Dissolved Organic Matter or Salts Change the Bioavailability

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Dissolved organic matter or salts change the bioavailability processes and toxicity of the nanoscale tetravalent lead corrosion product PbO2 to medaka fish Chun-Wei Chiang, Ding-Quan Ng, Yi-Pin Lin, and Pei-Jen Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02072 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Environmental Science & Technology

Dissolved organic matter or salts change the bioavailability processes and toxicity of the nanoscale tetravalent lead corrosion product PbO2 to medaka fish

Chun-Wei Chiang1, Ding-Quan Ng1, Yi-Pin Lin2 and Pei-Jen Chen1*

1

Department of Agricultural Chemistry, College of Bio-Resources and Agriculture,

National Taiwan University, Taipei, Taiwan 2

Institute of Environmental Engineering, College of Engineering, National Taiwan

University, Taipei, Taiwan

Submitted to Environmental Science & Technology

*To whom correspondence should be addressed at the Department of Agricultural Chemistry, National Taiwan University, R319, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan. Fax

+886-2-33669684, Telphone +886-2-33669817. E-mail:

[email protected]

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Graph TOC

2 3

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Abstract

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Nanoscale lead dioxide (nPbO2(s)) is a corrosion product formed from the chlorination

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of lead-containing plumbing materials. This metal oxide nanoparticle (NP) plays a

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key role in determining lead pollution in drinking water and receiving water bodies.

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This study uses nPbO2(s) and medaka fish (Oryzias latipes) as surrogates to investigate

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the aqueous fate and toxicological risk of metal oxide NPs associated with water

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matrices. The larvae of medaka were treated with solutions containing nPbO2(s) or

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Pb(II)aq in different water matrices for 7 to 14 days to investigate the in vivo toxic

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effects of NPs. Ionic strength enhanced aggregation and sedimentation of nPbO2(s) in

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water, leading to increased lead contents in fish bodies. However, the presence of

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dissolved organic matter in water enhanced particle stability and accelerated the lead

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dissolution, thus changing the bioavailability processes (bioaccessibility) of particles.

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Oxidative stress response and neurotoxicity in exposed fish was greater for nPbO2(s)

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solution with increased salinity than dissolved organic matter. We predict the

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bioavailability processes and toxicity of nPbO2(s) in medaka from the aqueous particle

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behavior under environmentally relevant exposure conditions. Our investigation

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suggests a toxicological risk of metal oxide NP pollution in the aquatic environment.

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Keywords: nanoscale lead dioxide, metal oxide nanoparticles, dissolved organic

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matter, bioavailability processes, toxicological risk

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Introduction

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With rapid development in nanotechnology, nanomaterials (< 100 nm) are

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increasingly being used in a variety of products to improve our daily life.

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Nanomaterials are considered a category of emerging contaminants;1 recent studies

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indicate that globally, approximately 66,000 metric tons of engineered nanomaterials

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are directly released to surface waters every year with the use and consumption of

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related products.2 Metal oxide nanoparticles (NPs) such as TiO2, CuO, and iron oxides

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are a class of the most extensively applied NPs because of their unique physical and

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chemical properties for both industrial or household applications.3 Although the actual

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environmental concentrations of most NPs are largely unknown, emerging evidence

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reveals that several metal or metal oxide NPs have been detected or are likely present

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in natural waterways.4-6 The environmental fate and potential ecotoxicological effects

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of such NPs need to be investigated.7-9

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Nanoscale lead dioxide PbO2(s) (nPbO2(s)) particles is formed inside

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lead-bearing pipes as a tetravalent solid corrosion product in the drinking water

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distribution system when free chlorine is used as the disinfectant.10 In 2003, a

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dramatic increase of lead concentration in drinking water of Washington, DC because

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of a change in disinfectant from free chlorine to chloramine in 200010, 11, causing lead

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poisoning for children.12 Chlorination of Pb(II) solids, including massicot (PbO),

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cerussite (PbCO3), and hydrocerussite (Pb3(OH)2(CO3)2) resulted in the formation of

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nPbO2.13-17 However, the change in disinfectant and the redox environments altered

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the stability of nPbO2(s), contributing to the lead contamination problem.10, 11, 14-16, 18, 19

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Lead is a toxic metal that causes multiple adverse effects including neurotoxicity,

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nephrotoxicity, deleterious effects on the hematological and cardiovascular systems

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and even increased incidences of some cancers.20, 21 Although nPbO2(s) particles are a

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stable sink for soluble lead in chlorinated drinking water due to its low solubility, they

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may also be detached and released into drinking water.22 However, the current

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regulatory action of lead in drinking water is based on the toxicity of lead ion, because

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the toxicity of nPbO2(s) is unknown; thus, the hazardous risk of nPbO2(s) exposure may

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be underestimated. No study has reported the presence of lead dioxide in the natural

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environment, except in water distribution systems where PbO2 was qualitatively

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detected in pipe scaling using x-ray diffraction.13, 23 It should be noted that the method

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for the quantitative measurement of PbO2 was only recently being developed.24, 25

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Metal oxide NPs often interact with water matrices (e.g., salts, clay colloids) and

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may eventually aggregate to the bottom of water bodies.26,

27

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organic matter (DOM) in natural water may change NP chemical and physical

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behaviors and affect the bioaccessibility, bioavailability and toxicity to aquatic

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organisms.28, 29 Because the environmental processes of NPs may not be relevant to

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the behavior of traditional contaminants, elucidating the aqueous fate and

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toxicological risk of metal oxide NPs such as nPbO2(s) is urgently needed before lead

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pipes or lead-containing NPs and applications are completely replaced, restricted or

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carefully evaluated.

However, dissolved

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Several in vitro and in vivo studies have demonstrated that soluble metal NPs

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such as Ag NPs cause cytotoxicity, which is often equivalent to the toxicity of the

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dissolved ion.30, 31 For highly reactive NPs such as nanoscale zerovalent Fe (nZVI),

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surface-modified nZVI decreases NP neurotoxicity in cultured rodent microglia or

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neurons by reducing sedimentation, which limits the cells’ exposure to particles.32

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Similarly, bioavailability and larval mortality is lower with surface-modified nZVI

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than the uncoated form in medaka fish (Oryzias latipes).33 In contrast, sperm of

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Mytilus

galloprovincialis

exposed

to

stabilized

nZVI

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showed

enhanced

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spermiotoxicity in vivo as compared with the uncoated nZVI.34 Such contradictory

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results for stabilized and unstabilized nZVI may originate from specific particle

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characteristics (e.g., changes in physicochemical properties after surface modification)

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that often determine the aqueous behavior of NPs and affect bioavailability processes

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or toxicity.7, 35

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Metal oxide NPs have less or the least water solubility, which often results in

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lower acute or sublethal toxicity than for the releasing ions.36, 37 Adsorption of humic

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acid (HA) increased suspension stability and decreased TiO2 NP exposure to

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developing zebrafish, but toxicity was greater with TiO2 NPs in the presence of HA.38

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We need more investigation on the bioavailability processes (representing

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bioaccessibility) that addresses individual physical, chemical, and biological

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interactions of aggregated versus suspended metal oxide NPs for fish exposures under

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ecologically relevant exposure conditions.39, 40 It helps to understand other related

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issues concerning their uptake of NPs across a cellular membrane, biodistribution and

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toxicity in organisms.39, 40

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Medaka fish is extensively used as an alternative animal model for studying

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embryonic development, mutagenesis, carcinogenesis, endocrinology, toxicity, and

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environmental pollution.41,

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biomarkers that reflect bioavailability processes (bioaccessibility), oxidative stress

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and neurotoxicity in larvae treated with well-characterized solutions of nPbO2(s) in

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different water matrices for 7-14 days under environmentally relevant exposure

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conditions. We correlated the particle behavior with the in vivo toxic effect(s) of

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nPbO2(s) in fish and discuss the environmental fate and potential toxicological impact

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of metal oxide NPs in the natural water environment. Effects on lower vertebrate

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species may provide valuable information on the toxic action of NPs, thus unraveling

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In this study, we used medaka to examine sublethal

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or adding evidence for effects that are suspected in humans.

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Materials and Methods

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nPbO2(s), stocks and fish dosing solutions

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The nPbO2(s) was synthesized by chlorination of 0.1M Pb(NO3)2 solution with

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NaOCl at a NaOCl/Pb2+ molar ratio of 1.1 as described previously.43 White solids

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precipitated rapidly and slowly turned reddish-black. The solutions were mixed for 24

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hr. The slurry was transferred to 50 mL polypropylene tubes and centrifuged at 6000

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rpm for 6 min. The solids were collected and dialyzed in ultrapure water for 3 days,

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freeze-dried and stored before use. The freshly prepared stock of nPbO2(s) was

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suspended in N2 saturated deionized (DI) water with 30-min sonication and analyzed

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by dynamic light scattering (DLS, Delsa Nano C; Beckman Coulter, CA, USA) for

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size distribution. The particle morphologic features were examined by transmission

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electron microscopy (TEM; JEM1200EXII; Jeol Ltd., Tokyo) and X-ray diffraction

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(XRD, X'pert PRO, PANalytical, Almelo, Netherlands).

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Seven fish-dosing solutions with different levels of total organic carbon (TOC)

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and electric conductivity (EC) were used (see Table 1 and Table S1). The chemical

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parameters of selected waters were follow the reasonable ranges of EPA synthetic

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water and reported natural waters44 and should provide suitable habitat environments

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for

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http://www.oecd.org/home/). The nPbO2(s) stock was spiked into the solutions to reach

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the expected concentrations (5 and 10 mg/L-Pb equivalent concentrations) and used

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immediately for fish toxicity assay and solution characterization.

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concentrations were chosen based on larval mortality results (LC50 >25 mg/L for

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nPbO2(s), data not shown), which did not show any significant increase in larval death

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during exposure as compared with the control group. The stock solution of Pb(II)aq

freshwater

fish

species

(according

to

OECD

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Guidelines

(TG203);

Tested

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was prepared by dissolving Pb(NO3)2 in embryo rearing medium (ERM; containing 1

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g NaCl, 0.03 g KCl, 0.04 g CaCl2·2H2O, and 0.163 g MgSO4·7H2O in 1 L DI water,

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adjusted to pH 7.2).45 All chemicals and reagents were of analytical grade.

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Monitoring particle behavior in fish dosing solutions

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Temporal changes in particle size and sedimentation in nPbO2(s)-containing fish

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dosing solutions (20 mg/L) were monitored ex situ for 24 hr by DLS and UV–vis

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spectrophotometery (U-1900, Hitachi, Japan) at 300 nm, the wavelength of maximum

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absorption for nPbO2(s). The zeta-potential of nPbO2(s) in dosing solutions at different

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pH values was measured by using the Delsa Nano C. The pH value, dissolved oxygen,

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EC, residual chlorine and other water parameters were monitored under ambient

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conditions. Total lead concentration and nPbO2(s) dissolution in fish dosing solutions

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were quantified before and after daily water renewal and in the presence or absence of

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fish, as described in SI. The lead speciation in different water matrices was calculated

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with Visual MINTEQ (http://vminteq.lwr.kth.se/download/).

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Total lead quantification in larvae

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The medaka fish were bred at the Department of Agricultural Chemistry,

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National Taiwan University, according to the animal research protocol approved by

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the Institutional Animal Care and Use Committee. One day before chemical exposure,

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free swimming larvae (20 fish per beaker) were randomly divided into 250 mL glass

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beakers containing 100 mL of clean ERM for 1-day acclimation. Fish (7 days

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post-hatching [dph]) were continuously exposed for 7 days to 100 mL solutions of

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nPbO2(s) (0.25-5.0 mg/L Pb-equivalent concentrations) or Pb(II)aq (0.10 and 0.25 mg/L

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Pb-equivalent concentrations) prepared with ERM or dechlorinated tap water (TW) (4

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replicates per concentration). The blank controls include clean ERM or TW without

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any lead spiked. All dosing solutions were freshly prepared and 100% was renewed

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every 24 hr. Fish were sufficiently fed daily with Otohime (Nisshin Co, Tokyo) before

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water renewal. Total lead concentration of lead spiked solutions was quantified by

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KI-assisted photospectrometry and inductively coupled plasma-mass spectrometry

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(ICP-MS; Agilent Technologies, SC, USA) for nPbO2(s) and Pb(II)aq respectively.25 At

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the end of exposure, larvae were rinsed with ERM, removed exceed water and then

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weighed. The tissue was digested in 65% lead-free HNO3 solution (180 °C, 2 hr) and

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30% H2O2 solution (100 °C, 1 hr). The homogenate was heated to 250 °C in the

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digestion tube until completely dry. To reach complete reduction of nPbO2(s), acidified

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KI solution (4 g/L, pH 2) was added to the tube, followed by 10-min sonication and

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the addition of 10 mL of 0.5 N HNO3 solution to reconstitute the homogenate.25, 46

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The total lead content in fish tissues was quantified by ICP-optical emission

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spectrometry (ICP-OES; PerkinElmer, Optima 2000, CT, USA). The bioconcentration

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factor (BCF) was defined as the ratio of the concentration of total lead in fish bodies

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to the concentration of total lead in dosing solutions (Table S3, SI).47

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To investigate how water matrices (e.g., dissolved organic matter [DOM] or

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ionic strength [IS]) affected the bioaccessibility of nPbO2(s) to fish, larvae were treated

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with nPbO2(s) (5.0 mg/L) spiked in ERM or TW with or without 0.5 mg/L humic acid

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(HA, Sigma-Aldrich, MO, USA) or ERM salts for 7 days. At the end of exposure,

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total lead content in fish bodies was quantified as previously described.

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Sublethal toxicity tests (oxidative stress responses and AChE activity)

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Larvae (7 dph) were continuously exposed to nPbO2(s) (0-25 mg/L, prepared in

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ERM or TW) for 7-14 days, harvested and homogenized in phosphate buffered saline,

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then centrifuged (12,000×g, 30 min) at 4℃. The supernatant was immediately used

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for measuring total protein level (BCA Protein Assay Kit, Thermo Fisher Scientific,

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USA), intracellular level of reactive oxygen species (ROS) by 2’7’-dichlorofluorescin

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diacetate assay48 and activity of the antioxidants superoxide dismutase (SOD)49 and

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catalase (CAT)50 and the lipid peroxidation product malondialdehyde (MDA) by using

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the TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Activity of the

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neurotoxic biomarker acetylcholinesterase (AChE) was quantified by using the

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Amplite Colorimetric AChE Assay Kit (AAT Bioquest, Sunnyvale, CA, USA).

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Statistical Analysis

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Each experiment was performed with three or four replicates. Data are presented

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as mean ± SD. Statistical analyses involved one-way ANOVA followed by the

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Duncan's multiple range test with SAS Learning Edition 4.1 (SAS Inst., Cary, NC).

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Differences were considered statistically significant at p < 0.05.

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Results

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Characteristics of nPbO2 particles

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The mean hydro-diameter of synthetic nPbO2(s) (post-suspended in carboxymethyl

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cellulose [CMC]) was 50.6 ± 14.7 nm (n=3) as measured by DLS. This result agreed

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with single particle morphology observed in TEM and SEM images (Figure S1a and

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S1b). Without the CMC stabilization, the original nPbO2 particles tended to

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agglomerate in DI water because their sizes ranged from 85 to 105 nm within 24 hr

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(Figure 1). The XRD spectrum showed that synthesized nPbO2 is plattnerite (β-PbO2)

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(Figure S1c).

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Physical and chemical behaviors of nPbO2(s) in different water matrices

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Figure 1 shows the effects of agglomeration or aggregation on the dynamic change

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in particle size and sedimentation rate of nPbO2(s) dispersion in seven water samples (see

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Table 1 and Table S1 for water quality parameters). The particle size of nPbO2(s)

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increased immediately to 232 nm when particles were added to ERM (W2 with TOC=0

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mg/L and EC=2.12 mS/cm) and grew to 25,960 nm in 24 hr but remained at 121-180 nm

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in TW (W3 with TOC=0.21 mg/L and EC=0.12 mS/cm) at 24 hr (Figure 1a). With the

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addition of HA in ERM (W4 with TOC=0.14 mg/L and EC=2.08 mS/cm) or TW (W5

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with TOC=0.29 mg/L and EC=0.12 mS/cm), the particle size remained around 100 nm

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at 24 hr, which suggests that HA could stabilize nPbO2(s).

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The rate of nPbO2(s) sedimentation increased with time within 24 hr for all water

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samples (Figure 1b), with the greatest sedimentation rate in ERM, followed by TW or

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DI, then ERM or TW with HA. This observation was consistent with the change in sizes

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or zeta potential of particle agglomeration or dispersion in these water matrices. The

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greatest change in zeta potential between pH 3.0 and 10.0 was found for nPbO2(s) in DI

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water (from 27.5 to -47.5 mV), followed by TW (from 11.4 to -28.8 mV) and ERM

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(from 2.23 to -15.2 mV) (Figure S2). The addition of HA in ERM enhanced the

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dispersal stability of nPbO2 (between -25.7 and -35.7 mV at pH 3-7).

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Table 1 shows the lead dissolution from nPbO2(s) particles in 7 fish dosing solutions

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(with fish) before and after daily water renewal. In general, nPbO2(s) (5 mg/L) increased

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lead dissolution with exposure time for all water samples. In ERM (no TOC), nPbO2(s)

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contributed 0.025 to 0.085 mg/L of Pb(II)aq from 0 to 24 hr. Lead dissolution was

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significantly greater in TW (containing 0.21 mg/L TOC) than ERM solution at 24 hr

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(0.165 vs 0.085 mg/L). Higher HA or TOC concentration in water significantly

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increased lead dissolution (e.g., [Pb2+]24-hr: W3>W2; W4>W2; W5>W2; W7>W2;

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W7>W6). The absence of fish produced a similar trend with less lead dissolution from

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nPbO2(s) in the same water samples. Table S2 (SI) shows lead speciation in different

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water matrices with Visual MINTEQ. The concentrations of Pb2+ and Pb(OH)2(aq) are not

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different among these waters and Pb(SO4)22-, PbCl2(aq) and PbCl3- species are only

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present in ERM(1x)-containing waters (W2, W4, W6 and W7). The concentrations of

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PbCl+ and PbSO4(aq) in ERM(1x)-containing waters are much greater than those in TW

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and TW+HA, while that of PbOH+ presents an opposite trend among these waters. This

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was attributed to the high salt content in ERM solutions (W2, W4, W6 and W7). In

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non-ERM solutions (W3 and W5), complexation with organic matter contributed at least

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30% of total Pb.

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Lead quantification in larvae and bioconcentration factor (BCF) from nPbO2(s)-

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or Pb(II)aq exposure

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The total lead concentration of dosing solutions was about 90% and 102% of their

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nominal values for nPbO2(s), and 88% and 90% for Pb(II)aq, respectively (Table S3).

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The total lead quantification for nPbO2 solutions measured by KI-assisted

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photospectrometry agreed well with KI-assisted ICP-OES results (Table S3).

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Lead concentration in treated larvae increased with increasing concentration of

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nPbO2(s) or Pb(II)aq solutions prepared in ERM or TW after 7-day exposure (Figure

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2a). The BCF value was determined as the total lead content in fish divided by the

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total lead concentration in dosing solutions (Table S3). However, the BCF showed an

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opposite trend, with high BCF found at low nPbO2(s) or Pb(II)aq concentration. The

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total lead concentration in fish body was higher in nPbO2(s)-spiked ERM solutions

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than TW (0.06-0.56 vs 0.06-0.24 μg/mg-body weight [μg/mg-bw]) (p