ZnS quantum dot nanoparticles by Schoenoplectus tabernaemontani in hydroponic mesocosms

Ecological Engineering 70 (2014) 114–123

Contents lists available at ScienceDirect

Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng

Uptake and accumulation of CuO nanoparticles and CdS/ZnS quantum dot nanoparticles by Schoenoplectus tabernaemontani in hydroponic mesocosms Dongqing Zhang a,∗ , Tao Hua a , Fei Xiao b , Chunping Chen b , Richard M. Gersberg c , Yu Liu a , Wun Jern Ng a , Soon Keat Tan a a Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, School of Civil and Environmental Engineering, Nanyang Technological University, 1 CleanTech Loop, #06-10, Singapore 637141, Singapore b School of Chemical and Biological Engineering, N1.2-B1-03, Nanyang Technological University, Singapore, Singapore c Graduate School of Public Health, San Diego State University, Hardy Tower 119, 5500 Campanile, San Diego, CA 92182-4162, USA

a r t i c l e

i n f o

Article history: Received 8 December 2013 Received in revised form 1 April 2014 Accepted 19 April 2014 Keywords: Nanoparticles CuO NPs CdS/ZnS QDs Uptake Schoenoplectus tabernaemontani

a b s t r a c t Rapid growth in production and use of nanoparticles (NPs) will inevitably result in the introduction of NPs deliberately or unintentionally into industrial and urban wastewater streams. Efficient removal of NPs from wastewater is particularly important in view of increasing concerns over potential ecotoxicity and hazards of NP exposure to humans and the environment over the long term. For the first time, we report the fate of copper oxide (CuO) NPs and cadmium sulphide/zinc sulphide (CdS/ZnS) quantum dots (QDs) in aquatic mesocosms planted with Schoenoplectus tabernaemontani. S. tabernaemontani was more sensitive to toxic effects of CdS QDs than CuO NPs and the reduction in plant biomass was significant (p < 0.05) in the presence of CdS QDs at the levels of either 5 or 50 mg L−1 after exposure period of 21 d. Levels of Cu2+ under CuO NP treatment in the root ranged from 14 ␮g g−1 (0.5 mg L−1 treatment) to 4,057 ␮g g−1 (50 mg L−1 treatment), while the values for Cd2+ under CdS QDs treatment were 9 to 1518 ␮g g−1 , respectively. Root uptake percentage (of the total initial NP mass) for CuO NP treatment ranged from 40.6 to 68.4%, while the values were 8.7 to 21.3% for CdS QDs. The low value (<0.2) of the translocation factors for both CuO NPs and CdS QDs implied the limited translocation potential from the roots to the shoots. Transmission electron microscopy confirmed that the CuO NPs and CdS/ZnS QDs could penetrate the cell walls of S. tabernaemontani roots. To our knowledge, this is the first evidence of NP uptake and accumulation by the higher aquatic plant S. tabernaemontani. These findings provide impetus for future investigation on NP behaviour and removal, as well as the complex interaction between NPs and plants in aquatic plant-based treatment systems. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nanotechnolgy, the designing and manufacturing of nanoscale (<100 nm) materials, opens up a tremendous field of new applications in consumer products, chemical and medical equipment, information technology and energy (Domingos et al., 2011; Yuan et al., 2013). As one of the most important engineered types, copper oxide (CuO) NPs exhibit unique optical, electrical and catalytic properties, and are used intensively in electronics, ceramics, films, polymers inks, and coatings (Yang et al., 2006; Shi et al., 2011).

∗ Corresponding author. Tel.: +65 6790 6619/+65 8165 6212; fax: +65 6790 6620. E-mail addresses: [email protected] (D. Zhang), [email protected] (S.K. Tan). http://dx.doi.org/10.1016/j.ecoleng.2014.04.018 0925-8574/© 2014 Elsevier B.V. All rights reserved.

Quantum dots (QDs) are inorganic semiconductor nanocrystals which range from 2 to 10 nm in diameter and consist of a cadmium core (i.e., CdSe, CdS) with or without an outer shell of zinc that is passivated by an additional layer or organic ligands (Domingos et al., 2011; Navarro et al., 2012). Because of their unique sizedependent optical and photophysical properties, QDs are being extensively developed in the field of solid-state LED lighting, solar energy conversion, as well as for biological applications (Anikeeva et al., 2009; Luque et al., 2007). Despite the bright future of nanotechnology, the rapidly increasing number of consumer products containing engineered nanoparticles (NPs) has resulted in exponentially increasing quantities of NPs being discharged into the environment (Stampoulis et al., 2009; Klaine et al., 2008). In particular, NPs exhibit physical

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

and chemical properties significantly different from those of their macrosize analogs; therefore concern has been growing regarding to their fate and ecotoxicity in environmental compartments (Ma et al., 2010; Ganesh et al., 2010). Results from material flow analysis have suggested that the majority of NPs in consumer products may be released into sewer systems and thus reach wastewater treatment plants (Mueller and Nowack, 2008; Gottschalk et al., 2009). Given the proliferation of nanomaterials and their unforeseen potential for significant impacts to the aquatic environment, demands for NP removal from wastewater streams have been rapidly increasing (Limbach et al., 2008; Brar et al., 2010). The use of constructed wetlands (CWs) for removal of a wide variety of contaminants has been well documented, highlighted by their low-cost, simple operation, and environmental and wildlife friendliness (Vymazal, 2005; Imfeld et al., 2009). Plants have versatile detoxification systems to counter phytotoxicity of xenobiotics and these cellular detoxification systems have been termed “green liver” (Sandermann, 1994). In particular, higher aquatic plants are considered to be a critical component of aquatic ecosystems that will directly interact with NPs, and plant uptake could be an important transport and exposure pathway for NPs (Navarro et al., 2012; Wang et al., 2012). However, despite the increasing literature on plant uptake and accumulation of NPs in recent years, knowledge in this field is still rudimentary. The possibility of trophic NP transfer has initiated studies, but these have been almost exclusively in a range of edible plants or crops (Rico et al., 2011; Nair et al., 2010), such as Cu NPs in mung bean and wheat (Lee et al., 2008), Ag NPs in crop plant (Lee et al., 2012), and CeO2 NPs in wheat and pumpkin (Schwabe et al., 2013), etc. To date there have been no studies assessing the potential for uptake of NPs by aquatic plants. Fundamental concerns exist about the behaviour of NPs in aquatic systems and the interaction between NP and aquatic plants, as well as the effect of plant species, size and physicochemical composition of NPs on uptake kinetics (Ma et al., 2010). The main objective of this study was to investigate the potential for accumulation and elimination of CuO NPs and CdS QDs by the wetland plant Schoenoplectus tabernaemontani cultivated in hydroponic mesocosms. The specific research aims were: (i) to investigate the phytotoxicity caused by the exposure of CuO NPs and CdS QDs to S. tabernaemontani; (ii) to evaluate the potential for uptake and bioaccumulation of CuO NPs and CdS QDs NPs in the tissues of S. tabernaemontani; and (iii) to examine plant cell uptake and internalization of CuO NPs and CdS QDs.

2. Materials and methods 2.1. Preparation of CuO NPs and CdS QDs suspensions CuO NPs (>99.8% purity) were purchased from Sigma-Aldrich (Singapore) with an average particle size of 40 ± 5 nm (CuO NPs). CdS/ZnS QDs were provided by the School of Chemical and Biological Engineering, Nanyang Technological University. Prior to use, CdS QDs were dialyzed in deionized water using a biotech cellulose ester dialysis membrane (MWCO, Spectrum Laboratories Inc.) to remove free ions and unbound ligands. Cu NPs and CdS QDs suspensions at the nominal concentrations of 0 (control), 0.5, 5.0 and 50 mg L−1 were prepared by weighing dry CuO NPs and QDs into Milli-Q water (Millipore, Bedford, USA) with constant stirring. Ultrasonic treatment in a water bath (VWR 75T Aquasonic sonicator, 30 ◦ C, 100 W, 40 kHz) was carried out for 1 h to increase dispersion of the NPs. The pH value of the solution was then adjusted to 6.7 ± 0.2.

115

Particles in the nutrient solution (description in Section 2.2) were extracted using a vacuum probe and filtered using nylon membrane filters with a pore size of 0.2 ␮m (Whatman, USA). Specific surface area was measured by the Brunauer–Emmett–Teller (BET) method (Autosorb 1, Quantachrome, USA), based on the adsorption–desorption isotherm of N2 . The zeta potential of the particles in the suspensions was estimated using a Zetasizer Nano Z90 (Malvern Instruments, UK). The presence of NPs in the plant tissues was analysed by scanning electron microscopy (SEM, JEOL, JCM-6000). Particle characterization and morphology was visualized using transmission electron microscopy (TEM, JEOL 2010) operated at 200 kV. Samples for TEM were prepared by slow evaporation of a drop of aqueous solution placed onto 400 mesh carbon/formvar grid. Dissolution kinetics of CuO NPs were determined in the nutrient solutions. CuO NP suspensions at the maximum exposure concentration (50 mg L−1 ) were used after sonication. At 4, 8, 16, 24 and 72 h, the suspension was centrifuged (10,000 rpm for 30 min) and filtered using a nylon membrane filter with 0.2 ␮m pore size (Whatman, USA). The filtered suspensions were acidified using nitric acid (>90%, Sigma-Aldrich, Singapore) and then digested in an Anton Paar Microwave Reaction System (Multiwave 3000, Alpha Analytical USA) following the USEPA 3051 method. Concentrations of released Cu2+ in the supernatant were determined using inductively coupled plasma optical emission spectroscopy (ICPOES, Perkin-Elmer, Optima 2100, USA). For QA/QC of the ICP-OES, a series of calibration standards (National Institute of Standards and Technology (NIST)), were prepared daily for instrument calibration. Sample testing did not begin until the calibration standards and check standards were in agreement. After each set of 10 samples, a blank was analyzed to ensure that no residue was carried over from the previous sample. Recovery efficiencies were periodically tested resulting in values in an acceptable range of 92–98%. Each sample was analyzed three times by ICP-OES and the mean concentration was then calculated. 2.2. Hydroponic culture S. tabernaemontani 0.2 m in height, was purchased from Uvaria Tide (Singapore). The rhizomes were thoroughly washed to remove any soil particles attached to the plants. A 25% strength Hoagland nutrient solution was prepared with the following chemical composition (in mmol L−1 ): 0.75 K2 SO4 , 0.65 MgSO4 ·7H2 O, 2.0 Ca(NO3 )2 , 0.1 KCl, 0.25 KH2 PO4 , 1 × 10−3 H3 BO3 , 1 × 10−3 MnSO4 ·H2 O, 1 × 10−3 ZnSO4 ·7H2 O, 5 × 10−6 (NH4 )6 Mo7 O24 , 0.1 FeEDTA. All of these chemicals were purchased from Sigma-Aldrich (Singapore) and the chemicals were of >98% purity. The plants were then acclimatized to Hoagland nutrient solution for four weeks. Thereafter, plants of uniform size were transferred to 2 L vessels (four plants per vessel corresponding to the four exposure periods studied, i.e., 3, 7, 14 and 21 d) which contained Hoagland nutrient solution spiked with CuO NPs and CdS QDs. A total set of 24 assays was established. Treatment 1 was set up including nine vessels (with three replicates) spiked with CuO NPs. In the second treatment, CdS QDs was spiked into another nine vessels (with three replicates) containing nutrient solution. Initial concentrations for both CuO NPs and CdS QDs were 0.5, 5.0 and 50 mg L−1 , respectively. Controls (three vessels) were also performed where neither CuO NPs nor CdS QDs were spiked into the nutrient solutions. Additionally, since CuO NPs may release cupric ions during sonication process and the aqueous dissolved Cu dissolve from CuO NPs can be toxic to plants at high concentrations (Wang et al., 2012; Shi et al., 2011). The level of Cu2+ released from CuO NPs during NP suspension preparation was measured in order to investigate if the phytotoxicity was mainly due to CuO

116

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

Fig. 1. CuO NPs (A–C) and CdS/ZnS QDs (D) in deionized water at different resolution (i.e., 500, 100 and 50 nm for CuO NPs, while 20 nm for CdS/ZnS QDs) characterized by TEM. These are representive images of particles after drying the suspension on the microscope grid which resulted in aggregation.

NPs themselves (or the level of dissolved copper ion). Our results indicated that the CuO NP suspension with the concentration of 50 mg L−1 released 0.06 mg L−1 Cu2+ (Fig. S1 in SI). In this regard, an additional set of three vessels spiked with Cu2+ at the concentration of 0.06 mg L−1 was included in the experimental design. All the mesocosms were wrapped with tinfoil to prevent from light exposure. Four plants corresponding to the four exposure periods studied (i.e., 3, 7, 14 and 21 d) were cultured in the nutrient solutions. Roots of the plants were submerged in the nutrient solution, and shoots of the plants were kept above the surface of nutrient solution and were never in direct contact with the exposure solution. All the assays were placed randomly outside of the building of School of Civil and Environmental Engineering at Nanyang Technological University, Singapore (133◦ 48 0 E, 1◦ 22 0 N). Owing to its geographical location and maritime exposure, the climate of Singapore is classified as a tropical rainforest climate with uniform temperature (ranging from 23 ◦ C (73.4 ◦ F) to 32 23 ◦ C (89.6 ◦ F)), high humidity (81% in average) and abundant rainfall (2340 mm y−1 ). Moreover, Singapore with its rather constant degree of solar radiation and long daylight (from 7 am sunrise to 7 pm sunset) is without marked seasonal variation throughout the whole year. Therefore the environmental conditions (e.g., temperature, humidity, and solar radiation etc.) were nearly the same throughout the experiment. 2.3. Quantification of Cu2+ and Cd2+ concentration in plant tissues At the end of each exposure period, the plants were washed with tap water followed by rinsing with deionized water three times. Plant biomass was measured before and after drying at 70 ◦ C for 24 h to evaluate the phytotoxicity caused by CuO NPs and CdS QDs. Subsequently, the roots and shoots (0.5 g of plant tissues) were digested with 6 mL trace-pure HNO3 (Sigma-Aldrich, Singapore) in an Anton Paar Microwave Reaction System following the USEPA 3051 method. The samples after microwave acid digestion were

diluted to 40 mL using Milli-Q water and then filtered using a 0.45 ␮m nylon membrane (Whatman, USA). The concentrations of Cu and Cd in the plant tissues were determined using ICP-OES. 2.4. Electron microscopy Fresh roots of S. tabernaemontani were thoroughly washed with Milli-Q water. The rootlets from both control and two of the treatments were cut and coated with gold for 60 s (ca. 1 nm thickness of gold) by using a sputter coater. CuO NPs and CdS QDs in both suspension and plant tissues were observed under a scanning electron microscopy (SEM, JEOL-6700). The morphology of CuO NPs and CdS QDs in plant tissues were characterized using TEM. The root tissues were further examined under TEM to determine if CuO NPs and CdS QDs could penetrate the cell wall and be taken up by plant cells. Root samples for TEM were prepared following procedures below. In brief, S. tabernaemontani root segments (diameter, ∼1 mm; height, 2 mm) were taken from roots above the apical part of the root tip. They were prefixed in 4% glutaraldehyde diluted with 0.1 M cacodylate buffer for 4 h. Then the tissues were rinsed twice with 0.1 M phosphate buffer (pH 7.5) for 10 min, and postfixed in 1% osmium tetroxide and 0.1 M cacodylate buffer for 1 h. The tissues were rinsed with 0.1 M cacodylate buffer and dehydrated in a graded ethanol series (50, 70, 80 90, and 99.9%). Resin infiltration and embedding were in a graded resin: 30, 50 and 70% for 2 h and 100% overnight. The tissues were embedded in beam capsules and polymerized at 60 ◦ C for 72 h. After semi-thin section (thickness ≈ 150 nm) and ultrathin sections (thickness < 70 nm), they were then stained with uranyl acetate (45 min) and lead citrate (3 min). The sections were observed by TEM. 2.5. Statistical analysis All metal concentration measured for the nanoparticle treatments were conducted in triplicate. Tests to determine statistical differences between treatments were carried out by comparing

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

the critical value through one-way analysis of variance (ANOVA). Comparisons were considered significantly different at p < 0.05. All statistical analyses were carried out using SPSS statistical software package (SPSS, Version 17.0).

The TEM image of CuO NPs and CdS QDs in deionized water is shown in Fig. 1. These are images of particles after drying the suspension on the microscope grid which resulted in aggregation. Due to their small size and huge surface area, NPs tend to aggregate in the aqueous phase. Under TEM image, individual NPs were observed in the solution in near-spherical shape. The size distribution of CuO NPs was measured and showed (Fig. S2 in SI), a size range of 23–50 nm with a mean size of 38 ± 7 nm, evidencing the similarity to information provided by producer (35 nm). The surface area of CuO NPs was measured to be 12.8 ± 0.4 m2 g−1 . Electrophoretic mobility and surface charge measurements indicated that the CuO NPs are negatively charged with a mean zeta potential of −2.8 ± 0.2 mV at pH 6.2 (at the end of experiment). The CdS QDs used in this study has a mean size of 4.3 ± 0.3 nm (Fig. S3 in SI). Due to the carboxylate groups on the QD surface, their dispersion in deionized water showed a negative zeta potential with value of −9.8 ± 0.3 mV at pH 6.0. A summary of the characteristics of the CuO NPs and CdS QDs used in this study is shown in Table 1. 3.2. Effect of CuO NPs and CdS QDs on S. tabernaemontani growth

Control

30

a a a

0.06 Cu ions

28 Biomass of S. tabernaemontani (g)

3.1. Characterization of CuO NPs and CdS QDs

32

0.5 Cu NPs 26 5.0 Cu NPs

A

a

24 50 Cu NPs 22

a

20 18 16 14 12 10 0

5

10

15

20

25

Days 32

Biomass of S. tabernaemontani (g)

3. Results and discussion

117

30

Control

28

0.5 CdS QDs

26

5.0 CdS QDs

24

50 CdS QDs

B a

ac 22 20

bc

18 16

bc

14 12 10

Fig. 2a shows the phytotoxicity to the growth of S. tabernaemontani by exposure to CuO NPs. By d 21, the biomass of S. tabernaemontani exposed to CuO NPs at concentrations of 5.0 and 50 mg L−1 was reduced by 9% and 18%, respectively, compared to the unexposed control plants. Toxicity was observed to be dependent upon the concentrations of CuO NPs in the nutrient solution as well as exposure time. However, there were no significant differences (p > 0.05) in biomass between control and CuO NP treatments. In comparison, Wang et al. (2012) observed similar phytotoxicity of CuO NPs, and reported that the CuO NPs at 100 mg L−1 inhibited seedling growth of maize and reduced the fresh weight of plant tissues by 34%. Our results also indicated that Cu2+ treatments (at the concentration of 0.06 mg L−1 which was released from CuO NP suspension) did not show significant impact on the growth of S. tabernaemontani (p > 0.05) (see Fig. 2a). Therefore, the phytotoxicity in NP-exposed S. tabernaemontani was apparently caused by CuO NPs themselves. Wang et al. (2012) also reported that the concentration of solubilized Cu2+ released from CuO NPs was not sufficient to cause toxicity to maize and the observed negative physiological effects are solely due to the CuO NPs. In similar way, Shi et al. (2011) also showed that while a CuO–NP suspension only released 0.16 mg L−1 of soluble copper to the growth medium, it inhibited frond production in Lemna minoras

0

5

10

15

20

25

Days Fig. 2. Phytotoxicity exposed by CuO NPs (A) and CdS QDs (B) to S. tabernaemontani growth. The growth response of S. tabernaemontani exposure to CuO NPs and CdS QDs for 21 days at external concentrations of 0.5, 5.0 and 50 mg L−1 , respectively, was recorded.). Different letters indicate significant difference of biomass between control and NP treatments at different concentrations, significant at p < 0.05 (ANOVA).

(duckweed) to approximately the same extent as 0.60 mg L−1 dissolved copper (or nearly four-fold higher than would be expected based on the level of dissolved copper released). The phytotoxicity of CdS QDs to S. tabernaemontani growth is shown in Fig. 2b. S. tabernaemontani was more sensitive to CdS QDs than CuO NPs, and the reduction in biomass yield was significant (p < 0.05) in the presence of CdS QDs at the concentrations of both 5 and 50 mg L−1 . At the end of the experiment, CdS QDs reduced the biomass of S. tabernaemontani by 16%, 31% and 44% at the concentration of 0.5, 5.0 and 50 mg L−1 , respectively, compared to the unexposed control plants. In addition, CdS QDs showed more pronounced evidence (the shoots became yellow, withered and dry) of toxicity than did the CuO NPs. Several reports have demonstrated toxicity of either the composite core or the surface coating of CdS/ZnS QDs (Priester et al., 2009; Domingos et al., 2011).

Table 1 Characterization of CuO NPs and CdS QDs used in this study. Particle

Size (nm)

Purity (%)

Surface area (m2 g−1 )

Zeta potential (mV)

CuO NPs CdS QDs

38 ± 7 4.3 ± 0.3

99.8 99.5

12.84 ± 0.41 –

−2.8 ± 0.2 −9.8 ± 0.3

Note: 1. The size distribution was examined using transmission electron microscopy (TEM, JEOL 2010). 2. The purity information is from producer. 3. The measurements of CuO NPs surface area were conducted by the Brunauer–Emmett–Teller (BET) method. 4. Zeta potential is measure by Zetasizer Nano Z90 instrument (Malvern Instruments, UK).

118

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

A 25

a

a

20

a 15

b

b b

b

10 5

400 350

5000

5.0 mg L-1 a a

450

a

a

300 250 200

150 100

b

b

b b

50

0

Concentraons in the root (μg L-1)

Concentraons in the roots (μg g-1)

a

0.5 mg L-1

Concentraons in the root (μg L-1)

500

30

4000

d-14 d-21 CdS nanoparcles

a

a

a

3500 3000

a

2500

b

2000 1500

b

b

b

1000 500

0 d-3 d-7 CuO nanoparcles

50 mg L-1

4500

0 d-3 d-7 CuO nanoparcles

d-14 d-21 CdS nanoparcles

d-3 d-7 CuO nanoparcles

d-14 d-21 CdS nanoparcles

0.5 mg L-1 3 2.5

a

2 1.5

a a

a a

a

a a

1 0.5

7

5.0 mg L-1

b

6

b

5

a

3

2

a

b

b

4

a a

1 0

0 d-3 d-7 CuO nanoparcles

d-14 d-21 CdS nanoparcles

d-7 d-3 CuO nanoparcles

d-14 d-21 CdS nanoparcles

Concentraons in the shoot (μg L-1)

Concentraon in the shoot (μg g-1)

3.5

Concentraons in the shoot (μg L-1)

B 24

50 mg L-1 20

a

a a

16

a

12

b b

8 4

b

b

0 d-3 d-7 CuO nanoparcles

d-14 d-21 CdS nanoparcles

Fig. 3. The concentrations of Cu2+ and Cd2+ under the treatments of CuO NPs and CdS QDs in the tissues of S.tabernaemontani. (A) In the roots; and (B) in the shoots (unit: ␮g g−1 dry weight). For each exposure time, significant at p < 0.05 (ANOVA), different letters indicate significant difference between Cu2+ and Cd2+ concentrations in the plant tissues.

Due to photolysis and oxidation, this loss of the protective coating of QDs may occur. The subsequent toxicity of bare CdS QDs and dissolved cadmium is considered as major environmental concern (Priester et al., 2009). The prominent inhibition of S. tabernaemontani growth in the present study might be attributed to both of the intact CdS QDs and dissolved Cd; however, which form plays the major role in eliciting the phytotoxicity remains mostly unassessed (Priester et al., 2009; Navarro et al., 2012).

3.3. Uptake and translocation of Cu and Cd 3.3.1. Uptake and assimilation of Cu and Cd in the roots Accumulated levels of Cu and Cd in the roots of S. tabernaemontani under the treatments of CuO NPs and CdS QDs are shown in Fig. 3a. The levels of total Cu in the roots for the CuO NP treatments ranged from 14 to 4057 ␮g g−1 , while the values were from 9 to 1518 ␮g g−1 for total Cd under CdS QD treatment (dry weight). Compared to Cd, Cu showed significant (p < 0.05) potential to be taken up by the roots of S. tabernaemontani. At the end of the experiment, the accumulated concentrations of Cu in the roots at the concentrations of 0.5, 5.0 and 50 mg L−1 , respectively, were 2.3, 3.8 and 2.7 times higher than those of Cd at the same concentrations. The concentrations of both Cu and Cd in the roots of S. tabernaemontani were time and concentration dependent. Statistical analysis showed that there was a significant (p < 0.05) correlation between the levels of both Cu and Cd in the roots and the concentrations of NPs in the nutrient solutions. This finding is consistent with previous studies (Lee et al., 2008; Zhou et al., 2011; Shi et al., 2011). Lee et al. (2008) presented a linear relationship showing that accumulation of Cu NPs in plant tissues of bean and wheat plants was related to the concentration of NPs in growth media. Zhou et al. (2011) investigated the adsorption and uptake of CuO NPs in wheat

roots, and concluded that both adsorption and uptake of CuO NPs increased with increasing exposure concentrations of CuO NPs. Bioavailability was estimated by calculating the bioaccumulation factors (BAFs) (defined as the ratio of total Cu concentration in plant tissues to the initial Cu concentration of CuO NPs in nutrient solutions). Table 2 shows the estimated BAFs in the roots of S. tabernaemontani for CuO NPs and CdS QDs. The BAFs of CuO NPs for the roots ranged from 27.1 to 81.1, while the corresponding values for CdS QD NPs were from 11.6 to 30.4. All of the BAFs for CuO NPs were higher than those for CdS QDs, implying that S. tabernaemontani had a greater potential to assimilate Cu compared to Cd. Table 3 shows the assimilated mass (mg) and uptake percentage (%) of NPs in the roots, which is defined as the ratio of the assimilated mass (mg) of NPs in the plant tissues to the initial mass (mg) of NPs in the nutrient solution. The root uptake percentage for Cu under CuO NP treatment ranged from 40.6 to 68.4%, while the values were 8.7 to 21.3% for Cd under CdS QD treatment. The efficient removal (especially for CuO NPs) by this higher aquatic plant suggests an approach for economically remediating wastewaters containing these metal nanoparticles. The metals are mostly stored in the roots of the plant, which may then be harvested and incinerated with appropriate disposal of the ashes. Recovery of the original metals from the ashes has also been investigated (Laznicka, 2006). In comparison, Navarro et al. (2012) investigated the uptake of water-dispersible CdS/ZnSe QDs by Arabidopsis thaliana plants, and reported that 7–11% of the CdS/ZnSe QDs could be estimated to be presented in the roots during 7-d exposure period. It is worth noting that many previous studies have suggested that the potential of plant uptake and translocation of NPs may vary significantly due to dissimilarity of plants (e.g., species and age), and nanoparticle characteristics (e.g., particle size, specific surface area, and physic-chemical properties) (Rico et al., 2011; Ma et al., 2010; Nair et al., 2010). Zhou et al. (2011) quantified the uptake of

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

119

Table 2 Estimated BAFsa for CuO nanoparticles and CdS quantum dot in the roots.

2+

Cu

Cd2+

a b

Nanoparticle concentrations in the solution (mg L−1 )

Exposure time

Day-3

Day-7

Day-14

Day-21

0.5 5.0 50 0.5 5.0 50

27.1 67.8 45.1 17.4 11.6 13.4

37.2 74.7 66.6 17.9 19.7 17.9

39.7 78.0 69.3 20.1 20.8 19.9

50.0 83.0 81.1 21.6 21.9 30.4

Statistical analysisb

a b b c c ac

Bioaccumulation factors (BAFs) = detected concentrations of NPs in the roots/initial concentrations of NPs in the nutrient solution. Different letters indicate significant difference of BAF at different concentrations between Cu and Cd, significant at p < 0.05 (One-way ANOVA).

CuO NPs by wheat roots and reported that the levels ranged from 27 to 250 ␮g g−1 as compared to the higher values of CuO NPs in the roots (ranging from 14 to 4057 ␮g g−1 ) that we measured. This discrepancy in the uptake capability of CuO NP may be largely due to the difference in plant species. Because aquatic plants absorb the majority of their nutrients from the water column, it is possible that they may absorb NPs in suspension, potentially facilitating trophic transfer (Glenn et al., 2012). Moreover, since the cell types that make up xylem tissue show great variability across different plant groups, plants with different xylem structures may demonstrate different uptake kinetics of NPs (Ma et al., 2010). In a similar manner, López-Moreno et al. (2010) investigated the fate of CeO2 NPs in different crop plants and reported that at 4000 mg L−1 of CeO2 , the concentrations of Ce (␮g g−1 ) varied significantly between species (approximately 300 for corn, 400 for soybean, 3000 for tomato, and 6000 for alfalfa). 3.3.2. Translocation of Cu and Cd from roots to shoots Fig. 3b shows the concentrations of Cu and Cd in the shoots. The levels of Cu in the shoots ranged from 0.8 to 17.7 ␮g g−1 , while the values were from 0.8 to 8.7 ␮g g−1 (dry weight) for Cd. The levels of Cu and Cd remained much lower in the shoots than those in the roots (14–4057 ␮g g−1 for CuO NPs and 9–1518 ␮g g−1 for CdS QDs), implying that the translocation potential of S. tabernaemontani for both Cu and Cd was limited and NPs cannot move easily into the shoots. The present finding is consistent with previous study of Lin and Xing (2008) which indicated that metal NPs tended to be adsorbed so strongly to the surface of the roots that few NPs could be transported to the shoots. Similarly, Navarro et al. (2012) reported that the translocation of CdS/ZnSe QDs from root to leaves was negligible (with concentration of 0.015 ␮g g−1 in the leaves). The translocation factor (TF) is defined as the shoot to root concentration ratio (Cshoot /Croot ). The low TF values for both CuO NPs and CdS QDs (<0.2) implies that the translocation potential was limited. The BAFs for the shoots of S. tabernaemontani are shown in

Table 4. The BAFs of CuO NPs for the shoots ranged from 0.2 to 6.0, while the values were from 0.1 to 2.9 for CdS QDs. Table 5 shows the assimilated mass (mg) and uptake percentage (% of mass initially in solution) of NPs in the shoots. The shoot uptake percentage for Cu ranged from 0.39 to 9.01%, while the values ranged from 0.14 to 4.37% for Cd. Our finding is in good agreement with previous studies which indicated that NPs concentrations in shoots were less than 10% of the total assimilated NP mass (Navarro et al., 2012; Lin and Xing, 2008). 3.4. SEM and TEM results SEM images of the root tissues of S. tabernaemontani are shown in Fig. 4. No particles were observed on the root epidermis of the control (Fig. 4a). However, individual and aggregated NPs are clearly visible in the epidermis of the roots exposed to CuO NPs and CdS QDs (Fig. 4B and C) after 21 d at the concentration of 50 mg L−1 , implying that the adsorption and adherence of CuO NPs to the root epidermis were apparent. The spectrum of energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of CuO NPs and CdS QDs in the roots of S. tabernaemontani (see Fig. 5). Further analysis was performed to confirm the presence of CuO NPs and CdS QDs in the cells of plant roots. The TEM image in Fig. 6 shows the uptake of CuO NPs by a cell of the root of S. tabernaemontani, which was exposed to nutrient solutions enriched with NPs at the concentration of 50 mg L−1 . After an exposure period of 21 d, CuO NPs were adsorbed and aggregated on the cell membrane (Fig. 6b). In addition, CuO NPs could not only penetrate into the cell walls of plant tissue, but also were taken up by the cell and could clearly be seen around an organelle within the cell (Fig. 6c). It seemed that NPs crossed the cell membrane and formed agglomerates, either with themselves or with other cellular materials within the cell. Currently, cellular penetration is the most accepted mode of action by which NPs interact with plants (Lee et al., 2008; Chen et al., 2010). The presence of NPs in plant cells has been

Table 3 The assimilated mass (mg) and uptake percentage (%) of Cu and Cd in the roots. Nanoparticle concentrations in the solution (mg L−1 )

Cu2+

Cd2+

a b c d

0.5 5.0 50 0.5 5.0 50

Assimilated mass (mg)a

Statistical analysisb

Day-3

Day-7

Day-14

Day-21

0.28 2.53 20.28 0.05 0.43 6.04

0.30 2.77 23.71 0.06 0.73 6.37

0.33 2.95 26.5 0.07 0.79 7.60

0.34 3.23 28.5 0.09 0.85 10.7

a a b a a c

Uptake percentage (%)c

Statistical analysisd

Day-3

Day-7

Day-14

Day-21

55.3 50.7 40.6 11.5 8.7 12.1

59.7 55.3 47.4 13.3 14.6 12.7

65.7 59.0 52.9 14.2 15.8 15.2

68.4 64.6 56.9 15.8 17.0 21.3

a ac bc d d d

Assimilated mass of NPs (mg) = root weight (g) × detected concentrations of NPs in roots (␮g g−1 ). Different letters indicate significant difference of assimilated mass at different concentrations between Cu and Cd, significant at p < 0.05 (one-way ANOVA). Uptake percentage (%) = assimilated mass of NPs (mg) in the roots/initial NPs in the nutrient solution (mg) × 100. Different letters indicate significant difference of uptake percentage at different concentrations between Cu and Cd, significant at p < 0.05 (one-way ANOVA).

120

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

Table 4 Estimated BAFsa for CuO nanoparticles and CdS quantum dot in the shoots.

CuO

CdS

a b

Zn2+ concentrations in the solution (mg L−1 )

Exposure time

Day-3

Day-7

Day-14

Day-21

0.5 5.0 50 0.5 5.0 50

1.6 0.2 0.2 1.6 0.7 0.1

3.4 0.3 0.3 1.7 0.8 0.1

5.4 0.5 0.3 2.4 0.8 0.1

6.0 0.7 0.4 2.9 1.1 0.2

Statistical analysisb

a bc bc b bc c

Bioaccumulation factors (BAFs) = detected concentrations of NPs in the shoots/initial concentrations of NPs in the nutrient solution. Different letters indicate significant difference of BAF at different concentrations between Cu and Cd, significant at p < 0.05 (one-way ANOVA).

Table 5 The assimilated mass (mg) and uptake percentage (%) of Cu and Cd in the shoots. Nanoparticle concentrations in the solution (mg L−1 )

2+

Cu

Cd2+

a b c d

0.5 5.0 50 0.5 5.0 50

Assimilated mass (mg)a

Statistical analysisb

Day-3

Day-7

Day-14

Day-21

0.02 0.02 0.26 0.01 0.06 0.07

0.03 0.03 0.27 0.02 0.07 0.07

0.04 0.05 0.30 0.02 0.08 0.12

0.04 0.07 0.31 0.03 0.10 0.15

ad ad b a cd c

Uptake percentage (%)c

Statistical analysisd

Day-3

Day-7

Day-14

Day-21

3.32 0.39 0.53 2.61 1.22 0.14

5.50 0.62 0.55 3.19 1.43 0.15

8.16 0.89 0.60 4.03 1.56 0.25

9.01 1.41 0.62 4.37 2.18 0.31

a b b cd bd b

Assimilated mass of NPs (mg) = shoot weight (g) × detected concentrations of NPs in shoots (␮g g−1 ). Different letters indicate significant difference of assimilated mass at different concentrations between Cu and Cd, significant at p < 0.05 (one-way ANOVA). Uptake percentage (%) = assimilated mass of NPs (mg) in the shoots/initial NPs in the nutrient solution (mg) × 100. Different letters indicate significant difference of uptake percentage at different concentrations between Cu and Cd, significant at p < 0.05 (one-way ANOVA).

reported by several researchers (Torney et al., 2007; Chen et al., 2010). In a recent study by Kim et al. (2010), TEM images showed the presence of CuO NPs in the endodermis of the root tissue of Cochliobolus sativus after exposure to CuO NPs at the concentration of 1000 mg L−1 . Bali and Harris (2010) showed that both Medicago sativa (alfalfa) and Brassica juncea (mustard green) demonstrated the formation of colloidal Au particles inside live specimens; however, the mechanistic aspects of such NP formation inside the live plants have not yet been fully elucidated. However, the possibility of biogenic synthesis of NPs in vivo in these plants cannot be ruled out. The presence of CdS QDs in a cell of the root of S. tabernaemontani was also confirmed by TEM image (Fig. 7). The present study also demonstrated that CdS QDs penetrated the cell wall (Fig. 7b) and aggregated around organelles within the cell and can be seen as beads on the string (Fig. 7c and d). The uptake and internalization of QDs within plant cells was also reported by Etxeberria et al. (2006) who investigated the endocytic pathway for solute uptake of fluorescent CdS/ZnSe/ZnS QDs (20 nm) by cultured cells of Platanus

occidentalis. Using confocal microscopy, the authors observed that the 40 nm nano-spheres were stored in the vacuoles of sycamore cells as most other solutes, but the 20 nm CdS/ZnSe/ZnS QDs were mostly contained in larger spherical organelles after 18 h. The authors demonstrated that protoplasts from sycamore-cultured cells could effectively take up 40 nm polystyrene nano-spheres and 20 nm CdS/ZnSe/ZnS QDs. Recent studies have already demonstrated that NPs can penetrate the cell wall and membranes of different plants (Lee et al., 2012; Schwabe et al., 2013). It was assumed that the diameters of NPs relative to the pore diameters of the plant cell wall (3.5–5 nm) would already restrict the ability of NPs to penetrate the plant cell via active/passive transport (Carpita et al., 1979). However, NPs can penetrate a cell wall even when they are larger than the cell-wall pores, and they may be then further internalized, depending on their properties. Several mechanisms have been identified which would allow NPs to penetrate plant cells: (i) resembling endocytosis or nonendocytic penetration allowing absorption of these materials which would ordinarily be too big for the cell wall

Fig. 4. SEM image of S. tabernaemontani root epidermis under treatment of (A) control; (B) 50 mg L−1 CuO NPs; and (C) 50 mg L−1 CdS QDs. Nanoparticles were clearly shown on the root epidermis of S. tabernaemontani after exposure to CuO NPs and CdS QDs for 21 days. Nanoparticles were pointed by arrows.

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

A

121

001

1000 900 800 700

Counts

600

200 100

CuKb

CuLl CuLa

300

PKa

400

CuKa

CKa NKa OKa

500

0

B

0.00

1000

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

6.00

7.00

8.00

9.00

10.00

keV

001

900 800

600 500

CdLb

CdLb2

100

CdLa

200

SKb

300

SKa

400

CKa NKa OKa

Counts

700

0 0.00

1.00

2.00

3.00

4.00

5.00 keV

Fig. 5. EDS profile of CuO NPs (A) and CdS QDs (B) at the concentration of 50 mg L−1 .

(Etxeberria et al., 2006); (ii) inducing the formation of new and large size pores which allow the internalization of large NPs to penetrate cell walls (Navarro et al., 2008; Lin and Xing, 2008); and (iii) binding to carrier proteins and connecting with ion channels, or binding to organic chemicals (Rico et al., 2011; Nair et al., 2010). Once inside a plant cell, internalization can occur via fluid-phase endocytosis, which is the incorporation of solutes from the apoplast to the organelle via vesicles generated at the plasma membrane (Etxeberria et al., 2006). In cell suspensions, NPs can be transported apoplastically or symplastically through plamodesmata or intercellular bridges (Lucas and Lee, 2004), which were reported to be cylindrical channels with approximate diameters of 40 nm (Tilney

et al., 1991). Indeed, this may well explain why the individual CuO NPs (with particle size of 38 nm) and CdS/ZnS QDs (with particle size of 4 nm) in the present study could readily pass through the plant cell wall after being absorbed to the plant root epidermis, suggesting that endocytosis path ways may be one mode, through which NPs penetrate into the cells of S. tabernaemontani. These active-transport mechanisms are involved in key cellular processes such as nutrient uptake and the regulation of plasma membrane receptors, as well as plasma membrane recycling and signalling (Etxeberria et al., 2006). The existence of NPs around the organelle within the plant cell demonstrated that NPs may be transported within the cells through the plasmodesmata.

Fig. 6. Transmission electron microscopy. (A) Detection of CuO NP aggregation in the root tissues of S. tabernaemontani after 21 days; (B) CuO NPs were aggregated around plasma membrane of the root cell; (C) CuO NPs have penetrated both the cell wall and plasma membrane in the root of S. tabernaemontani. CuO NPs could clearly be seen within the cell and aggregated around organelle. Nanoparticles were pointed by arrows.

122

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123

Fig. 7. TEM image of the roots of S. tabernaemontani exposed to CdS QDs for 21 days at external concentration of 50 mg L−1 . (A) CdS QDs in the root tissue; (B) CdS QDs could not only penetrate the cell wall, but also be taken up by the plant cell; (C) CdS QDs were aggregated around a single organelle within the cell and seen as beads on the string after 21 d; (D) CdS QDs attached on the membrane of the same organelle and from enlarged imaging of C. NPs were pointed by arrows.

4. Conclusion The present study provides the first evidence of the ability of higher aquatic plants to take up and translocate CuO NPs and CdS QDs. The results presented in this study demonstrate adverse effects of both CuO NPs and CdS QDs on the growth of S. tabernaemontani. Biomass was significantly reduced by CdS QDs at the concentration of 5 and 50 mg L−1 during the 21-d exposure period. Adherence of CuO NPs and CdS QDs to the root epidermis of S. tabernaemontani was observed. S. tabernaemontani showed higher potential to take up CuO NPs into the roots, compared to CdS QDs. The results of SEM, EDS and TEM analysis confirmed the presence of CuO NPs and CdS QDs in the root tissues of S. tabernaemontani, and demonstrated that NPs could penetrate the cell wall and reach organelles. Future in-depth research is needed on nanoparticle behaviour related to the aquatic ecotoxicity, as well as the complex soil-nanoparticle-plant interactions in an actual treatment wetland. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ecoleng. 2014.04.018. References Anikeeva, P.O., Halpert, J.E., Bawendi, M.G., Bulovic, V., 2009. Quantum dots lightemitting devices with electroluminescence tunable over the entire visible spectrum. Nano Lett. 9, 2532–2536.

Bali, R., Harris, A.T., 2010. Biogenic system of Au nanoparticles using vascular plants. Ind. Eng. Chem. Res. 49 (24), 12762–12772. Brar, S.K., Verma, M., Tygi, R.D., Surampalli, R.Y., 2010. Engineered nanoparticles in wastewater and wastewater sludge—evidence and impacts. Waste Manage. 30, 504–520. Carpita, N., Sabularse, D., Montezinos, D., Delmer, D.P., 1979. Determination of the pore-size of cell walls of living plant cells. Science 205, 1144–1147. Chen, R., Ratnikova, T.A., Stone, M.B., Lin, S., Lard, M., Huang, G., Hudson, J.S., Ke, P.C., 2010. Differential uptake of carbon nanoparticles by plant and mammalian cells. Small 6, 612–617. Domingos, R.F., Simon, D.F., Hauser, C., Wilkinson, K.J., 2011. Bioaccumulation and effects of CdTe/CdS/ZnS QDss on Chlamydomonas reinhardtii–nanoparticles or the free ions? Environ. Sci. Technol. 45, 7664–7669. Etxeberria, E., Gonzalez, P., Baraja-Fernandez, E., Romero, J.P., 2006. Fluid phase endocytic uptake of artificial nano-spheres and fluorescent quantum dots by sycamore cultured cells: evidence for the distribution of solutes to different intracellular compartments. Plant Signal. Behav. 1, 196– 200. Ganesh, R., Smeraldi, J., Hosseini, T., Khatib, L., Olson, B.H., Rosso, D., 2010. Evaluation of nanocopper removal and toxicity in municipal wastewaters. Environ. Sci. Technol. 44, 7808–7813. Glenn, J.B., White, S.A., Klaine, S.J., 2012. Interactions of gold nanoparticles with freshwater aquatic macrophytes are size and species dependent. Environ. Toxicol. Chem. 31 (1), 194–201. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2 , ZnO, Ag, CNT, Fullerenes) for different regions. Environ. Sci. Technol. 43, 9216–9222. Imfeld, G., Braeckevelt, M., Kuschk, P., Richnow, H.H., 2009. Monitoring and assessing processes of organic chemicals removal in constructed wetlands. Chemosphere 74, 349–362. Kim, B., Park, C.S., Murayama, M., Hochella, M.F., 2010. Discovery and characterization of silver sulphide nanoparticles in final sewage sludge products. Environ. Sci. Technol. 44, 7509–7514. Klaine, S.J., Alvarez, P.J.J., Batley, G.E., Fernandes, T.F., Handy, R.D., Lyon, D.Y., Mahendra, S., Mclaughlin, M.J., Lead, J.R., 2008. Nanomaterials in the environment: behaviour, fate, bioavailability and effects. Environ. Toxicol. Chem. 27, 1825–1851. Laznicka, P., 2006. Giant Metallic Deposits: Future Sources of Industrial Metals. Springer-Verlag Berlin Heidelberg, Germany.

D. Zhang et al. / Ecological Engineering 70 (2014) 114–123 Lee, W.M., An, Y.J., Yoon, H., Kwbon, H.S., 2008. Toxicity and bioavailability of copper nanoparticles to the terrestrial plants mung bean (Phaseolus radiates) and wheat (Triticum aestrivum): plant agar test for water-insoluble nanoparticles. Environ. Toxicol. Chem. 27, 1915–1921. Lee, W.M., Kwak, J.I., An, Y.J., 2012. Effect of silver nanoparticles in crop plants Phaseolus radiatus and Sorghum bicolor: media effect on phytotoxicity. Chemosphere 86, 491–499. Limbach, L.K., Bereiter, R., Müller, E., Krebs, R., Gälli, R., Stark, W.J., 2008. Removal of oxide nanoparticles in a model wastewater treatment plant: influence of agglomeration and surfactants on clearing efficiency. Environ. Sci. Technol. 42, 5828–5833. Lin, D.H., Xing, B.S., 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ. Sci. Technol. 42, 5580–5585. López-Moreno, M.L., Rosa, G.D.L., Hernández-Viezcas, J.A., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2010. X-ray absorption spectroscopy (XAS) corroboration of the uptake and storage of CeO2 nanoparticles and assessment of their differential toxicity in four edible plant species. J. Agric. Food Chem. 58, 3689–3693. Lucas, W.J., Lee, J.Y., 2004. Plasmodesmata as a supracellular control network in plants. Nat. Rev. Mol. Cell Biol. 5, 712–726. Luque, A., Marti, A., Nozik, A.J., 2007. Solar cells based on quantum dots: multiple exciton generation and intermediate bands. MRS Bull. 32, 236–241. Ma, X., Geiser-Lee, J., Deng, Y., Kolmakv, A., 2010. Interaction between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci. Total Environ. 408, 3053–3061. Mueller, N.C., Nowack, B., 2008. Exposure mdeling of engineered nanoparticles in the environment. Environ. Sci. Technol. 42, 4447–4453. Nair, R., Varghese, S.H., Nair, B.G., Maekawa, T., Yoshida, Y., Kumar, D.S., 2010. Nanoparticulate material delivery to plants. Plant Sci. 179, 154–163. Navarro, E., Baun, A., Behra, R., Hartmann, N.B., Filser, J., Miao, A.J., Quigg, A., Santschi, P.H., Sigg, L., 2008. Environmental behaviour and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 17, 372–386. Navarro, D.A., Bisson, M.A., Aga, D.S., 2012. Investigating uptake of water-dispersible CdS/ZnSe/ZnS quantum dots nanoparticles by Arabidopsis thaliana plants. J. Hazard. Mater. 211–212, 427–435.

123

Priester, J.H., Stoimenov, P.K., Mielke, R.E., Webb, S.F., Ehrhardt, C., Zhang, J.P., Stucky, G.D., Holden, P.A., 2009. Effects of soluble cadmium salts versus CdS/ZnSe quantum dots on the growth of planktonic Pseudomonas aeruginosa. Environ. Sci. Technol. 43, 2589–2594. Rico, C.M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2011. Interaction of nanopaticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 59, 3485–3498. Sandermann, H., 1994. Higher plant metabolism of xenobiotics: the green liver concept. Pharmacogenetics 4, 225–241. Schwabe, F., Schulin, R., Limbach, L.K., Stark, W., Bürge, D., Nowack, B., 2013. Influence of two types of organic matter on interaction of CeO2 nanoparticles with plants in hydroponic culture. Chemosphere 91, 512–520. Shi, J., Abid, A.D., Kennedy, I.M., Hristova, K.R., Silk, W.K., 2011. To duckweeds (Landoltia punctate), nanoparticulate copper oxide is more inhibitory than the soluble copper in the bulk solution. Environ. Pollut. 159, 1277–1282. Stampoulis, D., Sinha, S.K., White, J.C., 2009. Assay-dependent phytotoxicity of nanoparticles to plants. Environ. Sci. Technol. 43, 9473–9479. Tilney, L.G., Cooke, T.J., Connelly, P.S., Tilney, M.S., 1991. The structure of plasmodesmata as revealed by plasmolysis, detergent extraction, and protease digestion. J. Cell Biol. 112, 739–747. Torney, F., Trewyn, B.G., Lin, V.S.-Y., Wang, K., 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotechnol. 2, 295–300. Vymazal, J., 2005. Horizontal sub-surface flow and hybrid constructed wetlands systems for wastewater treatment. Ecol. Eng. 25, 478–490. Wang, Z., Xie, X., Zhao, J., Liu, X., Feng, W., White, J.C., Xing, B., 2012. Xylem- and phloem-based transport of CuO nanoparticles in maize (Zea mays L.). Environ. Sci. Technol. 46, 4434–4441. Yang, J.G., Okamoto, T., Ichino, R., Bessho, T., Sarake, S., Okido, M., 2006. A simple way for preparing antioxidating nano-copper powers. Chem. Lett. 35, 648– 649. Yuan, Z., Li, J., Cui, L., Xu, B., Zhang, H., Yu, C., 2013. Interaction of silver nanoparticles with pure nitrifying bacteria. Chemosphere 90, 1404–1411. Zhou, D., Jin, S., Li Li Wang, Y., Weng, N., 2011. Quantifying the adsorption and uptake of CuO nanoparticles by wheat root based on chemical extraction. J. Environ. Sci. 11, 1852–1857.