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Degradation of chlorpyrifos and inactivation of Escherichia coli O157:H7 and Aspergillus niger on apples using an advanced oxidation process
Food Control 109 (2020) 106920
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Short communication
Degradation of chlorpyrifos and inactivation of Escherichia coli O157:H7 and Aspergillus niger on apples using an advanced oxidation process
T
J. Hoa, R. Prosserb, M. Hasania, H. Chena, B. Skanesb, W.D. Lubitzc, K. Warrinera,∗ a
Department of Food Science, University of Guelph, Guelph, Ontario, Canada School of Environmental Science, University of Guelph, Guelph, Ontario, Canada c School of Engineering, University of Guelph, Guelph, Ontario, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Chlorprifos Escherichia coli O157:H7 Apples Advanced oxidation process UV-C Hydrogen peroxide Response surface methodology
Chlorpyrifos is a widely used insecticide in apple production but there are concerns that residues ingested by consumers can lead to chronic neurological conditions. In the following, a method based on Advanced Oxidation Process (AOP) was validated for the degradation of chlorpyrifos on apples and inactivating Escherichia coli O157:H7, along with Aspergillus niger spores. AOP is a process that generates free-radicals from the UV-C (at 254 nm) mediated degradation of hydrogen peroxide thereby leading to highly oxidative species. By using Response Surface Methodology (RSM) it was found that the degradation of chlorpyrifos was primarily dependent on UV-C dose and synergistically enhanced by applying the hydrogen peroxide at temperatures up to 66 °C. However, the degradation of chlorpyrifos was independent on the hydrogen peroxide concentration up to 6% v/ v. Maximal degradation of chlorpyrifos on apples was achieved through an AOP treatment applying 68.4 kJ/m2 UV dose and 1.22% v/v H2O2 at 66 °C that resulted in 118 μg degradation (representing 59% of the original level) of the pesticide with < 20 ppb accumulation of the degradative photoproduct, CPY-O. The same treatment supported a > 6.6 log CFU reduction in E. coli O157:H7 and > 4.7 log CFU reduction of A. niger spores. Applying UV alone in the case of E. coli O157:H7 or hydrogen peroxide alone against A. niger spores supported a lower level of lethality compared to AOP. In conclusion, the study has demonstrated that an AOP based process can support the degradation of chlorpyrifos and address microbiological hazards associated with apples.
1. Introduction Chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridylphosphorothioate), is a broad spectrum organophosphate (OP) pesticide that is widely used in agricultural for the control of insects (Gomez, 2009; Mladenova & Shtereva, 2009; Solomon et al., 2014). The pesticide is one of the most commonly applied pesticides in North America with 10% of usage being applied in tree fruit (e.g. apples) production (Atwood & Paisley-Jones, 2017; Eaton et al., 2008). Although beneficial to agriculture for pest control, there have been health concerns in relation to chlorpyrifos residue exposure on foods and in the environment. Specifically, there is accumulating evidence that chlorpyrifos and other organophosphates, exhibit neurotoxic activity in children (Bouchard et al., 2011; Rauh et al., 2012; Trasande, 2017). For example, Bouchard et al. (2011) conducted a 7-year birth cohort study (n = 329) and reported that children exposed to high levels of OPs had lower IQ scores compared to control groups. Mitigation strategies to reduce exposure of the population to chlorpyrifos have been to ban the pesticide, as in the case of Hawaii,
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and devise approaches to reduce carriage on fresh produce. With regards to the latter, the post-harvest washing of produce has been shown to reduce chlorpyrifos levels on fruit by approximately 50% (Rawn et al., 2008). Washing produce with low concentrations of acetic acid, citric acid and potassium permanganate have also been shown to reduce chlorpyrifos concentrations in date fruit (Osman, Al-Humaid, AlRedhaiman, & El-Mergawi, 2014). However, washing of produce within tanks results in a redistribution of pesticides between produce batches and further dissemination within the environment via wastewater disposal (Burkul, Ranade, & Pangarkar, 2015; Köck-Schulmeyer et al., 2013). A more effective approach is to use water-free technologies to decontaminate fruit and to degrade chlorpyrifos thereby reducing levels entering down-stream washing processes. One potential approach is based on Advanced Oxidation Process (AOP) that has previously been shown to inactivate Listeria monocytogenes on apples (Murray, Moyer, Wu, Goyette, & Warriner, 2018). AOP involves generating hydroxyl radicals from the degradation of ozone and/or hydrogen peroxide. The generation of free radicals can be achieved through the interaction
Corresponding author. E-mail address: [email protected] (K. Warriner).
https://doi.org/10.1016/j.foodcont.2019.106920 Received 7 July 2019; Received in revised form 24 September 2019; Accepted 25 September 2019 Available online 26 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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between ozone and hydrogen peroxide, or more rapid via UV-C (254 nm) mediated decomposition of the oxidants (Abramovic, Banic, & Sojic, 2010; Atwood & Paisley-Jones, 2017; Behin & Farhadian, 2017; Femia, Mariani, Zalazar, & Tiscornia, 2013; Marican & Durán-Lara, 2018). The use of AOP for degrading pesticides, including chlorpyrifos, has previously been demonstrated in wastewater (Abramovic et al., 2010; de Oliveira et al., 2014; Ikehata & Gamal El-Din, 2005; Marican & Durán-Lara, 2018; Thind, Kumari, & John, 2018). It was found that UVC with a dose delivered from an 8 Watt low pressure lamp over 4 h at 45 °C resulted in a 39.7% degradation of chlorpyrifos compared to no decrease when 2 g/l hydrogen peroxide was applied alone (de Oliveira et al., 2014). However, by combining UV-C with hydrogen peroxide it was possible to achieve a 62.3% decrease which was independent of the water temperature (de Oliveira et al., 2014). In a further example, an AOP process based on using a combination of UV-C, hydrogen peroxide and titanium oxide supported 73% chlorpyrifos degradation in water compared to 41% when hydrogen peroxide (up to 50 ppm) was applied alone (Thind et al., 2018). Therefore, the AOP based process holds promise as a means of degrading chlorpyrifos although has yet to be studied directly on apple fruit. In the following study the AOP originally developed to decontaminate apples was applied for chlorpyrifos degradation. The process is based on the apples being sprayed with hydrogen peroxide at a set temperature then transferred to a chamber housing UV-C lamps to degrade the pesticide in addition to direct inactivation of microbes (Murray et al., 2018). A treatment of 6% v/v hydrogen peroxide applied at 48 °C in combination with UV-C dose of 54 mJ/cm2 supported a 3 log reduction of L. monocytogenes both on the surface and subsurface of apple fruit (Murray et al., 2018). Therefore, the potential to inactivate pathogens on apples and simultaneously degrade chlorpyrifos offers a potential of a one-step process to reduce the biological and chemical hazards linked to apples. In the current study the selected pathogen of concern was Escherichia coli O157:H7 due to the previous links to foodborne illness outbreaks linked to apples (Kenney, Burnett, & Beuchat, 2001). Aspergillus niger was selected as a test microbe due to its inherent resistance to UV-C in the spore form, in addition to acting as a surrogate for potential mycotoxin producing molds (Taylor-Edmonds, Lichi, Rotstein-Mayer, & Mamane, 2015). A key part of the AOP is optimizing the hydrogen peroxide concentration and temperature along with UV-C dose. Excess hydrogen peroxide can potentially result in a decrease in the efficacy of generating radicals due to self-neutralization that terminates the reaction (de Oliveira et al., 2014). Excess UV-C can also contribute to photobleaching of produce along with loss of sensitive nutrients (Koutchma, Popović, Ros-Polski, & Popielarz, 2016). In the current study response surface methodology (RSM) was used to determine which factors and their settings affect the degradation of chlorpyrifos. A full factorial central composite RSM design was used in this study (Thind et al., 2018). The design is composed of axial points between a selected central point whereby different combinations of the processing parameters are applied and response (i.e. extent of chlorpyrifos degradation) recorded (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). From the response curves it is possible to identify the optimal working parameters of the AOP, in addition to identifying the contribution from each component to the observed pesticide degradation reaction and pathogen log reduction.
Table 1 RSM trials (20) with amount of chlorpyrifos reduction (μg) observed using various test parameters of UV-C dose (kJ/m2), temperature (°C) and concentration (%) of hydrogen peroxide. Reduction from each trial was corrected using the average positive control recovery of 89.4% (n = 177). Trial
UV dose (kJ/m2) (X1)
Temperature of H2O2 (°C) (X2)
H2O2 (%) (X3)
Chlorpyrifos Reduction (μg) (Y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
34.8 22.5 54.0 37.0 22.4 38.3 39.4 39.4 11.7 38.9 56.1 50.3 37.9 21.8 39.3 37.2 62.3 38.5 22.3 53.3
46.5 32.5 32.5 46.5 60.5 70.0 46.5 46.5 46.5 46.5 60.5 32.5 46.5 32.5 46.5 46.5 46.5 23.0 60.5 60.5
3.5 5.0 5.0 1.0 5.0 3.5 3.5 3.5 3.5 3.5 2.0 2.0 6.0 2.0 3.5 3.5 3.5 3.5 2.0 5.0
50 32 52 60 54 58 52 54 28 56 88 74 54 30 52 62 92 58 52 74
replicate centre points (Table 1). Runs were performed in triplicate involving the three previously mentioned factors. The response variable can be expressed as a function (Eqn. (1)) of the independent process variables according to the following response surface quadratic model. 3
Y = a0 +
3
i=1
2
3
aii x 2 +
ai x i + i=1
aij x i xj + i=1 j=i+1
(Equation 1)
where Y is the predicted response, xi and xj are factors influencing the predicted response, αi, αii, and αij are the coefficient of linear, quadratic, and interaction terms respectively. Lastly, ε is a random error. Regression analysis for the quadratic equation model was determined using Design Expert® 11.1.0 software. First, the model fit was tested to determine the quality of the model using the coefficient of determination (R2). ANOVA analysis was conducted to determine whether the test variables listed are significant or not. This was done using F-test and P-values with 95% confidence level. 2.2. Advanced oxidation process reactor The advanced oxidation process (AOP) reactor (Clean Works Corporation, Beamsville, ON, CA) was constructed from stainless steel with frame dimensions of 122 × 38 × 20 cm (length x width x height). The light source was located at the top of the frame as described by Murray et al. (2018). The reactor was fitted with 4 × 25 W UV-C lamps (60 cm length) emitting at 254 nm (Sani-Ray, Hauppauge, NY, USA). The UV intensity was varied through altering the distance of the lamps from the samples from 10 to 16 cm. A radiometer (Trojan Technologies Inc, London, ON, CA) was used to measure the UV-intensity with treatment times being used to deliver a defined UV-dose. Prior to introducing the apple samples into the UV reactor, the hydrogen peroxide (0.2 ml) of defined concentration and temperature was dispensed onto sample.
2. Materials and methods 2.1. Response surface methodology
2.3. Deposition and recovery of chlorpyrifos on apples
RSM was used in the experimental design of this study. Design Expert® Software 11.1.0 (Stat-Ease Inc, Minneapolois, USA) was used to produce a full factorial central composite design investigating the effect of UV-C dose (kJ/m2), temperature of hydrogen peroxide (°C), and concentration of hydrogen peroxide (% v/v) on the degradation of chlorpyrifos (Table 1). The experimental design of this study included a total of 20 runs with six
Crispin apples were provided by Moyer's Apple Products (Beamsville, ON, CA) and stored at 4 °C until required. The apples were prepared by initially washing under running municipal water then towel drying. Only apples showing no signs of spoilage or damage were 2
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used in the study. The peel of the apples was removed using a peeler, then peels were cut into 2.5 cm × 3.0 cm pieces. A working stock solution of chlorpyrifos (> 99% purity; Sigma-Aldrich, St Louis, MO, USA) was prepared by dissolving 10 mg of the pesticide in 10 ml acetonitrile (HPLC grade, Sigma Aldrich). Aliquots (0.1 ml) of chlorpyrifos (200 μg) was introduced onto the surface peel of the sample and allowed to dry at room temperature in the dark until dry. The apple samples were treated with one of the 20 treatments listed in Table 1 with non-treated samples acting as positive controls.
voltage was maintained at 4.5 kV and the drying gas temperature of 300 °C with a flow rate of 12 l/min. Nebulizer pressure was 40 psi. Nitrogen was used as both nebulizing and drying gas while helium was used as collision gas at 60 psi. The mass spectrometer was set on enhanced resolution positive ion scan mode from 200 to 400 m/z. The instrument was externally calibrated with the ESI TuneMix (Agilent). Quantitation of the CPY-O was determined using the Quant Analysis software (Bruker Daltonics) monitoring the intensity of the parent peak at 33.9 m/z with a retention time of 6.9 min. Calibration curves were created with serial dilutions of standard CPY-O in solution between 0.31 and 40 ppm and the concentration of unknowns was determined from the calibration function.
2.4. Chlorpyrifos analysis Chlorpyrifos was recovered by transferring the apple peel sample to a 50-ml tube containing 20 ml of acetonitrile then transferred to a sonication bath (Crystal Electronics, Newmarket, Canada) for 20 min. The rinsate was decanted onto a fresh tube and stored at 4 °C for a maximum period of 24 h before quantification. High performance liquid chromatography (HPLC) was performed with an Agilent 1100 Series HPLC instrument equipped with a photodiode array detector operating at 290 nm (Agilent, Fair Lawn, CA, USA). An Agilent Zorbax Eclipse Plus C18 analytical column (4.6 × 150 mm, 3.5 μm) attached to a Zorbax Eclipse Plus-C18 analytical guard column (4.6 × 12.5 mm, 5 μm) was used as a stationary phase with a flow rate of 1.0 ml/min and the column temperature was uncontrolled with an average room temperature of 23 °C. The mobile phase was in isocratic mode with a composition of 85:15 (%, v/v) acetonitrile/water with 0.1% trifluoroacetic acid (Millipore Sigma, St Louis, MO, USA).
2.7. Inactivation of Escherichia coli O157:H7 and Aspergillus niger Escherichia coli O157:H7 (stx2, eae, nalidixic acid resistant) originally isolated from a clinical case and A. niger spores were applied in the study. E. coli O157:H7 was cultivated in tryptic soy broth (TSB, Oxoid, Hampshire, UK) at 37 °C for 16 h. The cells were harvested by centrifugation and resuspended in saline and optical density at 600 nm adjusted to 0.2 (approximately 8 log CFU/ml). The suspension was held at 4 °C until required. Aspergillus niger spores were produced by inoculating the mold onto Potato Dextrose Agar (PDA) and incubating for 14 days at 25 °C. The spores were harvested by flooding the plate with sterile distilled water and scraping with spreader. The spore suspension was then harvested by centrifugation and pellet resuspended in sterile distilled water prior to storing at 4 °C until required. Apple peel sections were inoculated with either E. coli O157:H7 or A. niger spores to give a final cell density of 8 and 6 log CFU/sample respectively. The inoculated apple samples were then placed within a biosafety hood and allowed to dry at room temperature for 3.5 h. Three skin samples were treated with UV-C and 0.2 ml of 1.2% v/v hydrogen peroxide (diluted from 30% v/v hydrogen peroxide with milli-Q water; Sigma-Aldrich, Oakville, Ontario, Canada), three other samples were treated with only UV and another 3 samples were treated with hydrogen peroxide only. Optimal treatment parameters were used (1.2% hydrogen peroxide heated to 69 °C and 29.2 min of UV exposure). The treated samples and non-treated controls were placed in 20 ml of saline and vortexed to release microbes from the apple peel samples. A dilution series was prepared in saline and plated onto the appropriate agar. E. coli O157:H7 was plated onto tryptic soy agar (TSA) supplemented with 50 μg/ml nalidixic acid that was subsequently incubated at 37 °C for 24 h. Aspergillus niger was enumerated on PDA plates incubated at 25 °C for 5 days.
2.5. Q-ToF analysis of degradation products Untreated spiked samples and treated samples prepared using the optimal treatment along with analytical standards of chlorpyrifos, CPY-O and TCP were submitted for Q-ToF analysis to determine the presence or absence of these compounds. Liquid chromatography–mass spectrometry analyses were performed on an Agilent 1200 HPLC liquid chromatograph interfaced with an Agilent UHD 6530 Q-Tof mass spectrometer at the Mass Spectrometry Facility of the Advanced Analysis Centre (University of Guelph). A C18 column (Agilent Poroshell 120, EC-C18 50 mm × 3.0 mm 2.7 μm) was used for chromatographic separation with the following solvents water with 0.1% v/v formic acid (A) and acetonitrile with 0.1% v/v formic acid (B). The mobile phase gradient was as follows: initial conditions were 10% B hold for 1 min then increasing to 100% B in 29 min followed by column wash at 100% B for 5 min and 20 min re-equilibration. The flow rate was maintained at 0.4 ml/min. The mass spectrometer electrospray capillary voltage was maintained at 4.0 kV and the drying gas temperature at 250 °C with a flow rate of 8 L/min. Nebulizer pressure was 30 psi and the fragmentor was set to 160 °C. Nitrogen was used as both nebulizing and drying gas. The mass-to-charge ratio was scanned across the m/z range of 50–1500 m/z in 4 GHz (extended dynamic range) positive and negative ion modes. The acquisition rate was set at 2spectra/s. The instrument was externally calibrated with the ESI TuneMix (Agilent). The sample injection volume was 10 μl. Mass spectrometer control, data acquisition and data analysis were performed with MassHunter® Workstation software (B.04.00).
3. Results 3.1. Response surface methodology (RSM) and precision The average recovery of chlorpyrifos from apple peel using the applied methodology was 89.4% ± 3.8%. The mean recovery was used as a recovery correction factor for AOP treated samples. The percent degradation of chlorpyrifos across the various AOP treatments ranged from 30 to 88% (Table 1). The extent of chlorpyrifos degradation was analyzed using Design Expert® 11.1.0 software and determined to fit a linear model (Supplementary Table 1). The linear terms X1 (UV dose) and X2 (H2O2 temperature) were shown to be significant (P < 0.05) whereas X3 (H2O2 concentration) was not significant (Supplementary Table 1). Surface plots illustrate the inter-dependency of UV-C dose, hydrogen peroxide concentration and temperature (Fig. 1). A synergistic effect between hydrogen peroxide temperature and UV-C dose on the degradation of chlorpyrifos was observed (Fig. 1A). In contrast, there was no significant (P > 0.05) interaction between hydrogen peroxide concentration and the applied UV-C dose (Fig. 1B). In a similar manner, there was a relatively negligible interaction between the concentration and temperature of hydrogen peroxide on chlorpyrifos degradation (Fig. 1C). From the interaction between the three parameters it was possible to
2.6. Quantification of chlorpyrifos metabolites CPY-O and TCP Samples were injected into a Dionex UHPLC UltiMate 3000 liquid chromatograph interfaced to an amaZon SL ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). An Eclipse Plus C18 column (5 μm particle size, 150 mm × 2.1 mm, Agilent) was used for chromatographic separation. The initial mobile phase conditions were 60% v/v water (0.1% v/v formic acid) and 40% v/v acetonitrile (0.1% v/v formic acid) for 1 min followed by a single step gradient to 100% acetonitrile (0.1% v/v formic acid) in 8 min. The flow rate was maintained at 0.4 ml/min. The mass spectrometer electrospray capillary 3
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Fig. 1. 3D surface graph of different interaction effects of an Advanced Oxidation Process for degrading chlorpyrifos on apples. Chlorpyrifos was introduced onto apple peel and treated with various combinations of hydrogen peroxide and UV dose with the decrease in pesticide levels being recorded. The plots illustrate UV dose and H2O2 temperature (A), UV dose and H2O2 concentration (B) & H2O2 concentration and H2O2 temperature (C). Table 2 Log count reduction of Escherichia coli O157:H7 and Aspergillus niger spores inoculated onto apple peel sections then treated with UV-C (at 254 nm), hydrogen peroxide (1.22% v/v at 66 °C) or a combination of UV-C and hydrogen peroxide. Microbe
Treatment
Log CFU/Sample Initial Loading
Escherichia coli O157:H7
Aspergillus niger
Control (Non-treated) H2O2 (1.22% v/v) UV-C (69 kJ/m2) UV-C + H2O2 (69 kJ/m2 and 1.22% v/v) Control (Non-treated) H2O2 (1.22% v/v) UV-C (69 kJ/m2) UV-C + H2O2 (69 kJ/m2 and 1.22% v/v)
8.6 ± 0.5
7.0 ± 0.4
Post-Treatment
LCR
< 2.3a 2.7 ± 0.4 < 2.3a
> 6.6a 5.9 ± 0.4b > 6.6a
5.3 ± 0.4 3.8 ± 0.6 < 2.3a
1.7 ± 0.4a 3.2 ± 0.6b > 4.7c
Mean values for each of the test microbes, followed by the same letter are not significantly (P > 0.05) different. a Limit of enumeration = 2.3 log CFU/Sample.
produce an equation (Eqn. (2)) used to describe the observed reduction of chlorpyrifos. Reduction of chlorpyrifos from the six centre points ranged from 82 to 90 μg with a standard deviation of 3.6 μg that demonstrates reproducibility of the predictive model (Supplementary Table 2). This shows that the data is replicable with minimal variation between samples. In addition, through verification, a 118 μg (59%) degradation of chlorpyrifos was achieved using an optimal AOP treatment of 69 kJ/m2 UV-C dose and 1.22% v/v H2O2 at 66 °C that agreed with a predictive value of 126 μg suggested by Design Expert software. The photoproduct of chlorpyrifos degradation (CPY–O) was detected but found to be < 20 ppb in the AOP treated samples and absent in controls.
TCP was not detected in any treated samples or controls.
Y = 5.742 + 1.071 X 1 + 0.390 X 2
2.241 X 3 @ 36
s
J m ˆ2
(Equation 2)
3.2. Microbial inactivation The AOP treatment with a UV-C dose of 69 kJ/m2and 1.22% v/v H2O2 at 66 °C to maximise chlorpyrifos degradation also supported a > 7 log cfu reduction of E. coli O157:H7 (Table 2). With 1.22% v/v 4
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hydrogen peroxide alone applied at 66 °C also supported > 8 log cfu reduction of the pathogen (Table 2). A UV-C does of 69 kJ/m2alone resulted in a > 6.6 log CFU/sample decrease in E. coli O157:H7 numbers. In comparison, A. niger spores on apple surfaces were decreased by > 4.7 log CFU/sample with the applied AOP treatment with UV-C alone supporting a 3.2 ± 0.6 log CFU/sample reduction. In contrast to E. coli O157:H7, hydrogen peroxide alone supported a 1.7 log CFU/ sample log reduction of A. niger spores (Table 2).
In the current study the degradation of chlorpyrifos could be positively correlated to temperature which is in agreement with others (Cengiz, Catal, Erler, & Bilgin, 2015; Oliveria et al., 2014). The mechanism of thermal decomposition of chlorpyrifos involves a series of stepwise elimination reactions that cleaves ethylene residues to leave 3,5,6,-trichloro-2-pyridinol as the main degradation product (Kennedy & Mackie, 2018). Evidently the presence of free radicals with UV-C promotes the degradation reaction although given the temperature sensitivity of fruit it would have less relevance within commercial processing. A UV-C process alone could be used to degrade chlorpyrifos but for inactivation of E. coli O157:H7 there was a significant contribution of hydrogen peroxide. The results are in agreement with other reports on the inactivation of microbes on fresh produce and eggs shell surfaces (Hadjok, Mittal, & Warriner, 2008; Murray et al., 2018; Rehkopf, Byrd, Coufal, & Duong, 2017; Xie, Hajdok, Mittal, & Warriner, 2008). Here, it is thought that the free radicals generated of the surface of sample can access microbes shielded from UV-C by surface structures. It is possible that the proportion of free radicals required to inactive microbes is less than that for chlorpyrifos oxidation. From a practical prospect, the results would suggest that hydrogen peroxide has a greater importance in antimicrobial action of AOP with photo-oxidation of chlorpyrifos being more effected by UV-C that is enhanced by treatment temperature. In this respect the optimum AOP treatment parameters can collectively act to reduce microbial and chemical hazards in fresh produce.
4. Discussion Through following RSM approach it was demonstrated that AOP could degrade chlorpyrifos via a process that was primarily dependent on the UV-C dose and temperature. It has been reported that chlorpyrifos in aqueous solutions is relatively stable to UV-C light with the addition of hydrogen peroxide or Fe (Fenton) required to support degradation of the pesticide (Gandhi, Lari, Tripathi, & Kanade, 2016). Further studies have confirmed the finding that chlorpyrifos UV-C degradation is enhanced when applied as part of an Advanced Oxidation Process (Gandhi et al., 2016; Savic et al., 2019; Thind et al., 2018; Utzig et al., 2019). Thind et al. (2018) reported that hydrogen peroxide within the range of 0.5–1.5% v/v supported a 42% decrease in chlorpyrifos levels but could be increased to 53% reduction when used in combination with UV-C. A further increase in chlorpyrifos degradation up to 74% was obtained by using a combination of hydrogen peroxide, titanium dioxide and UV-C therefore suggesting a synergistic effect. Utzig et al. (2019) reported the degradation of chlorpyrifos in acetonitrile with UV supporting complete degradation within 180 min treatment with the inclusion of hydrogen peroxide accelerating the process. However, the authors reported the generation of toxic byproducts when the AOP was applied but not when UV-C was applied alone (Utzig et al., 2019). Yet, unlike the current study, previous reports on the AOP mediated degradation of chlorpyrifos have focused on aqueous or ethanolic solutions. In the current study where the AOP process was performed on the apple surface it was evident that hydrogen peroxide contributed a minor role on the overall degradation process compared to when applied in a aqueous phase. Indeed, the presence of increasing levels of hydrogen peroxide in the AOP reduced the degree of chlorpyrifos degradation by UV-C light. A further difference between the AOP being applied on the surface of fruit vs aqueous solutions is the generation of toxic by-products in the latter from chlorpyrifos degradation. For example (Utzig et al., 2019), reported that CPY-O was formed by the AOP mediated degradation of the pesticide. However, in the current study the concentration of the oxon form was < 20 ppb which reflects the low contribution of hydrogen peroxide within the overall AOP process. The underlying differences observed in the degradation of chlorpyrifos on the surface of apples compared to in aqueous solution could be based on several factors. For example, it is known that the concentration of hydrogen peroxide has to be balanced with regards generating sufficient free-radicals to support the chlorpyrifos reaction but not in excess to impeded the degradative action (de Oliveira et al., 2014). In the case of aqueous solutions there is diffusion of hydrogen peroxide and generation of hydroxyl radicals to prevent the inhibition of the reaction. It is also conceivable that in air the UV-C transmission to the surface of apples would be greater than that of water, especially if absorbing constituents are present. Therefore, in aqueous solutions the hydrogen peroxide would constitute the primary element of the AOP given the absorption of UV-C by the water constituents. By directly illuminating the surface of apples, the generation of radicals can occur although direct reaction of chlorpyrifos with UV-C photons predominating in the degradative action. Additional possibilities are the aqueous phase forms a relative constant matrix to support the reaction of radicals with chlorpyrifos. For example, high alkaline pH and presence of Fe can promote the chlorpyrifos degradation but which could be less controlled on the apple surface (Behin & Farhadian, 2017). Regardless of this fact, the results indicate that AOP studies performed in aqueous phase do not always translate to treating whole fruit.
Acknowledgements The authors wish to thank the Ontario Agri-Food Innovation Alliance for their generous financial support for the project (# 030302). We also wish to thank Moyers Apple Products for the donation of apples and Clean Works Corporation for the laboratory scale Advanced Oxidative Process reactor. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106920. References Abramovic, B. F., Banic, N. D., & Sojic, D. V. (2010). Degradation of thiacloprid in aqueous solution by UV and UV/H2O2 treatments. Chemosphere, 81(1), 114–119. https:// doi.org/10.1016/j.chemosphere.2010.07.016. Atwood, D., & Paisley-Jones, C. (2017). Pesticide industry sales and usage 2008 - 2012. Behin, J., & Farhadian, N. (2017). Response surface methodology for ozonation of trifluralin using advanced oxidation processes in an airlift photoreactor. Applied Water Science, 7(6), 3103–3112. https://doi.org/10.1007/s13201-016-0443-y. Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965–977. https://doi.org/10.1016/j.talanta.2008.05.019. Bouchard, M., F., Chevrier, J., Harley Kim, G., Kogut, K., Vedar, M., et al. (2011). Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children. Environmental Health Perspectives, 119(8), 1189–1195. https://doi.org/10.1289/ehp.1003185. Burkul, R. M., Ranade, S. V., & Pangarkar, B. L. (2015). Removal of pesticides by using various treatment method: Review. International Journal of Emerging Trend in Engineering and Basic Sciences, 2(2), 88–91. Cengiz, M. F., Catal, M., Erler, F., & Bilgin, K. (2015). The effects of heat treatment on the degradation of the organophosphate pesticide chlorpyrifos-ethyl in tomato homogenate. Quality Assurance and Safety of Crops & Foods, 7(4), 537–544. https://doi.org/ 10.3920/Qas2013.0301. Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L. G., et al. (2008). Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment. Critical Reviews in Toxicology, 38(sup2), 1–125. https://doi.org/ 10.1080/10408440802272158. Femia, J., Mariani, M., Zalazar, C., & Tiscornia, I. (2013). Photodegradation of chlorpyrifos in water by UV/H2O2 treatment: Toxicity evaluation. Water Science and Technology, 68(10), 2279–2286. https://doi.org/10.2166/wst.2013.493. Gandhi, K., Lari, S., Tripathi, D., & Kanade, G. (2016). Advanced oxidation processes for the treatment of chlorpyrifos, dimethoate and phorate in aqueous solution. Journal of Water Reuse and Desalination, 6(1), 195–203. https://doi.org/10.2166/wrd.2015.062. Gomez, L. E. (2009). Use and benefits of chlorpyrifos in US agriculture. Dow AgroSciences LLC. Hadjok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of human pathogens and
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J. Ho, et al. spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide. Journal of Applied Microbiology, 104, 1014–1024. Ikehata, K., & Gamal El-Din, M. (2005). Aqueous pesticide degradation by ozonation and ozone-based advanced oxidation processes: A review (Part I). Ozone: Science & Engineering, 27(2), 83–114. https://doi.org/10.1080/01919510590925220. Kennedy, E. M., & Mackie, J. C. (2018). Mechanism of the thermal decomposition of chlorpyrifos and formation of the dioxin analog, 2,3,7,8-Tetrachloro-1,4-dioxino-dipyridine (TCDDpy). Environmental Science & Technology, 52(13), 7327–7333. https:// doi.org/10.1021/acs.est.8b01626. Kenney, S. J., Burnett, S. L., & Beuchat, L. R. (2001). Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing. Journal of Food Protection, 64(9), 1328–1333. Köck-Schulmeyer, M., Villagrasa, M., López de Alda, M., Céspedes-Sánchez, R., Ventura, F., & Barceló, D. (2013). Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact. The Science of the Total Environment, 458–460, 466–476. https://doi.org/10.1016/j.scitotenv.2013.04.010. Koutchma, T., Popović, V., Ros-Polski, V., & Popielarz, A. (2016). Effects of Ultraviolet Light and high-pressure processing on quality and health-related constituents of fresh juice products. Comprehensive Reviews in Food Science and Food Safety, 15(5), 844–867. https://doi.org/10.1111/1541-4337.12214. Marican, A., & Durán-Lara, E. F. (2018). A review on pesticide removal through different processes. Environmental Science and Pollution Research, 25(3), 2051–2064. https:// doi.org/10.1007/s11356-017-0796-2. Mladenova, R., & Shtereva, D. (2009). Pesticide residues in apples grown under a conventional and integrated pest management system. Food Additives & Contaminants Part A Chem Anal Control Expo Risk Assess, 26(6), 854–858. https://doi.org/10.1080/ 02652030902726060. Murray, K., Moyer, P., Wu, F., Goyette, J. B., & Warriner, K. (2018). Inactivation of Listeria monocytogenes on and within apples destined for caramel apple production by using sequential forced air ozone gas followed by a continuous advanced oxidative process treatment. Journal of Food Protection, 81(3), 357–364. https://doi.org/10. 4315/0362-028X.JFP-17-306. Oliveira, A., Pereira Ribeiro, J., Oliveira, J., De Keukeleire, D., Saldanha Duarte, M., & Nascimento, R. (2014). Degradation of the pesticide chlorpyrifos in aqueous solutions with UV/H2O2: Optimization and effect of interfering anions. Journal of Advanced Oxidation Technologies, 17(1), 133–138. de Oliveira, A. G., Ribeiro, J. P., de Oliveira, J. T., De Keukeleire, D., Duarte, M. S., & do Nascimento, R. F. (2014). Degradation of the pesticide chlorpyrifos in aqueous solutions with UV/H2O2: Optimization and effect of interfering anions. Journal of Advanced Oxidation Technologies, 17(1), 133–138.
Osman, K. A., Al-Humaid, A. I., Al-Redhaiman, K. N., & El-Mergawi, R. A. (2014). Safety methods for chlorpyrifos removal from date fruits and its relation with sugars, phenolics and antioxidant capacity of fruits. Journal of Food Science & Technology, 51(9), 1762–1772. https://doi.org/10.1007/s13197-012-0693-0. Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X., et al. (2012). Brain anomalies in children exposed prenatally to a common organophosphate pesticide. Proceedings of the National Academy of Sciences, 109(20), 7871–7876. https:// doi.org/10.1073/pnas.1203396109. Rawn, D. F. K., Quade, S. C., Sun, W.-F., Fouguet, A., Bélanger, A., & Smith, M. (2008). Captan residue reduction in apples as a result of rinsing and peeling. Food Chemistry, 109(4), 790–796. https://doi.org/10.1016/j.foodchem.2008.01.061. Rehkopf, A. C., Byrd, J. A., Coufal, C. D., & Duong, T. (2017). Advanced Oxidation Process sanitization of hatching eggs reduces Salmonella in broiler chicks. Poultry Science, 96(10), 3709–3716. https://doi.org/10.3382/ps/pex166. Savic, J. Z., Petrovic, S. Z., Leskovac, A. R., Pasti, T. D. L., Nastasijevic, B. J., Tanovic, B. B., et al. (2019). UV-C light irradiation enhances toxic effects of chlorpyrifos and its formulations. Food Chemistry, 271, 469–478. https://doi.org/10.1016/j.foodchem. 2018.07.2017. Solomon, K. R., Williams, W. M., Mackay, D., Purdy, J., Giddings, J. M., & Giesy, J. P. (2014). Properties and uses of chlorpyrifos in the United States. In J. P. Giesy, & K. R. Solomon (Eds.). Ecological risk assessment for chlorpyrifos in terrestrial and aquatic systems in the United States (pp. 13–34). Cham: Springer International Publishing. Taylor-Edmonds, L., Lichi, T., Rotstein-Mayer, A., & Mamane, H. (2015). The impact of dose, irradiance and growth conditions on Aspergillus niger (renamed A. brasiliensis) spores low-pressure (LP) UV inactivation. Journal of Environmental Science and Health - Part A: Toxic/Hazardous Substances & Environmental Engineering, 50(4), 341–347. https://doi.org/10.1080/10934529.2015.987519. Thind, P. S., Kumari, D., & John, S. (2018). TiO2/H2O2 mediated UV photocatalysis of Chlorpyrifos: Optimization of process parameters using response surface methodology. Journal of Environmental Chemical Engineering, 6(3), 3602–3609. https://doi. org/10.1016/j.jece.2017.05.031. Trasande, L. (2017). When enough data are not enough to enact policy: The failure to ban chlorpyrifos. PLoS Biology, 15(12), e2003671. https://doi.org/10.1371/journal.pbio. 2003671. Utzig, L. M., Lima, R. M., Gomes, M. F., Ramsdorf, W. A., Martins, L. R. R., Liz, M. V., et al. (2019). Ecotoxicity response of chlorpyrifos in Aedes aegypti larvae and Lactuca sativa seeds after UV/H2O2 and UVC oxidation. Ecotoxicology and Environmental Safety, 169, 449–456. https://doi.org/10.1016/j.ecoenv.2018.11.003. Xie, Y., Hajdok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of MS2 F (+) coliphage on lettuce by a combination of UV light and hydrogen peroxide. Journal of Food Protection, 71, 903–907.
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Short communication
Degradation of chlorpyrifos and inactivation of Escherichia coli O157:H7 and Aspergillus niger on apples using an advanced oxidation process
T
J. Hoa, R. Prosserb, M. Hasania, H. Chena, B. Skanesb, W.D. Lubitzc, K. Warrinera,∗ a
Department of Food Science, University of Guelph, Guelph, Ontario, Canada School of Environmental Science, University of Guelph, Guelph, Ontario, Canada c School of Engineering, University of Guelph, Guelph, Ontario, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Chlorprifos Escherichia coli O157:H7 Apples Advanced oxidation process UV-C Hydrogen peroxide Response surface methodology
Chlorpyrifos is a widely used insecticide in apple production but there are concerns that residues ingested by consumers can lead to chronic neurological conditions. In the following, a method based on Advanced Oxidation Process (AOP) was validated for the degradation of chlorpyrifos on apples and inactivating Escherichia coli O157:H7, along with Aspergillus niger spores. AOP is a process that generates free-radicals from the UV-C (at 254 nm) mediated degradation of hydrogen peroxide thereby leading to highly oxidative species. By using Response Surface Methodology (RSM) it was found that the degradation of chlorpyrifos was primarily dependent on UV-C dose and synergistically enhanced by applying the hydrogen peroxide at temperatures up to 66 °C. However, the degradation of chlorpyrifos was independent on the hydrogen peroxide concentration up to 6% v/ v. Maximal degradation of chlorpyrifos on apples was achieved through an AOP treatment applying 68.4 kJ/m2 UV dose and 1.22% v/v H2O2 at 66 °C that resulted in 118 μg degradation (representing 59% of the original level) of the pesticide with < 20 ppb accumulation of the degradative photoproduct, CPY-O. The same treatment supported a > 6.6 log CFU reduction in E. coli O157:H7 and > 4.7 log CFU reduction of A. niger spores. Applying UV alone in the case of E. coli O157:H7 or hydrogen peroxide alone against A. niger spores supported a lower level of lethality compared to AOP. In conclusion, the study has demonstrated that an AOP based process can support the degradation of chlorpyrifos and address microbiological hazards associated with apples.
1. Introduction Chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridylphosphorothioate), is a broad spectrum organophosphate (OP) pesticide that is widely used in agricultural for the control of insects (Gomez, 2009; Mladenova & Shtereva, 2009; Solomon et al., 2014). The pesticide is one of the most commonly applied pesticides in North America with 10% of usage being applied in tree fruit (e.g. apples) production (Atwood & Paisley-Jones, 2017; Eaton et al., 2008). Although beneficial to agriculture for pest control, there have been health concerns in relation to chlorpyrifos residue exposure on foods and in the environment. Specifically, there is accumulating evidence that chlorpyrifos and other organophosphates, exhibit neurotoxic activity in children (Bouchard et al., 2011; Rauh et al., 2012; Trasande, 2017). For example, Bouchard et al. (2011) conducted a 7-year birth cohort study (n = 329) and reported that children exposed to high levels of OPs had lower IQ scores compared to control groups. Mitigation strategies to reduce exposure of the population to chlorpyrifos have been to ban the pesticide, as in the case of Hawaii,
∗
and devise approaches to reduce carriage on fresh produce. With regards to the latter, the post-harvest washing of produce has been shown to reduce chlorpyrifos levels on fruit by approximately 50% (Rawn et al., 2008). Washing produce with low concentrations of acetic acid, citric acid and potassium permanganate have also been shown to reduce chlorpyrifos concentrations in date fruit (Osman, Al-Humaid, AlRedhaiman, & El-Mergawi, 2014). However, washing of produce within tanks results in a redistribution of pesticides between produce batches and further dissemination within the environment via wastewater disposal (Burkul, Ranade, & Pangarkar, 2015; Köck-Schulmeyer et al., 2013). A more effective approach is to use water-free technologies to decontaminate fruit and to degrade chlorpyrifos thereby reducing levels entering down-stream washing processes. One potential approach is based on Advanced Oxidation Process (AOP) that has previously been shown to inactivate Listeria monocytogenes on apples (Murray, Moyer, Wu, Goyette, & Warriner, 2018). AOP involves generating hydroxyl radicals from the degradation of ozone and/or hydrogen peroxide. The generation of free radicals can be achieved through the interaction
Corresponding author. E-mail address: [email protected] (K. Warriner).
https://doi.org/10.1016/j.foodcont.2019.106920 Received 7 July 2019; Received in revised form 24 September 2019; Accepted 25 September 2019 Available online 26 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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between ozone and hydrogen peroxide, or more rapid via UV-C (254 nm) mediated decomposition of the oxidants (Abramovic, Banic, & Sojic, 2010; Atwood & Paisley-Jones, 2017; Behin & Farhadian, 2017; Femia, Mariani, Zalazar, & Tiscornia, 2013; Marican & Durán-Lara, 2018). The use of AOP for degrading pesticides, including chlorpyrifos, has previously been demonstrated in wastewater (Abramovic et al., 2010; de Oliveira et al., 2014; Ikehata & Gamal El-Din, 2005; Marican & Durán-Lara, 2018; Thind, Kumari, & John, 2018). It was found that UVC with a dose delivered from an 8 Watt low pressure lamp over 4 h at 45 °C resulted in a 39.7% degradation of chlorpyrifos compared to no decrease when 2 g/l hydrogen peroxide was applied alone (de Oliveira et al., 2014). However, by combining UV-C with hydrogen peroxide it was possible to achieve a 62.3% decrease which was independent of the water temperature (de Oliveira et al., 2014). In a further example, an AOP process based on using a combination of UV-C, hydrogen peroxide and titanium oxide supported 73% chlorpyrifos degradation in water compared to 41% when hydrogen peroxide (up to 50 ppm) was applied alone (Thind et al., 2018). Therefore, the AOP based process holds promise as a means of degrading chlorpyrifos although has yet to be studied directly on apple fruit. In the following study the AOP originally developed to decontaminate apples was applied for chlorpyrifos degradation. The process is based on the apples being sprayed with hydrogen peroxide at a set temperature then transferred to a chamber housing UV-C lamps to degrade the pesticide in addition to direct inactivation of microbes (Murray et al., 2018). A treatment of 6% v/v hydrogen peroxide applied at 48 °C in combination with UV-C dose of 54 mJ/cm2 supported a 3 log reduction of L. monocytogenes both on the surface and subsurface of apple fruit (Murray et al., 2018). Therefore, the potential to inactivate pathogens on apples and simultaneously degrade chlorpyrifos offers a potential of a one-step process to reduce the biological and chemical hazards linked to apples. In the current study the selected pathogen of concern was Escherichia coli O157:H7 due to the previous links to foodborne illness outbreaks linked to apples (Kenney, Burnett, & Beuchat, 2001). Aspergillus niger was selected as a test microbe due to its inherent resistance to UV-C in the spore form, in addition to acting as a surrogate for potential mycotoxin producing molds (Taylor-Edmonds, Lichi, Rotstein-Mayer, & Mamane, 2015). A key part of the AOP is optimizing the hydrogen peroxide concentration and temperature along with UV-C dose. Excess hydrogen peroxide can potentially result in a decrease in the efficacy of generating radicals due to self-neutralization that terminates the reaction (de Oliveira et al., 2014). Excess UV-C can also contribute to photobleaching of produce along with loss of sensitive nutrients (Koutchma, Popović, Ros-Polski, & Popielarz, 2016). In the current study response surface methodology (RSM) was used to determine which factors and their settings affect the degradation of chlorpyrifos. A full factorial central composite RSM design was used in this study (Thind et al., 2018). The design is composed of axial points between a selected central point whereby different combinations of the processing parameters are applied and response (i.e. extent of chlorpyrifos degradation) recorded (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). From the response curves it is possible to identify the optimal working parameters of the AOP, in addition to identifying the contribution from each component to the observed pesticide degradation reaction and pathogen log reduction.
Table 1 RSM trials (20) with amount of chlorpyrifos reduction (μg) observed using various test parameters of UV-C dose (kJ/m2), temperature (°C) and concentration (%) of hydrogen peroxide. Reduction from each trial was corrected using the average positive control recovery of 89.4% (n = 177). Trial
UV dose (kJ/m2) (X1)
Temperature of H2O2 (°C) (X2)
H2O2 (%) (X3)
Chlorpyrifos Reduction (μg) (Y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
34.8 22.5 54.0 37.0 22.4 38.3 39.4 39.4 11.7 38.9 56.1 50.3 37.9 21.8 39.3 37.2 62.3 38.5 22.3 53.3
46.5 32.5 32.5 46.5 60.5 70.0 46.5 46.5 46.5 46.5 60.5 32.5 46.5 32.5 46.5 46.5 46.5 23.0 60.5 60.5
3.5 5.0 5.0 1.0 5.0 3.5 3.5 3.5 3.5 3.5 2.0 2.0 6.0 2.0 3.5 3.5 3.5 3.5 2.0 5.0
50 32 52 60 54 58 52 54 28 56 88 74 54 30 52 62 92 58 52 74
replicate centre points (Table 1). Runs were performed in triplicate involving the three previously mentioned factors. The response variable can be expressed as a function (Eqn. (1)) of the independent process variables according to the following response surface quadratic model. 3
Y = a0 +
3
i=1
2
3
aii x 2 +
ai x i + i=1
aij x i xj + i=1 j=i+1
(Equation 1)
where Y is the predicted response, xi and xj are factors influencing the predicted response, αi, αii, and αij are the coefficient of linear, quadratic, and interaction terms respectively. Lastly, ε is a random error. Regression analysis for the quadratic equation model was determined using Design Expert® 11.1.0 software. First, the model fit was tested to determine the quality of the model using the coefficient of determination (R2). ANOVA analysis was conducted to determine whether the test variables listed are significant or not. This was done using F-test and P-values with 95% confidence level. 2.2. Advanced oxidation process reactor The advanced oxidation process (AOP) reactor (Clean Works Corporation, Beamsville, ON, CA) was constructed from stainless steel with frame dimensions of 122 × 38 × 20 cm (length x width x height). The light source was located at the top of the frame as described by Murray et al. (2018). The reactor was fitted with 4 × 25 W UV-C lamps (60 cm length) emitting at 254 nm (Sani-Ray, Hauppauge, NY, USA). The UV intensity was varied through altering the distance of the lamps from the samples from 10 to 16 cm. A radiometer (Trojan Technologies Inc, London, ON, CA) was used to measure the UV-intensity with treatment times being used to deliver a defined UV-dose. Prior to introducing the apple samples into the UV reactor, the hydrogen peroxide (0.2 ml) of defined concentration and temperature was dispensed onto sample.
2. Materials and methods 2.1. Response surface methodology
2.3. Deposition and recovery of chlorpyrifos on apples
RSM was used in the experimental design of this study. Design Expert® Software 11.1.0 (Stat-Ease Inc, Minneapolois, USA) was used to produce a full factorial central composite design investigating the effect of UV-C dose (kJ/m2), temperature of hydrogen peroxide (°C), and concentration of hydrogen peroxide (% v/v) on the degradation of chlorpyrifos (Table 1). The experimental design of this study included a total of 20 runs with six
Crispin apples were provided by Moyer's Apple Products (Beamsville, ON, CA) and stored at 4 °C until required. The apples were prepared by initially washing under running municipal water then towel drying. Only apples showing no signs of spoilage or damage were 2
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used in the study. The peel of the apples was removed using a peeler, then peels were cut into 2.5 cm × 3.0 cm pieces. A working stock solution of chlorpyrifos (> 99% purity; Sigma-Aldrich, St Louis, MO, USA) was prepared by dissolving 10 mg of the pesticide in 10 ml acetonitrile (HPLC grade, Sigma Aldrich). Aliquots (0.1 ml) of chlorpyrifos (200 μg) was introduced onto the surface peel of the sample and allowed to dry at room temperature in the dark until dry. The apple samples were treated with one of the 20 treatments listed in Table 1 with non-treated samples acting as positive controls.
voltage was maintained at 4.5 kV and the drying gas temperature of 300 °C with a flow rate of 12 l/min. Nebulizer pressure was 40 psi. Nitrogen was used as both nebulizing and drying gas while helium was used as collision gas at 60 psi. The mass spectrometer was set on enhanced resolution positive ion scan mode from 200 to 400 m/z. The instrument was externally calibrated with the ESI TuneMix (Agilent). Quantitation of the CPY-O was determined using the Quant Analysis software (Bruker Daltonics) monitoring the intensity of the parent peak at 33.9 m/z with a retention time of 6.9 min. Calibration curves were created with serial dilutions of standard CPY-O in solution between 0.31 and 40 ppm and the concentration of unknowns was determined from the calibration function.
2.4. Chlorpyrifos analysis Chlorpyrifos was recovered by transferring the apple peel sample to a 50-ml tube containing 20 ml of acetonitrile then transferred to a sonication bath (Crystal Electronics, Newmarket, Canada) for 20 min. The rinsate was decanted onto a fresh tube and stored at 4 °C for a maximum period of 24 h before quantification. High performance liquid chromatography (HPLC) was performed with an Agilent 1100 Series HPLC instrument equipped with a photodiode array detector operating at 290 nm (Agilent, Fair Lawn, CA, USA). An Agilent Zorbax Eclipse Plus C18 analytical column (4.6 × 150 mm, 3.5 μm) attached to a Zorbax Eclipse Plus-C18 analytical guard column (4.6 × 12.5 mm, 5 μm) was used as a stationary phase with a flow rate of 1.0 ml/min and the column temperature was uncontrolled with an average room temperature of 23 °C. The mobile phase was in isocratic mode with a composition of 85:15 (%, v/v) acetonitrile/water with 0.1% trifluoroacetic acid (Millipore Sigma, St Louis, MO, USA).
2.7. Inactivation of Escherichia coli O157:H7 and Aspergillus niger Escherichia coli O157:H7 (stx2, eae, nalidixic acid resistant) originally isolated from a clinical case and A. niger spores were applied in the study. E. coli O157:H7 was cultivated in tryptic soy broth (TSB, Oxoid, Hampshire, UK) at 37 °C for 16 h. The cells were harvested by centrifugation and resuspended in saline and optical density at 600 nm adjusted to 0.2 (approximately 8 log CFU/ml). The suspension was held at 4 °C until required. Aspergillus niger spores were produced by inoculating the mold onto Potato Dextrose Agar (PDA) and incubating for 14 days at 25 °C. The spores were harvested by flooding the plate with sterile distilled water and scraping with spreader. The spore suspension was then harvested by centrifugation and pellet resuspended in sterile distilled water prior to storing at 4 °C until required. Apple peel sections were inoculated with either E. coli O157:H7 or A. niger spores to give a final cell density of 8 and 6 log CFU/sample respectively. The inoculated apple samples were then placed within a biosafety hood and allowed to dry at room temperature for 3.5 h. Three skin samples were treated with UV-C and 0.2 ml of 1.2% v/v hydrogen peroxide (diluted from 30% v/v hydrogen peroxide with milli-Q water; Sigma-Aldrich, Oakville, Ontario, Canada), three other samples were treated with only UV and another 3 samples were treated with hydrogen peroxide only. Optimal treatment parameters were used (1.2% hydrogen peroxide heated to 69 °C and 29.2 min of UV exposure). The treated samples and non-treated controls were placed in 20 ml of saline and vortexed to release microbes from the apple peel samples. A dilution series was prepared in saline and plated onto the appropriate agar. E. coli O157:H7 was plated onto tryptic soy agar (TSA) supplemented with 50 μg/ml nalidixic acid that was subsequently incubated at 37 °C for 24 h. Aspergillus niger was enumerated on PDA plates incubated at 25 °C for 5 days.
2.5. Q-ToF analysis of degradation products Untreated spiked samples and treated samples prepared using the optimal treatment along with analytical standards of chlorpyrifos, CPY-O and TCP were submitted for Q-ToF analysis to determine the presence or absence of these compounds. Liquid chromatography–mass spectrometry analyses were performed on an Agilent 1200 HPLC liquid chromatograph interfaced with an Agilent UHD 6530 Q-Tof mass spectrometer at the Mass Spectrometry Facility of the Advanced Analysis Centre (University of Guelph). A C18 column (Agilent Poroshell 120, EC-C18 50 mm × 3.0 mm 2.7 μm) was used for chromatographic separation with the following solvents water with 0.1% v/v formic acid (A) and acetonitrile with 0.1% v/v formic acid (B). The mobile phase gradient was as follows: initial conditions were 10% B hold for 1 min then increasing to 100% B in 29 min followed by column wash at 100% B for 5 min and 20 min re-equilibration. The flow rate was maintained at 0.4 ml/min. The mass spectrometer electrospray capillary voltage was maintained at 4.0 kV and the drying gas temperature at 250 °C with a flow rate of 8 L/min. Nebulizer pressure was 30 psi and the fragmentor was set to 160 °C. Nitrogen was used as both nebulizing and drying gas. The mass-to-charge ratio was scanned across the m/z range of 50–1500 m/z in 4 GHz (extended dynamic range) positive and negative ion modes. The acquisition rate was set at 2spectra/s. The instrument was externally calibrated with the ESI TuneMix (Agilent). The sample injection volume was 10 μl. Mass spectrometer control, data acquisition and data analysis were performed with MassHunter® Workstation software (B.04.00).
3. Results 3.1. Response surface methodology (RSM) and precision The average recovery of chlorpyrifos from apple peel using the applied methodology was 89.4% ± 3.8%. The mean recovery was used as a recovery correction factor for AOP treated samples. The percent degradation of chlorpyrifos across the various AOP treatments ranged from 30 to 88% (Table 1). The extent of chlorpyrifos degradation was analyzed using Design Expert® 11.1.0 software and determined to fit a linear model (Supplementary Table 1). The linear terms X1 (UV dose) and X2 (H2O2 temperature) were shown to be significant (P < 0.05) whereas X3 (H2O2 concentration) was not significant (Supplementary Table 1). Surface plots illustrate the inter-dependency of UV-C dose, hydrogen peroxide concentration and temperature (Fig. 1). A synergistic effect between hydrogen peroxide temperature and UV-C dose on the degradation of chlorpyrifos was observed (Fig. 1A). In contrast, there was no significant (P > 0.05) interaction between hydrogen peroxide concentration and the applied UV-C dose (Fig. 1B). In a similar manner, there was a relatively negligible interaction between the concentration and temperature of hydrogen peroxide on chlorpyrifos degradation (Fig. 1C). From the interaction between the three parameters it was possible to
2.6. Quantification of chlorpyrifos metabolites CPY-O and TCP Samples were injected into a Dionex UHPLC UltiMate 3000 liquid chromatograph interfaced to an amaZon SL ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). An Eclipse Plus C18 column (5 μm particle size, 150 mm × 2.1 mm, Agilent) was used for chromatographic separation. The initial mobile phase conditions were 60% v/v water (0.1% v/v formic acid) and 40% v/v acetonitrile (0.1% v/v formic acid) for 1 min followed by a single step gradient to 100% acetonitrile (0.1% v/v formic acid) in 8 min. The flow rate was maintained at 0.4 ml/min. The mass spectrometer electrospray capillary 3
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Fig. 1. 3D surface graph of different interaction effects of an Advanced Oxidation Process for degrading chlorpyrifos on apples. Chlorpyrifos was introduced onto apple peel and treated with various combinations of hydrogen peroxide and UV dose with the decrease in pesticide levels being recorded. The plots illustrate UV dose and H2O2 temperature (A), UV dose and H2O2 concentration (B) & H2O2 concentration and H2O2 temperature (C). Table 2 Log count reduction of Escherichia coli O157:H7 and Aspergillus niger spores inoculated onto apple peel sections then treated with UV-C (at 254 nm), hydrogen peroxide (1.22% v/v at 66 °C) or a combination of UV-C and hydrogen peroxide. Microbe
Treatment
Log CFU/Sample Initial Loading
Escherichia coli O157:H7
Aspergillus niger
Control (Non-treated) H2O2 (1.22% v/v) UV-C (69 kJ/m2) UV-C + H2O2 (69 kJ/m2 and 1.22% v/v) Control (Non-treated) H2O2 (1.22% v/v) UV-C (69 kJ/m2) UV-C + H2O2 (69 kJ/m2 and 1.22% v/v)
8.6 ± 0.5
7.0 ± 0.4
Post-Treatment
LCR
< 2.3a 2.7 ± 0.4 < 2.3a
> 6.6a 5.9 ± 0.4b > 6.6a
5.3 ± 0.4 3.8 ± 0.6 < 2.3a
1.7 ± 0.4a 3.2 ± 0.6b > 4.7c
Mean values for each of the test microbes, followed by the same letter are not significantly (P > 0.05) different. a Limit of enumeration = 2.3 log CFU/Sample.
produce an equation (Eqn. (2)) used to describe the observed reduction of chlorpyrifos. Reduction of chlorpyrifos from the six centre points ranged from 82 to 90 μg with a standard deviation of 3.6 μg that demonstrates reproducibility of the predictive model (Supplementary Table 2). This shows that the data is replicable with minimal variation between samples. In addition, through verification, a 118 μg (59%) degradation of chlorpyrifos was achieved using an optimal AOP treatment of 69 kJ/m2 UV-C dose and 1.22% v/v H2O2 at 66 °C that agreed with a predictive value of 126 μg suggested by Design Expert software. The photoproduct of chlorpyrifos degradation (CPY–O) was detected but found to be < 20 ppb in the AOP treated samples and absent in controls.
TCP was not detected in any treated samples or controls.
Y = 5.742 + 1.071 X 1 + 0.390 X 2
2.241 X 3 @ 36
s
J m ˆ2
(Equation 2)
3.2. Microbial inactivation The AOP treatment with a UV-C dose of 69 kJ/m2and 1.22% v/v H2O2 at 66 °C to maximise chlorpyrifos degradation also supported a > 7 log cfu reduction of E. coli O157:H7 (Table 2). With 1.22% v/v 4
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hydrogen peroxide alone applied at 66 °C also supported > 8 log cfu reduction of the pathogen (Table 2). A UV-C does of 69 kJ/m2alone resulted in a > 6.6 log CFU/sample decrease in E. coli O157:H7 numbers. In comparison, A. niger spores on apple surfaces were decreased by > 4.7 log CFU/sample with the applied AOP treatment with UV-C alone supporting a 3.2 ± 0.6 log CFU/sample reduction. In contrast to E. coli O157:H7, hydrogen peroxide alone supported a 1.7 log CFU/ sample log reduction of A. niger spores (Table 2).
In the current study the degradation of chlorpyrifos could be positively correlated to temperature which is in agreement with others (Cengiz, Catal, Erler, & Bilgin, 2015; Oliveria et al., 2014). The mechanism of thermal decomposition of chlorpyrifos involves a series of stepwise elimination reactions that cleaves ethylene residues to leave 3,5,6,-trichloro-2-pyridinol as the main degradation product (Kennedy & Mackie, 2018). Evidently the presence of free radicals with UV-C promotes the degradation reaction although given the temperature sensitivity of fruit it would have less relevance within commercial processing. A UV-C process alone could be used to degrade chlorpyrifos but for inactivation of E. coli O157:H7 there was a significant contribution of hydrogen peroxide. The results are in agreement with other reports on the inactivation of microbes on fresh produce and eggs shell surfaces (Hadjok, Mittal, & Warriner, 2008; Murray et al., 2018; Rehkopf, Byrd, Coufal, & Duong, 2017; Xie, Hajdok, Mittal, & Warriner, 2008). Here, it is thought that the free radicals generated of the surface of sample can access microbes shielded from UV-C by surface structures. It is possible that the proportion of free radicals required to inactive microbes is less than that for chlorpyrifos oxidation. From a practical prospect, the results would suggest that hydrogen peroxide has a greater importance in antimicrobial action of AOP with photo-oxidation of chlorpyrifos being more effected by UV-C that is enhanced by treatment temperature. In this respect the optimum AOP treatment parameters can collectively act to reduce microbial and chemical hazards in fresh produce.
4. Discussion Through following RSM approach it was demonstrated that AOP could degrade chlorpyrifos via a process that was primarily dependent on the UV-C dose and temperature. It has been reported that chlorpyrifos in aqueous solutions is relatively stable to UV-C light with the addition of hydrogen peroxide or Fe (Fenton) required to support degradation of the pesticide (Gandhi, Lari, Tripathi, & Kanade, 2016). Further studies have confirmed the finding that chlorpyrifos UV-C degradation is enhanced when applied as part of an Advanced Oxidation Process (Gandhi et al., 2016; Savic et al., 2019; Thind et al., 2018; Utzig et al., 2019). Thind et al. (2018) reported that hydrogen peroxide within the range of 0.5–1.5% v/v supported a 42% decrease in chlorpyrifos levels but could be increased to 53% reduction when used in combination with UV-C. A further increase in chlorpyrifos degradation up to 74% was obtained by using a combination of hydrogen peroxide, titanium dioxide and UV-C therefore suggesting a synergistic effect. Utzig et al. (2019) reported the degradation of chlorpyrifos in acetonitrile with UV supporting complete degradation within 180 min treatment with the inclusion of hydrogen peroxide accelerating the process. However, the authors reported the generation of toxic byproducts when the AOP was applied but not when UV-C was applied alone (Utzig et al., 2019). Yet, unlike the current study, previous reports on the AOP mediated degradation of chlorpyrifos have focused on aqueous or ethanolic solutions. In the current study where the AOP process was performed on the apple surface it was evident that hydrogen peroxide contributed a minor role on the overall degradation process compared to when applied in a aqueous phase. Indeed, the presence of increasing levels of hydrogen peroxide in the AOP reduced the degree of chlorpyrifos degradation by UV-C light. A further difference between the AOP being applied on the surface of fruit vs aqueous solutions is the generation of toxic by-products in the latter from chlorpyrifos degradation. For example (Utzig et al., 2019), reported that CPY-O was formed by the AOP mediated degradation of the pesticide. However, in the current study the concentration of the oxon form was < 20 ppb which reflects the low contribution of hydrogen peroxide within the overall AOP process. The underlying differences observed in the degradation of chlorpyrifos on the surface of apples compared to in aqueous solution could be based on several factors. For example, it is known that the concentration of hydrogen peroxide has to be balanced with regards generating sufficient free-radicals to support the chlorpyrifos reaction but not in excess to impeded the degradative action (de Oliveira et al., 2014). In the case of aqueous solutions there is diffusion of hydrogen peroxide and generation of hydroxyl radicals to prevent the inhibition of the reaction. It is also conceivable that in air the UV-C transmission to the surface of apples would be greater than that of water, especially if absorbing constituents are present. Therefore, in aqueous solutions the hydrogen peroxide would constitute the primary element of the AOP given the absorption of UV-C by the water constituents. By directly illuminating the surface of apples, the generation of radicals can occur although direct reaction of chlorpyrifos with UV-C photons predominating in the degradative action. Additional possibilities are the aqueous phase forms a relative constant matrix to support the reaction of radicals with chlorpyrifos. For example, high alkaline pH and presence of Fe can promote the chlorpyrifos degradation but which could be less controlled on the apple surface (Behin & Farhadian, 2017). Regardless of this fact, the results indicate that AOP studies performed in aqueous phase do not always translate to treating whole fruit.
Acknowledgements The authors wish to thank the Ontario Agri-Food Innovation Alliance for their generous financial support for the project (# 030302). We also wish to thank Moyers Apple Products for the donation of apples and Clean Works Corporation for the laboratory scale Advanced Oxidative Process reactor. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2019.106920. References Abramovic, B. F., Banic, N. D., & Sojic, D. V. (2010). Degradation of thiacloprid in aqueous solution by UV and UV/H2O2 treatments. Chemosphere, 81(1), 114–119. https:// doi.org/10.1016/j.chemosphere.2010.07.016. Atwood, D., & Paisley-Jones, C. (2017). Pesticide industry sales and usage 2008 - 2012. Behin, J., & Farhadian, N. (2017). Response surface methodology for ozonation of trifluralin using advanced oxidation processes in an airlift photoreactor. Applied Water Science, 7(6), 3103–3112. https://doi.org/10.1007/s13201-016-0443-y. Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965–977. https://doi.org/10.1016/j.talanta.2008.05.019. Bouchard, M., F., Chevrier, J., Harley Kim, G., Kogut, K., Vedar, M., et al. (2011). Prenatal exposure to organophosphate pesticides and IQ in 7-year-old children. Environmental Health Perspectives, 119(8), 1189–1195. https://doi.org/10.1289/ehp.1003185. Burkul, R. M., Ranade, S. V., & Pangarkar, B. L. (2015). Removal of pesticides by using various treatment method: Review. International Journal of Emerging Trend in Engineering and Basic Sciences, 2(2), 88–91. Cengiz, M. F., Catal, M., Erler, F., & Bilgin, K. (2015). The effects of heat treatment on the degradation of the organophosphate pesticide chlorpyrifos-ethyl in tomato homogenate. Quality Assurance and Safety of Crops & Foods, 7(4), 537–544. https://doi.org/ 10.3920/Qas2013.0301. Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L. G., et al. (2008). Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment. Critical Reviews in Toxicology, 38(sup2), 1–125. https://doi.org/ 10.1080/10408440802272158. Femia, J., Mariani, M., Zalazar, C., & Tiscornia, I. (2013). Photodegradation of chlorpyrifos in water by UV/H2O2 treatment: Toxicity evaluation. Water Science and Technology, 68(10), 2279–2286. https://doi.org/10.2166/wst.2013.493. Gandhi, K., Lari, S., Tripathi, D., & Kanade, G. (2016). Advanced oxidation processes for the treatment of chlorpyrifos, dimethoate and phorate in aqueous solution. Journal of Water Reuse and Desalination, 6(1), 195–203. https://doi.org/10.2166/wrd.2015.062. Gomez, L. E. (2009). Use and benefits of chlorpyrifos in US agriculture. Dow AgroSciences LLC. Hadjok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of human pathogens and
5
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J. Ho, et al. spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide. Journal of Applied Microbiology, 104, 1014–1024. Ikehata, K., & Gamal El-Din, M. (2005). Aqueous pesticide degradation by ozonation and ozone-based advanced oxidation processes: A review (Part I). Ozone: Science & Engineering, 27(2), 83–114. https://doi.org/10.1080/01919510590925220. Kennedy, E. M., & Mackie, J. C. (2018). Mechanism of the thermal decomposition of chlorpyrifos and formation of the dioxin analog, 2,3,7,8-Tetrachloro-1,4-dioxino-dipyridine (TCDDpy). Environmental Science & Technology, 52(13), 7327–7333. https:// doi.org/10.1021/acs.est.8b01626. Kenney, S. J., Burnett, S. L., & Beuchat, L. R. (2001). Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing. Journal of Food Protection, 64(9), 1328–1333. Köck-Schulmeyer, M., Villagrasa, M., López de Alda, M., Céspedes-Sánchez, R., Ventura, F., & Barceló, D. (2013). Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact. The Science of the Total Environment, 458–460, 466–476. https://doi.org/10.1016/j.scitotenv.2013.04.010. Koutchma, T., Popović, V., Ros-Polski, V., & Popielarz, A. (2016). Effects of Ultraviolet Light and high-pressure processing on quality and health-related constituents of fresh juice products. Comprehensive Reviews in Food Science and Food Safety, 15(5), 844–867. https://doi.org/10.1111/1541-4337.12214. Marican, A., & Durán-Lara, E. F. (2018). A review on pesticide removal through different processes. Environmental Science and Pollution Research, 25(3), 2051–2064. https:// doi.org/10.1007/s11356-017-0796-2. Mladenova, R., & Shtereva, D. (2009). Pesticide residues in apples grown under a conventional and integrated pest management system. Food Additives & Contaminants Part A Chem Anal Control Expo Risk Assess, 26(6), 854–858. https://doi.org/10.1080/ 02652030902726060. Murray, K., Moyer, P., Wu, F., Goyette, J. B., & Warriner, K. (2018). Inactivation of Listeria monocytogenes on and within apples destined for caramel apple production by using sequential forced air ozone gas followed by a continuous advanced oxidative process treatment. Journal of Food Protection, 81(3), 357–364. https://doi.org/10. 4315/0362-028X.JFP-17-306. Oliveira, A., Pereira Ribeiro, J., Oliveira, J., De Keukeleire, D., Saldanha Duarte, M., & Nascimento, R. (2014). Degradation of the pesticide chlorpyrifos in aqueous solutions with UV/H2O2: Optimization and effect of interfering anions. Journal of Advanced Oxidation Technologies, 17(1), 133–138. de Oliveira, A. G., Ribeiro, J. P., de Oliveira, J. T., De Keukeleire, D., Duarte, M. S., & do Nascimento, R. F. (2014). Degradation of the pesticide chlorpyrifos in aqueous solutions with UV/H2O2: Optimization and effect of interfering anions. Journal of Advanced Oxidation Technologies, 17(1), 133–138.
Osman, K. A., Al-Humaid, A. I., Al-Redhaiman, K. N., & El-Mergawi, R. A. (2014). Safety methods for chlorpyrifos removal from date fruits and its relation with sugars, phenolics and antioxidant capacity of fruits. Journal of Food Science & Technology, 51(9), 1762–1772. https://doi.org/10.1007/s13197-012-0693-0. Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X., et al. (2012). Brain anomalies in children exposed prenatally to a common organophosphate pesticide. Proceedings of the National Academy of Sciences, 109(20), 7871–7876. https:// doi.org/10.1073/pnas.1203396109. Rawn, D. F. K., Quade, S. C., Sun, W.-F., Fouguet, A., Bélanger, A., & Smith, M. (2008). Captan residue reduction in apples as a result of rinsing and peeling. Food Chemistry, 109(4), 790–796. https://doi.org/10.1016/j.foodchem.2008.01.061. Rehkopf, A. C., Byrd, J. A., Coufal, C. D., & Duong, T. (2017). Advanced Oxidation Process sanitization of hatching eggs reduces Salmonella in broiler chicks. Poultry Science, 96(10), 3709–3716. https://doi.org/10.3382/ps/pex166. Savic, J. Z., Petrovic, S. Z., Leskovac, A. R., Pasti, T. D. L., Nastasijevic, B. J., Tanovic, B. B., et al. (2019). UV-C light irradiation enhances toxic effects of chlorpyrifos and its formulations. Food Chemistry, 271, 469–478. https://doi.org/10.1016/j.foodchem. 2018.07.2017. Solomon, K. R., Williams, W. M., Mackay, D., Purdy, J., Giddings, J. M., & Giesy, J. P. (2014). Properties and uses of chlorpyrifos in the United States. In J. P. Giesy, & K. R. Solomon (Eds.). Ecological risk assessment for chlorpyrifos in terrestrial and aquatic systems in the United States (pp. 13–34). Cham: Springer International Publishing. Taylor-Edmonds, L., Lichi, T., Rotstein-Mayer, A., & Mamane, H. (2015). The impact of dose, irradiance and growth conditions on Aspergillus niger (renamed A. brasiliensis) spores low-pressure (LP) UV inactivation. Journal of Environmental Science and Health - Part A: Toxic/Hazardous Substances & Environmental Engineering, 50(4), 341–347. https://doi.org/10.1080/10934529.2015.987519. Thind, P. S., Kumari, D., & John, S. (2018). TiO2/H2O2 mediated UV photocatalysis of Chlorpyrifos: Optimization of process parameters using response surface methodology. Journal of Environmental Chemical Engineering, 6(3), 3602–3609. https://doi. org/10.1016/j.jece.2017.05.031. Trasande, L. (2017). When enough data are not enough to enact policy: The failure to ban chlorpyrifos. PLoS Biology, 15(12), e2003671. https://doi.org/10.1371/journal.pbio. 2003671. Utzig, L. M., Lima, R. M., Gomes, M. F., Ramsdorf, W. A., Martins, L. R. R., Liz, M. V., et al. (2019). Ecotoxicity response of chlorpyrifos in Aedes aegypti larvae and Lactuca sativa seeds after UV/H2O2 and UVC oxidation. Ecotoxicology and Environmental Safety, 169, 449–456. https://doi.org/10.1016/j.ecoenv.2018.11.003. Xie, Y., Hajdok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of MS2 F (+) coliphage on lettuce by a combination of UV light and hydrogen peroxide. Journal of Food Protection, 71, 903–907.
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