Relative Contributions of Copper Oxide Nanoparticles and Dissolved

Jan 12, 2017 - The toxicity of soluble metal-based nanomaterials may be due to the uptake of metals in both dissolved and nanoparticulate forms, but t...
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Relative Contributions of Copper Oxide Nanoparticles and Dissolved Copper to Cu Uptake Kinetics of Gulf Killifish (Fundulus grandis) Embryos Chuanjia Jiang, Benjamin T. Castellon, Cole W. Matson, George R. Aiken, and Heileen Hsu-Kim Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04672 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017

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Relative Contributions of Copper Oxide Nanoparticles and Dissolved Copper to Cu Uptake Kinetics of Gulf Killifish (Fundulus grandis) Embryos Chuanjia Jiang 1,2†, Benjamin T. Castellon 2,3, Cole W. Matson 2,3, George R. Aiken 4, Heileen Hsu-Kim 1,2*

1

Department of Civil and Environmental Engineering, Duke University, Durham, NC 27708

2

Center for the Environmental Implications of NanoTechnology (CEINT), Duke University,

Durham, NC 27708 3

Department of Environmental Science, Institute of Biomedical Studies, Center for Reservoir

and Aquatic Systems Research (CRASR), Baylor University, Waco, TX 76798 4

U.S. Geological Survey, Boulder, CO 80303

*Corresponding author: Heileen Hsu-Kim. Email: [email protected]. Phone +1-919-660-5109



Present address: State Key Laboratory of Advanced Technology for Materials Synthesis and

Processing, Wuhan University of Technology, Luoshi Road 122, Wuhan 430070, China

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Abstract: The toxicity of soluble metal-based nanomaterials may be due to the uptake of metals

2

in both dissolved and nanoparticulate forms, but the relative contributions of these different

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forms to overall metal uptake rates under environmental conditions are not quantitatively defined.

4

Here we investigated the linkage between the dissolution rates of copper(II) oxide (CuO)

5

nanoparticles (NPs) and their bioavailability to Gulf killifish (Fundulus grandis) embryos, with

6

the aim of quantitatively delineating the relative contributions of nanoparticulate and dissolved

7

species for Cu uptake. Gulf killifish embryos were exposed to dissolved Cu and CuO NP

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mixtures comprising a range of pH values (6.3–7.5) and three types of natural organic matter

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(NOM) isolates at various concentrations (0.1–10 mg-C L−1), resulting in a wide range of CuO

10

NP dissolution rates that subsequently influenced Cu uptake. First-order dissolution rate

11

constants of CuO NPs increased with increasing NOM concentration and for NOM isolates with

12

higher aromaticity, as indicated by specific ultraviolet absorbance (SUVA), while Cu uptake rate

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constants of both dissolved Cu and CuO NP decreased with NOM concentration and aromaticity.

14

As a result, the relative contribution of dissolved Cu and nanoparticulate CuO species for the

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overall Cu uptake rate was insensitive to NOM type or concentration but largely determined by

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the percentage of CuO that dissolved. These findings highlight SUVA and aromaticity as key

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NOM properties affecting the dissolution kinetics and bioavailability of soluble metal-based

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nanomaterials in organic-rich waters. These properties could be used in the incorporation of

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dissolution kinetics into predictive models for environmental risks of nanomaterials.

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Graphic for the Table of Contents

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INTRODUCTION

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With the rapid development of nanotechnology in the last few decades, there have been

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increasing concerns over the implications of engineered nanomaterials to human health and the

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environment.1-6 To elicit toxicity in an organism, nanomaterials and associated toxic species

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must first be taken up by the organism or approach its immediate vicinity.7 Therefore,

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quantifying the bioavailability of nanomaterials and constituents that may be released from

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nanomaterials is a key step in explaining the toxic effects of nanomaterials in various

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environmental and physiological media.

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After release into the environment, nanomaterials can undergo a range of processes that

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alter their bioavailability and toxicity. These processes include homoaggregation and

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heteroaggregation, dissolution and release of ionic species, surface modifications, and

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transformation into materials with a different chemical composition (e.g., sulfidation).8-14 The

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relative rates of these concurrent processes, and not necessarily their reaction potential at

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thermodynamic equilibrium, will control mobilization of nanomaterials in the aquatic

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environment and exposure to target organisms. For soluble metal-based nanomaterials such as

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silver (Ag), zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs), both nanoparticulate

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and dissolved forms of the metals can be taken up by the organisms and typically at different

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rates.15-19 Moreover, the dissolution and bioavailability of metal-based nanomaterials can be

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strongly influenced by the physico-chemical properties of the media, notably pH,11, 20 ionic

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strength/salinity,21, 22 hardness,23 and inorganic and organic ligands (e.g. chloride, amino acids,

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extracellular polymeric substances and humic substances).11, 12, 24-30 Due to these influencing

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factors as well as differences in nanomaterial properties and test organisms, there have been

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apparently inconsistent reports where, in some studies, the undissolved nanomaterials are

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bioavailable and responsible for the observed toxicity,31-33 while in other cases, the toxic effects

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could be attributed solely to the uptake of dissolved metal ions.34-36 These apparent

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inconsistencies highlight a major gap in the field of nanoecotoxicology, in which the relative

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contributions of nanoparticles and dissolved ions released from the NPs have not been

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effectively determined. In this respect, the relative uptake rates of NPs and corresponding

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dissolved metal ions need to be quantified under environmentally relevant conditions.

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While the bioavailability of metal ions has been extensively studied,37, 38 efforts have

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recently been made to quantify uptake kinetics of NPs by experimentation (e.g. radioactive and

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stable isotope labeling)22, 39 and modeling (e.g. biodynamic models).15, 17, 22, 40 To increase the

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accuracy of measured biodynamic parameters of soluble metal-based NPs, it is desirable to

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account for the dissolution of the NPs prior to41 or during uptake experiments,22, 39 using

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measured or assumed dissolved metal concentrations.22, 39 Moreover, other constituents of the

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aquatic environment such as dissolved natural organic matter (NOM) are known to influence

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nanoparticle surface chemistry and bioavailability in complex ways, e.g. through adsorption of

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NOM to particle surfaces, change in colloidal aggregation rates, enhanced dissolution of

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nanoparticles, and metal-ligand complexation of metal ions released from the nanoparticles.42

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These processes have been extensively studied, but rarely in combination and generally without

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quantification of reaction rates that are needed to delineate the relative importance of

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nanoparticles and dissolved metals for organismal exposures.

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This study aimed to quantify the relative rates of nanoparticle and dissolved metal uptake

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and the importance of NOM type and concentration for controlling this balance. Herein, we

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evaluated the copper uptake kinetics in Gulf killifish (Fundulus grandis) embryos exposed to

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CuO NPs in diluted artificial seawater (ASW). CuO NPs were selected as a model soluble metal-

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based nanomaterial, for their moderate solubility, which allows for relatively low exposure doses,

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as well as the wide application of CuO NPs and other Cu-based nanomaterials in biocidal agents

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and coatings.11, 12, 43, 44 While zebrafish and their embryos have been widely used as model

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organisms in nanotoxicological studies,12, 18, 25, 34 Gulf killifish and its closely related sister

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species Atlantic killifish (Fundulus heteroclitus) are widely distributed in estuarine habitats on

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the eastern coasts of the United States. Because of their wide ecological distribution as well as

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their tolerance of salinity and other environmental gradients, these species are commonly used as

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model teleosts in ecotoxicological and, most recently, nanotoxicological studies.21, 45, 46

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MATERIALS AND METHODS

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Overall Experimental Design. The addition of CuO NPs into the test media results in a

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mixture of NPs and dissolved Cu, the concentration ratio of which changes with time. Thus, in

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addition to CuO NP uptake experiments, separate positive control experiments with dissolved Cu

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were performed in order to quantify uptake rates of dissolved Cu. Copper exposure experiments

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were performed with three types of NOM and a range of NOM concentrations and pH values

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(Table 1) that were selected to produce a wide spectrum of nanoparticle aggregation and

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dissolution rates. Dissolution kinetics of CuO NPs were determined at high time resolution using

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anodic stripping voltammetry (ASV),47, 48 under identical conditions as the uptake experiments.

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Collectively these data were used to differentiate uptake of dissolved Cu and nanoparticulate

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CuO in the CuO NP exposures, in order to determine the relative contributions of dissolved and

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nanoparticulate species to Cu uptake kinetics across a wide range of water chemistry conditions.

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Materials and Test Organism. Purity and sources of chemicals are provided in Table S1

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in Supporting Information (SI). CuO NPs (nominally 25 min prior to use, resulting in Z-average hydrodynamic diameter

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of 250±30 nm via dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS).

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Three aquatic NOM isolates were used in the study, including Suwannee River humic

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and fulvic acids (SRHA, 2S101H; SRFA, 2S101F) from the International Humic Substances

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Society and Williams Lake hydrophobic acid (WLHpoA). These three NOM isolates were

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selected because our previous work has shown a wide range of NOM-enhanced dissolution rates

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for ZnO NPs.48 These NOM isolates also span the typical range of specific UV absorbance at

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280 nm (SUVA280), a parameter that is positively correlated with dissolution rates:

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4.92±0.34 L mg-C−1 m−1 for SRHA, 3.34±0.22 L mg-C−1 m−1 for SRFA and 1.22±0.11 L mg-C−1

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m−1 for WLHpoA.48 Dried powders of the NOM isolates were dissolved in ultrapure water, and

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the pH was adjusted to 6.5–7.5 with 0.1 N NaOH. The stocks were then filtered through 0.2 µm

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nylon-membrane syringe filters (VWR), and the concentration of the NOM stocks (typically 170

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to 400 mg-C L−1) was determined using a Total Organic Carbon (TOC) Analyzer (TOC-L CPN,

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Shimadzu).

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Embryos used in the study were produced by wild-type F. grandis populations collected

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from two reference sites on Galveston Bay, Texas: Smith Point (29°32'37.26"N, 94°47'08.12"W)

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and Gangs Bayou (29°15'30.34"N, 94°54'45.00"W). These populations were established and

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maintained as previously described.49 Briefly, the killifish were kept in recirculating 10‰ ASW

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(Instant Ocean) on a 14:10 h light cycle at 22–25 ºC and were fed twice daily (Purina AquaMax).

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After spawning, the fertilized embryos were disinfected with 0.3% hydrogen peroxide in 5‰

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ASW, rinsed twice, and incubated at 28°C in 5‰ ASW prior to use in uptake experiments at

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48±2 h post-fertilization (hpf). The embryos were on average 2.0–2.1 mm in diameter and 6 mg

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wet weight. Average dry weight of the embryos at 48 hpf was 0.85±0.04 mg, as measured after

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drying at 60 ºC overnight (> 12 h). The maintenance and handling of Gulf killifish were

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performed in compliance with the relevant laws and institutional guidelines and approved by the

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Baylor University Institutional Animal Care and Use Committee.

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CuO NP and Dissolved Copper Uptake Experiments. All uptake experiments were

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performed in dilute ASW (5‰ salinity) buffered to pH 6.3–7.5 with 2 mM MOPS and filtered

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through a 0.2-µm polycarbonate membrane. Experiments were performed with or without an

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NOM isolate at 0.1–10 mg-C L−1. In each experiment, buffered ASW and NOM stocks were

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pipetted into five 60-mL flat-bottom polypropylene tubes (SC480-W, Environmental Express),

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and then 4 viable embryos at 48±2 hpf were placed into each tube. Subsequently, CuO NP stock

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or dissolved Cu(NO3)2 solution (1.0 or 0.1 mM) was added, achieving a total volume of 40 mL in

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each tube. The suspensions were mixed end over end twice and incubated at 27–28 ºC in the dark

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for up to 48 h (IsotempTM, Model 6841, Fisher Scientific). Total nominal Cu concentration in the

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samples was 30 µM for CuO NP exposure and 1.0 or 0.4 µM (for experiments at pH 7.5) for

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dissolved Cu exposure. A blank control sample was prepared in the same way as described above

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except that no Cu was added.

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Each copper test mixture (dissolved Cu or CuO NP; with or without NOM) was

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performed in duplicate, with a few exceptions in triplicate. Copper content in the embryos was

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measured at different times post dosing (ranging from 2 h to 48 h). At each time point, aliquots

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of the exposure media were taken from above the embryos for measurements of total Cu

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concentration in the media (i.e. [Cu]T) by ICP-MS and, for CuO NP exposure, hydrodynamic

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diameter (dh) and zeta potential (ζ-potential) of the CuO NPs (Malvern Zetasizer Nano ZS). Then

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the 4 embryos in the sample were removed and rinsed in 5 mL of 5‰ ASW for 2 min,

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transferred to a clean 60-mL polypropylene tube (SC480-W, Environmental Express) and stored

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at −20 ºC. In some experiments, the embryos were dechorionated mechanically after exposure,21

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and the removed chorions (i.e., the acellular outer membrane enveloping the embryo) were

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collected in a 60-mL polypropylene tube. At the 48-h time point (i.e. 96±2 hpf), the embryos

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during rinsing were examined under a light microscope (AmScope, SE305R-PZ) to ensure that

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the embryos were still viable, as indicated by cardiac rhythm. The embryos or removed chorions were digested in 2 mL of concentrated nitric acid (~70%

156 157

as HNO3) at 90 ºC for 3 h on a hot block (Environmental Express, SC100), prior to the

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determination of Cu content in the embryos/chorions ([Cu]org, in unit of molCu per embryo;

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justification given in the SI) by ICP-MS. More details of the uptake experiment are provided in

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the SI.

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CuO NP Dissolution Experiments. The dissolution kinetics of CuO NPs were

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monitored over 48 to 72 h in duplicate or triplicate experiments, under the same conditions as the

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uptake experiments, but typically with no embryos in the samples. In each experiment, 10 or 11

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CuO NP samples (with 30 µM nominal total Cu) were prepared in 60-mL flat-bottom

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polypropylene tubes using the same method as the CuO NP uptake experiments, and total

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dissolved Cu concentration (i.e. [Cu]d) was measured by ASV at multiple time points (from 1 to

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72 h). A few experiments were performed with embryos to test if they affected the dissolution

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kinetics of CuO NPs. In addition to the water chemistry conditions in Table 1, CuO NP

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dissolution was also measured at additional pH values of 6.5, 6.8 and 7.2, with 1 mg-C L−1

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SRHA. The ASV measurement was performed on a Metrohm 663 VA Stand and Ecochemie

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µAutolab Type III potentiostat system,47, 48 with detailed procedures provided in the SI.

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Model of CuO NP Dissolution Kinetics. Dissolution kinetics of CuO NPs were modeled

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as surface-controlled dissolution, and the change in [Cu]d over time (t) was described using

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Equation 1:

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 d [Cu]d [Cu]d  = k d ([CuO]0 − [Cu]d )1 −  [Cu]  dt d,eq  

(1)

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where kd is the apparent first-order dissolution rate constant (in molCu molCuO−1 s−1) of CuO NP,

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[CuO]0 is the initial total concentration of CuO NP (i.e., 30 µM), and [Cu]d,eq is the dissolved Cu

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concentration measured at equilibrium (defined at t = 48 or 72 h). The rate constant kd was

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estimated by fitting Equation 1 to the measured dissolved Cu data for time points between 1 and

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24.2 h, where [Cu]d had not reached dissolution equilibrium. All data fitting was performed with

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Berkeley Madonna software v.8.3.18), using the Runge-Kutta (RK4) method with a step size of

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0.02 h. Initial dissolved Cu concentration in the reaction mixture (0.3–1.7 µM) was designated

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by the [Cu]d measured at t = 1 h or the average of [Cu]d measured at 1 and 2 h, if the former was 10 ACS Paragon Plus Environment

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higher than the latter. For dissolution experiments performed at pH 7.5 or with 0.1 mg-C L−1

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WLHpoA, the first 12 h of the dissolution curves were linearly fitted instead of using Equation 1

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(reasons given below in the subsection CuO NP Dissolution Rates).

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Model of Dissolved Cu and CuO NP Uptake Kinetics. The change in Cu content in

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Gulf killifish embryos ([Cu]org, in molCu per embryo) exposed to dissolved Cu and CuO NP over

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time was due to uptake/excretion of dissolved Cu ions and/or undissolved CuO NPs. This

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process was described by Equation 2 for organisms exposed to dissolved Cu and Equation 3 for

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organisms exposed to CuO NPs (which partly dissolved in the test matrices):

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d [Cu]org dt

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d [Cu]org dt

= k u,d [Cu]T − k e [Cu]org

(2)

= k u,NP ([Cu]T − [Cu]d ) + k u,d [Cu]d − k e' [Cu]org

(3)

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where ku,d and ku,NP are the uptake rate constants (L embryo−1 h−1) of dissolved Cu and CuO NPs,

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respectively, while ke and k’e are the elimination rate constants (h−1) of Cu in embryos exposed to

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dissolved Cu and CuO NPs, respectively. Herein, we did not distinguish between the elimination

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rates of dissolved Cu and nanoparticulate CuO, since their respective concentrations in the

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embryos could not be measured. Neither did we account for the possibility of biphasic metal

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elimination, which features fast and slow elimination phases.17

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Thus, the model was fit to the first 24 or 12 h of experimental data, when [Cu]org was

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relatively low and elimination of Cu from the embryos was assumed to be negligible. This

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assumption resulted in modifications of Equations 2 and 3 to the following:

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d [Cu]org dt

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t WLHpoA, as shown in Table 1) were more effective at

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promoting the dissolution of the CuO NPs, which is consistent with previous findings on NOM-

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promoted dissolution of bulk CuO50 as well as ZnO NPs.48 At a low concentration of 0.1 mg-C

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L−1, WLHpoA inhibited CuO NP dissolution within the first 12 h, which is consistent with

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decreased dissolution of ZnO NP (6.14 mM total concentration) by relatively low concentration

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(2.6 mg-C L−1) of SRFA.51

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It should be noted that all dissolution data were obtained by ASV. This approach for

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dissolved Cu ([Cu]d) measurement was validated in a subset of mixtures by ultracentrifugation of

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the CuO NP suspensions and quantification of Cu in the supernatant by ICP-MS. For example,

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[Cu]d values measured by ASV was 109% and 88% of the ICP-MS values, for CuO NP

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suspensions in buffered 5‰ ASW with no NOM and with 5 mg-C L−1 SRFA, respectively

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(Figure S3). The presence of 4 embryos in the CuO NP suspensions did not appear to affect the

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dissolution kinetics of CuO NPs, as shown in controls with buffered 5‰ ASW with and without

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1 mg-C L−1 SRHA (Figure S4) (These are conditions that could be expected to be most

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susceptible to dissolution changes caused by the embryos). It should also be noted that the

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starting dissolved Cu concentration (at time 0) could not be measured, due to protocol limitations.

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Fast initial dissolution was inferred from the measurements at 1 h, where the first measured [Cu]d

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could be a quarter of maximum [Cu]d under the same conditions (e.g. with 10 mg-C −1 SRHA).

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Such patterns, that is, faster initial dissolution followed by slower dissolution was also observed

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for CuO NPs in DI water or mesocosm freshwater, as measured by membrane dialysis.52

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Table 1. CuO NP dissolution rate constants and uptake rate constants for F. grandis embryos exposed to mixtures of dissolved Cu and CuO NPs under a variety of water chemistry conditions that included three types of natural organic matter (NOM) isolates with a spectrum of values for specific UV absorbance at 280 nm (SUVA280). Uptake rate Apparent CuO Uptake rate constant of dissolution rate constant of nanoparticulate NOM type and CuO constant dissolved copper No. pH (range) concentration [DOC]×SUVA280 kd ku,d ku,NP −1 −1 −1 −1 −1 (m ) (molCu molCuO s ) (L embryo h ) (L embryo−1 h−1) 1 7.0 (7.0–7.1) no NOM 0 0.71×10−6 64.6 (±3.7) ×10−6 3.9 (±1.5) ×10−6 2 7.0 (7.0–7.1) 0.1 mg-C L−1 WLHpoA 0.12 n/a 71.1 (±29.5) ×10−6 4.8 (±0.4) ×10−6 3 7.0 (7.0–7.1) 5 mg-C L−1 WLHpoA 6.1 0.65×10−6 43.2 (±4.6) ×10−6 2.0 (±0.1) ×10−6 4 6.3 (6.3–6.4) 1 mg-C L−1 SRHA 4.9 2.5×10−6 36.3 (±6.9) ×10−6 3.0 (±0.4) ×10−6 5 7.0 (7.0–7.1) 1 mg-C L−1 SRHA 4.9 0.56×10−6 30.1 (±2.1) ×10−6 1.4 (±0.5) ×10−6 6 7.5 (7.4–7.5) 1 mg-C L−1 SRHA 4.9 n/a 25.8 (±8.1) ×10−6 1.3 (±0.2) ×10−6 7 7.0 (7.0–7.1) 5 mg-C L−1 SRFA 16.7 1.1×10−6 24.3 (±4.0) ×10−6 1.2 (±0.3) ×10−6 8 7.0 (7.0–7.1) 5 mg-C L−1 SRHA 24.6 2.6×10−6 17.7 (±1.7) ×10−6 1.4 (±0.9) ×10−6 9 7.0 (7.0–7.1) 10 mg-C L−1 SRHA 49.2 2.2×10−6 11.2 (±0.6) ×10−6 0.8 (±0.3) ×10−6

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(a)

Dissolved Cu [Cu]d (µM)

5

4

1 mg-C L-1 SRHA pH 6.3, kd=2.5×10-6

3

pH 6.5, kd=1.8×10-6 pH 6.8, kd=1.1×10-6 pH 7.0, kd=5.6×10-7

2

pH 7.2, kd=5.7×10-7 pH 7.5, kd: n/a

1

-1

Unit of kd: molCu molCuO

s

-1

0 0

10

20

30

40

50

60

70

Time (h)

253

Dissolved Cu [Cu]d (µM)

5

(b)

4 10 mg-C L-1 SRHA 5 mg-C L-1 SRHA 5 mg-C L-1 SRFA 1 mg-C L-1 SRHA no NOM 5 mg-C L-1 WLHpoA 0.1 mg-C L-1 WLHpoA

3

2

1

pH = 7.0

0 0

254 255 256 257 258 259

10

20

30

40

50

60

70

Time (h)

Figure 1. Measured (symbols) and modeled (lines) dissolution of CuO NPs in 5‰ ASW (a) buffered to pH 6.3–7.5 in the presence of 1 mg-C L−1 SRHA, and (b) buffered at pH 7.0 in the presence or absence of different NOM isolates (SRHA, SRFA and WLHpoA) at 0.1–10 mg-C L−1. Ionic strength ~90 mM, T = 28 ºC. Experiments were performed in the dark. Error bars are standard deviation of replicate experiments (N = 2 or 3).

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Colloidal Stability of CuO NPs. In 5‰ ASW (~90 mM ionic strength) with no NOM or

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a very low NOM concentration (e.g. 0.1 mg-C L−1 WLHpoA), the CuO NPs quickly aggregated,

262

with Z-average hydrodynamic diameter (dh) reaching 2 µm within 12 h (Figure 2a). The addition

263

of higher SUVA280 NOM (e.g. SRHA at 1 mg-C L−1 and greater) or at higher concentration of

264

low SUVA280 NOM (e.g. 5 mg-C L−1 WLHpoA) stabilized the NPs against aggregation (Figure

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2a and complete data set in Figure S5). Expectedly, the larger NP aggregates settled at faster

266

rates, with less total copper remaining in the water column (Figures 2b and S6). For example,

267

after 24 h of incubation, 16±2 and 23±1 µM Cu remained in media without or with 1 mg-C L−1

268

SRHA, respectively, as measured at 9 mm above the bottom of the containers (Figure 2b). When

269

SRHA concentration increased from 1 to 10 mg-C L−1, the aggregation and sedimentation

270

behaviors of the CuO NPs were not further affected. The increased colloidal stability of the CuO

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NPs was likely due to the steric hindrance of aggregation by the NOM,48, 53, 54 and, to a lesser

272

extent, the more negative surface charge. The surface charge of the CuO NPs became more

273

negative as NOM concentration or SUVA280 increased (Figures 2c and S7). With no NOM, the ζ-

274

potential of the CuO NPs was on average −5±1 mV in the 1 h to 24 h timeframe. With the

275

addition of 1 and 10 mg-C L−1 SRHA, the ζ-potential was reduced to −10±1 and −14±1 mV,

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respectively. Considering the different dissolution kinetics and colloidal stabilities of CuO NPs,

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we expected the uptake kinetics of CuO NPs to vary between the tested water chemistry

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conditions.

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2000 1500

dh (nm)

1000 500 300 200 100 0

280 281 282 283 284

0

10

20

30

Time (h)

40

50

0

(b)

30 25

(c)

-4 ζ−potential (mV)

no NOM 0.1 mg-C L-1 WLHpoA 5 mg-C L-1 WLHpoA 5 mg-C L-1 SRFA 1 mg-C L-1 SRHA 5 mg-C L-1 SRHA 10 mg-C L-1 SRHA

(a)

2500

Total Cu concentration in media [Cu]T (µM)

279

20 15 10

-8

-12 5 0

0

10

20

30

40

Time (h)

50

-16

0

10

20

30

40

50

Time (h)

Figure 2. (a) Z-average hydrodynamic diameter (dh), (b) total suspended Cu concentration ([Cu]T) and (c) zeta potential (ζ-potential) of the CuO NPs in 5‰ ASW buffered to pH 7.0 with 2 mM MOPS, in the absence or presence of different NOM isolates at different concentrations. Ionic strength ~90 mM, T = 28 ºC. Experiments were performed in the dark. Error bars are standard deviations of replicate CuO NP uptake experiments (N = 2 to 6).

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Uptake and Distribution of Dissolved Cu and CuO NPs. The copper content in Gulf

286

killifish embryos increased with time after exposure to dissolved Cu (Figure S8) or CuO NPs

287

(Figure S9). The ratio of Cu content in chorions to that measured in whole embryos (i.e., the

288

chorion and the contents enclosed within) was typically > 0.4 (Figure S10), suggesting that a

289

major portion of the Cu was associated with the chorions. This observation is consistent with

290

silver uptake in Atlantic killifish and zebrafish embryos exposed to Ag NPs or AgNO3, where the

291

majority (> 0.6) of measured Ag in exposed embryos was also associated with chorions.18, 21

292

The Cu distribution in the embryos was time-dependent. For both dissolved Cu and CuO

293

NP exposures, Cu content measured in whole embryos typically increased throughout the 48-h

294

dosing period, while Cu content in chorions increased at slower rates after 12 or 24 h post dosing

295

(Figures S8 and S9). Consequently, the ratio of Cu content in chorions to that measured in whole

296

embryos typically decreased after 12 or 24 h (Figure S10), indicating that Cu became saturated in

297

the chorions over time and more passed through into the interior compartments.

298

We note that at some time points the calculated ratios were >1, which was due to

299

biological variability and measurement uncertainty. We also recognize that Cu accumulation in

300

the chorion may not be directly accessible for specific organism functions or will induce toxic

301

effects. In fact, the chorion can often serve as a barrier for nanoparticles.18 However, uptake into

302

the chorion is a first step towards bioaccumulation, and the chorion loaded with nanoparticles

303

can become a site for long-term metal exposure and in situ biotransformations.21 Thus, the

304

differentiation of dissolved and nanoparticulate Cu uptake pathways provide useful insights to

305

understanding the bioavailability of metals originating from nanomaterials.

306 307

Uptake Kinetics of Dissolved Cu and CuO NPs. The modeled Cu uptake kinetics generally agreed with measured data within the first 24 or 12 h post dosing (Figures S8 and S9).

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In modeling the uptake kinetics (Equations 4 and 5), both input parameters (as continuous

309

functions of time) of dissolved Cu concentration ([Cu]d) and total Cu concentration in suspension

310

([Cu]T) changed with time. The decrease in [Cu]T for CuO NP uptake experiments was mainly

311

due to the sedimentation of the NPs (Figures 2b and S6), while the change for the dissolved Cu

312

uptake experiments (Figure S11) was due to Cu uptake into embryos. [Cu]d versus time was

313

typically modeled according to Equation 1. The [Cu]T values in both CuO NP and dissolved Cu

314

uptake experiments were assumed to change linearly with time within the first 24 h, and the

315

linearly fitted equations of [Cu]T versus time were used as input parameters in modeling the

316

uptake kinetics.

317

The pH of the exposure media had moderate effects on the uptake rate constants of

318

dissolved Cu (ku,d) (Figures S8d-f and Table 1), while NOM concentration and type more

319

strongly influenced ku,d. At pH 7.0, copper uptake was inhibited as SRHA concentration

320

increased (Figures 3a-c), with ku,d decreasing from (64.6±3.7)×10−6 with no NOM to

321

(11.2±0.6)×10−6 L embryo−1 h−1 with 10 mg-C L−1 SRHA (Table 1). At a constant NOM

322

concentration (5 mg-C L−1), the NOM isolate with the higher SUVA280 value decreased

323

dissolved Cu uptake (Figures 4a-c). Thus, the uptake rate constant of dissolved Cu was

324

dependent upon both NOM concentration and NOM type, with ku,d negatively correlated to

325

[DOC] multiplied by SUVA280 (Figure S12a). This result is consistent with previous findings

326

that NOM with higher aromaticity and SUVA tends to bind more strongly with metal ions (e.g.

327

Zn2+ and Cu2+) and are more effective in decreasing dissolved metal bioavailability.55, 56 This

328

combined parameter ([DOC]×SUVA280) reflects both NOM type and concentration and could be

329

interpreted to represent the concentration of aromatic carbon associated with dissolved NOM.

330

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331 332 333 334 335 336 337 338 339 340

Figure 3. Cu uptake in Gulf killifish embryos exposed to dissolved Cu (a, b, and c) or CuO NPs (d, e, and f) with SRHA at different concentrations. Gulf killifish embryos (48 hpf) were exposed to mixtures of 5‰ ASW (~90 mM ionic strength) buffered to pH 7.0 with 2 mM MOPS and containing Cu and NOM combinations as shown. Solid and open symbols represent Cu content measured in whole embryos and chorions, respectively, and dash lines represent modeled uptake kinetics. Circle, square and triangle symbols represent experimental replicates from different batches of embryos (replicate number N = 2 or 3). The Cu contents are averages of 4 embryos/chorions in each sample.

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Figure 4. Cu uptake in Gulf killifish embryos exposed to dissolved Cu (a, b, and c) or CuO NPs (d, e, and f) with different NOM isolates at 5 mg-C L−1. Exposure mixtures comprised of 5‰ ASW (~90 mM ionic strength) buffered to pH 7.0 with 2 mM MOPS, and the following NOM isolate: (a, d) WLHpoA, (b, e) SRFA or (c, f) SRHA. Different symbols correspond to experimental replicates from different batches of embryos (replicate number N = 2 or 3).

349

related to aromatic carbon concentration from the NOM and fairly straightforward to interpret,

350

overall Cu uptake rates involving the CuO NPs yielded more complicated relationships with

351

NOM type and concentration. For example, the overall Cu uptake rates in exposure media spiked

352

with 30 µM CuO NPs were similar among different NOM isolates at 5 mg-C L−1 (Figures 4d-f).

353

These observations were due to NP dissolution that occurred at the same time as uptake and the

354

fact that two forms of Cu (dissolved and nanoparticulate) were bioavailable to the organism.

355

Thus, overall Cu uptake was modeled by discretizing the process to dissolved Cu and

356

nanoparticulate CuO uptake pathways (Equation 5). Values of ku,d from the dissolved Cu-only

357

uptake experiments and the dissolution data (Figure 1, Table 1) were used to estimate NP-

While the overall uptake of copper in the dissolved Cu-only experiments was directly

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specific uptake rate constant ku,NP. It should be noted that this approach was based on the

359

assumption that the presence of NPs did not affect the uptake kinetics of dissolved metal ions;

360

thus the contributions of NPs and ions could be added in a linear fashion.22 This assumption

361

would be valid if Cu accumulation is low and CuO NPs attached to the surface of embryos do

362

not block entrance to pore canals for ion diffusion or transport. The values of ku,NP were also

363

negatively correlated with [DOC]×SUVA280 (Figure S12b), while ku,NP and ku,d were

364

significantly correlated (Figure S13).

365

The importance of aromatic carbon concentration from NOM for nanoparticle uptake and

366

ku,NP values is not as well-known as it is for dissolved metal uptake rates and ku,d. NOM with

367

greater SUVA values are known to also impart increased colloidal stability for nanoparticle

368

suspensions,48, 54 which was also observed in this study (Figure 2). Thus, ku,NP was decreased for

369

CuO NP suspensions with smaller hydrodynamic diameter, and a possible explanation is that

370

larger CuO NP aggregates can settle on embryo surfaces more easily. Quantitative analysis of the

371

relationship between aggregate size and ku,NP was not feasible, mostly due to the polydispersity

372

of the aggregation data. On the other hand, ζ-potential of CuO NPs in the media shifted

373

negatively as [DOC]×SUVA280 increased (Figures 2c and S7, Table 1). This enhanced

374

electrostatic barrier may also decrease the bioavailability of the CuO NPs, as demonstrated for

375

humic acid-alleviated toxicity of Ag NPs to aquatic organisms of different trophic levels (algae,

376

water fleas and zebrafish larvae).57 In sum, both smaller aggregate size (and thus slower settling

377

velocity) and lower surface affinity (i.e., likely due to increased electrostatic barrier and possibly

378

due to NOM-induced steric hindrance) may have contributed to the decrease in ku,NP at higher

379

aromatic carbon concentration.

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Relative Contributions of CuO NPs and Dissolved Cu for Cu Uptake. The relative

381

contribution of dissolved Cu uptake and CuO NP uptake in Equation 5 can be described by

382

taking the ratio of the individual terms that contribute to the overall change in Cu concentration

383

in the organism (d[Cu]org/dt) as shown in the following:

384

k u,d [Cu]d Uptake rate dissolved Cu = Uptake rate CuO NP k u,NP ([Cu]T − [Cu]d )

(6)

385

The calculated Cu uptake rate ratio as shown in Equation 6 depends mainly on the extent

386

of CuO NP dissolution (Figure S14). For experiments performed at pH 6.3, and with 5 or 10 mg-

387

C L−1 SRHA, the Cu uptake ratio was >1 most of the time (Figures S14d, h, i). With 5 mg-C L−1

388

SRFA, the ratio increased above 1 after 12 h (Figure S14g), but under the other water chemistry

389

conditions the ratio was < 1. In particular, for experiments with 0.1 mg-C L−1 WLHpoA or at pH

390

7.5, where only 1–2% of the CuO NPs dissolved within the first 12 h, the ratio remained at 0.2–

391

0.3 (Figure S14b, f).

392

This trend was extrapolated over a broad range of [DOC]×SUVA280 values and

393

percentage of CuO NP dissolution (Figure 5), using the ku,d and ku,NP values obtained from the

394

experiments (Figure S12). The relative contributions of dissolved Cu and nanoparticulate CuO to

395

overall Cu uptake rates were mainly dependent upon the percentage of CuO NP that dissolved in

396

the media, and were insensitive to the [DOC]×SUVA280 parameter. The reason for the

397

insensitivity is that the uptake rate constants of both dissolved Cu and nanoparticulate CuO were

398

decreased roughly proportionally with higher aromatic dissolved carbon (Figures S12 and S13).

399

Over the tested [DOC]×SUVA280 range of 0.1–50 m−1, uptake rates of dissolved Cu and

400

nanoparticulate CuO were equal when 4.5−6.1% of the CuO NPs had dissolved. When CuO NPs

401

dissolved to a higher degree (e.g. in response to pH changes or undersaturation conditions58),

402

uptake rate of dissolved Cu will exceed that of nanoparticulate CuO. 23 ACS Paragon Plus Environment

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403

404 405 406 407 408 409

Figure 5. Relative contributions of dissolved Cu uptake and CuO NP uptake as influenced by [DOC]×SUVA280 and percentage of CuO NP dissolution. The relative rates of uptake (shown in color) were extrapolated from measurement data (black symbols; N = 81) from all CuO NP exposure experiments.

410

Environmental Implications. The behavior and bioavailability of metal-based NPs and

411

metal ions released from the NPs in aquatic environments depend upon not only the

412

concentration of NOM but also the characteristics of NOM. This study quantitatively

413

demonstrated that NOM with higher SUVA and at higher concentrations decreased the uptake

414

rate constants of both dissolved Cu and nanoparticulate CuO. However, this study also indicated

415

that the ratio of these uptake rate constants did not change with NOM type and concentration.

416

Hence, the bioavailability of CuO NPs is in proportion to that of dissolved Cu under a variety of

417

solution conditions.

418

Aromatic carbon concentration, as indicated by the combined parameter of

419

[DOC]×SUVA280, is a determinant of the bioavailability of both NPs and dissolved metal ions.

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This parameter [DOC]×SUVA280 is essentially the decadal absorption coefficient at 280 nm (i.e., 24 ACS Paragon Plus Environment

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a280) of the water matrices, and may be applicable for environmental settings where dissolved

422

NOM is the dominant UV absorbing constituent. Exceptions to this might be waters containing

423

dissolved ferric ions, which can contribute to UV absorbance.59

424

The relative contributions of dissolved Cu and nanoparticulate CuO to overall Cu uptake

425

kinetics are mainly determined by the extent of CuO NP dissolution, which in turn is influenced

426

by multiple environmental and physiological parameters, including pH and organic ligands such

427

as NOM. While it is imperative to continue mechanistic studies investigating the effects of

428

complex water chemistry conditions on the dissolution rates of metal-based nanomaterials, our

429

findings support the use of dissolution rate as one of the critical functional assays for forecasting

430

nanomaterial risk in complex and varied environmental systems.60 Moreover, since the

431

bioavailability of dissolved metal has been extensively investigated, with uptake rates

432

determined under various conditions, findings of this study suggest that existing knowledge on

433

dissolved metal bioavailability and toxicity is instrumental in constructing models for predicting

434

the environmental and human health risks of nanomaterials.

435 436 437

Acknowledgments To our dear friend and mentor George Aiken, who not only helped to unlock the

438

mysteries of dissolved organic matter but also generously taught us the joys of fellowship in

439

scientific discovery.

440

We are also grateful for the insights and assistance from Drs. Marc Deshusses, Amrika

441

Deonarine, Brett Poulin, Joe Ryan, and Keith Lucey, and to Alexis Carey for the image of the

442

Gulf killifish embryo. The work was supported by the National Science Foundation (NSF)

443

(CBET-1066781) and the Center for the Environmental Implications of NanoTechnology (DBI-

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444

1266252), which is funded by the NSF and the U.S. Environmental Protection Agency (EPA).

445

Partial support was provided by the C. Gus Glasscock, Jr. Endowed Fund for Excellence in

446

Environmental Sciences at Baylor University. Additional support was provided by the U.S.

447

Geological Survey National Research and Toxic Substances Hydrology Programs. Any use of

448

trade, firm, or product names is for descriptive purposes only and does not imply endorsement by

449

the U.S. Government. This paper has not been subjected to EPA review; therefore, the opinions

450

expressed in this paper are those of the authors and do not necessarily reflect the views of the

451

EPA.

452

Supporting Information Available: Additional information related to the preparation of

453

CuO NP stock suspensions, procedures of the uptake and dissolution experiments, purity and

454

sources of chemicals (Table S1), CuO NP characterization (Figure S1), pH of uptake exposure

455

media (Figure S2), validation of CuO NP dissolution experiments by ASV (Figures S3 and S4),

456

Z-average hydrodynamic diameter (Figure S5) and zeta potential (Figure S7) of CuO NPs, total

457

copper concentration in the media of CuO NP (Figure S6) and dissolved Cu uptake experiments

458

(Figure S11), complete data sets for uptake kinetics of dissolved Cu (Figure S8) and CuO NPs

459

(Figure S9), distribution of Cu in embryos (Figure S10), ku as a function of [DOC]×SUVA280

460

(Figure S12), correlation between ku of dissolved Cu and nanoparticulate CuO (Figure S13), ratio

461

of instantaneous uptake rates of dissolved Cu and CuO NP (Figure S14), and ASV calibration

462

curves (Figure S15). This material is available free of charge via the Internet at

463

http://pubs.acs.org.

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Winn, R. N.; Crawford, D. L. Fundulus as the premier teleost model in environmental biology: Opportunities for new insights using genomics. Comp. Biochem. Physiol. D 2007, 2, 257-286. 46. Dubansky, B.; Whitehead, A.; Miller, J. T.; Rice, C. D.; Galvez, F. Multitissue molecular, genomic, and developmental effects of the deepwater horizon oil spill on resident Gulf killifish (Fundulus grandis). Environ. Sci. Technol. 2013, 47, 5074-5082. 47. Jiang, C. J.; Hsu-Kim, H. Direct in situ measurement of dissolved zinc in the presence of zinc oxide nanoparticles using anodic stripping voltammetry. Environ. Sci.-Process Impacts 2014, 16, 2536-2544. 48. Jiang, C. J.; Aiken, G. R.; Hsu-Kim, H. Effects of natural organic matter properties on the dissolution kinetics of zinc oxide nanoparticles. Environ. Sci. Technol. 2015, 49, 11476-11484. 49. Oziolor, E. M.; Bigorgne, E.; Aguilar, L.; Usenko, S.; Matson, C. W. Evolved resistance to PCB- and PAH-induced cardiac teratogenesis, and reduced CYP1A activity in Gulf killifish (Fundulus grandis) populations from the Houston Ship Channel, Texas. Aquat. Toxicol. 2014, 150, 210-219. 50. Gao, Y.; Korshin, G. Effects of NOM properties on copper release from model solid phases. Water Res. 2013, 47, 4843-4852. 51. Miao, A. J.; Zhang, X. Y.; Luo, Z. P.; Chen, C. S.; Chin, W. C.; Santschi, P. H.; Quigg, A. Zinc oxide-engineered nanoparticles: Dissolution and toxicity to marine phytoplankton. Environ. Toxicol. Chem. 2010, 29, 2814-2822. 52. Vencalek, B. E.; Laughton, S. N.; Spielman-Sun, E.; Rodrigues, S. M.; Unrine, J. M.; Lowry, G. V.; Gregory, K. B. In Situ Measurement of CuO and Cu(OH)2 nanoparticle dissolution rates in quiescent freshwater mesocosms. Environ. Sci. Technol. Lett. 2016, 3, 375380. 53. Domingos, R. F.; Tufenkji, N.; Wilkinson, K. J. Aggregation of titanium dioxide nanoparticles: Role of a fulvic acid. Environ. Sci. Technol. 2009, 43, 1282-1286. 54. Deonarine, A.; Lau, B. L. T.; Aiken, G. R.; Ryan, J. N.; Hsu-Kim, H. Effects of humic substances on precipitation and aggregation of zinc sulfide nanoparticles. Environ. Sci. Technol. 2011, 45, 3217-3223. 55. Baken, S.; Degryse, F.; Verheyen, L.; Merckx, R.; Smolders, E. Metal complexation properties of freshwater dissolved organic matter are explained by its aromaticity and by anthropogenic ligands. Environ. Sci. Technol. 2011, 45, 2584-2590. 56. Smith, K. S.; Ranville, J. F.; Lesher, E. K.; Diedrich, D. J.; McKnight, D. M.; Sofield, R. M. Fractionation of fulvic acid by iron and aluminum oxides-Influence on copper toxicity to Ceriodaphnia dubia. Environ. Sci. Technol. 2014, 48, 11934-11943. 57. Wang, Z.; Quik, J. T. K.; Song, L.; Van den Brandhof, E. J.; Wouterse, M.; Peijnenburg, W. Humic substances alleviate the aquatic toxicity of polyvinylpyrrolidone-coated silver nanoparticles to organisms of different trophic levels. Environ. Toxicol. Chem. 2015, 34, 12391245. 58. Kent, R. D.; Vikesland, P. J. Dissolution and persistence of copper-based nanomaterials in undersaturated solutions with respect to cupric solid phases. Environ. Sci. Technol. 2016. 59. Poulin, B. A.; Ryan, J. N.; Aiken, G. R. Effects of iron on optical properties of dissolved organic matter. Environ. Sci. Technol. 2014, 48, 10098-10106. 60. Hendren, C. O.; Lowry, G. V.; Unrine, J. M.; Wiesner, M. R. A functional assay-based strategy for nanomaterial risk forecasting. Sci. Total Environ. 2015, 536, 1029-1037.

30 ACS Paragon Plus Environment

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