Environ. Sci. Technol. 2008, 42, 8959–8964
Toxicity of Silver Nanoparticles to Chlamydomonas reinhardtii E N R I Q U E N A V A R R O , †,‡ FLAVIO PICCAPIETRA,† BETTINA WAGNER,† FABIO MARCONI,† RALF KAEGI,† NIKSA ODZAK,† L A U R A S I G G , † A N D R E N A T A B E H R A * ,† Eawag, Swiss Federal Institute of Aquatic Science and ¨ berlandstrasse 133, Technology, U P.O. Box 611, 8600 Du ¨ bendorf, Switzerland, and Instituto Pirenaico de Ecolog´ia (CSIC), Avda. Montan ˜ ana 1005, Apdo. 202, 50080 Zaragoza, Spain
Received February 15, 2008. Revised manuscript received August 26, 2008. Accepted August 27, 2008.
Silver nanoparticles (AgNP) are likely to enter the aquatic environment because of their multiple uses. We have examined the short-term toxicity of AgNP and ionic silver (Ag+) to photosynthesis in Chlamydomonas reinhardtii using fluorometry. AgNP ranged in size from 10 to 200 nm with most particles around 25 nm. As determined by DGT (diffusive gradients in thin films), by ion-selective electrode, and by centrifugal ultrafiltration, about 1% of the AgNP was present as Ag+ ions. Based on total Ag concentration, toxicity was 18 times higher for AgNO3 than for AgNP (in terms of EC50). However, when compared as a function of the Ag+ concentration, toxicity of AgNP appeared to be much higher than that of AgNO3. The ionic Ag+ measured in the AgNP suspensions could not fully explain the observed toxicity. Cysteine, a strong Ag+ ligand, abolished the inhibitory effects on photosynthesis of both AgNP and Ag+. Together, the results indicate that the interaction of these particles with algae influences the toxicity of AgNP, which is mediated by Ag+. Particles contributed to the toxicity as a source of Ag+ which is formed in presence of algae.
Introduction Developments of nanotechnology are leading to a rapid proliferation of nanomaterials that are likely to become a source of many different engineered nanoparticles (NP) in the environment. The unique NP properties, such as high specific surface area and mobility, could potentially lead to unexpected health or environmental hazards (1, 2). Because of their widespread use in various consumer products, it is expected that nanoparticles are released in the aquatic environment, where their fate and behavior are largely unknown. Toxic effects of nanoparticles on aquatic organisms remain to be evaluated. Silver nanoparticles (AgNPs) are already in use in numerous consumer products including textiles, personal care products, food storage containers, laundry additives, home appliances, paints, and even food supplements (1). AgNPs are added to all these products because of their bactericidal effects. On the basis of these uses, it is likely that AgNPs will * Corresponding author phone: +41 44 823 5119; fax: +41 44 823 5311; e-mail:
[email protected]. † Eawag, Swiss Federal Institute of Aquatic Science and Technology. ‡ Instituto Pirenaico de Ecologı´a (CSIC). 10.1021/es801785m CCC: $40.75
Published on Web 10/01/2008
2008 American Chemical Society
be released to the aquatic environment, be a source of dissolved silver, and possibly exert toxic effects on aquatic organisms (3). Toxicity of AgNPs to bacteria has been examined (4-10), but the mechanisms of toxicity have not been fully elucidated. In particular it is unclear whether toxicity is specifically related to nanoparticle properties or is due to the effects of Ag+ ions. Toxic effects of AgNPs may be related to damages at cell membranes, to oxidative stress, or to interactions of Ag+ ions with proteins and enzymes (4, 7, 11). Toxicity of ionic silver to a variety of aquatic organisms, such as algae, fish, and Daphnia, has been studied and shown to be significant (12-14). From an evaluation of the literature, Ag+ displays toxicity to aquatic organisms in the nanomolar concentration range (12, 15, 16). Uptake of Ag+ by Chlamydomonas reinhardtii has been observed to occur rapidly (17), being hypothesized to occur via a Cu (I)-transporter through the cell membrane (16). Ag+ uptake strongly depends on Ag speciation in the media, with different effects of chloride and thiosulfate as ligands (16-18). No information is available on the uptake and toxicity of AgNPs to algae. The present study aims at examining whether AgNPs are toxic to freshwater algae, using Chlamydomonas reinhardtii as a model organism, and whether toxicity of AgNPs is related to release of Ag+ from the AgNPs, or to specific effects of AgNPs. Toxic effects of AgNPs and Ag+ on the photosynthesis of Chlamydomonas reinhardtii are assessed and compared. A ligand (cysteine) is used to decrease the Ag+ concentration and thus to disentangle the contribution of Ag+ and AgNPs to the overall toxicity. The toxicity mechanisms of AgNPs to Chlamydomonas reinhardtii are discussed on the basis of this experimental evidence.
Materials and Methods Materials. All experiments were carried out using carbonatecoated AgNP which were provided by NanoSys GmbH (Wolfhalden, Switzerland) as an aqueous suspension. Carbonate coating serves to maintain AgNP in suspension by avoiding aggregation. The original AgNP suspension (1 g L-1, 9.27 mM, based on the silver mass) was kept in the dark and experimental concentrations were prepared using MOPS (3morpholine propanesulfonic acid, buffer at pH 7.52) or synthetic media (0.5 mM NaHCO3, 0.09 mM MgCl2, 0.5 mM TES, N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, buffer at pH 7.4). Nanopure water was used to prepare stock solutions of 10 mM silver nitrate and 0.1 mM MOPS. Before each experiment, fresh stock solutions of cysteine were prepared in nanopure water and kept on ice to preserve it from oxidation. AgNO3 and AgNP concentrations were freshly prepared before use. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich. Particle Characterization. AgNP were characterized under experimental conditions (927 µM AgNP in 10 mM MOPS, pH 7.5) and in nanopure water suspensions for size and Zeta potential by Dynamic Light Scattering (DLS) using a Zeta Sizer (Nano ZS, Malvern Instruments). In addition, influence of pH on the AgNP aggregation was studied in 10 mM MOPS at pH 5.3 (by addition of HNO3) and in acetate buffer at pH 4.2. In addition, particles were visually investigated using a transmission electron microscope (TEM, CM30, FEI). The microscope was operated in bright field mode at an acceleration voltage of 300 kV. Images were recorded with a CCD camera attached to the microscope. Metal Measurements. The total silver concentration (isotope 107Ag) was measured in acidified solutions (0.1 M HNO3) by ICP-MS (Element 2 High Resolution Sector Field VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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ICP-MS; Thermo Finnigan). Reliability of the measurements was controlled using specific water references (National Water Research Institute, Burlington, Canada). Prior to the ICP-MS measurements, AgNP suspensions were digested with HNO3 in a microwave oven. Recovery of Ag was 88-95%. Measurement of Ag+ in the AgNP Suspensions by Diffusive Gradients in Thin Films (DGT), Ion-Selective Electrode (ISE), and Centrifugal Ultrafiltration. DGT (19) performance to measure Ag+ was tested (Supporting Information, Tables S2 and S3). Ag+ concentrations in synthetic media containing AgNP (52 ( 8 nM and 66 ( 9 nM AgNP) were measured by deploying DGT devices. Also, total Ag concentrations were measured at the beginning and at the end of an 8-day period at 25 °C, using ICP-MS. Free Ag+ was also measured using a Ag-ISE (Metrohm 6.0502.180), following procedures recommended by ref 20. The potential was measured using a voltmeter Orion 720A and an Orion reference electrode (Ag/AgCl) with double junction, with 10% KNO3 in the outer junction. All measurements were carried out in a glass vessel protected from light at constant temperature 25 °C under purging with N2. The ionic medium for calibration and measurements was 0.03 M KNO3 and 0.01 M MOPS at pH 7.5. The ISE was preconditioned before each experiment by immersion for 3 h in a solution containing 0.01 M Ag+. The calibration curve was constructed from diluted AgNO3 solutions in the Ag+ range 1 × 10-6 to 1 × 10-3 M and was extended to the range 5 × 10-9 to 3 × 10-7 M by addition of chloride as a ligand. Linear calibration was obtained over the whole range with a slope of 59.3 mV/log [Ag+] (Supporting Information, Figures S4 and S5). For measurements in AgNP suspensions, dilutions of the original suspension (9.27 mM Ag) were prepared in the range 1:10 to 1:105. Ag+ concentrations were calculated from the obtained potential using the linear calibration. The influence of time, cysteine, and algae on the extent of silver dissolution of 5 µM AgNP in 0.01 M MOPS at pH 7.5 was examined by centrifugal ultrafiltration (Millipore Amicon Ultra-4 3K) through a membrane with a nominal molecular weight limit of 3 kDa. Suspensions were centrifuged for 30 min at 3000g (Megafuge 1.0R, Heraeus Instruments). The concentration of Ag in the filtrate was related to the Ag concentration before ultrafiltration as determined by ICPMS (Supporting Information, Table S4). Speciation Calculations. Ag+ present in the AgNP exposure media at the beginning of the experiments was assumed to be equal to the Ag+ present in suspensions without algae (measured by ISE, DGT, and centrifugal ultrafiltration). Calculations of the Ag+ concentrations upon cysteine addition to AgNO3 or AgNP were done using the software ChemEQL 3.0 (21) using the corresponding equilibrium constants (20), displayed in the Supporting Information (Table S1). Calculated Ag+ refers to either the free ions in AgNO3 solutions in equilibrium with cysteine, or to the free ions present in the AgNP suspensions, with or without cysteine. In diluted AgNO3 solutions, Ag+ concentrations were equal to the total Ag concentrations measured by ICP-MS. Algal Culture and Photosynthetic Measurement. Chlamydomonas reinhardtii was cultured according to the procedures described in ref 22. Cell numbers were determined using a cell counter (Z2 Coulter Counter, Beckman Coulter). The algal photosynthetic yield of the photosystem II in light was measured by fluorometry using a PHYTO-PAM (Heinz Walz GmbH) equipped with an Optical Unit ED-101US/MP. This parameter reflects the efficiency of the photochemical energy conversion process (23). Effects of Ag+ and AgNP to Algae. Exponentially growing algae in culture media were first centrifuged (2000g, 10 min) and then resuspended in 10 mM MOPS media, to a final volume of 50 mL in plastic Erlenmeyers, to obtain a final density of 2 × 105 cells mL-1. Photosynthetic yield was 8960
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neither affected by the algal translocation into MOPS, nor by exposure to cysteine (Supporting Information, Figure S1). Toxicity of AgNP and AgNO3 to photosynthesis was assessed by concentration-response experiments, exposing algae to increasing concentrations of AgNO3 (from 100 to 10,000 nM) and AgNP (from 10 to 100,000 nM). Photosynthetic yield of the algae was measured in several experiments over 1-5 h exposure time. To examine the effects of Ag+ to toxicity, cysteine was added as a silver ligand. Algae were exposed for 1 h to 500 nM AgNO3 under different cysteine concentrations from 1 to 500 nM. Photosynthetic yields expressed as % of control were plotted as a function of the calculated Ag+ (ranging from 10-2 to 499 nM). The role of Ag+ in the toxicity of AgNP to algal photosynthesis was examined by exposing algae for 1 h to 5 or 10 µM AgNP and to different cysteine concentrations (10, 50, 100, and 500 nM). To compare toxicity from both AgNP concentrations, results were plotted as a function of the calculated Ag+ (ranging from 10-3 to 100 nM). The photosynthetic values of all the experiments were represented as percentage of the respective controls. Values were plotted as a function of the measured values of total Ag and of Ag+ and fitted to a four-parameters logistic curve (described in Table 1) using Sigma Plot 9.0 (SPSS Inc., USA), to obtain the corresponding EC50 values. Statistics. All errors are expressed as standard deviations (SD) except for EC50 results which are expressed as standard errors (SE). Differences between treatments for the different measured variables were tested using simple and repeated measures ANOVA, followed by Fisher LSD posthoc test when significant differences were found (p < 0.05). Data were logtransformed if required to meet assumptions of ANOVA, and the homogeneity of variances was tested using the Levene test. In other cases, differences were tested using the Dunnett test (to assess differences versus a control), or Kruskal-Wallis ANOVA of ranks (nonparametric test used when assumptions of homogeneity of variances were not reachable). All statistical analyses were computed using Statistica 6.0 (Statsoft, Inc., Tulsa, OK). Toxicity curves have been adjusted to exponential decay or four-parameters logistic curves using Sigma Plot 9.0 (Systat Software Inc., San Jose, CA), which has also been used to calculate the EC50.
Results AgNP Characterization. Particle size measurements in 1/10 AgNP nanopure water dilutions showed that the particle size ranged from 10 to 200 nm, with a median particle diameter of 40 nm. However, in terms of volume, 98% of the AgNP were within 25 ( 13 nm. The TEM analysis confirmed these results, and also revealed particles smaller than 10 nm (Figure 1). These differences are explained by a bias of the DLS measurements toward larger particles. Under experimental conditions (MOPS, light and agitation, Supporting Information Figure S3), AgNP showed no aggregation. AgNP showed a similar averaged size (44 nm) and distribution range but a less negative Zeta potential (-36.6 ( 3.2 mV). At pH 5.33 a decrease of the Zeta potential (-21.2 ( 4.38 mV) and particle aggregation (1200 nm) were observed. Even larger aggregates (6143 nm) were produced in acetate buffer (final pH 4.2); this suspension also showed the less negative Zeta potential: -10.5 ( 0.6 mV (Supporting Information Figure S2). Cysteine concentrations in the range from 1 to 2000 nM did not affect the aggregation state of AgNP (Supporting Information, Figure S3). As measured by DGT, maximum labile Ag (mostly Ag+) concentrations present in the AgNP suspensions were 0.9-1.0% of the total Ag concentration (Supporting Information, Tables S2 and S3). Using the Ag-ISE, Ag+ was determined to be 0.7-1.2% of the total Ag in the AgNP solutions. Dissolved
TABLE 1. AgNO3 and AgNP EC50 Values to the Photosynthetic Yield of C. reinhardtii, Expressed on the Basis of Total Ag Concentrations or Free Ag+a treatment
time
parameter
value (nM)
as a function of total Ag EC50 188 EC50 184
AgNO3
1h 2h
AgNO3 + cysteine
1h
EC50
200
AgNP
1h 2h 3h 4h 5h
EC50 EC50 EC50 EC50 EC50
3300 1049 879 801 829
AgNP
std. error
number of experiments
61 81
3 3
3
1
572 396 159 84 69
3 2 1 1 1
as a function of free Ag+ at the beginning of the experiment 1h EC50 33 5 2h EC50 10 4 3h EC50 9 2 4h EC50 8 0.8 5h EC50 8 0.7
3 2 1 1 1
5 µM AgNP + cysteine
1h
EC50
57
n.s.
1
10 µM AgNP + cysteine
1h
EC50
61
n.s.
1
Experimental data were fitted to a logistic model: %photosynthesis ) min + ((max - min)/1+([Ag]/EC50)Hillslope). In the case of AgNP with cysteine, no standard errors associated were calculated because of the lack of points in the last part of the curve. a
FIGURE 1. TEM image showing AgNP nanoparticles. Ag was also determined to be 0.7-0.8% of the total Ag in the AgNP suspension by centrifugal ultrafiltration (Table S4), and was time-independent (for 3 h). Based on these results, we estimated that in AgNP suspensions, 1% of the Ag was in form of free Ag+. No increases of dissolved Ag were detected in presence of cysteine concentrations in the range applied in the toxicity experiments (10-500 nM). However, at higher cysteine concentrations, dissolved Ag increased somewhat (Table S4). Differently, dissolved Ag was found to drop to 0.1% after 1 h in presence of algae. Effects of Ag+ and AgNP to Algal Photosynthesis. Increasing concentrations of AgNO3 and AgNP reduced the algal photosynthetic yield (Figure 2A). The concentrationresponse curves followed a logistic model (r2 > 0.99). AgNO3 showed similar EC50 values upon 1 or 2 h of exposure: 188
( 61 (n ) 3) and 184 ( 81 nM (n ) 3), respectively (Table 1). Differently, AgNP toxicity was time-dependent during the first 2 h, showing an averaged EC50 of 3300 ( 572 (n ) 3) after 1 h, and 1049 ( 396 nM (n ) 2) after 2 h. In one experiment where photosynthetic yield was monitored over 5 h, toxicity stabilized after 2 h (Table 1). Based on total Ag concentration, AgNO3 displayed higher toxicity than AgNP, even though this difference in toxicity declined over time. However, based on Ag+, AgNP appeared to be more toxic than AgNO3 (Figure 2B). Cysteine decreased the inhibitory effects of 500 nM AgNO3 in a concentration-dependent manner (Figure 3). Equimolar concentrations of cysteine completely abolished the toxicity of AgNO3 to the photosynthetic yield, and EC50 values were similar to those obtained in absence of cysteine, (Table 1), indicating that cysteine was a suitable ligand to decrease Ag+ availability. The role of Ag+ in determining the toxicity of 5 and 10 µM AgNP suspensions was assessed in the presence of cysteine (Figure 4). When plotted as a function of the Ag+ concentrations all photosynthetic yield values followed the same concentration-response curve, independently of the total AgNP concentration. At the highest cysteine concentration (500 nM), photosynthetic yield was comparable to the control values. Inhibition of photosynthesis was similar at cysteine concentrations between 10 and 100 nM.
Discussion We studied the toxicity of AgNP to the photosynthesis of Chlamydomonas reinhardtii with the aim of contributing to the understanding of effects of nanomaterials in the environment. Since we hypothesized that particle toxicity depends on Ag+ associated with or released from particles, we also examined the toxicity of AgNO3. Toxicity of AgNP and AgNO3 to the photosynthetic yield in C. reinhardtii was examined upon short-term exposures in order to minimize accumulation of algal products in the exposure media, and thus changes in the silver speciation. AgNO3 proved to be toxic using photosynthesis as an end point, decreasing the photosynthetic yield of algae in a VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Concentration-response curves of photosynthetic yield upon exposure (1 h) to 500 nM AgNO3 plus cysteine. The Ag+ values are calculated from addition of different cysteine concentrations (secondary x axis) to 500 nM AgNO3.
FIGURE 2. Concentration-response curves of photosynthetic yield after 1 and 2 h of exposure to AgNP and AgNO3; only two of the experiments are shown. Photosynthetic values are expressed as the percentage of the control, where Ag+ concentration corresponds to the detection limit of the method. Graph (A) displays the results as a function of the total Ag present in the suspensions, whereas (B) displays results as a function of the free Ag+ concentrations. concentration-dependent manner. From the concentrationresponse curves, the calculated EC50 values were similar upon 1 and 2 h of exposure (188 and 184 nM AgNO3, respectively), indicating a fast cellular uptake of silver. Uptake of Ag+ by Chlamydomonas reinhardtii has been observed to occur rapidly and to depend on silver speciation (18, 24). Silver has previously been shown to inhibit algal photosynthesis (25, 26) but comparisons are limited to total silver concentrations. Nevertheless, EC50 values determined in our study are 2-13 times higher than Ag concentrations determined to inhibit growth in various algal species (16). These differences are expectable considering that in our study EC50 values reflect the acute toxicity of Ag to photosynthesis in contrast to the other studies where the long-term toxicity to growth has been examined. Since nanoparticles are prone to display different aggregation states depending on their chemical environment, 8962
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FIGURE 4. Concentration-response curves of photosynthetic yield expressed as a function of the calculated Ag+, obtained upon addition of increasing amounts of cysteine to 5 and 10 µM AgNP suspensions. The cysteine concentrations (nM) are indicated beside every data point. their characterization in the media used for toxicity experiments is particularly important (27, 28). The median diameter of the AgNP was around 40 nm in the original particle suspension and was maintained in the experimental media. The three methods used (DGT, Ag-ISE, and centrifugal ultrafiltration) all indicated that about 1% of total Ag in AgNP suspensions was present as Ag+, already in the original provided suspension. A typical concentration-response curve was observed for the effects of the AgNP to photosynthesis (Figure 2A). Time course experiments showed the inhibitory response to increase with time up to 2 h in contrast to the experiments with AgNO3. However, EC50 values did not further decrease with increasing exposure time over 2 h. Considering that toxicity of Ag+ to algal growth depends on intracellularly accumulated silver (16), the time-dependent
toxicity might reflect the contribution of the AgNP to the toxicity. This conclusion is also corroborated by the finding that when toxicity data were related to the Ag+ concentration (Figure 2B) AgNP appeared to be more toxic than AgNO3 suggesting that toxicity cannot be explained solely by the concentration of Ag+ ions in the original suspensions. Cysteine, a strong silver ligand, proved useful to examine the contribution of Ag+ to the overall toxicity of AgNP. In case of AgNO3 experiments, in which the concentration of AgNO3 was fixed to 500 nM and the free Ag+ concentration was modified by addition of cysteine, the calculated EC50 value was similar to that obtained in absence of cysteine (Table 1). The complete abolishment of toxicity to photosynthesis at equimolar concentration of cysteine (Figure 3) indicates that protective effects of cysteine are due to complexation of Ag+ in the exposure media resulting in a reduced silver bioavailability, thus validating the Free Ion Activity Model for Ag+ toxicity (29). In case of AgNP the role of Ag+ in explaining toxicity was evidenced by toxicity experiments carried out with two different AgNP concentrations and in the presence of varying cysteine concentrations. When plotted as a function of the Ag+ concentration (calculated using the Ag+ measured by ISE), experimental points fitted to one concentrationresponse curve with toxicity of AgNP completely abolished at the highest applied cysteine concentration (Figure 4). A striking result was the observation that inhibitory effects to photosynthesis appeared at Ag+ concentrations by far lower than the EC50 for AgNO3 and that similar inhibition levels (about 60%) occurred in the range of 10 to 100 nM cysteine. These findings together with the fact that cysteine did not influence silver dissolution up to 100 nM cysteine (Table S4), suggest that besides the presence of Ag+ in the exposure AgNP solution at the beginning of the experiment, additional Ag+ is likely formed during exposure in presence of algae. Inhibitory effects are then observed if this additional Ag+ exceeds the cysteine concentration, but not in the presence of an excess of cysteine. Since algae efficiently decreased Ag+ concentration from the AgNP exposure medium (Table S4) reflecting the efficient silver accumulation by algae (18, 24), all together, the results present indirect evidence that toxicity of AgNP is mediated by Ag+. Particles contribute to toxicity serving as sources of Ag+. The release and uptake of Ag+ result upon interaction of particles with algae. Therefore, the efficient assimilation of Ag may enhance the Ag dissolution and at the same time hinder its detection in the exposure medium. Whether Ag+ formation occurs at the algal interface or in the exposure medium upon interaction with secreted algal products cannot be sorted out. Release of Ag+ from the AgNP implies oxidation, which may be more efficient in the presence of algae, e.g., due to H2O2 production. H2O2 is very reactive with Ag yielding hydroxyl radicals, thus resulting in dissolution of Ag+ as a byproduct of the reaction (30). H2O2 is a metabolic product of algae which may be secreted in the exposure medium (31-34), and upon reaction with AgNP may lead to the release of Ag+. Published rates on H2O2 production by algae (33, 34) are in excess of the estimated Ag+ release. Our results differ from those obtained in a study on the effects of AgNP to bacteria (6). There, bactericidal properties of the nanoparticles were related mainly to direct effects based on particles found to accumulate intracellularly and at the cell membrane. However, ionic or dissolved silver were not measured. In addition, the acute toxicity of copper nanoparticles to zebrafish was attributed mainly to the effects of particles (35). Soluble copper was found to produce morphological effects and gene expression profiles different from those induced by copper nanoparticles, indicating that nanoparticles effects on gills are not mediated merely by dissolution. Similar to our results, the toxicity of nanopar-
ticulate zinc oxide to the alga Pseudokirchneriella subcapitata was found to depend on the concentration of soluble ZnO, even though no measurements of ionic Zn2+ were made (36). Likewise, ZnO was found to display higher acute cytotoxicity than less soluble nanoparticles such as TiO2 and CeO2 (37). Finally, the role of metal dissolution for particle-induced cytotoxicity was also evidenced in case of CdSe quantum dots (38). Our results have demonstrated the determinant role of free Ag+ for the toxicity of AgNP and the occurrence of significant interactions of nanoparticles with algae. Thus, not only abiotic factors affecting their dissolution rates, such as size and surface area (39) or the chemical conditions of the environment (40), but also biotic interactions should be considered to assess the risks posed by nanomaterials entering in the aquatic ecosystems.
Acknowledgments We thank David Kistler and Ursula Lindauer for ICP-MS measurements, Brian Sinnet for TEM analysis, and J. Kramer for advising on ISE measurements.
Supporting Information Available Results from aggregation of the AgNP, their solubility, and photosynthetic yield of the algae under experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org.
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