Ecotoxicology

https://doi.org/10.1007/s10646-020-02328-8

Effects of carboxylated multi-walled carbon nanotubes

on bioconcentration of pentachlorophenol and hepatic damages
in goldfish
Haifeng Kan1,2 Haiyun Zhang3 Mingxia Lu1 Fuzheng Zhao2 Shixiang Gao2 Guiyang Yan1 Jitao Huang1
● ● ● ● ● ● ●

Xu-Xiang Zhang 2

Accepted: 27 November 2020

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
Carboxylated multi-walled carbon nanotubes (MWCNT-COOH) exerts strong adsorption capacity for pentachlorophenol
(PCP) and they inevitably co-occur in the environment, but few studies have characterized the effects of MWCNT-COOH on
the bioavailability of PCP and its oxidative and tissue damages to fish. In this work, we assessed the PCP accumulation in
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different organs and the induced oxidative and tissue damages of goldfish following 50-d in vivo exposure to PCP alone or
co-exposure with MWCNT-COOH. Our results indicated that PCP bioaccumulation in goldfish liver, gill, muscle, intestine
and gut contents was inhibited after co-exposure with MWCNT-COOH in uptake phase. PCP exposure alone and co-
exposure with MWCNT-COOH evoked severe oxidative and tissue damages in goldfish bodies, as indicated by significant
inhibition of activities of antioxidant enzymes, remarkable decrease in glutathione level, simultaneous elevation of
malondialdehyde content, and obvious histological damages to liver and gill. The decreased accumulation of PCP in the
presence of MWCNT-COOH led to the reduction of PCP-induced toxicity to liver tissues, as confirmed by the alleviation of
hepatic oxidative damages. However, co-exposure groups had higher concentrations of PCP in the tissues than PCP
treatment alone (p < 0.05 each) in the depuration phase, revealing that MWCNT-COOH-bound pollutants might pose higher
risk once desorbed from the nanoparticles. These results provided substantial information regarding the combined effects of
PCP and MWCNT-COOH on aquatic species, which helps to deeply understand the potential ecological risks of the
emerging pollutants.
Keywords Bioaccumulation Hepatic oxidative stress Joint toxicity MWCNT-COOH Pentachlorophenol
● ● ● ●

Supplementary information The online version of this article (https:// Introduction

doi.org/10.1007/s10646-020-02328-8) contains supplementary
material, which is available to authorized users.
* Shixiang Gao
ecsxg@nju.edu.cn
* Xu-Xiang Zhang
zhangxx@nju.edu.cn
1
Fujian Provincial Key Laboratory of Featured Materials in
Biochemical Industry, Fujian Province University Key Laboratory
of Green Energy and Environment Catalysis, College of Chemistry
and Materials, Ningde Normal University, Ningde, Fujian 352100,
China
2 greater than that single-walled carbon nanotubes
State Key Laboratory of Pollution Control and Resource Reuse,
School of the Environment, Nanjing University, Nanjing 210023,
China
3
Institute of Eco-environmental Protection, Shanghai Academy of
Agricultural Sciences, Shanghai 201403, China
Carbon nanotubes (CNTs) have been gaining greater
interest over the past two decades due to their unique
physical-chemical properties. Apart from the pristine forms,
CNTs have been functionalized with different hydrophilic
moieties that allow them to be well dispersed in water.
These “water-dispersible” functionalized CNTs promise
many potential applications (Chen and Jafvert 2010;
Davood et al. 2017). In addition, multi-walled carbon
nanotubes (MWCNTs) with a low price have been widely
used in recent years, with annual production 3-4 times
(SWCNTs) (Cho et al. 2008). This inevitably results in
significant emissions to the environment during production,
consumption and disposal (Nowack and Bucheli 2007),
which may induce health and ecological risks to organisms

and human beings. For example, carboxylated multi-walled
carbon nanotubes (MWCNT-COOH) can be used as
adsorption filters to remove contaminants from drinking
water (Rajabi et al. 2016), facilitating the exposure of CNTs
to human beings through drinking water. Although the oral
toxicity study reported no death or abnormal clinical
symptoms in rats after exposure to SWCNTs (2000 mg/kg
body weight) and mice (1000 mg/kg body weight) (Miya-
waki et al. 2008; Kolosnjaj et al. 2010), MWCNTs have
been found to affect antioxidant enzyme activity, serum
hormone and biochemical stress biomarkers, hematological
parameters, histopathology, and growth performance of
carp (Rezaei Tavabe et al. 2020). However, both MWCNTs
and SWCNTs can be easily accumulated in fish gut (Maes
et al. 2014), and functionalized CNTs can enter the skull of
a fish (Soubaneh et al. 2019). It has been found that
SWCNTs are toxic to respiratory system of fish but cannot
induce severe damages to anti-oxidative system and
osmoregulatory physiology (Smith et al. 2007; Freixa et al.
2018).
As a pesticide, pentachlorophenol (PCP) has been widely
used in China to fight against schistosomiasis. The con-
centrations of PCP in the Yangtze River reach 0.6 mg/L
(Gao et al. 2008), and its maximum level is about 103.7 mg/
L in Dongting Lake (Zheng et al. 2000). High concentra-
tions of PCP in water may lead to high risk of exposure and
adverse health effects on aquatic organisms and human
beings (Yu et al. 2014). It is known that exposure to PCP at
low levels (no more than 30 μg/L) can induce production of
malondialdehyde (MDA) in zebrafish larvae (Fang et al.
2015). In the offspring of PCP-exposed zebrafish, the rate of
malformation (spinal curvature) was significantly increased
after exposure to PCP at even lower levels (9 μg/L) (Yu
et al. 2014; Ge et al. 2017). The induction of high toxicity
by low-dose exposuremay result from the strong bio-
concentration capacity of PCP in animal bodies, as Kondo
et al. (2005) indicated that the bioconcentration factors
(BCFs) of PCP ranged from 2.1 × 103 to 4.9 × 103 for
Japanese medaka after 60-d exposure.
Many studies have shown that CNTs can strongly adsorb
organic chemicals, such as 1, 2-dichlorobenzene (Peng et al.
2003), trihalomethane (Lu et al. 2005), and xylene (Chin
et al. 2007). Due to the strong sorption affinity for a wide
range of organic contaminants, CNTs can inevitably affect
their bioavailability and toxicity once released to the
environment. For example, SWCNTs significantly
increased the accumulation of pre-adsorbed phenanthrene in
a Japanese medaka by 2.1 fold after 72-h exposure (Su et al.
2013). In contrast, fluoranthene pre-adsorbed on MWCNTs
was no longer bioavailable to fathead minnow, even after
the CNT-fluoranthene was ingested and retained in the
digestive tract (Linard et al. 2015). Park et al. (2010) also
observed significant reduction in bioavailability of 17α-
H. Kan et al.
ethinylestradiol to zebrafish under 3.0–12.1% of C60 co-
exposure. However, limited information is currently avail-
able for the effects of CNTs on bioavailability of PCP and
their health damages to aquatic animals.
In this study, goldfish were exposed to relatively low
concentrations of PCP (~50–100 μg/L) for 64 d, which is a
reasonable simulation of natural aquatic environments due
to the relatively long exposure phase and the low con-
centrations of PCP. It is reasonable to expect that the
interactions of PCP with MWCNT-COOH can lead to dif-
ferent effects on organisms from what would be observed
from the individual chemicals. The objectives of the study
were (1) to investigate the impact of MWCNT-COOH on
tissue distribution and depuration of PCP in goldfish; (2)
and to assess the effect of PCP and MWCNT-COOH co-
exposure on hepatic oxidative stress and histopathological
damages in goldfish. This study may extend our knowledge
regarding the combined toxicities of CNTs and PCP in
water environment, which may provide a scientific basis for
the establishment of environmental standards for the
emerging pollutants.
Materials and methods
Materials
MWCNT-COOH was purchased from Nanotech Port Co.,
Shenzhen, Guangdong, China. The nanoparticles are char-
acterized with diameter of 20–40 nm, length of less than
5 μm, ash content of less than 3%, purity of 97%, and
special surface area of 30–50 m2 g−1. PCP (98% purity) was
purchased from Sigma Aldrich, and PCP solution (1 g/L)
was prepared from dimethyl sulfoxide.
Preparation and characterization of MWCNT-COOH
samples
The nanoparticles were ultrasonic dispersed in deionized
water for 8–9 h (electrified for 5 s, disconnected for 3 s,
60–80 W), and the MWCNT-COOH water suspension was
prepared using ultrasonic cell interferers (Nanjing Emma-
nuel Instrument Equipment Co., China), until the absor-
bance at 520 nm remained unchanged. A further sonication
for 1 h was performed immediately prior to use. High-
concentration MWCNT-COOH solution was diluted
directly with deionized water. Microscopic analysis of
carbon nanotubes was performed using transmission elec-
tron microscopy (TEM) with a JEM-200CX electron
microscope (JEOL Co., Tokyo, Japan). The content of
carboxyl groups on the surface was measured using the
Boehm titration method (Oickle et al. 2010) (details shown
in Method S1 of the Supplementary Materials). Zeta
Effects of carboxylated multi-walled carbon nanotubes on bioconcentration of pentachlorophenol and. . .
potential of MWCNT-COOH suspensions was determined
using a Zeta potential analyzer (Zeta PALS, Brookhaven
Instruments Corp) (details shown in Method S2 of the
Supplementary Materials). UV-visible spectroscopy was
used to quantify the dispersibility of CNTs following the
method recommended by Shieh et al. (2007). To avoid the
interference of matrices, a wavelength of 520 nm was
selected for the quantification of MWCNT-COOH in the
test water, according to the absorption spectrum (Fig. S1).
The suspended concentration of MWCNT-COOH was
quantified by standard solutions with gradient concentra-
tions (Fig. S2).
Adsorption and desorption experiments
Before the exposure assay, the adsorption and desorption of
PCP on MWCNT-COOH were investigated at 25 °C by
batch experiments (details shown in Method S3 of the
Supplementary Materials). The adsorption experiment was
carried out in a 40 ml spiral-covered glass vial lined with
polytetrafluoroethylene. A weighing adsorbent (4 mg of
MWCNT-COOH) and sufficient volume of the background
solution were added to the vial. Then it was sealed with a
spiral cap lined with aluminum foil and oscillated at
150 rpm and 25 °C for 5 d to achieve adsorption equili-
brium. The concentration of soluble PCP was determined by
centrifugation at 3000 g for 5 min (details shown in Method
S4 of the Supplementary Materials). Bottles without
MWCNT-COOH showed a PCP loss of less than 5% of the
initial concentration throughout the process.
Fish culture and exposure experiments
A total of 150 juvenile Carassius auratus were purchased
from Fisheries Research Institute of Nanjing (Nanjing,
China), and the fish feeding prior to exposure experiment
was conducted following the patterns of our previous report
(Kan et al. 2015). The fish were divided into five treatments:
[1] control, no PCP and no MWCNT-COOH; [2] PCP(50),
50 μg/L PCP and no MWCNT-COOH; [3] PCP(50) + CNT
(100), 50 μg/L PCP and 100 μg/L MWCNT-COOH; [4]
PCP(100), 100 μg/L PCP and no MWCNT-COOH; and [5]
PCP(100) + CNT(100), 100 μg/L PCP and 100 μg/L
MWCNT-COOH. After the absorption period of 50 d, the
fish were transferred to another tank for purification for 14
d, and the fresh water was renewed every day. Throughout
the bioaccumulation period (50 d) and purification period
(14 d), fish particles were fed at a rate of 1% of body weight
per day, and abnormal behavior, disease and mortality were
checked daily. On Days 1, 3, 6, 15, 28, 50, 57 and 64, three
fish were randomly selected from each group, and their
weights and lengths were recorded before they were killed
by a blow to the head. Liver, muscle, intestine, gill and gut
content were sampled for all the fish before removing the
intestinal contents. All tissue samples were rinsed in deio-
nized water and immediately stored at −20 °C for sub-
sequent analysis.
Biochemical assays, PCP analysis and
histopathological observation
On Days 7, 14, 21 and 28, five fish were randomly sampled
from each group, and dissected on ice to obtain liver tissue.
Hepatic activities of total antioxidants-superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GPx),
and the content of glutathione (GSH) and malondialdehyde
(MDA) were determined using the Diagnostic Reagent Kits
(Nanjing Jiancheng Bioengineering Institute, China)
according to the manufacturer’s instructions. Sample pre-
treatment, biochemical analysis and activity/content calcu-
lation were described in detail in a previous study (Wang
et al. 2015). The concentrations of PCP in the fish tissue
(liver, gill, muscle, intestine and gut content) were also
determined for three fish randomly selected from each
group on Days 1, 3, 6, 15, 28, 50, 57, and 64. Details about
tissue collection, sample treatment and instrumental analy-
sis are shown in Method S5 of the Supplementary Materi-
als. Tissue sections of liver and gill were prepared to
investigate the histopathological damages following the
method recommended by Kan et al. (2015).
Statistical analysis
Tissue PCP concentrations were calculated from triplicate
samples, expressed as mean and standard deviation (n = 3).
BCFs is expressed as the ratio of the cumulative PCP
concentration in selected tissues to the concentration in the
water under the pseudo-stable state. The homogeneity of
variance was examined and a one-way analysis of variance
(ANOVA) was hereafter performed to evaluate the sig-
nificance of the differences in PCP levels and biochemical
indices between any two different groups, any two different
tissues, or any two different exposure durations. The dif-
ference with p < 0.05 was considered statistically
significant.
Results
Characterization of MWNT-COOH in the test water
and PCP adsorption and desorption
TEM images of MWNT-COOH nanoparticles before and
after sonication showed that the bundled bulk MWNT-
COOH aggregates were broken up and well dispersed as
smaller aggregates or even a single CNT after 8-h probe

Fig. 1 Adsorption-desorption isotherms (Langmuir model) of PCP by
MWCNT-COOH. Data points are mean values that were calculated
from triplicate samples
sonication (Fig. S3). The outer diameter of the tubulars was
20–40 nm and the length ranged from several hundreds of
nanometers to several micrometers. No aggregates or sedi-
mentation in the MWCNT-COOH solution were observed
after two weeks. Table S1 shows the actual concentration of
MWCNT-COOH in the test water, which confirmed the
stable dispersibility of MWCNT-COOH. This may account
for the enhanced degree of surface carboxylization, as
confirmed by the much higher surface carboxyl content after
8 h high-power sonication compared to the control (Fig.
S4). Isothermal data of PCP adsorption on MWCNT-
COOH fit the Langmuir model well (R2 = 0.956). The
constants Q0 and Kd were 54016.3 ± 1569.9 mg/g and
1.0107 ± 0.1028 mg/L, respectively. The isotherms of
MWCNT-COOH did not show significant adsorption-
desorption hysteresis (Fig. 1).
Effect of co-exposure with MWCNT-COOH on PCP
accumulation in fish
PCP was not detected in fish from the control on any
sampling day throughout the 50-d exposure (uptake) period.
It should be noted that the concentration of PCP in liver
reached the maximum level on Day 15 in each exposure
group, while PCP uptake reached a pseudosteady-state on
Day 28 in other tissues (Fig. 2). PCP in intestines and gut
contents had significantly higher concentrations than those
in gills, muscle and liver. The addition of MWCNT-COOH
notably inhibited the bioconcentration of PCP in different
tissue samples (two-way ANOVA test, p < 0.05, Fig. 2), and
BCFs of PCP in all types of tissues showed a decreasing
trend after the co-exposure with MWCNT-COOH, but
statistical analysis revealed that the co-exposure posed the
significant inhibition effect on the PCP bioconcentration in
only liver and gill (Table 1). Aqueous concentration of PCP
H. Kan et al.
in test water maintained relatively constant throughout the
exposure phase, and the addition of MWCNT-COOH
notably reduced its aqueous concentration by 13.8% and
22.9% in the low- and high-dose exposure media, respec-
tively (Fig. 2). Interestingly, during the depuration period,
PCP concentrations of all tissues co-exposed with
MWCNT-COOH were significantly higher than those
exposed to PCP alone, especially for the high-dose group
(Fig. 2).
Hepatic oxidative and histological damages induced
by the PCP exposure alone or co-exposure with
MWCNT-COOH
Histopathological observation showed that 14-d exposure to
50 μg/L PCP induced multifocal inflammation and cell
swelling in livers, and focal necrosis and cytoplasmic
vacuolization. More severe and irreversible type of damages
appeared in high frequency in PCP(100) group (Fig. 3). The
hepatic tissue damages were also observed in the co-
exposure groups (Fig. 3). Both PCP exposure alone and co-
exposure with MWCNT-COOH were found to induce
aneurisms, oedema, epithelial lifting, and hypertrophy of
mucus cells in gills (Fig. 4).
This study also investigated the hepatic oxidative
damages caused by PCP exposure alone or PCP and
MWCNT-COOH co-exposure for 7, 14, 21 and 28 d.
Hepatic SOD, CAT and GPx activities and GSH level were
significantly inhibited in the groups treated with 50 or
100 μg/L PCP alone (Figs. 5 and 6). Compared with the
control, PCP(50) group had notably increased MDA con-
tents in liver on Days 21 and 28, and PCP(100) group
showed the similar changes at any sampling time points
(Fig. 6). The addition of MWCNT-COOH seemed to
obviously alleviate the hepatic oxidative damages induced
by PCP exposure, because SOD, CAT and GPx activities
and GSH level in the co-exposure groups were higher than
those exposed with PCP alone (Figs. 5 and 6), and the
addition of MWCNT-COOH caused obvious increases of
MDA contents in comparison with PCP treatment alone
(Fig. 6).
Discussion
This study revealed that the addition of MWCNT-COOH
significantly reduced PCP bioaccumulation at pseudosteady
state in all tissues. The high adsorption capacity of
MWCNT-COOH for PCP indicates that MWCNT-COOH
may exert strong effects on the fate of PCP once released to
the environment. Previous studies have demonstrated strong
sorption capacity of CNTs for a wide range of organic
contaminants (Yang et al. 2006b; Wang et al. 2009).
Effects of carboxylated multi-walled carbon nanotubes on bioconcentration of pentachlorophenol and. . .
Fig. 2 Accumulation and depuration of pentachlorophenol (μg g−1 dry
weight, dw) in liver (A), muscle (B), gut content (C), intestine (D) and gill
(E) of goldfish (Carassius auratus) with or without addition of MWNT-
COOH and the actual concentration of PCP in the exposure tanks (F).
Mean and standard deviation values were calculated from triplicate
samples. PCP(50): 50 μg/L pentachlorophenol; PCP(100): 100 μg/L
pentachlorophenol; PCP(50) + CNT(100): 50 μg/L pentachlorophenol
The reduced bioavailability of PCP in test water mainly
accounts for the inhibition of PCP bioaccumulation in
goldfish by MWCNT-COOH, since it has been reported that
CNTs are capable of reducing bioaccumulation factors of
PAHs in Eisenia fetida (Petersen et al. 2009), Streblospio
benedicti (Ferguson et al. 2008), and biota-sediment (Shen
and 100 μg/L MWNT-COOH; PCP(100) + CNT(100): 100 μg/L penta-
chlorophenol and 100 μg/L MWNT-COOH. Bars with different letters
above them are significantly different among the different treatments with
same exposure time (one-way ANOVA with Tukey’s multiple compar-
ison test, p < 0.05). Values that do not share the same superscript letters
(a, b, c or d) are significantly different (p < 0.05)
et al. 2012). Furthermore, the adsorption of PCP on
MWCNT-COOH appeared to reach an equilibrium within
few days, indicating the fast sorption rates of PCP on the
amended MWCNT-COOH. Salam and Burk 2010 found
that PCP in aqueous solutions was eliminated by MWCNTs
within a very short time due to great sorption affinity. The
 H. Kan et al.

Table 1 Estimated
Exposure groups Liver Muscle Gut content Gill Intestine
bioconcentration factors (BCFs)
of PCP in different types of PCP(50) 518.2 ± 45.9a 270.8 ± 59.6a 1184.5 ± 115.5a 491.9 ± 55.0a 583.8 ± 282.9
tissues of the goldfish after PCP
exposure alone or co-exposure PCP(50) + CNT(100) 374.5 ± 47.9 ab
226.4 ± 26.7 989.6 ± 193.3 350.2 ± 59.0a 762.6 ± 290.9
a a a b
with MWCNT-COOH PCP(100) 246.3 ± 50.0 163.1 ± 44.3 793.5 ± 114.2 361.5 ± 28.2 958.6 ± 228.6
PCP(100) + CNT(100) 223.2 ± 30.4b 110.6 ± 41.9 765.5 ± 270.5 321.3 ± 7.6b 808.0 ± 235.3
Mean and standard deviation values were calculated from triplicate samples. PCP(50): 50 μg/L
pentachlorophenol; PCP(100): 100 μg/L pentachlorophenol; PCP(50) + CNT(100): 50 μg/L pentachloro-
phenol and 100 μg/L MWNT-COOH; PCP(100) + CNT(100): 100 μg/L pentachlorophenol and 100 μg/L
MWNT-COOH
The numbers with the same superscript letter in each column indicate that BCFs are significantly different
among the exposure groups for each type of tissues (p < 0.05)

Fig. 3 Histopathological
observation of liver damages
after 14-d exposure to no PCP
and no CNT (a), 50 μg/L PCP
and no CNT (b), 50 μg/L PCP
and 100 μg/L CNT (c), 100 μg/L
PCP and no CNT (d), and
100 μg/L PCP and 100 μg/L
CNT (e). Multifocal
inflammation (green arrow), cell
swelling (black arrow), and
vacuolization (yellow arrow),
and necrosis (red arrow) are
indicated

strong sorption of PCP on MWCNT-COOH may make it
quite difficult to desorb in vivo, which decreased the
bioavailability of PCP to goldfish. On the other hand, the
zeta potential of MWCNT-COOH in test water at the pH of
7.8 was negative (ζ = −43 ± 3.5 mV). Zhu et al. (2012)
indicated that the negatively charged Au nanoparticles were
taken up and digested by Japanese medaka more easily due
to the electronic repulsion between Au nanoparticles and the
intestinal mucus layers.
In the depuration phase, the concentrations of PCP in all
tissues of the co-exposure groups were much higher than
those of PCP treatment alone, especially for the high-dose
group. After the exposure, MWCNT-COOH was unlikely to
be completely eliminated by fish, and finally a small amount
of them could remain in the gills, intestines, and the surface
of fish. Petersen et al. (2011) have indicated that SWCNT
have appreciable absorption into tissues of the intestine
tract, and Smith et al. (2007) and De et al. (2014) reported
Effects of carboxylated multi-walled carbon nanotubes on bioconcentration of pentachlorophenol and. . .
Fig. 4 Histopathological
observation of gill damages after
14-d exposure to no PCP and no
CNT (a), 50 μg/L PCP and no
CNT (b), 50 μg/L PCP and
100 μg/L CNT (c), 100 μg/L
PCP and no CNT (d), and
100 μg/L PCP and 100 μg/L
CNT (e). Aneurisms (red arrow)
and oedema (black arrow),
epithelial lifting (orange arrow),
and hypertrophy of mucus cells
(green arrow) are indicated
that secreted fish mucus rapidly aggregated previously
dispersed CNT on the surface of the gills, which carried
various pollutants. Therefore, the enhanced concentrations
of PCP in the tissues of co-exposure groups ascribed to its
desorption from CNT particles. Sun et al. (2014) reported
that MWCNTs had a significantly strong sorption capacity
for PCP, and PCP desorption in the simulated digestive
fluids was fast (Salam and Burk 2010). In this study, the
digestive fluid in goldfish gut could drive the desorption of
PCP from adhered CNTs, thus facilitating PCP uptake by
intestine tissues. Wu et al. (2013) indicated that desorption
of toxic chemicals from CNTs made these chemicals mobile
and bioavailable in the environment, and consequently led
to increasing risk of both toxic chemicals and CNTs.
The bio-accumulated PCP is more likely to be metabo-
lized in liver, which makes the maximum uptake of PCP
show up earlier than that in other tissues, and liver is the
most important organ for detoxification and metabolism of
xenobiotics (Zhang et al. 2012). This study revealed that
PCP exposure inhibited the activities of antioxidant
enzymes (SOD, CAT, and GPx) and the level of GSH, but
increased the level of MDA in liver, which were intimately
associated with oxidative damages of goldfish liver. These
results are in agreement with the data obtained from the
in vivo assays of earthworm by Zhang et al. (2014). A
number of studies have reported the direct toxicities of low-
dose PCP exposure on fish, including oxidative damages
(Kan et al. 2015; Luo et al. 2009). However, in the presence
of MWCNT-COOH, the activities of SOD, CAT, GPx and
the level of GSH were less suppressed whereas MDA levels
were less enhanced. The reason may be that the decreased
accumulation of PCP led to reduced toxicity to liver tissues
due to adsorption of PCP on the co-existed MWCNT-
COOH. Zhang et al. (2014) also indicated that the toxicity
of PCP-Na on E. fetida may be alleviated by the presence of
MWCNTs.
MWCNT-COOH was likely to be captured and aggre-
gate on the mucus layer covering the surface of gills, which
might alleviate the adverse effects induced by PCP. How-
ever, the protective role of gill mucus secretion cannot be
sustained for longer time, as the severe damages such as
aneurisms, oedema and epithelial lifting were observed in
the gills after the co-exposure for 14 d. Gills are multi-
functional and complex organs through which fish make
 H. Kan et al.

Fig. 5 Activities of antioxidant enzymes (SOD and CAT) in liver of

goldfish after PCP exposure alone or co-exposure with MWCNT-
COOH for 7, 14, 21, and 28 d. Values with different superscript letters
(a–d) at each exposure time point are significantly different from each
other (p < 0.05)

intimate contact with the surrounding water and they

account for over half of the body surface area. Only a few
microns of delicate gill epithelium separate the internal
environment from a continually flowing external environ-
ment, making branchial function very sensitive to envir-
onmental toxic substances (Flores and Thomaz 2011). The
gills were more sensitive to MWCNT than the other organs
(Lee et al. 2015). Gill injury was induced in the exposure
Fig. 6 The GPx activity (A), GSH level (B) and MDA content (C) in
media in this study, which would reduce respiratory effi- liver of goldfish after 7, 14, 21, and 28 d of exposure to PCP,
ciency such as gas transfer rates and water flow between the MWCNT-COOH, or their mixture. The results are presented as mean
lamellae. ± SD (n = 5). Values that do not share the same superscript letters (a, b
or c) are significantly different among the different treatments with
same exposure time (p < 0.05)

Conclusions
groups in uptake phase. The decreased accumulation of
Bioaccumulation of PCP in goldfish bodies could be alle- PCP led to the reduction of PCP toxicity to organisms, as
viated by the presence of MWCNT-COOH for all exposure confirmed by the response of antioxidant enzymes. In the
Effects of carboxylated multi-walled carbon nanotubes on bioconcentration of pentachlorophenol and. . .
depuration phase, co-exposure with MWCNT-COOH
showed significantly higher concentrations of PCP in all
tissues than PCP exposure alone. Our findings suggest that
future toxicology studies should not only focus on the
inherent toxicity of individual chemicals, but also consider
the possible interactions with existing environmental con-
taminants and their effects on the behavior and fates of
pollutants in natural environments.
Data availability
Some important data are available in the Supplementary
Materials.
Acknowledgements This work was supported by the Natural Science
Foundation of Fujian Province (No. 2018J01438), the National Natural
Science Foundation of China (21377050), the State Key Lab of Pol-
lution Control and Resource Reuse Research (PCRRF16008) and the
Scientific Research and Development Foundation of Ningde Normal
University (Nos. 2016Y04, 2016Q44 and 2017ZDK02).
Author contributions HK, SG and XXZ conceived and designed the
study. HK, HZ, ML and GY conducted the experiments. FZ and SG
performed the data analysis. HK, JH and XXZ wrote the manuscript.
All authors have read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethics statement The fish were humanely treated in accordance with
the National Institutes of Health Laboratory Animal Care and Use
Guidelines (China).
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Chen CY, Jafvert CT (2010) Photoreactivity of carboxylated single-
walled carbon nanotubes in sunlight: Reactive oxygen species
production in water. Environ Sci Technol 44:6674–6679. https://
doi.org/10.1021/es101073p
Chin CJ, Shih LC, Tsai HJ, Liu TK (2007) Adsorption of o-xylene and
p-xylene from water by SWCNTs. Carbon 45:1254–1260. https://
doi.org/10.1016/j.carbon.2007.01.015
Cho HH, Smith BA, Wnuk JD, Fairbrother DH, Ball WP (2008)
Influence of surface oxides on the adsorption of naphthalene onto
multiwalled carbon nanotubes. Environ Sci Technol
42:2899–2905. https://doi.org/10.1021/es702363e
Davood AF, HD MH, Chung TS (2017) Cross-linked mixed matrix
membranes consisting of carboxyl-functionalized multi-walled
carbon nanotubes and P84 polyimide for organic solvent nano-
filtration (OSN). Sep Purif Technol 186:243–254. https://doi.org/
10.1016/j.seppur.2017.06.021
De SF, Matsubara EY, Franchi LP, Martins IP, Ramires Rivera LM
(2014) Evaluation of carbon nanotubes network toxicity in zeb-
rafish (Danio rerio) model. Environ Res 134:9–16. https://doi.
org/10.1016/j.envres.2014.06.017
Fang Q, Shi X, Zhang L, Wang Q, Wang X, Guo Y (2015) Effect of
titanium dioxide nanoparticles on the bioavailability, metabolism,
and toxicity of pentachlorophenol in zebrafish larvae. J Hazard
Mater 283:897–904. https://doi.org/10.1016/j.jhazmat.2014.10.039
Ferguson PL, Chandler GT, Templeton RC, Demarco A, Scrivens
WA, Englehart BA (2008) Influence of sediment-amendment
with single-walled carbon nanotubes and diesel soot on bioac-
cumulation of hydrophobic organic contaminants by benthic
invertebrates. Environ Sci Technol 42:3879–3885. https://doi.org/
10.1021/es702830b
Flores LF, Thomaz AT (2011) Histopathologic alterations observed in
fish gills as a tool in environmental monitoring. Braz J Biol
71:179–188. https://doi.org/10.1590/S1519-69842011000100026
Freixa A, Acuna V, Sanchis J, Farre M, Barcelo D (2018) Ecotox-
icological effects of carbon based nanomaterials in aquatic
organisms. Sci Total Environ 619-620:328–337. https://doi.org/
10.1016/j.scitotenv.2017.11.095
Gao J, Liu L, Liu X, Zhou H, Huang S (2008) Levels and spatial
distribution of chlorophenols, 2,4-dichlorophenol, 2,4,6-tri-
chlorophenol, and pentachlorophenol in surface water of China.
Chemosphere 71:1181–1187. https://doi.org/10.1016/j.chemosp
here.2007.10.018
Ge T, Han J, Qi Y, Gu X, Ma L, Zhang C, Naeem S, Huang D (2017)
The toxic effects of chlorophenols and associated mechanisms in
fish. Aquat Toxicol 184:78–93. https://doi.org/10.1016/j.aquatox.
2017.01.005
Kan H, Zhao F, Zhang XX, Ren H, Gao S (2015) Correlations of gut
microbial community shift with hepatic damage and growth
inhibition of Carassius auratus induced by pentachlorophenol
exposure. Environ Sci Technol 49:11894–11902. https://doi.org/
10.1021/acs.est.5b02990
Kolosnjaj TJ, Hartman KB, Boudjemaa S, Ananta JS, Morgant G,
Szwarc H, Wilson LJ, Moussa F (2010) In vivo behavior of large
doses of ultrashort and full-length single-walled carbon nanotubes
after oral and intraperitoneal administration to Swiss mice. ACS
Nano 4:1481–1492. https://doi.org/10.1021/nn901573w
Kondo T, Yamamoto H, Tatarazako N, Kawabe K, Koshio M (2005)
Bioconcentration factor of relatively low concentrations of
chlorophenols in Japanese medaka. Chemosphere 61:1299–1304.
https://doi.org/10.1016/j.chemosphere.2005.03.058
Lee JW, Choi YC, Kim R, Lee SK (2015) Multiwall carbon nanotube-
induced apoptosis and antioxidant gene expression in the gills,
liver, and intestine of Oryzias latipes. Biomed Res Int
2015:485343. https://doi.org/10.1155/2015/485343
Linard EN, van den HP, Karanfil T, Apul OG, Klaine SJ (2015)
Influence of carbon nanotubes on the bioavailability of fluor-
anthene. Environ Toxicol Chem 34:658–666. https://doi.org/10.
1002/etc.2853
Lu CS, Chung YL, Chang KF (2005) Adsorption of trihalomethanes
from water with carbon nanotubes. Water Res 39:1183–1189.
https://doi.org/10.1016/j.watres.2004.12.033
Luo Y, Wang XR, Ji LL, Su Y (2009) EPR detection of hydroxyl
radical generation and its interaction with antioxidant system in
Carassius auratus exposed to pentachlorophenol. J Hazard Mater
171:1096–1102. https://doi.org/10.1016/j.jhazmat.2009.06.132
Maes HM, Stibany F, Giefers S, Daniels B, Deutschmann B, Baum-
gartner W, Schaeffer A (2014) Accumulation and distribution of
multiwalled carbon nanotubes in zebrafish (Danio rerio). Environ
Sci Technol 48:12256–12264. https://doi.org/10.1021/es503006v
Miyawaki J, Yudasaka M, Azami T, Kubo Y, Iijima S (2008) Toxicity
of single-walled carbon nanohorns. ACS Nano 2:213–226.
https://doi.org/10.1021/nn700185t
Nowack B, Bucheli TD (2007) Occurrence, behavior and effects of
nanoparticles in the environment. Environ Pollut 150:5–22.
https://doi.org/10.1016/j.envpol.2007.06.006

Oickle AM, Goertzen SL, Hopper KR, Abdalla YO (2010) Standar-
dization of the Boehm titration: Part II. Method of agitation,
effect of filtering and dilute titrant. Carbon 48:3313–3322. https://
doi.org/10.1016/j.carbon.2010.05.004
Park JW, Henry TB, Menn FM, Compton RN (2010) No bioavail-
ability of 17 alpha-ethinylestradiol when associated with nC(60)
aggregates during dietary exposure in adult male zebrafish (Danio
rerio). Chemosphere 81:1227–1232. https://doi.org/10.1016/j.
chemosphere.2010.09.036
Peng XJ, Li YH, Luan ZK, Di ZC, Wang HY (2003) Adsorption of 1,2-
dichlorobenzene from water to carbon nanotubes. Chem Phys Lett
376:154–158. https://doi.org/10.1016/S0009-2614(03)00960-6
Petersen EJ, Pinto RA, Landrum PF (2009) Influence of carbon
nanotubes on pyrene bioaccumulation from contaminated soils by
earthworms. Environ Sci Technol 43:4181–4187. https://doi.org/
10.1021/es803023a
Petersen EJ, Zhang L, Mattison NT, O’Carroll DM, Whelton AJ,
Uddin N, Tinh N (2011) Potential release pathways, environ-
mental fate, and ecological risks of carbon nanotubes. Environ
Sci Technol 45:9837–9856. https://doi.org/10.1021/es201579y
Rajabi M, Mirza B, Mahanpoor K, Mirjalili M, Najafi F, Moradi O
(2016) Adsorption of malachite green from aqueous solution by
carboxylate group functionalized multi-walled carbon nanotubes:
Determination of equilibrium and kinetics parameters. J Ind Eng
Chem 34:130–138. https://doi.org/10.1016/j.jiec.2015.11.001
Rezaei Tavabe K, Yavar M, Kabir S, Akbary P, Aminikhoei Z (2020)
Toxicity effects of multi-walled carbon nanotubes (MWCNTs)
nanomaterial on the common carp (Cyprinus carpio L. 1758) in
laboratory conditions. Compar Biochem Physiol Toxicol Phar-
macol 237:108832. https://doi.org/10.1016/j.cbpc.2020.108832
Salam MA, Burk RC (2010) Thermodynamics and kinetics studies of
pentachlorophenol adsorption from aqueous solutions by multi-
walled carbon nanotubes. Water Air Soil Pollut 210:101–111.
https://doi.org/10.1007/s11270-009-0227-1
Shen M, Xia X, Wang F, Zhang P (2012) Influences of multiwalled
carbon nanotubes and plant residue chars on bioaccumulation of
polycyclic aromatic hydrocarbons by Chironomus plumosus lar-
vae in sediment. Environ Toxicol Chem 31:202–209. https://doi.
org/10.1002/etc.722
Shieh YT, Liu GL, Wu HH, Lee CC (2007) Effects of polarity and pH
on the solubility of acid-treated carbon nanotubes in different
media. Carbon 45:1880–1890. https://doi.org/10.1016/j.carbon.
2007.04.028
Smith CJ, Shaw BJ, Handy RD (2007) Toxicity of single walled
carbon nanotubes to rainbow trout (Oncorhynchus mykiss):
respiratory toxicity, organ pathologies, and other physiological
effects. Aquat Toxicol 82:94–109. https://doi.org/10.1016/j.aqua
tox.2007.02.003
H. Kan et al.
Soubaneh YD, Pelletier E, Desbiens I, Rouleau C (2019) Radi-
olabeling of amide functionalized multi-walled carbon nanotubes
for bioaccumulation study in fish bone using whole-body auto-
radiography. Environ Sci Pollut Res 27:3756–3767. https://doi.
org/10.1007/s11356-019-05794-8
Su Y, Yan X, Pu Y, Xiao F, Wang D (2013) Risks of single-walled
carbon nanotubes acting as contaminants-carriers: potential
release of phenanthrene in Japanese medaka (Oryzias latipes).
Environ Sci Technol 47:4704–4710. https://doi.org/10.1021/
es304479w
Sun H, Ruan Y, Zhu H, Zhang Z, Zhang Y (2014) Enhanced bioac-
cumulation of pentachlorophenol in carp in the presence of multi-
walled carbon nanotubes. Environ Sci Pollut Res Int
21:2865–2875. https://doi.org/10.1007/s11356-013-2234-4
Wang X, Tao S, Xing B (2009) Sorption and competition of aromatic
compounds and humic acid on multiwalled carbon nanotubes.
Environ Sci Technol 43:6214–6219. https://doi.org/10.1021/
es901062t
Wang XH, Qu RJ, Huang Q, Wei Z (2015) Hepatic oxidative stress
and catalyst metals accumulation in goldfish exposed to carbon
nanotubes under different pH levels. Aquat Toxicol
160:142–150. https://doi.org/10.1016/j.aquatox.2015.01.015
Wu W, Jiang W, Zhang W, Lin D (2013) Influence of functional
groups on desorption of organic compounds from carbon nano-
tubes into water: Insight into desorption hysteresis. Environ Sci
Technol 47:8373–8382. https://doi.org/10.1021/es401567g
Yang W, Gan J, Hunter W, Spurlock F (2006b) Effect of suspended
solids on bioavailability of pyrethroid insecticides. Environ
Toxicol Chem 25:1585–1591. https://doi.org/10.1897/05-448R.1
Yu LQ, Zhao GF, Feng M, Wen W, Li K, Zhang PW, Peng X, Huo WJ
(2014) Chronic exposure to pentachlorophenol alters thyroid hor-
mones and thyroid hormone pathway mRNAs in zebrafish. Environ
Toxicol Chem 33:170–176. https://doi.org/10.1002/etc.2408
Zhang L, Hu C, Wang W, Ji F, Cui Y (2014) Acute toxicity of multi-
walled carbon nanotubes, sodium pentachlorophenate, and their
complex on earthworm Eisenia fetida. Ecotoxicol Environ Saf
103:29–35. https://doi.org/10.1016/j.ecoenv.2014.01.041
Zhang Y, Zhang X, Wu B, Cheng S (2012) Evaluating the tran-
scriptomic and metabolic profile of mice exposed to source
drinking water. Environ Sci Technol 46:78–83. https://doi.org/10.
1021/es201369x
Zheng MH, Zhang B, Bao ZC, Yang H, Xu XB (2000) Analysis of
pentachlorophenol from water, sediments, and fish bile of
Dongting lake in China. Bull Environ Contam Toxicol 64:16–19.
https://doi.org/10.1007/s001289910003
Zhu X, Tian S, Cai Z (2012) Toxicity assessment of iron oxide
nanoparticles in zebrafish (Danio rerio) early life stages. Plos
ONE 7:e46286. https://doi.org/10.1371/journal.pone.0046286