Article Cite This: J. Agric. Food Chem. 2017, 65, 9880-9886
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Tracking Changes of Hexabromocyclododecanes during the Refining Process in Peanut, Corn, and Soybean Oils Peng Zhang,†,‡ Chunmei Li,†,‡ Fen Jin,*,‡ Hang Su,‡ Hua Shao,‡ Maojun Jin,‡ Shanshan Wang,‡ Yongxin She,‡ Lufei Zheng,‡ Jing Wang,‡ and Yuwei Yuan*,§ ‡
Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards and Testing Technology for Agro-products, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China § Key Laboratory for Pesticide Residue Detection, Ministry of Agriculture, Institute of Quality and Standards for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, People’s Republic of China S Supporting Information *
ABSTRACT: Hexabromocyclododecanes (HBCDs) are harmful compounds, which could be taken up by plants and occur in vegetable oils. In this study, we systematically tracked the changes of HBCDs during different refining processes in peanut, corn, and soybean oils in China. The refining processes were efficient at removing the concentrations of total HBCDs (∑HBCDs), although the levels did increase for peanut and corn oils during the neutralization and bleaching steps. Quite significant reductions in the ∑HBCD concentrations were observed for soybean oils (71−100%) through refining. α-HBCD and ∑HBCD levels were significantly and positively correlated with the peroxidation value (PV), suggesting that PV might be an indicator reflecting the changes of α-HBCD and ∑HBCDs during the oil-refining processes. HBCD intakes from vegetable oils represented a low concern for public health. The results might be helpful for quality and process control with a view to minimize the levels of HBCDs in vegetable oils. KEYWORDS: hexabromocyclododecanes, peanut oil, corn oil, soybean oil, refining process
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INTRODUCTION Hexabromocyclododecanes (HBCDs), mainly used in electronic products, upholstery textiles, and thermal insulation building materials, are additive brominated flame retardants.1 Commercial HBCD mixtures mainly consist of three diastereoisomers, termed α-HBCD (10−13%), β-HBCD (1− 12%), and γ-HBCD (70%).2 The annual global production of HBCDs was approximately 28 000 tons, of which approximately 9000−15 000 tons were produced in China.3 Environmental data demonstrate persistence of HBCDs in various environmental matrices as well as bioaccumulation in numerous living organisms.4,5 Toxicological studies have shown that HBCDs can cause thyroid hormone disruption, reproductive disorders, and neurotoxicity in mammals.6−10 In view of the environmental and toxicological impact, HBCDs were added to the list of persistent organic pollutants in annex A of the Stockholm Convention in May 2013.3 Many studies have shown that HBCDs are ubiquitous in environments, especially with high concentrations detected in soil and sediment.11−13 Recently, Li et al.14 found that HBCDs could be taken up by plants from contaminated soil, such as by cabbage and radish. However, there is insufficient information on HBCDs in plant-derived foods, especially in oilseed crops and their products. Eljarrat et al.15 and Goscinny et al.7 investigated the occurrence of HBCDs in various food groups, and they found that HBCDs were detected in all analyzed vegetable oils, with concentrations ranging from below the limits of detection (LODs) to 42 ng/g. In a previous study by our group,16 the presence of HBCDs in seven edible vegetable oils (peanut, corn, soybean, olive, sunflower seed, sesame, and © 2017 American Chemical Society
blend oils) were determined. The results showed that HBCDs were detected in all of the peanut, corn, and sunflower seed oils, with the highest concentrations in peanut oils (0.349 ng/g). To obtain an edible oil, the refining processes, including neutralization, bleaching, and deodorization, were a necessary step to remove further undesirable compounds that contribute undesirable flavors, color, and aromas that are disagreeable to consumers, affect the stability of the product, and/or are toxic in nature.17 Some studies have assessed the impact of refining on polycyclic aromatic hydrocarbons, 4-hydroxy-trans-alkenals, metals, total contents of phenolics and carotenoids, etc.18−27 However, to the best of our knowledge, no previous studies have examined the changes of HBCDs during the oil-refining processes. Therefore, the study presented here was to systematically track the changes of HBCDs during the different oil-refining processes in peanut, corn, and soybean oils in China. The removal rates of HBCDs were estimated, and the peroxidation value (PV), acidity value (AV), and carbonyl value (CV) were also determined to help understand the potential factors influencing the removal of HBCDs during the refining processes. Finally, the human health risk assessments of HBCDs were estimated. Received: Revised: Accepted: Published: 9880
August 3, 2017 October 17, 2017 October 23, 2017 October 23, 2017 DOI: 10.1021/acs.jafc.7b03606 J. Agric. Food Chem. 2017, 65, 9880−9886
Article
Journal of Agricultural and Food Chemistry
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30 °C) to dryness and reconstituted with 10 mL of cyclohexane/ethyl acetate (1:1, v/v) solvent for purification by an AccuPrep MPS−GPC system (J2 Scientific, Columbia, MO, U.S.A.). The gel permeation chromatography (GPC) system is comprised of an autosampler, an injector with a 5 mL loop, an ultraviolet (UV) detector at 254 nm, a fraction collector, and a GPC column (400 × 30 mm) containing 50 g of polymer resin styrene−divinylbenzene BioBead SX-3 (Bio-Rad, Hercules, CA, U.S.A.). Cyclohexane/ethyl acetate (1:1, v/v) was used as the mobile phase at a flow rate of 4.7 mL/min. The fraction containing target compounds between 18 and 30 min was collected in a 100 mL flask. After GPC purification, the eluate was evaporated to dryness with a rotary evaporator (Heidolph, Schwabach, Germany) at 150 rpm and 30 °C. The residue was reconstituted in acetonitrile (1 mL) and then transferred to a glass centrifuge tube containing C18 (100 mg) as the dispersive solid-phase extraction sorbent. The mixture was shaken on a vortex vigorously for 2 min, centrifuged for 5 min (3000 revolutions/min and 4 °C), and then filtered through a 0.22 μm filter before analysis. HPLC Conditions. Chromatographic analysis was conducted using an Agilent 1200 series HPLC system (Agilent, Santa Clara, CA, U.S.A.) equipped with a vacuum degasser, a column oven, a quaternary pump, and an autosampler. The separation was performed using an XBridge C18 column (150 mm × 2.1 mm × 3.5 μm, Waters, Wexford, Ireland) at 40 °C and an injection volume of 5 μL. The gradient mobile phase consisted of A (0.1% formic acid aqueous solution) and B (acetonitrile), with a gradient elution of 0−5 min, 60−70% B; 5−15 min, 70−90% B; 15−17 min, 90−95% B; 17−18 min, 95−60% B; and 18−23 min, 60% B. Tandem Mass Spectrometry (MS/MS) Conditions. Applied Biosystems−SCIEX API 5000 (Foster City, CA, U.S.A.) tandem quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was performed in the negative ionization mode with multiple reaction monitoring (MRM) for mass spectrometric detection. Typical parameters of the ESI source were as follows: ion spray voltage, −4500 V; atomization air pressure, 65 psi; auxiliary gas pressure, 45 psi; curtain gas pressure, 25 psi; dwell time, 100 ms; resolution Q1, low; and resolution Q2, unit. The parameters of precursor and qualifier ions for MRM detection, declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), and retention time (RT) are listed in Table S1 of the Supporting Information. All system control, data acquisition, and data analysis were performed using AB SCIEX Analyst 1.4.2 software (Applied Biosystems). Quality Assurance and Quality Control. The identification was based on the RT, the transitions, and their ion ratio using MRMs of HBCDs.28,29 Concentrations of all target HBCDs were quantified by the area of peaks. All glassware was heated to 450 °C for 4 h and then rinsed with pure water and LC−MS-grade methanol before use to avoid potential contamination. During the analysis process, the laboratory blank and matrix spike were incorporated in the analytical procedures for every batch of six samples. The recoveries were evaluated using olive oil samples spiked with target compounds at three levels, including 0.1, 1, and 10 ng/g. Olive oil consisted predominantly of 18-carbon unsaturated fatty acids, as do most other commonly consumed edible oils (including soybean, corn, and peanut oils), and especially, it is free of HBCDs. Therefore, olive oil was chosen as the representative oil in this study. Furthermore, the matrix effects for the HBCDs were evaluated, and the results showed that matrix effects for the HBCDs were small, showing suppression or enhancement in the range of 2−16% (see Table S2 of the Supporting Information). The LODs of α-HBCD, β-HBCD, and γ-HBCD were 0.03, 0.003, and 0.03 ng/g, respectively, defined as the concentration at a peak with a signal-to-noise ratio of 3. Determination of the PV. The PV of the oil sample was determined according to Chinese National Standard GB/T 5009.372003. A vegetable oil sample (2 g) was dissolved in 30 mL of chloroform/glacial acetic acid (3:2, v/v). Then, 1 mL of saturated solution of potassium iodide was added. The mixture was shaken manually for 0.5 min and then kept in the dark for 3 min. After the addition of 100 mL of distilled water, the mixture was titrated against
MATERIALS AND METHODS
Chemicals and Reagents. Three HBCD standards, including αHBCD, β-HBCD, and γ-HBCD diastereoisomers (purity > 98%), were purchased from AccuStandard, Inc. (New Haven, CT, U.S.A.). Liquid chromatography−mass spectrometry (LC−MS)-grade methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). High-performance liquid chromatography (HPLC)-grade formic acid (99.7%) was purchased from Dikma Technology, Inc. (Richmond Hill, NY, U.S.A.). Ultrapure water was produced using a Milli-Q RC apparatus (Millipore, Bedford, MA, U.S.A.). Octadecyl (C18) dispersive solid-phase extraction sorbent was purchased from Agela Technologies (Tianjin, China). Sampling. On the basis of the market sales survey, four vegetable oils were studied in this work, including peanut oil, corn oil, pressed soybean oil, and extracted soybean oil. The vegetable oil samples were collected from commercial plants from August 2014 to January 2015 in China. The yield of the four plants ranged from 600 to 2000 tons/ day. All of the raw materials used for oil extractions originated from China, except for the soybeans used for extracted soybean oil extraction, which were imported from the U.S.A., Brazil, and Argentina. All of the crude oils were extracted from the corresponding raw materials by No. 6 solvent oil (a mixture of light alkanes), except for the pressed soybean oil, which was extracted by the cold press process. The generalized flowchart of oil-refining process and the sites of sampling points are described in Figure 1. In the oil-refining
Figure 1. Generalized flowchart of the oil-refining process and the sites of sampling (∗). process, the neutralization was made at 85−95 °C and the bleaching was made at 100−105 °C with both activated earth and activated charcoal. The deodorization was accomplished at 230−270 °C and 100−200 mbar to eliminate volatile compounds and pigments. For each kind of oil, the crude, neutralized, bleached, and deodorized oils processed from the same batch were kindly provided by the oil plant (n = 3). More than 500 mL of each type of oil sample was collected and stored at −20 °C until further analysis. Sample Preparation. A homogenized vegetable oil sample (5 g) was weighed into a 50 mL glass centrifuge tube. After equilibrium for 20 min, acetonitrile (10 mL) was added and the tube was shaken on a vortex vigorously for 3 min and centrifuged for 5 min (3000 revolutions/min and 4 °C). The supernatant was transferred to a 50 mL glass bottle, and these procedures were repeated again. The combined supernatants were rotoevaporated (15 kPa, 110 r/min, and 9881
DOI: 10.1021/acs.jafc.7b03606 J. Agric. Food Chem. 2017, 65, 9880−9886
Article
Journal of Agricultural and Food Chemistry
Figure 2. Concentrations of HBCDs in four kinds of oils during different refining processes (n = 3): (a) peanut oil, (b) corn oil, (c) pressed soybean oil, and (d) extracted soybean oil. Risk Assessment. The margin of exposure (MOE) on the basis of the the European Union Risk Assessment Report was calculated. The daily intake (DI) of HBCD was calculated by the following equation:
sodium thiosulfate (2 mM) until the yellow color nearly disappeared. Finally, titration was continued until the blue color just disappeared after 1 mL of starch indicator solution (1%) was added. PV (g/100 g) was calculated according to the following equation: PV = C × (V − Vk) × 12.69 × 78.8/m
DI = (C × IR j × EF × ED)/(BWj × AT)
(1)
where C is the concentration of HBCD in oil, IRj is the intake of people (40.2 g/day),30 EF is the exposure frequency (365 days/year), ED is the exposure duration (30 years), BWj is the body weight (63 kg),30 and AT is the averaging time (EF × ED). According to European Food Safety Authority (EFSA),31 the chronic human dietary exposure (Dr,h) of HBCDs has been calculated using a one-compartment pharmacokinetic model at a steady state and set at 3 μg kg−1 of body weight day−1. The following equation was used for estimating the MOE:31
where C is the concentration of sodium thiosulfate (M), V and Vk are the volumes of sodium thiosulfate exhausted by the sample and blank, respectively (mL), and m is the mass of the oil sample (g). Determination of the AV. The AV of the oil sample was determined according to Chinese National Standard GB/T 5530-2005. A vegetable oil sample (3 g) was dissolved in 50 mL of neutral diethyl ether/ethanol (2:1, v/v), and then the mixture was shaken by hand. After cooling to room temperature, the mixture was titrated against potassium hydroxide (0.05 M) using phenolphthalein solution as an indicator. AV (mg/g) was calculated according to the following equation:
AC = (V × C × 56.11)/m
MOE = Dr,h /DI
(5)
Data Analysis. Statistical analysis was carried out using the statistical software SPSS 13.0 for Windows. The statistical significance of differences was assessed by applying the one-way analysis of variance (ANOVA). The data set was analyzed using regression analysis and Pearson’s correlation coefficient to examine the strength of the relationships. A probability of 0.05 or lower (p ≤ 0.05) was considered as significant.
(2)
where V is the volume of potassium hydroxide exhausted by samples (mL), C is the concentration of potassium hydroxide (M), and m is the mass of the oil sample (g). Determination of the CV. The CV of the oil sample was determined according to Chinese National Standard GB 5009.2302016. A vegetable oil sample (0.5 g) was dissolved in 25 mL of benzene in a volumetric flask. A total of 5 mL of the solution was added to 3 mL of trichloroacetic acid and 5 mL of 2,4-dinitrobenzene hydrazine, and then the mixture was shaken by hand. Then, it was placed in the water bath (60 °C), heating for 30 min. After cooling to room temperature, the mixture was added to 10 mL of potassium hydroxide−ethyl alcohol (4 g/mL) and then shaken vigorously. After deposited for 10 min, the absorbance was determined at a wavelength of 440 nm. The CV (mequiv/kg) was determined according to the following equation:
CV = A /(854 × m × V2/V1) × 1000
(4)
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RESULTS AND DISCUSSION Changes of HBCD Concentrations during Refining and Diastereoisomer Profiles. The concentrations of HBCDs in the peanut, corn, pressed soybean, and extracted soybean oils during each process are presented in Figure 2. The concentrations of total HBCDs (∑HBCDs) in the crude oils of peanut, corn, pressed soybean, and extracted soybean were 0.04, 0.06, 0.52, and 0.10 ng/g, respectively. After refining processes, the ∑HBCD concentrations in the vegetable oils decreased to less than 0.15 ng/g, which were far lower than those in vegetable oils in Spain (0.45 ng/g)15 and Belgian (0.425 ng/g).7 As expected, ∑HBCD concentrations fluctuated along with the oil-refining processes. For peanut oil (Figure 2a), only α-
(3)
where A is the absorbance at 440 nm, m is the mass of oil samples (g), V1 is the total volume after the dilution of samples (mL), and V2 is the volume of samples for the test (mL). 9882
DOI: 10.1021/acs.jafc.7b03606 J. Agric. Food Chem. 2017, 65, 9880−9886
Article
Journal of Agricultural and Food Chemistry
reductions were observed in the α-HBCD and β-HBCD concentrations for pressed soybean oil (90 and 82%, respectively) through the refining process. The concentration of α-HBCD was continuously decreased along the refining processes by 14−83%, and the bleaching step removed the most α-HBCD in pressed soybean oil (83%), indicating that the bleaching step is more capable of removing α-HBCD. Different from the case for α-HBCD, the concentration of βHBCD decreased by neutralization (35%) and deodorization (80%) but increased after the bleaching step (36%). The results suggest that deodorization is the more effective procedure contributing to the β-HBCD decrease. Changes of PV, AV, and CV during Refining. The PVs, AVs, and CVs of the peanut, corn, pressed soybean, and extracted soybean oils after each processing step are shown in Table 1. It can be seen that, after refining processes, the PVs
HBCD was detected in the oil samples. The mean concentration of α-HBCD was reduced by 25% after the refining processes, although the neutralization step significantly increased the level by 14 times in crude oil. There was a significant difference in the α-HBCD levels during the refining of peanut oil (p < 0.05), reducing slightly by bleaching (22%) and rapidly by deodorization (93%). Similar to the case for peanut oil, only α-HBCD was detected in the corn oil samples, with the concentration of α-HBCD reducing by 17% through the refining process (Figure 2b). The neutralization and bleaching steps continuously and significantly increased the levels of α-HBCD both by 100%. However, after the deodorization step, the level was greatly reduced by 79%. Similar to the case for peanut and corn oils, only α-HBCD was detected in the extracted soybean oil samples (Figure 2d). The concentrations of α-HBCD were continuously and significantly decreased by neutralization (60%) and bleaching (100%), which last below the LOD after the bleaching step. Different to the case for the above oils, three diastereoisomers were detected in all of the pressed soybean oil samples (Figure 2c), and the sustained reduction of ∑HBCD concentrations (up to 71%) was significant (p < 0.05) during the refining processes. In addition, each refining process had a significant effect on the levels of ∑HBCDs, with the levels decreasing by 15−50%, and the deodorization process removed the most ∑HBCDs in the pressed soybean oil (50%). To the best of our knowledge, there is no available literature that has examined the effect of the refining process on the levels of ∑HBCDs. The reasons to account for the decreases of HBCDs in the studied vegetable oils during refining processes might be that the absorbent used in the bleaching step absorbed HBCDs or the HBCDs were removed in the steam-stripping process under the vapor pressure at a high temperature. However, the α-HBCD levels significantly increased in peanut and corn oils after the neutralization step, which might be related with the amount of sodium hydroxide, temperature, and time used in the oil plants, and the types of oils may also have an effect. Besides, the bleaching step significantly increased the levels of α-HBCD in corn oil, which might be related to the different types of oils or the amount of bleaching earth and temperature being different. As for the profiles of HBCDs, α-HBCD was dominant during the oil-refining processes for peanut, corn, and extracted soybean oils, except for the pressed soybean oil, with concentrations ranging from
