Distinct biokinetic behavior of ZnO nanoparticles in Daphnia magna quantified by synthesizing 65Zn tracer

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Distinct biokinetic behavior of ZnO nanoparticles in Daphnia magna quantified by synthesizing 65Zn tracer Wei-Man Li, Wen-Xiong Wang* Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

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abstract

Article history:

ZnO nanoparticles (nZnO) are widely used in different fields and there are increasingly

Received 18 August 2012

concerns for their hazards in the environment. The biokinetic behavior of nZnO in aquatic

Received in revised form

organisms however remains essentially unknown. The aim of this study was to separate

5 November 2012

the uptake and depuration behavior of nZnO and Zn ions in a freshwater cladoceran

Accepted 12 November 2012

Daphnia magna. We for the first time radio-synthesized the nZnO and followed its uptake

Available online 20 November 2012

and depuration in D. magna. Two concentrations (0.5 mg/L and 2 mg/L) of nZnO were employed in this study, and the releases of nZnO into soluble Zn were 20e30% during the

Keywords:

experiments. We found that the uptake of nZnO by D. magna was related to nZnO

nZnO

concentration. The uptake of ionic Zn released from nZnO by D. magna followed a linear

Biokinetics

increase during the exposure period (40 min or 8 h). The nanoparticles could enter the body

Daphnia

of daphnids and reached a peak within a short time, followed by a rapid release. Uptake of

Uptake

nanoparticles was mainly by direct ingestion, with negligible nZnO absorption onto the

Depuration

carapace. The depuration of nZnO was also rapid and controlled by nZnO dissolution in the

Radiosynthesis

body of D. magna. Our study showed a distinctive uptake mode of nZnO and suggested that both dissolved Zn and nanoparticles should be considered in studying the toxicology of nZnO. ª 2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Semiconductor nanomaterials are now extensively manufactured and widely used in the fields of electronics, optics and sensors because of their novel properties. ZnO is an important group IIeVI semiconductor with a band gap of 3.37 eV and a large exciton binding energy of 60 meV at room temperature (Wang, 2004). ZnO nanocrystals have considerable potentials as materials for sensors, light-emitting diodes, transparent conductive films, solar cells, and UV blockers (Qi et al., 2008; Klingshirn, 2007; Ko¨nenkamp et al., 2004; Izaki and Omi, 1996; Law et al., 2005; Dhobale et al., 2008). Subsequently, the engineered nZnO will be inevitably discharged into the aquatic environment, and may be accumulated by the organisms through internal or external exposure routes. It is thus

important and imperative to understand the physiochemical and biological behavior of nZnO in the aquatic environment. Different from many other nanomaterials such as nTiO2, multiwalled carbon nanotubes, nCeO2, which are relatively stable in the aquatic environment, nZnO is easily soluble (Adams et al., 2006; Johnston et al., 2010; Saleh et al., 2008). Its solubility is dependent on the pH, natural organic compounds, and the concentration of nanomaterials (Miao et al., 2010). Hence, it is notoriously difficult to investigate the mechanisms underlying the nZnO toxicity. Recently, several studies have focused on the toxicities of nZnO. For example, Lin and Xing (2008) investigated the root uptake and phytotoxicity of nZnO to Lolium perenne, and found that nZnO were able to concentrate in the rhizosphere, entered the root cells, and inhibited the seedling growth of ryegrass. They concluded

* Corresponding author. Tel.: þ(852) 23587346; fax: þ(852) 23581559. E-mail address: [email protected] (W.-X. Wang). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2012.11.018

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that the phytotoxicity of ZnO nanoparticles was not caused primarily by their dissolution in the bulk nutrient solution or the rhizosphere. Some studies attributed the acute toxicities of nZnO to the zinc ions released from the nanoparticles (Wiench et al., 2009; Ma et al., 2009), while others argued that the toxicities of nanoparticles cannot be eliminated because of their small sizes and high surface areas (Wu et al., 2010). In any aquatic toxicity study, a key obstacle is the lack of method to separate Zn ions and nanoparticles in the nZnO solution. As a result, it is important to distinguish the mechanisms of uptake and depuration behavior between nZnO and ionic Zn released from the nanoparticles. The incorporation of Zn into the bodies of aquatic organisms can be either from dissolved uptake or dietary ingestion, but the relative importance of each route is still unclear. Consequently, it is necessary to investigate the biokinetics of nZnO such as the uptake rate constant from the dissolved phase (ku) and the efflux rate constant (ke) in order to quantify the bioavailability of nZnO. One approach to determine the biokinetics is to radiolabel the nanomaterials with the isotopes. For example, Zhao and Wang (2010) quantified the kinetics of a commercially available AgNP in Daphnia magna by radiolabelling the AgNP with 110mAg radiotracer. Carbon-14 is also widely used to radiolabel the carbon nanotubes to investigate the accumulation of carbon nanotubes in the organisms (Petersen et al., 2008a,b; 2009; Ferguson et al., 2008). While for the nZnO, it may be difficult to use the radiolabeling methodology, considering the relatively high solubility and the competition between the increased Zn ions and the radioactive Zn (Brunner et al., 2006; Secco and Moore, 1957). In fact, in our earlier attempt, we used 65Zn to radiolabel the nZnO, but found that almost all the radiolabeled 65Zn was released back into the water following resuspension of these radiolabeled nZnO. Therefore, adding 65Zn into the precursor to synthesize the radioactive nZnO would be more useful and an advanced method. Dybowska et al. (2011) synthesized the isotopically enriched (89.6%) nanoparticles with a rare isotope of 67ZnO and measured the uptake of 67Zn by freshwater snails (Lymnaea stagnalis) exposed to diatoms amended with the particles. In the present study, we therefore quantified the biokinetics (e.g., the uptake and loss) of synthesized nZnO in a sensitive zooplankton Daphnia magna using the radiotracer methodology. Zn is an important essential metal for all organisms and play a critical role in metallic enzymes. The biokinetic process considered in this study included the uptake of nZnO from water and the elimination of nZnO. The biokinetic processes of nZnO and Zn2þ dissolved from nZnO were separated, to provide a better understanding of the uptake and elimination of nZnO by daphnids. This study can help to understand the hazard of nZnO apart from Zn2þ presented in aquatic environment and will supply a premise to thoroughly investigate the toxicities of nZnO to aquatic organisms.

mostly used in the synthesis of nZnO (Dybowska et al., 2011; Lin et al., 2005), while we amended the method to substitute DEG with ethylene glycol (EG), which was much simpler to perform in the laboratory. Zinc acetate dehydrate [Zn(OAc)22H2O] was used as the precursor. Since 65Zn (ZnCl2) dissolved in 0.1 M HCl was used as the tracer, a very small portion should be added to avoid the influence on the kinetics of forced hydrolysis, but to ensure sufficient radioactivity used in the following experiments. However, the ratio of water is important in determining the shape and size of the final products, thus the redundant water in ZnCl2 solution should be removed before the forced hydrolysis in polyol. To solve this problem, we designed the following synthesis procedures. A small portion of 65Zn in the form of ZnCl2 dissolved in 0.1 M HCl was mixed with stable Zn(OAc)22H2O to serve as the precursor of nZnO. The non-isotope modified nZnO were firstly synthesized to optimize the proper parameters and for the fundamental characterization such as transmission electron microscopy (TEM, JEOL 2010), X-ray diffraction (XRD, Powder X-ray diffractometer-PANalytical) as well as ZetaPALS (DLS, Brookhaven Instruments). The 0.0022 g stable ZnCl2 (extra pure reagent, Nacalai Tesque Inc, Kyoto, Japan) and 400 mL 11.6 mol/L HCl were dissolved in 50 ml Milli-Q water to mimic the 65Zn solution. In a typical synthesis, 4 ml as-prepared ZnCl2 solution, 400 mL 1 mol/L NaOH, and 4 ml ethylene glycol (Reagent European Pharmacopoeia, 99.5%) were mixed in a 100 ml two-neck bottle. The mixture was heated to about 120  C for 2 h to evaporate the water in the system. The solution was then allowed to cool in air to room temperature. The 0.0879 g Zn(OAc)22H2O (ACS reagent, 99.0%, SigmaeAldrich), 0.04 g PVP (MW ¼ 10,000, SigmaeAldrich) as well as 6 mL extra EG were then added, and the mixture was ultra-sonicated for 3 min to obtain a transparent solution. The solution was placed in an oil bath and heated to 190  C for 13 min. Immediately, 28 mL Milli-Q water was added into the system causing the transparent solution into cloudy. The solution was allowed to continue reacting for another 3 min. The molar ratio of 65Zn to total Zn (radioactive and stable Zn) was 0.0032 as calculated from the amount of precursor. The products were then washed by Milli-Q water and ethanol for several times to remove extra surfactants and solvents. Stock solution (10 mL) were measured for its radioactivity and the radiolabeling efficiency (compared with the radioactivity of the same volume of original 65Zn solution) in the system was 70%. Decrease of radiolabeling efficiency was probably due to nZnO loss in the washing procedure. The concentration of the final solution was quantified by repeating synthesis of stable nZnO with the same method, and measured by atomic absorption spectrometry (AAS). The efficiency of radiolabeled nZnO was also used to estimate the concentration of nZnO solution, which was similar among the repeated experiments. Finally, the synthesized nZnO were made to a 1.5 g/L stock solution.

2.

Materials and methods

2.2.

2.1.

Synthesis of radioactive nZnO

For the dissolution of synthesized nZnO, two concentrations (0.5 mg/L and 2 mg/L) were made from the stock solution at room temperature. And 1 ml solution was centrifuged using centrifugal filter devices (3 kD, Amicon Ultra-4) for 40 min with

Feldmann and Jungk (2001) synthesized the radioactive nZnO by forced hydrolysis in polyol. Diethylene glycol (DEG) is

Dissolution equilibrium analysis

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a speed of 4000 rpm. There were two replicates for each time point. The nZnO were retained in the upper chamber of the devices. The tubes were washed by 5% HNO3 for several times to collect the nanoparticles and to measure the concentrations of zinc in the form of nanoparticles. The filtrates were also measured for zinc concentrations. The percentages of dissolution were calculated as the free ions concentration in the whole mixture.

2.3.

Characterization

The 100 mg/L solution was made from diluting the stock solution with simplified M7 medium (SM7, containing only CaCl2, MgSO4, K2HPO4, KH2PO4, NaNO3, NaHCO3, Na2SiO3, H3BO3, and KCl and without disodium ethylenediaminetetraacetic acid, trace metals, or vitamins, pH8.2). The sample was ultra-sonicated before each characterization to minimize the aggregates. In the sample preparation for the dynamic lighting scattering (DLS, Brookhaven Instruments) to determine the hydration radius and zeta potential, ultra-sonicated samples were placed in clean cuvettes, and at least three consecutive measurements were conducted at room temperature, with each consisting of five runs of 10 s duration. Transmission electron microscopy (TEM, JEOL 2010) was also employed to characterize the morphology of synthesized samples. The samples were prepared by depositing a drop of the suspensions on a carbon-coated copper specimen grid and allowing the water to evaporate in the air. The nZnO powders were placed on a clean glass to characterize the phase of the samples by Powder X-ray diffraction (XRD, Cu-ka).

2.4.

Separation of Zn2þ from the mixture

As the Zn2þ in the nanoparticle solutions would influence the bioavailability of nZnO, it was necessary to develop a method to separate the dissolved Zn2þ from the nZnO. As nZnO could be re-dissolved after extraction and re-dispersion in the medium, we designed a method to investigate the uptake of the mixture and free ions in the solution respectively, and calculated the nZnO uptake. To achieve this goal, the stirred cell with 1 kDa membrane was employed. When the solutions reached equilibrium, they were placed in the stirred cell to separate the insoluble nZnO and the dissolved Zn2þ under 75 psi pressure of N2, which was then used for the uptake experiment (see below).

2.5.

Uptake of nZnO by daphnids from water

Daphnia magna was cultured in filtered unpolluted pond water with a density of 1 individual/10 mL. The animals were fed daily with green alga Chlamydomonas reinhardtii at 5  104 cells/ mL (neonates 3 days old) or 105 cells/mL (adults 3 days old). The green algae were cultured in an artificial WC medium (containing CaCl2: 0.25 mM, MgSO4: 0.15 mM, NaHCO3: 0.15 mM, NaNO3: 1 mM, K2HPO4: 0.05 mM, H3BO3: 0.1 mM, and trace metals and vitamins) (Guillard and Lorenzen, 1972). Both green algae and daphnids were cultured at 23.5  C and 14:10 h light: dark cycle. The green algae were collected at the exponential growth stage by centrifugation to remove the growth

897

medium and stored in pond water at 4  C. Seven days old D. magna were used in all kinetic measurements. After nZnO solutions (two concentrations: 0.5 and 2.0 mg/L, each with three replicates) reached equilibrium in dissolution with the solutions, we conducted the uptake experiments by D. magna. Previous study found that the 48-h 50% lethal concentration (LC50) of nZnO in D. magna was 1 mg/L (Wiench et al., 2009), and the 48-h LC50 of soluble Zn in D. magna was 0.87 mg/L (Wang and Guan, 2010). Upon acute exposure to 1 mg Zn/L, notable (5%) mortality of daphnids was only observed after 33 h of exposure (Wang and Guan, 2010). At the two exposed concentrations (0.5 and 2.0 mg/L), daphnids showed normal behavior within the short exposure periods (<8 h). The choice of these two concentrations was based on the measurable amount of Zn accumulation in the animals during the short exposure period. Before the exposure, daphnids were depurated in clean SM7 for 2 h to remove the food retained in their gut lines. Subsequently, they were transferred into different concentrations of solution at a density of 1 individual per 10 ml (total water volume of 100 ml). For the short time exposure, at the time of 10, 20, 30, 40 min, all daphnids were collected and rinsed with uncontaminated SM7 to remove the weakly absorbed nZnO on their carapaces. After washing for about 1 min, they were radioassayed by g-counter and returned back to the exposure medium immediately. While for the long time exposure (8 h), at the time of 2, 4, 6, 8 h, all the 10 daphnids were collected, washed and radioassayed with the same method mentioned above. The accumulated Zn concentrations were determined by the specific activities of radioisotope. The slope of the linear regression between the newly accumulated Zn and exposure time was defined as the influx rate (I, mg/g/h). The uptake rate constant (ku, L/g dry wt/h) was calculated as I/Cw, while Cw is the metal concentration in water (mg/L) (Tan and Wang, 2008).

2.6.

Depuration of nZnO by daphnids

D. magna were exposed in the solution of radio-synthesized nZnO (0.5 and 2 mg/L, with three replicates for each treatment) for 30 min with a density of 1 individual per 10 ml medium (100 ml total medium). At the end of nanoparticle exposure, daphnids were collected and rinsed with clean SM7 solution and radioassayed by g-counter. The daphnids were then transferred into clean SM7 solution. At 20 min, 40 min, 1, 2, 4, 6, 8, 12 and 24 h, the radioactivity remaining in the daphnids were quantified as above. The Zn concentrations in daphnids were determined by the specific activities of radioisotope. The depuration rate was calculated as the slope of linear regression between the natural logarithm of the percentage of retained Zn in daphnia (ln%) and the exposure time.

3.

Results

3.1.

Morphology and size characterization of nZnO

The TEM images (Fig. 1) shows that the synthesized nZnO had a size distribution of 10e30 nm, and were easy to aggregate

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Released 65Zn (%)

100 0.5 mg/L 2 mg/L 60

20 0

5

10

15

20

25

Time (h) Fig. 3 e The percentage of released 65Zn from nanoparticles in the suspension at two different nZnO concentrations. Mean ± SD (n [ 3).

determined as 425 and 1584 mg/L (for 0.5 and 2 mg/L, respectively), with 127.8 and 316.9 mg/L soluble Zn in filtration. Fig. 1 e TEM images of the synthesized nZnO nanoparticles.

3.3.

into larger particles. XRD investigations (Fig. 2) showed that nZnO were wurtzite structure. The results of DLS showed that the hydrodynamic diameters of synthesized nZnO were 46e56 nm. They were slightly larger than the size observed by TEM, implying aggregation in the medium. The zetapotentials were 26.4  0.98 mV, indicating that the nanoparticles were relatively well dispersed in SM7 medium.

3.2.

Dissolution rates

The dissolution analysis of synthesized nZnO (Fig. 3) showed that about 30% of ZnO dissolved in the medium at 0.5 mg/L nZnO, compared with 20% dissolution for the 2 mg/L solution. On the other hand, the dissolution reached equilibrium within 2 h. Based on these results, we conducted the uptake experiments 2e3 h after the solutions were freshly made. The actual concentrations of total Zn and Zn2þ in the medium were

When daphnids were exposed to the mixture of radioactively synthesized nZnO and Zn2þ, the accumulated nZnO and Zn2þ in daphnids exhibited irregular pattern. In the long-term experiment (8 h), the accumulated Zn in daphnids decreased with the exposure time (Fig. 4C and D). When the exposure time was shorten to 40 min, the uptake of Zn2þ by daphnids could be ignored compared with the uptake of the mixture (Fig. 4A and B). As a result, the uptake of nZnO was equal to that of the mixture. There were differences between 0.5 mg/L and 2 mg/L in our experiments. For the low nZnO concentration (0.5 mg/L), the accumulated Zn in the daphnids increased linearly with the exposure time (influx rate ¼ 49.9  17.7 mg/g/ min) and then reached a plateau. While for 2 mg/L experiment, uptake of nZnO by daphnids only occurred within the first 10 min (influx rate ¼ 190.9  178.8 mg/g/min). The uptake rate constant ku from water at both nZnO concentration treatments were 0.117  0.042 L/g/min and 0.120  0.112 L/g/ min, respectively. To remove the possibilities of absorption of nZnO on the exoskeletons of daphnids, we also exposed the freshly dead daphnids (by freezing in liquid N2 for 20 min) in the nZnO solutions and found no evidence of nZnO uptake.

800 101

0 20

40

103 112

200

110

102

400

3.4.

100 002

Intensity

600

60

80

2 theta Fig. 2 e XRD pattern of the synthesized nZnO.

Uptake of nanoparticles from water

Depuration of nZnO by Daphnia magna

The retained nZnO in daphnia decreased sharply within the first 8 h of depuration, and then leveled off for both 0.5 and 2 mg/L concentration treatments (Fig. 5). At the end of depuration, there was no significant difference between the two concentrations on the percentage of retained radioactivity in daphnids (12.1  3.7% for 0.5 mg/L and 19.9  10.6% for 2 mg/L, p > 0.05, t-test). The Zn elimination from daphnids in both treatments was a two-compartmental depuration. For the first compartment (0e8 h), ke1 was 5.427  2.790/day and 4.421  2.514/day for the 0.5 and 2 mg/L treatment, respectively. For the second compartment (8e24 h), ke2 was 0.393  0.240/day and 0.143  0.092/day for the two concentration treatments, respectively.

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0.5 mg/L

Newly accumulated Zn (µg/g dry weight)

3000

2 mg/L 9000

A

Experiment 1 Unfiltered Filtered

2000

Experiment 1

6000 3000

1000

0

0 10

1200

B

20

30

10

40

6000

C

Experiment 2

800

4000

400

2000

0 120

240

360

480

20

30

D

40

Experiment 2

0 120

240

360

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Time (minutes) Fig. 4 e The uptake of synthesized nZnO from the water by Daphnia magna (ZnO concentration, 0.5 mg/L and 2 mg/L). (A) and (B): exposure of 40 min; (C) and (D) exposure of 8 h. the filled circle represented the uptake of unfiltered solution and the blank circle represented the uptake of filtrate (i.e., only soluble Zn). Mean ± SD (n [ 3).

Discussion

4.1. Characterization, dissolution and radiolabeling efficiency A critical problem to be solved in the nZnO toxicology study is to distinguish the contribution of the free zinc ions and the nZnO nanoparticles in the suspension to the overall nZnO toxicity. As the dissolution rate of nZnO varied with the concentrations of nanoparticles as well as the water chemistry such as the pH and organic materials, each experimental system has to be identified for its ratio of dissolution. In earlier study, Franklin et al. (2007) used equilibrium dialysis to show the rapid dissolution of ZnO nanoparticles in a freshwater medium (pH-7.6), with saturation solubility in the milligram per liter range, similar to that of bulk ZnO. Miao et al. (2010) found that the initial nanoparticle concentration only slightly influenced the Zn2þ release, whereas the pH and aggregation state had the most primary effects on the dissolution. In our experiment (pH: 8.2e8.6), the initial concentration of nanoparticles had great influences on the released percentage of Zn2þ. The dissolution percentage decreased with concentration. In our preliminary experiment, we also radiolabeled 65Zn onto commercially available nZnO, but found that most of the 65 Zn were released from the nanoparticles when we dispersed the radiolabeled nanoparticles in clean SM7 solution. Such rapid dissolution may be caused by the high solubility of ZnO in aquatic environments, and we strongly highlighted the necessity of quantifying the partitioning of Zn in ionic and nanoparticle phases when studying the biokinetics and toxicity of nZnO in aquatic systems. For other metallic

nanoparticles such as AgNP, the dissolution appeared to be a less significant problem as compared to nZnO. Zhao and Wang (2010) firstly labeled 110mAg onto the stable Ag particles in the Milli-Q and obtained strongly absorbed nanoparticles. In earlier study, Wu et al. (2010) supplemented the culture medium with EDTA to deactivate Zn2þ, and the inhibition of Mycobacterium smegmatis growth caused by nZnO was significantly alleviated (from 38 to 17%) comparing to the control samples. In our experiment, EDTA (with a molar ratio of 1:1 with total zinc) was also used to chelate the free Zn ions in the system. However, we found that almost all zinc ions were released from the nanoparticles in the presence of EDTA, obviously suggesting that chelating agents such as EDTA can influence the dissolution equilibrium of nZnO. Based on the equilibrium constant of EDTA-Zn (K ¼ 16.5), we estimated that

%Zn retained in daphnids

4.

100

0.5 mg/L nZnO 2 mg/L nZnO

10

1 0

4

8

12 16 Time (h)

20

24

Fig. 5 e Retention of Zn in Daphnia magna during 24 h depuration after exposure to synthesized nZnO solution for 30 min. Mean ± SD (n [ 3).

900

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in the 5 mg/L and 10 mg/L solution, only 3% existed as free zinc ions. As a result, EDTA was not suitable to chelate the free Zn ions.

4.2.

Uptake of nZnO

As reported in previous literature, after the daphnids were exposed to the Zn2þ solutions, the zinc ions accumulated in the daphnids increased linearly with the exposure time (Yu and Wang, 2002). The synthesized nZnO exhibited different uptake kinetics with that of free zinc ions in water which was proportional to the Zn concentrations in ambient water. The calculated influx rate and uptake rate constant for the filtrates (only free zinc ions, 8 h exposure experiment) of synthesized nZnO were 14.9  2.47 mg/g/h and 0.087  0.014 L/g/h (for 0.5 mg/L nZnO) and 21.6  0.25 mg/g/h and 0.068  0.008 L/g/h (for 2 mg/L nZnO). In the low concentration range, the uptake rate constant, which can be compared among different species, metals and under various physiochemical conditions, was 0.086 L/g/h for Enteromorpha crinite (Chan et al., 2003), and 0.094-0.244 L/g/h for daphnids (Tan and Wang, 2008), similar to the uptake of filtrates of nanoparticles. From the long time exposure, the uptake of free zinc ions can be further separated from the uptake of the mixture. However, the accumulated Zn in daphnids exposed in the nanoparticle solutions decreased with exposure time. It appeared that the uptake of nZnO occurred within a short time. There may be three routes for nanoparticles to interact with the daphnids, including the uptake of the free zinc ions released by nanoparticles, direct uptake of nZnO, and absorption on the exoskeleton of daphnids (Poynton et al., 2011). In our present study, the last route was eliminated in our parallel experiment with the exposure of dead daphnids, which showed that the nanoparticles absorbed on the exoskeleton of daphnids over a short exposure time was negligible. The main difficulty was thus to distinguish the uptake of free zinc ions and nanoparticles. Over the 40 min exposure period, the calculated influx rate of Zn2þ was small enough to be ignored when compared with that of the nanoparticles. Consequently, the uptake of the mixture of free zinc ions and the nZnO was equal to that of the nanoparticles. Thus, we can calculate the parameters of the accumulated nZnO in daphnids. The influx rate of nZnO was 49.9  17.7 mg/ g/min and 190.9  178.8 mg/g/min, for 0.5 mg/L and 2.0 mg/L nZnO, respectively, much higher than that of the zinc ions (14.9  2.47 mg/g/h and 21.6  0.25 mg/g/h for the filtrates of 0.5 mg/L and 2 mg/L, respectively), another proof of the different mechanisms of nanoparticles. It is interesting to note that the concentration of nZnO in daphnids decreased in the long time exposure (8 h). One reason may be that nZnO itself lowered the uptake ability of the animals. Such reduced Zn uptake may also be caused by the reduced filtration activity of the animals. To test this hypothesis, we exposed the daphnids in 0 (control), 0.5, 2, 5, and 10 mg/L nZnO solution for 30 min. The animals were then transferred into clean SM7, and fed with C. reinhardtii at a cell density of 105 cells/ml. After 1 h feeding, the decrease of cell density was then quantified. Indeed, the filtration activity of the animals was reduced by 90% as compared to the control at nZnO concentrations >2 mg/L. At a lower concentration

(0.5 mg/L), the ingestion rate merely decreased to 75% compared with the control experiment. These data therefore suggested that nZnO can hinder the filtration activity of the animals and may thus reduce the intake of nZnO over time of exposure. Earlier, rapid uptake of ZnO nanowire aggregates in human monocyte macrophages by phagocytosis and intracellular dissolution within a membrane compartment was observed (Muller et al., 2010). Lin and Xing (2008) observed the presence of ZnO nanoparticles in the endodermal and vascular cells of the ryegrass, indicating that ZnO nanoparticles could enter the ryegrass cells and move to the stele. Zhu et al. (2010) reported that D. magna can accumulate TiO2 nanoparticles from ambient environment with a high bioconcentration factor (1.18  105 for the exposure concentration of 1 mg/L). The nanoparticles may be directly ingested by daphnids into the gut since the reported mean size of the filter meshes of D. magna were 240e260 nm, and the nanoparticles or the aggregates in the range of 100e200 nm can be seized as food (Brendelberger, 1991). In our short time exposure when the accumulated nZnO reached a peak, the possibility of the nanoparticles ingested by D. magna was bigger than that of phagocytosis.

4.3.

Depuration of nZnO

The large ke of the nZnO indicated a rapid elimination of nanoparticles from D. magna, showing that nanoparticles can be easily depurated from the organisms. In contrast, daphnids were unable to excrete the carbon nanotubes to either clean artificial freshwater or filtered lake water after 24 h of depuration, even though the lake water had a substantial concentration of natural organic matter (Petersen et al., 2009). Different from the slow depuration of carbon nanotubes in daphnia, the high depuration of nZnO may be caused by the higher solubility in the body of daphnia. Hasler (1935) estimated the pH inside the gut of daphnids based on the color changes of indicator dyes during processing through the gut. The revealed pH varied from 6.8 in the anterior end to 7.2 at the causal end. Later, Von Elert (2004) investigated the relative pH changes in the gut of Daphnia using the fluorescent dye, indicating that the proximal parts of the gut were neutral and distal parts were slightly more alkaline with pH values of 8e9. The Zn ions released from the nanoparticles may be transported in different parts of the organisms. The pH in the gut of D. magna was lower than SM7 medium in our experiments, especially in the proximal parts. To quantify the solubility percentage of ZnO in the gut, we conducted the solubility experiments in SM7 (pH-6.8) and observed that more than 90% ZnO was soluble in the solution. Guan and Wang (2004) reported that about 10% of Zn remained in the daphnids after 12 h of depuration, similar to our data. It is expected that nZnO taken up by daphnids may dissolve into Zn, and its efflux behavior may be comparable to the behavior of Zn ions, which can then explain the rapid depuration of nZnO by daphnids. The quantified influx rate and depuration rate can be used to calculate the bioconcentration factor (BCF) of nZnO by D. magna. Accordingly, the BCF was 1.21  106 L/kg, much higher than that of the free ionic Zn. Our study therefore showed

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 8 9 5 e9 0 2

a distinctive uptake mode of nZnO and Zn2þ, and suggested that either the Zn2þ or the nZnO themselves should be considered for the study of nZnO toxicology. Our study demonstrated that D. magna can accumulate nZnO through ingestions of these nanoparticles, different from that of the Zn ions. The inhibition in food uptake may cause a reduction in growth and reproduction dynamics (Hanazato, 2001). The nZnO can be easily depurated by the daphnids, mainly caused by the high solubility of nanomaterials in the gut of the animals. The toxicity of nZnO may be contributed both by the inhibition of food intake and the dissolved nZnO in the gut of daphnia, and cannot be solely attributed by the direct toxicity of nZnO or the Zn released by the nanoparticles.

Acknowledgment We thank the two anonymous reviewers for their comments. This study was supported by a General Research Fund (663011) from the Hong Kong Research Grants Council to W.-X. Wang.

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