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Toxicity of Oxidatively Degraded Quantum Dots Paige Wiecinski, Kevin M. Metz, Tisha C King Heiden, Kacie Louis, Andrew Mangham, Robert J Hamers, Warren Heideman, Richard E. Peterson, and Joel A. Pedersen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es304987r • Publication Date (Web): 01 Jul 2013 Downloaded from http://pubs.acs.org on July 8, 2013
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Environmental Science & Technology
Toxicity of Oxidatively Degraded Quantum Dots Paige N. Wiecinski1,† Kevin M. Metz,2,‡ Tisha C. King Heiden,1,§ Kacie M. Louis,3,# Andrew N. Mangham,3 Robert J. Hamers,3 Warren Heideman,1,4 Richard E. Peterson,1,4 and Joel A. Pedersen*,1-3 1
Molecular and Environmental Toxicology Center, 2 Environmental Chemistry and Technology Program, 3 Department of Chemistry, 4 School of Pharmacy and ‖ Department of Soil Science, University of Wisconsin-Madison, Madison Wisconsin 53706 *
Corresponding author: tel: (608) 263-4971; fax: (608) 265-2595; e-mail:
[email protected]. † Current address: Lancaster Laboratories, Lancaster, PA ‡ Current address: Department of Chemistry, Albion College, Albion, MI § Current address: Department of Biology, University of Wisconsin – La Crosse, La Crosse, WI #
Current address: AkzoNobel, Brewster, NY
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Abstract. Once released into the environment, engineered nanoparticles (eNPs) are
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subjected to processes that may alter their physical or chemical properties, potentially
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altering their toxicity vis-à-vis the as-synthesized materials. We examined the toxicity to
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zebrafish embryos of CdSecore/ZnSshell quantum dots (QDs) before and after exposure to
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an in vitro chemical model designed to simulate oxidative weathering in soil
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environments based on a reductant-driven Fenton’s reaction. Exposure to these oxidative
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conditions resulted in severe degradation of the QDs: the Zn shell eroded, Cd2+ and
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selenium were released, and amorphous Se-containing aggregates were formed.
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Weathered QDs exhibited higher potency than did as-synthesized QDs. Morphological
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endpoints of toxicity included pericardial, ocular and yolk sac edema, non-depleted yolk,
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spinal curvature, tail malformations, and craniofacial malformations. To better
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understand the selenium-like toxicity observed in QD exposures, we examined the
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toxicity of selenite, selenate and amorphous selenium nanoparticles (SeNPs). Selenite
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exposures resulted in high mortality to embryos/larvae while selenate and SeNPs were
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non-toxic. Co-exposures to SeNPs + CdCl2 resulted in dramatic increase in mortality and
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recapitulated the morphological endpoints of toxicity observed with weathered QD
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exposures. Cadmium body burden was increased in larvae exposed to weathered QDs or
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SeNP + CdCl2 suggesting the increased potency of weathered QDs was due to selenium
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modulation of cadmium toxicity. Our findings highlight the need to examine the toxicity
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of eNPs after they have undergone environmental weathering processes.
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INTRODUCTION
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Increased production and use of engineered nanoparticles (eNPs) makes their
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release into the environment inevitable. Once released into the environment, eNPs may
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be subjected to processes that alter their physical and chemical properties in ways that
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affect their toxicity.1-4 These processes may be collectively termed “weathering”.
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Aggregation/agglomeration of eNPs is recognized as an important process affecting eNP
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fate and has consequences on their toxicity.5-7 Depending on eNP composition and that of
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intentional coatings, eNPs may also be altered by simple dissolution, redox
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transformations, photolysis, and the loss or acquisition of organic or inorganic coatings.6,
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8-15
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induced alterations to eNPs have the potential to influence their bioavailability, uptake,
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and toxicity.17
These transformations may impact eNP suspension stability.6,12,14,16 Environmentally
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Most current knowledge about eNP toxicity stems from research focused on
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intact, as-synthesized NPs.2,18 However, organisms are unlikely to be exposed to NPs in
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their as-synthesized forms.1 Environmental alterations of eNPs is expected to alter uptake
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and toxicity. For example, exposure of CdSe quantum dots (QDs) to acidic or alkaline
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conditions increases toxicity to bacteria due to release of Cd2+ and SeO32−.8 Dissolution
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of ZnO NPs (i.e., release of Zn2+) has been shown to explain NP toxicity to bacteria,6 but
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release of Zn2+ alone could not explain toxicity to Daphnia magna.19 As zero-valent iron
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NPs oxidize to magnetite (Fe3O4) and maghemite (Fe2O3), the antimicrobial capacity of
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the particles diminishes as the oxidation state of Fe increases.20 The same trend has been
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observed in rodent microglial cells, where more oxidized forms of iron induce lower
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levels of oxidative stress.21 As a further example, exposure to an aged TiO2
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nanocomposite (T-LiteTM) used in sunscreens has a small effect on developing zebrafish
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(Danio rerio).22 To our knowledge, aside from this latter example, effects of weathered
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eNPs on aquatic vertebrates have not been reported.
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We previously developed an in vitro chemical model based on the extracellular
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chemistry of lignolytic fungi23,24 to allow investigation of the oxidative weathering of
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eNPs under conditions representative of oxic soil environments.9 The model consists of a
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methoxyhydroquinone (MHQ)-promoted Fenton’s reaction leading to the production of
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hydroxyl radicals (H2O2 + Fe2+ → •OH + OH− + Fe3+) and other reactive oxygen species
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(ROS). Exposure of PEGylated CdSecore/ZnSshell quantum dots (QDs) to this model
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system caused dissolution of the QDs, releasing Cd2+ from the nanoparticle core and
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producing amorphous Se-containing aggregates.9 The toxicity of the as-synthesized
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CdSecore/ZnSshell QDs has been investigated,25 but that of the products of oxidative
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weathering has not yet been reported.
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The objectives of this study were to (1) evaluate changes in toxicity induced by
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oxidative weathering vis-à-vis as-synthesized QDs; and (2) provide insight into the
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causes of observed changes in toxicity. To accomplish these objectives we employed the
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zebrafish embryo as a model organism. The zebrafish embryo is a well-established and
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widely used vertebrate model in developmental toxicity,26,27 and has recently been shown
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to have utility in assessing the toxicity of eNPs.25,28-33 Oxidative weathering of QDs
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results in a complex mixture of products, complicating the determination of mechanisms
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of toxicity. We therefore adopted a reductionist approach and examined the toxicity to
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zebrafish embryos of Se nanoparticles, dissolved cadmium, dissolved selenium species,
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and their mixtures.
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MATERIALS AND METHODS
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Chemicals. The following chemicals were purchased from Sigma-Aldrich
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Chemicals (Milwaukee, WI): CdO (99.5%), trioctylphosphine oxide (TOPO, technical
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grade, 90%), trioctylphosphine (TOP, technical grade, 90%), zinc stearate (technical grade),
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sulfur powder (99.98%), selenium powder (100 mesh, >99.5%), tetramethylammonium
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hydroxide solution (TMAH, ~2.2 M) in methanol, ZnCl2 (>98%), and CdCl2 (99%). Ferrous
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sulfate (ACS certified), hydrazine (99%), selenous acid (98%), sodium selenate (99%),
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sodium selenite (99%), and selenium tetrachloride (>98%) were obtained from Fisher
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Scientific (Waltham, MA). We procured 2,5-dimethoxyhydroquinone (97%) from Alfa Aesar
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(Ward Hill, MA) and PEG5000-OCH3 (>96%) from RAPP polymer (Germany).
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Nanoparticle Syntheses. Synthesis of the CdSecore/ZnSshell QDs was described
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previously.9,25 The CdSe core diameter was 2.6 ± 0.1 nm; the ZnS shell thickness did not
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exceed 1 nm.9 Coordinating ligands from the synthesis were exchanged with 5000-Da
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PEG-thiol (PEG5000) in chloroform. After functionalization, the solvent was evaporated,
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and QDs were suspended in ultrapure water (18 MΩ-cm resistivity, Barnstead
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NANOpure Ultrapure Water System, Dubuque, IA, USA). In the exposure medium (vide
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infra) the hydrodynamic diameter and ζ-potential of the PEG5000-QDs were 21 ± 1 nm
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and –2.0 ± 0.6 mV.25 After 24 h in exposure medium dissolved Cd concentrations were ≤
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28 µM.25 The characterization of the QDs used in this study are detailed elsewhere.9,25
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Amorphous SeNPs were synthesized via hydrazine (N2H4) reduction of selenous
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acid (H2SeO3) in ultrapure water similar to published methods.34 To keep the SeNP
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solutions similar to the QD solutions, we used PEG-thiol as the coordinating ligand rather
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than bovine serum albumin. Briefly, a 0.35 M N2H4 solution (5 mL) was reacted in 20
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mL ultrapure water at room temperature for 10 min. To this solution, 10 mL 0.07 M
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H2SeO3 and excess PEG5000-thiol was added. The solution was stirred for 2 h then stored
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at 4 °C.
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Oxidative Weathering of CdSecore/ZnSshell QDs. PEGylated QDs were
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subjected to the MHQ-driven Fenton’s reaction.9 Solutions containing 2 µM QDs (7 ×
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1015 QD·L-1), 200 µM H2O2, 20 µM Fe2+ and 20 µM MHQ in 10 mM acetate buffer (pH
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4.1) were allowed to react overnight (16-18 h) in the dark. The reaction was quenched by
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adjusting pH to 6 with NaOH. Oxidative weathering of the PEG5000-CdSecore/ZnSshell QDs
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by the MHQ-promoted Fenton’s reaction resulted in the erosion of the ZnS shell, release
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of Cd from the core, and formation of amorphous selenium aggregates ~10s to 100s of
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nm in diameter (as determined by transmission electron microscopy, selected area
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electron diffraction, and energy dispersive X-ray spectroscopy).9 These findings are
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reported in detail elsewhere.9 Amounts of dissolved and (nano)particulate Cd, Zn, and Se
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present after weathering are provided in Table S1 in the Supporting Information. We
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operationally defined dissolved and (nano)particulate species as those passing and
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retained by a 10 kDa MWCO filter (pore size ~2.8 nm).9,25 Solutions were diluted to the
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desired QD concentrations for zebrafish experiments in embryo medium immediately
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before dosing (see below for composition).
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Scanning Electron Microscopy (SEM). SeNPs were spin-cast onto doped
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silicon wafers for imaging by a Leo Supra55 VP SEM. Data were obtained using 2 kV
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incident electron energy using the standard in-lens detector.
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Ultraviolet-visible (UV-Vis) Spectroscopy. UV-Vis spectra of NP
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suspensions were obtained using a Shimadzu PC-2401 UV-Visible spectrophotometer.
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The core diameter and number concentration of as-synthesized QDs was estimated from
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the position and absorbance of the first exciton peak centered between 525 and 530
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nm.9,35 While the accuracy of UV-visible absorbance measurements have received
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criticism,36 such estimates are considered acceptable for comparative analyses.
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Concentrations of SeNPs were determined by matching spectra from 450-600 nm to UV-
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Vis spectra obtained from MHQ-driven Fenton’s reaction exposed QDs.9
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Raman Spectroscopy. Raman spectra were recorded on a Thermo Scientific
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DXR Raman microscope using a 780 nm excitation from a diode-pumped, solid-state
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laser. Scans (32) were accumulated at 3 cm-1 resolution using 0.1-1 mW laser power.
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Care was taken to not alter chemical compounds due to laser power. Samples were first
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run at low power to assess any changes from interaction with the laser prior to increasing
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power to produce spectra with higher S/N ratio.
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Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Digests of
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zebrafish larvae (vide infra) were analyzed by magnetic-sector ICP-MS (Thermo
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Finnigan Element 2) at the Wisconsin State Lab of Hygiene to determine Cd and Se body
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burden. Unexposed control larvae had Cd and Se concentrations of 0.0055 ± 0.0004 µg·g-
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1
and 0.14 ± 0.01 µg·g-1, respectively.
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Zebrafish Exposures. Beginning 4-6 hours post fertilization (hpf) zebrafish
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embryos (AB strain) were exposed to graded concentrations (corresponding to 0.2 to 200
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µM Cd) of as-synthesized or weathered QDs suspended in embryo medium (58 mM
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CaCl2, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 0.5 mM HEPES, pH 7; ionic
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strength (I) = 0.18 M). For comparison, we also exposed fish to CdCl2, Na2SeO3,
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Na2SeO4, SeNPs and PEG5000-thiol. Exposures were conducted in a 96-well plate format
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with a single embryo per well. Dosing solutions (100 µL) were renewed daily, and
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zebrafish embryos/larvae were monitored for mortality and morphological signs of
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toxicity until 120 hpf. Experiments were conducted in triplicate, and data are presented as
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the mean ± SE.
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Morphological endpoints of toxicity were evaluated at 120 hpf following
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exposure to QD concentrations equivalent to 20 µM Cd, a concentration that produced
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consistent morphological endpoints for both weathered and as-synthesized QDs. Larvae
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(n = 12) were immobilized in 3% methylcellulose and imaged live (2.5×) in lateral
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orientation. Images were analyzed for total body length, yolk sac edema and pericardial
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edema.
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To determine cadmium and selenium body burdens, larvae (15-20 per replicate)
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were collected at 120 hpf after exposure to 20 µM Cd or Se equivalents as-synthesized
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QDs, weathered QDs, or controls (viz. embryo media, CdCl2, Na2SeO3, SeNP or SeNP +
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CdCl2), euthanized with 3-aminobenzoic acid ethylester, rinsed 3× and transferred to pre-
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weighed microcentrifuge tubes. Wet weights were measured following removal of excess
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water, and larvae were digested overnight in 5 M HNO3 at 60 °C. Cadmium and selenium
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concentrations were determined by ICP-MS (vide supra).
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Statistical Analyses. Statistical significance of differences between treatments
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and control was determined using Student’s t-test. Mortality data at 120 hpf were used to
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calculate median lethal concentrations (LC50 values) and 95% confidence intervals
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(CI95%) by probit analysis (USEPA Probit Analysis Program, Ver 1.5). Median effects
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concentrations (EC50 values) were determined for selected morphological endpoints of
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toxicity from incidence data using the trimmed Spearman-Karber method (USEPA
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Trimmed Spearman-Karber Analysis Program, Ver 1.5). LC50 and EC50 values were
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compared among treatments for statistical significance by two-way analysis of variance
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(ANOVA). The level of significance for all analyses was p ≤ 0.05.
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RESULTS AND DISCUSSION
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Toxicity of Oxidatively Weathered CdSecore/ZnSshell QDs. We obtained
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dose-response relationships for mortality induced by exposure to as-synthesized and
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oxidatively weathered PEG5000-CdSecore/ZnSshell QDs (Figure 1a). The LC50 for the as-
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synthesized PEG5000 QDs was 42 (CI95%: 24-75) µM Cd equivalents as we previously
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reported.25 Weathering produced a leftward shift in dose-response curves (Figure 1a) and
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a decline in LC50 value to 14 (CI95%: 9-21) µM Cd equivalents. The mortality induced by
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both the as-synthesized and oxidatively weathered QDs was higher than that produced by
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an equivalent concentration of CdCl2 (Figure 1a). The higher toxicity of intact QDs
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relative to CdCl2 is consonant with our previous findings.25
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The types of morphological endpoints of toxicity observed did not differ between
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as-synthesized and weathered QDs (Figure 1b). Morphological endpoints included
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pericardial, ocular and yolk sac edema (i.e., swelling due to fluid retention), low
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absorption of the yolk, spinal curvature, tail malformation, opaque tissues (particularly in
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the head region), craniofacial malformation and failure to inflate the swim bladder. Some
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of these endpoints fit the profile of Cd toxicity (i.e., spinal curvature, pericardial edema,
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ocular edema),37,38 while others correspond to those associated with Se toxicity (i.e.,
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pericardial edema, yolk sac edema, low absorption of the yolk).39
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Weathered
PEGylated
QDs
displayed
increased
potency for
inducing
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morphological endpoints of toxicity relative to as-synthesized QDs. That is, weathered
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QDs produced morphological malformations both at lower concentrations and in more
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larvae than did as-synthesized QDs. Exposure to weathered PEG5000-QDs at a
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concentration of 20 µM Cd equivalents (i.e., 6.7 × 1016 QD·L-1) significantly increased
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the incidences of pericardial edema, yolk sac malformations and craniofacial
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malformations relative to the as-synthesized QDs (Figure 2). Increases in incidence of
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pericardial edema and craniofacial malformations were also observed upon exposure to 2
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µM Cd equivalents weathered vs. as-synthesized QDs (data not shown). We calculated
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EC50 values for pericardial edema, yolk sac malformations, and craniofacial
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malformations for as-synthesized and weathered QDs. For as-synthesized PEG5000-QDs,
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EC50 values for pericardial edema, yolk sac malformations and craniofacial
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malformations were 18 (CI95%: 12-28), 9 (CI95%: 6-12) and 9 (CI95%: 5-14) µM Cd
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equivalents; for weathered PEG5000-QDs, the EC50 values for these responses decreased
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to 8 (CI95%: 6-11), 4 (CI95%: 3-5) and 3 (CI95%: 2-4) µM Cd equivalents, respectively.
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We quantified three morphological endpoints of toxicity following exposure to 20
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µM Cd equivalents as-synthesized or weathered QDs: total body length, pericardial
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edema and yolk sac edema (Figure 3). Total body length and pericardial edema differed
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between as-synthesized and weathered PEG5000-QDs (p ≤ 0.05; Figure 3 a,c), while yolk
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sac malformations (i.e., edema, lack of adsorption) did not (p > 0.05; Figure 3b). These
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data indicate that exposure to the weathered QDs produced more severe effects for some
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morphological endpoints further demonstrating that weathered QDs were more toxic than
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their as-synthesized counterparts. Toxicity of Dissolved Inorganic Selenium Species. Few prior studies
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have
reported
the
toxicity
of
inorganic
selenium
compounds
to
zebrafish
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embryos/larvae.40,41 Furthermore, the morphological endpoints of toxicity caused by
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selenium compounds have not been described. Selenium was expected to contribute to
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the toxicity of oxidatively weathered CdSecore/ZnSshell QDs due to its oxidation to soluble
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species during the weathering process. We therefore investigated the toxicity to zebrafish
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embryos/larvae of three dissolved inorganic selenium compounds (i.e., sodium selenate,
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sodium selenite and selenium tetrachloride).
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Dose-response relationships were obtained for Na2SeO4, Na2SeO3 and SeCl4 at
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120 hpf (Figure 4a). Sodium salts of selenium anions are water-soluble. Selenium
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tetrachloride decomposes in water to yield selenous acids (pH was adjusted to 7 in
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exposure solutions with NaOH). The most potent selenium compound tested was SeO32−
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(LC50 = 5.1 (CI95%: 3.7-7.1) µM Se) followed by SeCl4 (LC50 = 240 (CI95%: 193-280) µM
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Se). No mortality above baseline was observed following exposure to Na2SeO4 over the
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concentration range tested (0-200 µM Se). Prevalent morphological effects observed after
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exposure of zebrafish embryos/larvae included yolk sac edema, failure to adsorb the yolk,
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yolk discoloration, and pericardial edema. These endpoints of toxicity were produced at
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much lower concentrations of SeO32− than SeCl4 (Figure 4b). Morphological endpoints of
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toxicity produced by exposure to SeO32− were similar to those described for other fishes
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exposed to this selenium oxyanion.39 These data suggest that dissolved Se species may
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play a role in the toxicity. However, dissolved Se cannot account for the total toxicity of
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weathered QDs because, at a given dose, only a fraction (~76%) of Se was dissolved
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(Table S1). Additionally, larvae exposed to inorganic selenium species do not show all
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endpoints of toxicity of those exposed to weathered QDs.
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Cadmium and Selenium Body Burdens. We measured Cd and Se body
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burden by ICP-MS in larvae exposed to 20 µM Cd equivalents of as-synthesized or
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weathered QDs. For comparison, we measured body burden in larvae exposed to CdCl2,
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Na2SeO3, SeNPs and SeNP + CdCl2. Cadmium body burden in exposed larvae increased
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in the following order: as-synthesized QDs < weathered QDs < CdCl2 < SeNPs + CdCl2
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treatment groups (Figure 5a). The difference in Cd body burden between larvae exposed
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to weathered and as-synthesized QDs was small but significant (p = 0.05). Cadmium
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body burden was higher in larvae co-exposed to SeNPs + CdCl2 than in those exposed to
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CdCl2 alone (Figure 5a). Selenium body burden in exposed larvae increased in the
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following order: weathered QDs < as-synthesized QDs < SeNPs + CdCl2 < SeNPs
