In situ quantitative and visual investigation of the retention of polycyclic aromatic hydrocarbons on the root surface of Kandelia obovata using a microscopic fluorescence spectral analysis method

Author’s Accepted Manuscript In situ quantitative and visual investigation of the retention of polycyclic aromatic hydrocarbons on the root surface of Kandelia obovata using a microscopic fluorescence spectral analysis method Huadong Tan, Ruilong Li, Yaxian Zhu, Yong Zhang www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(17)30178-9 http://dx.doi.org/10.1016/j.talanta.2017.01.068 TAL17250

To appear in: Talanta Received date: 9 November 2016 Revised date: 14 January 2017 Accepted date: 25 January 2017 Cite this article as: Huadong Tan, Ruilong Li, Yaxian Zhu and Yong Zhang, In situ quantitative and visual investigation of the retention of polycyclic aromatic hydrocarbons on the root surface of Kandelia obovata using a microscopic fluorescence spectral analysis method, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.01.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

In situ quantitative and visual investigation of the retention of polycyclic aromatic hydrocarbons on the root surface of Kandelia obovata using a microscopic fluorescence spectral analysis method

Huadong Tana, Ruilong Lia, Yaxian Zhub, Yong Zhanga* a

State Key Laboratory of Marine Environmental Science of China (Xiamen

University), College of the Environment & Ecology, Xiamen University, Xiamen 361102, China. b

Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen

University, Xiamen 361005, China. *

Correspondence to: State Key Laboratory of Marine Environmental Science of China

(Xiamen University), College of the Environment & Ecology, Xiamen University, Xiamen 361102, China. [email protected]

Abstract

A novel approach for in situ determination of individual benzo[a]pyrene (B[a]P), pyrene (Pyr) and anthracene (Ant) on the root surface micro-zone (0.960 mm2) of Kandelia obovata (K. obovata) was established using a microscopic fluorescence

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spectral analysis (MFSA) system. The detection limits for the three polycyclic aromatic hydrocarbons (PAHs) were 44.2, 59.7 and 36.3 ng g-1 on lateral roots and were 42.8, 62.4 and 39.1 ng g-1 on taproots of the K. obovata root micro-zone. Using the established MFSA method, retention of the PAHs on the K. obovata lateral root and taproot surface micro-zone were investigated in situ. The retention of PAHs on the lateral root and taproot surface of K. obovata showed uneven distribution, and both of the retained quantities showed significant differences, which was related to both passive uptake patterns or active uptake patterns of the PAH and the polarity index ((O+N)/C) of the root surface. In addition, increased quantities of retention of the PAHs on both lateral root and taproot surfaces of K. obovata were observed in the order of Ant < Pyr < B[a]P in the presence of graphene oxide (10 mg L-1) for 7 days. The results of this work provided an in situ method for the investigation on the retention of PAHs on plant lateral root and taproot surfaces at the microscopic scale, contributing to the understanding of the mechanisms of plant root uptake of PAHs.

Keywords: In situ, micro-zone, PAHs, mangrove root, graphene oxide, microscopic fluorescence spectral analysis

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1. Introduction

Polycyclic aromatic hydrocarbons (PAHs), a typical type of persistent organic pollutant (POP), are widely distributed in the environment [1]. PAHs have been a major concern because of their carcinogenicity, mutagenicity, teratogenicity, and endocrine disruption [2]. Over 90 % of PAHs in the environment exist on the sediment/soil surface [3]. Once the uptake of PAHs occurs by plant roots, PAHs can then be translocated to aerial parts [4-6]. This translocation poses a potential risk to human health through food chains and the food web. Thus, it is critically important for the risk assessment of human exposure to understand the mechanisms of plant root uptake of PAHs.

Extensive studies have underlined a series of passive partitioning processes from soil and sediment to pore water, and from pore water to plant roots, which were closely influenced by multiple factors (including the matrix compartment properties and the physicochemical properties of PAHs) [7-10]. Zhan et al showed that the specific surface and lipid content of plant root tissue were two predominant factors contributing to uptake of phenanthrene (a model PAH) using stepwise multiple linear regression analysis [10]. Noticeably, most PAHs are still retained on the root surface (e.g., the ratio of retention and absorption had a mean value of 14.4 times for acenaphthene) during the PAH uptake process by the sequential extraction method,

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wherein a portion of PAHs retained on the root surface may be the potential source into the root interior and subsequent translocation within other plant parts [7, 11].

However, there exists no evidence that the plant root components present the same properties as those before extraction and isolation. Therefore, the uptake process of PAHs on intact plant root should be investigated in situ [9, 12]. With the development of in situ microscopic techniques (including fluorescence microscope (FM), laser confocal fluorescence microscopy (LCSM) and two photon laser confocal fluorescence microscopy (TPLSCM)), Wild et al showed the retention and distribution of phenanthrene and anthracene with the highly focused “streams” on living unmodified wheat (Triticum aestivum L.) and maize (Zea mays L.) root epidermis at the microscopic scale, which significantly differed from the results acquired by isolation and sequential extraction methods [13]. Therefore, the retention mechanism and distribution of PAHs on the plant root surface at the microscopic scale should be specially investigated.

The lack of suitable analytical methods for in situ micro-zone quantification and visualization of PAHs limits the investigation of the retention mechanism and distribution of PAHs on the root surface at the microscopic scale. The sensitive and accurate chromatography approaches (including GC, LC and HPLC) for analysing PAHs on and in the plant inevitably destroy the originally existing forms of PAHs due to their destructive pretreatment process, and thus are unable to investigate the

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retention and distribution of PAHs on the plant root surface micro-zone in situ [14-15]. Alternatively, some in situ methods, such as UV-Vis and Fourier transform infrared spectrometry, have only been used to qualitatively analyse PAHs owing to the shortcomings of related instruments [16]. Therefore, these techniques were not applicable for in situ quantifying and visualizing of the retention of PAHs on the plant root surface micro-zone.

Considering that PAHs have high fluorescence quantum yield, it has been demonstrated that the fluorescence spectral technique has the ability in situ to determine and investigate environmental behaviours, for example photolysis [15, 17] and depuration [18-20] of PAHs on the plant surface. Solid surface fluorimetry (SSF) and fiber-optic fluorimetry (FOF) methods for in situ determination of PAHs adsorbed on mangrove leaf surfaces were established by Wang et al [14] and Chen et al [17]. Later, to further achieve the field detection of PAHs in actual root samples, Li et al established an in situ method using a laser-induced fluorescence (LIF) technique with time-gated fluorescence to investigate the transport mechanisms of PAHs from the mangrove surface to its root tissue [21, 22]. However, these techniques do not meet the requirements to quantify and image the distribution of PAHs on the plant root surface micro-zone. Fortunately, many in situ microscopic techniques, such as FM, LSCM and TPLSCM techniques, have been used to trace the environmental behaviour of PAHs in and on plants [13, 23-24]. The transport and storage of PAHs within living poaceae (Zea mays L. and Triticum aestivum L.) and mangrove 5

(Aegiceras corniculata L.) roots were observed using the TPLSCM technique by Wild et al [13] and Wang et al [23]. Nevertheless, these in situ microscopic techniques, without high-sensitivity quantitative measures, are limited to investigating the heterogeneous distribution of PAHs within the plant, which were significantly different from their uniform behaviours based on the traditional isolation and sequential extraction methods [13, 25]. Therefore, a new microscopic fluorescence spectral analysis (MFSA) system, combining high-sensitivity fluorescence spectrometry and fluorescence microscopic techniques, has been established and was utilized to try to in situ quantify and visualize PAHs on the mangrove root surface micro-zone.

In this study, a novel method for in situ determination and visualization of the PAHs on K. obovata root micro-zone was established by MFSA. Then, the method was used in situ to determine and visualize the retention of PAHs on the K. obovata lateral root and taproot surface micro-zone. The root surface morphology and surface element compositions were investigated by scanning electron microscopy coupled with an energy dispersive X-ray detector (SEM-EDX) to try to illustrate the mechanisms of the PAHs retained on the K. obovata root surface micro-zone. Graphene oxide (GO), an important derivative of graphene-family nanomaterials, can affect the fate and transport of PAHs in the water and sediment environment by π-π interaction and the hydrophobic effect [26-27]. Moreover, the indirect toxicity of GO has been observed by improving the level of co-contaminants in wheat (Triticum 6

aestivum L.) root tissue [28]. Thus, the implications of GO on the retention of the PAHs on the K. obovata lateral root and taproot surface micro-zone were also in situ observed.

2. Materials and methods

2.1 Reagents and apparatus

The Benzo[a]pyrene (B[a]P), pyrene (Pyr) and anthracene (Ant) with different physicochemical properties (benzene rings, steric hindrance effect and Kow) were selected as model compounds of PAHs due to their prevalence in both sediment/soil and roots of mangrove [29]. These PAHs with a purity of 99 % were purchased from Sigma-Aldrich Co. Ltd., UK. GO (purity: 98 %, height: 0.55 - 1.2 nm, diameter: 0.5 3 μm) was obtained from Chengdu Organic Chemicals Co. Ltd (Chengdu, China). All of the other chemicals used in the study were acquired from Sinopharm Chemical Reagent Co. Ltd. with analytical reagents (A.R.) grade. The stock solutions of the individual PAH in an acetone solution with 1000 mg L-1 were prepared according to ref. 18. Based on the method reported, the dispersion solution of GO in half-strength Hoagland’s solution was prepared [30].

The MFSA system, combining an inverted fluorescence microscope (Olympus IX73, Olympus Co. Ltd., Japan) and a high sensitivity fluorescence spectrometer

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(FLS920, Edinburgh Instruments Ltd., UK), was constructed in the lab. The schematic diagram of the MFSA system is shown in Figure 1. The detailed parameters are as follows: synchronous spectra scan mode; an optimized constant wavelength offset (Δλ= λem - λex) with the scanning range of the emission spectra between 405 - 475 nm; and a fluorescence filter of AT-DAPI/hoechst/AlexaFluor350 for the fluorescence microscope system. Microscopic images with the size of 2560 × 2160 pixels (width × height) were acquired using a digital fluorescence camera (Andor Zyla 4.2 sCMOS camera, Andor Co., UK). The exposure time of the microscopic images was 0.4 s. The micro-zone area for each observation of the K. obovata root surface was 0.960 mm2.

2.2 Sample collection and pretreatment

Mature K. obovata hypocotyls were collected from Zhangjiangkou Mangrove National Nature Reserve located in Zhangzhou, Fujian, China (east longitude: 117°25′; north latitude: 23°55′; altitude: 0 m above sea level). Then, the K. obovata hypocotyls were quickly transported to the laboratory and were cultured in a sand bed for 3 months with the same culture conditions as ref. [31]. The K. obovata mangrove plants with similar fresh weight and length (14.5 ± 0.6 g, 21.6 ± 0.7 cm) were cleaned carefully with Milli-Q water to remove sand, sediment and silts adsorbed onto the root surface prior to use [21].

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2.3 Quantification of PAHs adsorbed on the K. obovata root surface micro-zone by MFSA

To obtain the calibration curve of the PAHs adsorbed on root surface micro-zone, the roots of K. obovata seedlings were exposed to B[a]P, Pyr and Ant (3.5 × 10-7 g L-1, 1.0 × 10-5 g L-1 and 6.0 × 10-6 g L-1). The exposures were performed in a 1000 mL water solution containing 1 % mercuric chloride to inhibit microbial activity and were renewed every day. Before each time of the renewal, the contaminated root was put on a fixed quartz slit (48 mm × 15 mm × 1.25 mm, length × width × height) in the MFSA system. Each time, the same position on the root sample surface were located by the horizontal moving rulers with a minimum increment of 100 μm in the MFSA system, and then fluorescence spectra and microscopic images of PAHs adsorbed on the root surface micro-zone (n=9) were obtained by the MFSA system. Meanwhile, part of the roots of K. obovata were used to obtain the quantity of the PAH adsorbed on the root surface, which were extracted with 20 mL methanol shaken for 3 min according to a previously reported method [11]. Then the concentration of the PAH was measured using a fluorescence spectrophotometer (Cary Eclipse, Varian, USA). The linear regression curves between the quantity of the PAHs and their average fluorescence intensities adsorbed on the micro-zone of the K. obovata surface are presented in Figure S1 of the Supplementary Information (SI). There were three replications for each treatment.

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2.4 Retained quantities of PAHs on root surface micro-zones in the presence and absence of GO

Six groups of K. obovata seedlings were cultured in black plastic pots containing half-strength Hoagland’s solution with a continuous concentration of B[a]P, Pyr and Ant (2.0 × 10-7 g L-1, 2.0 × 10-6 g L-1 and 1.0 × 10-6 g L-1, respectively) with and without the presence of GO (10 mg L-1). Then, all the pots were stored in a controlled climate chamber (light/dark regime: 16/8 h, temperature: 25/20 °C, relative humidity: 60 %, light intensity: 400 μmol m−2 s−1) [31]. The K. obovata roots and part of the stalk were immersed into the culture solution to keep all roots in the solution. After 7 days, the retained quantity of PAH on the K. obovata lateral root and taproot surface were measured using the MFSA system measurement method as described in Section 2.3. Our results confirmed that GO at 10 mg L-1had no effects on the in situ measurements of the PAHs on the root surface micro-zone (p >0.05, compared to their control).

2.5 Characterization of surface morphology and element distribution of K. obovata roots

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According to a previously reported method [32], a field emission scanning electron microscope with an energy dispersive X-ray spectroscope (SEM-EDX) (Carl Zeiss, Germany) was used to characterize the surface morphology and the main element composition of K. obovata lateral roots and taproots. Briefly, the K. obovata root samples were first fixed in 2.5 % glutaraldehyde in a 0.1 mmol L-1 phosphate buffer (pH 7.2) for 8 h. Subsequently, they were dehydrated in a graded ethanol series (30, 50, 70, 80, 90 and 100 %), and then the ethanol was exchanged using tertiary butyl alcohol. Later, the root samples were put in liquid N2 and a sterile knife was used to acquire a new intact surface. Lastly, the SEM and elemental mapping analysis of root samples were obtained by SEM-EDX with an accelerating voltage of 10 kV using Al Kα radiation.

2.6 Membrane permeability measurement of the K. obovata root surface in the presence and absence of GO

Electrolyte leakage, used to assess the membrane permeability of the K. obovata root surface, was carried out as in a previous report [28]. In brief, the contaminated roots were separated between lateral roots and taproots. The roots were then cleaned by Milli-Q water three times to remove surface adhered electrolytes. Later, the roots were put in vials with 10 mL of pure water, and then kept on a shaker at 500 rmp at 298.15 K. Lastly, the electrical conductivity (EC1) of the root surface was determined. 11

The root samples were then incubated at 393.15 K for 20 min. The final electrical conductivity (EC2) was obtained by a conductivity metre. The membrane permeability was represented as electrolyte leakage ((EC1/EC2) × 100 %).

2.7 Statistical analysis

The fluorescence intensity data of the K. obovata root surface micro-zone, measured nine times, were obtained by MFSA. Analysis of variance (ANOVA) at the 95 % confidence level was conducted to assess the significant difference of the concentration of PAHs retained on the K. obovata root surface micro-zones (n=15) with and without the presence of GO using SPSS version 19.0.

3. Results and Discussion

3.1 The synchronous fluorescence spectra of PAHs adsorbed onto the K. obovata root surface micro-zone

The emission spectra of PAHs adsorbed on the lateral root and taproot surface micro-zone were obtained by MFSA. As shown in Figure 2(a), the peak emission spectra of B[a]P adsorbed on the lateral root surface micro-zone was 476 nm with an excitation at 380 nm. However, strong auto-fluorescence from the root surface

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influenced the determination of B[a]P (357 ng g-1) adsorbed on the surface micro-zone with a low signal to noise ratio (S/N) (3.1 for lateral roots and 3.4 for taproots) due to root exudates, plant root cells and bacteria [33-34]. These results indicated that conventional emission spectra did not meet the requirements needed to determine PAHs adsorbed on the K. obovata root surface micro-zone. Li et al showed that the interference of the background fluorescence and scattered light from tea were effectively lowered using a synchronous fluorescence spectra technique with simple microwave-assisted pretreatment [35]. The synchronous fluorescence spectra technique was, thus, used in this study to determine lower concentrations of PAHs adsorbed on the root surface micro-zone.

The synchronous wavelength offset (Δλ) of PAHs adsorbed on the root micro-zone were first optimized. As shown in Figure 2(b), the highest S/N of synchronous fluorescence intensities of B[a]P (357 ng g-1), with a Δλ at 55.0 nm, was increased to 18.9 for lateral roots. The peak synchronous fluorescence spectra (λem) of B[a]P adsorbed on the lateral root surface micro-zone was 441 nm, with Δλ at 55.0 nm, as shown in Figure 2(c). Also, the optimal detection wavelengths of the Pyr and Ant adsorbed on the K. obovata root surface micro-zone were also obtained for Pyr (λem= 439 nm, Δλ = 90.0 nm) and Ant (λem = 442 nm, Δλ= 60.0 nm). Similar results were observed for the PAHs adsorbed on the taproot. These results showed that the synchronous fluorescence spectra method met the requirement needed to determine the PAHs adsorbed on the K. obovata root surface micro-zone. 13

Observation of PAHs on the root surface micro-zone was conducted to confirm the protocol for visualization of PAHs. Direct visualization (microscopic image, as shown in Figure 3) indicated that B[a]P appeared as some white patches on the root epidermis, as indicated by the yellow arrows in Figure 3. These patches showed the uneven distribution of B[a]P on the K. obovata lateral root and taproot surface micro-zone. Similar results were observed for Pyr and Ant. These results showed that the established MFSA could visualize PAHs on the K. obovata lateral root and taproot surface micro-zone.

3.2 Analytical merits and recovery of the established method

To determine in situ PAHs adsorbed onto the K. obovata root surface micro-zone, a series of PAHs quantities adsorbed on the root surface were determined by the MFSA system. The detailed merits of the analytical method for PAHs adsorbed on the K. obovata root surface micro-zone are listed in Table 1. The dynamic linear ranges of B[a]P, Pyr and Ant adsorbed on the lateral root surface micro-zone were 260-2520, 310-2870 and 170-2180 ng g-1, respectively, with correlation coefficients (R2) of 0.9861, 0.9857 and 0.9678, respectively. The limits of detection (LOD) of B[a]P, Pyr and Ant adsorbed on the lateral root surface micro-zone were 44.2, 59.7 and 36.3 ng g-1, respectively. Also, the limits of quantification (LOQ), as the concentration giving a signal equal to the blank plus ten times the standard deviation, of B[a]P, Pyr and Ant 14

were 132.6, 178.2 and 100.8 ng g-1 for lateral root, and 128.4, 189.1 and 117.3 ng g-1 for taproot, respectively. The precision of the method (Table 3) shown was determined as repeatability (n = 9) and also as reproducibility (n = 5, measurements of different days); the respective results were lower than 12.5 % and 15.0 %. Furthermore, the results obtained by the proposed MFSA method and the laser-induced time-resolved nanosecond fluorescence spectroscopy (LITRF) method reported by Li et al were shown in the Table 4 and were analysed by the paired t-test. The relative deviations of determination results of the two in situ methods were less than 10%. It was evident that there was no statistically significant difference between the results obtained by the proposed method and the LITRF method (p <0.05).

To verify the feasibility of the established MFSA method for in situ determination of PAH adsorbed on the K. obovata root surface micro-zone, a recovery experiment was conducted based on ref. [14]. The mangrove seedlings were exposed to a continuous concentration (C1) of PAH in half-strength Hoagland’s solution with the treatment described in Section 2.3. The adsorption quantity (Q1, ng g-1) of PAHs on the roots was measured using the above-mentioned method. Then, other similar roots of K. obovata were selected to be exposed to another continuous concentration (C2, C2=2C1), and the same method was used to determine the adsorption quantity (Q2) of PAH on the root surface. Finally, the recovery was calculated by p= ((Q2-Q1)/Q1) × 100 %. The results of the recovery experiment are shown in Table 2. For K. obovata lateral roots, the recoveries of B[a]P, Pyr and Ant were 72.7-118.8 %, 15

85.9-127.5 % and 85.8-109.1 %, respectively. The recoveries for taproots were 78.7-112.5 % for B[a]P, 78.4-121.8 % for Pyr and 67.3-125.0 % for Ant. Above results indicated that the new MFSA method can be used for the direct determination of PAHs on K. obovata lateral root and taproot surface and thus subsequent results of retention of PAHs were accurate and reliable.

Several methods for the in situ determination of PAHs on the plant root/leaves surface have been published [14-15, 18, 21-22]. Compared with the previous reports, the proposed method has combined the in situ micro-zone (0.960 mm2) quantitative determination and visualization of the PAHs with a spatial resolution of 10 μm on the root surface of K. obovata. The results marked a significant step toward in situ micro-zone determination and also visualization of PAHs in plant root.

3.3 The retention of PAHs and microscopic distribution of PAHs on the K. obovata root surface

The retained quantities of the PAHs on the K. obovata root surface after 7 days are shown in Figure 4(a). These results showed that the retained quantity of the individual PAH on the lateral root surface micro-zones followed the order of Ant (566 ng g-1) > Pyr (486 ng g-1) > B[a]P (470 ng g-1). Similar results were observed for the taproot. The distributions of the B[a]P retained on the K. obovata lateral root and taproot surface micro-zone are shown in Figure 5(a) and Figure 5(c), respectively. It 16

was observed that the B[a]P appeared as white patches on both the lateral root and taproot epidermis of K. obovata.

The uptake process of PAHs by plant roots mainly consists of the following two parts: (1) dissolved PAHs diffuse and adsorb on the root epidermis and (2) only a portion of the PAHs transport into the cortex root tissues or translocate to other tissues/organs. Many reports have emphasized that the uptake of PAHs by plant roots is dominated by their partition into plant roots [8-9]. Therefore, the relationship between the retained quantity of PAHs on the root surface and the partition process into root tissues needs to be examined. The octanol-water partitioning coefficient (Kow) has been commonly used to describe the uptake of PAHs in the water/soil-plant system. Wang et al observed a negative relationship between root uptake of PAHs and the Kow of PAHs [36]. In the present study, there was a negative linear correlation between the log Kow of PAHs and the retained quantities of PAHs on surface micro-zones (n=15) of the K. obovata root (R2=0.8008 for lateral roots and R2=0.7457 for taproots, p < 0.05). Further studies have shown that active uptake also exists in the plant [37-38]. By lowering the temperature from 298.15 K to 288.15 K, the linear relationship improved with a higher R2 value for both lateral root (R2 = 0.8965, p < 0.05) and taproots (R2 = 0.8235, p < 0.05) between the log Kow and the retained quantity of PAHs on the root surface micro-zones. Therefore, there was evidence that the active uptake of PAHs also contributed to the retention of PAHs on the root surface micro-zone. 17

Significant differences of the PAH uptake capacities in different root types were observed due to the heterogeneous system in structure and function [11, 39]. In addition, as Figure S2 and Table 5 show, the element distributions of carbon (C), oxygen (O), nitrogen (N), and sulphur (S) on the plant lateral root surface were 60.72 %, 33.37 %, 5.28 %, and 0.63 %, respectively. Similarly, the main elements on the taproot surface micro-zone were 58.54 % for C, 38.41 % for O, 2.71 % for N, and 0.34 % for S. Therefore, the role of the surface chemical composition from the K. obovata root surface micro-zone on retaining quantities of PAHs should be specifically investigated. Figure 4(a) shows that the retained quantity of B[a]P on the lateral root was 470 ng g-1 higher than that of the taproot (310 ng g-1). Similar results were observed for Pyr and Ant. As Table 5 shows, the polarity index ((O+N)/C) of the K. obovata taproot was 0.70 higher than that of the lateral root (0.64), indicating that the lateral roots of K. obovata had a lower polarity index than the taproot. As PAHs are hydrophobic substances, they have more affinity on a lower polarity ((C+O)/N) surface [40]. Therefore, PAHs possessed a higher retained capacity on the lateral root surface micro-zone than on the taproot.

3.3.3 Implication of GO on retained quantity and microscopic distribution of PAHs on the root surface micro-zones

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As Figure 4(b) shows, the retained quantities of B[a]P, Pyr and Ant adsorbed on the lateral root surface micro-zone increased from 470 ng g-1 to 557 ng g-1, from 486 ng g-1 to 548 ng g-1, and from 566 ng g-1 to 611 ng g-1 in the presence of GO, respectively. Similar results were observed for taproots. The increase in retained quantity (ΔQ) is calculated by ΔQ=QGO-Qs, where QGO (μg g-1) is the retained quantity of PAHs on the root surface in the presence of GO and Qs (ng g-1) is the retained quantity of PAHs on the root surface without GO. The ΔQ values following the order B[a]P > Pyr > Ant were 87, 63 and 45 ng g-1, respectively. The microscopic images (Figure 5(b) and Figure 5(d)) also indicated that the retained quantity of B[a]P on the K. obovata root surface micro-zone were visually increased in the presence of GO.

Many reports have shown that GO can adsorb and enrich HOCs (including PAHs) in the GO-water interface due to its delocalized π-electron system and large surface area [26, 41]. The π-π interactions and hydrophobic effect are regarded as the two most significant mechanisms for GO interaction with PAHs (GO-PAHs). In addition, GO was easily adsorbed on the plant cell surface due to its superior mechanical properties and hydrophilic nature, while also having high flexibility and ductility [30, 42]. GO-PAHs could thus co-adsorb on the root surface, which contributed to the increase of the retained quantity of PAHs on the root surface. Furthermore, Wang et al showed that the adsorption capacity of GO for PAHs increased with the number of benzene rings of the PAHs [26]. Therefore, the retained quantity on the root surface with the order of B[a]P > Pyr > Ant were related to the 19

adsorption capacity of GO for PAHs (B[a]P > Pyr > Ant) in this study. Compared with no GO treatment, higher retained quantities of PAHs on the K. obovata lateral root surface were observed, as shown in Figure 4(a) and 4(b). Similarly, a higher retained quantity of the PAHs was also observed for taproots in the presence of GO. Hu et al showed that the adhesion of GO on the plant cellular surfaces contributed to its surface nitrogen-containing chemicals [28, 42]. As Table 5 shows, the content of nitrogen (5.28 %) on the lateral root surface was higher than the taproot (2.71 %), suggesting that more PAHs might be retained on the lateral root surface micro-zone.

Recently, Hu et al showed that GO caused structural damage to the cell wall and affected membrane permeability of wheat root [28]. Hence, the effect of GO on the plant root surface physiology status (the permeability of plant root epidermis cell membranes) were specifically considered. Electrolytic leakage was used to monitor the permeability of K. obovata root cell membranes after exposure to GO. For lateral roots, the electrolytic leakage was improved from 6.7 % to 16.5 % for B[a]P, as shown in Figure S3. Compared to the control, significant differences (p < 0.05) for all PAHs were observed, indicating that the structure of the K. obovata lateral root and taproot epidermis cell membrane have changed after GO exposure, which may contribute to the uptake of PAHs. However, as Figure 6 shows, the microscopic images of the K. obovata root transverse section showed that there was less visually observable PAH entering both the lateral root and taproot.

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4. Conclusion

From the above results, in situ determination and visualization of retention on the lateral root and taproot surface micro-zone of K. obovata by MFSA was confirmed to be a simple and rapid method, with spatial resolution that was able to distinguish the microscopic distribution of PAHs on different root types of the same plant. The method had its own obvious advantages in that it could in situ observe the implications of GO on the retention of PAHs on the lateral root and taproot surface of K. obovata. Therefore, the established MFSA method has great potential ability to be developed as an in situ micro-zone determination and visualization of the PAHs retained on the surface of the plant (including mangrove) and distinguish their microscopic distribution on different surface types of the same plant.

More studies must be conducted in the near future. Indeed, the LOD (36.3 - 62.4 ng g-1) of the established method in this study was higher than the traditional chromatography approaches (GC, LC and HPLC) and the reported in situ methods [21-22, 43]. Due to the combinations of laser induced multi-dimension fluorescence spectra and LCSM, the deep UV multi-dimension laser confocal fluorescence spectral imagers that will be developed by our group have the potential ability to improve the sensitivity of the established method.

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Acknowledgements

The authors are grateful for the financial support from the Natural Science Foundation of China (Nos. 21627814, 21577110 and 21177102) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20130121130005).

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[32] H.L. Lu, B.B. Liu, Y. Zhang, J. Ye, C.L. Yan, Comparing analysis of elements sub-cellular distribution in Kandelia obovata between SEM-EDX and chemical extraction, Aquat. Bot. 112 (2014) 10-15. [33] X.L. Pan, J.Y. Yang, S.Y. Mu, D.Y. Zhang, Fluorescent properties and bifenthrin binding behavior of maize (Zea mays L.) seedling root exudates, Eur. J. Soil Biol., 50 (2012) 106-108. [34] X.Y. Liao, X. Ma, X.L. Yan, L.Y. Lin, P.L. Shi, Z.Y. Wu, Transportation and localization of phenanthrene and its interaction with different species of arsenic in Pteris vittata L., Chemosphere, 153 (2016) 307-314. [35] X.Y. Li, N. Li, H.D. Luo, L.R. Lin, Z.X. Zou, Y.Z. Jia, Y.Q. Li, A novel synchronous fluorescence spectroscopic approach for the rapid determination of three polycyclic aromatic hydrocarbons in tea with simple microwave-assisted pretreatment of sample, J. Agric. Food Chem., 59 (2011) 5899-5905. [36] Z.C. Wang, Z.F. Liu, Y. Yang, T. Li, M. Liu, Distribution of PAHs in tissues of wetland plants and the surrounding sediments in the Chongming wetland, Shanghai, China, Chemosphere, 89 (2012) 221-22. [37] X.H. Zhan, H.L. Ma, L.X. Zhou, J.R. Liang, T.H. Jiang, G.H. Xu, Accumulation of phenanthrene by roots of intact wheat (Triticum acstivnm L.) seedlings: passive or active uptake?, BMC Plant Boil., 10 (2010) 1-8. [38] X.H. Zhan, X. Yi, L. Yue, X.R. Fan, G.H. Xu, B.S. Xing, Cytoplasmic pH-Stat during phenanthrene uptake by wheat roots: a mechanistic consideration, Environ. Sci. Technol. 49 (2015) 6037-6044. [39] Z.X. Chen, H.G. Ni, X. Jing, W.J. Chang, J.L. Sun, H. Zeng, Plant uptake, translocation, and return of polycyclic aromatic hydrocarbons via fine root branch orders in a subtropical forest ecosystem, Chemosphere, 131 (2015) 192-200. [40] L. Chen, S.Z. Zhang, H.L. Huang, B. Wen, P. Christie, Partitioning of phenanthrene by root cell walls and cell wall fractions of wheat (Triticum aestivum L.), Environ. Sci. Technol., 43 (2009) 9136-9141. [41] F. Wang, J.J.H. Haftk, T.L. Sinnig, J.L.M. Hermens, W. Chen, Adsorption of polar, nonpolar, and substituted aromatics to colloidal graphene oxide nanoparticles, Environ. Pollut., 186 (2014) 226-233. [42] X.G. Hu, K.C. Lu, L. Mu, J. Kang, Q.X. Zhou, Interactions between graphene oxide and plant cells: regulation of cell morphology, uptake, organelle damage, oxidative effects and metabolic disorders, Carbon, 80 (2014) 665-676.

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[43] N. Ratola, S. Lacorte, A. Alves, D. Barceló, Analysis of polycyclic aromatic hydrocarbons in pine needles by gas chromatography–mass spectrometry: comparison of different extraction and clean-up procedures. J Chromatogr. A, 1114 (2006) 198-204.

Figure 1. Schematic diagram of MFSA system set in the lab.

Figure 2. Fluorescence spectra of the K. obovata lateral root surface micro-zone without and with B[a]P contamination (357 ng g-1): (a) conventional emission spectra; (b) the relationship of the relative fluorescence intensities and synchronous wavelength offset (Δλ); (c) synchronous fluorescence spectra.

Figure 3. The microscopic images of B[a]P adsorbed on the K. obovata lateral root surface micro-zone: (a) blank, (b) [B[a]P] = 357 ng g-1. The bright patches represented the B[a]P fluorescent signals.

Figure 4. Retained quantity of the individual PAH on the K. obovata lateral root and taproot surface: (a) without the exposure of GO; (b) with the exposure of 10 mg L-1 GO. The error bars represent the standard deviation of 15 micro-zones of the root surface.

Figure 5. Microscopic distribution images of B[a]P retained on the K. obovata lateral root and taproot surface in the absence/presence of GO: (a) lateral root without the exposure of GO; (b) lateral root with the exposure of 10 mg L-1 GO; (c) taproot root

26

without the exposure of GO; (d) taproot with the exposure of 10 mg L-1 GO. The white patches indicated by the yellow arrow represent the B[a]P fluorescent signal.

Figure 6. Microscopic distribution images of B[a]P retained on the K. obovata lateral root and taproot tissue (radial section) in the absence/presence of GO: (a) lateral root without the exposure of GO; (b) lateral root with the exposure of 10 mg L-1 GO; (c) taproot root without the exposure of GO; (d) taproot with the exposure of 10 mg L-1 GO. The white patches indicated by the yellow arrow represent the B[a]P fluorescent signal.

Table 1. The analytical merits of the established MFSA method (n=9).

Linear PAH

Correlatio Detection limit Quantificatio

Calibration range

n

a

n limits b

(ng g-1)

coefficient

(ng g-1)

(ng g-1)

260-2520

0.9853

44.2

132.6

Pyr y =41.5x + 30432 310-2870

0.9667

59.7

178.2

Ant y = 82.3x + 23680 170-2180

0.9678

36.3

100.8

Root s

equation

B[a]

y d =64.1x c +

P

31930

Lateral root

27

B[a] y = 69.5x + 27890 280-2580

0.9847

42.8

128.4

Pyr y =37.5x + 20391 330-2920

0.9789

62.4

189.1

Ant y = 88.1x + 22371 190-2210

0.9778

39.1

117.3

P Taproo t

a

Detection limits of the established method were calculated by 3Sb/m, where the “Sb”

represents the standard deviation of the blank (n=9) and the “m” represents the slope of the calibration curve. b

Quantification limits of the established method were calculated by 10Sb/m, where the

“Sb” represents the standard deviation of the blank (n=9) and the “m” represents the slope of the calibration curve. c

x represents the concentration of the B[a]P adsorbed on the root surface.

d

y represents the average relative fluorescence intensity of the B[a]P adsorbed on the

root surface micro-zone (n=9).

Table 2. The recoveries of PAHs adsorbed on the micro-zone of the K. obovata mangrove root surface (n=9). Root

PAHs

C1

Q1

-1

(g L )

(ng g ) −7

0.5 × 10 B[a]P

−7

1.0 × 10

−7

Lateral root

1.5 × 10

−6

Pyr

C2 -1

1.0 × 10

−6

2.0 × 10

268 569 671 387 493

28

Q2

-1

Recovery -1

(g L )

(ng g )

(%)

−7

463

72.7

−7

1098

92.9

−7

1468

118.8

−6

719

85.9

−6

1106

124.4

1.0 × 10 2.0 × 10

3.0 × 10 2.0 × 10 4.0 × 10

3.0 × 10−6 −6

0.5 × 10 Ant

−6

1.0 × 10

−6

2.0 × 10

−7

0.5 × 10 B[a]P

−7

1.0 × 10

−7

1.5 × 10

−6

1.0 × 10 Taproot

Pyr

−6

2.0 × 10

−6

3.0 × 10

−6

0.5 × 10 Ant

−6

1.0 × 10

−6

2.0 × 10

665

6.0 × 10−6

1513

127.5

368

−6

685

86.2

−6

1007

85.8

−6

1399

109.1

−7

708

78.7

−7

1035

91.0

−7

1437

112.5

−6

702

83.7

−6

1003

78.4

−6

1495

121.8

−6

624

67.3

−6

976

102.9

−6

1501

125.0

542 669 396 542 676 382 559 674 373 481 667

0.5 × 10 1.0 × 10 2.0 × 10 1.0 × 10 2.0 × 10 3.0 × 10 2.0 × 10 4.0 × 10 6.0 × 10 0.5 × 10 1.0 × 10 2.0 × 10

Table 3. Accuracy and precision of the established MFSA method.

Repeatability (n=9)

Spiking level Root type

PAHs (g L-1)

Lateral root

Taproot

RSD (%)

Reproducibility (n=5) RSD (%)

B[a]P

1.2 × 10−7

6.3

15.3

Pyr

2.5 × 10−6

8.1

6.6

Ant

1.2 × 10−7

9.6

14.0

B[a]P

1.2 × 10−7

11.0

13.1

Pyr

2.5 × 10−6

9.6

14.6

Ant

1.2 × 10−7

10.1

11.5

29

Table 4. Compared with experimental results for the in situ determination of PAHs on K. obovata root surface by the proposed method (MFSA) and LITRF method (n = 3). Relative Root type

PAHs

MFSA

LIRFT

(ng g -1)

(ng g -1)

deviation (%)

Lateral root

Taproot

B[a]P

350.7

377.5

7.1

Pyr

312.2

289.9

7.7

Ant

300.5

327.3

8.2

B[a]P

349.3

386.4

9.6

Pyr

404.1

381.9

5.8

Ant

389.3

416.4

6.5

Table 5. Surface elemental composition of the K. obovata lateral root and taproot. Root

C (%)

O (%)

N (%)

S (%)

O/C

(O+N)/C

Lateral root

60.72

33.37

5.28

0.63

0.55

0.64

Taproot

58.54

38.41

2.71

0.34

0.66

0.70

Highlights

·

An in situ method was developed for determining and visualizing PAHs on root surface. 30

·

The established method exhibited 10 μm spatial resolutions on mangrove root surface.

·

Retention of PAHs on the lateral root and taproot surface was investigated in situ.

·

The effects of GO on retention of the PAHs on the root surface were observed in situ.

Figure 1. Schematic diagram of MFSA system set in the lab.

31

(a)

(b)

(c)

Figure 2. Fluorescence spectra of the K. obovata lateral root surface micro-zone without and with B[a]P contamination (357 ng g-1): (a) conventional emission spectra; 32

(b) the relationship of the relative fluorescence intensities and synchronous wavelength offset (Δλ); (c) synchronous fluorescence spectra.

(a)

150 µm

(b)

150 µm Figure 3. The microscopic images of B[a]P adsorbed on the K. obovata lateral root surface micro-zone: (a) blank, (b) [B[a]P] = 357 ng g-1. The bright patches represented the B[a]P fluorescent signals.

33

(a)

(b)

Figure 4. Retained quantity of the individual PAH on the K. obovata lateral root and taproot surface: (a) without the exposure of GO; (b) with the exposure of 10 mg L-1 GO. The error bars represent the standard deviation of 15 micro-zones of the root surface.

34

(b)

(a)

150 µm

150 µm

(d)

(c)

150 µm

150 µm

Figure 5. Microscopic distribution images of B[a]P retained on the K. obovata lateral root and taproot surface in the absence/presence of GO: (a) lateral root without the exposure of GO; (b) lateral root with the exposure of 10 mg L-1 GO; (c) taproot root without the exposure of GO; (d) taproot with the exposure of 10 mg L-1 GO. The white patches indicated by the yellow arrow represent the B[a]P fluorescent signal.

(b)

(a)

35

cortex

epidermis

150 µm

150 µm

(c)

(d)

150 µm

150 µm

Figure 6. Microscopic distribution images of B[a]P retained on the K. obovata lateral root and taproot tissue (radial section) in the absence/presence of GO: (a) lateral root without the exposure of GO; (b) lateral root with the exposure of 10 mg L-1 GO; (c) taproot root without the exposure of GO; (d) taproot with the exposure of 10 mg L-1 GO. The white patches indicated by the yellow arrow represent the B[a]P fluorescent signal.

36

Fluorescence microscope

Optical fiber

Fluorescence spectrometry

MFSA system

In situ

Visualization Micro-zone

b Quantitation

Mangrove root

Cluster PAH

Graphene oxide

Root surface ce

Transport

Sorption