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To better understand the ecotoxicological effects of zinc oxide nanoparticles on aquatic plants, laboratory experiments were conducted using Salvinia natans All. as the test organism for 7-d exposure to the NPs. The activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and the content of chlorophyll were determined at the end of the exposure to evaluate the toxicity of the NPs in the culture medium on the plants. To investgate if the release of soluble Zn from ZnO NPs suspensions plays a key role in the toxicity, we investigated its aggregation and dissolution in the medium. Our results indicate that ZnO NPs could bring harmful effects to S. natans when its concentration is above 50 mg L-1 in the culture medium, and Zn2+ released from the NPs may be the main source of its toxicity to this species.
Keywords: ZnO nanoparticles; Salvinia natans; Toxicity; Aggregation; Dissolution
Nanoparticles (NPs) have been referred to new materials with at least two dimensions under 100 nm . The unique physicochemical properties of NPs are attributable to their small size, chemical composition, surface structure, solubility, shape, and aggregation . This makes them attractive for a wide range of novel applications in electronics, healthcare, cosmetics, technologies and engineering industries . According to conservative estimates, the number of consumer products on the market containing NPs or nanofibers now exceeds 1300 and is growing rapidly .
According to "The Nanotechnology Consumer Products Inventory" , the most common metal oxide NPs material mentioned in the product descriptions was titanium dioxide (TiO2), followed by Zinc oxide (ZnO). ZnO NPs are widely used in pigments, semiconductors, sunscreens, and food additives. Meanwhile, they may be released to the environment and become a threat to the ecosystem during the life cycle of these commercial products.
The extensive use of NPs will undoubtedly increase the potential risk for human and environmental exposures. Investigations on the ecotoxicology of NPs are now emerging, with a goal of assessing NPs' harmful effects to the ecosystem. Because water environment is the most important and maybe the ultimate destination of NPs released in the environment despite their sources, many studies focused on toxicity of NPs on aquatic organisms .
Toxicological effects of ZnO NPs upon a variety of aquatic organisms have recently been reported . It has also been shown to have toxic effects and can inhibit the growth of microalgae Pseudokirchneriella subcapitata , crustaceans Daphnia magna and Thamnocephalus platyurus and bacteria Vibrio fischeri . However, research on the toxicity of ZnO NPs is far from complete and needs more work to evaluate its release risk using other organisms, such as aquatic plants. In addition, influences of aggregation and dissolution on toxicity of the NPs remain to be determined.
Salvinia natans (L.) All. is a free-floating, aquatic heterosporous fern that reproduces vegetatively and forms rapidly expanding mats of foliage on still water surfaces in tropical and subtropical regions. The plant was previously selected for toxicity of heavy metals .
The present work aimed to evaluate the ecotoxicological effects of ZnO NPs on S. natans via changes of photosynthetic pigments and responses of antioxidant enzymes. Meanwhile, influences of aggregation and dissolution on toxicity of the NPs are investigated.
2. Materials and Methods
2.1 Characterization of ZnO Nanoparticles
ZnO NPs were purchased from Aipurui Co., Ltd., Nanjing, China. The surface area of the NPs was further determined using the multipoint Brunauer-Emmett-Teller (BET) method. The morphology of the NPs was examined using transmission electron microscopy (H-7500, HITACHI, Japan).
2.2 Cultivation and growth of S. natans
Salvinia natans (L.) All. was obtained from Linyi Fengheyuan Garden Co., Ltd (Linyi, China). The nutrient solution used for growth was prepared according to the OECD 221 guidelines (2006) and contains KNO3, Ca(NO3)2Â·4H2O, KH2PO4, K2HPO4, MgSO4Â·7H2O, H3BO3, ZnSO4Â·7H2O, Na2MoO4Â·2H2O, MnCl2Â·4H2O, FeCl3Â·6H2O, and EDTA Disodium-dihydrate. The pH was adjusted to 6.5 by addition of 0.1N NaOH.
The plants were rinsed with sterile water and were allowed to acclimatize to the medium for 7 days before the beginning of the NPs exposure. Three plants were cultivated in 500 mL beakers containing 350 mL of the culture medium. The beakers were placed in a thermostated chamber (at 24 Â± 2 oC) with a photoperiod of 16 h of light (180 Âµmol m-2 s-1) and 8 h of darkness. The duration of the exposure was 7 d. Exposures of the NPs were performed at four concentrations (mg L-1): 0 (Control), 1, 10 and 50. A positive control which was exposed to ZnSO4 with the concentration of 44 mg L-1 (Zn2+ 10 mg L-1) was also performed.
A stock suspension of ZnO at 100 mg L-1, obtained by addition of ZnO NPs to the culture medium was used to prepare various ZnO concentrations. The stock suspension was treated by a sonicator (Vibra-Cell TM, USA; 50 Hz, 10 s pulse and 5 s interval) for 10 min just before used. The culture media with different ZnO concentrations were prepared by adding the stock suspension, followed by sonication for 20 s.
After 7-d exposure, the plants were harvested, rinsed with distilled water, and their fresh weights were measured. The relative growth rate (g g-1 d-1) in each treatment was calculated by the formula: RGR= (lnW2 âˆ’lnW1)/t, where W1 and W2 are the initial and final fresh weight (g), and t is the incubating time (d). The dry weight of the plants was obtained by being oven dried at 80 oC for 48 h to constant weight.
2.3 Pigments assay
The contents of chl a, chl b and and carotenoid in the leaves of the plants were analysed according to Lichtenthaler . Freeze dried leaves were cut into small pieces and subsamples of approximately 5 mg were extracted with 8 mL 96% ethanol in the dark at room temperature. After 24 h, the pigment extracts were, after vigorously stirring, centrifuged for 3 - 5 min and the absorbance of the extracts measured at 665, 648, and 470 nm with a VIS-7220 spectrophotometer.
2.4 Antioxidative enzymes assay
Superoxide dismutase (SOD, EC 22.214.171.124) activity was determined by use of the ferric cytochrome c method with xanthine/xanthine oxidase used as the source of super-oxide radicals, and a unit of activity was defined as that described in McCord and Fridovich . Catalase activity (CAT, EC.126.96.36.199) was assayed spectrophotometrically with a Hitachi U-3000 by measuring the decrease of absorbance at 240 nm due to H2O2 decomposition . Peroxidase (POD, EC 188.8.131.52) activity was determined by use of the methods described by Li et al. .
2.5 Aggregation of the NPs and concentrations of Zn2+ assay
Prior to the test, ZnO NPs were added to the culture medium with the volume 350 mL in a 500 mL beaker and got a final concentration of 10 mg L-1. The suspension was treated by a sonicator as described above, and then the beakers were placed in the thermostated chamber (at 24 Â± 2 oC) with no disturbing. Particle size of the ZnO NPs in the suspension was determined with a Nanotrac 250 particle analyzer (Microtrac Inc., USA). The 10 mL water samples were taken from the upper layer of the suspension carefully to avoid disturbing at each hour of the initial 24 h for the test of the particle distribution.
The concentrations of Zn2+ in the culture media (with plants) at 7 d were detected by ICP-OES (VISTA-MPX, USA).
2.6 Statistical analysis
All samples were triplicated and three independent experiments were run. The results presented are the arithmetic means with their corresponding standard deviations. The differences between groups were tested for significance using one-way analysis of variance (ANOVA) using Origin 7.0, taking P < 0.05 as significant and P < 0.01 as highly significant.
3.1 Characteristics of ZnO NPs
The results show that the crystalline phase of ZnO NPs is monocrystalline, and the mean size of the single particle was about 30 nm (Fig. 1) with the surface area 90 m2 g-1.
3.2 Chlorophyll and carotenoid contents
Both chlorophyll and carotenoid were influenced by ZnO NPs and ZnSO4 (Table 1). The contents of chl a and chl b decreased with the increase of the dosage of ZnO NPs. The chl a content of the group 50 was about 2.57 mg g-1 DW (60% of the control), significantly lower than that of the control (P < 0.05). When treated with ZnSO4, both chl a and chl b were significantly lower than those of the control (P < 0.05), and also lower than the groups treated with ZnO NPs.
The content of carotenoid increased slightly when treated with low concentration of ZnO NPs (1 mg L-1), and then decreased with the increase of the dosage. The carotenoid contents of the group 50 and group ZnSO4 were significantly lower compared with that of the control.
3.3 Responses of the antioxidant enzymes
Responses of SOD, CAT and POD are shown in Fig. 2. The activities of SOD increased with increasing of the NP dosages and only the group 50 showed significant differences (P < 0.05) from the control. When exposed to 10 mg L-1 Zn2+, the SOD activity of S. natans was significantly higher (P < 0.01) than that of the control.
The responses of CAT were similar to SOD. When exposed to ZnO NPs for 7 d at the highest concentration (50 mg L-1), the CAT activity of S. natans was significantly higher (P < 0.05) than that of the control. The group ZnSO4 followed the trend of SOD.
As for POD, it increased first with the increase of the dosage of ZnO NPs, the dropped markedly (P < 0.05) at the concentration of 50 mg L-1. When exposed to ZnSO4 for 7 d, the POD activity of S. natans was significantly lower (P < 0.01) than that of the control.
3.4 Aggregation and dissolution of ZnO NPs
Figure 3 depicts particle size distribution (mean value) of 30-nm ZnO NPs in the culture medium (with no plants) over the initial 24 h. Figure 4 shows the size distribution at 0 h, 3 h, 6 h and 12 h after preparation. Although the suspension was treated with sonication, the mean value of the particle size was about 130 nm. Aggregation occurred immediately after preparation, and the particle size could reach more than 4 Âµm. Over the initial 6 h tested, there were no regular patterns of aggregation could be detected in both solutions. After 12 h, the particle sizes reduced to lower than 1 nm, suggesting that there were no ZnO NPs in the supernatants. The white aggregations could be found at the bottom of the beakers.
The concentrations of Zn2+ in the culture media were shown in Table 1. The medium of the control provided Zn2+ as one of the essential nutrients with the concentration 11.4 Âµg L-1 which could maintain the normal growth of S. natans. The portions of dissolution were the principal sources of the Zn2+ in the group 1, 10, 20 and 50, with the mean concentrations of 0.413, 1.874, 2.572 and 2.963 mg L-1, respectively. The concentration of Zn2+ in the group ZnSO4 was 8.712 mg L-1.
The results of the toxicological tests in this study indicate that ZnO NPs could generate harmful effects to S. natans when its initial concentration was above 50 mg L-1 in the culture medium. The results from aggregation and dissolution analyses suggest that Zn2+ released from the NPs may be the main source of toxicity of ZnO NPs to the species.
Antioxidant enzymes of living organisms possess antioxidative activities and can protect cells against adverse effects of reactive oxygen species (ROS). The enzymes SOD, CAT and POD can control cellular levels of ROS . The activities of these antioxidants determine the steady state levels of ROS in cells. POD plays a vital role in scavenging these ROS. SOD and CAT could work together to convert O2Â· and H2O2 into harmless H2O and O2, reduce the formation of hydroxyl free radical Â·OH as well as lower the overall free radical content of cells. In this study, SOD and CAT showed increased activities in the plants treated with the ZnO NPs, indicating better ROS scavenging in the system. According to the reports , Zn has a potentiality to increase the biosynthesis of antioxidant enzymes in S. polyrhiza. The activity of POD was significantly inhibited at the concentration of 50 mg L-1. This may be that the stress from the NPs or the Zn2+ was beyond the protective ability of the enzymes.
Colloid is the generic term applied to particles in the 1-nm to 1-Âµm size rangeIn aquatic systems. Therefore, ZnO NPs existed in the solutions as colloid first, and then aggregated to large particles and deposited. It was reported that colloidal fate and behavior are dominated by aggregation , and colloids will ultimately aggregate to particles (>1 Âµm) that are sufficiently large that their transport is dominated by sedimentation. It is important in the self-purification of water bodies and results in pollutant loss from surface waters and accumulation in the sediments and is analogous to the likely behavior of NPs, with aggregation and subsequent sedimentation an important process in their ultimate fate. Our results showed that the aggregation of the ZnO NPs is significant in the culture medium and that there were substantially no ZnO NPs in the supernatants over 12 h. It is consistent with the resent reports [10, 11], which evaluated the aggregation of the ZnO NPs in a freshwater system.
The dissolution of ZnO is highly pH-dependent. Acid could facilitate and accelerate the process, while alkaline hinders it. The pH value of the culture medium used in this study is 6.5. We observed that the Zn2+ concentration was 2.963 mg L-1 in the group 50, which was basically close to a resent report with a measured average Zn2+ concentration about 2.6 (0.8 mg L-1 in culture media (pH 7.0; ZnO NPs) 20 mg L-1). It has been assumed that the predominant bioavailable portion of the total contaminant was the soluble form . Meanwhile, Adams et al. and Franklin et al. also reported the close relationship between the dissolved portion and the toxicity of the ZnO NPs. Although other studies have shown that metal or metal oxide NPs may be more toxic than either their ionic forms or their parent compounds , solubility is likely to be an important aspect and should be considered in the further ecotoxicological researches of the NPs.
The dissolution rate of NPs is influenced by several properties of NPs, including size, surface area, surface curvature and roughness of the particle . Moreover, other factors, such as pH, ionic strength and aggregation may also affect the dissolution significantly. How aggregation affects the dissolution behavior of particles is not well understood. The formation of clumps or clusters of primary particles results in increased hydrodynamic size and reduced specific surface area, and may hinder dissolution by reducing the average equilibrium solubility of the particle system. The behavior may be further complicated by the aggregate volumes and packing factors, with larger, more densely packed aggregates exhibiting a slower dissolution . We detected that the Zn2+ concentration in the group 10 was 1.874 mg L-1, while 2.963 mg L-1 in the group 50. The reason might be the above mentioned.
NPs and natural colloids will interact and this will affect NP behavior in the natural environment. At present, no direct published data are available on the concentrations of NPs in natural waters, but a recent report using a simplified box model and known current uses suggested environmental concentrations is approximately 1 to 100 Âµg/L , whereas typical dissolved and colloidal organic matter in freshwaters may be found at 1 to 10 mg L-1 concentrations. Disturbing in the actual aquatic environment may also influence the aggregation of the NPs.
In general, toxicological effects of ZnO NPs to S. natans were investigated in the present study, and the results show that significant stress from the NPs at 50 mg L-1 could be observed. Because the NPs are apt to aggregation and dissolution in the medium, Zn2+ released from the NPs may be the main source of the toxicity. Research on aggregation in the natural aquatic environment, sediment toxicity and bioaccumulation of NPs may provide more valuable information.
Fig. 1 Transmission electron microscopic image of ZnO nanoparticles.
Fig. 2 The relative growth rate (RGR) of S. natans over 8 d exposure.
Fig. 3 The activities of SOD, CAT, and POD of S. natans after 7-d exposure to ZnO NPs and ZnSO4 with different concentrations.
Fig. 4 Variation of the particle sizes (mean Â± SD) of ZnO in the culture medium (with no plants) over the initial 24 h.
Table 1 Effects of ZnO nanoparticles and ZnSO4 on chl a, chl b and carotenoid (mg g-1 DW) in the leaves of Salvinia natans.
Table 2 The concentrations of Zn2+ in Salvinia natans and the culture media after 7 d exposure.
Roots (without rinsing) (mg/g DW)
0.015 Â± 0.006
0.018 Â± 0.005
0.016 Â± 0.006
0.011 Â± 0.002
0.45 Â± 0.13
0.33 Â± 0.19
0.49 Â± 0.15
0.41 Â± 0.08
2.61 Â± 0.72
1.56 Â± 0.38
3.45 Â± 0.53
1.87 Â± 0.32
3.17 Â± 0.56
1.93 Â± 0.66
6.88 Â± 1.22
2.57 Â± 0.19
3.65 Â± 0.81
1.97 Â± 0.71
8.18 Â± 1.35
2.93 Â± 0.29
4.28 Â± 0.83
3.82 Â± 0.67
3.64 Â± 0.57
8.71 Â± 0.56