Article pubs.acs.org/JAFC
Occurrence and Residue Pattern of Phthalate Esters in Fresh Tea Leaves and during Tea Manufacturing and Brewing Pingxiang Liu,†,§ Hongping Chen,*,†,# Guanwei Gao,†,§ Zhenxia Hao,†,# Chen Wang,†,# Guicen Ma,†,# Yunfeng Chai,†,# Lin Zhang,†,§ and Xin Liu*,†,# †
Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China # Key Laboratory of Tea Quality and Safety & Risk Assessment, Ministry of Agriculture, Hangzhou 310008, China §
S Supporting Information *
ABSTRACT: The residues of 16 phthalate esters (PAEs) in fresh tea leaves and made tea were determined via gas chromatography−tandem mass spectrometry to study their distribution and degradation characteristics during tea planting and processing. Five PAEs were detected in all fresh tea leaves, and higher concentrations were detected in mature leaves. The distribution of PAEs in fresh tea leaves ranged from 69.7 to 2244.0 μg/kg. The degradative percentages of ∑5PAEs during green tea manufacturing ranged from 61 to 63% and were significantly influenced by the drying process. The transfer rates of PAEs-D4 ranged from 5.2 to 100.6%. PAEs with a high water solubility showed the highest transfer coefficient in the range of 91.8− 100.6%, whereas PAEs with a high log Kow showed a low leaching efficiency below 11.9%. These results benefit the risk evaluation and establishment of a maximum residue limit for PAEs in tea. KEYWORDS: tea, phthalate ester, gas chromatography−tandem mass spectrometry, residue pattern, transfer rate
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INTRODUCTION Phthalate esters (PAEs), a group of diesters of o-phthalic acid, are important environmental endocrine disruptors widely used as plasticizers for polymers. PAEs can migrate from products to the immediate environment and enter the food chain under certain conditions because they are not chemically bound to the polymer matrix.1−3 Toxicological studies on PAEs have indicated their adverse health effects, including reproductive toxicity, embryonic developmental toxicity, and teratogenicity. The U.S. Environmental Protection Agency has classified six PAEs as priority pollutants.4 The European Food Safety Authority set the tolerable daily intakes of bis-butyl ester (DBP), benzylbutyl ester (BBP), bis-2-ethylhexyl ester (DEHP), and diisononyl phthalate to 0.01, 0.5, 0.05, and 0.15 mg/kg bw, respectively.5−8 Furthermore, the European Union established specific migration limits (SMLs) for commonly occurring PAEs in food contact materials; the SMLs for DBP, BBP, and DEHP are 0.3, 30, and 1.5 mg/kg, respectively.9 Tea is a popular nonalcoholic beverage worldwide, with its consumptive amount being second only to water. Tea has received increasing attention for its antioxidant,10 antimicrobial,11 antiobesity,12 and anticarcinogenic13 properties. However, the presence of poisonous substances, such as organic pollutants, heavy metals, and pesticides, in tea has also emerged as an urgent concern. Organic contaminants from the soil, water, and atmosphere can be transported over long distances before deposition and could accumulate in vegetation.14,15 For example, different types of plants can absorb DEHP from plastic mulch film and soil.16,17 Tea plants can absorb and accumulate polycyclic aromatic hydrocarbons (PAHs) from water and air.14 Organic pollution residues accumulating in tea © 2016 American Chemical Society
result from the pollutants emitted by machines or those present in air during manufacturing.18 In our previous study, the levels of 16 PAEs in tea samples ranged from 305.0 to 1210.0 μg/kg.19 Several studies also found PAEs in all monitored tea samples and tea beverages.20,21 However, information on the occurrence and residue pattern of PAEs in fresh tea leaves and during tea processing is lacking. The present study aims to investigate the presence of 16 PAEs in fresh tea leaves from different plant parts, determine the residue pattern of PAEs during tea manufacturing, and assess the transfer rates of PAEs from made tea to infusions. The study was designed to provide preliminary data for risk control and assessment of PAEs in tea planting and processing and to present information for the establishment of a maximum residue limit (MRL) for PAEs, as well as reference values of tea drinking among consumers.
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MATERIALS AND METHODS
Sample Collection. The tea variety Longjing 43, aged over 8 years, was chosen for the present study. Samples were collected on April 27, May 4, and May11, 2016. The sampling sites were located in highway and mountainous regions in Hangzhou, China. Five groups of fresh tea leaves were collected from the tea plants in the sampling sites to determine PAE content. Each group comprised a bud, one and two leaves, three and four leaves, upper mature leaves, and lower mature leaves (Figure 1). About 250 g of each group of fresh tea leaves was sampled each time and then transferred into glass bottles. All sample pretreatments were finished within 1 day. Received: Revised: Accepted: Published: 8909
August 30, 2016 October 20, 2016 October 26, 2016 October 26, 2016 DOI: 10.1021/acs.jafc.6b03864 J. Agric. Food Chem. 2016, 64, 8909−8917
Article
Journal of Agricultural and Food Chemistry
mL of absolute ethanol was added to break the emulsion. The hexane layer was collected in a 50 mL heart-shaped bottle after dehydration with anhydrous sodium sulfate. Then, the combined hexane layer was concentrated to dryness using a rotary evaporator at 40 °C and then dissolved with 1 mL of acetone. Quantification of PAEs-D4 in the tea infusion was performed using matrix-calibrated solutions and external standard method. Target compounds were analyzed by GC-MS/MS using a Varian 450 GC equipped with a Varian CP-8400 autosampler and a Varian 300 series GC-MS/MS triple-quadrupole system. The chromatograph was fitted with a VF-5 MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Varian, USA). The column temperature program was 80 °C (1 min), which was increased to 180 °C at a rate of 15 °C/min (2 min holding time) and then to 280 °C at a rate of 5 °C/ min (15.33 min holding time). Helium gas (99.999% pure) was used at a constant flow of 1.0 mL/min. Then, 1 μL of the sample was injected into the GC-MS/MS in splitless mode with an injector temperature of 250 °C and a GC-MS/MS transfer line temperature of 280 °C. The GC-MS/MS parameters for PAEs and PAEs-D4 analysis are listed in Table S-1 (Supporting Information). Quality Assurance and Quality Control. PAEs in solvents, sorbents, plastic consumables, glassware, and laboratory air and dust could result in cross-contamination.23 Therefore, in our experiment, we avoided contact with any plastic materials. All glassware and chinaware were soaked in methanol overnight, rinsed with hexane, and then dried at 120 °C for at least 4 h (glassware such as pipet and volumetric flask used in the quantitative analysis was dried at 40 °C). Anhydrous sodium sulfate, anhydrous magnesium sulfate, and sodium chloride were heated for 4 h in a muffle furnace before use. Furthermore, two procedural blanks and two solvent blanks were run with every batch of samples, and all experiments were conducted in triplicate. Method Validation. Method validation was performed with linearity, accuracy, precision, and limits of quantification (LOQs) (Tables S-2 and S-3, Supporting Information). Good linearities were obtained with r2 > 0.9913 for PAEs and r2 > 0.9928 for PAEs-D4 with different calibration levels ranging from 1 μg/L (except for the following: 2 μg/L for BBP; 5 μg/L for DEEP, DBEP, and DCHP; 10 μg/L for BMPP, DPhP, DNOP, and DNP; and 25 μg/L for DMEP) to 500 μg/L and from 1 to 100 μg/L, respectively. The accuracy of the method was validated by measuring the recovery of spiked PAEs in the fresh tea leaves, made tea, and tea infusion. The concentrations of the fresh tea leaves and made tea were 50, 200, and 500 μg/kg, whereas those of the tea infusion spiked with PAEs-D4 were 1, 5, and 20 μg/L. The recoveries of PAEs from fresh tea leaves and made tea ranged from 83.5 to 114.1% and from 84.5 to 109.9%, respectively, and the recovery of PAEs-D4 from the tea infusion ranged from 85.6 to 96.1%. Acceptable precision was obtained with relative standard deviations (RSDs) 42% during green tea manufacturing, respectively.38,39 Previous studies illustrated that the degradation mechanism of pesticides during green tea manufacturing depends on evaporation and thermodecomposition.40 Sood et al.41 found that deltamethrin is less degradable (13.8%) during thermal treatment than during evaporation (46.0%) in the drying process and that the loss of dicofol through thermal treatment (44.6%) exceeds the loss from evaporation (23.8%), thus indicating the thermal decomposition of dicofol. Therefore, PAE loss during green tea manufacturing may mainly result from evaporation because PAEs are thermostable. Among various manufacturing methods, drying is regarded as the key factor in the largest loss of PAEs (39−45%). This attribution could be associated with the long hours (at least 1 h) of hot blasting at 80 °C. The result of the current work is in line with the observations of Wu et al.42 and Grover et al.,43 who found that drying leads to the dissipation of 29% carbaryl and 59% acenaphthene during oolong and black tea manufacturing. The loss of PAEs during spreading may be attributed to the long processing time (10−12 h) and fugacity
Furthermore, DBP and DEP, especially DEP, had relatively low concentrations in the samples and showed no significant correlation with the maturation degree of the leaves. Hence, PAEs with a relatively high vapor pressure were likely to volatilize to the air and be absorbed by the aerial parts of the tea plant. This result is identical to the conclusion of Ryan et al.15 that chemicals with a high vapor pressure can easily move throughout the soil and be taken up by above-ground portions of the plant. PAE Residue Pattern during Tea Processing. One bud and two leaves obtained from the two sampling sites were subjected to traditional green tea processing. Five PAEs (∑ 5 PAEs) were detected in the samples from each manufacturing stage, and the concentrations of these PAEs are presented in Table 2 The fresh tea leaves were dehydrated during processing. The moisture content of the samples at each stage of manufacturing was determined to translate the PAE levels into concentrations of dry matter. The total loss of ∑5PAEs was 250.8−357.2 μg/ kg (61−63%) (Figure 3). These values are similar to those of some nonpolar and thermostable pesticides, such as fenazaquin 8912
DOI: 10.1021/acs.jafc.6b03864 J. Agric. Food Chem. 2016, 64, 8909−8917
Article
10.8 14.7 4.4 92.4 137.3 ± ± ± ± ± 121.4 162.0 252.0 1370.2 2244.0 ND ND ND 24.2 ± 3.8 130.2 ± 17.4
± ± ± ± ± 69.7 118.2 181.7 1598.1 2096.4
0.3 1.0 2.6 7.6 7.6 ± ± ± ± ±
Figure 3. Phthalate ester concentrations of dry matter (±SD (μg/kg)) in different stages of green tea manufacture (n = 3).
of PAEs with a high vapor pressure and small molecular weight. In this work, fixing showed a minimal effect on the loss of PAEs because the fixing time was short (4−6 min), and no blasting measure was taken. Although the pot temperature was high (about 200 °C), the PAEs did not degrade because of the stability of their chemical structures. We observed that rolling in green tea manufacturing exerted minimal influence on PAE level. The amount of ∑5PAEs found in rolled leaves marginally increased because of the accumulation of PAEs from the environment and equipment. This result can be attributed to the absence of a high-temperature treatment or other blast measures during this step. DMP, DiBP, DBP, and DEHP were the main PAEs detected in the tea samples. The dissipation rates during green tea processing were 91−92% for DMP, 69−73% for DBP, and 56− 62% for DiBP; that for DEHP increased by 10−22%. A possible increase in DEHP originating from the surface of processing equipment or from atmospheric deposition during long-term spreading may result in DEHP accumulation. This observation is aligned with the report of Lin et al.,18 who stated that tea leaves could absorb the PAHs from the environment during withering and rolling. The amounts of DMP, DiBP, and DBP, which possess a relatively high vapor pressure and low molecular weight, decreased obviously during tea manufacturing, especially during spreading and drying. Chen and Wan40 found that a high vapor pressure equates to the loss of more pesticide residues during manufacturing. In the present study, DEHP accumulated during spreading, fixing, and rolling, whereas it decreased during drying. Owing to the relatively low vapor pressure and high thermostability of DEHP, its loss
bud one and two leaves three and four leaves upper mature leaves lower mature leaves 2
11.0 18.3 18.0 14.3 13.9
33.7 42.7 50.3 37.3 45.7
± ± ± ± ± 1.0 2.9 2.6 1.2 1.0 ± ± ± ± ±
2.3 3.1 0.6 2.1 5.1
46.7 63.3 78.0 126.7 271.7
± ± ± ± ±
5.8 11.5 2.6 20.8 30.1
7.8 20.0 48.0 88.3 158.3
ND ND ND 923.3 ± 49.3 1070.0 ± 104.4 2.1 2.5 3.2 10.0 45.1 ± ± ± ± ± 22.3 17.7 57.7 110.0 353.3
ND ND ND 46.0 ± 6.0 201.0 ± 33.7
DPrP
ND ND ND 28.3 ± 2.9 121.7 ± 10.4 0.6 2.1 5.0 5.8 15.3 ± ± ± ± ±
DNP DMEP
ND ND ND 1203.3 ± 100.2 1166.7 ± 152.8
DEHP
10.7 14.3 55.0 96.7 213.3 1.2 1.2 1.0 11.0 10.0 ± ± ± ± ±
DiBP
7.7 13.7 31.0 72.7 110.0 2.9 10.1 7.2 11.5 15.3 ± ± ± ± ±
DMP
28.3 69.3 72.0 126.7 213.3 ± ± ± ± ±
1.5 2.5 2.1 1.7 1.5 DBP
± ± ± ± ±
16.3 17.7 14.3 17.5 18.7 1.2 1.0 1.2 1.0 0.8
DEP
1
6.7 3.2 9.3 11.0 5.8
sample
bud one and two leaves three and four leaves upper mature leaves lower mature leaves
sampling site
PAE
Table 2. Phthalate Ester Concentrations (± SD (μg/kg)) in Fresh Tea Leaves of Different Parts (n = 3)
ND ND ND 41.9 ± 3.3 246.9 ± 15.4
sum
2.1 5.0 12.4 121.7 187.3
Journal of Agricultural and Food Chemistry
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DOI: 10.1021/acs.jafc.6b03864 J. Agric. Food Chem. 2016, 64, 8909−8917
Article
Journal of Agricultural and Food Chemistry Table 3. Phthalate Ester Cncentrations (± SD (μg/kg)) in Different Stages of Green Tea Manufacturing (n = 3) PAE sampling site
sample
moisture content (%)
DEP
DBP
DMP
DiBP
DEHP
sum
1
fresh leaves spreading fixing rolling drying
79.1 71.2 60.7 60.7 3.4
± ± ± ± ±
0.3 0.6 2.7 2.7 0.2
4.0 5.7 5.3 6.3 4.7
± ± ± ± ±
0.9 0.6 0.6 0.6 0.6
15.8 13.3 31.7 33.3 22.7
± ± ± ± ±
4.9 5.8 5.8 5.8 2.5
34.2 27.5 21.2 26.3 12.5
± ± ± ± ±
4.7 2.5 1.3 1.5 2.5
17.7 23.5 24.0 23.7 36.3
± ± ± ± ±
2.4 2.8 0.9 2.5 2.3
13.5 20.8 35.4 45.0 76.7
± ± ± ± ±
5.1 4.7 5.9 2.5 11.2
85.2 90.8 117.6 134.7 152.9
± ± ± ± ±
13.0 10.4 11.7 8.0 12.7
2
fresh leaves spreading fixing rolling drying
79.4 70.5 61.6 60.3 3.3
± ± ± ± ±
0.3 0.5 2.3 2.5 0.2
5.2 6.3 6.7 7.8 7.7
± ± ± ± ±
0.8 0.6 0.6 0.3 1.5
17.4 18.5 41.2 40.3 22.2
± ± ± ± ±
4.4 3.0 4.7 2.5 2.4
48.2 37.5 35.8 35.7 20.0
± ± ± ± ±
3.3 6.6 5.8 2.1 5.0
25.0 30.4 31.5 31.8 44.6
± ± ± ± ±
3.8 2.3 2.6 2.0 0.9
21.0 41.0 51.5 54.5 108.0
± ± ± ± ±
5.6 8.5 15.0 3.1 13.5
116.7 133.7 166.7 170.2 202.4
± ± ± ± ±
5.5 7.8 23.0 5.5 18.5
Table 4. Transfer Rates of Seven Phthalate Esters-D4 during Tea Brewing content (μg/L) ± SD (transfer rate (%) ± SD) spike level (μg/kg)
detected concentration in made tea (μg/kg)
first infusion
second infusion
DMP-D4
50 200 500
40.0 206.5 473.8
219.5 ± 20.5 (82.3 ± 7.7) 1210.7 ± 269.5 (87.9 ± 19.6) 2561.2 ± 241.4 (81.1 ± 7.6)
25.2 ± 10.6 (9.5 ± 4.0) 143.9 ± 55.4 (10.5 ± 4.0) 457.8 ± 109.0 (14.5 ± 3.5)
DEP-D4
50 200 500
38.7 180.0 463.3
215.1 ± 20.5 (83.4 ± 7.9) 982.7 ± 83.1 (81.9 ± 6.9) 2515.1 ± 185.8 (81.4 ± 6.0)
36.4 ± 7.3 (14.1 ± 2.8) 200.8 ± 49.7 (16.7 ± 4.1) 592.4 ± 85.4 (19.2 ± 2.8)
DiBP-D4
50 200 500
38.2 194.0 464.2
49.4 ± 16.1 (19.4 ± 6.3) 292.1 ± 89.7 (22.6 ± 6.9) 712.1 ± 391.5 (23.0 ± 12.7)
30.4 ± 13.5 (11.9 ± 5.3) 87.7 ± 18.6 (6.8 ± 1.4) 428.2 ± 79.8 (13.8 ± 2.6)
31.4 ± 10.6 29.4 ± 7.2 36.9 ± 14.5
DBP-D4
50 200 500
38.3 183.5 473.8
53.0 ± 9.6 (20.8 ± 3.8) 364.1 ± 44.7 (21.6 ± 3.7) 709.5 ± 172.7 (22.5 ± 5.5)
36.5 ± 4.3 (14.3 ± 1.7) 198.6 ± 33.8 (16.2 ± 2.8) 497.6 ± 113.3 (15.8 ± 3.6)
35.0 ± 4.4 37.8 ± 2.6 38.2 ± 8.3
DnPP-D4
50 200 500
37.8 197.5 461.7
