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Influence of natural organic matter and surface charge on the toxicity and bioaccumulation of functionalized ceria nanoparticles in Caenorhabditis elegans. Blanche Collin, Emily Oostveen, Olga Tsyusko, and Jason M Unrine Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 29 Dec 2013 Downloaded from http://pubs.acs.org on January 6, 2014
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Influence of natural organic matter and surface charge on the toxicity and
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bioaccumulation of functionalized ceria nanoparticles in Caenorhabditis elegans.
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Blanche Collin*, Emily Oostveen, Olga V. Tsyusko, Jason M. Unrine*
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University of Kentucky, Department of Plant and Soil Sciences, Lexington KY, USA
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Total word count: 7043
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Text: 5543 words
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1 large figure: 600 words
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3 small figures: 900 words
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Supporting information
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*To whom correspondence may be addressed: [email protected], [email protected], Department of Plant and Soil Sciences, University of Kentucky, Agriculture Science Center North, N122-H, 1100 s. Limestone St., Lexington, KY 40546, USA; (859)-257-2467
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ABSTRACT
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The objective of this study was to investigate the role of CeO2 nanoparticle (NP) surface
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charge and the presence of natural organic matter (NOM) in determining bioavailability
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and toxicity to the model soil organism Caenorhabditis elegans. We synthesized CeO2-
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NPs functionalized with positively charged, negatively charged, and neutral coatings. The
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positively charged CeO2-NPs were significantly more toxic to C. elegans and
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bioaccumulated to a greater extent than the neutral and negatively charged CeO2-NPs.
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Surface charge also affected the oxidation state of Ce in C. elegans tissues after uptake.
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Greater reduction of Ce from Ce (IV) to Ce (III) was found in C. elegans, when exposed
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to the neutral and negatively charged relative to positively charged CeO2-NPs. The
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addition of humic acid (HA) to the exposure media significantly decreased the toxicity of
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CeO2-NPs, and the ratio of CeO2-NPs to HA influenced Ce bioaccumulation. When the
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concentration of HA was higher than the CeO2-NP concentration, Ce bioaccumulation
37
decreased. These results suggest that the nature of the pristine coatings as a determinant
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of hazard may be greatly reduced once CeO2-NPs enter the environment and are coated
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with NOM.
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Key words: humic acid, nematodes, speciation, toxicity, µ-XRF, µ-XANES.
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Environmental Science & Technology
INTRODUCTION
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Engineered nanomaterials are used in many commercial products and
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applications. Their production, use and disposal will inevitably lead to environmental
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releases.1, 2 CeO2 nanoparticles (CeO2-NPs) are among the most important nanomaterials
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with increasing uses as polishing media,3 catalysts in automotive fuel additives,4 gas
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sensors, and other applications.5 Ce in CeO2-NPs can reversibly transition between
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Ce(III) and Ce(IV) valence states and can act as a regenerative catalyst due to oxygen
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vacancies present at the surface of the nanoparticles.6 The magnitude of environmental
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releases of CeO2-NPs and subsequent behavior and biological effects are currently
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unclear. The need for assessment of the environmental effects of these nanomaterials was
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expressed by international organizations such as the Organisation for Economic
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Cooperation and Development (OECD).7 Given the limited number of ecotoxicological
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studies with CeO2-NPs, the extent of their toxicity in the environment is uncertain.
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Toxicity in Daphnia magna and Cophixalus riparius has been reported at 1 mg L-1 after a
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96h exposure8 but in other studies no acute toxicity was measured in D. magna after the
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same duration exposure at 10 mg L-1
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chronic exposures revealed higher toxicity in D. magna, with a threshold at 5.6 mg L-1 10
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and 100 % mortality after 7 days at 10 mg L-1 CeO2-NPs.9
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or 48h exposure at 1000 mg L-1.10 However,
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There is evidence suggesting that CeO2-NPs can act as a pro-oxidant or an
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antioxidant. Several investigations have reported that CeO2-NP toxicity is caused by
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oxidative stress due to the reduction of Ce(IV) to Ce(III).11, 12 In direct contrast, CeO2-
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NPs have also been found to possess biological antioxidant properties that can protect
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cells from damage caused by reactive oxygen species (ROS) both in vitro and in vivo.13-15
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While CeO2-NP size has been shown to influence reproduction and survival of
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Caenorhabditis elegans,16 the toxic effects of the NPs can also be affected by their
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surface coating. The surface coating interfaces the NP with its biological environment
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and can control the pathways of cellular internalization. Therefore, toxicity can be
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influenced not only by the composition of the core but also by the coating.17 For example,
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dextran coated CeO2-NPs induced positive effects in in vivo studies, such as anti-tumor
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and anti-oxidant effects18, 19 while non-coated CeO2-NPs induced toxicity.12, 20, 21 Surface
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charge is another key property, which can control the colloidal stability of NP dispersion
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and transport in environmental and biological systems. In the context of in vitro studies,
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the surface charge of CeO2-NPs has been shown to dictate NP subcellular localization in
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cells, and to influence their cytotoxicity.22, 23 Cationic NPs in general have been shown to
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be more toxic than their neutral or anionic counterparts.24-26
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An important aspect in risk assessment of manufactured nanomaterials is to
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understand their environmental interactions. The risks associated with exposure to NPs
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will be determined in part by the environmental processes that control NP fate, transport
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and transformation.27 Exposure modeling has shown that soils are important sinks for
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engineered NPs.1 Once in soils, NPs will interact with abundant organic ligands,
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including natural organic matter (NOM), which can result in the formation of a nanoscale
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coating on the NPs, changing their aggregation, sorption and biological activity.8, 28 The
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vast majority of studies that have investigated how surface charge affects the toxicity of
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manufactured did not to take into account how these environmental transformations can
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impact the toxicity of pristine nanomaterials.
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The objectives of this study were to investigate how charge of CeO2-NP polymer
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coatings influences interaction with NOM and how this in turn affects particle stability
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and CeO2-NP uptake, distribution and toxicity to the model soil nematode C. elegans.
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Nematodes are important members of the terrestrial mesofauna, with a crucial role as
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grazers of microorganisms;29 C. elegans have also been widely used as a model in
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aquatic/soil eco-toxicity testing of metals and NPs.29-31
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We hypothesized that, due to its higher affinity with negatively charged biological
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membranes, CeO2-NPs functionalized with a positively charged polymer would have
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increased toxicity and bioaccumulation in C. elegans compared to those functionalized
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with neutral or negatively charged polymers. We also hypothesized that toxicity of
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CeO2-NPs in C. elegans will be associated with a reduction of Ce valence of IV to III.
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Finally, we hypothesized that the addition of NOM to positively charged CeO2-NPs
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would decrease CeO2-NP accumulation and toxicity in C. elegans by conferring a net
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negative surface charge over a wide pH range. We tested these hypotheses by exposing
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C. elegans to CeO2-NPs of varying charge with or without NOM and measuring the
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effects on subsequent bioavailability and toxicity.
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MATERIALS AND METHODS
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Nanoparticle synthesis and characterization
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Cerium dioxide NPs with 3 different stabilizing agents were investigated in this
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study. Dextran coated CeO2-NPs with a nominal 4 nm primary particle diameter (DEX-
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CeO2(0)) were synthesized using a modification of the procedure described by Perez et
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al.18 This coating was then functionalized with diethylaminoethyl groups to confer either
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a net positive charge (diethylaminoethyl dextran; DEAE-CeO2(+)), or carboxymethyl
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groups to confer a net negative charge (carboxymethyl dextran; CM-CeO2(-)). Details of
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the synthesis and functionalization of the particles can be found in the supplemental
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information (SI). Primary particle diameter was characterized by depositing the particles
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on carbon coated copper grids and examining with transmission electron microscopy
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(TEM; Jeol 2010F, Tokyo, Japan). Freeze-dried NPs and coatings were analyzed for
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functionalization on a Thermo-Fisher Nicolet 6700 Fourier transform infrared
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spectrometer. Electrophoretic mobility titrations were performed on each type of coated
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CeO2-NP to gain insight into the intrinsic surface charge behavior imparted by the
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coatings as a function of pH in the exposure medium, which was moderately hard
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reconstituted water32 (MHRW) (Figure S4). Mean hydrodynamic diameters and
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electrophoretic mobilities of the NP treatment suspensions were measured using a Nano-
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ZS zetasizer (Malvern Instruments, Malvern, United Kindgom), at a suspension
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concentration of 100 mg Ce L-1 CeO2-NPs with and without 35 mg L-1 humic acid (HA),
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and 25 mg L-1 DEAE-CeO2(+) in the presence of 0, 7, 35, 70 mg HA L-1 (Figure S6, S7,
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S8). Suspensions were prepared using the CeO2-NP stock suspension and HA stock
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solution, adjusted at pH 8.1 and incubated at 25°C for 24h. The zeta potential was
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calculated using the Hückel approximation.
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Pahokee peat humic acid solution
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The soil humic acid Pahokee Peat (HA) obtained from the International Humic
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Substances Society (IHSS), was chosen as a soil HA model. HA was dissolved in 0.002N
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NaOH in DI (pH 7) at 500 mg HA L-1. The HA solution was stirred overnight and filtered
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(cellulose acetate, 0.2 µm). This stock solution was kept at 4°C. The total organic carbon
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measured in the stock solution, using a TOC-VCPN total organic carbon analyzer
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(Shimadzu Corporation), was 182.6 mg C L-1. According to the IHSS Pahokee peat HA is
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52.12% C, therefore the actual concentration of the stock solution was 350 mg HA L-1.
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Caenorhabditis elegans exposure
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Caenorhabditis elegans (wild type, Bristol strain N2) were obtained from the
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Caenorhabditis Genetics Center (Minneapolis, MN). The nematode culture maintenance,
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generation of age-synchronized worms and toxicity testing followed previously
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established protocols,33 except the use of MHRW as exposure medium. MHRW was
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chosen in order to provide a chemical environment close to that found in soil pore water
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and for some ecological relevance. Mortality was measured after a 48 hours of exposure
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at the L1 and L3 developmental stages. The exposure concentrations of DEAE-CeO2(+)
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for L1 nematodes ranged from 0 to 100 mg Ce L-1 (0, 1, 5, 10, 25, 50, 75, and 100), while
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the exposure concentrations of the same NPs used for L3 animals ranged from 0 to 1000
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mg Ce L-1 (0, 1, 5, 25, 100, 500, and 1000). A similar range of concentrations was also
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used for L1 nematodes exposed to DEX-CeO2(0) and CM-CeO2(-) (0, 100, 500, and 1000
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mg Ce L-1). All exposures for every concentration tested were conducted with and
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without 7 mg HA L-1. As a control, the DEAE-DEX coating alone was tested at a
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concentration of 50 mg C L-1, which would correspond to the concentration of C in a
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suspension of 200 mg Ce L-1 DEAE-CeO2(+). Escherichia coli (strain OP50) was
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supplied as a food source (10 µg ml-1, OD600 of 0.018) at the beginning and after 24h of
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the exposure. All tests were conducted in 24-well polycarbonate tissue culture plates.
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Three replicates were performed for each test. A 1.0 ml aliquot of test solution was added
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to each well, which was subsequently loaded with 10 (±1) nematodes. The plates were
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observed under a stereomicroscope and the nematodes were counted and scored.
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Following an established protocol, nematodes that did not move in response to a gentle
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prod by a Pt wire were counted as dead.34
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Synchrotron X-ray analysis
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In order to test the effect of CeO2-NPs surface charge and the presence of HA on
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Ce bioaccumulation, C. elegans at the L3 stage were exposed to 100 mg Ce L-1 of DEX-
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CeO2(0), CM-CeO2(-) and DEAE-CeO2(+) with and without 35 mg HA L-1 for 24h
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without feeding. The presence of food (E. coli) in the media can affect NPs behavior and
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oxidation state.12 We tested the effect of feeding on Ce bioaccumulation and speciation in
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C. elegans exposed to DEAE-CeO2(+) for 24h with food (E. coli strain OP50). To further
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test the effects of HA on Ce accumulation and speciation, L3 stage C. elegans were
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exposed to 25 mg Ce L-1 DEAE-CeO2(+) with 7, 35 and 70 mg HA L-1 for 48h. We
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selected Ce concentrations that were below LC10 to ensure the nematode’s survival
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during bioaccumulation experiment. After exposure, live nematodes were removed and
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preserved in 20% ethanol for up to a few days before X-ray analysis. The fresh samples
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were placed on metal-free polyimide film (Kapton; DuPont, Wilmington, DE) and
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immediately mounted in the X-ray beam. Several solutions were also prepared to analyze
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Ce speciation: NPs stock suspension, NPs in the MHRW, NPs after a 24h exposure, NPs
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with HA, and NPs with food (see Figure S10). Mixtures of 500 mg Ce L-1 CeO2-NPs and
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500 mg L-1 of ascorbic acid, tannic acid, albumin, glutathione, cysteine, collagen and
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Na2SO4 (potential reductants) adjusted to pH 7 were also analyzed (Figure S11). These
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suspensions were analyzed in a cell constructed from polyimide tubing.
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Scanning X-ray fluorescence microscopic measurements of Ce were collected at
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the Ce Lα1 emission line (5750 eV) using beamline X-26A at the National Synchrotron
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Light Source at Brookhaven National Laboratory (Upton, NY). We used Ce LIII-edge X-
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ray absorption near-edge spectroscopy (XANES) to determine the oxidation state of Ce in
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CeO2-NPs suspensions and on selected locations in C. elegans samples. Athena software
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was used to process the normalization and liner combination fitting (LCF) of the XANES
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spectra.35 C. elegans samples were fitted with 2 standards: Ce(III)sulfate as a Ce(III)
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standard and with the spectra of the NPs stock solution that they were exposed to, either
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DEX-CeO2(0), CM-CeO2(-), or DEAE-CeO2(+), as a Ce(IV) standard. For every
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treatment, the proportions of Ce(III) and Ce(IV) obtained by LCF for each individual
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spectrum (Table S2) were averaged. Details of the analytical methods are provided in the
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SI.
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Statistical analyses
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The SPSS statistic software package was used for statistical analyses. The data
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were tested for normality using the Shapiro-Wilk test and found to be non-normally
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distributed; therefore, we used non-parametric statistics. We tested for significant effects
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of treatments on Ce intensity, Ce speciation (proportion of Ce(III) or Ce(IV)) in C.
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elegans, and mortality using Kruskal-Wallis 1-way ANOVA followed by pairwise
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comparison using the Mann-Whitney U-test. Differences of p 2 µm)
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(Figure S6) may have settled onto the bottom of the well plates, locally increasing the Ce
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exposure concentration to C. elegans, which typically dwell near the bottom of the wells.
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The addition of E. coli did not modify the Ce accumulated by C. elegans exposed to
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DEAE-CeO2(+) without HA but decreased the Ce accumulated by C. elegans exposed to
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HA and DEAE-CeO2(+) compared to the same treatment without food (Figure 2). This
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may indicate that in the absence of food, the nematodes were filter feeding on the CeO2
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and HA aggregates, which led to a higher rate of ingestion and subsequent assimilation.
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The Ce oxidation state in animals exposed to 100 mg Ce L-1 DEAE-CeO2(+) was not
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significantly affected by the presence of HA or food, with a high proportion of Ce
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measured as Ce(IV): from 73 to 82 % (Figure 3).
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Addition of 7 mg HA L-1 to 25 mg Ce L-1 DEAE-CeO2(+) significantly increased
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Ce intensity measured in C. elegans from an average of 47.8 ± 15 to 77 ± 18 cps (Figure
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4). The addition of 35 mg HA L-1 induced a wide variation of Ce intensity, varying
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between 15.5 cps and 247 cps, with an average at 139 ± 108 cps. The addition of 70 mg
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HA L-1 concentration significantly decreased the Ce accumulation compared to other
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treatments, reaching 9.5 ± 4.5 cps. The higher Ce bioaccumulation with 7 mg HA L-1
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compared to that without HA may be partly explained by the absorption of small
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aggregates. At these HA and NPs concentrations, most of the aggregates had an average
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size of 11.7 nm, but two larger size fractions were formed at 40 nm and >2 µm (Figure
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S8). For the 35 mg HA L-1 treatment, the presence of µm size aggregates (Figure S8)
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explained this important variability. As we can observe on the µ-XRF map of this
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treatment (Figure 4), some aggregates can adhere to the nematode cuticle and drastically
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influence the Ce intensity. The samples (2 out of 5) where no aggregates were observed
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on the nematode had a lower Ce intensity than the control. This seems to indicate that the
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actual absorbed concentrations were lower. Moreover, most of the aggregates were too
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large to be absorbed by C. elegans. As a filter-feeder, its pharynx and buccal cavity
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exclude larger particles than 3 µm in young adults.64 The large size of aggregates may
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also partly explain the significant decrease of Ce accumulation in C. elegans in the 70 mg
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HA L-1 treatment. But for smaller aggregates, the low accumulation may be mainly
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explained by the change in ZP, which became negative.
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These results showed that the presence of HA greatly influenced Ce accumulation
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in C. elegans, increasing its accumulation for a high NP:HA ratio, and decreasing it for a
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low NP:HA ratio. Therefore, while the toxicity was always decreased in the presence of
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HA, Ce bioaccumulation is highly dependent on the ratio of NPs to HA. In all specimens
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exposed to 25 mg Ce L-1 DEAE-CeO2(+), Ce was mainly present as Ce(IV), with a
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proportion ranging from 19.3 % without HA to 38 % with 50 mg HA L-1 (Figure 3). The
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high Ce accumulation coupled with a low toxicity in C. elegans exposed to a high NP:HA
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ratio showed that the presence of HA reduced the inherent toxicity of DEAE-CeO2(+).
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Several processes can explain how HA could have alleviated toxicity despite increased
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bioaccumulation. The adsorption of HA at the surface of the NPs can form a physical
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barrier to NP interaction with the cell membrane and also reduce binding of NPs to
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important proteins and biomolecules.8, 65 In another study, aqueous organic matter may
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have also mitigated nC60 toxicity by coating the NPs, hindering direct contact with the
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cells and possibly altering nC60 surface chemistry through an undetermined
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mechanism.66 It is possible that in our study, the partial HA adsorption was sufficient to
442
decrease the toxicity by reducing the reactivity between cells and CeO2-NP surfaces, but
443
not to reverse the zeta potential so it did not decrease the NP absorption. The HA may
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also act as an antioxidant by reacting with ROS65, 67 which would mitigate the oxidative
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stress of CeO2-NPs. These effects cannot be confirmed in this study as no significant
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decrease in the amount of reduced Ce in C. elegans was measured with HA (Figure 3).
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However, HA may have intercepted reactive oxygen species that were generated without
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affecting the overall valence of the particles.
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Environmental implications
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This study showed that the surface chemistry of pristine CeO2-NPs, specifically
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their charge, had a profound effect on their toxicity. The positively charged DEAE-
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CeO2(+) were far more toxic to C. elegans and were bioaccumulated to a greater extent
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than the neutral and negatively charged DEX-CeO2(0) and CM-CeO2(-). Surface charge
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also influenced the oxidation state of CeO2-NPs once taken up by C. elegans. The
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addition of HA to the exposure medium decreased the toxicity of CeO2-NPs, and its
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concentration was shown to influence Ce bioaccumulation. For a higher ratio of HA to
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NPs, which is a more relevant scenario in the environment, the presence of HA
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significantly decreased Ce bioaccumulation. This work suggests that although initial
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surface chemistry had a profound impact on toxicity, environmental transformations
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(specifically coating with natural organic matter) reduced the effects of initial surface
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chemistry, and rendered ultra-small (2-5 nm), positively charged CeO2-NPs significantly
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less toxic for the endpoints tested at environmentally realistic ratios of HA to CeO2.
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Acknowledgements
466 467
This research was supported by the United States Environmental Protection Agency (U.S.
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EPA) through a Science to Achieve Results (STAR) grant (RD-83485701-0). J. Unrine
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and O. Tsyusko were supported by the National Science Foundation (NSF) through
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cooperative agreement EF-0830093 (Center for Environmental Implications of
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Nanotechnology). It has not been formally reviewed by EPA or NSF. The views
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expressed in this document are solely those of the authors. EPA and NSF do not endorse
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any products or commercial services mentioned in this publication. Portions of this work
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were performed at Beamline X26A, National Synchrotron Light Source (NSLS),
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Brookhaven National Laboratory. X26A is supported by the Department of Energy
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(DOE) - Geosciences (DE-FG02-92ER14244 to The University of Chicago - CARS). Use
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of the NSLS was supported by DOE under Contract No. DE-AC02-98CH10886. The
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authors gratefully acknowledge W. Rao, S. Wirick, D. McNear, J. Kupper, J. Nelson, P.
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Bertsch, A. Butterfield, U. Graham, S. Rathnayake and S. Shrestha.
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Supporting information
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Additional data on nanoparticle characterisation, µ-XANES analysis and C. elegans
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toxicity. This material is available free of charge via the Internet at http://pubs.acs.org.
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FIGURE CAPTIONS
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Figure 1. Mortality of Caenorhabditis elegans exposed to positively charged
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diethylaminoethyl-dextran coated CeO2 nanoparticles (DEAE-CeO2(+)) at L1 (A)
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and L3 (B) stages for 48 h with feeding. Diethylaminoethyl-dextran (DEAE)
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coating alone was also tested. Live Escherichia coli, strain OP50 bacteria were
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included as a nutrient source. Data are presented as the mean ± standard deviation
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(SD) (n=3). Treatment labeled with the same letter did not differ significantly
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(Kruskal–Wallis test followed by pairwise Mann–Whitney U-tests, p
Article
Influence of natural organic matter and surface charge on the toxicity and bioaccumulation of functionalized ceria nanoparticles in Caenorhabditis elegans. Blanche Collin, Emily Oostveen, Olga Tsyusko, and Jason M Unrine Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 29 Dec 2013 Downloaded from http://pubs.acs.org on January 6, 2014
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Influence of natural organic matter and surface charge on the toxicity and
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bioaccumulation of functionalized ceria nanoparticles in Caenorhabditis elegans.
3 4 5 6
Blanche Collin*, Emily Oostveen, Olga V. Tsyusko, Jason M. Unrine*
7 8
University of Kentucky, Department of Plant and Soil Sciences, Lexington KY, USA
9 10 11
Total word count: 7043
12
Text: 5543 words
13
1 large figure: 600 words
14
3 small figures: 900 words
15
Supporting information
16 17 18 19 20 21 22
*To whom correspondence may be addressed: [email protected], [email protected], Department of Plant and Soil Sciences, University of Kentucky, Agriculture Science Center North, N122-H, 1100 s. Limestone St., Lexington, KY 40546, USA; (859)-257-2467
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ABSTRACT
24 25
The objective of this study was to investigate the role of CeO2 nanoparticle (NP) surface
26
charge and the presence of natural organic matter (NOM) in determining bioavailability
27
and toxicity to the model soil organism Caenorhabditis elegans. We synthesized CeO2-
28
NPs functionalized with positively charged, negatively charged, and neutral coatings. The
29
positively charged CeO2-NPs were significantly more toxic to C. elegans and
30
bioaccumulated to a greater extent than the neutral and negatively charged CeO2-NPs.
31
Surface charge also affected the oxidation state of Ce in C. elegans tissues after uptake.
32
Greater reduction of Ce from Ce (IV) to Ce (III) was found in C. elegans, when exposed
33
to the neutral and negatively charged relative to positively charged CeO2-NPs. The
34
addition of humic acid (HA) to the exposure media significantly decreased the toxicity of
35
CeO2-NPs, and the ratio of CeO2-NPs to HA influenced Ce bioaccumulation. When the
36
concentration of HA was higher than the CeO2-NP concentration, Ce bioaccumulation
37
decreased. These results suggest that the nature of the pristine coatings as a determinant
38
of hazard may be greatly reduced once CeO2-NPs enter the environment and are coated
39
with NOM.
40 41
Key words: humic acid, nematodes, speciation, toxicity, µ-XRF, µ-XANES.
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INTRODUCTION
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Engineered nanomaterials are used in many commercial products and
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applications. Their production, use and disposal will inevitably lead to environmental
47
releases.1, 2 CeO2 nanoparticles (CeO2-NPs) are among the most important nanomaterials
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with increasing uses as polishing media,3 catalysts in automotive fuel additives,4 gas
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sensors, and other applications.5 Ce in CeO2-NPs can reversibly transition between
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Ce(III) and Ce(IV) valence states and can act as a regenerative catalyst due to oxygen
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vacancies present at the surface of the nanoparticles.6 The magnitude of environmental
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releases of CeO2-NPs and subsequent behavior and biological effects are currently
53
unclear. The need for assessment of the environmental effects of these nanomaterials was
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expressed by international organizations such as the Organisation for Economic
55
Cooperation and Development (OECD).7 Given the limited number of ecotoxicological
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studies with CeO2-NPs, the extent of their toxicity in the environment is uncertain.
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Toxicity in Daphnia magna and Cophixalus riparius has been reported at 1 mg L-1 after a
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96h exposure8 but in other studies no acute toxicity was measured in D. magna after the
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same duration exposure at 10 mg L-1
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chronic exposures revealed higher toxicity in D. magna, with a threshold at 5.6 mg L-1 10
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and 100 % mortality after 7 days at 10 mg L-1 CeO2-NPs.9
9
or 48h exposure at 1000 mg L-1.10 However,
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There is evidence suggesting that CeO2-NPs can act as a pro-oxidant or an
63
antioxidant. Several investigations have reported that CeO2-NP toxicity is caused by
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oxidative stress due to the reduction of Ce(IV) to Ce(III).11, 12 In direct contrast, CeO2-
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NPs have also been found to possess biological antioxidant properties that can protect
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cells from damage caused by reactive oxygen species (ROS) both in vitro and in vivo.13-15
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While CeO2-NP size has been shown to influence reproduction and survival of
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Caenorhabditis elegans,16 the toxic effects of the NPs can also be affected by their
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surface coating. The surface coating interfaces the NP with its biological environment
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and can control the pathways of cellular internalization. Therefore, toxicity can be
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influenced not only by the composition of the core but also by the coating.17 For example,
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dextran coated CeO2-NPs induced positive effects in in vivo studies, such as anti-tumor
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and anti-oxidant effects18, 19 while non-coated CeO2-NPs induced toxicity.12, 20, 21 Surface
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charge is another key property, which can control the colloidal stability of NP dispersion
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and transport in environmental and biological systems. In the context of in vitro studies,
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the surface charge of CeO2-NPs has been shown to dictate NP subcellular localization in
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cells, and to influence their cytotoxicity.22, 23 Cationic NPs in general have been shown to
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be more toxic than their neutral or anionic counterparts.24-26
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An important aspect in risk assessment of manufactured nanomaterials is to
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understand their environmental interactions. The risks associated with exposure to NPs
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will be determined in part by the environmental processes that control NP fate, transport
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and transformation.27 Exposure modeling has shown that soils are important sinks for
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engineered NPs.1 Once in soils, NPs will interact with abundant organic ligands,
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including natural organic matter (NOM), which can result in the formation of a nanoscale
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coating on the NPs, changing their aggregation, sorption and biological activity.8, 28 The
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vast majority of studies that have investigated how surface charge affects the toxicity of
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manufactured did not to take into account how these environmental transformations can
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impact the toxicity of pristine nanomaterials.
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The objectives of this study were to investigate how charge of CeO2-NP polymer
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coatings influences interaction with NOM and how this in turn affects particle stability
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and CeO2-NP uptake, distribution and toxicity to the model soil nematode C. elegans.
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Nematodes are important members of the terrestrial mesofauna, with a crucial role as
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grazers of microorganisms;29 C. elegans have also been widely used as a model in
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aquatic/soil eco-toxicity testing of metals and NPs.29-31
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We hypothesized that, due to its higher affinity with negatively charged biological
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membranes, CeO2-NPs functionalized with a positively charged polymer would have
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increased toxicity and bioaccumulation in C. elegans compared to those functionalized
98
with neutral or negatively charged polymers. We also hypothesized that toxicity of
99
CeO2-NPs in C. elegans will be associated with a reduction of Ce valence of IV to III.
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Finally, we hypothesized that the addition of NOM to positively charged CeO2-NPs
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would decrease CeO2-NP accumulation and toxicity in C. elegans by conferring a net
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negative surface charge over a wide pH range. We tested these hypotheses by exposing
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C. elegans to CeO2-NPs of varying charge with or without NOM and measuring the
104
effects on subsequent bioavailability and toxicity.
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MATERIALS AND METHODS
107 108
Nanoparticle synthesis and characterization
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Cerium dioxide NPs with 3 different stabilizing agents were investigated in this
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study. Dextran coated CeO2-NPs with a nominal 4 nm primary particle diameter (DEX-
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CeO2(0)) were synthesized using a modification of the procedure described by Perez et
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al.18 This coating was then functionalized with diethylaminoethyl groups to confer either
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a net positive charge (diethylaminoethyl dextran; DEAE-CeO2(+)), or carboxymethyl
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groups to confer a net negative charge (carboxymethyl dextran; CM-CeO2(-)). Details of
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the synthesis and functionalization of the particles can be found in the supplemental
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information (SI). Primary particle diameter was characterized by depositing the particles
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on carbon coated copper grids and examining with transmission electron microscopy
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(TEM; Jeol 2010F, Tokyo, Japan). Freeze-dried NPs and coatings were analyzed for
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functionalization on a Thermo-Fisher Nicolet 6700 Fourier transform infrared
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spectrometer. Electrophoretic mobility titrations were performed on each type of coated
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CeO2-NP to gain insight into the intrinsic surface charge behavior imparted by the
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coatings as a function of pH in the exposure medium, which was moderately hard
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reconstituted water32 (MHRW) (Figure S4). Mean hydrodynamic diameters and
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electrophoretic mobilities of the NP treatment suspensions were measured using a Nano-
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ZS zetasizer (Malvern Instruments, Malvern, United Kindgom), at a suspension
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concentration of 100 mg Ce L-1 CeO2-NPs with and without 35 mg L-1 humic acid (HA),
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and 25 mg L-1 DEAE-CeO2(+) in the presence of 0, 7, 35, 70 mg HA L-1 (Figure S6, S7,
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S8). Suspensions were prepared using the CeO2-NP stock suspension and HA stock
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solution, adjusted at pH 8.1 and incubated at 25°C for 24h. The zeta potential was
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calculated using the Hückel approximation.
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Pahokee peat humic acid solution
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The soil humic acid Pahokee Peat (HA) obtained from the International Humic
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Substances Society (IHSS), was chosen as a soil HA model. HA was dissolved in 0.002N
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NaOH in DI (pH 7) at 500 mg HA L-1. The HA solution was stirred overnight and filtered
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(cellulose acetate, 0.2 µm). This stock solution was kept at 4°C. The total organic carbon
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measured in the stock solution, using a TOC-VCPN total organic carbon analyzer
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(Shimadzu Corporation), was 182.6 mg C L-1. According to the IHSS Pahokee peat HA is
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52.12% C, therefore the actual concentration of the stock solution was 350 mg HA L-1.
140 141
Caenorhabditis elegans exposure
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Caenorhabditis elegans (wild type, Bristol strain N2) were obtained from the
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Caenorhabditis Genetics Center (Minneapolis, MN). The nematode culture maintenance,
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generation of age-synchronized worms and toxicity testing followed previously
145
established protocols,33 except the use of MHRW as exposure medium. MHRW was
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chosen in order to provide a chemical environment close to that found in soil pore water
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and for some ecological relevance. Mortality was measured after a 48 hours of exposure
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at the L1 and L3 developmental stages. The exposure concentrations of DEAE-CeO2(+)
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for L1 nematodes ranged from 0 to 100 mg Ce L-1 (0, 1, 5, 10, 25, 50, 75, and 100), while
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the exposure concentrations of the same NPs used for L3 animals ranged from 0 to 1000
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mg Ce L-1 (0, 1, 5, 25, 100, 500, and 1000). A similar range of concentrations was also
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used for L1 nematodes exposed to DEX-CeO2(0) and CM-CeO2(-) (0, 100, 500, and 1000
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mg Ce L-1). All exposures for every concentration tested were conducted with and
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without 7 mg HA L-1. As a control, the DEAE-DEX coating alone was tested at a
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concentration of 50 mg C L-1, which would correspond to the concentration of C in a
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suspension of 200 mg Ce L-1 DEAE-CeO2(+). Escherichia coli (strain OP50) was
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supplied as a food source (10 µg ml-1, OD600 of 0.018) at the beginning and after 24h of
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the exposure. All tests were conducted in 24-well polycarbonate tissue culture plates.
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Three replicates were performed for each test. A 1.0 ml aliquot of test solution was added
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to each well, which was subsequently loaded with 10 (±1) nematodes. The plates were
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observed under a stereomicroscope and the nematodes were counted and scored.
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Following an established protocol, nematodes that did not move in response to a gentle
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prod by a Pt wire were counted as dead.34
164 165
Synchrotron X-ray analysis
166
In order to test the effect of CeO2-NPs surface charge and the presence of HA on
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Ce bioaccumulation, C. elegans at the L3 stage were exposed to 100 mg Ce L-1 of DEX-
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CeO2(0), CM-CeO2(-) and DEAE-CeO2(+) with and without 35 mg HA L-1 for 24h
169
without feeding. The presence of food (E. coli) in the media can affect NPs behavior and
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oxidation state.12 We tested the effect of feeding on Ce bioaccumulation and speciation in
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C. elegans exposed to DEAE-CeO2(+) for 24h with food (E. coli strain OP50). To further
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test the effects of HA on Ce accumulation and speciation, L3 stage C. elegans were
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exposed to 25 mg Ce L-1 DEAE-CeO2(+) with 7, 35 and 70 mg HA L-1 for 48h. We
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selected Ce concentrations that were below LC10 to ensure the nematode’s survival
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during bioaccumulation experiment. After exposure, live nematodes were removed and
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preserved in 20% ethanol for up to a few days before X-ray analysis. The fresh samples
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were placed on metal-free polyimide film (Kapton; DuPont, Wilmington, DE) and
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immediately mounted in the X-ray beam. Several solutions were also prepared to analyze
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Ce speciation: NPs stock suspension, NPs in the MHRW, NPs after a 24h exposure, NPs
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with HA, and NPs with food (see Figure S10). Mixtures of 500 mg Ce L-1 CeO2-NPs and
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500 mg L-1 of ascorbic acid, tannic acid, albumin, glutathione, cysteine, collagen and
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Na2SO4 (potential reductants) adjusted to pH 7 were also analyzed (Figure S11). These
183
suspensions were analyzed in a cell constructed from polyimide tubing.
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Scanning X-ray fluorescence microscopic measurements of Ce were collected at
185
the Ce Lα1 emission line (5750 eV) using beamline X-26A at the National Synchrotron
186
Light Source at Brookhaven National Laboratory (Upton, NY). We used Ce LIII-edge X-
187
ray absorption near-edge spectroscopy (XANES) to determine the oxidation state of Ce in
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CeO2-NPs suspensions and on selected locations in C. elegans samples. Athena software
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was used to process the normalization and liner combination fitting (LCF) of the XANES
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spectra.35 C. elegans samples were fitted with 2 standards: Ce(III)sulfate as a Ce(III)
191
standard and with the spectra of the NPs stock solution that they were exposed to, either
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DEX-CeO2(0), CM-CeO2(-), or DEAE-CeO2(+), as a Ce(IV) standard. For every
193
treatment, the proportions of Ce(III) and Ce(IV) obtained by LCF for each individual
194
spectrum (Table S2) were averaged. Details of the analytical methods are provided in the
195
SI.
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Statistical analyses
198
The SPSS statistic software package was used for statistical analyses. The data
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were tested for normality using the Shapiro-Wilk test and found to be non-normally
200
distributed; therefore, we used non-parametric statistics. We tested for significant effects
201
of treatments on Ce intensity, Ce speciation (proportion of Ce(III) or Ce(IV)) in C.
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elegans, and mortality using Kruskal-Wallis 1-way ANOVA followed by pairwise
203
comparison using the Mann-Whitney U-test. Differences of p 2 µm)
397
(Figure S6) may have settled onto the bottom of the well plates, locally increasing the Ce
398
exposure concentration to C. elegans, which typically dwell near the bottom of the wells.
399
The addition of E. coli did not modify the Ce accumulated by C. elegans exposed to
400
DEAE-CeO2(+) without HA but decreased the Ce accumulated by C. elegans exposed to
401
HA and DEAE-CeO2(+) compared to the same treatment without food (Figure 2). This
402
may indicate that in the absence of food, the nematodes were filter feeding on the CeO2
403
and HA aggregates, which led to a higher rate of ingestion and subsequent assimilation.
404
The Ce oxidation state in animals exposed to 100 mg Ce L-1 DEAE-CeO2(+) was not
405
significantly affected by the presence of HA or food, with a high proportion of Ce
406
measured as Ce(IV): from 73 to 82 % (Figure 3).
407
Addition of 7 mg HA L-1 to 25 mg Ce L-1 DEAE-CeO2(+) significantly increased
408
Ce intensity measured in C. elegans from an average of 47.8 ± 15 to 77 ± 18 cps (Figure
409
4). The addition of 35 mg HA L-1 induced a wide variation of Ce intensity, varying
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between 15.5 cps and 247 cps, with an average at 139 ± 108 cps. The addition of 70 mg
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HA L-1 concentration significantly decreased the Ce accumulation compared to other
412
treatments, reaching 9.5 ± 4.5 cps. The higher Ce bioaccumulation with 7 mg HA L-1
413
compared to that without HA may be partly explained by the absorption of small
414
aggregates. At these HA and NPs concentrations, most of the aggregates had an average
415
size of 11.7 nm, but two larger size fractions were formed at 40 nm and >2 µm (Figure
416
S8). For the 35 mg HA L-1 treatment, the presence of µm size aggregates (Figure S8)
417
explained this important variability. As we can observe on the µ-XRF map of this
418
treatment (Figure 4), some aggregates can adhere to the nematode cuticle and drastically
419
influence the Ce intensity. The samples (2 out of 5) where no aggregates were observed
420
on the nematode had a lower Ce intensity than the control. This seems to indicate that the
421
actual absorbed concentrations were lower. Moreover, most of the aggregates were too
422
large to be absorbed by C. elegans. As a filter-feeder, its pharynx and buccal cavity
423
exclude larger particles than 3 µm in young adults.64 The large size of aggregates may
424
also partly explain the significant decrease of Ce accumulation in C. elegans in the 70 mg
425
HA L-1 treatment. But for smaller aggregates, the low accumulation may be mainly
426
explained by the change in ZP, which became negative.
427
These results showed that the presence of HA greatly influenced Ce accumulation
428
in C. elegans, increasing its accumulation for a high NP:HA ratio, and decreasing it for a
429
low NP:HA ratio. Therefore, while the toxicity was always decreased in the presence of
430
HA, Ce bioaccumulation is highly dependent on the ratio of NPs to HA. In all specimens
431
exposed to 25 mg Ce L-1 DEAE-CeO2(+), Ce was mainly present as Ce(IV), with a
432
proportion ranging from 19.3 % without HA to 38 % with 50 mg HA L-1 (Figure 3). The
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high Ce accumulation coupled with a low toxicity in C. elegans exposed to a high NP:HA
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ratio showed that the presence of HA reduced the inherent toxicity of DEAE-CeO2(+).
435
Several processes can explain how HA could have alleviated toxicity despite increased
436
bioaccumulation. The adsorption of HA at the surface of the NPs can form a physical
437
barrier to NP interaction with the cell membrane and also reduce binding of NPs to
438
important proteins and biomolecules.8, 65 In another study, aqueous organic matter may
439
have also mitigated nC60 toxicity by coating the NPs, hindering direct contact with the
440
cells and possibly altering nC60 surface chemistry through an undetermined
441
mechanism.66 It is possible that in our study, the partial HA adsorption was sufficient to
442
decrease the toxicity by reducing the reactivity between cells and CeO2-NP surfaces, but
443
not to reverse the zeta potential so it did not decrease the NP absorption. The HA may
444
also act as an antioxidant by reacting with ROS65, 67 which would mitigate the oxidative
445
stress of CeO2-NPs. These effects cannot be confirmed in this study as no significant
446
decrease in the amount of reduced Ce in C. elegans was measured with HA (Figure 3).
447
However, HA may have intercepted reactive oxygen species that were generated without
448
affecting the overall valence of the particles.
449 450
Environmental implications
451
This study showed that the surface chemistry of pristine CeO2-NPs, specifically
452
their charge, had a profound effect on their toxicity. The positively charged DEAE-
453
CeO2(+) were far more toxic to C. elegans and were bioaccumulated to a greater extent
454
than the neutral and negatively charged DEX-CeO2(0) and CM-CeO2(-). Surface charge
455
also influenced the oxidation state of CeO2-NPs once taken up by C. elegans. The
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addition of HA to the exposure medium decreased the toxicity of CeO2-NPs, and its
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concentration was shown to influence Ce bioaccumulation. For a higher ratio of HA to
458
NPs, which is a more relevant scenario in the environment, the presence of HA
459
significantly decreased Ce bioaccumulation. This work suggests that although initial
460
surface chemistry had a profound impact on toxicity, environmental transformations
461
(specifically coating with natural organic matter) reduced the effects of initial surface
462
chemistry, and rendered ultra-small (2-5 nm), positively charged CeO2-NPs significantly
463
less toxic for the endpoints tested at environmentally realistic ratios of HA to CeO2.
464 465
Acknowledgements
466 467
This research was supported by the United States Environmental Protection Agency (U.S.
468
EPA) through a Science to Achieve Results (STAR) grant (RD-83485701-0). J. Unrine
469
and O. Tsyusko were supported by the National Science Foundation (NSF) through
470
cooperative agreement EF-0830093 (Center for Environmental Implications of
471
Nanotechnology). It has not been formally reviewed by EPA or NSF. The views
472
expressed in this document are solely those of the authors. EPA and NSF do not endorse
473
any products or commercial services mentioned in this publication. Portions of this work
474
were performed at Beamline X26A, National Synchrotron Light Source (NSLS),
475
Brookhaven National Laboratory. X26A is supported by the Department of Energy
476
(DOE) - Geosciences (DE-FG02-92ER14244 to The University of Chicago - CARS). Use
477
of the NSLS was supported by DOE under Contract No. DE-AC02-98CH10886. The
478
authors gratefully acknowledge W. Rao, S. Wirick, D. McNear, J. Kupper, J. Nelson, P.
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Bertsch, A. Butterfield, U. Graham, S. Rathnayake and S. Shrestha.
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Supporting information
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Additional data on nanoparticle characterisation, µ-XANES analysis and C. elegans
483
toxicity. This material is available free of charge via the Internet at http://pubs.acs.org.
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FIGURE CAPTIONS
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Figure 1. Mortality of Caenorhabditis elegans exposed to positively charged
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diethylaminoethyl-dextran coated CeO2 nanoparticles (DEAE-CeO2(+)) at L1 (A)
489
and L3 (B) stages for 48 h with feeding. Diethylaminoethyl-dextran (DEAE)
490
coating alone was also tested. Live Escherichia coli, strain OP50 bacteria were
491
included as a nutrient source. Data are presented as the mean ± standard deviation
492
(SD) (n=3). Treatment labeled with the same letter did not differ significantly
493
(Kruskal–Wallis test followed by pairwise Mann–Whitney U-tests, p
