Article pubs.acs.org/JAFC
Production of Insecticide Degradates in Juices: Implications for Risk Assessment Samantha A. Radford,*,§,†,‡ Parinya Panuwet,‡ Ronald E. Hunter, Jr.,‡,∥ Dana Boyd Barr,‡ and P. Barry Ryan†,‡ §
Department of Chemistry, Saint Francis University, Loretto, Pennsylvania 15940, United States Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States ‡ Laboratory for Exposure Assessment and Development for Environmental Research, Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, Georgia 30322, United States ∥ Global Analytical Services, Coca-Cola Company, Atlanta, Georgia 30313, United States †
ABSTRACT: This study was designed to observe the production of degradates of two organophosphorus insecticides and one pyrethroid insecticide in beverages. Purified water, white grape juice, apple juice, and red grape juice were fortified with 500 ng/g malathion, chlorpyrifos, and permethrin, and aliquots were extracted for malathion dicarboxylic acid (MDA), 3,5,6-trichloro-2pyridinol (TCPy), and 3-phenoxybenzoic acid (3-PBA) several times over a 15 day period of being stored in the dark at 2.5 °C. Overall, first-order kinetics were observed for production of MDA, and statistically significant production of TCPy was also observed. Statistically significant production of 3-phenoxybenzoic acid was not observed. Results indicate that insecticides degrade in food and beverages, and this degradation may lead to preexisting insecticide metabolites in the beverages. Therefore, it is suggested that caution should be exercised when using urinary insecticide metabolites to assess exposure and risk. KEYWORDS: pesticides, degradation, risk assessment, biomarkers of exposure, LC−MS/MS
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INTRODUCTION
metabolized before excretion, their presence in a subject’s urine could cause overestimation of insecticide exposure. While there are several studies of insecticide degradation as required by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR), many of them take place in sterile water, which does not reflect conditions found in food storage.11−13 There are few studies of insecticide degradate production in food; however, these few studies offer evidence of its occurrence. Lu et al.6 observed dialkyl phosphates (DAPs), which are nonspecific OP insecticide metabolites, in both organic and conventional orange juice and apple juice. They also found an increase in DAP concentration in orange juice and apple juice 3 days after an OP insecticide spike caused by degradation of the parent insecticide. Zhang et al.14 also found DAPs in produce that was already known to contain OP insecticides. This study also followed malathion degradation in strawberries by observation of both malathion loss and DAP production after a routine field application. More recently, both OP and pyrethroid insecticide degradates were found in produce samples which were known to contain parent insecticide.15 Of the three studies discussed here, only one accounted for analytic degradation, or the possibility of insecticides producing degradates during the extraction, cleanup, or detection processes. Therefore, the interpretation of these results must be limited.
Since the Food Quality Protection Act of 1996, there has been a large focus on children’s pesticide exposure and resulting health effects.1,2 Evidence is building that prenatal and early childhood exposure to some commonly used organophosphorus (OP) and pyrethroid insecticides can lead to childhood tremor, behavioral problems, and hyperactivity.3−5 Children’s unique characteristics, such as their small size, higher food intake to body mass ratio, lower detoxifying enzymatic activity, and immature neurological and reproductive systems, make them more susceptible to long lasting effects from insecticide exposure. Children also tend to have a preference for foods, such as fruit juices, that often contain higher than average insecticide concentrations, further increasing their risk.6 Therefore, it is critical that we have accurate methods to assess children’s exposures to these hazardous compounds. Exposure to nonpersistant insecticides such as pyrethroids and OP insecticides is often assesed using urinary biomarkers of exposure (BOEs).7 These BOEs are often metabolites formed by hydrolysis at a central ester linkage of the parent insecticide compound. They are then excreted through urine, from which they may be extracted and quantified.7−9 The use of these urinary metabolites as biomarkers of exposure is predicated upon an assumption of a quantitative correlation between insecticide exposure and metabolite output. However, these insecticides may degrade in food before ingestion. Because OP and pyrethroid insecticides are both esters, they are susceptible to hydrolysis, the same mechanism used by the human body for insecticide detoxification.9,10 If these insecticides degrade in food before ingestion, and if these degradates are not further © XXXX American Chemical Society
Received: March 10, 2016 Revised: May 20, 2016 Accepted: May 23, 2016
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DOI: 10.1021/acs.jafc.6b01143 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
A 3.0 mL sample was used, and since the matrix was liquid, it was loaded directly onto the Oasis HLB cartridge with no filtration after cartridge preconditioning. The method has previously been shown to be free of significant analytic degradation.22 Separation by LC−MS/MS. An Agilent 6460 Triple Quad LC− MS/MS equipped with a negative mode ESI interface was used to separate and analyze target compounds. A Zorbax Eclipse Plus PhenylHexyl column (3.0 × 100 mm, 3.5 μm particle size, Agilent, USA) was used for separation and kept at 45 °C. Solvent A was H2O with 1% acetic acid (v/v), and solvent B was MeOH with 1% acetic acid (v/v). The flow rate and the gradient program are shown in Table 1. The
In this work, the degradates of chlorpyrifos, malathion, and permethrin were chosen for analysis using an LC−MS/MS method. These three compounds are of particular interest because of their current frequency of detection in fruits, vegetables, and grains.16 In the 2009 USDA Pesticide Data Program annual summary, chlorpyrifos was found in 12 different types of produce, while malathion was found in six types of produce. Permethrin was also frequently detected in produce, particularly compared to other pyrethroids.16 In this study, malathion dicarboxylic acid (MDA), the specific metabolite of malathion, 3,5,6-trichloro-2-pyridinol (TCPy), the specific metabolite of chlorpyrifos and chlorpyrifos-methyl, and 3-phenoxybenzoic acid (3-PBA), a degradation product that is formed from several pyrethroids, are used to follow degradation of malathion, chlorpyrifos, and permethrin in juice stored in typical household conditions (i.e., a refrigerator) over the course of 2 weeks. These three degradates were chosen for analysis because they are also compounds analyzed as urinary insecticide metabolites and are often used as BOEs.17−19
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Table 1. Flow Rate through Column for Separation
MATERIALS AND METHODS
Reagents and Materials. Milli-Q (Millipore, Billerica, MA, USA) water, which has been treated to a resistance of 18.2 MΩ·cm, was chosen as a simple beverage matrix. The same water was also used for all laboratory experiments and procedures. Apple juice and red and white grape juice were all chosen for analysis due to children’s preference for fruit juices.6 They were obtained from a local grocery store. Methanol (HPLC grade) and glacial acetic acid were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Oasis HLB extraction cartridges (200 mg, 6 mL) were purchased from Waters Corporation (Milford, MA). Standards. MDA was purchased from Crescent Chemical Company (Islandia, NY, USA), TCPy was purchased from SigmaAldrich, and 3-PBA was purchased from Acros Organics (New Jersey, USA). A stock solution containing 10 ng/g MDA, TCPy, and 3-PBA in acetonitrile (ACN) was used to create standard dilutions in solvent from 5 to 2000 ng/g. These solvent standards were then used to create matrix-based calibration curves ranging from 0.25 to 100 ng/g. This range was based on amounts of insecticide degradation products found previously in foods and juices.6,20 Isotopically labeled standards (MDA-D6, DCCA-13C3, and 3PBA-13C6) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). These standards were diluted to 1000 ng/g in ACN and used as internal standards (ISTDs). Identification and Quantification of Analytes. Standards were prepared in matrix using increasing concentrations of analyte. Calibration curves ranged from 0.5 to 100 ng/mL and were calculated using a linear curve. Peaks were manually integrated using the Agilent Quantitative software (Agilent Technologies, Santa Clara, CA), and data was analyzed using Microsoft Excel 2011 (Microsoft, Redmond, WA, USA). Fortification and Extraction Protocol. To create stock solutions for extraction, 70.0 mL of water, apple juice, white grape juice, and red grape juice were fortified to 500 ng/mL malathion, chlorpyrifos, and permethrin. This concentration was chosen to improve the detectability of resulting degradates as discussed in EPA method 161-1 for the evaluation of pesticides’ environmental fates.21 Fortified beverages were then stored in amber glass jars in a refrigerator at 2.5 °C. This temperature was chosen to protect the integrity of the juices over the two week period. Extraction and cleanup were performed immediately after fortification and then 1, 3, 6, 8, 10, 12, and 14 days later (n = 2 for each matrix each day). Nonfortified juices were extracted as well (1.0 mL). The extraction protocol followed a previously published method for insecticide degradate extraction from food22 with a few modifications.
minute
%B
flow rate (mL/min)
0 1.5 3 8 8.8 10 11 13
30 35 50 60 100 100 80 80
0.8 0.8 0.8 0.8 0.9 0.9 0.9 0.9
mass spectrometric parameters were set as follows: the source temperature was 250 °C, the vaporizer gas flow (N2) was 5 L/min, the nebulizer gas flow was set to 35 psi, and the corona voltage was 3500 V. Each metabolite was matched to its own isotopically labeled internal standard except for TCPy, whose internal standard was isotopically labeled DCCA. Ions were analyzed in MRM mode, and their optimized fragmentor and collision energies are shown in Table 2.
Table 2. Precursor and Product Ions for Insecticide Degradatesa compound
precursor ion
product ion
FE
CE
tR
MDA-ISTD MDA MDA TCPy TCPy DCCA-ISTD DCCA DCCA 3-PBA-ISTD 3-PBA 3-PBA
280 273 273 198 196 210 207 209 219 213 213
147 141 157 198 196 210 207 209 99 93 169
80 80 80 96 96 90 90 90 98 122 122
1 1 12 0 0 0 0 0 20 16 8
3.3 3.9 3.9 7.3 7.3 6.7 8.2 8.2 7.4 9.0 9.0
a
Fragmentor energies (FE) and collision energies (CE) are expressed in volts, and retention times (tR) are expressed in minutes.
A separate calibration curve was made for water, but a mixed-matrix curve was made for apple juice and the grape juices. The curves contained nine points from 0.25 ng/g to 100 ng/g. Statistical Analysis. Analyte concentrations were determined using the calibration curves described. Each concentration was logarithmically transformed, and these transforms were averaged for each day’s sample. If possible, linear trend lines relating log concentrations and time in days were determined, and error bars denote standard deviation of the log transform. p-values were determined from the linear regression results, and half-lives were determined using the slope (m) and the relationship t1/2 = −log(2)/m. The criterion for significance was predetermined to be a p-value
