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]

ACS Paragon Plus Environment


1

Graph TOC

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Abstract

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

behavior under environmentally relevant exposure conditions. Our investigation

20

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

21

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

22

matter, bioavailability processes, toxicological risk

23



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Introduction

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

environmental concentrations of most NPs are largely unknown, emerging evidence

34

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

35

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

36

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

39

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

41

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

42

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

43

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

44

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

45

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

46

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

47

nephrotoxicity, deleterious effects on the hematological and cardiovascular systems

48

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


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

42

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

111

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


Guidelines

(TG203);

Tested


124

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

125

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

126

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

131

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

132

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

134

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

135

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

137

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

138

Total lead quantification in larvae

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

140

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

147

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

148

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

150

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

151

KI-assisted photospectrometry and inductively coupled plasma-mass spectrometry

152

(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

154

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

156

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

157

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

159

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

160

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

161

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

164

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

165

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

166

(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

172

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

173

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



174

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

175

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

176

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

177

neurotoxic biomarker acetylcholinesterase (AChE) was quantified by using the

178

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

181

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

182

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

183

Differences were considered statistically signiﬁcant at p < 0.05.

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Results

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Characteristics of nPbO2 particles

186

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

187

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

188

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

189

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

196

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

197

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

198

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

200

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

201

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

202

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

204

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

205

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

206

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

208

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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



224

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

225

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

226

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

227

30% of total Pb.

228

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

229

or Pb(II)aq exposure

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

231

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

232

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

233

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

234

Lead concentration in treated larvae increased with increasing concentration of

235

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

236

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

237

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

238

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

239

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

240

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

Dissolved Organic Matter or Salts Change the Bioavailability

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