Toxicity of ZnO Nanoparticles to Escherichia coli: Mechanism and the

Jan 31, 2011 - The generation of precipitates (Zn3(PO4)2 in PBS) and zinc complexes (of zinc with citrate and amino acids in MD and LB, respectively) ...
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Toxicity of ZnO Nanoparticles to Escherichia coli: Mechanism and the Influence of Medium Components Mei Li,†,‡ Lizhong Zhu,*,†,‡ and Daohui Lin†,‡ † ‡

Department of Environmental Science, Zhejiang University, Hangzhou, 310058, China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou, 310028, China

bS Supporting Information ABSTRACT: Water chemistry can be a major factor regulating the toxicity mechanism of ZnO nanoparticles (nano-ZnO) in water. The effect of five commonly used aqueous media with various chemical properties on the toxicity of nano-ZnO to Escherichia coli O111 (E. coli) was investigated, including ultrapure water, 0.85% NaCl, phosphate-buffered saline (PBS), minimal Davis (MD), and Luria-Bertani (LB). Combined results of physicochemical characterization and antibacterial tests of nano-ZnO in the five media suggest that the toxicity of nano-ZnO is mainly due to the free zinc ions and labile zinc complexes. The toxicity of nano-ZnO in the five media deceased as follows: ultrapure water > NaCl > MD > LB > PBS. The generation of precipitates (Zn3(PO4)2 in PBS) and zinc complexes (of zinc with citrate and amino acids in MD and LB, respectively) dramatically decreased the concentration of Zn2þ ions, resulting in the lower toxicity in these media. Additionally, the isotonic and rich nutrient conditions improved the tolerance of E. coli to toxicants. Considering the dramatic difference of the toxicity of nano-ZnO in various aqueous media, the effect of water chemistry on the physicochemical properties of nanoparticles should be paid more attention in future nanotoxicity evaluations.

’ INTRODUCTION With the rapid development of nanotechnology, a diverse range of nanomaterials and nanoproducts are emerging.1 ZnO nanoparticles (nano-ZnO), as one common engineered nanomaterial, have been widely used in many fields such as sunscreen products, textiles, paintings, industrial coatings, and antibacterial agents.2,3 Despite the excellent advantages, the ecological risk arising from their release during the production and application process has received increasing attention in many reports. NanoZnO has been found to be toxic to algae,2,4,5 crustaceans,6-8 fish,5 bacteria,9-13 nematodes,14 and plants15,16 at various levels. However, the toxicity mechanism of nano-ZnO is still controversial. The generation of reactive oxygen species (ROS) was once considered as the main cause of the nanotoxicity9-13 but lacked solid supporting evidence. Recently, many studies showed that the released zinc ions from nano-ZnO were responsible for the nanotoxicity.2,4-7,15-18 The toxicity of nano-ZnO correlated well with the concentration of the free hydrated Zn2þ ions or labile zinc complexes.2 Other studies ascribed the toxicity of nano-ZnO to both the dissolved zinc ions and the attachment of nanoparticles or their aggregates to the organism.19-22 The main toxicity mechanism of nano-ZnO may be different in various media because the physicochemical properties of nano-ZnO and the species of dissolved zinc can be changed by the medium components. Bacteria are the foundation of aquatic ecosystems and are very sensitive to low concentrations of toxic substances. Toxicity of nano-ZnO to bacteria has caused many concerns. The reported antibacterial effects of nano-ZnO differed in different studies3,6,9,10,22,23 with the inhibition concentration ranging from several to hundreds of mg L-1 (Table S1, Supporting Information). r 2011 American Chemical Society

The test condition was possibly the main reason for the different levels of toxicity, especially the medium used in the toxicity test. The chemical properties of the medium such as pH, ionic compositions, ionic strength (IS), and organic matter can affect the aggregation, surface charge, and chemical forms of nanoparticles. Some researchers found that the dispersion of TiO2 nanoparticles, the aggregation, and dissolution of Ag nanoparticles were influenced by the concentration of protein and ionic composition in the culture media.24,25 In addition, bacteria have a higher tolerance to toxicants under isotonic condition.14,26 Although the water chemistry is a crucial factor influencing the toxicity of nano-ZnO, very few related studies are available. In most studies, the toxicity of nano-ZnO was evaluated in the culture medium, in which the toxicity could be dramatically different from that in natural waters. The effect of the test medium on the physicochemical properties and the toxicity of nano-ZnO should be paid more attention. In this study, five commonly used aqueous media were chosen to investigate the potential effect of water chemistry on the toxicity of nano-ZnO to Escherichia coli O111 (E. coli). E. coli O111 was chosen as the test organism because it is a common bacterium (also pathogen) in water and widely used in toxicity studies. The results of this study are expected to help to understand the antibacterial mechanism of nano-ZnO and other nanotoxicity evaluations. Received: August 2, 2010 Accepted: January 4, 2011 Revised: December 15, 2010 Published: January 31, 2011 1977

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’ MATERIALS AND METHODS Materials. Nano-ZnO and bulk-ZnO were purchased from Hongsheng Material Sci & Tech Co., Zhejiang, China. Their properties are listed in the Supporting Information (Table S2). ZnSO4 3 7H2O (AR, Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) was used as the source of Zn2þ. E. coli O111 (Genbank access no. GU237022.1) isolated from sewage water was used as the test organism. The bacterium was kept in a LB solid plate at 4 °C in a refrigerator. Five aqueous media with different components were chosen as the test media, including (i) ultrapure water, (ii) 0.85% (w/v) NaCl solution, (iii) 5 mM phosphate-buffered saline (PBS), (iv) minimal Davis medium (MD),27 and (v) Luria-Bertani medium (LB), which contains 10 g L-1 NaCl, 10 g L-1 tryptone, and 5 g L-1 yeast extract. The water chemistry of the five aqueous media is shown in Table S3. Characterization of Nano-ZnO in Aqueous Media. The stock suspensions of 500 mg L-1 nano-ZnO were prepared by sonication (300 W) for 20 min in the corresponding filtrationsterilized aqueous media. Different concentrations of nano-ZnO suspensions were obtained by diluting the stock suspensions with the aqueous media. Then, the samples were kept on a constant temperature shaker incubator (150 rpm) at 25 °C overnight to reach relative dissolution equilibrium (Figure S1, Supporting Information) before the following characterizations. High-resolution transmission electron microscopy (HRTEM, JEM 2010, JEOL Ltd., Japan) was used to observe the morphologies of nano-ZnO (100 mg L-1) in ultrapure water, NaCl, and PBS solutions. The hydrodynamic diameters and zeta potentials of particles (50 and 100 mg L-1) in the five media were determined with a Zetasizer (Nano-ZS, Malvern Instruments, U.K.). The precipitates in PBS, MD, and LB media with nano-ZnO or Zn2þ ions were collected, thoroughly washed, freeze dried, and analyzed with Fourier transform infrared (FTIR, Bruker Vector 22, Germany) spectra, with nano-ZnO and Zn3(PO4)2 as controls. Zinc Concentration Determination and Species Analysis. The samples were centrifugated at 10 000g for 30 min and then filtered with a 0.22 μm mixed cellulose ester membrane. Zinc remaining in the supernatants was considered as the dissolved zinc,16,18,22 which was determined after digestion with 0.2% HNO3 using atomic absorption spectrometry (AAS, AAnalyst 700, Perkin-Elmer, USA) at 213.9 nm. Also, the dissolved zinc data were compared with those obtained by centrifugal ultrafiltration with a 3 kDa membrane. Considering the effect of the test temperature and pH change on the dissolution of ZnO, the dissolved zinc concentrations before and during the antibacterial tests were determined. The species of dissolved zinc of 50 mg L-1 nano-ZnO in the test media were calculated with Visual MINTEQ (version 2.61).28 For ultrapure water and NaCl solution, the determined dissolved zinc concentrations were used in the model to calculate the species of zinc. Nano-ZnO in PBS and MD media was assumed to dissolve into Zn2þ first and then react with phosphates and Na citrate, and the total zinc concentrations were used in the model. Antibacterial Tests. Considering the difference of the aqueous media, two types of antibacterial tests were used. For nutrient-free media (ultrapure water, 0.85% NaCl, and PBS solution), where bacteria could not grow and their activity would reduce with time, 3 h antibacterial tests were carried out. Twelve hour growth inhibition tests were used to characterize the toxicity of nano-ZnO to bacteria in MD and LB media with rich nutrients. In addition, the effect of components of MD and LB media on

Figure 1. Equilibrium solubility of nano-ZnO with different initial concentrations in various aqueous media. Error bars are standard deviations, n = 3.

the toxicity of nano-ZnO (50 mg L-1) was examined with 3 h antibacterial tests. The concentration of each component was the same as that in MD and LB media. The methods for the 3 and 12 h tests are detailed in the Supporting Information. TEM Analysis of E. coli Cells. The E. coli was exposed to nano-ZnO or Zn2þ (50 mg L-1) for 3 h on a shaker incubator (150 rpm) at 37 °C; then the ultrastructural characteristics of E. coli were observed using the TEM (JEM-1230, JEOL Ltd., Japan) with a method detailed in the Supporting Information.

’ RESULTS Characteristics of Nano-ZnO in Aqueous Media. TEM images show the morphologies of nano-ZnO in ultrapure water, NaCl, and PBS solutions (Figure S2, Supporting Information). The shape of pristine nano-ZnO was sphere-like with a mean diameter measured (using TEM) to be 19 ( 7 nm (n = 152) (Table S2, Supporting Information). Clear crystal lattices were observed for the nano-ZnO in ultrapure water and NaCl solution (Figure S2b and S2d, Supporting Information). However, dendritic solids with very few crystal lattices and no primary spherelike particles were observed in PBS solution (Figure S2e and S2f, Supporting Information), which suggested that nano-ZnO had been transformed into other chemical forms, such as Zn3(PO4)2. The hydrodynamic diameter could indicate the aggregation state of nano-ZnO in aqueous media. Nano-ZnO (50 and 100 mg L-1) was poorly dispersed, readily aggregated, and precipitated in the test media without shaking after sonication, except for LB where 50 mg L-1 nano-ZnO dissolved completely, which was confirmed by determination of dissolved zinc (Figure 1). The order of hydrodynamic diameter of nano-ZnO (100 mg L-1) in the test media was PBS > NaCl (1439 nm) > MD (1160 nm) > LB (692 nm) > ultrapure water (486 nm) (Table S4, Supporting Information). Large precipitates formed in PBS, and the sizes were out of the measuring range of the zetasizer. Zeta potential indicates the surface charge of the particles. The zeta potentials of particles (50 and 100 mg L-1) in ultrapure water were positive. The ions present in other media decreased the zeta potentials of particles to various levels (Table S4, Supporting Information), especially for MD where more anions such as PO43- and SO42- may be adsorbed on the surface of particles.29-31 FTIR spectra of nano-ZnO untreated or treated with PBS, MD, and LB are shown in Figure S3, Supporting Information. 1978

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Table 1. Zinc Species and Their Concentrations in Aqueous Media with 50 mg L-1 Nano-ZnO As Calculated Using Visual MINTEQ (pH was assumed to be 7.0) ultrapure water zinc species

NaCl -1

%

mg L

PBS -1

%

mg L

MD -1

%

mg L

mg L-1

%

dissolved zinca

100

6.91 ( 0.09

100

14.07 ( 1.79

100

0.35 ( 0.13

100

14.61 ( 0.97

Zn2þ

99.00

6.84

86.72

12.20

63.46

0.22

0.55

0.08

ZnOHþ Zn(OH)2 (aq)

0.89 0.11

0.06 0.01

0.39 -

0.05 -

-

-

-

-

ZnClþ

N

N

11.79

1.66

8.51

0.03

N

N

ZnCl2

N

N

0.90

0.13

-

-

N

N

ZnHPO4

N

N

N

N

26.97

0.09

0.56

0.08

Zn-Citrate-

N

N

N

N

N

N

90.57

13.23

Zn-(Citrate)24-

N

N

N

N

N

N

7.99

1.17

a The total dissolved zinc concentration was determined in the tests. N means no content. - means minimal content. The dissolved zinc in LB could be the zinc complexes with amino acids and polypeptides, but they could not be calculated with the model because of the complexity of organic components.

The peak at 450-460 cm-1 is the stretching vibration of the Zn-O bond in ZnO particles.12 The broad absorption bands at 3420-3440 cm-1 in all samples are possibly assigned to the stretching vibration of the hydroxyl group, which may come from the adsorbed H2O.12 Also, the peaks at 1500-1510 and 13901410 cm-1 can be seen in all the samples containing nano-ZnO, which are attributed to the symmetric and asymmetric O-C-O stretching vibration of the adsorbed carbonate anion during the preparation process of nano-ZnO.32,33 The peaks at 10401050 cm-1 from the stretching vibration of P-O34 were observed for nano-ZnO treated with PBS, MD, and LB but not for the pristine nano-ZnO (Figure S3, Supporting Information). It indicates that phosphates in the media have reacted with zinc or attached to the surface of ZnO particles strongly. Moreover, the peak of the Zn-O bond is much weaker for 100 mg L-1 nanoZnO than that for 500 mg L-1 nano-ZnO in PBS, and its FTIR spectrum is similar to that of Zn3(PO4)2 (Figure S3b, Supporting Information), which indicates that most of the ZnO particles at 100 mg L-1 have been transformed into Zn3(PO4)2. In contrast, there is no significant difference in the relative intensity of the Zn-O bond for various concentrations of nano-ZnO in MD and LB media (Figures S3c and S3d, Supporting Information). The peaks at 1636, 1403, and 1069 cm-1 in tryptone and yeast extract are, respectively, assigned to the stretching vibration of CdO, C-H, and P-O. CdO and C-H are from amino acids and polypeptides, while P-O is from nucleic acids containing phosphates. These peaks are also present in nano-ZnO treated with LB medium, which indicates the above organic molecules have reacted with zinc or attached to the surface of ZnO particles. Dissolution of Nano-ZnO and Chemical Species of Zinc. The dynamic dissolution curves of nano-ZnO and bulk-ZnO in ultrapure water were nearly the same (Figure S1, Supporting Information). Nano-ZnO and bulk-ZnO dissolved rapidly in the first 6 h and reached the equilibrium at the 10th hour. The dissolution rate was similar to the reported data.2 In addition, there was no significant difference between the dissolved zinc determined after filtration with a 0.22 μm membrane and that with a 3 kDa membrane. Figure 1 shows the equilibrium solubility of nano-ZnO at different concentrations in the five media. The dissolved zinc concentration initially increased with increasing nanoZnO in the test media except PBS and then leveled off. The maximum dissolved zinc concentration of nano-ZnO (0-500 mg L-1) varied

with the test media with an order of LB (68 mg L-1) > MD (38 mg L-1) > NaCl (14 mg L-1) > ultrapure water (6.9 mg L-1) > PBS (0.57 mg L-1). The effect of temperature and pH on the dissolution of nanoZnO during the antibacterial tests was also investigated (Figure S4, Supporting Information). Surprisingly, the dissolved zinc concentration of nano-ZnO (>10 mg L-1) in ultrapure water and NaCl solution decreased when the temperature increased from 25 to 37 °C. The maximum dissolved zinc concentration decreased to about 5 and 10 mg L-1 after 3 h in ultrapure water and NaCl solution, respectively. However, the dissolved zinc in PBS was not influenced by the increasing temperature. The growth of bacteria treated with nano-ZnO in MD or LB changed the pH little, and there was no significant change in dissolved zinc concentration. Table 1 shows the chemical species of dissolved zinc for 50 mg L-1 nano-ZnO in aqueous media except LB. Zn2þ ions were the main dissolved zinc species in ultrapure water, NaCl, and PBS solution, whereas it was Zn citrate- in MD, in which the Zn2þ and labile zinc complexes (ZnSO4, ZnHPO4) accounted for approximately 1% of dissolved zinc. Although the dissolved zinc species in LB medium could not be calculated because of the complexity of the organic components, the main dissolved zinc species were speculated to be complexes of zinc with amino acids and polypeptides from tryptone and yeast extract. Toxicity of Nano-ZnO to E. coli in the Aqueous Media. Figures 2 and 3 show the toxicity of nano-ZnO with various initial concentrations to E. coli in the five media. In order to investigate the toxicity mechanism of nano-ZnO, the toxicity of bulk-ZnO and Zn2þ ions in ultrapure water was evaluated simultaneously. It was observed that the dose-effect curves of nano-ZnO, bulkZnO, and Zn2þ ions were almost the same (Figure 2a). They all exhibited strong toxicity to E. coli with 3 h LC50 (concentration resulting in 50% mortality) lower than 0.1 mg Zn L-1. The bacterial mortality all exceeded 90% at concentrations of zinc higher than 1.0 mg L-1. The 3 h LC50 of nano-ZnO in NaCl solution was 1-5 mg L-1, and the 3 h LC90 was up to 10 mg L-1. No significant dose-effect relationship was observed for nano-ZnO in PBS solution, and the bacterial mortality was lower than 20% even for 50 mg L-1 nano-ZnO (Figure 2b). In MD and LB media, growth of E. coli was inhibited when the initial concentration of nano-ZnO was higher than 50 1979

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Figure 2. Toxicity of nano-ZnO to E. coli in (a) ultrapure water and (b) 0.85% NaCl and 5 mM PBS solutions. Error bars are standard deviations, n = 3.

Figure 3. Growth curves of E. coli in (a) MD and (b) LB media containing different initial concentrations of nano-ZnO. Error bars are standard deviations, n = 3.

and 100 mg L-1, respectively (Figure 3). However, the bacteria could still reach the stationary growth stage at concentrations of nano-ZnO less than 200 mg L-1. When the concentrations of nano-ZnO in MD and LB media increased to 500 mg L-1, the number of E. coli hardly increased within 12 h.

-33 3Zn2þ þ 2PO34 S Zn3 ðPO4 Þ2 Ksp ¼ 9:0  10 11:4 Zn2þ þ C6 H5 O37 S ZnC6 H5 O7 Kf ¼ 10

’ DISCUSSION

ZnO þ Hþ S Zn2þ þ OH-

ð1Þ

Zn2þ þ Cl -S ZnClþ

ð2Þ

ð4Þ

Zn2þ þ R-CHðNH2 Þ-COO- S R-CHðNH2 Þ-COO-Znþ

Effect of Medium Components on Physicochemical Properties of Nano-ZnO. In general, the aggregation and stability of

particle dispersions are controlled by the sum of attractive and repulsive forces between particles. Increasing ionic strength or decreasing surface charge would decrease the repulsive force and consequently increase the aggregation and hydrodynamic size of nanoparticles.35 Also, the surface charge of particles is mainly controlled by surface ionization and adsorption of multiply charged ions.29 In ultrapure water, the aggregation of ZnO particles could be attributed to the weaker electrostatic repulsive forces among particles under lower surface charges, whereas the larger hydrodynamic diameter in 0.85% NaCl solution was attributed to its higher ionic strength (0.145 M). In PBS solution, the generation of Zn3(PO4)2 was probably responsible for the occurrence of large precipitates. In addition to the physical properties, the chemical properties of nano-ZnO can also be influenced by the medium components. The possible main chemical reactions in the test media are shown by eqs 1-5

ð3Þ

ð5Þ where Ksp and Kf stand for the solubility product constant and the complexation constant, respectively, and R-CH(NH2)COO- stands for amino acids in LB medium. The solubility of nano-ZnO in ultrapure water was relatively low, and the main zinc species was Zn2þ under neutral condition. The higher dissolved zinc concentration in NaCl solution than that in ultrapure water may be caused by the complexation of Zn2þ with Cl- (eq 2). Phosphates in PBS, MD, and LB media could form Zn3(PO4)2 with zinc released from ZnO, which would reduce the concentration of dissolved zinc in the media (eq 3). Simultaneously, as multiply charged anion, PO43- may be also adsorbed on the surface of ZnO particles to inhibit the dissolution of ZnO. On the other hand, the citrate in MD and the organic components such as amino acids and polypeptides in LB could complex with zinc to enhance the dissolution of ZnO (eqs 4 and 5). Combined with the dissolution curves and FTIR spectra (Figures 1 and S3, Supporting Information), nano-ZnO can dissolve completely at low concentrations in MD and LB media, while ZnO and Zn3(PO4)2 are likely to coexist at high concentrations. 1980

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Possible Toxicity Mechanism of Nano-ZnO to E. coli in Ultrapure Water. In two recent studies, the toxicity of nano-

ZnO, bulk-ZnO, and Zn2þ ions was found to be similar at the same dissolved zinc concentration and Zn2þ ions dissolved from ZnO were therefore considered as the primary cause for ZnO ecotoxicity.2,18 A similar result was also observed in our study. The 3 h LC50s of nano-ZnO, bulk-ZnO, and Zn2þ ions were all lower than 0.1 mg Zn L-1 in ultrapure water (Figure 2a). Besides, nano-ZnO and bulk-ZnO almost completely dissolved at concentrations lower than 5 mg L-1, and thus, Zn2þ ions were the only toxicity source of nano-ZnO with concentrations lower than 5 mg L-1 in ultrapure water. For higher concentrations of nanoZnO, although a few ZnO particles may attach to the bacterial cells, it was difficult to determine the contribution of nano-ZnO itself considering the high toxicity of coexisting Zn2þ. In addition, the bacteria could release the solutes in response to osmotic down-shock in ultrapure water, resulting in the damage on the normal physiological functions and the decrease of tolerance of bacteria to toxicants.26 Therefore, the toxicity of nano-ZnO at 1 mg L-1 in ultrapure water was much higher than that in 0.85% NaCl solution as shown in Figure S5, Supporting Information. To further verify the toxicity mechanism of nano-ZnO, the ultrastructural characteristics of normal E. coli cells and those treated with ultrapure water, nano-ZnO, and Zn2þ ions were observed with TEM (Figure S6, Supporting Information). The morphologies of E. coli cells treated with nano-ZnO or Zn2þ ions were significantly different from those of normal E. coli cells. It was observed that the cytoplasmic membranes deformed, wherein some cells swelled and the intracellular substances leaked out under both Zn stress and osmotic stress (Figures S6b and S6c, Supporting Information). Combined with the toxicity results of nano-ZnO, bulk-ZnO, and Zn2þ ions in ultrapure water, it was concluded that the toxicity of nano-ZnO to E. coli was mainly attributed to the released Zn2þ ions. Effect of Medium Components on the Toxicity of NanoZnO. The toxicity of metals depends on their species and bioavailability. The colloidal metals and those complexed to strong organic ligands are considered biologically inactive.36,37 Zinc may have various species in different media, which could cause different toxicity to the E. coli in this study. As discussed above, Zn2þ was the main toxicity source of nano-ZnO in ultrapure water. The main species of the dissolved zinc in 0.85% NaCl solution was Zn2þ as shown in Table 1. Compared with those in ultrapure water, the larger aggregates and lower surface charges of ZnO particles in NaCl solution were more unfavorable to the attachment of ZnO particles to bacteria (Table S4, Supporting Information). Therefore, the toxicity of nano-ZnO in NaCl solution was also attributed to the Zn2þ ions. However, the toxicity of nano-ZnO in 0.85% NaCl was significantly lower than that in ultrapure water (Figure 2). The possible reason was that 0.85% NaCl was isotonic, in which the bacteria were more tolerant to zinc ions. Figure S5, Supporting Information, shows the decrease of bacterial mortality induced by 1 mg L-1 nano-ZnO with increase concentration of NaCl ( 0.2). In MD medium, both PO43- and citrate can react with zinc to form Zn3(PO4)2 and the citrate complexes, respectively. Figure 4 shows the effect of phosphates (K2HPO4 and KH2PO4) and sodium citrate on the toxicity of nano-ZnO (50 mg L-1). Similar to that in PBS solution, the phosphates dramatically decreased the toxicity of nano-ZnO. Nano-ZnO (50 mg L-1) dissolved completely in sodium citrate solution because of the strong complexation of zinc with citrate, and the toxicity was similar to that in ultrapure water (Figure 4a). However, no toxicity was observed for 50 mg L-1 nano-ZnO in the presence of sodium citrate and phosphates, where the dissolved zinc was about 12 mg Zn L-1 (Figure 4a). The toxicity of nano-ZnO (0-50 mg L-1) in sodium citrate solution was evaluated. Simultaneously, the species of dissolved zinc were calculated with Visual MINTEQ (data not shown). It was observed the toxicity was consistent with the concentration of Zn2þ, even though its concentration was much lower than Zn-citrate- (Figure S7, Supporting Information). The bacterial mortality was about 90% when the initial concentration of nanoZnO was 50 mg L-1 (Zn2þ 0.1 mg L-1). In MD containing nano-ZnO, besides Zn2þ there were labile complexes such as ZnSO4 and ZnHPO4, which could result in the toxicity to the E. coli. Moreover, MD medium could supply nutrients to bacteria and promote their growth. Many bacteria including E. coli can excrete large amounts of extracellular polymer substances (EPS) 1981

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Environmental Science & Technology during growth to resist toxicity.18,38 Therefore, the E. coli could still survive with low concentrations of nano-ZnO in MD. The concentration of Zn2þ and labile organic complexes increased with the increasing concentration of nano-ZnO and thus resulted in the higher toxicity to E. coli. In LB medium, amino acids and peptides can form complexes with zinc while nucleic acids containing phosphates can form the precipitates with zinc. Figure 4b shows the effect of LB components on the toxicity of nano-ZnO. Tryptone and LB media containing 50 mg L-1 nano-ZnO could still promote growth of bacteria, while the number of bacteria that survived significantly decreased in yeast extract medium. Nano-ZnO (50 mg L-1) completely dissolved in tryptone or LB medium, whereas only two-thirds of nano-ZnO dissolved in yeast extract medium (Figure 4b). Similar to MD medium, free Zn2þ, labile, and stable zinc complexes coexisted in the above three media. It was speculated that the concentration of Zn2þ ions and labile zinc complexes in yeast extract medium was higher than that in tryptone or LB medium. Although the stable zinc complexes were the dominating species of dissolved zinc in LB medium, the concentration of Zn2þ and labile organic complexes increased as nano-ZnO increased. When the concentration of nano-ZnO was lower than 100 mg L-1, the bacteria could resist the toxicity of Zn2þ and labile zinc complexes because the nutrients in LB could promote growth of bacteria. With the further increase of nano-ZnO in LB, the growth of E. coli was inhibited. In summary, the free zinc ions and labile zinc complexes are mainly responsible for the toxicity of nano-ZnO in aqueous media. The medium components such as PO43-, citrate, and organic matter can influence the dissolution of nano-ZnO and change the chemical species of zinc and thus indirectly affect the toxicity. Additionally, the ionic strength and nutrient condition also influence the tolerance of bacteria to toxicants. Therefore, the effect of water chemistry on physicochemical properties of nanoparticles and the physiological functions of organisms should be given more attention in future nanotoxicity evaluations. The mechanism underlying the effect of environmental conditions on the toxicity of nanoparticles warrants more specific investigations.

’ ASSOCIATED CONTENT

bS

Supporting Information. Methods for antibacterial tests and TEM analysis of E. coli cells; Tables S1-S4; Figures S1-S7. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone (fax): 86-571-88273733; e-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (20890111, 20737002, 40973067, 21077089) and Zhejiang Provincial Innovative Research Team of Water Treatment Functional Materials and their Application. Thanks to the anonymous reviewers for their comments which greatly improved the manuscript. ’ REFERENCES (1) Handy, R. D.; von der Kammer, F.; Lead, J. R.; Hassell€ ov, M.; Owen, R.; Crane, M. The ecotoxicology and chemistry of manufactured nanoparticles. Ecotoxicology 2008, 17, 287–314.

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