Abstract

This study aimed to assess the uptake of diclofenac, a widely used nonsteroidal anti-inflammatory pharmaceutical, by a macrophyte Cyperus alternifolius in a mesocosm-scale free water surface (FWS) constructed wetland. Quantitative analysis of diclofenac concentrations in water solution and plant tissues was conducted by high performance liquid chromatography analysis after sample pre-treatment with solid-phase extraction and liquid extraction, respectively. The FWS with Cyperus alternifolius obtained a maximum 69.3% diclofenac removal efficiency, while a control system without plant only had a removal efficiency of 2.7% at the end of the experiment period of 70 days. Based on mass balance study of the experimental system, it was estimated that plant uptake and in-plant conversion of diclofenac contributed about 21.4% of the total diclofenac removal in the mesocosm while the remaining 78.6% diclofenac was eliminated through biotic and abiotic conversion of diclofenac in the water phase. Diclofenac on the root surface and in roots, stems and leaves of Cyperus alternifolius was found at the concentrations of 0.15–2.59 μg/g, 0.21–2.66 μg/g, 0.06–0.53 μg/g, and 0.005–0.02 μg/g of fresh weight of plant tissues, respectively. The maximum bioaccumulation factor of diclofenac was calculated in roots (21.04) followed by root surface (20.49), stems (4.19), and leaves (0.16), respectively. Diclofenac translocation potentiality from root to stem was found below 0.5, suggesting a slow and passive translocation process of diclofenac. Current study demonstrated high potential of Cyperus alternifolius for phytoremediation of diclofenac in FWS and can be applied in other engineered ecosystems.

INTRODUCTION

The continued discharging of pharmaceuticals via excretion of humans, effluents from hospital, drug manufacture or even wastewater treatment plants (WWTPs) is an emerging global environmental concern for their widespread presence and potential toxicity in aquatic ecosystems (Zhang et al. 2012, 2013a). Most of the current WWTPs have shown insufficient capacity to eliminate pharmaceuticals (Luo et al. 2014). Diclofenac, a commonly used anti-inflammatory pharmaceutical, has been detected at concentrations of nanograms to micrograms per liter in WWTP effluent, rivers, lakes, underground water and even in drinking water (Zhang et al. 2013a; Luo et al. 2014). Oliveira et al. (2015) found aquatic diclofenac-driven toxicological effects in the cladoceran species Daphnia magna even at 0.5 μg/L, and Shultz et al. (2004) reported its liability for the declining different vulture species in the Indian subcontinent.

As an alternative to the conventional WWTPs, constructed wetlands (CWs) have been applied in treating various types of wastewater due to their advantages of low cost in both construction and operation, low maintenance, and generating aquatic plant products such as fertilizer or animal feed (Ávila et al. 2014). CWs were also studied as an alternative technology to remove pharmaceuticals with relatively better performance than WWTPs (Zhang et al. 2012; Verlicchi & Zambello 2014). Removal of diclofenac by using CWs has been investigated (Ávila et al. 2013) and previous results obtained moderate to high elimination performance (Hijosa-Valsero et al. 2010a; Ávila et al. 2015). Based on various designs, diclofenac removal ranged from 54 to 71% in vertical flow (Ávila et al. 2014) while horizontal subsurface flow (HSSF) showed 32–70% in pilot-scale CWs (Ávila et al. 2013). However, efficiency was found comparatively higher in full-scale systems. For instance, in full-scale hybrid CWs, Ávila et al. (2015) recently reported 89% of diclofenac removal, whereas earlier, Hijosa-Valsero et al. (2010a) estimated about 65–87% removal from municipal wastewater.

Aquatic plants in wetlands possess complex physiological and biochemical reactions to treat different kinds of xenobiotics (Huber et al. 2012; Zhang et al. 2014). Macrophytes can supply organics and oxygen through the rhizosphere and thus enhance microbial degradation (Carvalho et al. 2014). Previous studies demonstrated caffeine, clofibric acid, ibuprofen, naproxen, ketoprofen, etc. uptake by Scirpus validus (Zhang et al. 2013a, 2013b), Phragmites australis (Hijosa-Valsero et al. 2010b; Ávila et al. 2014, 2015), Typha angustifolia (Hijosa-Valsero et al. 2010b), and Typha latifolia (Hijosa-Valsero et al. 2010a; Bartha et al. 2014), suggesting that these plants can play a significant role in the removal of pharmaceutical and personal care products (PPCPs). However, information on studied macrophytes and PPCPs is still limited. In the case of diclofenac, which possesses low biodegradability, plant uptake might be a potential strategy in CWs. Zhang et al. (2011) investigated pharmaceutical removal in a laboratory-scale HSSF receiving synthetic wastewater, and found better diclofenac removal efficiency in the system planted with Typha angustifolia (55%) compared with the unplanted HSSF at 4 days' hydraulic retention time (HRT) and 26 °C. Similarly, in a study of PPCP removal from urban wastewater by using various types of mesocosm-scale CWs, Hijosa-Valsero et al. (2010b) observed that diclofenac removal was higher in the free water surface (FWS) CWs planted with Phragmites australis (57%), whereas unplanted gravel bed CWs in the open air showed less than 40% diclofenac removal during the summer season.

Results from previous research clearly indicated that plant existence favored diclofenac removal in CWs. But still few studies were carried out on diclofenac uptake within the aquatic macrophyte. Zhang et al. (2012) studied diclofenac uptake by Scirpus validus in hydroponic solutions for 21 days, where root accumulation ranged from 0.17 to 1.49 μg/g (fresh weight) and the concentration of diclofenac in shoot was found to be 0.13–0.49 μg/g (fresh weight) at the end of the experiment. In addition, rapid plant uptake and metabolism of diclofenac within Typha latifolia was reported (Bartha et al. 2014), suggesting better phytoremediation capacity. However, photodegradation was the dominant diclofenac removal pathway for all of the above mentioned studies. Thus, the potential plant uptake capacity and distribution in macrophyte still need further study.

Therefore, the current study investigated the role of Cyperus alternifolius, a commonly used macrophyte in CWs in South China, for diclofenac removal. The primary objectives of this study were to quantify uptake, translocation and conversion of diclofenac in Cyperus alternifolius in a FWS mesocosm.

MATERIALS AND METHODS

Chemicals

Diclofenac (C14H11Cl2NO2, CAS No. 15307-86-5) was purchased from Tokyo Kasei Kogyo Co., Ltd (Shanghai, China) with purity >98%. The stock diclofenac solution of 1,500 mg/L was prepared by dissolving 75 mg of diclofenac in 50 mL high performance liquid chromatography (HPLC)-grade methanol. Other chemicals used during investigation were analytical grade. Millipore water (18.2 MΩ resistivity) was used for preparing all aqueous solutions. All collected samples were analyzed in triplicate.

Experiment set-up

The experiment was performed in two FWS mesocosms, where one was the experimental system with 60 evenly distributed plants and the other was a control system without any plants, established in a rooftop greenhouse at a laboratory of the Chongqing University, China. Air temperature in the greenhouse varied between 30 and 35 °C during the experimental period from August to October 2014. Two square, flat-bottomed FWS mesocosms (120 cm width × 120 cm length × 20 cm height) were installed using low-density polyethylene material (Figure 1). Both mesocosms were covered with 2 mm thick plastic sheet to avoid photo-degradation and restrain evaporation. Shallow set-up (water level 16 cm) was installed to maintain proper distribution of plant roots and to understand the optimum impact of roots for diclofenac uptake. The hydroponic culture was prepared by adding 19 mL 1.5 g/L methanol dissolved diclofenac into 230 L nutrient solution. The nutrient solution was prepared with the following composition: 4.2 mg/L NH4+-N, 0.2 mg/L PO43–-P, 6.0 mg/L K+, and the pH value was adjusted to 7.1. The concentration of dissolved diclofenac was 0.13 mg/L. To minimize detection error and simulate conditions near but below visually evident toxicity (Zhang et al. 2012), a relatively higher concentration of diclofenac than that in a natural water body was selected. This concentration is much lower than its water solubility (2.4 mg/L) and diclofenac will not crystallize from the solution. Feeding of nutrient solution was conducted once at the beginning of the experiment. To keep a stable water level during the experiment, the water level was checked (Figure 1, point 2) before the day of each sampling date and the water level was adjusted by adding pure water. There was no recirculation of nutrient solution throughout the investigation to promote aeration in the studied mesocosms.

Figure 1

Schematic diagram of two laboratory-scale experimental FWSs. Both experimental (left side) and control (right side) systems have four points for water sampling (points 1, 3, 4) and water level monitoring (point 2).

Figure 1

Schematic diagram of two laboratory-scale experimental FWSs. Both experimental (left side) and control (right side) systems have four points for water sampling (points 1, 3, 4) and water level monitoring (point 2).

Cyperus alternifolius, an ornamental emergent perennial plant, was selected as the study macrophyte in the experiment. Cyperus alternifolius with the average height of 85 cm was collected from Huahuiyuan CW WWTP, Chongqing. The rhizomes were thoroughly washed to removal any sediment particles, and then submersed in tap water for two weeks before the experiment. Before installation of plants in the experimental FWS, each plant's roots were banded loosely using thread and inserted into the hole. A steel net was used under the plastic sheet of the experimental system to secure the plants to the bottom of the mesocosm. The distance between plants of the experimental system was kept at 15 cm to minimize the disturbance during plant sampling.

Water samples for temperature, dissolved oxygen (DO), turbidity, and pH were analysed onsite through points 1, 3, and 4 (Figure 1) with portable sensors as described by Zhai et al. (2012). Total organic carbon (TOC) was analysed by combustion oxidation – non-dispersive infrared absorption method using Elementar Liqui TOC II. The water samples for diclofenac concentration were drawn through points 1, 3, and 4 and analysed by HPLC after solid phase extraction (SPE) pretreatment. Plant samples were also collected simultaneously with water samples. During each sampling period, three plants were selected randomly for sample pretreatment and analysis. The sampling campaign was conducted on days 0, 8, 14, 28, 35, 42, 51 and 70 after the FWS was set up.

Extraction of diclofenac from plant samples

In the laboratory, Cyperus alternifolius samples were dried by nitrogen gas flush at room temperature. First, methanol was used to extract diclofenac from the root surface. This extraction operation was repeated three times. Each time the roots were submerged in 150 mL methanol for 15 min. All extracts were mixed and evaporated to ca. 50 μL under nitrogen gas. Then the vial was reconstituted by using 5 mL of a mixture of water (with 3% ice acetic acid) and acetonitrile (80/20, v/v), the same composition as the HPLC mobile phase, and filtered through a 0.22 μm membrane. The diclofenac concentration in the final solution was tested using HPLC. Results derived from HPLC were used to calculate the concentration of diclofenac (μg/g) adsorbed to the root surface (crs).

Second, diclofenac in plant tissues was extracted and determined. The plant was separated and the mass of each part weighed separately: roots (mr), stems (ms) and leaves (ml). Afterwards, extraction of diclofenac from roots, stems, and leaf tissues was conducted separately in parallel using the same extraction method used for root surface. About 8 g of plant tissues was placed in an agate mortar and ground using a glass pestle. The blend was then transferred into a conical flask. Methanol (150 mL) was injected into the flask as extractor. The extracted solution was shaken for 120 min, and kept still for 24 h. Later, the solution was centrifuged at 5,600 rpm for 10 minutes. All supernatant was filtered and collected using a 0.22 μm membrane. All filtrate was then evaporated to ca. 50 μL under nitrogen gas, the vial was reconstituted using 5 mL of HPLC mobile phase, and HPLC analysis was run to detect diclofenac concentration. The values originating from HPLC results were used to calculate concentration of diclofenac (μg/g) in roots (cr), stems (cs) and leaves (cl).

SPE for water samples

The raw water samples (500 mL) were filtered through a 0.45 μm membrane filter. To each sample (500 mL of the filtrate), 4 g EDTA was added. The SPE cartridges (Waters Sep-Pak Vac C18, 200 mg, 6 mL) were conditioned with 10 mL of both methanol and Milli-Q water at a flow-rate of approximately 3 mL/min. Samples were percolated to the SPE cartridges at a flow-rate of approximately 5 mL/min. The cartridge filter was then rinsed with methanol/Milli-Q water (10/90, v/v). Thereafter, the cartridges were allowed to dry under a continuous flow of nitrogen gas for 30 min and then eluted with 5 mL methanol. The elution was collected in 15 mL calibrated centrifuge tubes. The extract was then evaporated to ca. 50 μL under a gentle nitrogen stream and was then reconstituted to 2 mL with HPLC mobile phase, and then analysed by HPLC. The HPLC results were used to calculate diclofenac concentration in the water solution (C).

Chromatographic analysis

Chromatographic analysis was applied to quantify diclofenac concentrations in the extracted samples. The flow rate of mobile phase was 0.8 mL/min and the injection volume was 50 μL. Retention time for diclofenac was 3.5 min. Chromatographic analysis was performed on an Agilent 1260 HPLC system (Agilent, USA) with an Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm, Agilent) HPLC column. The recovery rates varied from 92.19 to 96.19% for water samples and 90.79 to 94.30% for macrophyte samples.

Data analysis

According to Zhang et al. (2011), a first order kinetics model was applied for analysis of the volumetric decay rate (kv) of pharmaceuticals in hydroponic mesocosm. Diclofenac adsorbed by plant tissues was mobile and could get desorbed to the water phase (Riederer 1990). Moreover, the continuously released root exudates, which are relatively more biodegradable, would accumulate in water and compete with diclofenac for microbial degradation. Therefore, when the concentration of diclofenac in water (C) decreased to a certain degree, an equilibrium concentration (Ce) would be reached.  
formula
(1)

Moreover, bioaccumulation factors (BAFs) and translocation factors (TFs) of diclofenac were estimated based on the previous study (Zhang et al. 2012, 2013c).

BAFs were assessed as the ratio of diclofenac concentrations in the plant tissues and the initial nutrient solutions.  
formula
(2)
Diclofenac translocation potentiality from root to stem was calculated by using TFs.  
formula
(3)
The percentage of diclofenac retained in plant was calculated as the ratio of assimilated diclofenac (in mg) in different parts of the plant to the initial diclofenac in the water solution (mg). The retained percentage of diclofenac (Rrs, Rr, Rs, Rl) in plants can be calculated as in Equations (4)–(7).  
formula
(4)
 
formula
(5)
 
formula
(6)
 
formula
(7)
where n means the number of sampling events, n = 1,2,…8.
To study the mass balance of diclofenac in the experimental system, a cascade reaction model was applied to analyse the kinetics of plant uptake and conversions of diclofenac in plant. In this cascade model, both the plant uptake process and the in-plant conversion process were treated as first order reactions. The governing equation for the cascade reaction model is:  
formula
(8)
where C is the concentration of diclofenac in water phase, CB is the concentration of diclofenac in plant body, and k1 and k2 are the reaction rate constants of the two first order reactions. The concentration of diclofenac in water phase can be calculated in the first order kinetics according to Equation (1). Substituting Equation (1) into Equation (8), one obtains:  
formula
(9)
After integration of Equation (10), the concentration of diclofenac in plant body can be calculated as below:  
formula
(10)
Considering the different units of CB (μg/g) and C0 (μg/L), the total mass of diclofenac in different phases was utilized in the calculation. The total mass of diclofenac in and on plant body can be calculated by Equation (11).  
formula
(11)

Here, V is the total volume of nutrient solution (230 L), C0 is the initial concentration of diclofenac (126.4 μg/L), Ce is the equilibrium concentration of diclofenac and kv is the observed decay rate of diclofenac in water. Equation (11) is the cascade reaction model to describe the uptake and in-plant conversion processes.

One-way analysis of variance (ANOVA) with Fisher's least significant difference post hoc (ɑ-0.05) tests were used to determine the significant differences between diclofenac concentrations in different mesocosms and diclofenac concentrations in different parts of the plant. The calculations were carried out with Origin 8.5 software.

RESULTS AND DISCUSSION

Diclofenac elimination from nutrient solutions

Diclofenac was eliminated throughout the experimental period in the FWS planted with Cyperus alternifolius and the removal efficiency reached 69.3 ± 0.2% after 70 days (Figure 2). In contrast, the diclofenac removal efficiency in the control system without plants was only 2.7 ± 1.3%. Based on Equation (1), the volumetric decay rate constant, kv, was 0.0279 d−1 and the equilibrium concentration Ce was 0.0187 mg/L in the present experiment. During investigation, more than one third of the initial plants were removed for sampling purposes, which might have an impact on the overall performance of the experimental system. The removal efficiency will be higher than the existing results if we consider no plants removed during the experimental period.

Figure 2

Removal of diclofenac from nutrient solution in both experimental and control FWS over time. Error bar indicates ± SD (sampling point, n = 3).

Figure 2

Removal of diclofenac from nutrient solution in both experimental and control FWS over time. Error bar indicates ± SD (sampling point, n = 3).

Diclofenac removal rate in present study was relatively lower than in the previous investigation by Zhang et al. (2012), where more than 98% removal efficiency was found in hydroponic solutions with initial diclofenac concentration ranging from 0.5 to 2 mg/L and planted with Scirpus validus for 21 days. The high elimination rate obtained in the previous study was due to the photodegradation that served as a major contribution (above 80%) to diclofenac removal in surface water (Zhang et al. 2012, 2013d). In our study we covered both experimental (with plant) and control (without plant) mesocosms to reduce photodegradation of diclofenac in water. Negligible diclofenac removal (2.7%) was observed in the control mesocosms. Therefore, the contribution of photodegradation in the present studied FWS was insignificant. Zhang et al. (2013d) reported 12–21% depletion of diclofenac in a 4 L vessel covered and planted with four Scirpus validus for 21 days, while at the same time in our experiment we observed around 37% removal.

Diclofenac retention in Cyperus alternifolius

Diclofenac was first detected on root surface at day 8, followed by in roots at day 14 and in stems and leaves at day 21 (Table 1). The concentrations of diclofenac in each part of the plant increased to a peak value: 2.59 ± 0.27 μg/g on root surface at the 14th day, 2.66 ± 0.78 μg/g in roots and 0.53 ± 0.03 μg/g in stems at the 28th day and 0.08 ± 0.02 μg/g in leaves at the 42nd day, then the concentrations in different parts of the plant all decreased to almost zero. Based on the results from one-way ANOVA analysis, the concentrations of diclofenac in root tissues were significantly higher (P < 0.05) than those in stems and leaves. The phenomenon that the concentrations of diclofenac in plant tissues all decreased to almost 0 μg/g demonstrated that diclofenac could be metabolized in Cyperus alternifolius. Rapid metabolic biodegradation of diclofenac in Typha latifolia has been reported by Bartha et al. (2014).

Table 1

The masses concentration (Conc.) and percentages of accumulated diclofenac retained (RE) and BAF values in different parts of Cyperus alternifolius; TF of studied plant along with study duration

Time (d)0814212835425170
Root surface Conc. (μg/g) 1.51 ± 0.27 2.59 ± 0.53 2.04 ± 0.03 0.43 ± 0.04 0.22 ± 0.07 0.15 ± 0.0 
RE (%) 1.31 1.81 2.52 0.56 0.35 0.27 
BAF 11.95 20.49 16.14 3.4 1.74 1.19 
Roots Conc. (μg/g) 1.22 ± 0.01 1.62 ± 0.53 2.66 ± 0.78 1.35 ± 0.62 0.73 ± 0.47 0.47 ± 0.12 0.21 ± 0.02 
RE (%) 0.85 3.45 2.16 1.31 0.92 0.43 
BAF 9.65 12.82 21.04 10.68 5.78 3.72 1.66 
Stems Conc. (μg/g) 0.06 ± 0.01 0.53 0.03 0.42 ± 0.05 0.31 ± 0.08 0.11 ± 0.0 
RE (%) 0.23 2.06 2.23 1.85 0.72 
BAF 0.47 4.19 3.32 2.45 0.87 
Leaves Conc. (μg/g) 0.01 ± 0.01 0.03 ± 0.01 0.05 ± 0.02 0.08 ± 0.02 
RE (%) 0.01 0.01 0.02 
BAF 0.04 0.08 0.12 0.16 
TF 0.04 0.2 0.31 0.43 0.23 
Time (d)0814212835425170
Root surface Conc. (μg/g) 1.51 ± 0.27 2.59 ± 0.53 2.04 ± 0.03 0.43 ± 0.04 0.22 ± 0.07 0.15 ± 0.0 
RE (%) 1.31 1.81 2.52 0.56 0.35 0.27 
BAF 11.95 20.49 16.14 3.4 1.74 1.19 
Roots Conc. (μg/g) 1.22 ± 0.01 1.62 ± 0.53 2.66 ± 0.78 1.35 ± 0.62 0.73 ± 0.47 0.47 ± 0.12 0.21 ± 0.02 
RE (%) 0.85 3.45 2.16 1.31 0.92 0.43 
BAF 9.65 12.82 21.04 10.68 5.78 3.72 1.66 
Stems Conc. (μg/g) 0.06 ± 0.01 0.53 0.03 0.42 ± 0.05 0.31 ± 0.08 0.11 ± 0.0 
RE (%) 0.23 2.06 2.23 1.85 0.72 
BAF 0.47 4.19 3.32 2.45 0.87 
Leaves Conc. (μg/g) 0.01 ± 0.01 0.03 ± 0.01 0.05 ± 0.02 0.08 ± 0.02 
RE (%) 0.01 0.01 0.02 
BAF 0.04 0.08 0.12 0.16 
TF 0.04 0.2 0.31 0.43 0.23 

The percentages of diclofenac retained in plant tissues, and the BAF and TF values, were calculated according to Equations (2)–(7) and illustrated in Table 1. Percentage diclofenac retained in different parts of the plant increased to a peak value (2.52% on root surface at day 21, 3.45% in roots at day 28, 2.23% in stems at day 35 and 0.02% in leaves at day 42) and then decreased to almost 0 at day 70. Peak BAFs in different parts of the plant were observed in roots (21.04) at day 28 followed by the root surface (20.49) at day 14 and stems (4.19) at day 28, respectively. Current BAF values of diclofenac in roots and stems of Cyperus alternifolius were higher than these values in Scirpus validus reported by Zhang et al. (2012), with values ranging from 0.40 to 1.36 for roots and 0.17 to 0.51 for shoots by 21 days, suggesting Cyperus alternifolius has better diclofenac uptake potentiality. Moreover, TFs increased to reach the peak value of 0.43 at day 42 and then decreased to 0 at day 51. The low translocation rate of diclofenac in the studied plant tissues was due to its lipophilicity, which results in partition to root epidermis. Afterwards, diclofenac molecules are not drawn into inner root or xylem, resulting in limited translocation potential from root to stem (Zhang et al. 2012).

Mass balance of diclofenac in the experimental FWS

Mass balance was analysed based on the kinetic study of the experimental FWS. As outlined in Figure 3, there are two possible removal pathways for diclofenac in nutrient solution: one, biotic and abiotic conversion in water solution and the other, uptake and conversion in plants.

Figure 3

Model reaction schematic for the uptake and degradation.

Figure 3

Model reaction schematic for the uptake and degradation.

According to Equation (11), the mass balance (MB) values of diclofenac in the experimental system were fitted to the self-developed nonlinear expression using nonlinear curve fit (Figure 4). The uptake rate constant (k1 = 0.0057 d−1) and in-plant conversion rate constant (k2 = 0.0611 d−1) were obtained with a correlation coefficient (r = 0.783, P = 0.017 < 0.05). It is difficult to get stable results when detecting low concentrations of micropollutants extracted from plant tissues. For this reason, relatively high variations in diclofenac values were observed at days 21 to 35 and at the end of the experiment. However, based on the experimental data, current model results are still acceptable and can roughly describe diclofenac retained in plant. Moreover, the present model results will be helpful for readers to understand the dynamics of diclofenac uptake by plants.

Figure 4

Nonlinear fitting of diclofenac in plant tissues to cascade reaction model.

Figure 4

Nonlinear fitting of diclofenac in plant tissues to cascade reaction model.

Based on the fitted parameters, the total masses percentage of diclofenac uptake in/on plant body, in-plant conversion, and biotic and abiotic conversion were estimated (Figure 5). Mass balance study revealed that total diclofenac uptake and conversion reached 14.9% at day 70, in which in-plant diclofenac conversion gradually increased with the investigated time and found from 0.7% at day 8 to 13.1% of diclofenac conversion at the end of the experiment (Figure 5).

Figure 5

Mass balance of diclofenac concentration percentage (%) in the experimental FWS along with the studied period.

Figure 5

Mass balance of diclofenac concentration percentage (%) in the experimental FWS along with the studied period.

Based on kinetic study of the plant uptake and conversion, the calculated biotic and abiotic degradation of diclofenac in water solution was subsequently increased to 54.8% at the end of the experiment. The gap between k1 and kv refers to the km, which is the reaction rate constant of biotic and abiotic diclofenac conversion, and the km value was 0.0222 d−1. Mass balance analysis of the experimental FWS showed that the percentage of diclofenac retained in/on plant increased from 0 at day 1 to 4.11% at day 21 and then decreased to 1.88% at day 70, which indicates that diclofenac uptake by plant was converted along with the study period. Considering diclofenac retained in plant, in-plant conversion, and biotic and abiotic conversion, the total calculated diclofenac removal (69.7%) was similar to the experimental removal efficiency (69.3%) at the end of the experiment. It is evident that, from the total calculated diclofenac removal, 21.4% was removed by plant uptake and in-plant conversion, while the remaining 78.6% was attributed to biotic and abiotic conversion in water solution at day 70.

In the experimental mesocosm, the coexistence of organic carbon, diclofenac and its metabolites can enhance microbial activity, which subsequently improves the removal of refractory contents from the system. TOC was measured periodically and found to be on average 46.8 and 19.0 mg/L in the experimental and control systems, respectively. DO values were almost the same throughout the experiment in the control system and ranged from 6.3 to 6.5 mg/L, while for the experimental system, DO improved with time and finally increased from 5.4 to 8.0 mg/L at day 70. The exudates released by plant roots can serve as an organic carbon source and then can enhance microbial activity in the rhizospheric zone (Lange et al. 2015) with increased DO level (Hijosa-Valsero et al. 2010b). The current study indicated that the presence of plants indirectly influenced the removal of diclofenac in water phase by enhancing microbial activity in the experimental system, while in the control system with low TOC and stable DO, microbial activity was limited. The mass balance analysis confirmed biotic and abiotic degradation of diclofenac in the hydroponic solution of the experimental mesocosm.

CONCLUSION

This is the first report that estimates the potential diclofenac uptake and in-plant conversion capacity of Cyperus alternifolius in a FWS. In this study, we constructed a mesocosm-scale FWS with Cyperus alternifolius and demonstrated the ability of the studied wetland macrophyte to uptake and convert diclofenac. The removal of diclofenac in water solution followed first-order kinetics, and a removal efficiency of 69.3 ± 0.2% was achieved at the end of experiment. The plants significantly increased the removal efficiency of diclofenac. Kinetic study showed that plant uptake and in-plant conversion of diclofenac contributed 21.4% of the total diclofenac removal, while biotic and abiotic conversion of diclofenac in water phase contributed 78.6%. Diclofenac was first detected on the root surface, and then in the roots, stems and leaves of Cyperus alternifolius, which indicates that the plant could take up the diclofenac from hydroponic solution and subsequently translocate it to the stems and leaves during the whole assay period of 70 days. The diclofenac taken up can be converted in the plant body. The current study demonstrated high potential of Cyperus alternifolius for phytoremediation of diclofenac in FWS and can be applied as engineered ecosystems to address diclofenac removal in aquatic ecosystems. Further research is still necessary to check the efficiency of Cyperus alternifolius in full-scale systems for uptake and metabolic pathway of diclofenac as well as other common pharmaceuticals in municipal wastewater.

ACKNOWLEDGEMENTS

Funding for this study was provided by the National Natural Science Foundation of China under grant no. 51208533 and 51478062, and by the Fundamental Research Funds for the Central Universities under grant no. CDJZR13215501.

REFERENCES

REFERENCES
Ávila
C.
,
Nivala
J.
,
Olsson
L.
,
Kassa
K.
,
Headley
T.
,
Mueller
R. A.
,
Bayona
J. M.
&
García
J.
2014
Emerging organic contaminants in vertical subsurface flow constructed wetlands: influence of media size, loading frequency and use of active aeration
.
Science of the Total Environment
494–495
,
211
217
.
Ávila
C.
,
Bayona
J. M.
,
Martín
I.
,
Salas
J. J.
&
García
J.
2015
Emerging organic contaminant removal in a full-scale hybrid constructed wetland system for wastewater treatment and reuse
.
Ecological Engineering
80
,
108
116
.
Carvalho
P. N.
,
Basto
M. C.
,
Almeida
C. M.
&
Brix
H.
2014
A review of plant-pharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands
.
Environmental Science and Pollution Research International
21
(
20
),
11729
11763
.
Hijosa-Valsero
M.
,
Matamoros
V.
,
Martin-Villacorta
J.
,
Becares
E.
&
Bayona
J. M.
2010a
Assessment of full-scale natural systems for the removal of PPCPs from wastewater in small communities
.
Water Research
44
(
5
),
1429
1439
.
Hijosa-Valsero
M.
,
Matamoros
V.
,
Sidrach-Cardona
R.
,
Martin-Villacorta
J.
,
Becares
E.
&
Bayona
J. M.
2010b
Comprehensive assessment of the design configuration of constructed wetlands for the removal of pharmaceuticals and personal care products from urban wastewaters
.
Water Research
44
(
12
),
3669
3678
.
Huber
C.
,
Bartha
B.
&
Schröder
P.
2012
Metabolism of diclofenac in plants – hydroxylation is followed by glucose conjugation
.
Journal of Hazardous Materials
243
,
250
256
.
Lange
M.
,
Eisenhauer
N.
,
Sierra
C. A.
,
Bessler
H.
,
Engels
C.
,
Griffiths
R. I.
,
Mellado-Vazquez
P. G.
,
Malik
A. A.
,
Roy
J.
,
Scheu
S.
,
Steinbeiss
S.
,
Thomson
B. C.
,
Trumbore
S. E.
&
Gleixner
G.
2015
Plant diversity increases soil microbial activity and soil carbon storage
.
Nature Communications
6
,
6707
,
doi:10.1038/ncomms7707
.
Luo
Y.
,
Guo
W.
,
Ngo
H. H.
,
Nghiem
L. D.
,
Hai
F. I.
,
Zhang
J.
,
Liang
S.
&
Wang
X. C.
2014
A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment
.
Science of the Total Environment
473–474
,
619
641
.
Oliveira
L. L.
,
Antunes
S. C.
,
Goncalves
F.
,
Rocha
O.
&
Nunes
B.
2015
Evaluation of ecotoxicological effects of drugs on Daphnia magna using different enzymatic biomarkers
.
Ecotoxicology and Environmental Safety
119
,
123
131
.
Shultz
S.
,
Baral
H. S.
,
Charman
S.
,
Cunningham
A. A.
,
Das
D.
,
Ghalsasi
G. R.
,
Goudar
M. S.
,
Green
R. E.
,
Jones
A.
,
Nighot
P.
,
Pain
D. J.
&
Prakash
V.
2004
Diclofenac poisoning is widespread in declining vulture populations across the Indian subcontinent
.
Proceedings. Biological Sciences/The Royal Society
271
(
Suppl. 6
),
S458
S460
.
Zhang
D. Q.
,
Tan
S. K.
,
Gersberg
R. M.
,
Sadreddini
S.
,
Zhu
J.
&
Tuan
N. A.
2011
Removal of pharmaceutical compounds in tropical constructed wetlands
.
Ecological Engineering
37
(
3
),
460
464
.
Zhang
D. Q.
,
Hua
T.
,
Gersberg
R. M.
,
Zhu
J.
,
Ng
W. J.
&
Tan
S. K.
2012
Fate of diclofenac in wetland mesocosms planted with Scirpus validus
.
Ecological Engineering
49
,
59
64
.
Zhang
D. Q.
,
Hua
T.
,
Gersberg
R. M.
,
Zhu
J.
,
Ng
W. J.
&
Tan
S. K.
2013a
Fate of caffeine in mesocosms wetland planted with Scirpus validus
.
Chemosphere
90
(
4
),
1568
1572
.
Zhang
D. Q.
,
Hua
T.
,
Gersberg
R. M.
,
Zhu
J.
,
Ng
W. J.
&
Tan
S. K.
2013b
Carbamazepine and naproxen: fate in wetland mesocosms planted with Scirpus validus
.
Chemosphere
91
(
1
),
14
21
.
Zhang
D. Q.
,
Gersberg
R. M.
,
Hua
T.
,
Zhu
J.
,
Ng
W. J.
&
Tan
S. K.
2013c
Assessment of plant-driven uptake and translocation of clofibric acid by Scirpus validus
.
Environmental Science and Pollution Research International
20
(
7
),
4612
4620
.
Zhang
D. Q.
,
Gersberg
R. M.
,
Hua
T.
,
Zhu
J.
,
Goyal
M. K.
,
Ng
W. J.
&
Tan
S. K.
2013d
Fate of pharmaceutical compounds in hydroponic mesocosms planted with Scirpus validus
.
Environmental Pollution
181
,
98
106
.
Zhang
D.
,
Gersberg
R. M.
,
Ng
W. J.
&
Tan
S. K.
2014
Removal of pharmaceuticals and personal care products in aquatic plant-based systems: a review
.
Environmental Pollution
184
,
620
639
.