Uptake and Metabolism of Phthalate Esters by Edible Plants

Jun 19, 2015 - Citing Articles; Related Content. Citation data is made available by participants in ... Physicochemical and microbial soil quality ind...
3 downloads 10 Views 2MB Size
Download PDF
Article pubs.acs.org/est

Uptake and Metabolism of Phthalate Esters by Edible Plants Jianqiang Sun,†,‡ Xiaoqin Wu,† and Jay Gan*,† †

Department of Environmental Sciences, University of California, Riverside, California 92521, United States College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China

‡

S Supporting Information *

ABSTRACT: Phthalate esters (PAEs) are large-volume chemicals and are found ubiquitously in soil as a result of widespread plasticulture and waste disposal. Food plants such as vegetables may take up and accumulate PAEs from soil, potentially imposing human health risks through dietary intake. In this study, we carried out a cultivation study using lettuce, strawberry, and carrot plants to determine the potential of plant uptake, translocation, and metabolism of di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP) and their primary metabolites mono-n-butyl phthalate (MnBP) and mono(2-ethylhexyl) phthalate (MEHP). All four compounds were detected in the plant tissues, with the bioconcentration factors (BCFs) ranging from 0.16 ± 0.01 to 4.78 ± 0.59. However, the test compounds were poorly translocated from roots to leaves, with a translocation factor below 1. Further, PAEs were readily transformed to their monoesters following uptake. Incubation of PAEs and monoalkyl phthalate esters (MPEs) in carrot cell culture showed that DnBP was hydrolyzed more rapidly than DEHP, while the monoesters were transformed more quickly than their parent precursors. Given the extensive metabolism of PAEs to monoesters in both whole plants and plant cells, metabolism intermediates such as MPEs should be considered when assessing human exposure via dietary intake of food produced from PAE-contaminated soils.

■

INTRODUCTION Phthalate esters (PAEs) are high-production volume chemicals that are widely used as plasticizers in numerous products, including various plastic films, packing materials, textiles, medical equipment, and electronics, among others.1 The worldwide consumption of PAEs was estimated at about 5 million tons per year.2 A finished plastic product may contain up to 60% of PAEs by weight, especially in PVC plastic films.3 As PAEs are not chemically bound to the plastic product matrix, they can be emitted into the environment during manufacturing, use, and disposal.3,4 In particular, plastic films in agricultural production (i.e., plasticulture) have become an integral part of modern agriculture. In plasticulture, plastic sheets are extensively used as surface mulch, soil tarps after fumigation, and row covers. In addition, the use of plastic greenhouses serves many functions, such as extending the growing season, conserving water, controlling weeds, and maintaining high quality produce. The estimated amounts of plastic mulch films and greenhouse covers are 0.7 and 1.0 million tons per year, respectively.5 As a result of the extremely widespread use, PAEs have become ubiquitous environmental contaminants and have been found in tissues and fluids of wildlife and humans.2,6 Animal tests suggest that PAEs are endocrine disrupting chemicals (EDCs),7 and the U.S. Environmental Protection Agency has further classified di-nbutyl phthalate (DnBP), di(2-ethylhexyl) phthalate (DEHP), © XXXX American Chemical Society

dimethyl phthalate, diethyl phthalate, butyl benzyl phthalate, and di-n-octyl phthalate as priority pollutants.8 Many studies have shown occurrence of PAEs in soils around the world, especially in agricultural soils.9 Among the detected PAEs, DEHP and DnBP are the most abundant congeners.9 Selected PAEs have also been detected in plants such as vegetables at levels of milligrams per kilogram (dry mass).10 To date, several groups have considered plant uptake of PAEs in the context of phytoremediation or absorption through air in enclosed environments, such as uptake of gaseous PAEs by wax gourds in enclosed greenhouses.11,12 However, so far, relatively little effort has been directed to understanding uptake, translocation, and accumulation processes of different PAE congeners by plants from soil. In a few published studies, the bioconcentration factor (BCF) was calculated from the survey data of PAEs in soils and vegetables.10,13,14 It is unclear whether plant uptake of PAEs differs among PAE congeners that have different properties, or among different plant species for the same PAEs. Monoalkyl phthalate esters (MPEs) are the primary degradation or biotransformation intermediates of PAEs via the hydrolysis of an ester bond.15 Studies show that metabolism Received: March 10, 2015 Revised: June 8, 2015 Accepted: June 19, 2015

A

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

0.1. Carrot seedlings were obtained after germination and cultivation in sand before transplanting. Plant containers without chemicals were included for each plant species and used to determine contamination emanating from other sources during the experiment. In addition, two separate groups of containers without plants but spiked with PAEs or MPEs were used to assess degradation of these compounds in the growth media. Both the control group with plants and the group without plants were added the same amount of nutrient solution. Each treatment, including the controls, consisted of three replicates. The plants were grown in a growth chamber operating with a 16 h light/8 h dark cycle, a constant 65% relative air humidity, and a gradual increase and then decrease of photosynthetic photon flux density that peaked daily at 350 μmol/m2s. The air in the growth chambers was freely exchangeable with the ambient air. To maintain plant health and minimize disturbance to the growth media, containers were bottom-watered twice a week with 50 mL of distilled water each time. After 28 days of growth, plants were removed from the containers and were separated into roots and leaves. The plant samples were rinsed with deionized water, freeze-dried, and ground to a fine powder using a stainless steel coffee grinder. The dried plant tissues were stored at −20 °C until extraction. The sand in each container was thoroughly mixed, and an aliquot was removed, dried, and stored at −20 °C before analysis. Carrot Cell Culture Experiment. Carrot callus was initiated from germinated Scarlet Nantes carrot seeds (Daucus carota Var. Sativus).24 The carrot cell suspensions were grown from the callus culture under sterile conditions in the absence of light in Narayan culture medium at 26 °C and 130 rpm.20,25 The metabolism experiment was carried out at the beginning of the subcultivation interval, starting with 15 parallel bottles each with 1.0 g of cell material suspended in 10 mL of culture medium. The initial concentration of either PAEs or MPEs in the growth media was 500 μg L−1. Three vials were sacrificed after 0, 2, 24, 48, and 120 h of incubation. The cell material was separated from the culture medium by centrifugation for 15 min at 12 000 rpm, washed twice with deionized water, wrapped in aluminum foil, immediately frozen, and then dried in a freeze drier (Labconco, Kansas City, MO). Two 0.1-mL aliquots of the cell culture medium were withdrawn and dried in 2 mL glass tubes for analysis of PAEs or MPEs. The extraction and analysis methods for PAEs and MPEs in the plant cell material were the same as those for plant tissues, but without the cleaning step. Analysis of Phthalate Esters and Phthalate Monoesters. To determine the concentrations of PAEs, the dried plant tissue samples were extracted and analyzed using a recently published method with some modifications.26. The analysis method was validated through preliminary experiments, and the results, including limit of detection (LOD), limit of quantification (LOQ), linearity, and recoveries, are given in Table S1. Briefly, a 0.2-g aliquot of plant sample was placed in a 50-mL glass centrifuge tube, spiked with deuterated DnBP and DEHP (as recovery surrogates) and then extracted with 15 mL of n-hexane and dichloromethane mixture (1:1, v/ v) in an ultrasonic water bath (50/60 Hz, Fisher) for 15 min, followed by centrifugation at 3000 rpm for 30 min. The supernatant was decanted into a 40-mL glass vial, and the residue was extracted one more time using 15 mL fresh solvent mixture. A glass chromatographic column (10 mm i.d. packed with silica gel and alumina oxide) was used for cleanup of the

of phthalates does not usually lead to the detoxification of PAEs, and that mono-n-butyl phthalate (MnBP) may contribute to the observed developmental toxicity of DnBP in rats.16 Likewise, mono(2-ethylhexyl) phthalate (MEHP), a primary metabolite of DEHP, may account for toxicity effects of orally ingested DEHP.17 However, even though MPEs likely coexist with PAEs in soil,2,18,19 at present there is no information on plant uptake of MPEs. Studies on pesticides have highlighted the capacity of plant metabolism after uptake.20 During the phase I plant metabolism, xenobiotics may undergo hydroxylation or hydrolysis,21 where reactions such as hydrolysis lead to the formation of the corresponding MPEs from PAEs. Therefore, consideration of only the unaltered parent may result in an underestimation of plant accumulation of PAEs from soil, and consequently inaccurate prediction of potential human exposure. In this study, we used whole plants of lettuce, strawberry, and carrot to evaluate the uptake and translocation of DEHP and DnBP and their corresponding monoester metabolites (MEHP and MnBP). To highlight the plant-based metabolism, we further explored the metabolism of PAEs and MPEs in a carrot cell culture.

■

MATERIALS AND METHODS Chemicals. Standards of DnBP, DEHP, MnBP, and MEHP were obtained from AccuStandard (New Haven, CT, USA), and internal standards DnBP-d4, DEHP-d4, MnBP-d4, and MEHP-d4 were purchased from C/D/N Isotopes (PointeClaire, Quebec, Canada). The stock solutions of PAEs and MPEs were prepared in n-hexane and methanol separately and stored in amber glass vials at −20 °C before use. All organic solvents used, i.e., n-hexane, dichloromethane, acetone, and methanol, were of HPLC grade (Fisher Scientific, Fair Lawn, NJ). Deionized water was prepared using a Barnstead E-Pure water purification system (Thermo Scientific, Dubuque, IA). Anhydrous sodium sulfate (Na2SO4), silica gel (60−100 mesh) and alumina oxide (100−200 mesh) used for sample cleanup were baked at 400 °C for 4 h before use to eliminate potential contamination. The #16 Silver sand was obtained from P. W. Gillibrand (Simi valley, CA) and used for plant cultivation in place of soil to provide controlled experimental conditions. Plant Cultivation and Treatments. Seedlings of Romaine lettuce (Lactuca sativa L.) and Quinault strawberry (Fragaria x ananassa.) with two to four leaves and seeds of Little Finger carrot (Daucus carota Var. Sativus) were purchased from the Certified Plant Growers (Temecula, CA) through a local retail nursery. The sand was washed with deionized water and heated overnight at 150 °C. Each glass jar (Kerr 1 Quart Wide Mouth) was filled with an aliquot of 1 kg sand (dry weight) and spiked with 1.0 mL of n-hexane containing PAEs or 1.0 mL of methanol containing MPEs. The nominal spiked concentration was 500 μg kg−1 (dry weight) for each congener, which was in the range of the frequently reported PAE concentrations in agricultural soils.22 Each container was mixed manually and placed in a fume hood for 4 h to evaporate the solvent. Each plant seedling was carefully removed from its original container, and the roots were rinsed with distilled water. The plant was then transplanted into each glass jar, followed by the addition of 300 mL of nutrient solution. Hydroponic nutrient solution was prepared according to Pedler et al.,23 and nutrients were supplied at the following concentrations (μM): NO3−, 4900; Ca, 1900; K, 1080; Mg, 500; S, 500; Cl, 191; Si, 187; NH4+, 100; P, 80; Fe, 20; B, 10; Zn, 8; Cu, 2; Mn, 0.6; Mo, 0.1; Ni, B

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

Figure 1. PAE and MPE concentrations in plant tissues. (A) PAE-treated group; (B) MPE-treated group; (C) MPEs produced in the PAE-treated group. Mean concentrations (dry weight) and the standard error (n = 3).

sample extract.27 After elution with 40 mL of acetone/hexane (1:4 v/v), the eluent was reduced to about 1 mL and then quantified using an Agilent 6890 gas chromatograph coupled to a 5973 mass spectrometer.27 Details of instrumental analysis using GC-MSD are provided in the Supporting Information. The PAE concentrations in the dried cell samples and soil samples were similarly measured using the above method, except that the sample extracts were not subjected to cleaning before analysis. To determine the concentrations of MPEs, the dried plant tissue samples from both PAE- and MPE-treated groups, the soil samples, and cell material samples were spiked with deuterated MnBP and MEHP as recovery surrogates and then extracted in a sonication water bath with 15 mL of deionized water twice, 15 min for each extraction.19 The extracts were centrifuged at 3000 rpm for 30 min. The supernatants were combined and acidified to pH 2 with glacial acetic acid and then liquid−liquid extracted using 3 mL of methylene chloride (three consecutive times, each for 10 min). The emulsion of organic solvent and water was separated by centrifuge at 1500 rpm for 5 min. The methylene chloride extracts were concentrated to near dryness under a gentle stream of nitrogen, and the residues were redissolved in 0.5 mL of methanol, followed by analysis on a Waters ACQUITY ultraperformance liquid chromatography (UPLC) equipped with a Waters Micromass electrospray ionization tandem mass spectrometer (ESI-MS/MS). Details of instrumental analysis for MPEs are provided in the Supporting Information (Table S2). Quality Assurance and Quality Control. Several practices were considered to ensure the quality and integrity of data. A deuterated standard for each target compound was added to all samples to correct for the potential analyte loss during sample preparation, matrix-induced signal suppression or enhancement, and variations in the instrument response. The surrogate recoveries in all samples were within acceptable limits ranging from 75−110%. To reduce contamination in blanks, care was exercised to avoid any contact with plastics throughout the experiment, including steps used for extraction, transfer, and storage. Samples were stored in aluminum foil or glass tubes. Procedural or method blanks (n = 2) and a sample

duplicate (n = 1) were included for every batch of 10 samples to monitor for potential background contamination and reproducibility. The LODs for individual PAEs and MPEs were calculated as 3 times the signal-to-noise level from the low-level spiked samples. No DnBP, MnBP, and MEHP were detected in the blanks. Only a small concentration of DEHP was sometimes found in the nonspiked controls but at much lower concentrations (generally lettuce (P < 0.05). The differences in the plant uptake of PAEs between plant species may be attributed to differences in plant lipid contents, among other factors.21 Phthalates are hydrophobic compounds (log Kow = 4.45 for DnBP and 7.50 for DEHP, Table S5) and their sorption to plant roots is likely influenced by the lipid content in roots. In plant roots, accumulation of DnBP (1126−2712 μg kg−1) appeared to be greater than that of DEHP in carrot and strawberry, and the concentrations of both DnBP and DEHP in roots were significantly higher than those in leaves (Figure 1A). Roots with a higher lipid content than most other plant tissues may preferentially accumulate hydrophobic compounds.28 Also, root lipid content has previously been found to be a good indicator for root uptake potential of other hydrophobic compounds.26 The mean BCF values of the leaf or root of the three species ranged from 0.26 to 4.78 for DnBP and 1.31 to 2.74 for DEHP (Table 1). The highest BCF values of both DnBP and DEHP were found in the roots of carrots, while the BCF values of DEHP were greater than 1 in the leaves and roots of all three species. The BCF values derived in the present study were slightly larger than those in previous studies surveying occurrence of PAEs in contaminated soils and plants grown in the soils, where BCF values were found to be generally below 1.10,14 It must be noted that the BCF values of DnBP in roots of strawberry and carrot were larger than those of DEHP, whereas the BCF values of DnBP in leaves were smaller than those of DEHP. This pattern was in agreement with Cai et al.14 Chemicals are usually taken up into plants from the soil pore water, and root uptake of organic chemicals from soil water is D

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

Figure 2. Amounts of PAEs in control and culture medium (A) and cell material (B), and amounts of MPEs produced in cell material (C, D) versus incubation time (h). Data shown are means ± standard deviation of three replicates.

generally crosses biomembranes (e.g., plasma membrane, tonoplast) at a slower rate than its corresponding neutral molecule.35 Given that MPEs are weak acids with the same estimated pKa value of 4.2 (Table S5), they are primarily in the ionized form within the normal soil pH range.36 Under the experimental conditions used in this study, the pH adjusted log Kow values for MnBP and MEHP were 0.07 and 1.66 (Table S5). Anions of MPEs should be difficult for plant uptake, due to the fact that plant cells have a negative electrical potential at the cell membrane (−71 to −174 mV) leading to repulsion of the negatively charged anion.37 The TF values of MnBP were also greater than those of MEHP in the plant species (Table 2), indicating a higher tendency for MnBP to translocate after uptake than MEHP. This result was consistent with previous observations that translocation of weak acids from roots to leaves was negatively related to pH-adjusted log Kow.26 Metabolism of Phthalate Esters in Whole Plants. Previous research has demonstrated that a large portion of hydrophobic chemicals taken up by plants may be transformed in vivo,20,38,39 and that transformation intermediates may exhibit different biological activity profiles than their parent forms.40 In general, the metabolic pathway of xenobiotics in plants may begin with activation of the molecule through reactions such as hydroxylation or hydrolysis.21 For the carboxylic acid ester structure of PAEs, hydrolysis (de-esterification) in plant cells may be the primary transformation pathway, leading to the formation of MPEs. Occurrence of MPEs in plant tissues was analyzed from the PAE-treated group. The monoester metabolites MnBP and MEHP were detected in all leaf and root samples of the tested plant species (Figure 1C). The levels of MnBP were found to be higher than those of MEHP in both leaves and roots, and the difference was more pronounced in the leaves. While the

derived in this study likely reflected the minimum values, as the initial spiked concentration was used in the calculation. To better depict the fractions of spiked PAE amounts in plant tissues, sand, and unaccounted dissipation, mass balance was estimated using the plant biomass and the concentrations in plant and sand at the end of the experiment (Table S7). Although plants can take up PAEs from a contaminated soil, the relative contribution of plant uptake to the total dissipation of PAEs from soil was estimated at less than 1% and therefore was negligible.33 Uptake of Monoalkyl Phthalate Esters by Plants. As the primary degradation and/or biotransformation products of PAEs, MPEs are increasingly detected in various environmental matrices.2,18,19 When spiked in the growth medium, uptake of MnBP and MEHP was also observed in the three plant species (Figure 1B). The MnBP concentrations in both leaves and roots of carrots were slightly higher than the other species (Figure 1B). The concentration of MEHP was also higher in carrot leaves, while the root of lettuce showed the highest MEHP accumulation. In addition, concentrations of MnBP in leaves and roots of all three plant species were consistently higher than those of MEHP, and the difference may be attributed to their physicochemical properties, such as Kow and pKa (Table S5). The BCF values of MnBP were similarly calculated and were found to be consistently greater than 1, whereas the BCF values of MEHP were always smaller than 1 (Table 1). Root uptake of neutral compounds was positively related to log Kow, but for weak acids, such correlation may not be valid.26 In general, weak acids undergo partial dissociation under environmental pH conditions and are therefore present in two forms, i.e., the neutral molecule and ionized species. Molecular dissociation may lead to reduced bioaccumulation by roots because an ion E

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

(Figure 2B). However, the levels of DnBP and DEHP associated with the cell mass gradually decreased thereafter to a final level of about 200 μg kg−1 after 120 h of incubation (Figure 2B). The decrease in accumulation of PAEs in the cell mass may be attributed to metabolism and/or formation of conjugates.20,21 To further evaluate metabolism of DnBP and DEHP in the carrot cells, time-dependent concentrations of the monoester metabolites MnBP and MEHP were also determined (Figure 2C,D). Corresponding to the rapid depletion of DnBP and DEHP from the growth medium, formation of MnBP and MEHP quickly increased within the first 2 h, and then at a more gradual pace thereafter. The level of MnBP at each time point was about 20 times higher than that of MEHP. Analysis of the medium showed no detectable MPEs, indicating that PAEs were metabolized in the cells and that the formed MPEs were not released into the solution. The transformation of PAEs to MPEs was calculated using a mass balance approach. Due to the significant dissipation of DEHP in the control medium, the remaining PAE concentrations calculated as the initial PAE concentration minus the measured MPE concentration were used to analyze the kinetics and the half-life (t1/2). In general, transformation of DnBP or DEHP in the cell suspension followed the second-order kinetics, and the plot of the inverse of the remaining PAE concentration versus the incubation time t was linear for both DnBP (r2 = 0.96) and DEHP (r2 = 0.74). From the linear regression, the reaction rate constant k was estimated to be 2 × 10−6 ng−1 h−1 for DnBP and 4 × 10−8 ng−1 h−1 for DEHP under the experimental conditions, suggesting that DnBP was metabolized significantly faster than DEHP in the carrot cell culture. Under the experimental conditions, the t1/2 was estimated to be 112 h for DnBP, but over 5000 h for DEHP, suggesting a relative recalcitrance to plant metabolism for DEHP. Macherius et al. studied the metabolism of triclosan, a common antibacterial agent, in plant cells, and obtained a t1/2 of 9 h, also demonstrating the ability of plant cells to metabolize certain organic pollutants.20 The metabolism of MnBP and MEHP was further explored after the addition of these metabolites into the carrot cell culture. In the control containers without carrot cells, MnBP and MEHP remained stable in the solution (Figure 3A). Similar to DnBP and DEHP, the levels of MnBP and MEHP in the culture medium quickly decreased initially, followed by more gradual decreases thereafter (Figure 3A). The MnBP and MEHP levels associated with the cell material increased rapidly within the first 2 h and then steadily decreased (Figure 3B). The plot of the inverse of the remaining concentration versus the incubation time t was also linear for MnBP (r2 = 0.98) and MEHP (r2 = 0.99), indicating that metabolism of MPEs to subsequently products also followed second order kinetics in the cell culture system. The reaction rate constant was calculated to be 2 × 10−5 ng−1 h−1 for MnBP and 1 × 10−4 ng−1 h−1 for MEHP. Therefore, the metabolism of MPEs in the carrot cells was considerably faster than that of PAEs. Results from this study clearly demonstrated that various vegetable plants were capable of taking up phthalates in both the di- and monoester forms. However, the translocation of PAEs from roots to above-ground tissues was relatively poor. From both the whole plant and carrot cell experiments, once in the plant, phthalate diesters were readily metabolized into their monoesters, while monoalkyl esters were further transformed. The efficient formation of monoesters from phthalates

mean concentrations of MEHP in the root and leaf samples were similar to those of DEHP, the produced MnBP, with a mean concentration of 3192.8 μg kg−1, was apparently higher than that of its parent form (i.e., DnBP). Therefore, similar to the dissipation of PAEs in the growth medium, DnBP appeared to be more susceptible to plant metabolism than DEHP,41 and the difference may be attributed to differences in their molecular structures such as alkyl chain length and hydrophobicity (Table S5). It is also likely that PAEs were degraded to MPEs in the growth media before being taken up by plant roots. The levels of MPEs in the sand were measured at the end of the cultivation experiment. The levels of MnBP were found to be 35.2−158 μg kg−1, and those of MEHP were 26.4−42.8 μg kg−1 in the growth media (Table S6). The plant uptake potential of the MPEs produced by degradation in the PAE-spiked sand media may be estimated by MPE concentrations in the planted sand at the end of the experiment (Table S6) multiplied by the BCF value of MPEs (Table 1). The derived values were much smaller than the measured MPE levels in plants from the PAEspiked treatment (Figure 1C). These results suggested that the contribution of MPEs formed from degradation in the growth media should be relatively insignificant, and that the majority of MPEs in the plant may be attributed to in-plant metabolism following uptake. However, degradation of PAEs to MPEs may be different in soil under field conditions, and the relative contribution of this process to the occurrence of MPEs in plants should be further evaluated under representative conditions. It must be noted that metabolites of organic compounds may be rapidly conjugated in plant tissues.42 Conjugated metabolites may be analyzed only after hydrolysis using specific enzymes, or quantified using radioactive isotope labeling (e.g., 14C).43 In the present study, since only the unconjugated form of monoesters was characterized, it is likely that the degree of metabolism was underestimated. For example, glucose conjugation is a frequent inactivation pathway in plants that yields glycosylate complexes that may be better absorbed and more bioavailable through food chains than their aglycon counterparts.44 Future research is needed to elucidate metabolism pathways of PAEs in plants and to explore the formation of metabolite conjugation after uptake. Metabolism in Carrot Cells. To better understand metabolism of PAEs in plants, a carrot cell culture experiment was subsequently carried out. Cell cultures represent a simpler model, as processes such as root uptake and translocation were excluded. In the abiotic control (e.g., without carrot cells), no apparent loss of DnBP was noticed over the 120 h of incubation (Figure 2A, hollow squares), while about 57.3% of DEHP was unaccounted for (Figure 2A, hollow circles), likely due to sorption to the container surfaces or degradation. In the presence of carrot cells, the overall level of DnBP in the culture medium decreased rapidly from the initial concentration of 503 ± 5.7 μg L−1 to 170 ± 13.1 μg L−1 after only 2 h (Figure 2A). Similarly, the level of DEHP also decreased quickly, from 503 ± 6.2 μg L−1 to 162 ± 24.0 μg L−1 (Figure 2A). After 48 h, DEHP was below the limit of quantification (5.0 μg L−1), while DnBP remained at a concentration of 87.7 μg L−1 (Figure 2A). Concurrent to the dissipation of PAEs in the cultivation medium, PAEs were detected in the cell mass, and the level increased rapidly within the first 2 h to 1833 ± 398 μg kg−1 for DnBP and 788 ± 71 μg kg−1 for DEHP, indicating adsorption of PAEs onto the cell material and/or absorption into the cells F

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

■

ACKNOWLEDGMENTS This work was supported by the NSFC (21307111). J.S. thanks the China Scholarship Council (CSC) for supporting his visit stay at UC Riverside.

■

(1) Staples, C. A.; Peterson, D. R.; Parkerton, T. F.; Adams, W. J. The environmental fate of phthalate esters: A literature review. Chemosphere 1997, 35 (4), 667−749. (2) Ikonomou, M. G.; Blair, J. D.; Kelly, B. C.; Surridge, B.; Gobas, F. A. P. C. Ultra-trace determination of phthalate ester metabolites in seawater, sediments, and biota from an urbanized marine inlet by LC/ ESI-MS/MS. Environ. Sci. Technol. 2009, 43 (16), 6262−6268. (3) Gomez-Hens, A.; Aguilar-Caballos, M. P. Social and economic interest in the control of phthalic acid esters. TrAC, Trends Anal. Chem. 2003, 22 (11), 847−857. (4) Fujii, M.; Shinohara, N.; Lim, A.; Otake, T.; Kumagai, K.; Yanagisawa, Y. A study on emission of phthalate esters from plastic materials using a passive flux sampler. Atmos. Environ. 2003, 37 (39− 40), 5495−5504. (5) Espi, E.; Salmeron, A.; Fontecha, A.; Garcia, Y.; Real, A. I. Plastic films for agricultural applications. J. Plast Film Sheet 2006, 22 (2), 85− 102. (6) Xie, Z. Y.; Ebinghaus, R.; Temme, C.; Caba, A.; Ruck, W. Atmospheric concentrations and air-sea exchanges of phthalates in the North Sea (German Bight). Atmos. Environ. 2005, 39 (18), 3209− 3219. (7) Meng, X. Z.; Wang, Y.; Xiang, N.; Chen, L.; Liu, Z. G.; Wu, B.; Dai, X. H.; Zhang, Y. H.; Xie, Z. Y.; Ebinghaus, R. Flow of sewage sludge-borne phthalate esters (PAEs) from human release to human intake: Implication for risk assessment of sludge applied to soil. Sci. Total Environ. 2014, 476, 242−249. (8) Keith, L.; Telliard, W. ES&T Special Report: Priority pollutants: I-a perspective view. Environ. Sci. Technol. 1979, 13 (4), 416−423. (9) Kong, S. F.; Ji, Y. Q.; Liu, L. L.; Chen, L.; Zhao, X. Y.; Wang, J. J.; Bai, Z. P.; Sun, Z. R. Diversities of phthalate esters in suburban agricultural soils and wasteland soil appeared with urbanization in China. Environ. Pollut. 2012, 170, 161−168. (10) Mo, C. H.; Cai, Q. Y.; Tang, S. R.; Zeng, Q. Y.; Wu, Q. T. Polycyclic aromatic hydrocarbons and phthalic acid esters in vegetables from nine farms of the Pearl River Delta, south China. Arch. Environ. Contam. Toxicol. 2009, 56 (2), 181−189. (11) Wu, Z. Y.; Zhang, X. L.; Wu, X. L.; Shen, G. M.; Du, Q. Z.; Mo, C. H. Uptake of di(2-ethylhexyl) phthalate (DEHP) by the plant Benincasa hispida and its use for lowering DEHP content of intercropped vegetables. J. Agric. Food Chem. 2013, 61 (22), 5220− 5225. (12) Fu, X. W.; Du, Q. Z. Uptake of di-(2-ethylhexyl) phthalate of vegetables from plastic film greenhouses. J. Agric. Food Chem. 2011, 59 (21), 11585−11588. (13) Ma, T. T.; Christie, P.; Luo, Y. M.; Teng, Y. Phthalate esters contamination in soil and plants on agricultural land near an electronic waste recycling site. Environ. Geochem. Health 2013, 35 (4), 465−476. (14) Cai, Q. Y.; Mo, C. H.; Wu, Q. T.; Zeng, Q. Y. Polycyclic aromatic hydrocarbons and phthalic acid esters in the soil-radish (Raphanus sativus) system with sewage sludge and compost application. Bioresour. Technol. 2008, 99 (6), 1830−1836. (15) Gavala, H. N.; Alatriste-Mondragon, F.; Iranpour, R.; Ahring, B. K. Biodegradation of phthalate esters during the mesophilic anaerobic digestion of sludge. Chemosphere 2003, 52 (4), 673−682. (16) Ema, M.; Kurosaka, R.; Amano, H.; Ogawa, Y. Developmental toxicity evaluation of mono-n-butyl phthalate in rats. Toxicol. Lett. 1995, 78 (2), 101−106. (17) Ito, R.; Seshimo, F.; Miura, N.; Kawaguchi, M.; Saito, K.; Nakazawa, H. High-throughput determination of mono- and di(2ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal. 2005, 39 (5), 1036−1041.

Figure 3. Amounts of MPEs in control and culture medium (A) and cell material (B) versus incubation time (h). Data shown are means ± standard deviation of three replicates.

highlights the importance to consider phthalate metabolites in their overall risk assessment, including potential human exposure via consumption of vegetables grown in phthalatecontaminated soils. Further research is needed to evaluate the occurrence of phthalate monoesters in vegetables and other food crops under field conditions with standard agronomic practices.

■

ASSOCIATED CONTENT

* Supporting Information S

Instrumental analysis methods for GC-MS and UPLC-MS/MS, physicochemical properties, chemical structures, method validation, plant biomasses, and concentrations and mass balance of PAEs and MPEs in plant and sand samples. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b01233.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: 951-827-2712. Fax: 951-827-3993. E-mail: [email protected]. Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology (18) Suzuki, T.; Yaguchi, K.; Suzuki, S.; Suga, T. Monitoring of phthalic acid monoesters in river water by solid-phase extraction and GC-MS determination. Environ. Sci. Technol. 2001, 35 (18), 3757− 3763. (19) Wang, A. L.; Cai, X. D.; Chi, J. Determination of Trace Phthalic acid monoesters in sediments and soils by GC-MS. Soil Sediment Contam. 2012, 21 (8), 996−1005. (20) Macherius, A.; Eggen, T.; Lorenz, W.; Moeder, M.; Ondruschka, J.; Reemtsma, T. Metabolization of the bacteriostatic agent triclosan in edible plants and its consequences for plant uptake assessment. Environ. Sci. Technol. 2012, 46 (19), 10797−10804. (21) Coleman, J. O. D.; BlakeKalff, M. M. A.; Davies, T. G. E. Detoxification of xenobiotics by plants: Chemical modification and vacuolar compartmentation. Trends Plant Sci. 1997, 2 (4), 144−151. (22) Zeng, F.; Cui, K. Y.; Xie, Z. Y.; Wu, L. N.; Liu, M.; Sun, G. Q.; Lin, Y. J.; Luo, D. L.; Zeng, Z. X. Phthalate esters (PAEs): Emerging organic contaminants in agricultural soils in peri-urban areas around Guangzhou, China. Environ. Pollut. 2008, 156 (2), 425−434. (23) Pedler, J. F.; Parker, D. R.; Crowley, D. E. Zinc deficiencyinduced phytosiderophore release by the Triticaceae is not consistently expressed in solution culture. Planta 2000, 211 (1), 120−126. (24) Mustafa, N. R.; de Winter, W.; van Iren, F.; Verpoorte, R. Initiation, growth and cryopreservation of plant cell suspension cultures. Nat. Protoc. 2011, 6 (6), 715−742. (25) Narayan, M. S.; Thimmaraju, R.; Bhagyalakshmi, N. Interplay of growth regulators during solid-state and liquid-state batch cultivation of anthocyanin producing cell line of Daucus carota. Process Biochem. 2005, 40 (1), 351−358. (26) Wu, X. Q.; Ernst, F.; Conkle, J. L.; Gan, J. Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by common vegetables. Environ. Int. 2013, 60, 15−22. (27) Sun, J. Q.; Huang, J.; Zhang, A. P.; Liu, W. P.; Cheng, W. W. Occurrence of phthalate esters in sediments in Qiantang River, China and inference with urbanization and river flow regime. J. Hazard. Mater. 2013, 248, 142−149. (28) Dodgen, L. K.; Li, J.; Parker, D.; Gan, J. J. Uptake and accumulation of four PPCP/EDCs in two leafy vegetables. Environ. Pollut. 2013, 182, 150−156. (29) Burken, J. G.; Schnoor, J. L. Predictive relationships for uptake of organic contaminants by hybrid poplar trees. Environ. Sci. Technol. 1998, 32 (21), 3379−3385. (30) Briggs, G. G.; Bromilow, R. H.; Evans, A. A. Relationships between lipophilicity and root uptake and translocation of non-ionized chemicals by barley. Pestic. Sci. 1982, 13 (5), 495−504. (31) Boxall, A. B. A.; Johnson, P.; Smith, E. J.; Sinclair, C. J.; Stutt, E.; Levy, L. S. Uptake of veterinary medicines from soils into plants. J. Agric. Food Chem. 2006, 54 (6), 2288−2297. (32) Su, Y. H.; Zhu, Y. G. Uptake of selected PAHs from contaminated soils by rice seedlings (Oryza sativa) and influence of rhizosphere on PAH distribution. Environ. Pollut. 2008, 155 (2), 359− 365. (33) Li, Y. W.; Cai, Q. Y.; Mo, C. H.; Zeng, Q. Y.; Lu, H. X.; Li, Q. S.; Xu, G. S. Plant uptake and enhanced dissipation of di(2-ethylhexyl) phthalate (DEHP) in spiked soils by different plant species. Int. J. Phytorem. 2014, 16 (6), 609−620. (34) Wang, J. L.; Xuan, Z.; Wu, W. Z. Biodegradation of phthalic acid esters (PAEs) in soil bioaugmented with acclimated activated sludge. Process Biochem. 2004, 39 (12), 1837−1841. (35) Trapp, S. Modelling uptake into roots and subsequent translocation of neutral and ionisable organic compounds. Pest Manage. Sci. 2000, 56 (9), 767−778. (36) Alzaga, R.; Pena, A.; Bayona, J. M. Determination of phthalic monoesters in aqueous and urine samples by solid-phase microextraction-diazomethane on-fibre derivatization-gas chromatographymass spectrometry. J. Sep. Sci. 2003, 26 (1−2), 87−96. (37) Trapp, S. Bioaccumulation of polar and ionizable compounds in plants. In Ecotoxicology Modeling; Devillers, J., Ed.; Springer: New York, 2009; pp 299−353.

(38) Yu, M.; Liu, J. Y.; Wang, T.; Sun, J. T.; Liu, R. Z.; Jiang, G. B. Metabolites of 2,4,4 ′-tribrominated diphenyl ether (BDE-28) in pumpkin after in vivo and in vitro exposure. Environ. Sci. Technol. 2013, 47 (23), 13494−13501. (39) Huang, H. L.; Zhang, S. Z.; Christie, P.; Wang, S.; Xie, M. Behavior of decabromodiphenyl ether (BDE-209) in the soil-plant system: uptake, translocation, and metabolism in plants and dissipation in soil. Environ. Sci. Technol. 2010, 44 (2), 663−667. (40) Celiz, M. D.; Tso, J.; Aga, D. S. Pharmaceutical metabolites in the environment: analytical challenges and ecological Risks. Environ. Toxicol. Chem. 2009, 28 (12), 2473−2484. (41) Xu, G.; Li, F. H.; Wang, Q. H. Occurrence and degradation characteristics of dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) in typical agricultural soils of China. Sci. Total Environ. 2008, 393 (2−3), 333−340. (42) Calderon-Preciado, D.; Renault, Q.; Matamoros, V.; Canameras, N.; Bayona, J. M. Uptake of organic emergent contaminants in spath and lettuce: an in vitro experiment. J. Agric. Food Chem. 2012, 60 (8), 2000−2007. (43) Li, J. Y.; Dodgen, L.; Ye, Q. F.; Gan, J. Degradation kinetics and metabolites of carbamazepine in soil. Environ. Sci. Technol. 2013, 47 (8), 3678−3684. (44) Pascal-Lorber, S.; Alsayeda, H.; Jouanin, I.; Debrauwer, L.; Canlet, C.; Laurent, F. Metabolic fate of [C-14]diuron and [C14]linuron in wheat (Triticum aestivum) and radish (Raphanus sativus). J. Agric. Food Chem. 2010, 58 (20), 10935−10944.

H

DOI: 10.1021/acs.est.5b01233 Environ. Sci. Technol. XXXX, XXX, XXX−XXX