Biokinetic Uptake and Efflux of Silver Nanoparticles in - American

Sep 10, 2010 - Daphnia magna, including the uptake from water, dietary assimilation, and elimination of AgNP. We found that the uptake of AgNP was ...
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Environ. Sci. Technol. 2010, 44, 7699–7704

Biokinetic Uptake and Efflux of Silver Nanoparticles in Daphnia magna CHUN-MEI ZHAO AND WEN-XIONG WANG* Section of Marine Ecology and Biotechnology, Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

Received May 3, 2010. Revised manuscript received August 24, 2010. Accepted August 27, 2010.

Silver nanoparticles (AgNP) are widely used as antibacterial products, and there are increasing concerns for their potential environmental risks in aquatic ecosystems. The biokinetics of AgNP in aquatic organisms has not yet been determined. In the present study, we employed a radiotracer methodology to quantify the biokinetics of AgNP in a freshwater cladoceran Daphnia magna, including the uptake from water, dietary assimilation, and elimination of AgNP. We found that the uptake of AgNP was concentration dependent and governed by two phases. The uptake rate constant (ku) was 0.060 L/g/h at low AgNP concentrations (2, 10, and 40 µg/L), which was 4.3 times lower than that of the Ag free ion. At a higher AgNP concentration (160 and 500 µg/L), the uptake rate increased disproportionately, likely as a result of direct ingestion of these nanoparticles by the daphnids. When the AgNP were associated with the algal food, their dietary assimilation efficiency (AE) was in the range of 22-45%, which was much higher than the dietary assimilation of Ag quantified under the same food conditions. The efflux rate constants of AgNP in daphnids were also much lower than those of the Ag, again suggesting the difficulty of eliminating AgNP by the daphnids. Water excretion was the main elimination route for both AgNP and Ag, but a higher percentage of AgNP was lost through fecal production. Finally, we used a kinetic equation to compare the importance of aqueous and dietary uptake of AgNP using the quantified kinetic parameters. The biokinetic model showed that more than 70% of AgNP accumulated in the daphnids was through ingestion of algae, highlighting the importance of AgNP transport along the food chain. Our present study showed the unique characteristic of AgNP biokinetics and suggested that more attention should be paid to the dietborne AgNP toxicity in aquatic ecosystems.

Materials and Methods

Introduction Nanoparticles have been widely used in numerous electronic applications and commercial products. Among the nanoparticles, Ag nanoparticles (AgNP) have one of the most promising applications due to their antibacterial activity (1, 2). They are now frequently used as an alternative bactericide and have wide applications in medical and wastewater treatments, as well as in personal care products. Inevitably, these commercial nanoparticles may end up in the environment, and an understanding of their environmental trans* Corresponding author e-mail: [email protected]. 10.1021/es101484s

ports both in terms of physicochemical behavior and biological behavior is imperative. Several recent studies have quantified the acute toxicities of Ag nanoparticles in different groups of organisms (e.g., bacteria, algae, cladocerans, fish, and cell lines) (3-11). These studies demonstrated that AgNP produced reactive oxygen species, leading to lethality and failure of embryonic development or reproduction. However, the actual bioaccumulation of AgNP in the organisms has rarely been simultaneously quantified, rendering ambiguity in the interpretation of the toxicity results. In a recent study in Daphnia magna (Zhao and Wang, submitted), we found that AgNP had little acute toxicity even when the accumulation of AgNP reached 83.5 mg/g dry wt at 500 µg/L AgNP exposed concentration (or 9.3% of the total AgNP in 1 L medium). Nevertheless, AgNP apparently presented a chronic toxic effect on the growth and reproduction of daphnids. One of the possible mechanisms underlying such toxic effects was the retention of these nanoparticles in the digestive tracts, which subsequently affected the food ingestion and digestive process (12, 13). The bioavailability of AgNP should be an important consideration in studying the AgNP toxicity (2). For herbivores and carnivores in aquatic ecosystems, the incorporation of AgNP into their bodies could be either from water absorption or dietary ingestion. These two routes may have different contributions to nanoparticle bioaccumulation. In addition to the uptake, bioaccumulation is also dependent on the elimination of AgNP out of the organisms. Consequently, the biokinetic parameters including uptake rate constant from water (ku), assimilation efficiency (AE) and efflux rate constant (ke) should be quantified in order to predict the bioavailability of AgNP. However, these kinetic parameters have never been quantified for the AgNP in any aquatic organisms. A kinetic approach in understanding the bioaccumulation of metals in aquatic animals has been comprehensively developed over the past decades (14-16). There is also a substantial need to understand the biokinetics of these nanoparticles in the animals in order to facilitate the risk assessments of nanoparticles in the environments. In this study, we quantified the kinetics of a commercially available AgNP to a sensitive zooplankton Daphnia magna. We took advantage of a radiotracer methodology that is widely used to study the metal biokinetics by radiolabeling the AgNP with 110 mAg radiotracer. The biokinetic processes considered in this study included the uptake of AgNP from water, the dietary assimilation of AgNP from ingested food sources, as well as the elimination of AgNP. We also specifically quantified the fates of AgNP after its assimilation by the daphnids. Finally, based on these kinetic measurements, we modeled the contribution of different uptake routes toward AgNP accumulation in D. magna.

 2010 American Chemical Society

Published on Web 09/10/2010

Organisms, Medium, and Materials. Daphnia magna was cultured in the creek water and fed with green algae Chlamydomonas reinhardtii (cultured in WC medium, 17) at 5 × 104 cells/mL (neonates e3 d old) or 105 cells/mL (adults >3 days old) each day. The density of daphnids was 1 individual/10 mL. The growth conditions for the green algae and daphnids were 23.5 °C and 14: 10 h light: dark cycle. All the kinetic measurements were conducted for the 7 day old adults. We used a commercially available carbonate-coated AgNP obtained from NanoSys GmbH (Wolfhalden, Switzerland, about 0.5-1% of Ag was found to be Ag+) in our experiments. The AgNP dispersed in the medium were VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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spherical shapes, with a particle size of approximately 20 nm, as observed by transmission electron microscopy (TEM, JEOL 2010F). The size distribution of AgNP (with 1 µM cysteine addition) determined by dynamic light scattering with a ZetaPALS (DLS, Brookhaven Instruments) showed that the hydrodynamic diameter was mainly in the range of 40-50 nm. The zeta-potential of AgNP was -19.6 ( 3.5 mV, indicating that AgNP were relatively well dispersed in the medium. All the media used in the experiments were simplified M7 (SM7) artificial water (18). Radioactive AgNP Preparation. The radioactive AgNP suspension was prepared by spiking gamma radioisotope 110 m Ag (in the form of AgNO3 dissolved in 0.1% HNO3) into the AgNP suspension. Two stocks with different AgNP concentrations were prepared for the following biokinetic experiments. Initially, 50 µCi/L 110 mAg was mixed with two of 100 mL 0.22 µm-filtered AgNP suspensions at 250 and 5000 µg/L AgNP, but without cysteine addition, in Milli-Q water in a Teflon beaker, respectively. After stirring for 12 h, the mixed solutions were filtered through 1 kDa membrane in the stirring cell to remove the nonadsorbed 110 mAg under 75 psi pressure of N2. The retained solutions (10 mL, with a concentration factor of 10) were transferred into 50 mL Teflon beakers. The radioactivities of mixture (before the filtration), the retained solution and permeate were radioassayed by Wallac 1480 NaI (T1) gamma counter (Turku, Finland) to calculate the mass balance before and after diafiltration. The radiolabeling efficiency was calculated as the percentage of the radioactivity of retention in the whole mixed solution (the sum of radioactivity of retention and permeate after filtration). For the high concentration AgNP stock (5000 µg/ L), the efficiency was 99.1%, indicating that most of the radioactive 110 mAg was adsorbed onto AgNP. However, the efficiency of the AgNP stock with low concentration of AgNP (250 µg/L) was 53.2%. Such difference in the radiolabeling efficiency was mainly due to the different AgNP concentrations. High AgNP concentration increased the interactions between the Ag+ and the AgNP. In radioactive AgNP stocks (without addition of cysteine), Ag was presented both as free ion and AgNP. With addition of cysteine, it formed complexed with Ag+, and such complex was not bioavailable to the daphnids (see below). For AgNPs themselves, since cysteine could sorb to the surface of the AgNP, the adsorbed Ag+, cysteine and AgNP formed the whole complexation. Therefore, the adsorbed Ag+ (as 110 mAg) could be used to trace the behavior of AgNP. Uptake of AgNP from Water. With the newly prepared radioactive AgNP stocks, we conducted the AgNP uptake from water by the daphnids. The radiolabeled AgNP stocks were dispersed into SM7 medium to achieve 2, 10, 40, and 160 µg/L AgNP exposure concentrations. For the highest AgNP concentration (500 µg/L), nonradiolabeled AgNP (400 µg/L) was also added into the medium. In all AgNP treatments, 1 µM cysteine was added in order to complex with any presence of Ag+ in the AgNP exposure. Speciation analysis (MINEQL+ V4.5) showed that all of the Ag+ combined with cysteine. To verify whether Ag+ combined with cysteine was bioavailable, 0.9 µg/L of 110 mAg were exposed to daphnids in the presence of 1 µM cysteine (again all Ag+ was combined with cysteine). Before the exposure, daphnids were depurated in clean SM7 for 1-2 h to remove the food retained in their gut lines. Then, they were transferred into the SM7 medium containing the radiolabeled AgNP at a density of 1 individual/10 mL in each beaker. There were three replicates in each treatment. At 2, 4, 6, and 8 h, daphnids were collected and rinsed with uncontaminated SM7 to remove the weakly adsorbed AgNP on their carapaces. After washing for 1 min, they were radioassayed by γ-counter and then returned back to the exposure medium immediately. At the end of exposure, daphnids were collected to analyze the relative distribution 7700

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of AgNP on the soft tissue and exoskeleton, using the described method (19). The accumulated Ag concentrations were determined by the specific activity of radioisotope. The influx rate (I, µg/g/h) was the slope of linear regression between accumulated Ag in daphnids and exposure time. The uptake rate constant from the water (ku, L/g/h) was calculated as the slope of the linear regression between the influx rate and the AgNP concentration in the medium through the origin of the natural coordinate. Assimilation of AgNP. AgNP assimilation was investigated at different AgNP concentrations in the algal diet. When the algae grew at the log phase, they were transferred into WC medium with different radioactive AgNP concentrations (20, 100, and 500 µg/L with 1 µM cysteine) added at a cell density of 2 × 105 cells/mL. Our previous experiment demonstrated that the algae did not take up the Ag+-cysteine complex. After 12 h of radiolabeling, the algae were centrifuged and resuspended in SM7 medium twice to remove the weakly adsorbed AgNP on the cells. The cell density of the algae was counted by a hemocytometer. The daphnids were depurated in the clean SM7 for 1-2 h to clear the food in their guts and then transferred into the medium at a density of 1 individual/ 10 mL. After that, they were fed with the radiolabeled algae at a cell density of 2 × 104 cells/mL. After 15 min of pulsefeeding, the daphnids were collected and rinsed with clean SM7 and the radioactivity of accumulated AgNP in their body was measured. After the radioassay, they were depurated in clean SM7 containing 1 µM cysteine with food addition at a cell density of 105 cells/mL. At 3, 6, 9, 12, and 24 h of depuration, the radioactivity retaining in daphnids was determined. The water and food (without AgNP) were renewed at each time point after the radioactivity was counted. The assimilation efficiency (AE) was the percentage of ingested AgNP retained in the daphnids at 12 h of depuration. The ingestion rate (IR) of daphnids was estimated from the radioactivity in daphnids after 15 min of pulsefeeding. Efflux of AgNP. AgNP elimination from daphnids was quantified at low and high AgNP concentrations and contrasted with the elimination of radioactive 110 mAgNO3 (control, 0.9 µg/L). Initially, daphnids were exposed to radioactive AgNP (at a concentration of 5 µg/L, 0.48 µCi/L; and 500 µg/L, 0.97 µCi/L) in SM7 medium at a cell density of 105 cells/mL for 2 days. For AgNP, 1 µM cysteine was added to complex with the Ag+. There were three replicates in each treatment and 20 individuals were used in each replicate at a density of 1 individual/10 mL. After 2 days of radiolabeling, the radioactivity in daphnids was measured. They were then depurated in clean SM7 medium with 1 µM cysteine at the same cell density. The depuration lasted for 4 days. For the first 2 days, the radioactivity in the daphnids was determined every 12 h, and then every 24 h for the following 2 days. Water and food were both refreshed when the radioactivity was counted. Compartmental analysis was then conducted to calculate the rate constant of loss from different compartments. The biological retention half-lives (t1/2) were calculated as t1/2 ) 0.693/ke. To investigate the fates of AgNP incorporated into the daphnids, an AgNP budget was also quantified at both AgNP concentrations (5 and 500 µg/L) and the control in a separate experiment with more intensive periods of sampling. Daphnids were radiolabeled and depurated as described above. At 0.5, 1, 1.5, 2, 3, and 4 days of depuration, a 10 mL medium, molts (using wide-mouth pipet), neonates (using narrowmouth pipet) and feces (filtered through 40 µm mesh) were collected for radioassay. All the media (including the food) were subsequently renewed at each time point. The relative percentage of AgNP distribution in each compartment of loss (excretion into water, molts, neonates, and feces) was then calculated.

FIGURE 1. (A) Accumulated Ag in Daphnia magna exposed to 110 mAg (0.9 µg/L) combined with cysteine (1 µM) and low AgNP concentrations (2, 10, and 40 µg/L) over 8 h period. (B) Accumulated Ag in Daphnia magna exposed to high AgNP concentrations (160 and 500 µg/L) over 8 h period. (C) Relationship between Ag influx rate (I) and AgNP concentration in the dissolved phase (Cw). (D) AgNP distribution on exoskeleton of Daphnia magna after 8 h exposure. Data are mean ( SD (n ) 3).

Results and Discussion Uptake of AgNP from Water. After the daphnids were exposed to a series of AgNP concentrations for 8 h, the accumulated AgNP in daphnids increased linearly with the exposure time (Figure 1A and B). When 110 mAg was complexed with cysteine, there was no uptake of Ag by the daphnids during the exposure, suggesting that this complex was not incorporated into the daphnids at 0.9 µg/L Ag and that cysteine was extremely efficient in complexing with Ag (Figure 1A). At the high exposure concentrations (160 and 500 µg/L AgNP), the accumulated AgNP increased significantly to 1.77 and 11.6 mg/g dry wt after 8 h of exposure, compared with those of low exposure concentrations (5.00, 19.9, and 57.9 µg/g dry wt for 2, 10, and 40 µg/L AgNP, respectively). The relationships between the influx rates (I) and the AgNP concentrations were biphasic (Figure 1C). At low exposure concentrations (2-40 µg/L AgNP), the I was in the range of 0.08 ( 0.04 and 2.39 ( 0.69 µg/g/h and the uptake rate constant ku1 from water was 0.060 L/g/h. At higher concentrations (160 and 500 µg/L), I increased significantly from 176 to 1156 µg/g/h. The calculated uptake rate constant ku2 was 2.2 L/g/h. No difference was observed on AgNP distribution between the exoskeleton and the soft tissue. Nearly 45% of AgNP was associated with the exoskeletons of the daphnids. A critical consideration in conducting the uptake of AgNP was the phase association of radiotracer during the uptake experiment, which had been recognized in experiments with the uptake of colloidal bound metals (20). In our study, we for the first time used the 110 mAg radiotracer to radiolabel the AgNP. We first used the stirrer cells at a concentration factor of 10 (21) to remove the 110 mAg which was not directly radiolabeled onto the AgNP. Any release or presence of free 110 m Ag was subsequently complexed with cysteine, which had a very high affinity with Ag as well as with other B-type metals. A recent study also indicated that Hg complexed with cysteine was essentially unavailable to a marine diatom (22). Our experiments provided direct evidence that 110 mAg complexed with cysteine was not accumulated by the daphnids. Consequently, 110 mAg adsorbed on AgNP could be used to track the behavior of AgNP.

In contrast with the uptake of Ag+, which was considered as a first-order kinetics (23), the kinetics of the uptake of AgNP appeared to be greatly dependent on the exposed AgNP concentrations. The uptake rate was proportional to the AgNP concentration (with a b coefficient of 1.15) at AgNP concentrations of 2 to 40 µg/L. The calculated ku1 was rather low, which may have been mainly due to the adsorption of AgNP onto daphnids. However, the influx rate increased significantly at a high AgNP concentration, with the b coefficient (2.47) much higher than 1, strongly suggesting that the uptake was no longer governed by the first order uptake kinetics. Such difference in the uptake indicated that AgNP was probably taken up by daphnids through different mechanisms. AgNP may have been directly ingested by the daphnids into the gut at a high AgNP concentration. It was reported that the mean sizes of the filter meshes of D. magna were 240-640 nm (24), and that food of dimensions as low as 100 nm (width) by 200 nm (length) could be seized (25). Endocytosis/pinocytosis has been implied for the uptake of nanoparticles (26). AgNP uptake at a low concentration may possibly be governed by endocytosis. Previously, there have been very few studies on the uptake of nanosized particles by the aquatic organisms. For zooplankton, Wang and Guo (20) first demonstrated that the colloidal-bound metals (in the size range of 1 kDa to 0.2 µm, Cd, Cr, and Zn) were bioavailable for biological uptake by copepods, although the uptake rate of these colloidal-bound metals was lower than the uptake of free metals. Pan and Wang (26) showed that the mussels (Perna viridis) were able to accumulate the colloidal-bound Ag, but also at a lower rate than the uptake of Ag+. Uptake of these colloidal metals may involve different mechanisms such as dissociation, permeation, cotransport, and endocytosis which were highly dependent on the functional physiologies of the studied organisms. Compared with the uptake of the Ag+ (0.260 L/g/h, Lam and Wang, 2006), the uptake of AgNP at the lower concentration range (0.060 L/g/h) was about 4.3-fold lower, again possibly as a result of different uptake mechanisms for Ag+ and AgNP. Bianchini and Wood (27) showed that Ag+ combined with proton ATPase could enter into the branchial epithelial cells in daphnids through Na+ channel. Because of the high affinity VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Retention of AgNP in Daphnia magna during 4 days depuration after being exposed to different concentrations of AgNO3 and AgNP. Data are mean ( SD (n ) 3).

FIGURE 2. (A) Retention of ingested AgNP in Daphnia magna fed with Chlamydomonas reinhardtii at different AgNP concentrations. (B) Relationship between AgNP assimilation efficiency (AE) in Daphnia magna and AgNP concentration in Chlamydomonas reinhardtii. Data are mean ( SD (n ) 3). of Ag+ with sulfur containing compounds (transporters), the process of Ag+ passing through the cell membranes would be rapid, which contributed to the high ku of Ag+. The y-intercept of the linear regression between the accumulated Ag and the time of exposure was operationally defined as the initial nonexchangeable adsorption. In our study, significant adsorption occurred for daphnids once they were immediately exposed to the AgNP (4.70-38.74 µg/g dry wt over the range 2-40 AgNP µg/L). Consistent with this result, AgNP were also found in the exoskeleton of daphnids. However, the detection of AgNP in the exoskeleton did not necessarily indicate the importance of surface sorption of AgNP by the daphnids, since these nanoparticles may also be affiliated with the internal surfaces of the animals. Assimilation. When the radiolabeled algae were resuspended into the SM7 medium, >90% of the radioactive AgNP still remained in/on the algae, indicating that nearly all of the AgNP incorporated into daphnids during the pulse feeding period were through dietborne ingestion. After 15 min of pulse-feeding, the percentage of ingested AgNP retained in daphnids decreased sharply within the first 3 h and then gradually leveled off (Figure 2A). With an increase in the AgNP concentration in the diet, the AgNP retention decreased. The calculated AEs decreased from 44.7% to 21.5% with an increase in AgNP concentration in algae from 0.216 to 9.04 mg/g (Figure 2B, r2 ) 0.999, p < 0.05). Our study presented the first measurements of AgNP dietary AE in any aquatic animal, and strongly suggested that AgNP could be highly assimilated by daphnids upon entering the trophic transfer processes. We can compare our measured AEs for AgNP with those determined for Ag+ in D. magna (23). Lam and Wang (23) demonstrated that the dietary Ag assimilation in daphnids was dependent on both the food concentration and the Ag concentration in the diet. With an increase in food concentration from 5 × 103 to 105 cells/mL, the AE dramatically decreased from 43.9% to 1% (23). In the present study, the AgNP AEs determined for the same type of food (C. reinhardtii) at a food concentration of 105 cells/ mL were 21.5-44.7%, which was 20-40 times higher than that of Ag determined under the same food conditions (23). 7702

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When the algae were spiked with different Ag concentrations (2.5-61.8 µg/g), the AEs decreased from 63% to 40% (23), but these results were determined at a lower food concentration (2 × 104 cells/mL). In the present study, AgNP concentrations in/on the algae were 0.216-9.035 mg/g (86.4-146.2 times higher than in the previous study), and the AE decreased only from 44.7% to 21.5%. These comparisons strongly suggest that AgNP was more efficiently assimilated by daphnids, and was more difficult to be depurated when they were ingested through the dietary route. The depuration of nanoparticles was also shown to be size-dependent. Rosenkranz et al. (12) used fluorescent carboxylated polystyrene beads as model nanoparticles and found that 20 nm particles ingested by daphnids were retained more efficiently (67% of the original particle burden) than 1000 nm particles (12.5% of the original particle burden) during 4 h depuration in the clean medium. In the present study, the dominant particle size was about 20 nm, which also contributed to their difficulty in being eliminated by the daphnids. Some studies demonstrated that adding algae could elevate the depuration of nanoparticles from the daphnids (13), but the nanoparticles could not be completely removed from daphnids in the end. These results were also consistent with our present finding. For example, during depuration with the presence of algal food at a cell density of 105 cells/mL, the ingested AgNP was rapidly eliminated by the daphnids within the first 3 h, after which the loss was at a much slower rate. Since the AgNP could not be completely depurated from the daphnids, there may be significant implications for the transport of AgNP along the aquatic food chains. Besides AgNP, other nanoparticles also showed potential dietary transfer. Holbrook et al. (28) found that carboxylated and biotinylated quantum dots could be transferred from ciliated protozoans to rotifers through dietary intake. Ferry et al. (29) reported the fates of gold nanoparticles in three laboratoryconstructed estuarine mesocosms containing seawater, sediment, sea grass, microbes, biofilms, snails, clams, shrimps, and fish. They found that the gold nanoparticles could be readily passed from the water column to the marine food webs in which the clams and biofilms accumulated the most nanoparticles on a per mass basis. Efflux. The daphnids were exposed to AgNP for 2 days to allow them to accumulate significant amounts of AgNP in their bodies, and were then depurated for 4 days. During the depuration, the accumulated Ag in daphnids from both AgNO3 and AgNP treatments were rapidly lost in the first 2 days, and then gradually lost in the following 2 days (Figure 3). At the end of depuration, there was no significant difference between low and high AgNP concentrations on the percentage of radioactivity retained in daphnids (6.7 ( 1.3% for 5 µg/L AgNP and 4.6 ( 0.8% for 500 µg/L AgNP; p > 0.05, t-test). However, the Ag retention in the AgNP treatment was significantly higher than that of AgNO3

(µg/L), If is the influx rate from the dietary phase (µg/g/d), IR is the ingestion rate (g/g/d) of the daphnids, AE is assimilation efficiency (%), and Cf is the AgNP concentration in the food (µg/g). Therefore, the fraction of AgNP accumulated from the dissolved phase (f) is calculated by: f ) Iw /(Iw + If) Since Cf can be calculated by the following equation: Cf ) CF × Cw FIGURE 4. The relative contributions the four routes in the total AgNO3 and AgNP efflux during the 4 day depuration. Data are mean ( SD (n ) 3). treatment (2.0 ( 0.1% for 0.9 µg/L AgNO3; p < 0.05, t-test). The Ag elimination from daphnids in both treatments was a two-compartmental depuration. For the first compartment, ke1 (1.83 ( 0.19/d) of AgNO3 treatment was higher than that of AgNP treatment (1.52 ( 0.13/d for 5 µg/L AgNP and 1.35 ( 0.10/d for 500 µg/L AgNP). A similar tendency was observed in the second compartment. The ke2 of AgNO3 treatment (0.34 ( 0.05/d) was also higher than that of AgNP treatment (0.13 ( 0.08 for 5 µg/L AgNP and 0.29 ( 0.02/d for 500 µg/L AgNP). Accordingly, the biological retention half-life of AgNP in daphnids would be 5.3 days for 5 µg/L AgNP and 2.4 days for 500 µg/L AgNP. The calculated Ag ke in the AgNO3 treatment in this study was similar to that measured by Lam and Wang (23; ke 0.36 ( 0.03 with dietary exposure). The lower AgNP ke indicated that they were difficult to be depurated from the daphnids. The retained AgNP could be transported and distributed all over the body. Lee et al. (30) found that AgNP could enter into the embryos during hatching and were retained by the embryos, and were subsequently observed in the retina, brain, heart, gill arches, and tail of the mature fish. Thus, the difficulty in depuration could lead to chronic toxicity and health risk during the entire life of these organisms. During the 4 day depuration, the excretion accounted for 62.8%, 70.0%, and 48.4% of the total Ag loss from daphnids of AgNO3, low and high AgNP treatments, respectively (Figure 4). No significant difference on molt loss was observed (14.3% for AgNO3, 14.8% and 12.4% for low and high AgNP). Only 2.6% for AgNO3, 3.7% and 1.8% for low and high AgNP were measured in the neonate, indicating that little assimilated Ag was transferred to the next generation. Maternal transfer was important for a few essential and toxic metals (e.g., Zn, Se, and Hg) (31). However, due to the difficulty of depuration, any AgNP transferred to the neonates may be retained for a long time. The loss by feces was significantly higher for the high AgNP treatment (37.2%) than for the AgNO3 treatment (20.3%), which may have been due to the higher tendency of excreted AgNP to bind with the feces. Modeling the Importance of AgNP Uptake from Water and Food. With the quantification of the biokinetic parameters such as the uptake rate constant of AgNP and the dietary assimilation of AgNP, it was then possible to separate the relative importance of these two uptake routes in daphnids, using the well established kinetic modeling approach. The respective influx of each route can be calculated by the following equations (Iw and If). Iw ) ku × Cw If ) IR × AE × Cf where Iw is the influx rate from the dissolved phase (µg/g/d), ku is the uptake rate constant from the dissolved phase (L/ g/d), Cw is the AgNP concentration in the dissolved phase

where CF represents the concentration factor in the algae (L/kg). Thus, the calculation of f could be written as follows: f ) ku /(ku + IR · AE · CF) Since the ku was dependent on the AgNP concentration in the water, we used the ku determined for the lower range of AgNP concentration (1.44 L/g/d at the AgNP concentration range of 2-40 µg/L) likely encountered in the environments in our calculation. Typical IR in daphnids was 0.91 g/g/d (32). CF was calculated by the ratio of AgNP accumulation in algae (both the adsorbed and absorbed) to the exposure concentration in the medium measured in the assimilation experiment (1.84 × 104 L/kg). AE varied over the range of 21.5-44.7%. Consequently, f was calculated to be in the range of 16-29%. Conversely, >70% of AgNP in daphnids was accumulated through the dietary route in the environment. Due to the large specific surface area, the most likely source of nanoparticles was from adsorption onto the inorganic or organic particles in the aquatic environment. Such adsorption would increase the chance of nanoparticles being transferred in the food chain. Indeed, a few recent studies have also noticed the significance of nanoparticle accumulation in the dietary phase (26, 27). Our study provides the first quantitative estimate of the fraction of AgNP uptake from the water. It is thus clear that further study should consider the potential dietborne toxicity of nanoparticles to aquatic ecosystem.

Acknowledgments We thank the anonymous reviewers for their helpful comments on this work. This study was supported by a RPC grant (RPC10.SC10) from HKUST to W.-X. Wang.

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