Journal of Hazardous Materials 283 (2014) 897–904

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Effect of titanium dioxide nanoparticles on the bioavailability,

metabolism, and toxicity of pentachlorophenol in zebrafish larvae
Qi Fang a,b , Xiongjie Shi c , Liping Zhang a , Qiangwei Wang a,b , Xianfeng Wang a,b ,
Yongyong Guo a , Bingsheng Zhou a,∗
a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
b
Graduate University of Chinese Academy of Sciences, Beijing 100039, China
c
College of Life Sciences, Wuhan University, Wuhan 430072, China

h i g h l i g h t s

• Effects of n-TiO2 on toxicity of PCP in zebrafish larvae were investigated.

• Co-exposure n-TiO2 enhanced metabolism of PCP to tetrachlorohydroquinone in larvae.
• Co-exposure n-TiO2 increased oxidative damage and developmental toxicity in larvae.
• NPs may influence toxicity of associated organic pollutants in the aquatic environment.

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated the influence of titanium dioxide nanoparticles (n-TiO2 ) on the bioavailability,
Received 8 August 2014 metabolism, and toxicity of pentachlorophenol (PCP) in fish. Zebrafish (Danio rerio) embryos or larvae
Received in revised form 14 October 2014 (2-h post-fertilization) were exposed to PCP (0, 3, 10, and 30 ␮g/L) alone or in combination with n-TiO2
Accepted 27 October 2014
(0.1 mg/L) until 6 days post-fertilization. Results showed that n-TiO2 treatment alone did not induce
lipid peroxidation, DNA damage, as well as the generation of reactive oxygen species (ROS) in the larvae.
Keywords:
As compared with PCP treatment, the co-exposure of PCP and n-TiO2 enhanced the induction of ROS
Pentachlorophenol
generation, eventually leading to lipid peroxidation and DNA damage. The nuclear factor erythroid 2-
Nano-titanium dioxide
Oxidative damage
related factor 2 gene transcriptions were significantly upregulated in both PCP treatment alone and in
Bioavailability and metabolism combination with n-TiO2 . Chemical analysis and histological examination showed that n-TiO2 adsorb
Zebrafish larvae PCP, and n-TiO2 are taken up by developing zebrafish larvae; however, PCP content was not enhanced in
the presence of n-TiO2 , but the metabolism of PCP to tetrachlorohydroquinone was enhanced in larvae.
The results indicate that n-TiO2 enhanced the metabolism of PCP and caused oxidative damage and
developmental toxicity, suggesting that NPs can influence the fate and toxicity of associated organic
pollutants in the aquatic environment.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction may influence the bioavailability, metabolism, fate, and toxicity of

The increasing production and use of manufactured nanopar-
ticles (NPs) have inevitably led to their release into the aquatic
environment, thereby posing a threat to aquatic organisms [1,2].
As a result, a mixture of NPs and other environmental toxicants is
present in the environment. In this regard, NPs are highly insoluble
and have a large surface area to volume ratio, suggesting that they
are likely to interact with other substances in water [2–4]. Thus, NPs
∗ Corresponding author. Tel.: +86 727768780042; fax: +86 727768780123.
E-mail address: bszhou@ihb.ac.cn (B. Zhou).
a toxicant to aquatic biota. Thus, interactions between the toxicant
and organisms are of particular concern for the environmental risk
assessment of organic contaminants.
Among NPs, titanium dioxide nanoparticles (n-TiO2 ) are one of
the most popular manufactured nanomaterials in various industrial
and environmental applications, such as, catalysts for the decom-
position of organic contaminants [5]. As a consequence, n-TiO2
are inevitably released into the environment and enter into the
aquatic environment [6]. Although it has been confirmed that n-
TiO2 exhibited low acute toxicity to fish species and other aquatic
invertebrates on the order of tens to hundreds of mg/L (e.g., Daph-
nia magna) [7–11], several studies have shown that n-TiO2 adsorb

http://dx.doi.org/10.1016/j.jhazmat.2014.10.039
0304-3894/© 2014 Elsevier B.V. All rights reserved.
898 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904
organic contaminants and enhances the uptake of organic pollu-
tants (e.g., tributyltin, polybrominated diphenyl ethers [PBDEs]) in
organisms [12,13]. Importantly, n-TiO2 are one of the common-
est materials used to degrade organic compounds, mainly on their
photocatalytic activity at the absorption of ultraviolet (UV) light
at specific wavelengths (especially at 300–388 nm), via generation
of reactive oxygen species (ROS) that can cause decomposition of
organic molecules [14–16]. Such interactions between n-TiO2 and
other toxicants may modify the environmental behavior and/or
toxicity of toxicants to aquatic organisms, and therefore, of par-
ticular concern for environmental risk assessments.
Pentachlorophenol (PCP) has been extensively used as her-
bicide, algaecide, wood preservative, defoliant, and fungicide
[17–20]. Owing to the past widespread application and persistent
properties, resistance to biodegradation, potential bioaccumu-
lation, and biomagnification, PCP resulted in environmental
contamination, and it is a common water contaminant [19–22].
The use of PCP in China was restricted in 1997. However, with
the re-emergence of schistosomiasis in the traditionally epidemic
areas, the production and use of PCP for snail elimination and
schistosomiasis control were warranted once again in some areas
[23,24]. As a consequence, PCP has resulted in widespread envi-
ronmental contamination in the local water environment where
PCP has been detected in surface waters in China (up to 1.15 ␮g/L)
[20,25]. A high PCP concentration has been detected in Dongting
Lake (up to 103.7 ␮g/L), which is attributed to its application for the
control of snailborne schistosomiasis [26–28]. Previous studies indi-
cate that principal mechanisms of action of PCP-induced toxicity
seem to be related to the uncoupling of oxidative phosphoryla-
tion in mitochondria and the generation of reactive oxygen species
(ROS) [17,29]. PCP undergoes oxidative dechlorination to form
tetrachlorohydroquinone (TCHQ) by cytochrome P450 enzymes,
resulting in oxidative stress on cells and tissues [30,31]. TCHQ
seems to be a more toxic metabolic product of PCP; it can induce
DNA strand breakage and deplete glutathione content [32,33].
This study, quantifies the potential effect of n-TiO2 on PCP with
respect to the uptake, bioconcentration, and metabolism of PCP in
the presence of n-TiO2 in zebrafish larvae. As PCP was reported
to cause ROS generation, oxidative stress, and DNA damage, we
investigated the potential effects on developmental toxicity as
well as oxidative damage and genotoxicity using the 8-hydroxy-
2-deoxyguanosine (8-OHdG) marker. Finally, responsive genes
related to ROS stress (e.g., nuclear factor erythroid 2-related factor
2, Nrf2) were also examined. Our results suggest that the presence
of n-TiO2 enhanced the metabolism of PCP and caused oxidative
damage, which lead to developmental toxicity in zebrafish larvae.
2. Materials and methods
2.1. Reagents
Pentachlorophenol (PCP) (>99%, CAS No. 87-86-5) and MS222
were purchased from Sigma (St. Louis, MO, USA). Stock solutions
were dissolved in dimethyl sulfoxide (DMSO) and were stored at
4 ◦ C. n-TiO2 were purchased from Hangzhou Wan Jing New Material
Company (CAS: 13,463-67-7; purity > 99.9%, China). The diameter
of n-TiO2 was 25 nm according to the manufacturer’s specifications.
All other chemicals used in this study were of analytical grade,
while HPLC grade and trace metal analysis grade chemicals were
used for the chemical analysis of PCP and for the measurement of
n-TiO2 , respectively.
2.2. Preparation and characterization of n-TiO2
Stock solutions of 1 mg/mL n-TiO2 (25 nm) were prepared by
dispersing the NPs in ultrapure water (Millipore, Billerica, MA) with
sonication (50 W/L, 40 kHz) for 20 min. Test n-TiO2 solutions were
prepared immediately prior to use by diluting the stock solutions
with fresh charcoal-filtered water and sonicating (50 W/L, 40 kHz)
for 20 min [34]. The test solution was renewed every 24 h with new
n-TiO2 to maintain relatively consistent levels of n-TiO2 exposure.
The properties of commercial n-TiO2 have been characterized
in a previous study [13]. The crystal structure was confirmed
by powder X-ray diffraction (XRD) using an X’Pert PRO XRD
instrument (PANalytical, Almelo, the Netherlands). The diameter
sizes were determined by transmission electron microscopy (TEM;
JEOL2010F) at an acceleration voltage of 100 kV. The surface area of
nano-TiO2 was measured by the Brunauer–Emmett–Teller method
using an ASAP 2020 physisorption analyzer (Micromeritics, Atlanta,
US). The average diameter and ␨ potential in water (0.1 mg/L) were
determined by dynamic light scattering using a Zetasizer Nano ZS
(Malvern instruments, Worcestershire, UK).
2.3. Zebrafish maintenance and experiment design
The maintenance of wild-type zebrafish (Danio rerio, AB strain)
and exposure to their embryos were performed as described pre-
viously [13]. Briefly, embryos that had developed normally and
reached the blastula stage (2 h post-fertilization) were selected for
the experiments. The zebrafish embryos were exposed to PCP alone
or in combination with n-TiO2 in the exposure solution, containing
0.2 mM of Ca(NO3 )2 , 0.13 mM of MgSO4 , 19.3 mM of NaCl, 0.23 mM
of KCl, and 1.67 mM of HEPES [35]. For PCP exposure, the embryos
were randomly distributed into glass beakers containing 500 mL
PCP solution at the environmental-related concentration (0, 3, 10,
and 30 ␮g/L). In parallel, experiments were conducted with a com-
bination of PCP and n-TiO2 (0, 3, 10, and 30 ␮g/L PCP; 0.1 mg/L
n-TiO2 ). There were three replicates for each exposure concen-
tration, and each beaker contained ≈400 embryos. The n-TiO2
exposure concentration was based on our previous findings: after
exposure to this n-TiO2 concentration (0.1 mg/L), neither devel-
opmental toxicity nor oxidative stress was observed in zebrafish
larvae [13]. A previous study also indicated that exposure to n-
TiO2 (20 nm; up to 500 mg/L; during 96 h exposure) neither affected
hatching nor induced deformity in zebrafish embryos [12]. In addi-
tion, with the increased production and application of n-TiO2 , levels
of n-TiO2 in the environment are expected to increase, and also
because of their persistent properties, 0.1 mg/L n-TiO2 is in the same
order of magnitude to the environmental concentration (predicted
as 16 ␮g/L in Switzerland) [36].
DMSO 0.01% (v/v) was added to both control and expo-
sure groups. The embryos or larvae were exposed until 6 days
post-fertilization (dpf); at this stage, the yolk was absorbed com-
pletely. During the experimental period, the exposure solution was
renewed daily. The temperature was maintained at 28 ± 0.5 ◦ C, and
the photoperiod was set to 14-h light/10-h dark throughout the
entire experiment. At 6 dpf, the larvae were randomly sampled,
immediately frozen in liquid nitrogen, and stored at −80 ◦ C for
subsequent gene, ROS, maliondialdehyde (MDA), 8-OHdG, super-
oxide dismutase (SOD), glutathione (GSH), and chemical analysis.
The developing toxicity (e.g., hatching, malformation, growth, and
survival) was also recovered.
2.4. Chemical analysis of PCP and its metabolites in water and
larvae
PCP could either undergo photolysis or be degraded by n-TiO2 ,
and the degradation products could be TCHQ and chlorophe-
nols (e.g. 2,3,4,6-tetrachlorophenol, 2,4,6-trichlorophenol, and
2,6-dichlorophenol) in water [37]. We measured PCP concen-
trations and the possible photocatalytic degradation products in
exposure media. A water sample (100 mL) was taken at 6 dpf before
 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904
renewing the exposed water in all treated groups (n = 3 replicates).
Briefly, the water sample was acidified to a pH of 4, and tribro-
mophenol (TBP) was added as a standard to check the recovery of
the extraction procedure, and the TBP concentration was 10 ␮g/L.
After being enriched by passing through an HLB column (Waters,
500 mg, 6 mL) [38], the solution was dried under nitrogen flow
and diluted in acetonitrile–ultrapure water (3:7) for analysis. The
recovery of this method ranged from 83% to 105%.
For the bioavailability of PCP in the larvae, the extraction, clean
up, analysis, and quality assurance and quality control were per-
formed according to China standard for determining PCP [39].
Briefly, 200 zebrafish larvae were collected, rinsed three times
with ultrapure water for 15 s to remove external n-TiO2 and PCP
that was adsorbed on the surface. Previous studies have shown
that little n-TiO2 was adhered to the surface of fish larvae [40,41].
After freeze-drying, the samples were weighed, added to 5 mL
5% triethylamine in acetonitrile–water, and ultrasonicated for
5 min. After centrifugation, the supernatant was collected. Through
enrichment by a MAX column (Oasis, 60 mg, 3 mL; Waters), the
solution was dried under nitrogen flow and diluted by formic
acid–methanol–water (v/v/v, 1:49:50) for analysis. Quantification
was conducted by a calibration curve created with a PCP standard.
Procedural blanks were analyzed simultaneously. The recovery of
standard addition in the blank was 85.92% (n = 6).
In this study, the TCHQ content was measured in the larvae
exposed to 30 ␮g/L PCP and co-exposure groups. The procedures
for extraction, clean up, and analysis were performed as previ-
ously described [42]. Briefly, 500 larvae were collected, cleaned,
freeze-dried, and weighed. After extraction with diethyl ether,
the solution was dried under nitrogen flow and diluted using
acetonitrile–ultrapure water (3:7). The recovery of standard addi-
tion in the blank was 80.33% (n = 3). Procedural blanks were
analyzed simultaneously. Quantification was conducted by a cal-
ibration curve created with a TCHQ standard.
The samples were analyzed for PCP, TCHQ, and several
chlorophenols using HPLC/MS (Agilent 1100HPLC/MSD Trap, CA)
employed with a DAD detector. The limit of detection was 1 ␮g/L,
which was calculated as three times the S/N from six runs for the
ongoing precision.
2.5. TEM examination of n-TiO2 in larvae
Transmission electron microscopy was performed to confirm
the uptake of n-TiO2 by zebrafish larvae. Larvae were collected
and euthanized in MS222, washed in phosphate buffer (PBS, 0.1 M,
pH 7.4), and fixed with 2.5% glutaraldehyde in the same PBS
overnight. They were then washed in the buffer, post-fixed with
1% osmium tetroxide, washed, dehydrated, and embedded in epoxy
resin (Spurr’s). Ultrathin sections (60–80 nm) were obtained using
an ultramicrotome (Leica EMUC6, Germany) and observed using a
Hitachi (HT-7700) TEM at 80 kV. The samples were not stained to
better reveal n-TiO2 particles [40].
2.6. Quantification of n-TiO2 in larvae by HPLC–ICP-MS
The quantification of n-TiO2 concentrations in larvae was per-
formed as previously described [13]. Briefly, 100 larvae from each
replicate were washed 5 times and weighed after
freeze-drying. Then, the samples were digested in 6 mL HNO3
using a microwave digestion system (Multiwave 3000, Anton
Paar, Austria) to decompose TiO2 into Ti4+ and evaporated
to dryness at 140 ◦ C. Nitric acid (2%) was added to the
samples to a constant volume to 5 mL. There were three
replicates (i = 3) for the measurement for both the water sam-
ples and larvae. The Ti4+ concentrations were determined by
HPLC–ICP-MS (NexION300X, PerkinElmer, MA). Quantification
899
was performed with a calibration cure with a Ti standard. Ti
concentrations were converted to TiO2 concentrations by molec-
ular weight conversion. The recovery for this TiO2 quantification
technique ranged from 94% to 107%. The measured n-TiO2 was
expressed as TiO2 .
2.7. Adsorption of PCP on n-TiO2
We examined the adsorption of PCP on n-TiO2 . PCP (30 ␮g/L)
was mixed with a suspension of n-TiO2 (0.1 mg/L). At 0, 2, 4, 8, 12,
20, and 24 h, 100 mL of the mixture was centrifuged for 5 min at
12,000 × g using a high-speed centrifuge to separate n-TiO2 par-
ticles. The supernatant was then collected and further analyzed
by HPLC–MS (Agilent 1100HPLC/MSD Trap, CA). The adsorption
amount of PCP was evaluated as the decrease in the PCP concen-
tration over time. This experiment was performed in a dark room
to avoid PCP degradation.
2.8. ROS, lipid peroxidation assessment, and antioxidant enzyme
assays
The generation of ROS in the larvae at 6 dpf was measured using
dichlorofluorescein diacetate as previously described [43]. Briefly,
20 larvae (n = 3) were washed two times with cold PBS (pH 7.4)
and then homogenized in cold buffer (0.32 mM sucrose, 20 mM
HEPES, 1 mM MgCl2 , and 0.5 mM) phenylmethylsulfonylfluoride
(pH 7.4). The homogenate was centrifuged at 15,000 × g at 4 ◦ C for
20 min, and the supernatant was transferred to new tubes for fur-
ther experiments. The fluorescence intensity was measured using
a microplate reader (Molecular Device, M2, Union City, CA) with
excitation and emission at 485 and 530 nm, respectively. The ROS
concentration was expressed in arbitrary units (DCF mg−1 protein).
Lipid peroxidation was assessed by measuring the amount of
malondialdehyde (MDA) (trace MDA assay kit, Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) according to the manu-
facturer’s introductions. Briefly, 100 larvae (n = 3) were collected
and homogenized with 200 ␮L normal saline. Then, the lysate was
centrifuged at 5000 × g for 5 min, and the supernatant was collected
for MDA detection. The level of MDA is expressed as nanomoles per
milligram of protein.
The activity of total SOD and GSH content was determined using
a diagnostic reagent kit (Nanjing Jiancheng Bioengineering Insti-
tute, Nanjing, China) according to the manufacturer’s introductions.
The larvae (100, n = 3) were collected and homogenized with 400 ␮L
normal saline. Then, the lysate was centrifuged at 5000 × g for
5 min, and the supernatant was collected for SOD and GSH detec-
tion. The absorbance was measured at 550 nm and 405 nm for SOD
and GSH, respectively. The level of GSH is expressed as micromoles
per gram of protein, and the activity of SOD is expressed as unit per
milligram of protein.
2.9. 8-OHdG detection
8-OHdG was measured by the enzyme-linked immunosorbent
assay (ELISA). The larvae (100, n = 3) were collected and homoge-
nized with 200 ␮L normal saline. Then, the lysate was centrifuged
at 5000 × g for 5 min, and the supernatant was collected for 8-OHdG
detection (FISH 8-OHdG ELISA Kit Nanjing Jiancheng Bioengi-
neering Institute, Nanjing, China) according to the manufacturer’s
introductions. The optical density was evaluated at 450 nm within
15 min. The 8-OHdG concentration was calculated according to the
standard curve and expressed in arbitrary units (ng/mg protein).
The protein content in our experiments was measured with a
Bradford protein assay kit (Beyotime Institute of Biotechnology,
Jiangsu, China).
900 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904

2.10. RNA isolation and quantitative real-time polymerase chain (A)

reaction (qRT-PCR)
12
PCP #
The total RNA was extracted from 30 homogenized zebrafish lar- PCP+n-TiO2

PCP content (µg/L)

vae (n = 3) using TRIzol Reagent (Invitrogen, Carlsbad, CA) according
to the manufacturer’s instructions. The extraction, purification, and 8
quantification of the total RNA and the synthesis of first-strand
cDNA were performed as described previously [43]. The primer
sequences of the selected genes were obtained using the online 4
program Primer3 (http://frodo.wi.mit.edu/; see Table S1). The ribo-
somal protein L8 (rpl8) was selected as the housekeeping gene,
which does not vary upon PCP exposure (data not shown). 0
0 3 10 30
2.11. Statistical analysis PCP conce ntration (µg/L)

All data were initially verified for normality and homogeneity

(B)
of variance using the Kolmogorov–Smirnov test and Levene’s test.
All data were expressed as the mean ± standard error (SEM). The 100
PCP
differences of the control and each exposure group were evaluated #

PCP content ( µg/g.dw)

PCP+n-TiO2
by one-way analysis of variance (ANOVA) followed by Dunett’s test 75
or two-way ANOVA followed by Tukey’s test using SPSS 13.0 (SPSS,
Chicago, IL). The differences between groups exposed to PCP alone
and co-exposure with n-TiO2 were also compared using the Stu- 50
dent’s t-test; a P value < 0.05 was considered to be a statistically
significant difference. 25

3. Results 0
0 3 10 30
3.1. Characterization of n-TiO2 PCP conce ntration (µg/L)

XRD revealed that peaks of n-TiO2 matched those expected
for the crystal phase of anatase (JCPDF No: 89-4921). The mea-
sured particle size was close to the manufacturer’s information
(mean ± SEM, n = 30, 27.73 ± 0.98 nm). The ␨ potential of n-TiO2 was
−26.6 mV, and the particle size in suspension was 164.07 ± 6.99 nm,
which revealed that n-TiO2 could aggregate in water (0.1 mg/L) (Fig.
S1). The surface area of n-TiO2 was measured as 48.7 m2 /g.
Fig. 1. Content of PCP: (A) in exposure water and (B) in larvae measured after
exposed to nominal PCP concentrations (0, 3, 10, and 30 ␮g/L) and/or in the presence
of 0.1 mg/L n-TiO2 for 6 days. Data are expressed as means ± SEM of three replicate
samples. Significance differences (P < 0.05) are indicated by # vs. the corresponding
PCP groups without n-TiO2 (two-way ANOVA followed by Tukey’s test).
3.5. Quantification of n-TiO2 in zebrafish larvae

In the larvae, the TiO2 concentration was measured as

3.2. Developmental toxicity
The overall hatching rates were over 90%, and survival rates
were over 85% in all groups. As compared to the control, there
was no significant influence on hatching and mortality rates in
both PCP and PCP with n-TiO2- treated groups. A significant increase
in the malformation was recorded in 30 ␮g/L PCP-treated group
(4.7 ± 0.29%) relative to the solvent control (2.5 ± 0.5%), while a
significantly higher malformation was further observed in the co-
exposure group (6.2 ± 0.6%) compared to the PCP-treated group
(4.7 ± 0.29%).
3.3. Adsorption of PCP on n-TiO2
The time-course adsorption kinetics is shown in Fig. S2. The
measured PCP concentration in water was stable over time. After
mixing PCP (30 ␮g/L) with 0.1 mg/L n-TiO2 , the measured PCP con-
centration in water decreased over time.
3.4. Transmission electron microscopy
103.3 ± 4.8, 107.4 ± 5.4, 103.0 ± 22.3, and 111.7 ± 13.4 ␮g/g dry
weight (dw) in 0, 3, 10, and 30 ␮g/L PCP co-exposure with 0.1 mg/L
n-TiO2 , respectively. There was no significant difference in the n-
TiO2 concentration between the n-TiO2 treatment alone and in
combination of PCP, indicating that the presence of PCP did not
affect the uptake of n-TiO2 in larvae.
3.6. PCP concentrations in water
No degradation products of PCP were detected in all the
exposure water. The PCP concentrations in exposure water were
measured before the renewal of the exposure solutions. The
measured PCP concentrations (n = 3 replicates) were 0.45 ± 0.01,
2.89 ± 0.50, and 8.91 ± 0.42 ␮g/L in the 3, 10, and 30 ␮g/L PCP expo-
sure groups, respectively (Fig. 1). In the groups exposed to PCP and
n-TiO2 , the measured PCP concentrations (n = 3 replicates) were
0.22 ± 0.01, 2.37 ± 0.32, and 7.11 ± 0.33 ␮g/L, respectively (Fig. 1).
The measured PCP concentration in the co-exposure group was sig-
nificant lower (25%) than that in the PCP-treated group (two-way
ANOVA followed by Tukey’s test, P < 0.001, Fig. 1).

TEM observation revealed that n-TiO2 aggregates were dis-
tributed in larval tissues with no tissue specificity and uniform
distribution. In addition, n-TiO2 aggregates were also observed in
mitochondria; TEM observation thus confirmed that n-TiO2 was
taken up by zebrafish larvae (Fig. S3).
3.7. Bioconcentration and metabolism of PCP in larvae
The bioconcentration of PCP was measured (5.50 ± 1.64,
18.30 ± 2.18, and 70.54 ± 6.89 ␮g/g, dry weight (dw)) in the larvae
exposed to 3, 10, and 30 ␮g/L PCP groups, respectively (Fig. 2A).
 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904
(A)
8 groups compared with the respective PCP treatment (two-way
TCHQ content ( µ g/g.dw)
# 30 ␮g/L, P < 0.05; Fig. 3A).
4 exposure of PCP and n-TiO2 resulted in a significantly higher MDA
0 alone (two-way ANOVA followed by Tukey’s test, P < 0.05; Fig. 3C).
PCP PCP+n-TiO2
(B)
0.18
# Dunnett’s test; P < 0.05; Fig. 3E); a further reduction of GSH content
0.12
TCHQ/PCP
0.06
0.00
PCP PCP+n-TiO2
Fig. 2. Content of TCHQ in larvae exposed to 30 ␮g/L PCP and/or in the presence of n-
TiO2 (0.1 mg/L) for 6 days: (A) TCHQ content and (B) TCHQ/PCP in larvae. Significance
differences (P < 0.05) are indicated by # vs. the corresponding PCP groups without
n-TiO2 (Student’s t-test).
In the groups co-exposed to PCP and n-TiO2 , PCP concentrations
showed a concentration dependence in the larvae (4.40 ± 1.02,
16.46 ± 3.39, and 46.74 ± 5.94 ␮g/g dw), respectively (Fig. 2A). We
compared the PCP concentrations in the larvae for PCP exposure
alone and in combination with n-TiO2 and revealed a significant
reduction of PCP concentrations in the 30 ␮g/L co-exposure groups
(two-way ANOVA followed by Tukey’s test, P < 0.05, Fig. 2A).
TCHQ is the major metabolite of PCP [42]. The TCHQ con-
tent was measured from the larvae exposed to 30 ␮g/L PCP alone
(4.56 ± 0.19 ␮g/g dw) and in combination with 0.1 mg/L n-TiO2
(3.66 ± 0.16 ␮g/g dw) (Fig. 2B). TCHQ in larvae for 30 ␮g/L PCP alone
was significantly higher than that in combination of PCP with n-
TiO2 (Student’s t-test, P < 0.05; Fig. 2B). However, the TCHQ/PCP
in the co-exposure group was significantly higher than that in the
PCP-exposed group (Student’s t-test, P < 0.05; Fig. 2C).
3.8. Oxidative damage
We observed oxidative damage effects including ROS content,
MDA content, 8-OHdG level, SOD activity, and GSH content (Fig. 3).
Exposure of n-TiO2 alone to zebrafish larvae did not induce genera-
tion of ROS, and alter MDA, 8-OHdG, SOD, and GSH compared to the
solvent control. ROS was significantly increased in all PCP exposure
groups relative to the solvent control (one-way ANOVA followed by
901
Dunnett’s test, 3 ␮g/L, P < 0.05; 10 ␮g/L, P < 0.05; 30 ␮g/L, P < 0.05;
Fig. 3A); ROS generation was further increased in the co-exposure
ANOVA followed by Tukey’s test, 3 ␮g/L, P < 0.05; 10 ␮g/L, P < 0.05;
A significant increase in the MDA level was observed in the
30 ␮g/L PCP group (one-way ANOVA followed by Dunnett’s test,
P < 0.01; Fig. 3B). Compared with PCP exposure alone, the co-
level (two-way ANOVA followed by Tukey’s test, 10 ␮g/L, P < 0.05;
30 ␮g/L, P < 0.05; Fig. 3B).
The 8-OHdG content was also significantly increased in 30 ␮g/L
PCP group (one-way ANOVA followed by Dunnett’s test, P < 0.05;
Fig. 3C). Likewise, the co-exposure of PCP and n-TiO2 resulted in a
significant higher 8-OHdG content, as compared to PCP treatment
The SOD activity was significantly decreased in 10 and 30 ␮g/L
PCP groups compared to the solvent control (one-way ANOVA fol-
lowed by Dunnett’s test; P < 0.05; Fig. 3D). The SOD activity was
further decreased in the co-exposure groups relative to PCP-treated
groups (two-way ANOVA followed by Tukey’s test, 3 ␮g/L, P < 0.05;
10 ␮g/L, P < 0.05; 30 ␮g/L, P < 0.05; Fig. 3D).
The reduction of GSH content was observed in 30 ␮g/L PCP alone
compared to the solvent control (one-way ANOVA followed by
was measured in the co-exposure group compared with the PCP-
treated group (two-way ANOVA followed by Tukey’s test, P < 0.05;
Fig. 3E).
3.9. Gene transcription profiles
We observed the gene transcription of nrf2 and sod1 after
exposure to PCP with and without n-TiO2 (Fig. 4). A significant
upregulation of nrf2 gene transcription (2.2-fold) was observed
in 30 ␮g/L PCP-treated groups compared to the solvent control
(one-way ANOVA followed by Dunnett’s test; P < 0.01; Fig. 4A).
In co-exposure groups, nrf2 gene transcription was significantly
upregulated compared with the respective PCP-treated group (two-
way ANOVA followed by Tukey’s test; 10 ␮g/L, P < 0.05; 30 ␮g/L,
P < 0.05; Fig. 4A).
A small but significant upregulation of sod1 gene transcription
was observed in 10 and 30 ␮g/L PCP exposure groups (1.5-fold
and 1.8-fold, respectively) (one-way ANOVA followed by Dunnett’s
test; 10 ␮g/L, P < 0.05; 30 ␮g/L, P < 0.01; Fig. 4B). The sod1 gene
was significantly upregulated in the co-exposure group relative to
the PCP-treated group (two-way ANOVA followed by Tukey’s test,
P < 0.05; Fig. 4B).
4. Discussion
This study demonstrated a reduction of PCP content by zebrafish
larvae in the presence of n-TiO2 . Furthermore, in combination with
n-TiO2 , the metabolism of PCP could be enhanced, leading to PCP-
induced oxidative damage and genotoxicity to zebrafish embryos
or larvae. PCP also caused the upregulation of genes related to
oxidative stress and glutathione metabolism (sod1).
An adsorption kinetics study showed that the PCP concentra-
tions in the exposure media were stable in water, while there was a
marked decrease in the PCP concentration in the exposure media in
the presence of n-TiO2 ; this result suggests that n-TiO2 can adsorb
PCP. Previous studies have reported that n-TiO2 can adsorb heavy
metals [4,34,44] or organic toxicants, including tributyltin, PBDEs,
DDT and tetrachlorodibenzo-p-dioxin [TCDD] [12,13,45,46], while
study showed the aggregation and subsequent sedimentation of n-
TiO2 with tributyltin [12]. At the same time, we did not measure
902 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904

(A)
9000
PCP

Fluorescence Intensity
PCP+ n-TiO2
# #

(DCF/µg prot)
#
** **
4500 ** ** **
*

0
0 3 10 30
PCP conce ntration (µg/L)

(B)
30
PCP
MDA (nmol/mg prot)

PCP+n-TiO2 #

# **
15 **
*

0
0 3 10 30
PCP conce ntration (µg/L)

Fig. 3. (A) ROS generation; (B) MDA content; (C) 8-OHdG level; (D) SOD activity; and (E) GSH content in larvae after exposure to PCP alone (0, 3, 10, and 30 ␮g/L) and in
the presence of n-TiO2 (0.1 mg/L) for 6 days. Significance differences (P < 0.05) are indicated by * vs. DMSO control (one-way ANOVA followed by Dunnett’s test); # vs. the
corresponding PCP groups without n-TiO2 ; P < 0.01 are indicated by ** vs. DMSO control (two-way ANOVA followed by Tukey’s test).

any metabolites of PCP in the exposure media. However, the pho-
tocatalytic degradation of PCP could occur in the presence of high
n-TiO2 concentrations (200 mg/L) under UV illumination condition
(450 W), via generation of ROS (e.g., hydroxyl radical) to attack para
position of the PCP ring to form a semiquinone radical, which in
turn disproportionates to yield detected metabolic products (e.g.,
p-chloranil and tetrachlorohydroquinone) [37]. Likewise, there is
ample evidence to indicate that photocatalytic degrade organic
compounds are most likely driven by a UV-induced (especially at
300–388 nm) formation of ROS by n-TiO2 [14,47,48]. For toxicity
assay using aquatic organisms, typical laboratory lighting cycle is
usually established by employing fluorescent lamps, which mainly
emit visible light. The light does not provide wavelengths of UV
radiation (below 400 nm) that trigger photoreactivity in most forms
of TiO2 [16], and no detectable UV light was measured from the fluo-
rescent lamps installed in the ceiling [49]. In our study, the intensity
of UV light is negligible from the fluorescent lamps (15 W/m2 :
UVA, 6.5 × 10−3 W/m2 ; UVB, 1.1 × 10−3 W/m2 ) [50]. Thus, the
photocatalytic degradation of PCP observed in the exposure media
did not occur in our experiment because of negligible UV illu-
mination. Hence, the decreased PCP in water was caused by
sedimentation and not by degradation.
In our study, the presence of n-TiO2 reduced PCP content
in larvae. In contrast, several previous studies have reported
enhanced bioavailability of organic toxicants to exposure organ-
isms (e.g., tributyltin, BDE-209, DDT, TCDD) in the presence of
n-TiO2 [12,13,45,46], as n-TiO2 acted as a carrier that enables com-
pounds to adsorb on the organism surface. At the same time,
adsorption and sedimentation also normally occurred in organic
toxicants with n-TiO2 in exposure media [12,46,51]. Thus, sedi-
mentation does not appear to be the major reason that affects the
bioavailability of organic toxicants by organisms. To further inves-
tigate the potential mechanism to account for the reduced PCP
content in co-exposed zebrafish, we measured the metabolite in
the larvae. We hypothesized that the observed PCP content in lar-
vae could be caused by the increased metabolism of PCP to other
 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904
Fig. 4. Gene transcription profiles in larvae after exposure to PCP (0, 3, 10, and
30 ␮g/L) alone or in the presence of 0.1 mg/L TiO2 for 6 days: (A) nrf2, (B) sod. The val-
ues are the means of three determinations on each of the three replicate exposures;
they are presented as the mean ± SEM of three replicates. Significance differences
(P < 0.05) are indicated by * vs. DMSO control (one-way ANOVA followed by Dun-
nett’s test); # vs. the corresponding PCP groups without n-TiO2 (two-way ANOVA
followed by Tukey’s test); P < 0.01 are indicated by: ** vs. DMSO control; P < 0.001
are indicated by *** vs. DMSO control.
products. TCHQ is a major metabolite of PCP in the mammalian sys-
tem and also in fish liver cells [42,52]. In our study, TCHQ/PCP in
larvae was significantly increased in the co-exposure group com-
pared with the PCP-treated group, supporting the evidence that
more PCP is transformed to TCHQ in the co-exposure group; how-
ever, the mechanism for the enhanced metabolism of PCP in the
presence of n-TiO2 is not known. As a consequence, the increased
production of TCHQ would generate a more potent source of ROS.
Previous studies have shown that TCHQ seems to be a more toxic
form of an organic toxicant, as it can autoxidize to its semiquinone
radicals under physiological conditions, which is the manner in
which PCP could generate a potent ROS source [32,33,53] and cause
oxidative damages to cells or tissues.
In our study, we observed a significant increase in ROS genera-
tion in the PCP treatment relative to the solvent control; in addition,
a significantly increased generation of ROS was measured in the co-
exposure groups, suggesting that an increased ROS amount could
be due to the presence of n-TiO2 . The generation of ROS would affect
the antioxidant system (e.g., by decreasing SOD and GSH content),
and if the oxidative stress overwhelmed the defending system,
oxidative damage would occur, which could cause lipid peroxida-
tion (measured as MDA) and genotoxicity (DNA damage, measured
as 8-OHdG). In agreement with this notion, we observed a decrease
in GSH content, reduced SOD activity, and increased MDA and 8-
OHdG, a biomarker for DNA damage [54], in the PCP-treated groups.
PCP exposed to Carassius carassius hepatocytes could induce ROS
903
generation, increase lipid peroxidation production (MDA), and
decrease GSH [55]. A further significant change of these biomark-
ers as compared to PCP treatment alone could be accounted for
by enhanced ROS generation, and thus oxidative stress induced by
co-exposure with n-TiO2 . In our study, we observed increased mal-
formation rates in the PCP and co-exposure groups, as compared
to the respective control, which can be attribute to the increased
ROS generation and thus induced oxidative stress. ROS-induced
oxidative stress is thought to contribute to abnormal development
during embryogenesis [56].
The gene nrf2 is presumably one of the most important path-
ways for cells to deal with oxidative stress generated from exposure
to exogenous and endogenous chemicals [57,58]. The transcription
factor nrf2, which regulates the induction of antioxidant enzymes
with an antioxidant response element, played a crucial role in medi-
ating the oxidative damage induced by PCP [59]. In our study,
we also observed an upregulation of nrf2 transcription, and this
alteration should be considered as a regulatory response to the
enhanced ROS-induced oxidative stress. In addition, nrf2 could
induce the transcription of sod gene transcription [57]. The sod gene
transcription was upregulated, which would likely encode more
SOD protein, to protect cells from ROS-induced oxidative damage.
In conclusion, this study demonstrated an increased metabolism
of PCP in zebrafish larvae in the presence of n-TiO2 , which induced
enhanced oxidative damage in early developing zebrafish larvae.
However, the interaction between n-TiO2 and PCP, e.g., the poten-
tial influences of n-TiO2 on uptake and bioavailability, are not well
known. However, this study emphasizes the fact that n-TiO2 could
modify the bioavailability and metabolism of PCP, thus suggest-
ing the consideration of other widespread engineered NPs in the
environment and interaction between other toxicants during envi-
ronmental risks assessment.
Acknowledgements
This work was supported by grants from the National Nat-
ural Science Foundation of China (No. 21237005), the Strategic
Priority Research Program of the Chinese Academy of Sciences
(No. XDB14040103), the National Environmental Protection Public
Welfare Science and Technology Research Program of China (No.
201309047) and the State Key Laboratory of Freshwater Ecology
and Biotechnology (No. 2011FBZ13).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jhazmat.
2014.10.039.
References
[1] S.J. Klaine, P.J. Alvarez, G.E. Batley, T.F. Fernandes, R.D. Handy, D.Y. Lyon, S.
Mahendra, M.J. McLaughlin, J.R. Lead, Nanomaterials in the environment:
behavior, fate, bioavailability, and effects, Environ. Toxicol. Chem. 27 (2008)
1825–1851.
[2] R.D. Handy, R. Owen, E. Valsami-Jones, The ecotoxicology of nanoparticles and
nanomaterials: current status, knowledge gaps, challenges, and future needs,
Ecotoxicology 17 (2008) 315–325.
[3] A. Baun, S.N. Sorensen, R.F. Rasmussen, N.B. Hartmann, C.B. Koch, Toxicity and
bioaccumulation of xenobiotic organic compounds in the presence of aqueous
suspensions of aggregates of nano-C60, Aquat. Toxicol. 86 (2008) 379–387.
[4] W.W. Yang, A.J. Miao, L.Y. Yang, Cd2+ toxicity to green alga Chlamydomonas
reinhardtii as influenced by its adsorption on TiO2 engineered nanoparticles,
PLoS One 7 (2012) 1–8.
[5] X. Chen, S.S. Mao, Titanium dioxide nanomaterials: synthesis, properties,
modifications, and applications, Chem. Rev. 107 (2007) 2891–2959.
[6] R. Kaegi, A. Ulrich, B. Sinnet, R. Vonbank, A. Wichser, S. Zuleeg, H. Simmler, S.
Brunner, H. Vonmont, M. Burkhardt, M. Boller, Synthetic TiO2 nanoparticle
emission from exterior facades into the aquatic environment, Environ. Pollut.
156 (2008) 233–239.
904 Q. Fang et al. / Journal of Hazardous Materials 283 (2014) 897–904
[7] G. Federici, B.J. Shaw, R.D. Handy, Toxicity of titanium dioxide nanoparticles
to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other
physiological effects, Aquat. Toxicol. 84 (2007) 415–430.
[8] X.S. Zhu, L. Zhu, Z.H. Duan, R. Qi, Y. Li, Y. Lang, Comparative toxicity of several
metal oxide nanoparticle aqueous suspensions to zebrafish (Danio rerio) early
developmental stage, J. Environ. Sci. Health A 43 (2008) 278–284.
[9] H. Ma, A. Brennan, S.A. Diamond, Phototoxicity of TiO2 nanoparticles under
solar radiation to two aquatic species: Daphnia magna and Japanese medaka,
Environ. Toxicol. Chem. 31 (2012) 1621–1629.
[10] V.K. Sharma, Aggregation and toxicity of titanium dioxide nanoparticles in
aquatic environment—a review, J. Environ. Sci. Health A 44 (2009) 1485–1495.
[11] C. Jacobasch, C. Völker, S. Giebner, J. Völker, H. Alsenz, T. Potouridis, H.
Heidenreich, G. Kayser, J. Oehlmann, M. Oetken, Long-term effects of
nanoscaled titanium dioxide on the cladoceran Daphnia magna over six
generations, Environ. Pollut. 186 (2014) 180–186.
[12] X.S. Zhu, J. Zhou, Z.H. Cai, TiO2 nanoparticles in the marine environment:
impact on the toxicity of tributyltin to abalone (Haliotis diversicolor
supertexta) embryos, Environ. Sci. Technol. 45 (2011) 3753–3758.
[13] Q. Chen Q. Wang, W. P. Zhou, J. Wang Li, X. Wang C. Huang, B. Zhou K. Lin,
Bioconcentration and metabolism of BDE-209 in the presence of titanium
dioxide nanoparticles and impact on the thyroid endocrine system and
neuronal development in zebrafish larvae, Nanotoxicology 8S1 (2014)
196–207.
[14] U.I. Gaya, A.H. Abdullah, Heterogeneous photocatalytic degradation of organic
contaminants over titanium dioxide: a review of fundamentals, progress and
problems, J. Photochem. Photobiol. C 9 (2008) 1–12.
[15] K. Nakata, T. Ochiai, T. Murakami, A. Fujishima, Photoenergy conversion with
TiO2 photocatalysis: new materials and recent applications, Electrochim. Acta
84 (2012) 103–111.
[16] H. Ma, A. Brennan, S.A. Diamond, Photocatalytic reactive oxygen species
production and phototoxicity of titanium dioxide nanoparticles are
dependent on the solar ultraviolet radiation spectrum, Environ. Toxicol.
Chem. 31 (2012) 2099–2107.
[17] D. Yin, Y. Gu, Y. Li, X. Wang, Q. Zhao, Pentachlorophenol treatment in vivo
elevates point mutation rate in zebrafish p53 gene, Mutat. Res. 609 (2006)
92–101.
[18] K. Farhadi, M.A. Farajzadeh, A.A. Matin, P. Hashemi, Dispersive liquid–liquid
microextraction and liquid chromatographic determination of
pentachlorophenol in water, Cent. Eur. J. Chem. 7 (2009) 369–374.
[19] W. Zheng, X. Wang, H. Yu, X. Tao, Y. Zhou, W. Qu, Global trends and diversity
in pentachlorophenol levels in the environment and in humans: a
meta-analysis, Environ. Sci. Technol. 45 (2011) 4668–4675.
[20] W. Zheng, H. Yu, X. Wang, W. Qu, Systematic review of pentachlorophenol
occurrence in the environment and in humans in China: not a negligible
health risk due to the reemergence of schistosomiasis, Environ. Int. 42 (2012)
105–116.
[21] J. Muir, G. Eduljee, PCP in the freshwater and marine environment of the
European Union, Sci. Total Environ. 236 (1999) 41–56.
[22] R.J. Letcher, W.A. Gebbink, C. Sonne, E.W. Born, M.A. Mckinney, R. Dietz,
Bioaccumulation and biotransformation of brominated and chlorinated
contaminants and their metabolites in ringed seals (Pusa hispida) and polar
bears (Ursus maritimus) from East Greenland, Environ. Int. 35 (2009)
1118–1124.
[23] M.H. Zheng, B. Zhang, Z.C. Bao, Analysis of pentachlorophenol from water
sediments, and fish bile of Dongting lake in China, Bull. Environ. Contam.
Toxicol. 64 (2000) 16–19.
[24] D. Tan, J.B. Zhang, Estimates of PCP-Na consumption in districts and provinces
in China by the top-down calculation method, Environ. Pollut. Control 30
(2008) 17–20.
[25] J.J. Gao, L.H. Liu, X.R. Liu, H.D. Zhou, S.B. Huang, Z.J. Wang, Levels and spatial
distribution of chlorophenols 2,4-dichlorophenol, 2,4,6-trichlorophenol, and
pentachlorophenol in surface water of China, Chemosphere 71 (2008)
1181–1187.
[26] M. Pavlica, G. Klobucar, N. Vetma, R. Erben, D. Papes, Detection of micronuclei
in haemocytes of zebra mussel and great ramshorn snail exposed to
pentachlorophenol, Mutat. Res. Fundam. Mol. Mech. 465 (2000) 145–150.
[27] Y. Guo, B. Zhou, Thyroid endocrine system disruption by pentachlorophenol:
an in vitro and in vivo assay, Aquat. Toxicol. 142-143 (2013) 138–145.
[28] T. Xu, J. Zhao, P. Hu, Z.J. Dong, J.Y. Li, H.C. Zhang, D.Q. Yin, Q.S. Zhao,
Pentachlorophenol exposure causes Warburg-like effects in zebrafish
embryos at gastrulation stage, Toxicol. Appl. Pharmacol. 277 (2014) 183–191.
[29] A.T. Proudfoot, Pentachlorophenol poisoning, Toxicol. Rev. 22 (2003) 3–11.
[30] Z. Mehmood, M.P. Williamson, D.E. Kelly, S.L. Kelly, Metabolism of
organochlorine pesticides: the role of human cytochrome P4503A4,
Chemosphere 33 (1996) 759–769.
[31] Y.J. Wang, Y.S. Ho, J.H. Jeng, H.J. Su, C.C. Lee, Different cell death mechanisms
and gene expression in human cells induced by pentachlorophenol and its
major metabolite, tetrachlorohydroquinone, Chem. Biol. Interact. 128 (2000)
173–188.
[32] Y.J. Wang, J.K. Lin, Estimation of selected phenols in drinking-water with
in-situ acetylation and study on the DNA-damaging properties of
polychlorinated phenols, Arch. Environ. Contam. Toxicol. 28 (1995) 537–542.
[33] Y.J. Wang, Y.S. Ho, S.W. Chu, H.J. Lien, T.H. Liu, J.K. Lin, Induction of glutathione
depletion p53 protein accumulation and cellular transformation by
tetrachlorohydroquinone, a toxic metabolite of pentachlorophenol, Chem.
Biol. Interact. 105 (1997) 1–16.
[34] C. Tan, W.H. Fan, W.X. Wang, Role of titanium dioxide nanoparticles in the
elevated uptake and retention of cadmium and zinc in Daphnia magna,
Environ. Sci. Technol. 46 (2012) 469–476.
[35] M. Westerfield, The Zebrafish Book, a Guide for the Laboratory Use of
Zebrafish (Danio rerio), third ed., University of Oregon Press, Eugene, OR, USA,
1995.
[36] C.O. Robichaud, A.E. Uyar, M.R. Darby, L.G. Zucker, M.R. Wiesner, Estimates of
upper bounds and trends in nano-TiO2 production as a basis for exposure
assessment, Environ. Sci. Technol. 43 (2009) 4227–4233.
[37] G. Mills, M.R. Hoffmann, Photocatalytic degradation of pentachlorophenol on
TiO2 particles: identification of intermediates and mechanism of reaction,
Environ. Sci. Technol. 27 (1993) 1681–1689.
[38] O. Jáuregui, E. Moyano, M.T. Galceran, Liquid chromatography-atmospheric
pressure chemical ionization mass spectrometry for chlorinated phenolic
compounds: application to the analysis of polluted soils, J. Chromatogr. A 823
(1998) 241–248.
[39] SN/T 2445-2010. Determination of Pentachlorophenol residues in food stuffs
of animal origin for import and export-LC–MS/MS method.
[40] O. Bar-Ilan, K.M. Louis, S.P. Yang, J.A. Pedersen, R.J. Hamers, R.E. Peterson, W.
Heideman, Titanium dioxide nanoparticles produce phototoxicity in the
developing zebrafish, Nanotoxicology 6 (2012) 670–679.
[41] S. Li, X. Pan, L.K. Wallis, Z. Fan, Z. Chen, S.A. Diamond, Comparison of TiO2
nanoparticle and graphene–TiO2 nanoparticle composite phototoxicity to
Daphnia magna and Oryzias latipes, Chemosphere 112 (2014) 62–69.
[42] G.R. Bruno, F.R. Jean, N.T. Thomas, Simultaneous assay of pentachlorophenol
and its metabolite tetrachlorohydroquinone, by gas chromatography without
derivatization, J. Chromatogr. B 533 (1990) 111–124.
[43] J. Deng, L.Q. Yu, C.S. Liu, K. Yu, X.J. Shi, B.S. Zhou,
Hexabromocyclododecane-induced developmental toxicity and apoptosis in
zebrafish embryos, Aquat. Toxicol. 93 (2009) 29–36.
[44] X. Zhang, H. Sun, Z. Zhang, Q. Niu, Y. Chen, J.C. Crittenden, Enhanced
bioaccumulation of cadmium in carp in the presence of titanium dioxide
nanoparticles, Chemosphere 67 (2007) 160–166.
[45] Y. Shi, J.H. Zhang, M. Jiang, L.H. Zhu, H.Q. Tan, B. Lu, Synergistic genotoxic1ity
caused by low concentration of titanium dioxide nanoparticles and p,p -DDT
in human hepatocytes, Environ. Mol. Mutagen. 51 (2010) 192–204.
[46] C.L. Laura, G. Frenzilli, T. Balbi, M. Bernadeschi, C. Ciacci, S. Corsolini, C.D.
Torre, R. Fabbri, C. Faleri, S. Focardi, Interactive effects of n-TiO2 and
2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis, Aquat. Toxicol.
153 (2014) 53–65, http://dx.doi.org/10.1016/j.aquatox.2013.11.002.
[47] D.S. Bhatkhande, V.G. Pangarkar, A. Beenackers, Photocatalytic degradation
for environmental applications—a review, J. Chem. Technol. Biotechnol. 77
(2002) 102–116.
[48] F. Seitz, M. Bundschuh, A. Dabrunz, N. Bandow, G.E. Schaumann, R. Schulz,
Titanium dioxide nanoparticles detoxify pirimicarb under UV irradiation at
ambient intensities, Environ. Toxicol. Chem. 31 (2012) 518–523.
[49] Z. Clemente, V.L.S.S. Castro, M.A.M. Moura, C.M. Jonsson, L.F. Fraceto, Toxicity
assessment of TiO2 nanoparticles in zebrafish embryos under different
exposure conditions, Aquat. Toxicol. 147 (2014) 129–139.
[50] Y. Tian, C.H. Dai, Y.X. Hao, C.P. Yao, J.L. Yu, H. Lu, W. Liu, Measurements of the
ultraviolet irradiation of artificial light sources in daily life, J. Clin. Dermatol.
35 (2006) 209–210 (in Chinese).
[51] L. Wang, Y. Huang, A.T. Kan, M.B. Tomson, W. Chen, Enhanced transport of
2,2 ,5,5 -polychlorinated biphenyl by natural organic matter (NOM) and
surfactant-modified fullerene nanoparticles (nC60), Environ. Sci. Technol. 46
(2012) 5422–5429.
[52] C. Pietsch, J. Hollender, F. Dorusch, P. Burkhardt-Holm, Cytotoxic effects of
pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCHQ)
on liver cells are modulated by antioxidants, Cell Biol. Toxicol. 30 (2014)
233–252.
[53] S. Naito, Y. Ono, I. Somiya, S. Inoue, K. Ito, K. Yamamaoto, S. Kawanishi, Role of
active oxygen species in DNA-damage by pentachlorophenol metabolites,
Mutat. Res. Fundam. Mol. Mech. 310 (1994) 79–88.
[54] A. Valavanidis, T. Vlahogianni, M. Dassenakis, M. Scoullos, Molecular
biomarkers of oxidative stress in aquatic organisms in relation to toxic
environmental pollutants, Ecotoxicol. Environ. Saf. 64 (2006) 178–189.
[55] Y.L. Dong, P.J. Zhou, S.Y. Jiang, X.W. Pan, X.H. Zhao, Induction of oxidative
stress and apoptosis by pentachlorophenol in primary cultures, Comp.
Biochem. Phys. C 150 (2009) 179–185.
[56] M. Yamashita, Apoptosis in zebrafish development, Comp. Biochem. Phys. B
136 (2003) 731–742.
[57] X.J. Shi, B.S. Zhou, The role of nrf2 and mapk pathways in pfos-induced
oxidative stress in zebrafish embryos, Toxicol. Sci. 115 (2010) 391–400.
[58] T.W. Kensler, N. Wakabayashi, S. Biswal, Cell survival responses to
environmental stresses via the Keap1-Nrf2-ARE pathway, Annu. Rev.
Pharmacol. 47 (2007) 89–116.
[59] T. Umemura, Y. Kuroiwa, Y. Kitamura, Y. Ishii, K. Kanki, Y. Kodama, K. Itoh, M.
Yamamoto, A. Nishikawa, M. Hirose, A crucial role of nrf2 in vivo defense
against oxidative damage by an environmental pollutant, pentachlorophenol,
Toxicol. Sci. 90 (2006) 111–119.