Supplementary Material
Effects of nano-TiO2 on perfluorooctanesulfonate bioaccumulation in fishes living in
different water layers: implications for enhanced risk of perfluorooctanesulfonate
Liwen Qiang†, Xiaoyu Pan‡, Lingyan Zhu†,* , Shuhong Fang†, Shengyan Tian‡
† College of Environmental Science and Engineering, Tianjin Key Laboratory of
Environmental Remediation and Pollution Control, Key Laboratory of Pollution
Processes and Environmental Criteria, Ministry of Education, Nankai University,
Tianjin, P.R. China 300071
‡ College of Marine Science of Engineering, Tianjin Key Laboratory of Marine
Resources and Chemistry, Tianjin University of Science and Technology, Tianjin, P.R.
China 300457
Summary
Number of Pages: 21
Page S2-S7: Additional Experimental Details
Page S8-S9: Table S1-Table S2
Page S10-S19: Figure S1–Figure S10
Page S20-S21: References
S1
Fish culture
Three kinds of test organisms including Danio rerio (D. rerio), Ctenopharyngodon idella
(C. idellus), and Hypostomus plecostomus (H. plecostomus) were obtained from local fish
market. The D. rerio is a tropical freshwater fish belonging to the minnow family of
the order Cypriniformes which likes to stay in upper water layer. It is a popular aquarium
fish, and also an important vertebrate model organism in scientific research. The rear
temperature was around 25 ℃ (Jia and Meng 2012). C. idella is one of the highest
production of aquaculture fish in the world and is very common in China. It usually
inhabits in middle and lower layers in freshwater. The C. idella has been widely used in the
study of biochemistry and molecular. We choose the juvenile C. idella in the experiment.
The rear temperature was around 25 ℃ (Shen et al. 2011). H. plecostomus is native to
South America and is one of tropical omnivorous fish which belong to siluridae. As a
demersal fish, it has strong hypoxia-resistant ability and adaptive capacity. H. plecostomus
is naturally nocturnal and accustomed to stay still and adhere to the wall of aquariums. H.
plecostomus is famous for its cleaning ability, which could suck residue in water. Thereby,
it is nicknamed of “scavenger”. The rear temperature was 22 ~ 30 ℃ (Pound et al. 2011;
Zhang et al. 2010). The fishes were cultured separately in dechlorinated tap water (pH
7.1-7.5) at 25 ± 1℃ (14:10h light/dark photo period) for at least one month and were fed
with fish feed twice a day. The average length and weight of the acclimatized D. rerio was
36 ± 4 mm and 180 ± 40 mg (n=225, including 45 backup fish). The average length
and weight of C. idella was 61 ± 6 mm and 1600 ± 500 mg (n=225, including 45
backup fish). The average length and weight of H. plecostomus was 70 ± 8 mm and 3600
S2
± 900 mg (n=225, including 45 backup fish).
PFOS adsorption and desorption experiments
PFOS adsorption. All PFOS sorption isotherms were obtained using batch experiments in
50 mL vials at 25 °C and pH 6.6 ± 0.3. The PFOS stock solution was diluted sequentially to
a series of solutions with the concentration varying over five orders of magnitude. Forty
mL of PFOS solution was added in a polypropylene (PP) centrifuge tube which contained
0.02 mg of nano-TiO2. The PP centrifuge tubes were sealed and shaken at 25 ℃ on a
temperature-controlled shaker at 200 rpm for 3 d to reach apparent equilibrium (a
preliminary experiment suggested that 3 d was enough for adsorption equilibrium, Figure
S7). The PP centrifuge tubes were centrifuged at 4000 rpm for 30 min. One mL of the
supernatant was used for measuring PFOS concentrations via a UPLC-MS/MS. The
solution was filtered through a 0.22-μm Teflon membrane (Agela Technologies, China) and
collected in an auto sampler vial with internal standard MPFOS. Parallel control
experiments without addition of nano-TiO2 were also performed to assess the loss of PFOS
due to the adsorption on the wall of PP tubes. The results indicated that the loss was less
than 3% of the initial mass (Su et al. 2013).
PFOS desorption. Desorption of PFOS from nano-TiO2 was conducted by
successively replacing approximately 75% of the supernatant with water or simulated
intestinal fluids (SIF) at 37 ℃. The tubes were resealed and placed on the shaker for
another 3 d to reach desorption equilibrium. After centrifugation at 4000 rpm for 30 min,
one mL of the supernatant was used for PFOS determination, and water (or SIF) was added
to initiate the next desorption step. All the adsorption and desorption experiments were
S3
repeated twice.
The simulated intestinal fluid (SIF) was prepared according to the United States
Pharmacopoeia (USP) guide (Bravo 2009). 6.8 g of monobasic potassium phosphate were
dissolved in 250 mL water, and then 77 mL of 0.2 M sodium hydroxide and 500 mL of
water were added and mixed along with 10.0 g of Trypsin. The SIF solution was adjusted
to pH 6.8 ± 0.1 with either 0.2 M sodium hydroxide or 0.2 M hydrochloric acid and then
diluted with water to 1000 mL, stored at 4 ℃.
Instrumental analysis for PFOS and Ti4+
PFOS was quantified on a UPLC-MS/MS (Waters, ACQUITY-UPLC/XEVO-TQS). Ten μL
of extracted samples was injected onto a ACQUITY UPLC ®BEH column (C18, 2.1×50
mm, 1.7 μm particle size, Waters, Ireland; VanGuardTMBEH C18, 1.7 μm particle size)
which was maintained at 50 ℃. The UPLC flow rate was 0.4 mL/min and the gradient was
as follows: initial composition was 25% B (methanol) and 75% A (3 mM formic aicd in
water, adjusted to pH 4.0 with ammonia), which was held for 0.5 min, ramped to 85% B by
5 min, increased to 100% B by 5.1 min, held until 7 min, and back to initial condition 75%
B by 9 min, and the column equilibrated for a further 20 min. The mass spectrometer was
operated in negative electrospay ionization multiple reaction monitoring (MRM) mode
with the following MS parameters: capillary voltage 2700 V, nebulizer gas flow at 7 bar,
desolvation gas flow at 800 L/h, cone gas flow at 150 L/h. Desolvation temperature was
350 ℃. The monitoring transition was 499/80, cone voltage was 45 V, collision energy was
40 V (Fang et al. 2014).
The quantification of Ti4+ was carried out by external seven-point-calibration (5-100
S4
μg/L) with the ICP-MS system (ELAN-DRC-e, Axial Field Technology Perkin Elmer
SCIEX, Waltham, MA, U.S.A.). The standard stock solution of titanium (Ti4+, 1000 mg/L)
was prepared by heating 0.417 g TiO2 nanoparticles in 30 ml of sulfuric acid–ammonium
sulphate solution (400 g ammonium sulphate in 700 ml concentrated sulfuric acid) and
finally diluted to 250 ml with water (Zhang et al. 2007). The stock solutions of Ti4+ were
diluted to relevant concentration levels. The concentrations of TiO2 in fish were based on
dry weight.
Assessment of nano-TiO2 size distribution in aquarium
The settling behavior of the nano-TiO2 particles were assessed in aquarium with the
disturbance of fish for 48 hours, nano-TiO2 size distribution in three layers were detected at
0, 2, 4, 6, 12, 24, 36 and 48 h, respectively. All the water samples were sampled in the
center of different water layers.
The size distribution of nano-TiO2 was examined by dynamic light scattering
(Zetapals/BI-200SM, Brookhaven, USA). In brief, 1.5 mL of water sample with nano-TiO2
particles (the water samples were analyzed immediately after sampling) was put in a clean
cuvette, and the DLS was operated with a scattering angle of 90° from the incident laser
beam. The zeta potential of the nanoparticles was measured at least seven times for each
sample. The intensity measurements were converted to hydrodynamic diameter number
distribution using Mie theory. Confidence in the precise in the number distribution is less
than the intensity-average diameter, but diameter number distribution can be used for
comparative purposes between treatments (Yang et al. 2013).
Characterization of TiO2 nanoparticles
S5
The morphologies and microstructures of the nano-TiO2 were characterized by field
emission scanning electron microscopy (FE-SEM, LEO, 1530 vp, Germany), and
transmission electron microscopy (TEM, JEM-2010FEF, JEOL, Japan) using 200 kV
accelerating voltage. The crystal structure of the nanoparticles was examined with an X-ray
diffraction (XRD, Rigaku, Japan) using a RINT 2000 vertical goniometer with Cu Kα (λ =
1.5406 Å) radiation at a scanning speed of 2°/min from 10° to 90° and at a voltage of 40
kV and potential current of 150 mA.
The number-averaged hydrodynamic radius of nano-TiO2 particles were obtained
using dynamic light scattering (DLS) on a ZETAPALS instrument (Zetapals/BI-200SM,
Brookhaven, USA).
Data Analysis
In the sorption experiments, the mass of PFOS adsorbed on nano-TiO2 could be calculated
using the following equation:
𝑞𝑒 =
(𝐶0 −𝐶𝑒 )×𝑉
𝑚
(S1)
where, qe is the mass of PFOS adsorbed on nano-TiO2 particles (mg/g), C0 and Ce are PFOS
concentrations in the aqueous phase at the beginning and end of adsorption experiment
(µg/L), V is the volume of the solution (40 ml), and m is the mass of nano-TiO2 particles
(mg).
For each desorption step, the mass of desorbed PFOS during the ith desorption step can
be calculated as follows:
∆𝑞𝑖 =
[𝐶𝑖 −𝐶𝑖−1 (1−𝑟)]×𝑉
𝑚
(S2)
where, ∆𝑞𝑖 is the mass of PFOS desorbed from nano-TiO2 (mg/g)，Ci and Ci-1 are the
PFOS concentrations in the aqueous phase at the end of the ith and (i-1)th desorption steps
S6
(µg/L), and r is the fraction of supernatant replaced at each desorption circle, which was
0.75 in this study. For the first desorption step, Ci-1 refers to the PFOS concentration in
aqueous phase at the end of adsorption experiment (Ce).
Langmuir equation was used to fit the adsorption isotherm:
𝑄 𝑏𝐶
0 𝑒
𝑞𝑒 = 1+𝑏𝐶
𝑒
(S3)
where, b is the equilibrium adsorption constant (L/µg), Q0 is the maximum adsorption
amount (mg/g), and the other parameters are as above (Weber Jr et al. 1991; Yang et al.
2006).
S7
Table S1. The average whole body PFOS levels in fish species after accumulation
equilibrium.
D. rerio
Equilibrium concentration
(ng/g)
Time to reach equilibrium
(day)
Increment (%)
C. idella
H. plecostomus
P
T-P
P
T-P
P
T-P
7325
±
222
18
11647
±
673
32
3366
±
219
40
5641
±
420
48
2705
±
217
18
3148
±
203
24
59.0
--
67.6
--
16.4
--
S8
Table S2. PFOS increment and TiO2 level in fish tissues with TiO2 in different water layers
Samples
Fish
scale/skin
Gill
T-P
Internal
organs
Head
Muscle
Bottom layer – H.
plecostomus
Upper layer – D. rerio
Middle layer – C. idella
PFOS
increment
(%)
TiO2
level
(μg/g)
PFOS
increment
(%)
TiO2
level
(μg/g)
PFOS
increment
(%)
TiO2
level
(μg/g)
815
48.9
398
32.0
225
143
912
79.6
797
41.3
421
86.9
1112
61.0
436
27.6
472
48.7
519
62.2
296
11.3
273
52.6
31.8
27.4
33.1
1.94
21.7
71.0
S9
Figure S1. Multilayer microcosm system of the experimental design (Danio rerio were in
upper layer, Ctenopharyngodon idella were in middle layer, Hypostomus plecostomus were
in the bottom layer)
S10
Figure S2. The arrange of sampling time and renewing time in the exposure experiment
S11
Figure S3. (A) SEM image of nano-TiO2 power in lower magnification; (B) SEM image of
nano-TiO2 power in higher magnification; (C) TEM image of nano-TiO2 power; (D) XRD
pattern of nano-TiO2 power.
S12
Figure S4. Hydrodynamic size variation of TiO2 in different water layers during 48h. (A)
Upper water layer; (B) Middle water layer; (C) Bottom water layer.
S13
Figure S5. SEM images of TiO2 in different water layers at 24 h. (A) TiO2 in the upper
layer; (B) TiO2 in the middle layer; (C) TiO2 in the bottom layer; (D) mucus-TiO2
complexes in the bottom layer.
S14
Figure S6. Concentrations of free PFOS and nano-TiO2 in vertical water column during 48
h exposure. (A) PFOS concentration in upper layer; (B) TiO2 concentration in upper layer;
(C) PFOS concentration in middle layer; (D) TiO2 concentration in middle layer; (E) PFOS
concentration in bottom layer; (F) TiO2 concentration in bottom layer. P represents the P
group which only includes PFOS, T-P represents the T-P group which includes PFOS and
nano-TiO2.
S15
Figure S7. Adsorption kinetics of PFOS on nano-TiO2 (dosage of nano-TiO2 was 0.5 ± 0.03
mg /L, initial PFOS concentration was 607 g ± 11 ng/L, temperature was 25 ℃，pH=6.8).
Adsorption kinetics of PFOS indicated that 12h was enough to reach adsorption
equilibrium for nano-TiO2.
S16
Figure S8. Adsorption-desorption isotherms of PFOS by nano-TiO2 (adsorption experiment
was conducted in water at 25 ℃, and adsorption isotherms were modeled using the
Langmuir equation, the Langmuir parameters were listed; desorption experiment was
conducted in water or simulated intestinal fluid at 37 ℃).
S17
Figure S9. 1% whole body protein concentration in different fish. Bars with different letters
above them are significantly different (p <0.05)
S18
Figure S10. Images of fishes feces at day 50. (A) Picture of fish feces in different test group;
(B) SEM of feces in T-P group.
S19
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