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Article pubs.acs.org/est Cellular Partitioning of Nanoparticulate versus Dissolved Metals in Marine Phytoplankton Gretchen K. Bielmyer-Fraser,*,† Tayler A. Jarvis,† Hunter S. Lenihan,‡ and Robert J. Miller§ † Valdosta State University, 1500 North Patterson Street, Valdosta, Georgia 31698, United States Bren School of Environmental Science and Management, University of California, Santa Barbara, California 93106, United States § Marine Science Institute, University of California, Santa Barbara, California 93106, United States ‡ ABSTRACT: Discharges of metal oxide nanoparticles into aquatic environments are increasing with their use in society, thereby increasing exposure risk for aquatic organisms. Separating the impacts of nanoparticle from dissolved metal pollution is critical for assessing the environmental risks of the rapidly growing nanomaterial industry, especially in terms of ecosystem effects. Metal oxides negatively affect several species of marine phytoplankton, which are responsible for most marine primary production. Whether such toxicity is generally due to nanoparticles or exposure to dissolved metals liberated from particles is uncertain. The type and severity of toxicity depends in part on whether phytoplankton cells take up and accumulate primarily nanoparticles or dissolved metal ions. We compared the responses of the marine diatom, Thalassiosira weissf logii, exposed to ZnO, AgO, and CuO nanoparticles with the responses of T. weissf logii cells exposed to the dissolved metals ZnCl2, AgNO3, and CuCl2 for 7 d. Cellular metal accumulation, metal distribution, and algal population growth were measured to elucidate differences in exposure to the different forms of metal. Concentration-dependent metal accumulation and reduced population growth were observed in T. weissf logii exposed to nanometal oxides, as well as dissolved metals. Significant effects on population growth were observed at the lowest concentrations tested for all metals, with similar toxicity for both dissolved and nanoparticulate metals. Cellular metal distribution, however, markedly differed between T. weissf logii exposed to nanometal oxides versus those exposed to dissolved metals. Metal concentrations were highest in the algal cell wall when cells were exposed to metal oxide nanoparticles, whereas algae exposed to dissolved metals had higher proportions of metal in the organelle and endoplasmic reticulum fractions. These results have implications for marine plankton communities as well as higher trophic levels, since metal may be transferred from phytoplankton through food webs vis à vis grazing by zooplankton or other pathways. ■ applications.10 Exposure of aquatic organisms to nanoparticles is difficult to quantify; however, some modeling efforts have reported the presence of metal oxides in aquatic systems at levels that may cause toxicity.11 Phytoplankton require the essential metals zinc (Zn) and copper (Cu) for enzyme functioning, but at elevated concentrations, these metals may exert toxicity.12−14 Most ecotoxicity work on phytoplankton has been done using dissolved metals, with a few recent studies on nanoparticles. For example, ZnO nanoparticles are known to reduce population growth rates in the diatoms Skeletonema marinoi, Thalassiosira pseudonana, and Thalassiosira weissf logii.13,14 Metal oxide nanoparticles dissolve in seawater to varying degrees, and release free metal ions,15 which may be the predominant cause of toxicity to aquatic organisms. However, the dynamics of dissolution and release of metal ions, and how these processes vary with concentration and material type is poorly understood. INTRODUCTION Diatoms are the dominant primary producers in the ocean, and because they are small and have a high surface-to-volume ratio, they can take up and accumulate substantial amounts of contaminants.1−4 This uptake may cause multiple impacts to marine food webs: reductions in population growth rate and possibly nutritional content of phytoplankton cells could reduce resources available for consumers,5 and accumulation of contaminants in phytoplankton can lead to trophic transfer and resulting toxic effects on consumers.6 Metals are an important class of such contaminants, and now are being discharged into coastal ecosystems as nanomaterials in addition to traditional bulk and dissolved forms. Nanomaterials are now widely utilized for their enhanced mechanical and optical properties, as well as their efficient electrical conductivity, relative to larger forms of similar materials.7 Because of their growing application in an array of fields including electronics, chemical, cosmetics, and biomedicine,7,8 nanomaterials are emerging as a new class of contaminants, with unknown environmental consequences.9 Metal oxide nanomaterials in particular are commonly used because they are synthesized relatively easily and have myriad industrial and consumer © 2014 American Chemical Society Received: Revised: Accepted: Published: 13443 March 25, 2014 October 13, 2014 October 22, 2014 October 22, 2014 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology Article prior to inoculation. Synthetic seawater was made by mixing Instant Ocean salt with 18 mΩ Milli-Q water and aerating at least 24 h before use.32 The sterilized media was inoculated with 2.5 × 105 algal cells and the algae were cultured for 7 d with continuous aeration. Algae were incubated under cool white fluorescent lights (12 h light: 12 h dark) at a light intensity of 36 μmol photons m−2 s−1 and a temperature of 20 °C. Algal densities were measured using a hemocytometer (Hausser Scientific, Horsham, PA) and a compound microscope. Nanoparticles. ZnO nanoparticles were obtained from Meliorum Technologies (Rochester, NY, USA) and characterized for size, morphology and chemical composition.33,34 ZnO nanoparticles were spheroid, 100% zincite, and 20−30 nm in diameter. CuO nanoparticles were obtained from SigmaAldrich (St. Louis, MO, USA) and described as <50 nm and 99.8% pure; characterization with ICP-OES and TEM showed that they were 84.8 ± 2.7% pure (impurities included Na, Ca, Si, and Mg) and 20−100 nm in diameter.35 AgO nanoparticles were obtained from QuantumSphere Inc. (Santa Ana, CA) and described as 20−30 nm in diameter. TEM characterization showed that the AgO particles were 20−70 nm in diameter, and no detectable impurities were found. Experimental Design. T. weissf logii was cultured as described above, but using F/2 nutrients without trace metals and without ethylenediaminetetraacetic acid (EDTA). T. weissf logii was exposed to ZnO, AgO, and CuO nanoparticles, as well as ZnCl2, AgNO3, and CuCl2 for 7 d. Nanomaterial stock suspensions were prepared by adding 10 mg of metal oxide nanoparticles to 10 mL of ultra pure 18 mΩ Milli-Q water, vortexing for 30 s, sonicating for 30 min, dilution (1:10) with 30 ppt synthetic seawater containing 10 mg L−1 alginate (previously made in a 1 mg L−1 stock in 18 mΩ Milli-Q water), and vortexing again for 30 s. Dissolved metal solutions were prepared by adding a 10 mg L−1 stock solution of each metal (Zn as ZnCl2, Ag as AgNO3, and Cu as CuCl2) to synthetic seawater (30 ppt salinity). Different volumes of each stock solution were added separately to the 1 L Erlenmeyer flasks to achieve the following nominal concentrations: 0.1, 0.5, and 1.0 mg L−1 of ZnO and ZnCl2, 1.0 and 10 mg/L of AgO and AgNO3, and 0.25 and 5.0 mg L−1 of CuO and CuCl2. A metalfree control treatment was also included in each experiment. Metal concentrations were chosen from past studies reporting growth effects in phytoplankton.6,12,14 Before inoculation with T. weissf logii and at 1, 3, 5, and 7 d during the experiment water samples were collected in 15 mL polypropylene centrifuge tubes from each flask, filtered (0.45 μm), and acidified with trace metal grade nitric acid (Fisher Scientific, Pittsburgh, PA) for later metal analysis. Measured dissolved metal concentrations are presented in Table 1. For the nanoparticle exposures, we took the difference between nominal and dissolved concentrations to represent the portion of the metal in nanoparticle form; thus the nanoparticle exposures represented a combination of dissolved and nanoparticulate metal. There were three replicate flasks per treatment. Approximately 5 × 108 cells L−1 were added to each flask at the start of the experiments. The cultures were continuously aerated with sterile 1 mL pipettes and stirred once a day by swirling. Algal cell density was measured at 0, 3, 5, and 7 d. The algae were exposed to the nanoparticles and dissolved metals throughout log phase growth. At the end of the exposure period, the algae were concentrated by centrifugation in 50 mL polypropylene centrifuge tubes at 3500 rpm for 15 min. Greater understanding of these dynamics is needed to elucidate differences in metal adherence, uptake, and accumulation patterns in phytoplankton exposed to nanoparticulate metals and dissolved metals. Metal ions typically traverse algal cell membranes through ion/voltage-gated channels16 and once inside the cell, exert toxicity by binding to metabolic sites normally occupied by other essential ions.17,18 Nanoparticles can directly diffuse across the membrane in some cases (i.e., small in size; positive ions on the surface of nanoparticles), pass through ion channels and transporter proteins, or enter the cell through endocytosis.19,20 Once inside the cell, nanoparticles can interact with oxidative organelles such as mitochondria, and cause oxidative stress by disrupting the balance between oxidant and antioxidant processes and generation of reactive oxygen species.21−23 Depressed growth of phytoplankton populations can cascade to reduce production of primary consumers, especially zooplankton and larval fish. In addition, accumulated metal in phytoplankton may be a significant source of dietary metals to primary consumers.12 However, pathways and patterns of metal accumulation and transfer to consumers are difficult to predict and thus not well understood. Several studies have shown that the chemical form of the metal within the prey can influence the assimilation of metals by consumers,24−29 and different subcellular fractions of accumulated trace metals vary in their trophic availability to consumers, perhaps reflecting ligand binding.24,25 Metals bound to different ligands, such as proteins or other organic molecules, could correspondingly differ in bioavailability.24,30 For example, Cheung and Wang29 demonstrated differences in the assimilation efficiency and transfer of cadmium (Cd), Zn, and silver (Ag) to the gastropod Thais clavigera depending on the subcellular compartmentalization of the metals in the prey (barnacles, oysters, mussels, snails, and limpets). Some researchers have suggested that the most bioreactive metal fraction in organisms is that which is bound to metallothioneins and possibly small peptides like glutathione.24,30 In a prior study, we found significantly reduced survival and reproduction of the copepod Acartia tonsa fed a diet of nanoparticulate ZnO-exposed diatoms.6 The dietary zinc concentrations causing effects to the copepod consumers (10−20 ug g−1 dw) were similar to those reported to cause effects in copepods fed diets (3−14 ug g−1 dw) previously exposed to dissolved zinc.6,12 However, the effects of the diets on copepods were different: nanoparticulate ZnO-exposed diets decreased copepod survival most severely, whereas dissolved zinc-exposed diets affected reproduction to the greatest degree.6,12 We hypothesized that this difference was due to differential partitioning of the two metal forms in the diatom cells. In this study, we examined the accumulation of nanoparticulate metal oxides (nZnO, nAgO, and nCuO) and corresponding dissolved metals (ZnCl2, AgCl, and CuCl2) in the widespread coastal marine diatom, Thalassiosira weissf logii, and compared the effects of the different materials on diatom population growth rates. ■ METHODS Algal Culturing. Thalassiosira weissf logii was obtained from AlgaGen LLC (Vero Beach, FL) and cultured in autoclaved 1 L Erlenmeyer flasks following APHA guidelines.31 The algal medium consisted of 300 mL of synthetic saltwater (30 ppt salinity) and F/2 nutrients, and was autoclaved and cooled 13444 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology Article Table 1. Mean ± Standard Deviations of Measured Dissolved Metal Concentrations in the Thalassiosira weissf logii Media during Exposure to Controls, ZnO, Zn as ZnCl2, AgO, Ag, as AgNO3, CuO, and Cu as CuCl2 for 7 d Methyldiaminetetraacetic acid (EDTA), to remove loosely bound metal from the cell wall, and then algal cells were centrifuged at 3500 rpm for 15 min. The supernatant fractions (representing loosely bound metals washed from the algal surface) were preserved in 15 mL centrifuge tubes. The remaining algal pellets were resuspended in 1 mL of purified 18 mΩ Milli-Q water and sonicated for approximately 1 min to lyse the algal cells, which was verified using a light microscope. The sample was then differentially centrifuged to separate the cells into cell wall, organelle, endoplasmic reticulum, and cytosol fractions, using previously established methods.12 Briefly, the lysed cells were centrifuged at 4000 rpm for 15 min to obtain the cell wall fraction. The organelle fraction was then obtained by centrifuging the remaining supernatant for 30 min at 10 000 rpm. The supernatant was then centrifuged at 100,000 rpm for 1 h to obtain the total membrane fraction (endoplasmic reticulum). The remaining supernatant was the cytosol fraction. Each fraction was digested by addition of 1 mL of trace metal grade nitric acid (Fisher Scientific, Pittsburgh, PA) and 1 mL of purified 18 mΩ Milli-Q water and then analyzed for metal concentration as described below. Data are presented as accumulated metal per gram algal dry weight (Table 2). This measurement includes metal in both dissolved and particulate form. Metal Analysis. Water samples and digested algae fractions were diluted with 18 mΩ Milli-Q water, and analyzed for metal concentration using graphite furnace atomic absorption spectrophotometry (GFAAS; PerkinElmer, AAnalyst 800). Certified 1 g/mL metal standards dissolved in 2% HCl (Fisher Chemical, Fairlawn, NJ) were used for GFAAS analysis of each metal. Detection limits for each metal were 1 μg L−1, based on these protocols. Standards were analyzed at the beginning and end of each analysis. Data Analysis. Statistical differences between treatments (p < 0.05; n = 3) were calculated using Analysis of Variance (ANOVA) followed by a posthoc Tukey’s test to determine measured metal (μg L−1) nominal metal concentration control Zn 10 μg L−1 ZnO 10 μg L−1 Zn 100 μg L−1 ZnO 100 μg L−1 Zn 500 μg L−1 ZnO 500 μg L−1 Zn 1000 μg L−1 ZnO 1000 μg L−1 Zn control Ag 1000 μg L−1 AgO 1000 μg L−1 Ag 10000 μg L−1 AgO 10000 μg L−1 Ag control Cu 250 μg L−1 CuO 250 μg/L Cu 5000 μg L−1 CuO 5000 μg L−1 Cu 3.92 9.41 8.40 94.2 94.4 203 461 301 920 1.80 321 828 1275 8152 2.97 78.0 173 769 2506 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.80 0.70 0.99 4.32 9.60 20.2 26.1 22.5 42.5 0.40 49.0 91.0 38.6 479 0.86 0.95 33.0 54.0 857 Subsamples of the concentrated algae were collected for the following additional procedures. First, the algae was filtered and washed with 0.5 M ammonium formate (to reduce the salt from the algae while maintaining the osmotic gradient for calculation of algal dry weight), and then dry weights were determined by drying multiple aliquots of algae from each treatment in preweighed aluminum weigh boats in an oven at 80 °C for 1 d. Second, Zn, Ag, and Cu distribution within the algal cells was determined. Metal Distribution in Algal cells. The intact algal cells from each treatment were washed with a metal chelator, 0.01 Table 2. Cellular Metal Distribution (Mean ± Standard Deviation × 10−9; μg Dissolved Metal/g Dry Weight) in Thalassiosira weissf logii after Exposure to Controls, Metal Oxide Nanoparticles (ZnO, AgO, and CuO), or Dissolved Metals (Zn as ZnCl2, Ag as AgNO3, and Cu as CuCl2) for 7 d (n = 3) nominal metal concentration control Zn 0.01 mg L−1 ZnO 0.01 mg L−1 Zn 0.1 mg L−1 ZnO 0.1 mg L−1 Zn 0.5 mg L−1 ZnO 0.5 mg L−1 Zn 1.0 mg L−1 ZnO 1.0 mg L−1 Zn control Ag 1.0 mg L−1 AgO 1.0 mg L−1 Ag 10 mg L−1 AgO 10 mg L−1 Ag control Cu 0.25 mg L−1 CuO 0.25 mg L−1 Cu 5.0 mg L−1 CuO 5.0 mg L−1 Cu a EDTA wash 0.58 17.2 5.49 19.4 16.0 26.7 58.7 50.9 120 1.15 6.08 4.27 14.6 16.1 1.02 5.78 6.79 14.9 17.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.1 14 1.6 18 2.8 8.4 15 0.3 19 0.4 1.2 0.7 4.0 3.7 0.2 2.2 1.9 0.2 2.4 cell wall 0.77 48.0 1.93 105 7.80 227 18.4 204 31.0 0.98 120 34.6 329 111 1.35 108 53.0 626 112 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 14 0.6 54 0.9 22 9.2 12 3.5 0.0 33 8.0 90 20 0.3 18 14 40 23 organelles 0.83 13.4 2.13 33.0 9.12 39.5 35.3 50.9 72.5 1.04 12.3 72.1 51.6 116 2.86 12.1 68.5 40.1 143 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.3 1.6 0.5 1.3 1.6 2.1 10 0.0 3.1 0.3 2.7 12 17 19 0.3 3.7 20 14 29 endoplasmic reticulum 0.68 10.6 2.50 19.0 6.78 16.7 13.8 26.4 24.1 0.93 5.99 19.8 10.1 47.4 0.88 4.46 46.6 26.0 85.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 3.1 0.3 2.2 1.0 0.5 6.3 0.8 3.0 0.1 1.5 2.6 2.1 8.4 0.3 1.5 14 8.3 16 cytosol totala ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.09 100 14.1 223 45.7 342 142 369 175 5.06 160 145 410 313 7.19 141 190 738 400 1.23 11.3 2.10 34.5 5.60 35.2 15.4 37.2 30.2 0.95 15.6 13.8 4.74 22.7 1.08 11.0 14.7 30.6 42.0 0.1 3.8 0.8 2.8 0.7 3.6 8.3 12 3.2 0.2 2.5 3.0 1.3 3.1 0.1 6.5 3.3 7.1 11 Indicates the calculated total. 13445 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology Article differences between treatments. Data were first assessed for normality and homogeneity of variances using the Shapiro− Wilk’s test and Bartlett’s test, respectively. ■ RESULTS Metal Concentrations. Concentrations of Zn, Ag, and Cu in the experimental media spiked with ZnCl2, AgNO3, and CuCl2 were similar to nominal concentrations, with values averaging 85% of nominal values (Table 1). In contrast, concentrations of dissolved Zn, Ag, and Cu in the experimental media spiked with nZnO, nAgO, and nCuO were considerably lower than nominal values, particularly at higher concentrations (Table 1). For nZnO, Zn concentrations were similar to nominal values at 0.1 mg L−1; however, at 0.5 and 1.0 mg L−1, values were two and 3-fold lower, respectively, than those values for ZnCl2 at the same nominal concentrations (Table 1). For 1 and 10 mg L−1 nAgO, Ag concentrations were approximately 3- and 7-fold lower than those observed for AgNO3. Likewise, for 0.25 and 5 mg L−1 nCuO, Cu concentrations were approximately 2- and 3-fold lower than those observed for CuCl2 (Table 1). Metal Toxicity to T. weissf logii. Exposure of T. weissf logii to increasing metal concentrations (both as nanoparticles and dissolved metals) resulted in significantly reduced population growth (Figures 1−3). Algal growth curves were similar between nAgO and AgNO3 exposures and between nCuO Figure 2. Thalassiosira weissf logii growth over 7 d after exposure to various concentrations of (A) AgO nanoparticles and (B) Ag as AgNO3. Asterisks indicate significant differences from control values (p < 0.05; n = 3). and CuCl2 exposures but some differences were observed for Zn (Figures 1−3). T. weissf logii exposed to ZnCl2 had a significantly greater growth reduction than algae exposed to nZnO by 7 d (Figure 1). Lowest observable effect concentrations (LOECs; dissolved metal concentrations only) were 94.2 and 94.4 μg L−1 for nZnO and ZnCl2 exposures, 321 and 828 μg L−1 for nAgO and AgNO3 exposures, and 78.0 and 173 μg L−1 for nCuO and CuCl2 exposures, respectively. Metal Accumulation and Distribution in T. weissf logii. Metals accumulated in a concentration-dependent manner in T. weissf logii after exposure to both metal oxide nanoparticles and dissolved metals. All cell fractions of metal-exposed algae were significantly elevated above the control cell fractions. Additionally, metal accumulation patterns were significantly different depending on the form of metal exposed (Figures 4−6; Table 2). In general, T. weissf logii exposed to metal oxide nanoparticles had higher proportions of metals in the cell wall fractions compared with organelle fractions. By contrast, higher proportions of metals were observed in the organelle fraction after exposure to dissolved metals (Figures 4−6; Table 2). Significantly higher Zn concentrations (p < 0.05; n = 3) were observed in nZnO-exposed cell fractions compared with the corresponding fractions from cells exposed to ZnCl2 (e.g., cell wall from ZnO-exposed algae compared to cell wall from ZnCl2-exposed algae), with only one exception (1 mg L−1, EDTA fraction). Significant differences (p < 0.05; n = 3) were also observed between all nAgO-exposed cell fractions and Figure 1. Thalassiosira weissflogii growth over 7 d after exposure to various concentrations of (A) ZnO nanoparticles (as seen in Jarvis et al. 2013) and (B) Zn as ZnCl2. Asterisks indicate significant differences from control values (p < 0.05; n = 3). 13446 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology Article Figure 5. Cellular metal distribution (mean ± standard deviation; μg metal/cell) in Thalassiosira weissf logii after exposure to AgO nanoparticles and Ag as AgNO3 for 7 d (n = 3). Asterisks indicate significant differences between a cellular fraction at a specific concentration of either metal oxide nanoparticles or dissolved metals (p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed fractions were significantly higher than controls. Figure 3. Thalassiosira weissflogii growth over 7 d after exposure to various concentrations of (A) CuO nanoparticles and (B) Cu as CuCl2. Asterisks indicate significant differences from control values (p < 0.05; n = 3). Figure 6. Cellular metal distribution (mean ± standard deviation; μg metal/cell) in Thalassiosira weissf logii after exposure to CuO nanoparticles and Cu as CuCl2 for 7 d (n = 3). Asterisks indicate significant differences between a cellular fraction at a specific concentration of either metal oxide nanoparticles or dissolved metals (p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed fractions were significantly higher than controls. weissf logii to AgNO3 and CuCl2 as compared with nAgO and nCuO (Figures 5 and 6; Table 2). The proportion of metal loosely bound to the cell wall (liberated from the EDTA wash) after exposure to nanoparticle or dissolved metals were similar or slightly lower when exposed to dissolved metals (Figures 4−6; Table 2). The total cellular concentrations were similar between nanoparticulate and dissolved Ag and Cu, yet significantly more Zn accumulated in algae exposed to nZnO compared with ZnCl2 (Figures 4−6; Table 2). The ratio of total Zn accumulated after exposure to ZnO as compared with exposure of ZnCl2 approximated 5, 2.5, and 2, for the 0.1, 0.5, and 1.0 mg L−1 treatments, and largely corresponded to Zn accumulation in the cell wall fractions (cell wall and EDTA washed; Table 2). Figure 4. Cellular metal distribution (mean ± standard deviation; μg metal/cell) in Thalassiosira weissf logii after exposure to ZnO nanoparticles and Zn as ZnCl2 for 7 d (n = 3). Asterisks indicate significant differences between a cellular fraction at a specific concentration of either metal oxide nanoparticles or dissolved metals (p < 0.05; n = 3). ER = endoplasmic reticulum. All metal-exposed fractions were significantly higher than controls. AgNO3-exposed cellular fractions except one (10 mg L−1, EDTA fraction) and between all nCuO-exposed cell fractions and CuCl2-exposed cellular fractions. For Ag and Cu, proportionally higher metal concentrations were observed in the endoplasmic reticulum fraction after exposure of T. 13447 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology ■ Article DISCUSSION The differences between dissolved and nominal metal concentrations demonstrated in Table 1 reflect the different exposure scenarios, with nanoparticulate ZnO, AgO, and CuO solutions containing nanoparticle and dissolved metals, and ZnCl2, CuCl2, and AgNO3 solutions containing solely dissolved metal. The dissolution of ZnO, AgO, and CuO nanoparticles was probably higher at the lower exposure concentration, a process well characterized in our previous study.6 Cytoxicity can result from both metal oxide nanoparticles and dissolved metal exposure.12,13,20 In the present study, cytotoxicity resulted in decreased population growth of T. weissf logii after exposure to both metal oxide nanoparticles and dissolved metals, similar to previous results.13 Miller et al.14 reported decreased population growth rates in the marine phytoplankton species Skeletonema marinoi, Isochrysis galbana, and Dunaliella tertiolecta after exposure to 1000 μg L−1 ZnO nanoparticles and in Thalassiosira pseudonana after exposure to 500 μg L−1 ZnO nanoparticles. Similarly, population growth of the algal species T. pseudonana, Chaetoceros gracilis, and Phaeodactylum tricornutum was reduced after exposure to 10 mg L−1 ZnO nanoparticles for 100 h.27 Franklin et al.13 reported toxicity values (72-h IC50−60 μg L−1 Zn) for both ZnO nanoparticles and ZnCl2 to the freshwater alga, Pseudokirchneriella subcapitata, and attributed toxicity solely to dissolved Zn. Likewise, Aruoja et al.36 reported an IC50 value of 0.71 mg Cu L−1 (similar to the results in this study) for growth of the algae Pseduokirchneriella subcapitata after exposure to CuO nanoparticles, where much of the toxicity was attributed to liberation of soluble Cu ions. At lower metal concentrations, metal nanoparticles may more quickly dissolve in solution, complicating results for short-term assays.6 Nevertheless, we found that toxicity levels were similar for nanoparticulate and dissolved forms of Zn, Cu, and Ag at the concentrations tested. This contradicts results of Shi37 who reported greater toxicity (i.e., decreased chlorophyll concentration) of nCuO than ionic Cu in duckweed, a freshwater plant, thus illustrating that results may be taxon- and media-dependent. More studies should be performed at lower exposure levels in the future to better elucidate differences in toxicity between the different forms of metals. Compared with controls, significantly higher metal concentrations were observed in T. weissf logii cells after exposure to metals in nanoparticulate or dissolved form. The Cu and Zn concentrations in the control algae were similar to past studies and likely reflect their essentiality.12 Silver is a nonessential metal, yet the control algae contained low concentrations, perhaps resulting from low Ag concentrations (∼2 ppb) in the dilution water and values near the detection limit of the GFAAS. These values were negligible and at least 2 orders of magnitude below the concentrations measured in the Agexposed algae. Metal accumulation in T. weissf logii cells were well within the range of values reported in the literature.6,12,38 DeSchamphelaere et al.37 reported a Zn concentration of 490 μg g−1 dw in the alga, Pseudokirchneriella subcapitata, after exposure to 60 μg L−1 and Guan and Wang39 reported Zn concentrations ranging from 27.2 to 280 μg g−1 dw in the alga, Chlamydomonas reinhardtii, after exposure to 2 to 200 μg L−1 Zn. The distribution of metal within the treated algal cells differed, depending on the form of metal in the exposure media. Exposure of T. weissf logii to metal oxide nanoparticles resulted in a relatively higher proportion of metal in the cell wall fraction, whereas exposure to dissolved metals resulted in a relatively higher proportion of metal in the organelle, and in some instances, endoplasmic reticulum fractions. Peng et al.40 reported Zn bioaccumulation in three algal species, Thalassiosira pseudonana, Chaetoceros gracilis, and Phaeodactylum tricornutum after exposure to 10 mg L−1 ZnO nanoparticles for 100 h. The ZnO-exposed algae exhibited apparent cell wall damage and internal degradation, characterized by a lack of recognizable organelles that was attributed to release of dissolved Zn upon partial dissolution. Histology was not assessed in the current study but it is plausible given the metal accumulation pattern that metal oxide nanoparticles may cause more damage to the cell wall than dissolved metals, and absorption of both forms of metals may cause damage to the organelle fraction. The greater proportion of metals in the cell wall fraction, as compared to the internal fractions, after nanoparticulate metal exposure may be due to size exclusion of the nanoparticles; dissolved metals may move relatively quickly as ions through channels into the cell.16 This may be exacerbated by aggregation of nanoparticles, which was not evaluated in this study but is expected to occur at levels that vary with the organic matter content of the media and other factors.15 The internal distribution of metals in the algal cells also varied to some degree, depending on the form of metal used in the exposure media. The control T. weissf logii and the algae exposed to ZnO nanoparticles had a higher proportion of Zn in the cytosol than in the endoplasmic reticulum. After exposure of T. weissf logii to dissolved Zn, the proportion of Zn in the endoplasmic reticulum increased relative to the cytosol. A similar pattern of metal accumulation was observed with dissolved Cu and to some extent dissolved Ag exposure. Consistent with the findings of this study, Xu et al.41 also reported a decline in the relative distribution of Zn in the cytoplasm of T. weissf logii after exposure to dissolved Zn. In mammalian cells, Zn concentrations in the cytosol are much higher than in the endoplasmic reticulum, and when in excess, Zn is generally sequestered by the endoplasmic reticulum.42 This may also be the situation for Zn, Cu, and Ag in algal cells. Differences in metal distribution in T. weissf logii after exposure to nanoparticles or dissolved metals likely resulted from differences in rates and/or modes of uptake. Additionally, depending on exposure concentration, various degrees of dissolution and release of metal ions from metal oxide nanoparticles apparently occurred over time,12,13,35 resulting in exposure of both dissolved and nanoparticulate metals, thus influencing the metal uptake and distribution in the algae. The patterns of metal distribution that we report have broad implications for food chain transfer and effects of metals in consumers. Bielmyer et al.12 previously reported a significant decrease in copepod reproduction after they consumed Thalassiosira pseudonana that were exposed to dissolved metals, resulting in accumulation values of 120 μg g−1 dw Ag, 3.05 μg g−1 dw Zn, or 31.9 μg g−1 dw Cu within the algal cells. These concentrations were lower than those shown to cause a decrease in copepod survival. Jarvis et al.6 demonstrated that feeding on nZnO-exposed T. weissf logii resulted in significantly reduced survival and reproduction of the copepod Acartia tonsa. The effect levels observed from copepods fed nanoparticleexposed diets were similar to those reported for dissolvedmetal-exposed diets.6,12 Contrary to past dietary studies with dissolved metals12,38,43,44 survival was a more sensitive end 13448 dx.doi.org/10.1021/es501187g | Environ. Sci. Technol. 2014, 48, 13443−13450 Environmental Science & Technology Article (5) Karl, D. M.; Bidigare, R. R.; Letelier, R. M. Long-term changes in plankton community structure and productivity in the North Pacific Subtropical Gyre: The domain shift hypothesis. Deep Sea Res., Part II 2001, 48.8, 1449−1470. (6) Jarvis, T. A.; Miller, R. C.; Lenihan, H. S.; Bielmyer, G. K. Toxicity of ZnO nanoparticles to the copepod, Acartia tonsa, exposed via a phytoplankton diet. Environ. Toxicol. Chem. 2013, 32, 1264− 1269. (7) Klaine, S. J.; Alvarex, P. J. J.; Batley, G. E.; Fernandes, T. F.; Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J. R. 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M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd, G. E.; Casey, P. S. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environ. Sci. Technol. 2007, 41, 8484−8490. (14) Miller, R. J.; Lenihan, H. S.; Muller, E. B.; Tseng, N.; Hanna, S. K.; Keller, A. A. Impacts of metal oxide nanoparticles on marine phytoplankton. Environ. Sci. Technol. 2010, 44, 7329−7334. (15) Keller, A. A.; Wang, H.; Zhou, D.; Lenihan, H. S.; Cherr, G.; Cardinale, B. J.; Miller, R. J.; Ji, Z. Stability and aggregation of metal oxide nanoparticles in natural aqueous matrices. Environ. Sci. Technol. 2010, 44, 1962−1967. (16) Campbell, P. G. C.; Errécalde, O.; Fortin, C.; Hiriart-Baer, V. P.; Vigneault, B. Metal bioavailability to phytoplankton-applicability of the biotic ligand model. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2002, 133, 189−206. (17) Sunda, W. G. 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Toxicol. 2009, 29, 69−78. (23) Toduka, Y.; Toyooka, T.; Ibuki, Y. Flow cytometric evaluation of nanoparticles using side-scattered light and reactive oxygen speciesMediated fluorescenceCorrelation with genotoxicity. Environ. Sci. Technol. 2012, 46, 7629−7636. (24) Bielmyer, G. K.; Grosell, M. Emerging issues in marine metal toxicity. In Essential Reviews in Experimental Biology, Bury, N., Handy, R., Eds.; Kings College: London, UK, 2011, Vol. 2, pp 129−158. point than reproduction after exposure to nanoparticle-exposed diets.6 The differences in metal accumulation and distribution in phytoplankton observed in the present study could contribute to the unique effects of ingestion of phytoplankton exposed to nanomaterials, compared with dissolved metals. Metal oxide nanoparticles bound to the cell walls or frustules of phytoplankton may facilitate rapid uptake of large amounts of metals by consumers, resulting in more acute toxicity effects. Metal ions that readily adsorb to algal surfaces may be released under changing pH within the digestive tract of consumers.45 Accumulation of metal oxide nanomaterials on phytoplankton cell walls may facilitate trophic transfer of these contaminants and cause reverberating effects on oceanic food webs. It is important to note that most of the metal concentrations in this study were higher than those presently found in most marine environments. These concentrations nevertheless enabled us to clearly measure decreases in T. weissf logii populations and significant differences in metal accumulation and distribution. We implicitly assume that patterns of metal distribution across cellular fractions do not depend heavily on concentration. More research is needed to examine accumulation and effects of metals at lower concentrations because ecosystem productivity can be affected by even small decreases in phytoplankton growth.46 Additionally, metal accumulation and distribution in this lower trophic level may have important implications up the food chain, decreasing fitness of consumers. Adult mortality rates of primary consumers such as zooplankton can modulate interannual variability of pelagic food webs and modify elemental cycling in the ocean;47 widespread contamination by metals, whether nanoparticulate or dissolved, may thus have complex and unpredictable effects on oceanic ecosystems. ■ AUTHOR INFORMATION Corresponding Author *Phone: (229) 333-5766 E-mail: [email protected]. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS Funding for the present study was provided in part by the University of California Center for Environmental Implications of Nanotechnology (UC CEIN) supported by the National Science Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-0830117, and by the Valdosta State University Graduate School (Valdosta, GA, USA). A VSU equipment grant provided the funding for the atomic absorption spectrophotometer. ■ REFERENCES (1) Trequer, P.; Nelson, D. M.; Van Bennekom, A. J.; DeMaster, D. J.; Leynaert, A.; Queguiner, B. The silica balance in the world ocean: A reestimate. Science 1995, 268, 375−379. (2) Field, C. B.; Behrenfeld, M. J.; Randerson, J. T.; Falkowski, P. Primary production of the biosphere, integrating terrestrial and oceanic components. Science 1988, 281, 237−240. (3) Lewis, M. A. Use of fresh-water plants for phytotoxicity testing A review. Environ. 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