J Nanopart Res (2010) 12:16451654 DOI 10.

1007/s11051-009-9740-9

RESEARCH PAPER

Toxicity of zinc oxide nanoparticles to zebrash embryo: a physicochemical study of toxicity mechanism
Wei Bai Zhiyong Zhang Wenjing Tian Xiao He Yuhui Ma Yuliang Zhao Zhifang Chai

Received: 1 January 2009 / Accepted: 8 August 2009 / Published online: 29 August 2009 Springer Science+Business Media B.V. 2009

Abstract The biological impact of engineered nanomaterials released into the aquatic environment is a major concern. In this work, the properties of ZnO nanoparticles (nano-ZnO, 30 nm) were characterized in a water suspension (E3 medium), and a zebrash 96-h post fertilization (hpf) embryolarval test was performed to assess the toxicity of nano-ZnO suspension. Nano-ZnO was found to readily form aggregates with different sizes; small aggregates (142.4517.7 nm) were still suspended in E3 medium, but large aggregates ([1 lm) quickly deposited on the bottom of 24-well plates; nano-ZnO was partially dissolved to Zn species (Zn(dis)) in E3 medium. In the nano-ZnO suspension, small aggregates, Zn(dis), and large aggregates might jointly exert inuence on the development of zebrash embryos. The embryo toxicity test revealed that nano-ZnO killed zebrash embryos (50 and 100 mg/L), retarded the embryo hatching (125 mg/L), reduced the body
W. Bai Z. Zhang (&) X. He Y. Ma Y. Zhao Z. Chai CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Key Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics, The Chinese Academy of Science, Beijing 100049, China e-mail: zhangzhy@ihep.ac.cn W. Tian College of Environment and Safety Engineering, Qingdao University of Science and Technology, Qingdao 266061, China

length of larvae, and caused tail malformation after the 96 hpf exposure. Zn(dis) only partially contributed to the toxicity of nano-ZnO. This research highlights the need to further investigate the ecotoxicity of nano-ZnO in the water environment. Keywords ZnO nanoparticles Solubility Aggregate Toxicity Zebrash embryo Environment EHS

Introduction Due to their unique properties and diverse nanostructures, ZnO nanoparticles (nano-ZnO) are widely applied in optoelectronics, cosmetics, catalysts, ceramics, pigments, etc. (EPA 2007; Wang 2004). Previous reports proposed that nano-ZnO was biosafe and biocompatible and could be applied in biomedical materials (Berube 2008). However, toxicological studies indicated that nano-ZnO had adverse impacts on human health and environmental species. The bio-safety of nano-ZnO is still a controversial issue. Nano-ZnO was recognized as a respiratory toxicant which caused metal fume fever (myalgia, cough, fatigue, etc.) (Beckett et al. 2005; Lam et al. 1985). Recently, in vivo cell experiments showed that exposure to nano-ZnO resulted in oxidative damage and inammation response in vascular/lung

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endothelial cells (Gojova et al. 2007; Lin et al. 2009). Animal experiments indicated that liver, spleen, heart, pancreas, and bone were target organs of oral exposure to 20- and 120-nm ZnO (Wang et al. 2008). Recent studies demonstrated the ecotoxicity of nanoZnO to some living organisms in the environment, such as higher plants (Lin and Xing 2007), algae (Franklin et al. 2007), bacteria (Brayner et al. 2006; Zhang et al. 2007), and daphnia (Zhu et al. 2008a). Nevertheless, it is still inadequate to comprehensively assess the toxicity of nano-ZnO and to understand its toxic mechanism. Compared with the conventional toxicology, the dose is no longer a sole factor in evaluating the toxicity of nanoparticles. The physicochemical properties of nanoparticles, such as size, shape, chemical composition, aggregation, etc., need to be taken into account in aquatic toxicity testing. Moreover, the pH and salinity of the aqueous exposure system are also important factors. Unlike insoluble nanoparticles such as nano-TiO2 and nano-SiO2, the solubility of nano-ZnO may play a more important role in its toxicity. However, inconsistent results on the solubility of nano-ZnO in water were presented in the literatures (Franklin et al. 2007; Lin and Xing 2007; Wang et al. 2008). The developing sh embryo or larvae is generally considered to be the most sensitive stage in the life cycle of a teleost, being particularly sensitive to lowlevel environmental pollutant (Westernhagen 1988). Zebrash, a small vertebrate species, can be rapidly and prolically bred and easily maintained in laboratory. Zebrash eggs (1.01.2 mm in diameter) are transparent and develop quite rapidly and synchronously, which facilitate direct optical observation of the toxic effects on their internal organs (Hallare et al. 2006, Samson and Shenker 2000). The eggs contain two distinct membranes, i.e., the outer chorionic membrane and the inner vitelline membrane, and between them there is a perivitelline space lled with a viscous uid. The chorion (3.5 lm in thickness) is the rst barrier, on which numerous pore canals of *0.50.7 lm in diameter are distributed (Lee et al. 2007; Rawson et al. 2000). Zebrash embryo has been proved to be a good model vertebrate to assess the toxicity of nanoparticles (Zhu et al. 2008b; Usenko et al. 2007; Lee et al. 2007; Cheng et al. 2007). The aim of this work is to explore in depth the physicochemical properties of nano-ZnO in water and

study its potential toxicity to aqueous organisms. The size distribution and solubility of nano-ZnO in water were measured. Zebrash embryo was adopted as a model to evaluate the toxicity of nano-ZnO, and toxic mechanism of nano-ZnO was discussed.

Materials and methods Fish maintenance and egg production Zebrash (Danio rerio) were maintained in overow containers lled with conditioned water (75 g NaHCO3, 18 g sea salt, 8.4 g CaSO4, per 1000 L) and were fed twice daily with live brine shrimps (Artemia salina) supplemented with dry shrimp akes (Brand et al. 2002). On the evening before spawning, male and female sh (ratio = 2:1) were placed in a hatching box. Spawning was triggered once the light was turned on in the next morning and was completed within 1 h. Viable eggs were collected and rinsed at least three times with E3 medium. E3 medium was the standard hatchery water for zebrash eggs (Brand et al. 2002), containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, pH 7.27.3, dissolved oxygen [6.3 mg/L, total hardness: 65 mg/L (as CaCO3), temperature: 28 1C. All of chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd, and were of analytical grade. In order to ensure developmental synchronization at the beginning of exposure, the embryos at the blastula stage of about 33.5 h post fertilization (hpf) were sorted under a stereo microscope and subjected to nano-ZnO exposure. Preparation and characterization of nano-ZnO suspensions ZnO nanoparticles (30 nm) used in this work were purchased from Hangzhou Wanjing New Material Co., Ltd. Its impurities (e.g., cadmium, nickel, copper, lead, manganese, chromium) were determined by ICP-MS (X7 ICP-MS, Thermo Electron Corp., Waltham, USA). The analytical results indicated that the purity was better than 99.9%. The size of nano-ZnO powder was measured by the scanning electron microscope (SEM, Hitachi S-4800, Tokyo, Japan). The stock suspension of nano-ZnO (100 mg/L) was prepared freshly by directly adding ZnO nanoparticles

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into E3 medium and dispersed by sonication (50 W, 40 kHz) for 30 min in an ice-water bath. A series of exposure suspensions (1, 5, 10, 25, 50, and 100 mg/L) were prepared by stepwise dilution with E3 medium. In order to characterize the properties of nano-ZnO in E3 medium, each dose of nano-ZnO suspensions was sampled at 0 and 24 h for analysis of size distribution, solubility, and pH. Size distribution was measured by dynamic light scattering (DLS, COMP ZL 380). Sampled nano-ZnO suspensions were centrifuged at 100,000g for 30 min, and the supernatant was taken for determination of dissolved zinc ions by ICP-MS. Here, strictly speaking, the dissolved Zn species (Zn(dis)) was not pure Zn2?, which was discussed in the later text. Based on the solubility results, contrastive Zn2? exposure experiment was performed, and Zn2? solutions with a series of concentrations (0.598, 2.152, 3.629, 4.066, 5.311, and 6.305 mg/L) were prepared by adding a certain quantity of ZnSO47H2O to E3 medium. The pH value of each concentration of nano-ZnO suspension was measured at 0 and 24 h (Sartorius pH Meter, PB-10, Germany). Embryo toxicity study The assay was mainly based on the embryo test procedure developed by Schulte and Nagel (1994). In brief, 24 eggs were transferred into a 24-well multiplates with one embryo per well. Twenty wells were lled with 2 mL of nano-ZnO suspension, and the remaining four wells were lled with 2 mL E3 medium to act as controls. All of 24-well multi-plates were covered with transparent plastic lm and placed in an illumination incubator at 28 1C with a 14/ 10 h light/dark photoperiod. The Zn2? exposure experiments were conducted in the same way as nano-ZnO exposure. Nano-ZnO suspensions and Zn2? solutions were renewed at 24-h intervals. The direct observation was performed in the well under a stereo microscope connected to a camera device at specied time points (8, 24, 32, 4896 hpf). The toxicological endpoints included mortality, gastrula development, tail detachment, eyes, somite formation, spontaneous movement in 20 s, circulatory system, heart frequency, pigmentation, otic capsule, hatching rate, malformations, and body length of larvae. After 96 hpf, the chorion was carefully removed from the unhatched eggs with small nipper. The larvae were positioned on the lateral side and

photographed. The tail curvature (lordosis, cyphosis) was estimated by drawing a straight line along the margin of the back axis, as described by Fraysse et al. (2006). The body length of larvae was measured using digital image analysis (Scion Image, ver. 4.0.3.2). The median hatching time (HT50) was calculated using Sigmoid Fit by Origin 8.0 (Origin Lab, USA). All experiments were repeated at least six times independently. Statistical analysis Statistical analysis was performed using the statistical package SPSS 10.0 for Windows (SPSS Inc., Chicago, USA). Data are presented as the average SEM. The data were tested for homogeneity and normality. If these assumptions were met, oneway analysis of variance (ANOVA) was performed, and otherwise, the non-parametric KruskalWallis test was performed. Signicance level was set at p \ 0.05.

Results Size distribution of nano-ZnO SEM image of nano-ZnO powder (Fig. 1) shows that most nano-ZnO powders were spherical and short-rod shape. The average size was 36.7 11.6 nm, and the particle sizes were basically homogeneous except individual large particles (80100 nm). The size distributions of nano-ZnO in E3 medium at 0 and 24 h were measured by DLS, as listed in Table 1. Generally, DLS measurement provides the hydrodynamic diameter of the particles suspended in water, named as effective size. At 0 h, the effective size of nano-ZnO at 1 mg/L was 47.3 12.9 nm, which was relatively close to the powder size. At the concentrations of 5100 mg/L, the effective sizes of nano-ZnO (47.31002.0 nm) increased with increasing concentrations, and they were obviously larger than the powder size. This phenomenon indicated that the increased concentrations led to a decrease in the dispersion of nano-ZnO particles in E3 medium. In addition, at 24 h, small nano-ZnO aggregates (142.4517.7 nm) were suspended in water, and the effective sizes of each concentration of nano-ZnO were found to be larger than those at 0 h, as shown in

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ZnO suspension, pH values remained constant at 0 and 24 h. Embryotoxicity of nano-ZnO and Zn2? The toxicity of nano-ZnO and Zn2? to zebrash embryos was studied by observing specic toxicological endpoints during the 96-hpf period. At 96 hpf, the embryonic mortalities induced by exposure to 50 and 100 mg/L nano-ZnO were recorded as 28.3 14.5% and 65.0 8.9%, respectively. Zn2? exposure did not lead to the embryo death over a concentration range of 0.598 to 6.305 mg/L. The hatching rates of zebrash embryos at 96 hpf are shown in Fig. 4(a). All of embryos hatched successfully in the control group. The hatching rates of embryos exposed to nano-ZnO decreased with the increasing concentrations of 110 mg/L. Zn2? at 0.598 mg/L had no effect on the hatching rate of embryos, but the increasing concentrations of Zn2? signicantly decreased the hatching rates. Comparative experiments showed that the hatching rate of embryos exposed to 10 mg/L nano-ZnO was markedly lower than that exposed to 3.629 mg/L Zn2?. In addition, no embryo hatched after exposure to nanoZnO over the concentration rage of 25 to 100 mg/L, suggesting that nano-ZnO led to a more severe inhibition of the embryonic hatch than Zn2? in the corresponding concentration range of 4.066 to 6.305 mg/L. Figure 4b illustrates the cumulative hatching rates of zebrash embryos from 48 to 96 hpf. Table 2 lists the median hatching time (HT50) calculated from the cumulative hatching rates in Fig. 4b. When the concentration of nano-ZnO was higher than 10 mg/L, HT50 was not calculated because of the lower hatching rates or no hatching. Figure 4b shows that cumulative hatching rates decreased with the increasing concentration of nano-ZnO in a dose-dependent pattern. Table 2 shows that nano-ZnO/Zn2? exposure induced a signicant increase in HT50, except 1 mg/L nano-ZnO and 0.598 mg/L Zn2?.

Fig. 1 SEM image of nano-ZnO powder

Table 1. Large aggregates ([1 lm) were visually deposited on the bottom of 24-well plates and were adsorbed to the outer surface of embryonic chorion with increasing concentrations of nano-ZnO, as illustrated in Fig. 2. Solubility of nano-ZnO Figure 3 shows the contents of Zn(dis) in different concentrations of nano-ZnO suspensions at 0 and 24 h determined by ICP-MS. Zn(dis) content of each concentration of nano-ZnO suspension at 24 h was higher than that at 0 h, and the dissolution of nanoZnO showed different change at 0 and 24 h. At 0 h, Zn(dis) contents increased quickly from 0.405 to 2.008 mg/L in the concentration range of 1 to 10 mg/L and then kept constant in the concentration range of 25 to 100 mg/L. At 24 h, however, Zn(dis) contents increased from 0.691 to 3.674 mg/L in the concentration range of 1 to 10 mg/L and then continued to increase to 6.074 0.202 mg/L with increasing concentrations of 25 to 100 mg/L. The pH values of nano-ZnO suspensions were measured at 0 and 24 h. The pH values increased from 7.13 to 7.60 with the increased nano-ZnO concentrations. For a certain concentration of nano-

Table 1 The size range and its cumulative intensity of nano-ZnO at different concentrations in E3 medium by DLS (n = 3, unit: nm) 1 mg/L 0h 24 h 47.3 12.9 142.4 36.8 5 mg/L 116.1 23.1 190.7 94.8 10 mg/L 101.4 26.4 260.8 41.9 25 mg/L 126.5 26.0 370.2 137.9 50 mg/L 377.1 310.3 487.0 433.1 100 mg/L 1002.0 259.2 517.7 374.6

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J Nanopart Res (2010) 12:16451654 Fig. 2 Photos of zebrash embryos exposed to different concentrations of nano-ZnO suspensions taken at 24 h. Amplication: 930

1649

Exposure to nano-ZnO shortened the body length of larvae and induced tail malformation, as shown in Figs. 5 and 6. Though nano-ZnO (\5 mg/L) and Zn2? (0.598 mg/L) did not affect the body length of larvae, both nano-ZnO ([10 mg/L) and Zn2? ([2.152 mg/L, except 4.066 mg/L) led to a

signicant decrease in body length of larvae. Comparative experiments indicated that nano-ZnO ([25 mg/L) induced a more severe decrease in body length of larvae than corresponding concentrations of Zn2? ([4.066 mg/L). In addition, no abnormality was observed in other developmental endpoints in the

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treated embryos, such as tail detachment, eyes, somite formation, heartbeat, circulatory system, pigmentation, otic capsule, etc.

Discussion Aggregation of nano-ZnO The aggregation of nanoparticles in water is an important factor in assessing their toxicity. The formation of aggregates leads to a change in size distribution of nanoparticles in water. Besides, the particle concentration and exposure time may affect the size distribution of nanoparticle. The present results demonstrated that the dispersion of nano-ZnO

Fig. 3 Content of Zn2? in the supernatant determined by ICPMS at 0 and 24 h (n = 3)

Fig. 4 a Effects of nanoZnO/Zn2? on hatching rates of zebrash embryos at 96 hpf (n = 6, * p \ 0.05). b Effects of nano-ZnO/ Zn2? on hatching rates of zebrash embryos from 48 to 96 hpf (n = 6, * p \ 0.05)

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J Nanopart Res (2010) 12:16451654 73.6 7.1*

1651

6.305 5.311 4.066

65.3 8.1*

67.9 14.2*

58.2 1.6*

Fig. 5 Effects of nano-ZnO/Zn2? on the length of larvae (n C 10, * p \ 0.05)

3.629

Median hatching time (HT50) of zebrash embryos at different concentrations of nano-ZnO or Zn2?

Note: All values are presented as the mean standard deviation

71.1 14.8*

in E3 medium was signicantly reduced at high concentrations, and nano-ZnO aggregates were formed and their effective sizes exceeded the dened nanoscale range (0.1100 nm). Moreover, the quantity of nano-ZnO aggregates increased with the exposure time. The aggregation might be ascribed to two reasons. First, no dispersant was added into the nano-ZnO suspension. Second, E3 medium was essentially a multivalent inorganic salt solution, and these ions could lead to the compression of the electrical double layer on the surface of nano-ZnO particle, which was responsible for the stability of nanoparticles (Zhang et al. 2008). Similar phenomenon was reported in the literatures (Adams et al. 2006; Franklin et al. 2007; Zhu et al. 2008b). Solubility of nano-ZnO The solubility of nanoparticles (especially metal/ metal oxide nanoparticles) in water plays an important role in the evaluation of their toxicity. Metal ions released from the nanoparticles possibly contribute, at least in part, to their toxicity (Brunner et al. 2006). Now only a few literatures investigated the solubility of nano-ZnO, and the results were discrepant due to the different experimental conditions, such as size, the suspending medium, pH, ultrasonic parameters, etc. (Franklin et al. 2007; Lin and Xing 2007; Wang et al. 2008; Yang and Xie 2006; Zhu et al. 2008b). The present study demonstrated that nano-ZnO was partially dissolved in E3 medium. Degen et al. reported that when ZnO particles (size: 1.18 mm)

Zn2? (mg/L)

C10 Nano-ZnO (mg/L) 5

52.7 0.7

58.3 7.2

0.598

63.0 10.1*

2.152

* p \ 0.05 versus the control group; (n = 6)

1 Concentration Control

Table 2

HT50 (h)

52.4 1.2

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1652 Fig. 6 Representative images of the a normal and b malformed larvae

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were immersed in water, their surface was hydrolyzed and a layer of hydroxide (Zn(OH)2(S)) was built up, and Zn(OH)2(S) was in equilibrium with the solution which contains Zn(dis) that could be represented by Zn2? , Zn(OH)? , and Zn(OH)2(aq) over (aq) (aq) the pH range 7 to 8 (Degen and Kosec 2000; Reichle et al. 1975). The possible reaction was shown as follows. ZnOH2S $ ZnOH2aq ZnOH2S $ Zn2 2OH aq ZnOH2aq $ ZnOH OH aq ZnOH $ Zn2 OH aq aq 1 2 3 4

any case, the aggregation of nanoparticles in water was a complicated issue and needed further studies. Embryonic toxicity For carbon-based nanoparticles, the dissolution does not occur without considering the residual impurities in preparation process. For metal-based nanoparticles, however, especial for nanoparticles compose of soluble metals, it is essential to take into account the effect of solubility on the toxicity of metal/oxide nanoparticles. Though the toxicity of nano-ZnO has been widely studied, its exact toxic mechanism is still unclear. The toxicity of nano-ZnO is, at least in part, ascribe to its solubility in water (Franklin et al. 2007; Gojova et al. 2007; Lin and Xing 2007; Lin et al. 2009; Zhu et al. 2008b). The generation of ROS is thought to be another reason for the toxicity of nano-ZnO, but it is still an open question. The toxicity of carbon-based nanoparticles to zebrash embryos is possibly due to the ROS (Cheng et al. 2007; Usenko et al. 2007; Zhu et al. 2007). In addition, Franklin et al proposed that the toxicity of nano-ZnO might be similar to that of nano-TiO2 because the band gap of ZnO (3.3 eV) was very close to TiO2 anatase (Franklin et al. 2007; Strehlow and Cook 1973). Generally, the toxicity of nano-TiO2 is ascribed to the ROS (Federici et al. 2007; Long et al. 2007). However, Adams et al. (2006) found that bacterial growth inhibition induced by nano-ZnO was observed with or without the light and proposed that the toxicity of nano-ZnO was not directly linked to the photocatalytic ROS production, and undetermined mechanisms were responsible for toxicity. Similar opinion was proposed by Zhu et al (Zhu et al. 2008b).

The above discussion suggests that nano-ZnO suspension was actually a three-component system containing small nano-ZnO aggregates suspended in water, dissolved Zn(dis) and large nano-ZnO aggregates deposited on the container bottom. These components might jointly exert inuences on zebrash embryos. Undoubtedly, Zn(dis) could easily penetrate into the eggs across the pore canals. Could nano-ZnO aggregates enter into eggs? For small nano-ZnO aggregates, their sizes were smaller than the chorion pore diameter (0.50.7 lm), so they might permeate into zebrash eggs directly through the pore canals. As for the large aggregates absorbed to the chorion, their sizes far exceeded the diameter of the pore canals, so the direct penetration was impossible. However, if the large aggregates were loosely formed, the aggregates could be easily separated and passed through the chorion. In addition, another possibility was that the pore canals of the chorion were jammed by large aggregates, so that no more nano-ZnO or Zn(dis) could enter into the eggs. In

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1653 zebrash strains. This research is supported by National Natural Science Foundation of China (Grant No. 10505024, 20707027, 10875136), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KJCX3.SYW.N3), and the Ministry of Science and Technology of China (Grant No. 2006CB705605).

Hatching retardation of zebrash embryos might be due to disturbance of the hatching enzyme and hypoxia induced by nano-ZnO. During the normal hatching process of teleost embryos, the chorion is digested by the hatching enzyme, which is a proteolytic enzyme secreted from hatching gland cells of the embryo. The hatching enzyme contains two constituent proteases: choriolysin H (HCE) and choriolysin L (LCE), which belong to the astacin protease family, a subfamily of zinc-proteases (Inohaya et al. 1997). Unfortunately, the transitory existence of the hatching enzyme made it difcult to observe the possible effect of chemical pollutant on its activity. Whereas, the structure and function of the zinc-protease were possibly disturbed to some degree by nano-ZnO and Zn(dis). In addition, as mentioned above, large nano-ZnO aggregates might block the pore canals of the chorion, resulting in the shortage of oxygen supply essential to the development of embryos, namely hypoxia, being similar to carbon nanotubes (Cheng et al. 2007). In summary, the physicochemical properties of nano-ZnO in the standard hatchery water for zebrash eggs (E3 medium) were in detail characterized, such as size distribution, solubility, and pH. Nano-ZnO particles in E3 medium readily formed aggregates with different sizes, and the quantity of aggregates increased with increasing concentrations. Nano-ZnO in E3 medium was partially dissolved to Zn(dis) represented by Zn2?, Zn(OH)?, and Zn(OH)2. Nano-ZnO suspension was actually a three-component system containing small nano-ZnO aggregates (142.4517.7 nm) suspended in water, dissolved Zn(dis), and large nano-ZnO aggregates ([1 lm) deposited on the container bottom. These components might jointly exert inuences on zebrash embryos. The embryo toxicity test revealed that nano-ZnO killed zebrash embryos (50 and 100 mg/L), retarded the embryo hatching (125 mg/L), reduced the body length of larvae, and caused tail malformation after the 96-hpf exposure. Comparative experiments showed that nano-ZnO at high concentrations of 10100 mg/L led to a more severe inhibition of embryonic development than the corresponding concentrations of Zn2? ([3.629 mg/L), suggesting that dissolved Zn species only partially contributed to the toxicity of nano-ZnO.
Acknowledgments The authors thank the National Zebrash Resources of China, Peking University, for kindly providing

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