Intracellular Silver Accumulation in Chlamydomonas reinhardtii upon

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Intracellular Silver Accumulation in Chlamydomonas reinhardtii upon Exposure to Carbonate Coated Silver Nanoparticles and Silver Nitrate Flavio Piccapietra,†,‡ Carmen Gil Allué,† Laura Sigg,†,‡ and Renata Behra*,† †

Eawag, Swiss Federal Institute of Aquatic Science and Technology, CH-8600 Duebendorf, Switzerland ETH, Institute of Biogeochemistry and Pollutant Dynamics IBP, CH-8092 Zurich, Switzerland

‡

S Supporting Information *

ABSTRACT: The intracellular silver accumulation ({Ag}in) upon exposure to carbonate coated silver nanoparticles (AgNP, 0.5−10 μM, average diameter 29 nm) and silver nitrate (20−500 nM) was examined in the wild type and in the cell wall free mutant of the green alga Chlamydomonas reinhardtii at pH 7.5. The {Ag}in was measured over time up to 1 h after a wash procedure to remove silver ions (Ag+) and AgNP from the algal cell surface. The {Ag}in increased with increasing exposure time and with increasing AgNP and AgNO3 concentrations in the exposure media, reaching steadystate concentrations between 10−5 and 10−3 mol Lcell−1. According to estimated kinetic parameters, high Ag+ bioconcentration factors were calculated (>103 L Lcell−1). Higher accumulation rate constants were assessed in the cell wall free mutant, indicating a protective role of the cell wall in limiting Ag+ uptake. The bioavailability of AgNP was calculated to be low in both strains relative to Ag+, suggesting that AgNP internalization across the cell membrane was limited.

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INTRODUCTION Determining the bioavailability of metals and their intracellular accumulation in aquatic organisms is essential for the evaluation of metal toxicity to aquatic life. Due to increasing production and use of engineered silver nanoparticles (AgNP) in consumer products,1−5 those could be potentially released as discrete particles or as composite colloids.6−8 According to the reported risk quotient between predicted AgNP concentration and their no effect concentration, a potential risk for the aquatic systems can be expected.9 Additionally, the detection of discrete and agglomerated AgNP was already reported under specific natural freshwater conditions.10,11 Consequently, aquatic organisms may be exposed to discrete AgNP, agglomerated AgNP, or to silver ions released from their surface. At present, only few studies have investigated the interactions of AgNP with algae. AgNP toxicity was examined for the green alga Chlamydomonas reinhardtii12 and for a coastal marine diatom.13 In both cases, the AgNP toxicity was reported to be primarily mediated by the free silver ions (Ag+). The intracellular accumulation of AgNP was reported only in one study for the freshwater alga Ochromonas danica.14 Thus, only a general hypothesis can be proposed for the AgNP internalization mechanisms in algae, mainly based on the comparison between the size of the nanoparticles and the size of the pores across the cell wall (5−20 nm),15 and assuming endocytotic uptake across the cell membrane.16 More information is available on interactions of Ag+ with algae. Bioaccumulation and Ag+ mediated toxicity of silver compounds in algae were reviewed by Ratte. 17 High © 2012 American Chemical Society

bioconcentration factors were reported for freshwater green algae (>105) and marine algae (>104).17 The silver uptake and toxicity to the green alga C. reinhardtii was also investigated.18−21 Examination of silver uptake in C. reinhardtii showed that the Ag+ uptake is very rapid and possibly mediated by a Cu(I) transport system.18 However, a systematic and quantitative comparison between intracellular silver accumulation in algae upon exposure to Ag+ and to AgNP needs to still be performed. Additionally, more information on the role of the cell wall is needed. According to comparative studies on the toxicity of Cd, Co, Cu, and Ni performed using the wild type and a cell wall free mutant of C. reinhardtii,22,23 the mutant was found to be more sensitive to metals, indicating that the cell wall plays a role in increasing the metal tolerance of the alga. However, the role of the cell wall as a barrier against silver uptake is still not clear. In the present study, the time and concentration dependence of the intracellular silver accumulation upon exposure to carbonate coated AgNP and to AgNO3 was examined in the model green alga C. reinhardtii. Silver concentrations were chosen according to the concentration range in which the photosynthetic yield of the wild type of this alga is affected.12 A comparison between the two different treatments based on the Received: Revised: Accepted: Published: 7390

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and 25 °C for the mutant, and 10 min, 3000 rpm, and 25 °C for the wild type). Cell number and volume were detected by an electronic particle counter (aperture 50 μm; Z2 Coulter Counter; Beckman Coulter, Fullerton, CA). The average diameter, volume and cell surface of wild type and mutant cells used for further evaluations and calculations were 5.76 μm, 100 fL and 104 μm2, and 4.15 μm, 37 fL and 55 μm2, respectively. Exposure Media and Conditions. To avoid silver complexation, all experiments were performed in 10 mM MOPS at pH 7.5. In this nutrient free medium, untreated algae maintain their maximal photosynthetic activity up to a few hours.12 Additionally, the exposure time was limited to 1 h to minimize silver complexation with algal exudates. To avoid the effect of the algal cell density on the intracellular silver accumulation (Supporting Information (SI) Figure S1 and Franklin et al.27), all experiments were performed with the same cell density of 1 × 106 cells mL−1. Before starting the exposure, algae were acclimatized for 15 min to 10 mM MOPS at pH 7.5, and samples of unexposed algae were taken to control for possible silver contamination. At the end of each experiment, the cell density in the suspension was checked again. All experiments were conducted under constant stirring at 25 °C and performed in three culture replicates. The errors bars presented in the figures represent their calculated standard deviation. Wash Experiments. To differentiate between total and intracellular silver content ({Ag}in), a washing procedure with a ligand was used to complex and remove the extracellular Ag+ adsorbed on the cell wall or the cell membrane of the algae.28,29 Based on preliminary experiments comparing the wash efficiency of EDTA, diethyldithiocarbamic acid ammonium salt (DDC) and cysteine in the wild type (SI Figure S2), cysteine was selected in this study as a wash ligand for silver due to its high removal efficiency and to the resulting stable {Ag}in measured over time (stability constant silver-cysteine complex AgCysH, logK = 22.7, SI Table S1).30 Thus, the {Ag}in is operationally defined as the noncysteine-removable silver. The time dependence of the cysteine wash efficiency was further examined by exposing the exponentially growing mutant to 30 nM AgNO3. After 1 h, algal samples were washed with 0.5 mM cysteine for 1−60 min, and finally filtered on cellulose nitrate filters by vacuum filtration using a PC filter holder (SM 16510, Sartorius AG; Göttingen, Germany). Aliquots of unwashed exposed algae were taken to measure the total silver content after 1 h of exposure. Similar experiments were performed for the wild type using 1 mM cysteine (SI Figure S2). In the case of exposure to AgNP, preliminary experiments for the optimization of the wash procedure used to remove the AgNP from the cell surface were performed by subsequent resuspension of the exposed algae in fresh media. Mutant algae were exposed to 1 μM AgNP and, after 1 h, cells were centrifuged (10 min, 1500 rpm, 25 °C) and resuspended in fresh 10 mM MOPS at pH 7.5. This wash procedure was repeated 1 to 5 times. Additionally, at the end of the last wash cycle, a 0.5 mM cysteine wash for 5 min was also performed to remove adsorbed Ag+. Finally, algae were filtered as previously described. To measure the total silver content after 1 h of exposure, samples of unwashed exposed algae were also taken. Similar experiments were performed for the wild type (SI Table S2).

Ag+ present in the exposure media was also performed to quantify the AgNP internalization in algae and the dissolution of Ag+ from the AgNP upon exposure. The silver uptake kinetics was further evaluated by modeling and calculating the silver uptake and release rate constants. To evaluate the role of the cell wall on silver accumulation, comparative studies were performed with the wild type and a cell wall free mutant of C. reinhardtii.

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MATERIALS AND METHODS Materials. Carbonate coated AgNP were provided by NanoSys GmbH (Wolfhalden, Switzerland) as an aqueous suspension with a nominal silver concentration of 1 g L−1 (9.27 mM), and were previously used and characterized by Piccapietra et al.11 and Navarro et al.12 The carbonate coating was chosen because it is not toxic to algae and as a representative coating of nanoparticles used in consumer products (similar chemical properties as citrate). According to previous analysis,12 1% (0.7−1.2%) of the total silver in the AgNP stock suspension was present as Ag+. Concentrations of AgNP are given as molarity of the total silver mass. To prevent redox reactions, all solutions containing silver were kept in the dark. AgNO3, L-cysteine, NaOH, 3-morpholine propanesulfonic acid (MOPS) and all compounds of the algal growth medium Talaquil24 were purchased from Sigma-Aldrich (purissimum grade; Buchs SG, Switzerland), and HNO3 (65%) and H2O2 (30%) from Merck (Suprapure chemicals; Darmstadt, Germany). Stock solutions of 0.1 M MOPS buffer (pH adjusted to 7.5) and 10 mM AgNO3 were prepared in deionized nanopure water (16−18 MΩ cm; Barnstead Nanopure Skan Ag, BaselAllschwil, Switzerland). All working AgNO3, AgNP, MOPS (10 mM) and cysteine solutions were freshly prepared prior to use. Cysteine was immediately stored on ice to prevent oxidation. To avoid metal contamination, all needed polycarbonate and Teflon materials were soaked at least for 24 h in 0.03 M HNO3, and then well rinsed with deionized nanopure water before use. Cellulose nitrate filters (pore size 0.45 μm; Sartorius AG, Goettingen, Germany) were first boiled into 0.03 M HNO3 and then dried at 50 °C for 24 h, without affecting their integrity.24,25 To avoid biological contaminations, all materials and media used for the algal growth were autoclaved prior to use. Nanoparticle Characterization. Z-average size and average zeta potential (ZP) of the AgNP were measured respectively by dynamic light scattering (DLS) and electrophoretic mobility using a Zetasizer (Nano ZS, Malvern Instruments) as described in Piccapietra et al.11 Analysis of dissolved silver was performed by ultrafiltration using Ultracel 3k Centrifugal Filter Devices (Amicon Millipore) with a molecular cutoff of 3 kDa (pore size 0 (SI Model S1). Consequently, the nonlinear k1

model was modified as Ae(−αt ) + B ⇄ {Ag}in . According to k −1

this model, the silver accumulation follows an exponential increase that rises to a maximum (steady-state) with a hyperbolic evolution over time:{Ag}in = ((k1B)/(k−1)) + ((k1A)/(k−1 −α))e(−αt) − (((k1B)/(k−1)) + ((k1A)/(k−1 −α)))e(−k−1t) (SI Model S2). Considered variables of this model are the measured intracellular silver accumulation ({Ag}in, mol Lcell−1 or mol m−2), the nonlinear uptake rate constant (k1, L Lcell−1 min−1 or L m−2 min−1) and the nonlinear release rate constant (k−1, min−1). The proposed equation was solved applying the constraint of k−1 > 0 and with the start point at x = 0. Similarly, the linear model was modified as k1

Ae(−αt ) + B → {Ag}in (solution: {Ag}in = ((k1A)/α)(1 − e(−αt)) + k1Bt, SI Model S3). Based on the kinetics data, a silver bioconcentration factor (BCF, L Lcell−1) was calculated as BCF = ((k1)/(k−1)).25,31 Additionally, a bioconcentration factor was also calculated based on the ratio between the intracellular steady-state silver concentration and the silver concentration in the exposure media (BCF*, L Lcell−1), as BCF* = (({Ag}in,steady−state)/ ([Ag]out)). Errors deriving from modeled parameters were calculated according to: ((stdx)/x) = (∑((stda)/a)2)1/2, where x is the value of the parameter calculated from the parameters a and 7392

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Figure 2. Intracellular silver content in wild type (A, C) and mutant (B, D) upon exposure to various concentrations of AgNO3 (A, B) and AgNP (C, D) over time.

Figure 3. Intracellular silver content in wild type (A, C) and mutant (B, D) as a function of the exposure AgNO3 (A, B) and AgNP (C, D) concentrations measured after various exposure times.

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Based on the exposure to similar total silver concentrations, higher {Ag} in was measured upon exposure to AgNO 3 compared with AgNP. A higher accumulation was observed in the wild type after 60 min of exposure to 0.5 μM AgNO3 (3.5 × 10−4 mol Lcell−1) compared with the exposure to 0.5 and 2 μM AgNP (4.1 × 10−5 and 1.1 × 10−4 mol Lcell−1, respectively). Higher values were also measured for 50 and 100 nM AgNO3 (6.6 × 10−5 and 8.3 × 10−5 mol Lcell−1, respectively) in comparison to 0.5 μM AgNP. Similar trends were found in exposure experiments using the mutant, where a higher value was measured upon exposure to 100 nM AgNO3 (3.2 × 10−4 mol Lcell−1) in comparison to 500 nM AgNP (2.1 × 10−4 mol Lcell−1). A comparison between the different treatments after 1 h of exposure was performed as a function of Ag+ (Figure 4). The

from its standard deviation (stda), and stdx is the standard deviation of x.

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RESULTS Nanoparticle Characterization. As reported by Piccapietra et al.,11 AgNP in the original suspension (pH 9.65) have a Z-average size of 29.0 ± 2.5 nm and an average ZP of −41.8 ± 3.1 mV. The analysis of the AgNP stability (size and ZP) in 10 mM MOPS at pH 7.5 revealed a constant Z-average size between 29 and 45 nm for AgNP concentrations between 9.27 mM and 10 μM. For highly diluted solutions (1 and 0.1 μM), an increase of the Z-average size up to 90 and 436 nm, and a ZP approaching to zero (due to dilution of the carbonate coating) was observed. Concerning AgNP dissolution (SI Table S3), similar percent of Ag+ compared to the AgNP stock solution (1%) was measured after 1 h of exposure in 10 mM MOPS (between 1.1% for 10 μM AgNP and 2.0% for 0.5 μM AgNP). Wash Experiments. The cellular silver content measured in the mutant with increasing cysteine wash time and with increasing number of resuspension cycles is reported in Figure 1. In the case of the AgNO3 treatment, the measured total silver content after 1 h of exposure to 30 nM AgNO3 was 3.2 × 10−4 mol Lcell−1. Approximately 50% of the silver was removed after 1 min of washing with 0.5 mM cysteine (1.5 × 10−4 mol Lcell−1). Longer wash times up to 1 h did not change this value, indicating that the adsorbed Ag+ was removed and that the Ag cysteine complex was not taken up by the cell. In the case of the AgNP treatment, a high fraction of silver (>95%) was removed by the first centrifugation and resuspension cycle, and a constant silver content between 6.4 × 10−4 and 1.5 × 10−4 mol Lcell−1 (approximately 3% of the total silver content of 8.7 × 10−3 mol Lcell−1) was measured with increasing number of subsequent wash cycles. Similar results were observed for the wild type (SI Figure S2 and Table S2). According to these results and based on the time optimization of the wash procedure, a 5 min cysteine wash and 3 subsequent resuspension cycles followed by a 5 min cysteine wash were selected as optimal for the determination of the intracellular silver content ({Ag}in) in the accumulation experiments upon exposure to AgNO3 and AgNP, respectively. Accumulation Experiments. The {Ag}in upon exposure to AgNO3 and AgNP increased over time in both strains (Figure 2). In the case of AgNO3, a fast uptake was observed in the first 10 min of exposure, and a steady-state was reached after 20 min in the mutant and after 60 min in the wild type. Maximal values of 3.5 × 10−4 and 3.2 × 10−4 mol Lcell−1 were measured in wild type and mutant after 1 h of exposure to 500 and 100 nM AgNO3, respectively. In contrast, the {Ag}in upon AgNP exposure followed a slow linear increase over time in the wild type and reached a constant value within 10 min in the mutant. At each time point, higher accumulation was observed upon exposure to 2 μM, as compared to 5 μM AgNP in the mutant. According to the concentration dependence of the intracellular silver accumulation (Figure 3A and B), higher {Ag}in was measured in both strains with increasing AgNO 3 concentrations in the exposure media, and a linear relationship was evident (R2 between 0.83 and 0.99). In the case of the AgNP treatment (Figure 3C and D), the {Ag}in showed a nonlinear increase that approached a maximum with increasing particle concentration in the exposure media. Above 2 and 5 μM AgNP, a constant accumulation level around 4.0 × 10−4 and 1.2 × 10−3 mol Lcell−1 was measured in the wild type and in the mutant, respectively.

Figure 4. Intracellular silver content in wild type (red circle and green upward triangle) and mutant (blue square and black downward triangle) after 1 h exposure to AgNO3 (red circle and blue square) and AgNP (green upward triangle and black downward triangle) as a function of the dissolved silver (Ag+) in the exposure media.

{Ag}in increased with increasing Ag+ concentration in the exposure media in both strains, and 4−8 and 8−18 times higher values were measured in wild type and mutant upon exposure to AgNP compared to AgNO3, respectively. In comparing the two strains, higher {Ag}in was always measured in the mutant upon exposure to the same AgNP and AgNO3 concentration. After 1 h of exposure to 100 nM AgNO3, the {Ag}in was to 8.3 × 10−5 for the wild type and 3.2 × 10−4 mol Lcell−1 for the mutant. The same trend was observed for the exposure to 5 μM AgNP, where the accumulation reached values of 5.4 × 10−4 and 1.1 × 10−3 mol Lcell−1 for the wild type and the mutant, respectively. Uptake Models and Kinetics. The uptake and release rate constants estimated using the data on the {Ag}in as a function of time upon exposure to AgNO3 (Figure 2) and based on the calculated decrease of [Ag]out (SI Model S1) are shown in Table 1. According to the R2 (between 0.76 and 0.98), the proposed uptake and release model assuming a decreasing [Ag]out described the experimental data well. Values of k1 between 25 and 69 L Lcell−1 min−1 were calculated for the wild type. Significantly higher (P < 0.05, t test) uptake rate constants were found for the mutant under the same exposure conditions, with k1 values ranging from 194 to 437 L Lcell−1 min−1. Similar difference between the two strains was observed for the uptake rate constants expressed on the basis of L m−2 min−1. The estimated release rate constants k−1 were always much lower compared to the correspondent uptake rate 7394

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Table 1. Estimated Uptake Rate Constants k1 (L Lcell−1 min−1 and L m−2 min−1), Release Rate Constants k−1 (min−1), Bioconcentration Factors Based on Kinetics Data (BCF, L Lcell−1), and Bioconcentration Factors Calculated According to the Internal Steady-State Silver Concentration (BCF* = {Ag}in [Ag]out−1, L Lcell−1) in the Wild Type and in the Mutant as a Function of Exposure Concentrations of AgNO3a

a

exposure

algae

20 nM AgNO3 50 nM AgNO3 100 nM AgNO3 500 nM AgNO3 20 nM AgNO3 50 nM AgNO3 75 nM AgNO3 100 nM AgNO3

wild type wild type wild type wild type mutant mutant mutant mutant

k1 (L Lcell−1 min−1) 69 61 25 45 437 248 194 366

± ± ± ± ± ± ± ±

21 8 5 13 63 27 47 76

k1 (L m−2 min−1) 0.07 0.06 0.02 0.04 0.30 0.17 0.13 0.25

± ± ± ± ± ± ± ±

0.02 0.01 0.00 0.01 0.04 0.02 0.03 0.05

k−1 (min−1) 0.03 0.03 0.02 0.06 0.10 0.08 0.06 0.09

± ± ± ± ± ± ± ±

0.01 0.01 0.01 0.02 0.02 0.01 0.02 0.02

BCF (L Lcell−1)

R2

BCF* (L Lcell−1)

± ± ± ± ± ± ± ±

0.90 0.98 0.97 0.90 0.88 0.96 0.81 0.76

2246 1782 1000 803 4692 3298 3567 4174

2480 1990 1410 793 4423 3155 3121 3945

1515 540 715 371 1014 548 1267 1311

The {Ag}in upon exposure to AgNP could not be described by both proposed uptake models.

constants (k1, L Lcell−1 min−1), and slightly higher values were observed for the mutant (approximately 0.08 min−1) compared to the wild type (approximately 0.04 min−1). High BCF (k1 k−1−1) were estimated, up to 4423 for the mutant and 2480 L Lcell−1 for the wild type. Based on the ratio between the measured intracellular silver steady-state concentrations and the exposure concentration, similar values (BCF*) up to 4692 and 2246 L Lcell−1 were calculated for the mutant and the wild type, respectively. Similar rate constants and BCF were calculated with a nonlinear uptake and release model assuming a constant [Ag]out (SI Model S4). Same procedure was applied for the linear model (SI Model S3). However, this model was not able to fit the considered data set.

the AgNP to C. reinhardtii under the considered exposure conditions. The intracellular silver accumulation upon exposure to AgNO3 was observed to be very high in both strains under all considered exposure conditions, following a time dependent increase that rose to a steady-state concentration (Figure 2A and B). For the mutant, the time needed to reach this steadystate was shorter (20 min). Relative to other studies, the {Ag}in measured in the wild type up to 60 min of exposure to 20 nM AgNO3 (0.005−0.03 μmol m−2) is 50 times lower compared to the values reported by Fortin and Campbell for the same alga exposed up to 60 min to 10 nM Ag+ (0.3−1.5 μmol m−2).18 This difference can be explained by the 100 times lower cell density used by Fortin and Campbell (1 × 104 cell mL−1), and indicates that the effect of the cell density on the intracellular metal accumulation has to be carefully considered (SI Figure S1).27 The experimental data were also well described by the uptake model, which considers a simultaneous silver uptake and release, but not by a simple linear uptake model. The estimated high uptake rate constants confirmed that the Ag+ uptake is a fast process in C. reinhardtii, as already reported by Fortin and Campbell.18 The estimated bioconcentration factors (BCF and BCF*) were very high and similar (>103 L Lcell−1), confirming that steady-state equilibrium was reached under the considered conditions. Upon comparison with other studies investigating intracellular bioaccumulation considering silver speciation, higher BCF can be derived from Fortin and Campbell for the same organism (between 104 and 105 L Lcell−1).18 Additionally, a linear relationship between the Ag+ concentration in the exposure media and the {Ag}in was determined (Figure 3A and B), confirming that the silver uptake was proportional to the Ag+ concentration in the media. This finding suggests that the influence of the algal exudates on the silver speciation was negligible for the considered exposure time. In the case of the AgNP exposure, the almost constant {Ag}in measured over time (Figure 2C and D) suggested that steadystate concentration was reached very quickly (less than 10 min). Based on total silver mass, a much lower {Ag}in was measured upon exposure to AgNP compared to AgNO3, confirming the limited bioavailability of AgNP relative to Ag+. According to the measured {Ag}in (Figure 3C and D, and Figure 4), the silver uptake was neither linearly proportional to the AgNP nor to the estimated Ag+ concentration, suggesting that further processes affect the bioavailability of silver in the exposure media. Upon comparison between the AgNP and AgNO3 treatments as a function of Ag+ concentrations (Figure 4), a 4−8 (wild type) and 8−18 (mutant) times increase in

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DISCUSSION The intracellular silver accumulation was systematically investigated in the wild type and in the cell wall free mutant of C. reinhardtii upon exposure to AgNO3 and AgNP. Cysteine at concentrations of 0.5 and 1 mM was used as a ligand in the wash procedure to discriminate between total and intracellular silver content ({Ag}in) in the mutant and in the wild type, respectively. According to the results (Figure 1A, and SI Figure S2), the Ag+ adsorbed on the cell wall and cell membrane was washed by complexation with cysteine, and silver-cysteine complexes were not taken up in either strain during the cysteine wash for up to 1 h. In the case of the AgNP exposure, the proposed wash procedure (Figure 1B, and SI Table S2) removed more than 95% of the silver from the cell surface. This result can be explained by the weak electrostatic interaction between the negatively charged AgNP (ZP approached to zero only for the highly diluted AgNP concentration) and the negatively charged algal cell surface. According to simple AgNP sorption and removal modeling (SI Table S4A), under the considered exposure conditions (0.5−10 μM AgNP) a maximal number of nanoparticles between 13 and 2134 was calculated to be available to each algal cell. Based on the measured total silver accumulation, only between 5 and 302 AgNP were calculated as being sorbed on the surface of each cell. After the wash procedure, the number of sorbed AgNP per cell decreased to a value between 0 and 12, that were enough to cover only 0.03− 0.46% of the algal surface (SI Table S4B). To cover 100% of the cells with nanoparticles, higher AgNP exposure concentrations between 40 μM and 3 mM would be needed, based on the AgNP size (SI Table S3C). Consequently, only few particles were sorbed on the algal cell surface after the wash procedure, providing a first indication of low bioavailability of 7395

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(5) Hendren, C. O.; Mesnard, X.; Droge, J.; Wiesner, M. R. Estimating production data for five engineered nanomaterials as a basis for exposure assessment. Environ. Sci. Technol. 2011, 45 (7), 2562− 2569. (6) Geranio, L.; Heuberger, M.; Nowack, B. The behavior of silver nanotextiles during washing. Environ. Sci. Technol. 2009, 43 (21), 8113−8118. (7) Benn, T. M.; Westerhoff, P. Nanoparticle silver released into water from commercially available sock fabrics. Environ. Sci. Technol. 2008, 42 (11), 4133−4139. (8) Kaegi, R.; Sinnet, B.; Zuleeg, S.; Hagendorfer, H.; Mueller, E.; Vonbank, R.; Boller, M.; Burkhardt, M. Release of silver nanoparticles from outdoor facades. Environ. Pollut. 2010, 158 (9), 2900−2905. (9) Gottschalk, F.; Sonderer, T.; Scholz, R. W.; Nowack, B. Possibilities and limitations of modeling environmental exposure to engineered nanomaterials by probabilistic material flow analysis. Environ. Toxicol. Chem. 2010, 29 (5), 1036−1048. (10) Chinnapongse, S. L.; Hackley, V. A.; MacCuspie, R. I. Persistence of singly dispersed silver nanoparticles in natural freshwaters, synthetic seawater, and simulated estuarine waters. Sci. Total Environ. 2011, 409 (12), 2443−2450. (11) Piccapietra, F.; Sigg, L.; Behra, R. Colloidal stability of carbonate-coated silver nanoparticles in synthetic and natural freshwater. Environ. Sci. Technol. 2012, 46 (2), 818−825. (12) Navarro, E.; Piccapietra, F.; Wagner, B.; Marconi, F.; Kaegi, R.; Odzak, N.; Sigg, L.; Behra, R. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 2008, 42 (23), 8959−8964. (13) Miao, A. J.; Schwehr, K. A.; Xu, C.; Zhang, S. J.; Luo, Z. P.; Quigg, A.; Santschi, P. H. The algal toxicity of silver engineered nanoparticles and detoxification by exopolymeric substances. Environ. Pollut. 2009, 157 (11), 3034−3041. (14) Miao, A. J.; Luo, Z. P.; Chen, C. S.; Chin, W. C.; Santschi, P. H.; Quigg, A., Intracellular uptake: A possible mechanism for silver engineered nanoparticle toxicity to a freshwater alga Ochromonas danica. Plos One 2010, 5 (12). (15) Navarro, E.; Baun, A.; Behra, R.; Hartmann, N. B.; Filser, J.; Miao, A. J.; Quigg, A.; Santschi, P. H.; Sigg, L. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008, 17 (5), 372−386. (16) Moore, M. N. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 2006, 32 (8), 967−976. (17) Ratte, H. T. Bioaccumulation and toxicity of silver compounds: A review. Environ. Toxicol. Chem. 1999, 18 (1), 89−108. (18) Fortin, C.; Campbell, P. G. C. Silver uptake by the green alga Chlamydomonas reinhardtii in relation to chemical speciation: Influence of chloride. Environ. Toxicol. Chem. 2000, 19 (11), 2769− 2778. (19) Fortin, C.; Campbell, P. G. C. Thiosulfate enhances silver uptake by a green alga: Role of anion transporters in metal uptake. Environ. Sci. Technol. 2001, 35 (11), 2214−2218. (20) Lee, D. Y.; Fortin, C.; Campbell, P. G. C. Contrasting effects of chloride on the toxicity of silver to two green algae, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. Aquat. Toxicol. 2005, 75 (2), 127−135. (21) Hiriart-Baer, V. P.; Fortin, C.; Lee, D. Y.; Campbell, P. G. C. Toxicity of silver to two freshwater algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata, grown under continuous culture conditions: Influence of thiosulphate. Aquat. Toxicol. 2006, 78 (2), 136−148. (22) Macfie, S. M.; Tarmohamed, Y.; Welbourn, P. M. Effects of cadmium, cobalt, copper, and nickel on growth of the green-alga Chlamydomonas ReinhardtiiThe Influences of the cell-wall and pH. Arch. Environ. Contam. Toxicol. 1994, 27 (4), 454−458. (23) Macfie, S. M.; Welbourn, P. M. The cell wall as a barrier to uptake of metal ions in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae). Arch. Environ. Contam. Toxicol. 2000, 39 (4), 413−419.

{Ag}in was measured for the AgNP compared to AgNO3. These differences were calculated to correspond with a maximal accumulation of 2−10 particles per cell, or to at least 0.4−2.1% increased Ag+ dissolution from the AgNP upon exposure (SI Table S4D). This calculation suggests that Ag+ is released from the AgNP surface during exposure, and that a limited number of AgNP are bioavailable to C. reinhardtii, which are internalized or strongly sorbed on the cell surface. The importance of free ions in NP uptake by C. reinhardtii was also reported for Cd based quantum dots.34 Comparison between {Ag}in in the wild type and in the mutant showed estimated uptake rate constants and bioconcentration factors for AgNO3 to be significantly higher in the mutant. Consequently, a limitation of the Ag + internalization due to a silver complexation on the cell wall surface and to a lower Ag+ diffusion through the cell wall may explain such results. The longer time required for the wild type to reach the steady-state concentration confirms this hypothesis. Additionally, the similar low number of accumulated AgNP calculated for the mutant indicated that the internalization of AgNP across the cell membrane is also limited. However, the investigation of the AgNP bioavailability to C. reinhardtii upon long-term exposure and to other algae with a different type of cell wall is further required.

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ASSOCIATED CONTENT

S Supporting Information *

Information on influence of cell density on accumulation, wash efficiency of the proposed procedures, silver-cysteine stability constant, AgNP dissolution, uptake models, and modeling of AgNP sorption and removal from algal surface. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 58 765 51 19; fax: +41 58 765 53 11; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank David Kistler for ICP-MS measurements, Bettina Wagner for help in the accumulation experiments and algae culturing, and Theodora Stewart for internal reviewing. This research was funded by the Swiss National Science Foundation (SNSF).

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REFERENCES

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Environmental Science & Technology

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