentages of chlorpyrifos removal were time-dependent and ranged from 30 83, 91 97, 80 92, 92 95, 87 97, 95 97, 64 100 and 90 97% for bell pepper, tomato, cucumber, carrot, arugula, parsley, cabbage and leek, respectively. In case of vegetables washed with OZW at 35oC, increasing of the contact time was not significantly effect on the removal percentages of chlorpyrifos except with arugula and cabbage. Likewise, the increasing of OZW temperature caused a negative consequence on the removal percentages of pesticide. The effect of these wash treatments on vegetable quality parameters indicated that the removing of chlorpyrifos by using OZW did not produce any significant undesirable effects on antioxidant capacity, total phenolic contents and vitamin C of the tested vegetables. Due to the large amount of vegetables consumed in fresh form, a higher risk of exposure to chlorpyrifos may occur and the search for a safety method to remove this pesticide with negligible residual deposits has always been preferred. Therefore, the present study validated that ozone technology as wash treatment is safe and promising processes for the removal of chlorpyrifos from the vegetable’s surface under domestic conditions to reduce the impact over consumer’s health. *Corresponding author: Khaled, A. O, Department of Pesticide Chemistry & Technology, Alexandria University, Alexandria, Egypt; Tel.: +2035901900; Fax: +2035922780; E-mail: kamosman@yahoo.com Received Date: January 23, 2017 Accepted Date: February 27, 2017 Published Date: April 04, 2017


Introduction
Pesticides are a group of artificially synthesized substances used in farms to control pests and to enhance agricultural production. However, the use of pesticides represents a risk, especially in the developing countries. In recent years, the scientific community has shown a great concern about the possible adverse effects of these pesticides in food. These residues cause detrimental effects on human health such as neurotoxicity, carcinogenesis, abnormal reproduction and cell development [1,2] (Burrows et al., 2002) especially in the developing countries where pesticide contamination is widespread [3] .
2 rotransmitter acetylcholine (ACh) in synaptic junctions leads to excessive stimulation of postsynaptic cells causing cholinergic toxicity. Also, chlorpyrifos may induce intracellular oxidative stress (Osman 1999), influence brain cell replication (Crumpton et al. 2000) and DNA synthesis (Dam et al. 2000) as well as disrupts normal cellular development and differentiation (Bebe and Panemangalore, 2003).
In fact, fruits and vegetables are basic ingredients of the highly demanded diet, associated with a beneficial and healthy function against numerous diseases [6,7] . With increasing global demand for vegetables, buyers demand these vegetables with lower pesticide residues. The development of efficient strategies is necessary to reduce pesticide residues from agricultural products [8] . Recently, many processes have been tested for degrading pesticides on various agricultural products. Some of these processes utilize powerful oxidizing agents such as O 3 /electron beam [9,10] , O 3 /H 2 O 2 , UV/O 3 [11] (Kuo, 1999), electrochemical oxidation processes [12,13] , UV, photo-Fenton system [14] , titanium dioxide catalytic treatment [15] , bio treatment [16] ., microwave irradiation [17] , UV/H 2 O 2 and ozonated water (Osman et al. 2014;Osman 2015). Hence, the use of such simple and non-toxic washing treatments to reduce such residues in fruit and vegetable samples can facilitate the commercialization and reduce the impact over the consumer health [18] . (Osman et al. 2014;Osman 2015). O 3 is a triatomic form of oxygen and is referred to as activated oxygen, allotropic oxygen or pure air and considered as a powerful oxidant, where it's higher oxidizing character (E 0 ) = 2.8V. It has a pungent, characteristic odor described as similar to ''fresh air after a thunderstorm [19] . It is an unstable gas with a half-life of in distilled water at 20 ºC is about 20 -30 min and degrades quickly into oxygen and thus leaves no residues in food [20] . Thus, it does not accumulate substantially without continual ozone generator [21] . These attributes make O 3 in gaseous or liquid form as an attractive candidate for controlling insects and fungi in stored products, used even in fruits and vegetables (Carletti et al. 2013;Osman 2015) and extend the storage life of fruits and products [8,22,23] Such advantages make ozone attractive to the food industry and therefore it has been affirmed as generally recognized as Safe (GRAS) for utilization in food processing [24] . Likewise, the preoxidation by ozone is an effective treatment for putting down the majority of the pesticides in many agricultural products [8,25,26]  . The removal efficiency of pesticides highly depends on the dissolved ozone level, temperature, pH level, type of pesticide and matrix [18,25,27] (Kusvuran et al., 2012).
Because there is a general trend in KSA to increase the production of vegetables, mainly due to their health properties and the demand to use chlorpyrifos for control of insects in vegetables. This leads to pesticide residues on (or in) the vegetables at harvest. These residue levels are generally well higher than the established tolerances [1] . Recently, the safety of vegetables, including contamination with agricultural pesticides is a major concern to both the producer and consumer, and the development of a method to remove the pesticides before marketing has been eagerly awaited. Thus, the present work was extended out to assess the potency of ozone (O 3 ) as a novel engineering science for different contact times as simple wash treatments for removal of chlorpyrifos residues from different sorts of vegeta-bles. The objective of this research was also to study the effect of these wash treatments on vegetable quality parameters such antioxidant capacity (AC), total phenolic contents (TP) and vitamin C in vegetables. ene reactor for 0, 15, 30, 45 and 60 min. Solution temperature was kept at either 25 or 35 ± 1ºC by water bath. The duration of dissolved ozone levels was controlled via adjusting the duration of bubbling. Excessive gaseous ozone was trapped in 2 % potassium iodide solution.

Sample preparation and solid-phase extraction
At the end of tested time intervals, vegetables were chopped and a subsample (10 g) was weighed into 50 ml glass tube and extracted with 20 ml acetone using a homogenizer (Euroturax, IKA Labortechnik Staufen, Germany) at full speed for 5 min. After addition of 10 g sodium chloride, the homogenate was centrifuged at 3,000 rpm for 5 min and the supernatant was transferred to a clean graduated cylinder.
SPE was carried out according to Štanbaher & Zupančič-Kralj, (2003) with slight modification. The columns were conditioned by passing 6 ml of ethyl acetate followed by 6 ml of methanol and then 8 ml of ultra-pure deionized water. The sorbent was never allowed to dry during the conditioning and sample loading steps. Then the extraction columns were fitted with detachable 70-ml polypropylene reservoirs to contain the diluted sample extract. The extract was transferred to the reservoir, which was partially filled with ultra-pure deionized water and then water was added to the top. Sample loading was performed under vacuum at flow rates of 5 ml min -1 . After the passage of the extract, the column was dried by vacuum aspiration under increased vacuum for 30 min. The pesticide was eluted with three 2-ml aliquots of ethyl acetate-acetone at the ratio of 90:10 (v/v). The eluates were collected in 12 ml tubes under gravity flow only. After all the elution solvent had passed through the extraction column, the residual solvent was forcibly removed from the column. The eluate was evaporated to less than 1 ml using a gentle stream of nitrogen and then the solvent was exchanged to isooctane by adding two 2-ml portions of isooctane and evaporated to low volume after each addition. The extract was quantitatively transferred to 2 ml clean vials, completed to 1 ml with isooctane and then analyzed by gas chromatography-mass spectroscopy (GC-MS).

Recovery studies
The vegetables used in the recovery test were confirmed to be free from chlorpyrifos. For recovery studies, subsamples of known blanks (10 g) were spiked prior to extraction by the addition of 2 ml of chlorpyrifos standard solution in acetone to give 0.00, 0.05, 0.25 or 0.50 mg/kg. They were then prepared according to the proposed procedure as described previously and then absolute recovery and precision (expressed as a relative standard deviation, RSD) were measured by analyzing three samples. The recovery values were 90 -101, 92 -95, 96 -105, 93 -102, 88 -95, 90 -94, 92 -99 and 82 -92% for bell pepper, tomato, cucumber, carrot, arugula, parsley, cabbage and leek, respectively with precision values ranged from 5 to 15%. The limits of detection (LOD) were calculated from the signal-to-noise ratios obtained by analyzing unspiked samples (n = 10); LOD was taken to be the concentrations of pesticide resulting in a signal-to-noise ratio of 3. The LOD values were 1, 0.5, 0.8, 0.5, 1, 1.2, 0.5 and 1.5 ppb, respectively with RSD ranged from 5 -15%.

Gas chromatography-mass spectrometry (GC-MS)
Gas Calibration was achieved by preparing matrix calibration standards from the extracts of blank samples in order to compensate for matrix effect. Analytes were quantified by using a 3-point calibration with those matrices matched calibration standards corresponding to the spiked concentration. Figures  1 and 2 represent the GC-MS chromatogram corresponding to standard chlorpyrifos (1 ppm) and tomatoes immersed in 2 mg/ kg chlorpyrifos and immersed in O 3 , respectively.

Effect of treatment on vegetable quality
The level of vitamin C in vegetables was measured according to the method of Klein and Perry (1982) using 2,6-Dichlorophenolindophenol (DCPIP) as an indicator at 515 nm. Vegetable sample was homogenized with blender at full speed for 2 min, weighed (1g) into 50 ml Teflon centrifuge tube containing 20 ml of 2% of oxalic acid, vigorously shacked for 1 h and centrifuged at 3,000 rpm for 10 min. Supernatant was used to measure the level of vitamin C and the results were expressed as mg vitamin C/100 g of fresh weight.

Extraction of total phenolics
One gram of sample was put into 50 ml Teflon centrifuge tube and extracted with 25 ml 80% ethanol using the homogenizer at full speed for 2 min. The extract was centrifuged at 4,000 rpm for 10 min and the supernatant was used to measure total phenolics (TP) and antioxidant capacity (AC).

Total phenolics
TP was determined according the method of Singleton and Rossi (1965) using the Folin-Ciocalteus reagent. In brief, 0.1 ml of extract was added to 7.9 ml of distilled water, 0.5 ml of Folin-Ciocalteus reagent, 1.5 ml of sodium carbonate solution (200 g/l) and then mixed vigorously. The mixture was allowed to stand for 1 h at the room temperature and then the absorbency was measured at a wavelength of 765 nm. Gallic acid (GAE) was used as a standard and the results were expressed as mg equivalents GAE/ 100 g of fresh weight.

Antioxidant capacity
AC or free radical scavenging activity was determined [28] using 1,1-diphenyl-2-picryl-hydrazil (DPPH) reagent. In brief, 1.5 ml of freshly prepared methanolic DPPH solution (0.02 mg/ml) was added to 0.75 ml of 80 % ethanol extract and then stirred. The decolourizing process was recorded after 5 min of reaction at a wavelength of 517 nm and compared with a blank control using the Spectrophotometer. The DPPH radical scavenging activity of the extracts was measured using the Trolox standard curve. Results were expressed as μmol Trolox equivalent (TE) antioxidant capacity/100 g of fresh weight.

Statistical analysis
Treatments were done in triplicate for each time. Data were calculated as mean ± SD analyzed using ANOVA. A probability of 0.05 or less was considered significant. The statistical package of the Costat Program (1986) was used for all chemo metric calculations.

Removing of chlorpyrifos by wash treatments
Postharvest treatments, such as the postharvest water wash and scrub that have been traditionally used to get rid of rubble and dirt, have been proven to reduce pesticide residues (El-Hadidi, 1993). The use of postharvest ozone dips has also demonstrated potential as an effective postharvest treatment in the reduction of pesticide residues on apple fruits. The use of ozonated water dips has similar potential as an alternative postharvest treatment method to remove pesticides from date fruits.
In the present study, the effects of DW and OZW wash treatments at 25 and 35 ºC for different dipping times on chlorpyrifos removal from different kinds of vegetables were investigated ( Table 1). The initial levels of residual chlorpyrifos varied with the kind of vegetables, where arugula had the highest level followed by parsley, leek, tomato, carrot, cucumber, cabbage, and then bell pepper. The levels of natural waxing and properties of vegetables impact the quantity of pesticide retained by vegetables [25] , where non-polar pesticides are tenaciously held in the waxy layer of peel of fruits and vegetables [3] .
In the present study, it was observed that the removal pesticide percentages by DW washing for 60 min meaningfully changed with the washing temperature increase. The percentages of chlorpyrifos removal ranged from 18 -42 and 48 -63% at 25 and 35 ºC, respectively. As a result, the results of this study demonstrated that the removal of the pesticide from the vegetables by washing with DW depended on the type of vegetables. Also, it was found that, the amount of chlorpyrifos residues was significantly decreased exponentially as the contact time increased in vegetables treated with ozonated water at 25 ºC; however, at 35 ºC the percentages of chlorpyrifos removal did not depend on the contact of time for all the tested vegetables except arugula. Compared with the control (no wash treatment), both DW wash and OZW treatments significantly (p < 0.05) reduced the tested pesticide residual levels on vegetable. However, in most cases when vegetables washed with OZW the residual levels reduced significantly (p < 0.05) compared with the DW treatment. When vegetables washed with DW for 60 min either at 25 or 35 ºC, the highest and lowest percentages of chlorpyrifos removal (42 and 18% ) was recorded for cabbage and parsley Int J Food Nutr Sci | Volume 3: Issue 2 www.ommegaonline.org Ozone as a Safety Post-Harvest Treatment for Chlorpyrifos when the temperature was 25 ºC, while the highest and lowest percentages of chlorpyrifos removal (63 and 49% ) was recorded for parsley and bell pepper, respectively. Ozone as a Safety Post-Harvest Treatment for Chlorpyrifos chlorpyrifos ethyl, tetradifon and chlorothalonil from the lemon, orange and grapefruit matrices, whereas increasing of ozonation temperature caused a negative effect on the removal percentages of pesticides (Kusvuran et al., 2012) and did not significantly (P < 0.05) increase the rate of degradation of azinphosmethyl by ozone [27] . (Ong et al., 1996) This may be due to the solubility of ozone in water is inversely proportional to temperature [25] . However, higher temperature enhanced the efficacy in the removal of methyl-parathion, parathion, diazinon and cypermethrin on vegetable surface (Brassica rapa) [25] .
The results from the present study were in agreement with those reported by [8,25] who found that tap water and ozonated water treatments significantly (p < 0.05) reduced pesticide the residual levels on vegetable, compared with the no wash treatment [8,25] . However, OZW wash further reduced the residual levels significantly (p < 0.05), compared with the tap water treatment. Azinphos-methyl, captan and formetanate hydrochloride in solution and on fresh and processed apples decreased by 50 -100% with ozone treatment [27] , mancozeb residues decreased by 56 -97% with ozone treatment at 1 and 3 ppm of ozone [30] . These differences could be attributed to a great variability in the conditions of the application, such as the feeding gas technique, the method used for ozone generation and application, the ozone concentration, and, above all, the exposure interval to the gas [31] . (1) showed that when vegetables contaminated with 2 mg/l of chlorpyrifos and then washed with OZW for 15 -60 min as contact times at 25 ºC, the percentages of removal were time-dependent and ranged from 30 -83, 91 -97, 80 -92, 92 -95, 87 -97, 95 -97, 64 -100 and 90 -97% for bell pepper, tomato, cucumber, carrot, arugula, parsley, cabbage and leek, respectively. In case of vegetables contaminated with 2 mg/l of chlorpyrifos and then washed with OZW at 35 ºC, the highest percentages of chlorpyrifos removal were recorded after 15 min for cabbage, parsley and carrot, 30 min for bell pepper, tomato, carrot and leek and 60 min for arugula as a contact time. No detectable amounts of chlorpyrifos were found in cabbage that was immersed in ozonated water for 45 and 60 min at a temperature of 25 ºC and for all the tested time intervals at 35 ºC. As the contact time increased and the temperature of OZW 35 ºC, ozone treatments did not improve degradation efficiency, showing that ozone concentrations were enough for the oxidization [29] . Although the high temperature benefits the chemical reaction between oxidants and substrates, it decreases the partial pressure of dissolved ozone in aqueous condition.

Data in Table
In many cases the increase in temperature of OZW from 25 to 35 ºC did not increase the removal percentages of chlorpyrifos from vegetables. Increasing of applied ozone dosage was not significantly effect on the removal percentages of www.ommegaonline.org Ozone as a Safety Post-Harvest Treatment for Chlorpyrifos

6
The quantity of pesticide being retained by vegetable highly depends on the levels of ozone and temperature [25] . From the results obtained in this research work and assuming the criterion that a treatment is efficient in degrading pesticides if a removal percentage of above 70% is obtained [26] , in the most cases OZW at 2 mg/l removed more than 70%. The present study revealed that removing of chlorpyrifos depends on the contact times when the temperature was 25 ºC. Nevertheless, the increased in temperatures did not significantly (P < 0.05) increase the rate of degradation of azinphosmethyl by ozone [27] . O 3 has a powerful oxidant having electrochemical oxidation potential of 2.0V, and therefore, can modify the chemical structure of the selected pesticides creating derived by-products. If these by-products are more toxic than the parent pesticide, such washing treatments should not be utilized to reduce pesticide residue levels in vegetables. It is well recognized that some organo phosphorus pesticides containing P = S bonds (actually organothiophosphorus pesticides) such as chlorpyrifos react with oxidative reagents producing its respective oxygen analogs (e.g. chlorpyrifos-oxon), which are more potent as mammalian acetyl cholinesterase inhibitors than the parent forms (Amdur et al. 1991). The possible formation of toxic by-products by either O 3 was investigated by gas chromatography mass spectrometry (GC-MS) in SCAN mode by monitoring m/z ions: 109, 197, 242, 270, 298 and 335 for chlorpyrifos-oxon. In the GC-MS analysis, chlorpyrifos appeared as a sharp and only a single peak at a retention time of 16.14 min corresponding to chlorpyrifos was observed in the GC-MS chromatogram and there is no intermediate or dead-end product detected using the analytical method described in the present study ( Figure 2). Products of chlorpyrifos degradation include 3, 5, 6-trichloro-2-pyridinol which subsequently breaks down to organ chlorine compounds and carbon dioxide (The Royal Society of Chemistry 1988). The present results are in accordance with many investigators who found that no toxic by-products such as chlorpyrifos-oxon, amitraz and dicofol were detected in date fruits ( [32] . O 3 selectively reacts with compounds containing heteroatoms such as S, N, O, and Cl via two different pathways, namely direct molecular and indirect radical chain-type reactions [33] . The reactivity of compounds with ozone varies largely due to their diverse structural features [8] . Thus, pesticides, which usually have some heteroatoms on the molecules, are often expected to be destroyed by ozonation [34] . So it is recommended to use O 3 as non-toxic washing treatment to reduce such residues in vegetables.

Effect of wash treatments on vegetables quality parameters
The effect of ozone wash treatments on quality parameters of vegetables is of interest since the wash treatment may be performed by vegetable' producers and consumers. Because vegetables are rich in antioxidant compounds, therefore, its consumption is considered to be one of the main factors of a healthy lifestyle [6,7] . Unfortunately, few studies dealt with the effect of ozone wash treatments on vegetable quality parameters [35][36][37][38][39][40][41][42][43][44][45] . Thus, research is needed to investigate the effect of current wash treatments on AC, TP and vitamin C, and ultimately devise ideal conditions of wash treatments suitable for vegetables.
Variations in the levels of AC (Table 2), TP (Table 3) and vitamin C (Table 4) contents were observed between the tested varieties, however tomatoes, arugula and parsley tended to have the highest values of AC, TP and vitamin C, respectively. Also, the present study revealed that in most cases the levels of AC, TP and vitamin C of the tested vegetables did not show significant variations either throughout the dipping times or the tested temperatures. The present study is in parallel to that found in tomatoes where O 3 -enriched atmosphere (concentration up to 1 µmol mol -1 ) did not attain statistical significance change in AC and TP [35] . In addition, the low ozone concentration did not affect organic acids, soluble sugars, lycopene and other micronutrients of vegetables and fruits [35] . (Ibanoglu, 2002;Mendez et al., 2003) or colour and pulling strength of persimmon leaves (Ikeura et al., 2013). Moreover, ozone is able to preserve the polyphenol and anthocyanin contents in grape and keeps the pectin methyl esterase and polygalacturonase activities [36] because it is changed into oxygen by autolysis (Li and Tsuge, 2006); therefore, it leaves no residues on treated commodities and this indicates that the penetration of ozone into the vegetable is unlikely. However, many reports have reported that quality of fruits and vegetables is greatly affected by post-harvest treatments [37] by affecting the nutritional and sensory quality of the product [38] . Plant pigment is bleached after treatment with ozonated water [8] (Badani et al., 1996;Klaiber et al., 2004;Lewis et al., 1996) and enhances the synthesis of resveratrol and of other bioactive phenolics in grapes [39] . The long-term ozone treatment greatly reduces the polyphenol content in grapes [36] (Botondi et al., 2015).
It is established that the presence of AC in the plant is due mainly to the presence of water-soluble compounds with potent free radical-scavenging effects, including phenolic compounds (mainly cinnamic acids) and flavonoids (flavones, flavonols and flavanones) [40][41][42][43][44][45] . Significant correlation between AC and TP in date palm fruits has been established by many investigators [45,46] confirming that these compounds play important role in antioxidant activities [47] .
This result indicated that using O 3 for pesticide removal did not produce any undesirable effect on AC, TP and vitamin C of the tested vegetables. The present investigations are in parallel with [48][49][50][51][52][53][54][55][56][57] Selma et al. (2008) who illustrated that there was no evidence of damage in melons treated with hot water, O 3 or their combination and they maintained initial texture and aroma. However, due to its strong oxidizing activity, O 3 may also cause physiological injury to fresh-cut produce [48] . Therefore, the possible negative impact of O 3 treatment on fruits sensory quality warrants further study [58][59][60][61][62][63][64][65][66][67][68][69][70] . In some cases ozone may promote oxidation degradation of chemical constituents present in the grains, discoloration or development (Mendez et al.  Each value is the mea ± S.D of three replicates. Means having different small letters in column or capital letters in row are significantly different (P < 0.05). Data are expressed as mg gallic acid equivalents (GAE)/100 g fresh weight of vegetable. Each value is the mean ± S.D of three replicates.
Means having different small letters in column or capital letters in row are significantly different (P < 0.05).

Conclusion
Food is the basic necessity of life and its contamination with pesticides is associated with severe effects on the human health. Hence it is pertinent to explore strategies that address this situation of food safety, especially in the developing countries where pesticide contamination is widespread due to indiscriminate usage.
Because vegetables are cultivated for culinary and salad purposes without heat treatments and are commonly consumed by people of different ages all over the world, a higher risk of exposure to pesticides especially in children and other vulnerable individuals may occur. Therefore, the search for a safety method to remove undesired pesticide residues in all the steps of the production and distribution of vegetables chain to decrease the intake of pesticide residues as well as to preserve the most of the essential vegetable nutrients has always been preferred.
In the present study, chlorpyrifos residues were significantly degraded and its residual levels in vegetables were reduced by water and/or O 3 at all the tested time intervals compared with unwashed-fruits and O 3 treatment was more potent than water wash treatment to remove these residues. By the end of the experiment about 83 -100% and 50 -100% of the initial levels of chlorpyrifos residues at 25 and 35 ºC were removed in O 3 -washed vegetables. When the temperature of ozonated water was 25 ºC, the efficacy in chlorpyrifos degradation was increased by prolonging the contact time, while when the temperature of ozonated water increased to 35 ºC, the efficacy of chlorpyrifos degradation was not increased by prolonging the contact time. Thus, the results of the present study indicate that it is unfeasible to use ozonated water at high temperature (e.g. 35 ºC) to degrade chlorpyrifos from vegetables. Also, the present study illustrates that the quantity of chlorpyrifos retained by vegetables varies with the kind of vegetables, where arugula had the highest initial level and bell pepper had the lowest one. The levels of natural waxing and properties of vegetables impact the quantity of pesticide retained by these vegetables.
Due to its high oxidability, high reaction rate, absence of intermediate or dead-end product detected, and instability with a large proportion of it would escape to the ambient or reduce to oxygen molecules in a few minutes without leaving a residue, ozonolysis technique should be used in pesticide-treated vegetables to remove residues adhering on the vegetable's surface without significant changes in antioxidant capacity, total phenolic and vitamin C. Therefore, the present study validated that ozone as wash treatment is safe and promising process for the removal of chlorpyrifos from the vegetable's surface. The results found in the present study must not be extrapolated to other pesticides, vegetables or conditions.