# Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO 2 ) Nanoparticles in Wetland Mesocosms

Nicholas Geitner, Jane Cooper, Astrid Avellan, Benjamin Castellon, Brittany

Perrotta, Nathan Bossa, Marie Simonin, Steven Anderson, Sayako Inoue,

Michael Hochella, et al.

► To cite this version:

Nicholas Geitner, Jane Cooper, Astrid Avellan, Benjamin Castellon, Brittany Perrotta, et al.  
Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO 2 ) Nanoparti-  
cles in Wetland Mesocosms. Environmental Science and Technology, 2018, 52 (17), pp.9768-9776.  
10.1021/acs.est.8b02040 . hal-03707279

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# Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO) Nanoparticles in Wetland Mesocosms

Nicholas K. Geitner, Jane L. Cooper, Astrid Avellan, Benjamin T. Castellon, Brittany G. Perrotta, Nathan Bossa, Marie Simonin, Steven M. Anderson, Sayako Inoue, Michael F. Hochella, Curtis John Richardson, Emily S. Bernhardt, Gregory V. Lowry, P. Lee Ferguson, Cole W. Matson, Ryan S. King, Jason M. Unrine, Mark R. Wiesner, and Heileen Hsu-Kim

Environ. Sci. Technol., **Just Accepted Manuscript** • DOI: 10.1021/acs.est.8b02040 • Publication Date (Web): 01 Aug 2018

Downloaded from http://pubs.acs.org on August 2, 2018

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Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria
(CeO₂) Nanoparticles in Wetland Mesocosms

Nicholas K. Geitner<sup>ab*</sup>, Jane L. Cooper<sup>ab</sup>, Astrid Avellan<sup>ad</sup>, Benjamin T. Castellon<sup>ah</sup>, Brittany G. Perrotta<sup>ai</sup>, Nathan Bossa<sup>ab</sup>, Marie Simonin<sup>ag</sup>, Steven M. Anderson<sup>ag</sup>, Sayako Inoue<sup>ac</sup>, Michael F. Hochella Jr.<sup>aef</sup>, Curtis J. Richardson<sup>ac</sup>, Emily S. Bernhardt<sup>ag</sup>, Gregory V. Lowry<sup>ad</sup>, P. Lee Ferguson<sup>ab</sup>, Cole W. Matson<sup>ah</sup>, Ryan S. King<sup>ai</sup>, Jason M. Unrine<sup>aj</sup>, Mark R. Wiesner<sup>ab</sup>, Heileen Hsu-Kim<sup>ab*</sup>

TOC Art

<figure><img src="image_2.png" /></figure>

<footnote><sup>a</sup>Center for the Environmental Implications of Nanotechnology, Duke University, Durham, NC, USA.</footnote>

<footnote><sup>b</sup>Civil and Environmental Engineering Department, Duke University, Durham, NC, USA.</footnote>

<footnote><sup>c</sup>Nicholas School of the Environment, Duke University, Durham, NC, USA</footnote>

<footnote><sup>d</sup>Civil & Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA, USA</footnote>

<footnote><sup>e</sup>Geosciences, Virginia Tech, Blacksburg, VA, USA</footnote>

<footnote><sup>f</sup>Energy and Environment Directorate Pacific Northwest National Laboratory Richland, WA, USA</footnote>

<footnote><sup>g</sup>Biology, Duke University, Durham, NC, USA</footnote>

<footnote><sup>h</sup>Environmental Science, Baylor University, Waco, TX, USA</footnote>

<footnote><sup>i</sup>Biology, Baylor University, Waco, Texas, USA</footnote>

<footnote><sup>j</sup>Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA</footnote>

<footnote>*Co-corresponding Authors: nicholas.geitner@duke.edu; hsukim@duke.edu</footnote>
## 1 ABSTRACT

Trace metals associated with nanoparticles are known to possess reactivities that are different from their larger-size counterparts. However, the relative importance of small relative to large particles for the overall distribution and biouptake of these metals is not as well studied in complex environmental systems. Here, we have examined differences in the long-term fate and transport of ceria (CeO₂) nanoparticles of two different sizes (3.8 vs. 185 nm), dosed weekly to freshwater wetland mesocosms over 9 months. While the majority of CeO₂ particles were detected in soils and sediments at the end of nine months, there were significant differences observed in fate, distribution, and transport mechanisms between the two materials. Small nanoparticles were removed from the water column primarily through heteroaggregation with suspended solids and plants, while large nanoparticles were removed primarily by sedimentation. A greater fraction of small particles remained in the upper floc layers of sediment relative to the large particles (31% vs 7%). Cerium from the small particles were also significantly more bioavailable to aquatic plants (2% vs 0.5%), snails (44 vs 2.6 ng), and insects (8 vs 0.07 µg). Small CeO₂ particles were also significantly reduced from Ce(IV) to Ce(III), while aquatic sediments were a sink for untransformed large nanoparticles. These results demonstrate that trace metals originating from nanoscale materials have much greater potential than their larger counterparts to distribute throughout multiple compartments of a complex aquatic ecosystem and contribute to the overall bioavailable pool of the metal for biouptake and tropic transfer.

20

## 21 INTRODUCTION

The rapidly increasing presence of nanomaterials in commercial products and industrial processes has caused significant concern over their potential environmental impact. An estimated

23
10000 tons of CeO₂ nanoparticles were produced in 2011, and have a wide range of applications in optical devices, silicon wafer polishing, catalytic converters, and diesel fuel additives.¹ In addition, as the tools to trace these materials have developed, it has become increasingly clear that naturally occurring or incidental nanoscale materials may play an even larger role in environmental processes, with significant implications to environmental and human health.²⁻⁷

Processes specific to nanoscale particles may include the movement of the materials themselves in ways that differ from macrocrystalline or dissolved phases,⁸,⁹ or by co-transport of other anthropogenic contaminants.¹⁰⁻¹³ In both cases of natural and engineered nanomaterials, a detailed and mechanistic understanding of the fate and transport of nanoscale materials, and how this compares to bulk materials, is critical to environmental modeling and risk forecasting.¹⁴,¹⁵

Nanomaterials possess immense variety in characteristics including core composition, surface functionality, size, and shape. Numerous studies have examined the transport of single nanoparticles,¹⁶⁻¹⁹ while others have illuminated differences in nanoparticle attachment, transport, and bio uptake due to surface chemistry in the laboratory.²⁰ For example, gold nanoparticles of smaller core diameter were taken up more by *Daphnia magna* than larger, otherwise similar particles.²¹ Once in contact with biological surfaces, uptake mechanisms and intra-organism transport of nanoparticles also depend strongly on size.²², ²³ Modeling of nanoparticle transport in the environment has also suggested that core size may be a critical parameter in these processes.²⁴ However, few studies have examined such factors of nanoparticle fate in complex environments outside of the laboratory. Despite the numerous laboratory studies on nanoparticle behavior in simulated environmental systems, the applicability of this knowledge for real natural systems remains understudied.
In the present work, we examined the environmental distribution and transformations of two similar CeO₂ nanoparticles of different primary core size added to outdoor wetland mesocosms. The first nanoparticle examined here was at the smallest range of typical nanomaterials (3.8 nm), while the second was much larger (185 nm) and likely pushes the boundary between nano and bulk material. Previous studies have found that 5-7 nm CeO₂ nanoparticles used as diesel fuel additives are transformed to single crystals in excess of 100 nm after combustion, making both size ranges highly relevant for environmental exposure.²⁵

This nine-month study consisted of chronic, weekly dosing of two sizes of CeO₂ nanoparticles into the aquatic portion of wetland mesocosms, which include both submerged aquatic, wetland, and dry terrestrial zones, over the course of nine months. While chronic dosing was selected to mimic reality, dosing concentrations were not intended to replicate expected environmental exposures, as the goal of this study was to determine material fate and transport, not to monitor toxicological effects. These large and complex systems captured a wide variety of environmental processes not possible in the lab. These include diel cycling of pH and dissolved oxygen, a depth gradient of redox potential, high biodiversity, a food web of multi-branch trophic chains, and gradual seasonal changes in conditions. The mesocosms supported a range of terrestrial and aquatic plants typical of North Carolina wetlands, algal communities, a variety of invertebrates, and a population of eastern mosquitofish (*Gambusia holbrooki*). Previous work in these mesocosms has revealed surprising results including nanoparticle transformations not previously seen in laboratory-scale studies,²⁶,²⁷ with significant implications in toxicity and fate.

The objective of this study was to test the hypothesis that a simple difference in core particle size distribution would result in significant variation in the fate and transport of these materials, manifesting in diverging mass distributions in the wetland ecosystem. We further examined
transport mechanisms of both nanoparticles with supplemental laboratory studies. Any observed
differences may then be utilized in support of a more comprehensive and predictive framework
of particulate matter transport in aquatic and soil systems.

## MATERIALS AND METHODS

### CeO₂ Particle Characterization

Stocks of small CeO₂ nanoparticles were obtained from Nyacol (CeO₂AC-30, Ashland, MA), and large particles from Alpha Aesar (NanoArc CE-6080, Tewksbury, MA). Each was characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and Fourier transform infrared spectroscopy (FTIR). TEM was performed with a FEI Tecnai G²Twin. Samples were prepared by dilution in DI water; a few drops were then applied to a Formvar-coated Cu grid (Ted Pella), and excess liquid was wicked away with filter paper. SEM was carried out on an FEI Quanta 600 FEG equipped with Energy Dispersive X-ray spectrometer (Bruker QUANTAX 400). The instrument was operated at 20 kV. DLS and zeta potential were performed with Malvern ZetaSize R ZS (Malvern, UK). For these, nanoparticle stock suspensions (as received) were diluted to 5 mg/L in freshly collected mesocosm water (details below), and pH adjusted to 8.8 using 0.1 M NaOH to mimic morning mesocosm pH values. These dilutions were bath sonicated for 30 minutes immediately prior to characterization. FTIR (Thermo Electron Nicolet 8700) was utilized in order to examine any molecules at the nanoparticle surfaces. Solid pellets were formed by mixing freeze-dried nanoparticles and an inert medium, boron nitride. Spectra were recorded from 4000 to 400 cm⁻¹ with 4 cm⁻¹ resolution. Results were recorded as an average of 64 scans.

### Mesocosm Setup
The constructed wetland ecosystems, or mesocosms, are open air, controlled release facilities, located in a clearing in the Durham division of the Duke Forest (North Carolina, USA). The structure of the mesocosms has been previously described.²⁸ In brief, these mesocosms consist of manufactured wooden boxes with dimension of 0.81 × 3.66 × 1.2 m (H × W × D) (Figure 1). The facility contains 30 of these mesocosms; this work utilized 9. These were randomly dosed with either small or large CeO₂ nanoparticles, or undosed controls, 3 replicates each. The bottom of each mesocosm comprised a flat bottom (1 m long) combined with a sloped bottom (13° inclination). This design created a permanent aquatic zone, a transition zone, and an upland or terrestrial zone, collectively providing a gradient of saturation and redox conditions in the system that were meant to simulate a freshwater wetland. The soil thickness throughout was 21 cm before rain, compaction and bio growth. The soil consisted of a commercial mix (Sands and Soil, Durham, NC) designed to mimic the composition of a local wetland (64% sand, 28% silt, 13% clay and 5% loss on ignition).

Approximately 250 L of local, untreated groundwater was added in June 2015 to each mesocosm such that the water table reached halfway up the transition zone slope. Plugs of an aquatic plant *Egeria densa* were planted 3 days later in aquatic zone. Additional species relevant to local North Carolina wetlands (*Lobelia elongate*, *Carex Lurida*, *Panicum Virgatum*, and *Juncus effusus*) were simultaneously planted as plugs in a uniform grid in the transition and terrestrial zones. Water was subsequently added only through natural precipitation. The site received average rainfall of 14.9 mm/day throughout Spring, Summer, and Fall. Small, recorded volumes of water were removed 4 times over the course of the experiment to prevent overflow after significant rain events. After 2 weeks of plant growth and prior to treatments, water was cycled between all mesocosms to ensure maximum uniformity in initial water chemistry.
Mesocosms were then allowed 6 weeks to establish before the introduction of eastern mosquitofish (Gambusia holbrooki, Triangle Pond Management, USA), 53 females and 15 males per mesocosm. At the beginning of dosing, water pH cycled daily between approximately 7.0 and 10.0, with an average pH of 8.8 in early morning. Water conductivity was an average of 111 ± 20 µS/cm and dissolved organic carbon an average of 11.9 ± 4.4 mg/L over the course of the experiment.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Cross-sectional view of the wetland mesocosm, with height and width dimensions and zone definitions noted. The depth of each mesocosm was 1.2 m. Dotted line indicates the water table.</figcaption></figure>

### Dosing

Once per week, each mesocosm received an assigned nanoparticle dose as a freshly prepared suspension in 1 L DI water. Each suspension was sonicated in a water bath for 10 minutes prior to dosing. Three mesocosm boxes were assigned to small CeO₂ particles, three boxes for large CeO₂, and three control boxes without treatment. Control boxes received 1 L DI water without nanoparticles at each dosing. Both large and small CeO₂ nanoparticle weekly doses consisted of 19.23 mg nanoparticles for 9 months from January to October 2016. Overall, a total of 0.75 g CeO₂ (0.61 g Ce) was added per mesocosm during the course of the 39-week experiment. Dosing concentrations were determined by inductively coupled plasma mass spectroscopy (ICP-MS)
analysis of stock solutions, as described below. Dosing suspensions were injected into the
aquatic zone of the mesocosms, 2 cm below the water surface in a moving grid pattern to ensure
even and consistent addition across the water surface.

Sampling and Digestion

For each water sample, 10 mL aliquots were collected weekly, immediately acidified with HNO3, and stored at room temperature. Whole plant roots or stems, as appropriate, were collected quarterly (Q1-3), dried at 60°C, and homogenized by grinding. At Q3 (39 weeks), the full plant biomass was similarly collected. Half of all E. densa samples were rinsed in 500 mL sterilized mesocosm water by shaking for 2 minutes followed by a final rinse by flowing water to remove particles from the exterior. Soil and sediment (aquatic and transition zones) samples were collected by submerging polypropylene tubes into the surface and extracting intact cores. Three cores were collected and composited in each zone of the mesocosm in order to better capture intra-mesocosm heterogeneity. These were subsequently separated into floc, 0-1 cm, and 1-5 cm depths. Plant roots were removed manually to the extent possible. The floc was assumed to exist in the top 0.5 cm of aquatic sediments. Each depth portion was then dried at 60°C, and homogenized by grinding. Fish were sampled non-invasively by trapping (n = 5 individuals per NP-treated mesocosm, n = 3 individuals per control mesocosm) and immediately euthanized with tricaine methanesulfonate and frozen (Duke University IACUC protocol # A135-16-06). As both snail species (Physella sp. and Lymnaea sp.) colonized the mesocosms incidentally with the E. densa, up to n = 5 individuals were hand collected from each mesocosm where available. Insects were collected from sediments at the end of Q3. The insects were then separated by
species, dried at 60°C, weighed, and homogenized by grinding. This work focuses solely on the
final distribution of materials at 39 weeks (Q3).

For each water sample digestion, a 5 mL aliquot was transferred to a 50 mL polypropylene tube. To this, 750 µL concentrated nitric acid (70%, trace metal grade) and 375 µL 30% hydrogen peroxide were added and allowed to react overnight at room temperature. Each tube was then heated to approximately 100°C on a heating block and held for 30 min. An additional aliquot of 750 µL nitric acid was added to water samples from the mesocosms dosed with large CeO₂ nanoparticles (to ensure complete digestion of these samples). Each sample was then cooled and diluted with ultra-pure water (18 MΩ-cm) resulting in approximately a 5% acid concentration.

The same procedure was followed for plant samples consisting of 25 mg dried and ground plant tissue. The digestion procedure was also followed for 25 mg dried and ground soil or sediment samples.

Whole fish were dried at 60° C and homogenized individually with mortar and pestle.
Homogenates (mean of 18 dry mg) were hydrated 24 h and digested in the same manner as the
other samples.

Elemental Analysis

Elemental analyses of acid digested samples were performed by ICP-MS with either Agilent 7900 (fish, snails, invertebrates, water) or Agilent 7700 (plants, soil) instruments. Analytes included Na, Mg, K, Ca, Mn, Fe, Cu, Zn, La, Ce, and Nd. La and Nd were measured in order to calculate background levels of Ce, and the remaining elements were measured as standard water
and soil chemistry parameters. Calibration was performed across a range of 0.1 – 500 µg/L for Ce, Nd, La, and Pb, up to 50,000 µg/L for the remaining analytes. Calibration for Ce in fish, snail, and invertebrate samples was performed down to 0.01 µg/L. Each sample run began with 2 blank samples. Additional method validation involved the analysis of metals drinking water standard (NIST 1643e), standard spikes, and a method blank solution of 2% nitric, 0.5% hydrochloric acid every 15 samples. Method limits of detection were calculated as the mean of 3 blank samples + 3 standard deviations of 10 replicate analyses of blank samples. The method limit of detection in water samples was found to be 1.2 and 0.22 µg/L for Ce and Nd, respectively. Limits of detection were similar in fish (0.99 µg/kg Ce), snails and insects (3.1 µg/kg Ce), and plants and soils (0.021 µg/kg Ce and 0.016 µg/kg Nd). Duplicate digests were conducted for 10.4% of the fish samples, and the mean relative percent difference [Ce] between samples was 11 ± 6%.

Background concentrations of Ce were accounted for by observing the ratio $[Ce]_{Bkrnd}/[Nd]_{Bkrnd}$ in each sample type. In each sample type, this ratio varied by less than 1% across samples. This method was used instead of subtracting Ce concentration values measured in the control (undosed) mesocosms due to spatial heterogeneity in soils and sediments, resulting in greater variability in control values. Both CeO₂ nanoparticles contained Nd below detection limits. We then utilized this ratio to calculate $[Ce]_{NP}$ within Ce treated samples by measuring $[Nd]$ in these samples:

$$[Ce]_{NP} = [Ce]_{tot} - [Nd] \frac{[Ce]_{Bkrnd}}{[Nd]_{Bkrnd}} \#1$$

For biological compartments including fish, snails, and insects, total Ce masses were calculated following organism body burden measurements after subtracting background Ce
concentrations as measured in control, untreated mesocosms. This was done by extrapolating the mass concentration found in each organism using the total mass of the particular organism found in that mesocosm. For fish, insects, and snails, this was accomplished by collecting all organisms present at the end of the experiment. Insect concentrations are based on collected dragonfly nymphs in the sediments, as these were present in all mesocosms and constituted the majority of the harvest.

## Speciation of cerium in plants and sediment

Ce oxidation states in samples was assessed by X-ray Absorption Near Edge Structure (XANES) at Ce LIII-edge (5.723 keV) at the beamline 20-ID of Advanced Photon Source facility (Chicago). Three young shoots (first 20 cm) of *Egeria densa* per mesocosm and composite samples of the floc layer and transition sediment per mesocosm were frozen just after collection. Prior to XANES analysis, these samples were freeze-dried at -55°C and 0.014mBar for 3 days, ground under N₂ atmosphere as one composite sample per treatment, pressed into pellets, sealed with Kapton® tape, and kept under anoxic environment until analysis.

During the analysis, samples were held in a He cryostat to prevent beam damage. XANES spectra were acquired from 5524 eV to 5684 eV at 10 eV increments, from 5684 eV to 5774 eV with a 0.3 eV increments, and then up to 6000 eV using 0.05 eV increments. XANES spectra were recorded in fluorescence mode using a vortex Si drift (4-element) and a Si(111) monochromator. For each sample, 2 scans were acquired on at least 3 different locations (minimum of 6 scans per pellet) to (i) prevent sample damage under the beam and (ii) check for the homogeneity of the pellet. During each scan, the XANES of a metallic Cr sheet was recorded (k edge=5898 eV) to verify beam stability. XANES spectra were calibrated in energy (if needed),
background subtracted with $E_0$ defined as the maximum of the 1st XANES derivative, and
normalized using Athena software.²⁹ Ce(III)-acetate and the commercial additive Nanobyk-3810
Ce(IV)O₂ nanoparticles were used as Ce(III) and Ce(IV) references, respectively. Linear
combination fits were performed in order to determine relative Ce(III) and Ce(IV) content.

## Sediment Depth Profile of Redox Relevant Species

Sediment cores were collected at the end of 9 months of dosing. Depth concentration
profiles of dissolved O₂, Mn²⁺, and Fe²⁺ concentrations at the sediment-water interface of these
cores were collected via voltammetry using solid state Au-Hg amalgam microelectrodes.²⁶ One
depth profile was taken from each core. Dissolved O₂ was calibrated against an air-saturated
sample of mesocosm surface water at room temperature (~22°C), assuming [O₂] = 272 µM.
Limits of quantification were 5 µM, 15 µM, and 15 µM for O₂, Mn²⁺, and Fe²⁺, respectively.

## Sedimentation and Heteroaggregation Studies

Nanoparticle heteroaggregation was quantified by measuring their attachment efficiency, $\alpha$, as described previously.⁸ Briefly, 2 g washed glass beads were acclimated with 1 g mesocosm floc in 40 mL mesocosm water for 72 hours under gentle mixing. The glass beads were then allowed to settle, and the solution was decanted. The glass beads were then gently washed three times in mesocosm water and dried in air; as a result of this process, the glass beads were coated by organics and other residual material from mesocosm floc, as evidenced by a darker color.

For experimental measurement of $\alpha$, 0.4 g of these beads were added to 30 mL unfiltered
mesocosm water with pH adjusted to 8.8 using 0.1 M NaOH. Under mixing with a magnetic stir
245 bar, 10 mL of either large or small CeO₂ nanoparticles was added to final suspension
246 concentrations of 10 mg/L CeO₂. Aliquots were collected over the course of a 60-minute mixing
247 experiment, and held static for 30 seconds, resulting in the settling of glass bead/CeO₂
248 heteroaggregates and separation from suspended, free nanoparticles. The suspended free
249 nanoparticle concentration was quantified by absorbance (305 nm for small particles, 610 nm for
250 large) A(t) for various mixing times t in a 1-cm pathlength cuvette (BMG SpectroStar Nano).

251 The same heteroaggregation experiment was performed under favorable attachment conditions
252 (α = 1) by spiking solutions with 6 mM NaNO₃, thus effectively eliminating electrostatic
253 stabilization of the nanoparticles.

254 The resulting measurements were fit to a first order kinetic equation where the slope of
255 ln (A(t=0)/A(t)) versus t scales with the product αβB.⁹ We assumed that B, the initial concentration of
256 background particles, and β, the collision frequency, were both constant across experiments.
257 Attachment efficiency α was determined by the linear slope value during unmodified attachment
258 conditions (αβB) divided by the same slope in the favorable attachment scenario (1 × βB).⁹

259 Sedimentation was assessed at the lab scale, simulating mesocosm conditions. 100 g of acid
260 washed glass beads (d=320 µm) were placed at the bottom of 750 mL glass bottles together with
261 500 mL of freshly collected mesocosm water. After 1 day of stabilization, nanoparticles were
262 injected with 100 µg/L final concentration. Glass beads were found to help prevent resuspension
263 of particles from lower regions of the bottle without affecting sedimentation rates. Samples were
264 taken at 5 cm depth from the water surface to measure the concentration of suspended
265 nanoparticles over time, and analyzed with ICP-MS as described above.

266
## Statistical Analysis

Where applicable, data are reported as the mean of all samples across 3 replicate mesocosms or 3 laboratory replicates. In all cases, error bars and reported uncertainties are standard deviations. In tests of statistical significance, a standard t-test of equal variance was employed where significance was defined as $p < 0.05$.

## RESULTS AND DISCUSSION

### Nanoparticle characterization

The large and small CeO₂ nanoparticles in pH 8.8 mesocosm water (to approximately mimic the mesocosm water pH in the mornings) were drastically different in size (hydrodynamic diameters less than 20 nm for small CeO₂; >100 nm for large CeO₂), while they both had similar magnitude of negative surface charge, as indicated by measured zeta potentials (Table 1). FTIR analysis confirmed manufacturer specifications of an acetate coating on small CeO₂ nanoparticles, with characteristic peaks at 1400 cm⁻¹ and 1530 cm⁻¹ from the carbonyl group (Supporting Information Figure S1).³⁰ Analysis of the large CeO₂ particles indicated the presence of chitosan. Identified peaks included 1660 cm⁻¹, attributed to C=O, 1570cm⁻¹, attributed to NH groups, and 1422 cm⁻¹, attributed to CH·OH.³¹

<table><caption>**Table 1.** Summary of nanomaterial characterization. DLS and Zeta potential measurements were carried out in mesocosm water.</caption><thead><tr><th></th><th>Size – TEM<br>(nm)</th><th>Size – DLS,<br>pH 8.8 (nm)</th><th>Zeta Potential,<br>pH 8.8 (mV)</th><th>Weekly<br>Particle<br>Dose (mg)</th><th>Total Ce<br>Dose (g)</th></tr></thead><tbody><tr><td>Small CeO<sub>2</sub></td><td>3.8 ± 1.1</td><td>15.8 ± 2.4</td><td>-23.3 ± 0.67</td><td>19.23</td><td>0.61</td></tr><tr><td>Large CeO<sub>2</sub></td><td>185 ± 60</td><td>281 ± 55</td><td>-25.8 ± 0.65</td><td>19.23</td><td>0.61</td></tr></tbody></table>
Distribution of CeO₂ Nanoparticles Across Compartments of the Mesocosms

The analysis of Ce in various compartments of the wetland mesocosms after the 9 month experiment indicated that most of the CeO₂ particles accumulated in the sediment, soil, and floc layers (Figure 2). Recovery of Ce relative to the total added Ce was 91 ± 18% for large and 134 ± 31% for small CeO₂ nanoparticles. The greater than 100% recovery for the small CeO₂ particles was possibly due to greater than expected heterogeneity in floc and sediment samples. Regardless, both particles had low residual aquatic concentrations over long timescales, and were also found primarily in various compartments of sediment and soil, compromising 94% and 98% for small and large particles, respectively.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Mass of Ce in major compartments of the wetland mesocosms after 9 months of weekly dosing with either large or small CeO₂ nanoparticles. Aquatic sediment was defined as the top 1 cm, while deep sediment was from a depth 1-5 cm of sediment in the aquatic zone. The dotted line indicates the total amount of Ce (610 mg) added as CeO₂ nanoparticles during this period. Each grouping represents the average (±1 std. dev.) for 3 replicate mesocosms. Compartments contributing less than 0.5% of the total are not shown for clarity.</figcaption></figure>
There were also significant size-based differences in the fate of these particles. First, accumulation of Ce in the floc was significantly greater for small particles (256 ± 57 mg) relative to the large particles (39.9 ± 4.2 mg) (p=0.03). Instead, large particles were found to accumulate in deeper (1-5 cm) layers of aquatic sediment, with 157 ± 110 mg detected for large particles vs 48.5 ± 24 mg for small, though not statistically significant due to variation between mesocosms (p=0.2). The presence of large CeO₂ nanoparticle aggregates in deeper sediment was confirmed by SEM and EDS (SI Figure S2). Further transport of large CeO₂ particles below 5 cm may account for some of the Ce mass not detected. Approximately 2% of small particles were detected in upland soil, while large particles in that compartment were below detection limits. The transport of small particles to upland soil is likely due to free particles still present in the water column during occasional flooding events.

Small particles also had significantly greater (p=0.002) total association with *E. densa* compared to the large particle dosings. This is in agreement with previous studies which found greater plant uptake in mesocosms dosed with nanoscale Ag and ZnO compared with mesocosms receiving larger scale materials of similar composition.³² We hypothesized that the difference in *E. densa* association may be due to greater attachment efficiencies of these smaller particles, resulting in greater initial interactions with plant surfaces. This hypothesis is explored further below.

Finally, by subtracting rinsed from unrinsed *E. densa* Ce concentrations, we were able to approximate internalized particles and those externally adsorbed to plant or associated periphyton surfaces. These results (Figure 3A) suggested that Ce originating from small CeO₂ nanoparticles were taken up significantly more by these aquatic plants, with internal Ce mass
more than twice external concentrations (23.3 ± 1.6 mg interior vs 9.5 ± 0.9 mg exterior, p=0.01). Large particles, in contrast, were approximately twice as likely to be found on the exterior than the interior of *E. densa* (3.0 ± 0.5 mg interior vs 6.0 ± 3.0 mg exterior, p=0.04). We argue that this is not an artificial result due to size differences in rinsing efficiency (i.e. greater shear experienced by larger particles), as such differences would actually be expected to give the opposite result.

The uptake of Ce in the aquatic animal compartments of the mesocosms at 9 months was a small fraction of the overall nanoparticle fate (Figure 3). However, understanding differences in bioavailability of small relative to large CeO2 is critical to understanding the ecological importance of nanoscale materials. In fish, no significant (p > 0.05) difference was observed in total internalized mass of Ce after dosing with small and large CeO2 nanoparticles. However, significant differences were observed in both snail (p=0.018) and insect (p < 0.0001) Ce masses. In insects, 8.0 ± 1.1 µg Ce was found when dosed with small particles. However, total mass in insects was over 120 times lower for those exposed to large particles, with just 0.065 ± 0.02 µg Ce detected. Snails comprised the lowest total masses (though highest organism concentrations of Ce) of the three compartments. Ce masses in the snails of mesocosms dosed with small and large particles were quantified at 44 ± 18 and 2.6 ± 0.4 ng, respectively, a 17-fold difference. In aggregate, these results suggest a higher bioavailability of the Ce originating from smaller nanoparticles to particular animal compartments in these mesocosms. However, no difference in accumulation was observed in fish. We hypothesize that this may be related to organism localization within a mesocosm as well as feeding habits. Verification of this hypothesis is left for future work.

349
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 3.** The calculated total mass of Ce from nanoparticles detected in *E* densa (A) and fish, snails, and insects (B) in the mesocosms for small (blue) and large (green) ceria nanoparticles. Data labels in B indicate the concentration of Ce detected per dry weight of organism, corrected for background concentrations.</figcaption></figure>
We also closely examined the speciation of Ce across the mesocosms over time. The relative abundance of Ce(III) and Ce(IV) has particular implications in understanding observed differences in Ce biouptake as well as nanoparticle toxicity and reactivity,¹,³³ and so is an important environmental endpoint. The XANES spectra for Ce in various compartments over time (Figure 4) highlight several important differences. Quality factors of the fits can be found in Table S1. First, in mesocosms receiving small CeO₂, 57% of the Ce on or within *E. densa* was in the Ce(III) form (Figure 4B), a drastic contrast to the original small CeO₂ particles that comprised fully of Ce(IV) (Figure 4A). For the *E. densa* exposed to large particles, a much smaller percentage of Ce (10%) was observed as the reduced form of Ce (Figure 4B).

<figure><img src="image_7.png" /><figcaption>**Figure 4.** X-ray absorption near edge structure (XANES) spectra for (A) Ce(IV)O₂ or Ce(III)-acetate reference compounds used for the combination fitting of the spectra of (B) *Egeria densa* exposed to CeO₂ small or large at 9 mo, or for the floc at 3, 6, or 9 mo exposed to (C) large CeO₂ or (D) small CeO₂, and (D) the surficial sediment exposed to either small or large CeO₂. Dotted lines indicate peak positions. Fitting results are shown on the plot.</figcaption></figure>
Further, Ce in flocs of the large CeO2 mesocosms underwent very limited reduction (Figure 4C), while in the small CeO2 mesocosms the Ce in the flocs were completely reduced to Ce(III) by the end of the experiment (9 months, Figure 4D). In fact, the speciation of Ce at 3 months in the floc is similar to the background Ce speciation found in the control mesocosm (data not shown). The fraction of Ce(III) found in the flocs of the large CeO2 mesocosms was mostly likely the native Ce in the wetland soil and the relative contribution of this native Ce decreased as CeO2 continued to be added during the 9 month experiment. Finally, the Ce detected in the top layer of the transition sediments (Figure 4E) at the 9 month time point indicate that approximately 20% of large CeO2 remains as Ce(IV) while the rest has been reduced.

The differential reduction of Ce(IV) to Ce(III) is thus dependent on particle properties, the compartment to which they accumulate within the mesocosm, and the residence time of the particles in each compartment. There are multiple possible explanations for these stark differences in Ce speciation over time. First, the clear size effect. It is expected that, due to lattice expansion at small crystal sizes, the smaller particles may have a higher percentage of defects and/or oxygen vacancies, giving them a higher propensity for reduction to Ce(III).³⁴ The small particles' surface/volume ratio was also much larger than those of the large particles. They were therefore expected to be more reactive, as atoms in the bulk of the material do not directly participate in redox reactions.³⁵

Second, the coatings of the two particles differed. The small CeO2 particles were coated with a low molecular weight organic acid (acetate) that may not have protected the particles from Ce reduction reactions while the large CeO2 were coated with high molecular weight molecules (long chitosan chains) can could be more protective of surface reactions. Coating also affects the
affinity of the nanoparticles for surfaces. Measurements of surface affinity discussed below, are also consistent with a lower rate of reduction of the Ce in the case of a lower surface affinity, seem to mirror results found for smaller rates of cerium nanoparticle reduction with lower surface affinities for the case of activated sludge reported previously.³⁶ However, differences in surface coatings are expected to be short lived compared to the time scale of the experiment, as neither stabilizer was covalently bound to the surface. Therefore, over time these surfaces are expected to exchange with various components of the local environment.³⁷⁻³⁹

Finally, differences in fate of the particles based on their size, as detailed in Figures 2 and 3, likely plays a secondary role. Accumulation in different mesocosm compartments results in different local chemical environments that would alter the extent of CeO₂ transformation. This is highlighted by measurements of redox conditions as a function of depth, which found 50-75% O₂ saturation just above the sediment surface, but 0% saturation immediately below the sediment-water interface (SI Figure S3). Also observed were increasing concentrations of Mn²⁺ and Fe²⁺ as a function of depth. We also observed similar profiles in mesocosms dosed with small and large CeO₂ nanoparticles. The presence of O₂ at the surface of the sediment is not detrimental to the presence of Ce(III), since the reduction of Ce(IV) is likely (Eh<sub>Ce4+/Ce3+</sub> = +1.5V, pH 8)⁴⁰ in oxic environments for the pH range found in the mesocosms (approx. 7 to 10). The drastic drop of oxygen on the top layer of the sediment can explain the further reduction of large CeO₂ NP on that compartment, where the proportion of Ce(III)/Ce(IV) originating from small or large CeO₂ tended to be similar. Therefore, differences in Ce oxidation state were primarily due to particle size and reactivity, not varying mesocosm conditions. However, Ce from both nanoparticles in the anoxic sediment environment were reduced in its majority. This reduction in soil environments is in agreement with previous studies of the reduction of small CeO₂ nanoparticles
in soil growth media.⁴¹ While no dissolution was observed in mesocosm water and sediments in the laboratory, it should be noted that under different environmental conditions, the release of Ce ions may significantly alter transport and bioavailability of smaller particles.

### Nanoparticle Transport Mechanisms

Following the observation of significant differences in overall fate of small and large CeO₂ nanoparticles, we further investigated their respective transport mechanisms. Specifically, we examined their sedimentation (Figure 5) and heteroaggregation in mesocosm water.

Heteroaggregation studies were performed on floc treated glass beads in order to more closely mimic the surface chemistry of the upper sediment layers. The zeta potential of these beads in pH 8.8 mesocosm water was -18.3 ± 1.1 mV. The sedimentation kinetics clearly show much more rapid settling of large particles. Heteroaggregation mixing studies resulted in α values of 0.72 ± 0.08 for small and 0.11 ± 0.04 for large CeO₂ nanoparticles, indicating much greater heteroaggregation of small particles with floc treated glass beads in mesocosm water. The difference in surface coating may also play a role in the different values of α, though these differences are likely mitigated by the complex water chemistry and presence of organic matter through sorption of organic matter on particle surfaces. However, autoaggregation is likely the dominant process in these experiments. Collision frequencies between nanoparticles and larger background particles (diameter on the order of several µm) decrease rapidly with increasing nanoparticle size.⁴¹ Settling rates increase as diameter to the second power.⁸, ⁹, ⁴² Based on Stokes settling alone, the time for the large and small particles to settle 5 cm (the sample height in these experiments) would be on the order of 10³ and 10⁶ minutes respectively. Thus, settling of individual nanoparticles based on their initial size alone cannot explain the removals observed in these experiments. We may therefore expect heteroaggregation or autoaggregation to play an
important role in reducing the concentration of CeO₂ particles over time. While heteroaggregation may have been favored for the smaller particles, heteroaggregation and subsequent settling, plus perhaps settling in the absence of heteroaggregation, favored more rapid removal of the larger CeO₂ nanoparticles (Figure 2). Together, differences in sedimentation and heteroaggregation clearly showed a stark contrast in transport mechanisms. These were manifested as significant differences in mesocosm fate. Specifically, large particles avoided heteroaggregation and sedimented to the deeper layers of aquatic sediment (Figure 2), while smaller particles remained in suspension and heteroaggregated with background surfaces, including floc. Smaller particles exhibiting more rapid surface attachment and resulting higher bioavailability is in agreement with previous mammalian studies.⁴³ This also provides an additional potential mechanism for differences in nanoparticle speciation. Previous studies of CeO₂ nanoparticles in activated sludge found that particles with higher values of α were more rapidly reduced to Ce(III) under aerobic conditions.³⁶ This was due to more rapid attachment to reducing bacteria, as was potentially the case in mesocosms. These results also provide possible insight for other exposure scenarios. For example, environments with higher organic matter content are expected to result in lower rates of heteroaggregation for both particles,⁸ and therefore both would be expected to have longer water lifetimes.
<figure><img src="image_8.png" /><figcaption>**Figure 5.** The sedimentation of an initial 100 µg/L aqueous suspension in mesocosm water of small (blue circles) or large (green squares) CeO₂ nanoparticles monitored over time</figcaption></figure>

## Environmental Implications

In this 9-month study with complex simulated wetland ecosystems, we found significant differences in the fate and transport of two nanoparticles of different size, with significant implications in understanding the environmental transport of nanomaterials, and of chemical and particulate environmental transport in general. While the two CeO₂ test materials were otherwise highly similar, including core composition and zeta potential, Ce originating from small particles was much more bioavailable, with significantly greater association with aquatic plants and macrofauna. Small particles accumulated in the upper floc layers of aquatic sediment, while larger particles settled in much deeper layers of sediment. These differences are consistent with relatively simple laboratory experiments of sedimentation and heteroaggregation, in which small particles possessed values of α approximately 6.5 times greater than large particles. Instead of heteroaggregating with other particles in the mesocosm surface water, large particles rapidly self-aggregated and settled out of solution.
Small CeO₂ nanoparticles were also more significantly transformed from Ce(IV) to Ce(III) by surface chemical reduction. This is most likely due to their higher reactivity by virtue of their small size, in addition to potential greater association with reducing microbes. Ce originating from the large particles did not experience the same extent reduction, as the much larger size of the particles rendered them less redox active.

Overall, trace elements such as Ce originating from smaller particles were much more likely to enter biological compartments due to transport properties as well as increased surface area for transformation reactions (e.g., reductive dissolution in the case of Ce). Larger particles transformed more slowly and settled quickly, making the upper sediment layers a sink for untransformed nanoparticles. We may therefore further expect long-range transport of large particles in flowing systems to be dominated by the motion of sediments, while smaller particles are more likely to move with the fluid and more loosely bound flocs. This work highlights that particle size not only influences the transport and transformation potential of trace elements originating from nanoparticles, but also measurably influences eventual biouptake and trophic transfer.

Acknowledgments

This material is based upon work supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093 and DBI-1266252, Center for the Environmental Implications of NanoTechnology (CEINT). Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been subjected to EPA review and no official endorsement should be inferred.
A portion of this research was performed using resources of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, and was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357, and the Canadian Light Source and its funding partners. We thank Dale L Brewe at APS (BL 20-ID) for his support.

## Supporting Information

Supporting information is available free of charge via the Internet at http://pubs.acs.org/. FTIR spectra, electron microscopy images, EDS spectra, XANES fitting parameters, and soil chemistry depth profiles.

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