1

In vivo evidence of intestinal lead dissolution from lead dioxide (PbO2) nanoparticles and

resulting bioaccumulation and toxicity in medaka fish

Ding-Quan Ng1,2,#

, Yao Chu1,#

, Shih-Wei Tan1, Shan-Li Wang

1, Yi-Pin Lin

3, -

4,

Yun-Liang Soo4, Yen-Fang Song

4 and Pei-Jen Chen

1, *

Submitted to Environmental Science Nano

Supplementary Materials content 12 pages:

Table A1 and Fig. A1-Fig. A10

Electronic Supplementary Material (ESI) for Environmental Science: Nano.This journal is © The Royal Society of Chemistry 2018

2

Table A1. Physical and surface properties of nanoscale PbO2 [nPbO2] and bulk PbO2 [bPbO2]

nPbO2 bPbO2

Primary particle diameter (nm), mean ± SDa 34.5 ± 11.4 132.4 ± 54.2

Specific surface area (m2/g)

b 28.2 2.3

Hydrodynamic diameter (nm), mean ± SDc

in deionized water 132.6 ± 36.9 217.3 ± 57.0

in tap water 186.8 ± 118.4 259.6 ± 68.2

Zeta potential (ζ , mV), mean ± SDd

in deionized water -29.8 ± 3.5 -27.0 ± 2.2

in tap water -22.1 ± 1.9 -23.6 ± 1.7 a Determined by TEM.

b Determined by BET.

c Determined by DLS.

d Determined by electrophoretic light scattering (ELS).

3

Fig. A1. TEM images of (left) nPbO2 and (right) bPbO2.

4

Fig. A2. X-ray diffraction (XRD) patterns of synthesized and purchased β-PbO2. Reference

standard for pl tt er te (β-PbO2) obtained from PDF card: 01-089-2805. The XRD pattern of

synthesized β-PbO2 was adapted from Chiang et al., 2017

synthesized

plattnerite

plattnerite

01-089-2805

purchased

plattnerite

5

Fig. A3. Pb L3 edge XANES images of different Pb-containing minerals. nPbO2 were

laboratory-synthesized and bPbO2 commercially purchased.

6

Fig. A4. Acute toxicity of (a) Pb(NO3)2 (20–120 mg/L-Pb) and (b) bPbO2 (125–1000 mg/L-Pb) in

medaka fish over 7 days.

(a)

(b)

7

(a)

(b)

Fig. A5. Dynamic changes in (a) size or (b) sedimentation rates of nPbO2 and bPbO2 particles (20

mg/L) in tap water (TW) and intestinal extract (IE). Sedimentation rate was determined as the

absorbance of nPbO2 at 300 nm in suspension at each time normalized by its initial absorbance at

time 0. Data are meanSD (n=3).

8

Fig. A6. Pb L3 edge XANES images of different Pb-containing minerals in (a) supernatant (after

air-drying) and (b) settled solids of dosing solutions after 24-h exposure in medaka fish. The

suspended solids and settled solids were collected by filtering the supernatant through a 0.2-μm–pore

size nylon membrane and freeze-dried before used for XANES analysis. Spectra represent a

combination of three repeated scans.

(a) (b)

9

Fig. A7. TXM images of digestive tracts of medaka fish in (a, b) blank control, and solutions with

(c, d, e) 20 mg/L nPbO2 and (f, g, h) 20 mg/L bPbO2. Only particle sizes > 500 nm were visible.

(a) (b)

(c) (d) (e)

(f) (g) (h)

10

Fig. A8. TXM images of gill tissue of medaka fish in (a, b) blank control, and solutions with (c,

d, e) 20 mg/L nPbO2 and (f, g, h) 20 mg/L bPbO2. Only particle sizes > 500 nm were visible.

(a) (b)

(c) (d) (e)

(f) (g) (h) (f)

11

Fig. A9. Total Pb (soluble + particulate) content in fish dosing solutions recovered after 24-h PbO2

(20 and 40 mg/L) exposure in adult fish. Data are meanSD. Fish consumed twice as much bPbO2

(6.2 and 4.3 mg/L) as nPbO2 (3.5 and 1.9 mg/L), because total Pb content recovered after 24 h-fish

exposure solutions was 16.5 and 38.1 mg/L for nPbO2 and 13.8 and 35.7 mg/L for bPbO2, assuming

PbO2 adsorption and PbO2 excretion in the fish were negligible

12

(a) Brain AChE activity

(b) Gill NaK-ATPase activity

13

(c) MDA content in liver

Fig. A10. (a) Brain acetylcholinesterase (AChE) activity, (b) gill NaKATPase activity and (c)

malondialdehyde (MDA) content in liver of adult male medaka fish with 7- to 14-day aqueous

exposure to bPbO2, nPbO2 (20 and 40 mg/L) and Pb2+

(325 μ /L). Different letters indicate p<0.05

among treatments. Data are mean SD (tissues from two fish were pooled as a subsample; n=3 for

each concentration in accordance with sub-lethal exposures)