Distribution of di-(2-ethylhexyl) phthalate in leaf cuticular waxes and leaf tissues of plants

Di-(2-ethylhexyl) phthalate (DEHP) as plasticizer is widely used in the modern plastic manufacturing industry, DEHP and its breakdown products have been identified as a global environmental contaminant. Vegetables and crops which are the energy sources of human beings are often exposed to DEHP, which enriched in humans through the food chain, resulting in many diseases. The content distribution of DEHP in leaf cuticular waxes and tissues of 14 plants including vegetables and crops, and in various parts of cells of 4 plants were investigated by gas chromatography–mass spectrometer (GC–MS). The results show the stronger the DEHP uptake ability of the plant the less ratio of DEHP in leaf cuticular wax occupying the total DEHP in the leaves of the plant. DEHP in atmosphere is adsorbed by leaf cuticular wax or stoma, then transferred to inner tissues through cell wall. Interestingly, we found that the leaf cuticular wax and cell wall of plants are possible barriers to uptake of DEHP for the plants possessing lower DEHP uptake ability. Our results will provide some information for further study on the mechanism of DEHP uptake by plants.


Introduction
Among the phthalate esters, di-(2-ethylhexyl) phthalate (DEHP) often used as plasticizer in the manufacture of plastics, varnishes, cosmetics and so on (Ghisari and Bonefeld-Jorgensen 2009;Lucas et al. 2012;Saeidnia and Abdollahi 2013). The general population is ubiquitously exposed to this chemical, which is posed a potential risk to human beings (Jobling et al. 1995;Tickner et al. 2001;Heudorf et al. 2007;Zarean et al. 2016). Health adverse impact of exposure to DEHP including effects on reproductive health, carcinogenesis, pregnancy outcome, and respiratory system (Zarean et al. 2016). The clinical manifestations are endocrine disorder, testicular feminization, ovarian dysplasia, neural lesion, hepatotoxicity, and cardiotoxicity (Rowdhwal and Chen 2018). DEHP and its decomposition products have been widely recognized as global environmental pollutants (Zarean et al. 2016;Zhang et al. 2019); therefore, this is a serious problem, which has aroused researchers' high attention.
DEHP possibly like polychlorinated biphenyl (PCB) accumulate in vegetation due to its contaminant affinity for lipids, and the accumulation is important in terms of fate of pollutants (Barber et al. 2003;Kania-Korwe and Lehmler 2016). Past efforts to understand the uptake mechanism of pollutants like DEHP by plants have been considerable, it was demonstrated that the major pathway is air-to-leaf (Wild et al. 2005a(Wild et al. , b, 2006. Vegetation can accumulate DEHP from the atmosphere may be by the means of deposition on the surface portion such as leaves, stems and bark (Smith and Jones 2000;Schuhmacher et al. 2006;Desalme et al. 2013), foliar uptake of DEHP may depend on physiological parameters of plants including specific area of leaves, surface hair, stomatal apparatus density, and cuticle structure (Howsam et al. 2000;Smith and Jones. 2000;Schuhmacher et al. 2006;Tao et al. 2006). A sequential extraction processes can separate chemicals that are adsorbed on the surfaces from those that are absorbed into the plant tissues, Kaupp et al. (2000) and Wang et al. (2008)  an in-depth understanding of the absorption processes of organic pollutants by successively extracting organic pollutants from the cuticles and inner tissue of leaves. Laboratory exposure experiments under controlled conditions provided a lot of information for a better understanding of the absorption of organic pollutants by leaves (Wild et al. 2005a, b). Vegetables and crops are the main sources which provide plant protein, dietary fiber, vitamins and energy to human beings. As the DEHP is widely used in modern industry, more and more DEHP is released into the environment, resulting in these vegetables and crops are often exposed to DEHP. Finally, DEHP through food chain enriched human body cause a variety of diseases. The present study investigates the partition of DEHP in cuticular waxes and in inner tissues of 14 plants including 11 kinds of vegetables (B. hispida, C. sativus, C. moschata, M. charantia, L. cylindrica, B. parachinensis, D. carota, L. sativa, C. frutescens, I. aquatica, and L. esculentum) and 3 kinds of crops (G. max, T. aestivum, and Z. mays), and compares the distribution of DEHP in various parts of cells of 4 plants (B. hispida, C. moschata, B. parachinensis, and I. aquatica) with different uptake ability of DEHP.

Solvents and reagents
In this experiment, all solvents and regents were of analytical grade and purchased from Huadong Chemicals Co. Ltd. (Hangzhou, China) except DEHP and chromatographically chloroform for dissolving DEHP standard were purchased from sigma Beijing Division (Beijing, China).

Sampling
Eleven vegetable plant seeds purchased from Shandong Academy of Agricultural Sciences (Jinan, China) including B. hispida (wax gourd), C. sativus (cucumber), C. moschata (pumpkin), B. parachinensis (flowering cabbage), M. charantia (bitter gourd), Lactuca sativa (lettuce), C. frutescens (hot pepper), L. cylindrical (towel gourd), I. aquatica (water spinach), D. carota (carrot) and L. esculentum (tomato) and three crops which are G. max (soy bean), Z. mays (maize) and T. aestivum (wheat) were utilized for experiment of foliar uptake of DEHP. Each plant was cultivated in a 30 cmdiameter-pot loaded 5 kg of soil (soil physical and chemical properties: pH (H 2 O) = 6.1, cation-exchange capacity (CEC) 14.5 cmol·kg −1 , organic matter 5.7%, total N 129 mg·kg −1 , available P 16.7 mg·kg −1 , or changeable K 133 mg·kg −1 ) and fertilizer. Each plant of a species with three replications (three pots) was randomly placed in a glass greenhouse with a 23 °C/18 °C light/dark temperature regime, and 65% relative humidity under natural light. DEHP treatment was carried out after each seedling grew 2 weeks. Four initial leaves were fixed upper four 4 petri dishes originally loaded 1 mL of DEHP, and added 1 mL of DEHP every week for a supplement. One ml of DEHP was equably distributed in the surface of the dish by volatilizing 10 mL of acetone solution of DEHP. After 4 weeks of foliar uptake of DEHP, the leaves of each plant were harvested and dip into liquid nitrogen immediately, and stored − 80 °C until DEHP determination in leaf cuticular waxes and leaf tissue, respectively.
Four plants B. hispida, C. moschata, B. parachinensis and I. aquatica, which stand for supper, strong, middle and weak foliar uptake of DEHP, were selected for study on distribution of DEHP in various cellar parts. The pot-cultivation and DEHP treatment of these four plants was the same as the above description.

Isolation of plant tissues
The isolation of leaf cuticular wax and leaf tissue followed a procedure modified from Riederer and Schonherr (1984). Fresh leaf samples weighing 5 g were extracted with 100 mL dichloromethane in 250-mL flask with gentle shaking for 2 min. The extract was filtered and used for measurement of DEHP in cuticular wax after clean-up. The extracted leaf samples were freeze-dried and pulverized to pass through a 40-mesh sieve. The dried samples were then extracted in a 50 mL vessel using 1:1 mixture of dichloromethane and acetone at room temperature for 24 h. The extraction solution was concentrated to near dryness in a vacuum rotary evaporator (RE-3000A, Shanghai Yarong Biochemical Instrument Co. Ltd, China).
Fresh leaves of B. hispida, C. moschata, B. parachinensis and I. aquatica were harvested. Fresh leaf samples weighing 10 g were extracted with 200 mL dichloromethane in 500-mL flask with gentle shaking for 2 min. DEHP in the extract was determined as DEHP in leaf cuticular wax. The extracted leaf samples were homogenized with 250 mL of cold extraction medium composed of 0.3 M sucrose, 25 mM tetrasodium pyrophosphate, 10 mM KH 2 PO 4 , 2 mM EDTA, 1% (w/v) and 20 mM ascorbate (pH 7.5) at 4 °C. The cell fragments isolation was performed according the method of Honda et al. (1966), with the modification as follows: the homogenate was sieved through a four-layer nylon cloth and then centrifuged for 10 min at 400×g, the precipitate was cell wall fraction. The supernatant was centrifuged for 20 min at 2000×g resulting chloroplast fraction and a supernatant which was centrifuged for 20 min at 50,000×g (Optima MAX-TL, Beckman Coulter, Inc., USA) giving mitochondrial fraction and a supernatant which was further centrifuged for 40 min at 100,000×g (Optima MAX-TL, Beckman Coulter, Inc., USA) yielding nucleoprotein fraction and soluble cytoplast fraction. The 5 fractions were freeze-dried, and the residues were subjected to DEHP extraction. The extraction was the same as the above procedure of the extraction of the extracted leaf samples.

Clean-up of extraction samples and determination of DEHP
The concentrated extracts were loaded on a combined column of silica gel and alumina. A glass chromatographic column (25 cm × 1 cm, length × I.D. Shanghai Shengong Co. Ltd, China), was packed with 3 cm alumina plus 10 cm silica, followed by 2 cm anhydrous sodium sulfate. Twenty mL of CHCl 2 were used for elution. The collected fraction was blown down to 0.5 mL under a gentle stream of nitrogen. The cleaned-up samples was analyzed by GC-MS (Sablayrolles et al. 2005) with a Hewlett-Packard 5890/5971 GC-MSD (Agilent Technologies, Palo Alto, CA, USA) equipped with an HP-5 trace analysis column (30 m, 0.32 mm i.d., 0.25 μm film thickness). The GC oven temperature was held at 150 °C for 3 min, and then a temperature program was performed: 20 °C min −1 to 300 °C and invariableness at 300 oC for 3 min. The temperatures of the injector and transfer line were 250 and 280 °C, respectively. Helium was the carrier gas at a linear flow rate of 20.7 cm·min −1 . The full scan electron impact data are obtained as follows: solvent delay 5 min, electron impact energy 70 eV, source temperature 200 °C, emission current 150 μA, scan rate 4 scan/s, detector voltage 350 V. The amounts of DEHP were calculated from a calibration curves y = 10 -6 5x + 0.2638 (concentration range 1-50 µg·mL −1 , R 2 = 0.9930) and y = 10 -6 4x + 3.4439 (concentration range 50-500 µg·mL −1 , R 2 = 0.9973). All samples were extracted and analyzed in triplicate.

Quality control
Standard DEHP was used to check the recoveries by doing through the entire procedure. Two procedural blanks were run with every set of extractions by going through the same extraction and cleanup procedures without leaf samples. Recoveries of DEHP were determined by spiking leaf samples of 14 plants with standards at both higher and lower concentrations. The recoveries of the measured DEHP varied from 81.1 to 108.2%.

Leaf stomatal characteristics
Optical microscope (Olympus, model STM6-LM, Japan) eyepiece 8×, objective lens 40× under field observation and measurement, 50 stomatal sizes were randomly measured and recorded. Stomatal sizes were mainly used to measure the length and width of guard cells, and the average value was calculated and the length-width ratio was calculated.

Statistical analysis
ANOVA Duncan's multiple range test was used for statistical analysis of each group. All statistical analyses were performed using SAS 9.4 (SAS Institute Inc., 2018). A probability p < 0.01 value is considered significant. The results of the DEHP experiment were expressed as mean ± SD with three repeated values for each potted plant.

DEHP in leaf cuticular waxes and in leaf tissue
The determination results of DEHP in leaf cuticular waxes and in leaf tissues of fourteen vegetables are shown in Table 1. Based on a statistical difference (p < 0.01) of DEHP in leaf cuticular waxes, the 14 plants could be classified into 3 groups. A highest content, 19.0 ± 2.95 μg·g −1 fresh mass (FM) was found in the leaves of B. hispida; higher contents, 7.88 ± 1.12 and 7.95 ± 0.87 μg·g −1 FM in the leaves of C. sativus and C. moschata; and lower contents, 1.49 ± 0.30 to 3.52 ± 0.84 μg·g −1 FM in the leaves of L. esculentum, Z. mays, L. cylindrical, I. aquatica, C. frutescens, T. aestivum, L. sativa, D. carota, M. charantia, G. max and B. parachinensis.
The situation of DEHP in leaf tissues was very similar to that of DEHP in leaf cuticular waxes though the plants should be classified into 4 groups according to a statistical difference (p < 0.01) of DEHP in leaf tissues. The percentage of DEHP in leaf cuticular waxes occupying DEHP in

DEHP in the various cellar parts of leaves
On the basis of the above results of DEHP in leaf cuticular waxes and in leaf tissues of fourteen plants and a consideration of easily obtaining enough leaf samples, B. hispida, B. parachinensis, C. moschata and I. aquatica were selected as representative plants with larger difference of foliar uptake of DEHP for the study of DEHP in the various cellar parts of leaves.  (Table 3), which indicate chloroplast possibly possess more fat-soluble components to fix DEHP than mitochondria and nucleoprotein. There was a larger amount in the soluble components (10.1-17.6%) ( Table 3) which exist a variety of carriers of DEHP for transfer in cells.

The relationship between DEHP accumulation and stomatal characteristics
The results showed that the stomatal length, stomatal width and stomatal device aspect ratio were not significantly correlated with DEHP accumulation, but positively correlated with stomatal density. The stomatal density of plants with strong DEHP accumulation was significantly higher than that of plants with weak DEHP accumulation (Table 4), indicating that DEHP could enter into plants through stoma.

Discussion
Generally, it is considered that the cuticular wax may be one of the major limiting factors in the uptake of chemicals into leaves (Barber et al. 2004;Schreiber et al. 2005;Smith et al. 2000). For example, the surface of the cuticle is believed to be the location of the barrier to uptake of persistent organic pollutants (POPs) in the leaf (Riederer and Schonherr 1984), and the composition of cuticles that controls cuticular permeability (Kerler and Schonherr 1988). Wax composition of plants, which is therefore probably the key factor regulating uptake of POPs into plants (Beattie and Marcell 2010). In present study, DEHP accumulation in leaf cuticular wax is higher than in tissues from 12 of the 14 species of plants, except for B. hispida and C. sativus (Table 1). This result suggests that B. hispida and C. sativus have a strong ability to transport DEHP across the cell wall, and perhaps stoma may also be involved in the absorption of DHEP (Table 4). We speculated that stomatal density of the nether epidermis of B. hispida and C. sativus may be higher than that of other 12 plants. B. hispida has a strong ability to absorb DEHP (Table 2), and the total DEHP content is much higher than other 13 plants (Table 1); therefore, it is a potential candidate for DEHP phytoremediation from the atmosphere. In fact, the study of Wu et al. (2013) show that B. hispida intercropping with leafy vegetables can reduce DEHP accumulation from the atmosphere, which is a good information for food safety, extrapolating from that, we think C. sativus might have the same effect.
A comparison between species showed a larger proportion of DEHP in cell wall for the plant with higher foliar uptake ability of DEHP (Table 3). Therefore, a saturated effect of foliar uptake of DEHP likely occurs in chloroplast, mitochondrial, nucleoprotein and the soluble cytoplast. A composite analysis of DEHP in leaf cuticular wax and in different cell components reveals the way that DEHP in atmosphere is adsorbed by leaf cuticular wax, then transfer to inner tissue through cell wall. It also can be concluded that cell wall is an effective transport barrier of DEHP into other inner tissues since a large amount of DEHP retains in cell wall.
A distinguishing point of this investigation indicated that the stronger the uptake ability of the plant the less ratio of DEHP in leaf cuticular waxes occupying the total DEHP in the leaves of the plant (Table 3). Inferred from this, the leaf cuticular wax is a barrier to uptake of DEHP for the plants possessing lower uptake ability of DEHP. The effect of surface of the cuticle that bars uptake of DEHP is possibly similar to PCBs (Barber et al. 2003), or POPs with low polarity and high fat solubility (Barber et al. 2004). Therefore, advanced research of composition and constructure of leaf cuticular waxes to the barrier of DEHP may help us to understand uptake of POPs of plants, which is a severe environmental problem. Based on our present research soluble cytoplast accumulated more DEHP than chloroplast, mitochondrial and nucleoprotein due to its high fat-soluble components, i.e. plasma membrane and tonoplast. High contents of DEHP in cuticular waxes and cell wall provide novel information for us to understand the mechanism of DEHP accumulation in plant leaves.
Author contribution statement SGM and WYL participated in the design of experiments, collected the data and drafted the manuscript. SGM, CZW and WYL participated in the design of experiments and helped write the manuscript. SGM and WYL coordinated the research and helped to finalize the manuscript. All authors read and approved the final manuscript.