metal accumulation and antioxidant defenses in the freshwater fish carassius auratus in response to...

Accepted Manuscript

Title: Metal accumulation and antioxidant defenses in thefreshwater fish Carassius auratus in response to single andcombined exposure to cadmium and hydroxylatedmulti-walled carbon nanotubes

Author: Ruijuan Qu Xinghao Wang Zunyao Wang ZhongboWei Liansheng Wang

PII: S0304-3894(14)00313-6DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2014.04.051Reference: HAZMAT 15892

To appear in: Journal of Hazardous Materials

Received date: 25-11-2013Revised date: 20-4-2014Accepted date: 22-4-2014

Please cite this article as: R. Qu, X. Wang, Z. Wang, Z. Wei, L. Wang,Metal accumulation and antioxidant defenses in the freshwater fish Carassiusauratus in response to single and combined exposure to cadmium andhydroxylated multi-walled carbon nanotubes, Journal of Hazardous Materials (2014),http://dx.doi.org/10.1016/j.jhazmat.2014.04.051

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http://dx.doi.org/doi:10.1016/j.jhazmat.2014.04.051

http://dx.doi.org/10.1016/j.jhazmat.2014.04.051

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Highlights

Cd and OH-MWCNTs have a synergistic effect on Carassius auratus.

OH-MWCNTs significantly increased Cd accumulation in liver after 12 d exposure.

Co-exposure to Cd and OH-MWCNTs evoked severe hepatic oxidative stress.

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Metal accumulation and antioxidant defenses in the freshwater fish

Carassius auratus in response to single and combined exposure to cadmium

and hydroxylated multi-walled carbon nanotubes

Ruijuan Qu, Xinghao Wang, Zunyao Wang*, Zhongbo Wei, Liansheng Wang

State Key Laboratory of Pollution Control and Resources Reuse, School of Environment,

Nanjing University, Jiangsu Nanjing 210023, P. R. China

* Corresponding author

Address: State Key Laboratory of Pollution Control and Resources Reuse, School of

Environment, Xianlin Campus, Nanjing University, Jiangsu Nanjing 210023, P. R. China.

Tel: +86-25-89680358;

Fax: +86-25-89680358.

E-mail: [email protected].

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Abstract

The effects of cadmium, hydroxylated multi-walled carbon nanotubes, and their mixture on

metal accumulation and antioxidant defenses were studied using the goldfish Carassius

auratus as the test organism. The fish were exposed to 0.1 mg/L Cd, 0.5 mg/L OH-MWCNTs,

or 0.1 mg/L Cd + 0.5 mg/L OH-MWCNTs for 3 and 12 days. Then, the Cd concentration was

determined in the gill, liver and muscle. Moreover, hepatic antioxidant enzyme activity

(superoxide dismutase, catalase and glutathione peroxidase), glutathione level and

malondialdehyde content were also measured. A continuous accumulation of Cd was

observed throughout the experimental period. Cd accumulation in tissues occurred in the

following order: gill > liver > muscle at 3 days and liver > gill > muscle at 12 days. The

concentrations of Cd in the livers of fish exposed to the combination of Cd + OH-MWCNTs

were significantly higher than those in fish exposed to either single chemical after 12 d of

exposure. Meanwhile, the mixture evoked severe oxidative stress in the exposed fish, as

indicated by significant inhibition of SOD, CAT and GPx activity, a remarkable decrease in

GSH level, and simultaneous elevation of MDA content. These results suggested that the

effect of the combined factors on metal accumulation and oxidative stress biomarkers was

more obvious than that of single factors at longer exposure durations.

Keywords: cadmium; hydroxylated multi-walled carbon nanotubes; metal accumulation;

oxidative stress biomarkers

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1. Introduction

Among the most promising and uniquely engineered nanomaterials, carbon nanotubes

(CNTs) have received a great deal of attention in the development of nanotechnology. They

are commercially available as single-walled carbon nanotubes (SWCNTs) or multi-walled

carbon nanotubes (MWCNTs). Because of their unique chemical and physical characteristics,

CNTs are widely used in fields ranging from mechanical engineering to biomedicine [1].

However, due to their extremely hydrophobic nature, CNTs can aggregate and do not disperse

evenly in water [2]. An effective way of overcoming this difficulty is to functionalize the

material surface by introducing different moieties such as carboxyl, carbonyl and hydroxyl

groups. In recent years, a common type of functionalized CNTs, hydroxylated multi-walled

carbon nanotubes (OH-MWCNTs), has attracted much research interest [3, 4].

The ability to interact with biological tissues and generate reactive oxygen species (ROS)

has been proposed as possible mechanisms involved in nanoparticle toxicity [5]. ROS such as

superoxides, hydrogen peroxide, hydroxyl and other oxygen radicals are capable of directly

oxidizing DNA, polyunsaturated fatty acids in lipids, and amino acids in proteins [6]. It is

reported that exposure to CNTs has caused increased generation of ROS in different types of

cultured cells [7, 8]. By contrast, some researches have suggested that CNTs also has a ROS

scavenging effect [9, 10]. The difference may be due to the metal catalyst residues in various

CNT samples. Although CNTs can enter aquatic environments through sources such as

general weathering, disposal of CNTs-containing consumer products [11, 12], accidental

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spillages, and waste discharges [13], there has been limited ecotoxicological study on the

oxidative stress inducing potential of CNTs in aquatic organisms.

Cadmium (Cd) is considered a priority hazardous substance in the field of water policy

by the European Commission as well as a metal of primary interest by USEPA. It may

accumulate in the aquatic environment as a result of industrial and mining practices [14].

High concentrations in a body of water can be very harmful to the organisms in that

ecosystem. As a non-essential element, cadmium can disrupt cellular homeostasis, resulting

in DNA damage, membrane depolarization and cytoplasm acidification [15]. Moreover, it

may stimulate production of ROS, leading to alterations in antioxidant enzyme systems and

oxidative stress in affected organisms [16, 17].

Like all aerobic organisms, fish are susceptible to the effects of ROS generated by the

metabolism of exogenous compounds. Under normal physiological conditions, ROS and

other pro-oxidants are continually reduced to less reactive species by the antioxidant defense

system, which consists of antioxidant enzymes such as superoxide dismutase (SOD), catalase

(CAT) and glutathione peroxidase (GPx), and low-molecular-weight non-enzymatic

antioxidants such as reduced glutathione (GSH) [18]. If the production of ROS overwhelms

the antioxidative capacity of cells, an imbalance between the generation and removal of ROS

can produce oxidative stress, resulting in oxidative damage to multiple cellular targets [19].

In addition to changes in antioxidant levels, a central measure of oxidative stress is lipid

peroxidation (LPO), as indicated by the accumulation of malondialdehyde (MDA) [20].

Many oxidant-mediated biomarkers, including both enzymatic and molecular parameters,

have been successfully used in the environmental risk assessment of xenobiotics [21].

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In natural environment, different types of toxic substances are present at various

concentrations. According to recent studies on co-effects of carbonaceous nanomaterials and

various contaminants [22-26], it is reasonable to expect that the interactions of Cd with CNTs

can lead to effects on organisms different from what would be observed from the individual

chemical. In this work, we assessed Cd accumulation in different organs of Carassius auratus

following in vivo exposure to Cd, OH-MWCNTs, or their mixture. Because liver is the most

important site for biochemical processes associated with detoxification, oxidative stress status

was evaluated in the liver by measuring enzyme activities (SOD, CAT, GPx), GSH level and

MDA content. The freshwater goldfish was chosen as the test organism due to its extensive

distribution in China and its widespread use in ecotoxicological research.

2. Experiment

2.1 Chemicals and reagents

Cadmium sulfate octahydrate (3CdSO4·8H2O) of analytical grade was obtained from

Sinopharm Chemical Reagent Co., Ltd. The powder of OH-MWCNTs (20-40 nm in diameter,

90-120 m2/g in special surface area, and > 97% in purity) was supplied by Shenzhen

Nanotech Port Co. Ltd. The nanoparticles were characterized using a Switzerland ARL

X'TRA X-ray diffraction-meter for X-ray diffraction (XRD) spectra and a Japan JEOL

JEM-200CX electron microscope for transmission electron microscopic (TEM) imaging (Fig.

1). The kits for the analysis of oxidative stress biomarkers were purchased from Nanjing

Jiancheng Bioengineering Institute. Deionized (DI) water was prepared from a Millipore

Milli-Q water purification system.

<Fig. 1>

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An aqueous suspension of OH-MWCNTs was prepared by dispersing the nanoparticle in

DI water with sonication for 12 h (80 W, 32 kHz) in an ultrasonic cell disruptor (Nanjing

Emmanuel Instrument Equipment Co., China) until the absorbance at 400 nm was unchanged,

and a further 30-min sonication was performed immediately prior to each use. The metal

stock was prepared by dissolving a defined amount of 3CdSO4·8H2O into one liter of DI

water, and the Cd concentration was measured to be 1.00 g/L by a flame atomic absorption

spectrophotometer (SOLLAR M6, Thermo, USA).

2.2 Characterizations of the CNTs material

Transmission electron microscopic (TEM) images of the sample before and after

sonication were obtained with a JEM-200CX electron microscope (JEOL Co., Tokyo, Japan).

X-ray diffraction (XRD) pattern was recorded on a X'TRA X-ray diffraction-meter (ARL Co.,

Switzerland). Since metal catalyst impurities may be introduced into the carbon nanotubes

during production, the OH-MWCNTs was treated by the method of Hu et al. [27] to

determine the contents of residual catalyst: An aliquot of 50 mg of MWCNTs was added into

25 ml of 3 mol/L HNO3 solution, and the mixture was ultrasonically stirred for 24 h. The

suspension was filtrated and then rinsed with deionized water until the pH of the suspension

reached about 6. The leachates were collected, filtered through 0.22 μm PTFE membranes,

and measured for catalyst residues using an Inductive Coupled Plasma Mass Spectrometer

(NexION 300 ICP-MS spectrometer, USA). This experiment was repeated three times.

Thermal gravimetric analysis (TGA) was performed using a STA 449C thermal analyzer

(Netzsch, Selb, Germany) to study the thermal stability of the CNTs material. The sample

were heated under air from 25 to 1000 oC at a heating rate of 10 oC/min. Brunauer–Emmett–

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Teller (BET) surface area of the sample was measured using the nitrogen adsorption method

on a Micromeritics ASAP 2020 (Micromeritics Instrument Co., Norcross, GA) at 77 K. The

content of hydroxyl groups grafted on the surface was measured by using the Boehm titration

method [28].

2.3 Adsorption of Cd by OH-MWCNTs

The interaction of Cd with OH-MWCNTs was characterized by performing the

traditional adsorption experiment. Cadmium adsorption at a specific concentration of

OH-MWCNTs (5.0 mg/l) was investigated at various metal concentrations (0.05, 0.10, 0.20,

0.50, 1.00, 1.50 and 2.50 mg Cd/l). The mixed solutions were shaken for 24 h to achieve the

adsorption equilibrium in a dark, temperature controlled (25 ± 0.5 ºC) shaker. We confirmed

that equilibrium conditions were reasonably well achieved during the 1-day experiments

through previously conducted kinetic experiments (data not shown). To examine whether

there was any significant precipitation of Cd ions and metal adsorption was thus

overestimated, a control treatment containing the same concentrations of Cd but no

OH-MWCNTs was also applied. All treatments were performed in duplicate.

After equilibration, the OH-MWCNTs were separated from the liquid phase by passing

the mixture through a 0.22 μm nylon filter. Control experiments using metal ions in solution

without MWCNTs showed that the sorption by nylon filters was less than 0.5%. Other control

experiments using uniform suspensions of OH-MWCNTs in the absence of cadmium

suggested that the nylon filters removed 99.8% of the OH-MWCNTs from solution. In these

experiments, the concentration of OH-MWCNTs in suspension was quantified by measuring

the UV absorption at 400 nm. Following solid/liquid separation, the filtrates were collected

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for analysis of residual Cd ions on a SOLLAR M6 flame atomic absorption spectrometer. The

amounts of Cd ions adsorbed on OH-MWCNTs were determined by subtracting the

equilibrium mass of ions in solution from the initial aqueous ion mass.

2.4 Exposure protocol

Carassius auratus (initial body mass: 38.25 ± 3.65 g, mean ± SD, n = 15) from a local

aquatic breeding base were initially acclimatized in aquaria containing 150 L dechlorinated

and aerated freshwater for at least 10 days before the experiment. During the acclimatization

period, fish were fed twice a day with commercial pellets, and food residues and metabolic

wastes were removed daily. The water quality parameters of the dechlorinated tap water used

for acclimation and subsequent exposure experiment were as follows: temperature: 20 ± 1 ºC,

pH: 7.25 ± 0.25, conductivity: 340.6 ± 16.4 μS/cm, total hardness: 135.5 ± 9.3 mg CaCO3/L,

alkalinity: 40.7 ± 5.2 mg CaCO3/L. Ion levels were also measured, with the following results:

Na+: 11.2 ± 0.2 mg/L, K+: 2.34 ± 0.07 mg/L, Mg2+: 7.74 ± 0.02 mg/mL, Ca2+: 41.07 ± 0.82

mg/L and Cl-: 28.3 ± 1.2 mg/L. The aquariums were aerated to oxygen saturation with air

stones attached to an air compressor. When the total mortality was less than 1%, the toxicity

test was started.

A total of 40 acclimated fish were randomly divided into 4 groups of 10 fish. The three

trial groups were exposed to 0.1 mg/L Cd, 0.5 mg/L OH-MWCNTs, and 0.1 mg/L Cd + 0.5

mg/L OH-MWCNTs, respectively, while the control group received no addition of cadmium

or OH-MWCNTs. The Cd exposure dosage was selected based on a series of toxicity tests,

particularly those involving antioxidant responses of fish to Cd exposure [29, 30], and the

OH-MWCNTs concentration was chosen according to previous studies investigating the

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effect of nanoparticles on oxidative stress status in aquatic species [31, 32]. All experimental

fish were fed twice a day, and the exposure water was renewed daily.

2.5 Sample Preparation

On days 3 and 12, five fish per group were randomly chosen and sacrificed in ice-water

using clean equipment. Gill, liver and muscle tissues were dissected out, rinsed with cold

physiological saline (0.9% NaCl) to remove the adherent blood, dried in filter paper, placed

in individual acid-washed high-density polyethylene vials, and freeze-dried in a freeze drier

(Advantage Freeze Dryer, USA) for metal measurement. In addition, approximately 0.30 g of

liver was homogenized (1:10, w/v) in cold saline using an IKA T10 homogenizer (IKA,

Germany). The homogenates were centrifuged (Eppendorf, Germany) at 4000 g for 15 min at

4 ºC. The supernatants were collected and diluted to various concentrations for biochemical

analysis.

2.6 Metal measurement

The freeze-dried gill, liver and muscle samples were digested in nitric acid and sulfuric

acid (4:1, v/v) at 120 ºC for at least 2 h. Cooled digestates were transferred into polyethylene

volumetric flasks and brought to a final volume of 10 mL with 3.5% ultrapure nitric acid.

Two blanks were digested simultaneously during each run. The concentrations of Cd in the

tissues were measured using a SOLLAR M6 flame atomic absorption spectrophotometer.

Calibration curves were constructed daily based on five standards. Digestion blanks indicated

negligible contamination. All measurements were performed in duplicate and Cd

concentrations are presented as μg/g dry weight (d.w.). The efficiency of the digestion

method was evaluated by analyzing a certified reference material, NIST SRM 1946 (Lake

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Superior fish tissue), which has a certified value of 2.08 ± 0.26 μg/kg. The value we obtained

was 2.01 ± 0.22 μg/kg.

2.7 Biochemical analysis

The protein level, enzyme activities (SOD, CAT, GPx), GSH level and MDA content in

the supernatants were measured using the Diagnostic Reagent Kits according to the

manufacturer´s instructions. All absorbances were recorded with a TU-1810 UV-Vis

spectrophotometer (Persee, China).

Protein concentration was assayed at 595 nm following the method of Bradford [33],

using bovine serum albumin as a standard.

SOD activity was determined at 550 nm according to McCord and Fridovich [34], which

is based on the measurement of the inhibition of the reduction rate of cytochrome c by the

superoxide radical. The activity was expressed as U/mg protein, with one U of SOD

corresponding to the quantity of enzyme that caused 50% inhibition of cytochrome c

reduction.

CAT activity was evaluated by monitoring residual H2O2 absorbance at 405 nm [35].

The activity was expressed as U/mg protein. One U of CAT is the enzyme that decomposes 1

mM of hydrogen peroxide per min.

GPx activity, estimated by the rate of NADPH oxidation, was assayed at 412 nm

according to Flohe and Gunzler [36]. Its activity was also expressed as U/mg protein, with

one U of GPx corresponding to the amount of enzyme that depleted 1 μmol GSH in one

minute.

GSH level, in μmol/g protein, was measured at 420 nm following the method of Tietze

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[37] with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) as the reagent. DTNB was reduced by

free sulfhydryl groups of GSH to form yellow-colored 5-thio-nitrobenzoic acid.

MDA content, as a biomarker for lipid peroxidation, was determined at 532 nm by the

thiobarbituric acid reactive species (TBARS) assay, which measures the amount of MDA that

reacts with thiobarbituric acid [38]. The unit was nmol/mg protein.

2.8 Statistical analysis

All values are expressed as the mean ± standard deviation (SD). The data were checked

for normality of distribution using the Shapiro-Wilk test and for homogeneity of variance

using the Levene test. Intergroup differences were evaluated using one-way analysis of

variance (ANOVA) followed by Duncan’s test (P < 0.05). All statistical analyses were

performed using the SPSS statistical package (ver. 17.0, SPSS Company, Chicago, USA).

Graphs were plotted with Origin 8.0 (Origin Lab, USA).

2.9 Integrated biomarker response

The Integrated Biomarker Response (IBR) [39], a method for combining all the

measured biomarker responses into one general stress index, was applied to assess the

potential toxicity of different exposure protocols to fish. The procedure of IBR calculation is

briefly described here: First, data were standardized as Y = (X – m) / s, where X is the value of

each biomarker response, m is the mean value of the biomarker, and s is the standard

deviation of the biomarker. Next, we computed Z = Y in the case of activation or Z = –Y in the

case of inhibition. The minimum value (Min) was obtained for each biomarker. Finally, the

score (S) was computed as S = Y + |Min|, where S ≥ 0 and |Min| is the absolute value of Min.

Star plots were used to display the biomarker results. A star plot radius coordinate

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represents the score of a given biomarker. When Si and Si+1 are assigned as two consecutive

clockwise scores of a given star plot, n is assigned as the number of radii corresponding to the

biomarkers, and the area Ai by connecting the ith and the (i+1)th radius coordinates can be

obtained as:

1sin ( cos sin )

2i

i i i

SA S Sβ β β

+= + ,

where

1

1

sintan( )

cosi

i i

SArc

S S

αβ

α+

+

=−

,

α is 2π/n radians, and Sn+1 is S1.

The total area corresponding to a given situation (IBR value) was obtained as:

1

IBRn

i

i

A=

= ∑ ,

where n is the number of biomarkers.

3. Results

3.1 Characterizations of OH-MWCNTs samples

TEM images of OH-MWCNTs nanoparticles before and after sonication are presented in

Fig. 1 A and B. As can be seen, these samples are almost entirely nanotubes with little to no

contamination with respect to other forms of carbon. The outer diameter of the tubular

structures is in the range of 20-40 nm and the length ranges from several hundreds of

nanometers to several micrometers. These values are largely consistent with those reported by

the manufacturer. No obvious difference in the morphology of OH-MWCNTs is observed

after ultrasonic dispersion. The crystal structures of as-received and ultrasound-treated

nanotubes were determined using XRD (Fig. 1 C and D). The diffraction peaks at 2θ = 26.5°,

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42.4°, and 54.7° can be attributed to the hexagonal graphite structures (002), (100), and (004)

[40]. Metal peaks are not identified, likely due to the low content and/or small particle sizes

of the metallic species in the sample. Ultrasonication treatment does not obviously change the

main characteristic diffraction peaks of OH-MWCNTs except that a weak peak is observed at

40.4°.

<Fig. 1>

The purity and thermal stability of OH-MWCNTs was evaluated by thermogravimetric

analysis under air atmosphere (Fig. 2). During the initial heat treatment stage, the sample

showed a slight mass loss and this was ascribed to the decomposition of amorphous carbon or

moisture/volatile material (such as residual solvent from the synthesis) degassing from the

material at the lowest temperatures [41, 42]. The CNTs was thermally stable up to the

temperature of 280 °C. Oxidation became rapid at about 325 °C, and approximately 7.20% of

the sample remained behind after performing TGA up to 1000 °C. The residues may be

metals and metal oxides, which reside inside the nanotubes before combustion.

<Fig. 2>

The metal concentrations in the leachates of the OH-MWCNTs sample as determined by

ICP-MS are listed in Table 1. The concentration of Ni, Fe, Pb and Cu was the highest,

followed by Cr, Co, Zn and Ti, and then Mo. It is noteworthy that Cd concentration in the

solution was too low to be quantified, which may suggest that the potential release of Cd was

not responsible for the toxicity of the OH-MWCNTs. Using N2-BET method, the specific

surface area of the raw OH-MWCNTs was estimated to be 103 m2/g. The concentration of the

surface hydroxyl groups on OH-MWCNTs was measured as 0.72 mmol/g, indicating that the

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carbonaceous material is soluble in pure water.

<Table 1>

3.2 Cadmium adsorption onto OH-MWCNTs

The amount of adsorbed Cd increased significantly in the range of low concentrations,

and it then increased gradually and reached 29.5 mg/g at an equilibrium Cd concentration of

2.45 mg/l (Fig. 3). The experimental data for Cd adsorption by OH-MWCNTs were analyzed

using the linear Langmuir sorption isotherm model as

qe = KaqmCe / (1 + KaCe)

where Ce is the equilibrium metal ion concentration (mg/l), qe is the amount adsorbed (mg/g),

qm is the maximum sorption capacity corresponding to complete monolayer coverage (mg/g)

and Ka is the Langmuir constant indirectly related to the energy of adsorption (l/mg).

The correlation coefficient r2 is 0.97, close to 1, suggesting that all data points could be

well fitted to the Langmuir sorption model and that the adsorbed cadmium ions form a

monolayer coverage on the surfaces of OH-MWCNTs. The qm value calculated by the

Langmuir equation is 32.89 mg/g. It is clear that OH-MWCNTs can concentrate Cd from

water.

<Fig. 3>

3.2 Cadmium accumulation in fish

When OH-MWCNTs nanoparticles were added into water, aqueous Cd concentration

decreased from 0.1 to 0.093 mg/l due to the weak adsorption onto the particles. Cadmium

accumulation in different fish tissues (gill, liver and muscle) of Carassius auratus after single

and combined exposure to Cd and OH-MWCNTs is shown in Fig. 4. From this figure we can

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see that cadmium concentration in all tissues increased steadily over time in the three

exposure groups. After 3 d of exposure, a significant accumulation of cadmium was observed

in gills of fish exposed to Cd or the mixture (1.75 and 2.01 times, respectively). However,

levels of cadmium in the liver and muscle, which were lower than in the gill, did not

significantly change across the different experimental groups. At the end of 12 d exposure,

cadmium concentrations in gill and liver tissues exposed to Cd or the mixture was

significantly higher than the respective control. The highest cadmium accumulation (4.87

times control) was observed in liver exposed to the mixture.

<Fig. 4>

3.2 Oxidative stress biomarkers in liver tissue

Activity values of the antioxidant enzymes are shown in Table 2.

<Table 2>

No significant change in SOD activity was observed after 3 d of exposure in any of the

groups. However, a longer exposure time caused significant inhibition (reduced by 18.1%) in

SOD activity for the co-exposure group. Still, the single exposure group exhibited no

significant difference from control.

The CAT activity was significantly inhibited (reduced by 14.8%) after exposure to the

mixture for 3 days. However, it did not significantly vary at the single exposure in respect to

the control. At day 12, both the OH-MWCNTs and the Cd + OH-MWCNTs group showed

significant decreases (reduced by 19.1% and 52.4%, respectively) in CAT activity.

Additionally, the presence of OH-MWCNTs at 0.1 mg/L Cd significantly decreased the CAT

activity by 17.4% and 55.2%, while the significant reduction caused by the addition of Cd at

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0.5 mg/L OH-MWCNTs was 12.1% and 41.2%, for 3 d and 12 d of exposure, respectively.

Similar to CAT, only the co-exposure group presented significantly lowered GPx

activity (reduced by 19.1%) compared to that in the control at 3 d. This situation was

unchanged on the 12th day, for which a significant difference from control was only observed

in the Cd + OH-MWCNTs group, with an inhibition of 18.4%. The two single-exposure

groups all showed GPx activities similar to the control values.

Changes in the hepatic GSH level and MDA content are shown in Fig. 5.

<Fig. 5>

There was no significant difference in GSH levels among experimental groups at day 3.

In contrast, GSH level in Carassius auratus exposed to the mixture (8.53±0.44 μmol/g

protein) was significantly decreased in relation to the control level (10.43±0.69 μmol/g

protein) after 12 d of exposure (see Fig. 4 A).

Compared to the control values, the MDA content was significantly increased in the

co-exposure group after 3 and 12 days. In the presence of 0.1 mg/L Cd, the addition of

OH-MWCNTs significantly increased MDA production from 0.74±0.18 to 0.99±0.19

nmol/mg protein for 3d and from 1.26±0.26 to 1.73±0.21 nmol/mg protein for 12d,

respectively. At each of the two sampling times, no significant difference in MDA content

was observed between the single-exposure groups and their respective controls except for the

12 d Cd group (see Fig. 4 B).

3.4 Integrated biomarker response

Based on the IBR, the ranking of the most affected groups was as follows: Cd +

OH-MWCNTs > OH-MWCNTs > Cd > Control for 3 days; Cd + OH-MWCNTs > Cd >

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OH-MWCNTs > Control for 12 days (Fig. 6). With the prolonged exposure time, this index

increased for the Cd single-exposure group but decreased for the OH-MWCNTs

single-exposure group. The largest IBR values were 9.20 and 12.35 for the 3 d and 12 d

exposures, respectively.

<Fig. 6>

4. Discussion

The Langmuir equation is widely used to model the adsorption processes of metal ions.

It assumes a monolayer coverage of adsorbate over a homogeneous adsorbent surface and the

adsorption of each molecule onto the surface has equal adsorption activation energy [43]. Up

to now, many researches have been conducted to investigate the adsorption of Cd2+ from

aqueous solution by raw and surface oxidized CNTs. Li et al. [44] suggested that Cd2+

adsorption by nitric acid treated multiwalled carbon nanotubes follows the Langmuir

equation and the maximum adsorption capacity qm was calculated to be 10.86 mg/g at an

equilibrium Cd2+ concentration of 10 mg/l. Cho et al. [45] applied the Langmuir isotherm

model to fit the Cd2+ adsorption equilibrium data and the qm value thus obtained was 5.60,

20.16 and 24.64 mg/g for the pristine, HNO3 and HNO3/H2SO4 treated MWCNTs,

respectively. Vukovic et al. [46] found that the Langmuir model better describes Cd2+

adsorption onto MWCNTs than the Freundlich model and the maximum Cd2+ adsorption

capacity on raw-MWCNTs, oxidized MWCNTs and ethylenediamine-functionalized

MWCNTs at 25 ºC reaches 1.26, 22.39 and 21.67 mg/g, respectively. These reported values

were generally lower than ours (32.89 mg/g). This could be explained by the difference in the

properties of the carbon sorbent, solution pH, ionic strength and temperature.

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It is known that cadmium is rarely distributed uniformly within the body tissues of fish,

but rather accumulates differentially [47]. Moreover, the distribution of the metal

accumulated in different organs can vary depending on the exposure route, the concentration

and duration of exposure to contaminants, and the fish species. In the present work, we

observed a continuous accumulation of Cd throughout the experimental period. Higher Cd

concentrations in gills than in liver and muscle were found in all the test animals after a 3 d

exposure period. This result is consistent with the finding of Kraal et al. [48] that cadmium

accumulation in different tissues in the carp (Cyprinus carpio) was decreased in the following

order intestine > gills > kidney > liver > muscle when the fish were exposed to

Cd-contaminated water. Because aqueous Cd ions that are in direct contact with gills may

bind in a non-specific manner to the mucopolysaccharides (constituents of mucoproteins,

which are glycoproteins) present on the outside of the gills, gills may be the first target for Cd

accumulation before its distribution to other organs [49]. After Cd absorption by the gills, the

metal is probably transferred to storage organs such as the liver or kidney at longer exposure

periods. Our data showed that Cd accumulation in liver was more evident than in gill and

muscle after 12 d of exposure. In this context, one may suppose that long-term exposure to

the metal permits transfer of Cd via blood transport from the gill to the liver which is

involved in storage and metal detoxification. In fact, the liver is considered the most

important organ for detoxification in acute exposure [50]. During the entire exposure period,

Cd concentration in fish muscle was always lower than in the other two tissues. This could be

explained by the large fraction of muscle tissue in comparison with the total fish weight, such

that the Cd accumulated in this tissue was diluted into a larger total mass than in organs such

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as liver and gill. No significant Cd accumulation in muscle was also reported in other studies

[51, 52].

When exposed to Cd in the presence of OH-MWCNTs, the cadmium concentrations in

gill, liver and muscle in the fish were always higher than those without OH-MWCNTs. A

significant difference between the two groups was found in liver on day 12. The enhanced

bioaccumulation of metals was also observed for titanium dioxide nanoparticles. For example,

Zhang et al. [53] showed that Cd concentration in carp exposed to Cd + P25 TiO2

nanoparticles reached 22.3 μg/g after 25 d of exposure, while the concentration in carp

exposed to Cd-contaminated water was only 9.07 μg/g. Besides, the existence of nano-TiO2

enhanced the copper accumulation in D. magna by 18-31% [54]. As indicated by the

adsorption isotherm experiment, a large amount of Cd was adsorbed onto OH-MWCNTs.

Thus, the free dissolved Cd concentration in water was decreased by the presence of

OH-MWCNTs. The underlying mechanism for the elevated cadmium concentration in the

co-exposure group seems to be that fish could directly ingest OH-MWCNTs particles from

water via gills and actively ingest the nanoparticles through intestines [55]. Inside the body of

the fish, the bound Cd on the surface of these particles may be released, distributed and

accumulated in liver, kidney or other organs. A similar ‘‘Trojan-horse effect’’ was reported in

Limbach’s research [56], where NPs served as carriers of ROS-inducing transition metals

such as cobalt into cells.

In this study, no mortality was observed during the single and combined exposures to

cadmium and carbon nanotubes for 12 days. Rodriguez-Ariza et al. [57] suggested that the

biochemical responses (e.g., oxidative stress) are often more likely than acute lethality for

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exposure to conventional chemicals. The ability of organisms to survive in contaminated

environments is principally due to their inducible defense mechanism that allows

detoxification and antioxidant protection. Antioxidant enzymes (e.g., SOD, CAT and GPx)

and numerous non-enzymatic antioxidants (e.g., GSH) have always been considered the

important components of the antioxidant defense system in animals. They can be induced as a

compensatory response to a mild oxidative stress. However, excess ROS produced by

xenobiotics can overwhelm the detoxifying or antioxidant mechanism, a condition that causes

significant oxidative damage and a loss of the compensatory mechanisms, resulting in the

suppression of antioxidant enzymes activities [58].

Since nanomaterials may be accumulated in the tissues of organisms and they can cause

artifacts due to the potential to confound toxicity assays [59], it is important to ensure that the

research results are not from an artifact. To assess if the presence of OH-MWCNTs in the

liver could influence our bioassays, an additional experiment was conducted following the

same procedures as described in the second section except that an appropriate amount of

OH-MWCNTs suspension was added before homogenization to make the final concentration

of OH-MWCNTs to be 0.05, 0.5 and 5 mg/l, respectively. From those data in Table 3, we can

clearly see that the five biomarkers in the three MWCNTs-added groups are not significantly

different from the control values. Moreover, to our knowledge, there have been no reports

suggesting that the presence of OH-MWCNTs will interfere with the enzyme bioassays.

These evidences allow us to conclude that our experimental results are reliable.

<Table 3>

The antioxidant enzymes SOD and CAT are considered the vital first-line defenses

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against oxygen toxicity. They have related functions and are essential for the conversion of

ROS to harmless metabolites. In particular, SOD works to catalyze dismutation of the

superoxide anion to H2O and H2O2, while CAT reduces H2O2 to non-toxic H2O and O2 [60].

An inverse relationship between the SOD and CAT activities was observed in most exposure

groups, i.e., stimulation of the SOD activity was accompanied by suppression of the CAT

activity, and vice versa. It is speculated that the increase in SOD may lead to the

accumulation of H2O2, which will restrain the function of CAT. In turn, CAT showed the

capacity to decompose H2O2 when SOD displayed low activity. In short, they functioned

together to maintain the balance between ROS production and antioxidant defense.

Following in vivo exposure to Cd, OH-MWCNTs and the mixture, a common decrease

in GSH and GPx was observed in the fish. As the second-line defenses against oxidative

damage, GSH and GSH-related enzymes play a major role in reduction of peroxides and

free-radical scavenging [20]. GSH is the main non-protein thiol in cells that quenches

oxyradicals through its sulfhydryl group, and it serves as an available co-substrate for GPx

which can also catalyze the reduction of hydroperoxides into hydroxyl compounds. Similar to

our findings, previous studies also showed lowered GSH levels after Cd exposure [61, 62].

This may be due to the high Cd accumulation in the cells, with Cd reacting with GSH to form

GSH–metal complexes, which may decrease the GSH content. Moreover, Reddy et al. [63]

reported GSH depletion in human embryonic kidney (HEK293) cells exposed to MWCNTs

(10-100 mg/l) for 48 hours. The inactivation of GPx may be caused by reduced GSH because

GPx is highly dependent on GSH concentration. Decreased intracellular GSH levels and

simultaneous inhibition of GSH-related antioxidant enzymatic activity could lead to oxidative

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imbalance and subsequent cell death, as suggested by Pandey et al. [64].

In addition, because the typical response to oxidative stress is peroxidative damage to

unsaturated fatty acids, an increase in the LPO level has been extensively used as a marker of

oxidative damage in organisms. The reactive carbonyls produced during lipid peroxidation

may diffuse from the original site of radical production, causing damage to inter- and

intra-cellular targets. The enhanced lipid peroxidation in the Cd-treated fish after 12 days is

probably due to the displacement of redox metal ions (i.e., iron) or to a decrease in

glutathione content [65, 66]. Moreover, Cd can act as a catalyst of the Fenton reaction,

prompting the conversion of the superoxide anion and of the hydrogen peroxide to a hydroxyl

radical; these oxygen species are proposed as a trigger of lipid peroxidation [67].

Compared with single exposure to Cd or OH-MWCNTs, the mixture of Cd and

OH-MWCNTs elicited more serious antioxidant responses. The depletion of SOD, CAT, GSH

and GPx activity and elevated levels of MDA is strongly correlated to ROS generation and

lipid peroxidation, indicating the induction of marked oxidative stress in fish following

co-exposure to the two chemicals. This finding was confirmed by the IBR result, which

provides a simple tool for the visualization of biological effects by combining different

biomarker signals. The usefulness of this index has been previously demonstrated in

environmental studies, regardless of the considerable variability in the biomarker sets,

contamination profiles and species used [68, 69]. Generally, the higher the IBR value is, the

more stressful the environment is. According to the IBR index, the toxicant-induced stress

was more serious in the co-exposure group.

Conclusions

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Based on these results, we could conclude that the co-exposure will affect the metal

accumulation and antioxidant defense systems of the goldfish in a way different from the

single exposure. The presence of OH-MWCNTs in Cd-contaminated water greatly enhanced

the accumulation of cadmium in the liver tissue after 12 d of exposure when compared with

the Cd or OH-MWCNTs exposure group. Correspondingly, changes in the five hepatic

oxidative stress biomarkers in Carassius auratus exposed to the mixture were more obvious

than in the fish exposed to only Cd or OH-MWCNTs. Our findings suggest that future

toxicology studies should focus not only on the inherent toxicity of individual chemicals, but

should also consider the possible interactions with existing environmental contaminants and

how these interactions may influence the behavior, effects, and fate of each other in a natural

environment. Such information will be critically important in the risk assessment of various

pollutants, and more research is required in this field.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of

China (No. 41071319, 21377051) and the Scientific Research Foundation of Graduate School

of Nanjing University (2013CL08).

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Table 1 Concentrations of metals in OH-MWCNTs samples as determined by ICP-MS analysisa.

Metals (μg/g) Cr Mo Co Ni Zn Cd Pb Fe Cu Ti

OH-MWCNTs 0.161 0.045 0.101 4.783 0.101 ndb 2.989 4.104 1.750 0.116

a The relative standard deviation was within ±5%.

b nd: not detectable.

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Table 2 Antioxidant enzymes (SOD, CAT and GPx) in liver of Carassius auratus after 3 and 12 d of

exposure to Cd, OH-MWCNTs and their mixture.

Enzymes Duration(d) Control Cd OH-MWCNTs Cd+OH-MWCNTs

SOD 3 52.02±4.61a 50.32±2.80a 54.84±8.23a 56.12±5.08a

12 55.63±5.69b 50.40±6.85ab 57.12±5.69b 44.32±3.01a

CAT 3 18.34±1.08b 18.90±2.44b 17.78±1.68b 15.62±0.56a

12 20.22±1.02c 21.46±1.62c 16.35±2.47b 9.62±0.61a

GPx 3 88.23±4.02b 84.27±2.15ab 83.37±5.88ab 79.89±4.17a

12 94.64±7.21b 97.51±6.15b 90.34±6.21b 77.18±4.53a

Values are mean ± SD, n = 5. Values that do not share the same superscript letter (a, b, c) are significantly

different (P < 0.05).

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Table 3 Values of the five biomarkers after incubation with the OH-MWCNTs material in fish liver.

Biomarkers Control 0.05mg/l-CNTs 0.5mg/l-CNTs 5mg/l-CNTs

SOD 38.91±1.61a 36.62±4.74a 38.45±3.23a 35.09±2.77a

CAT 27.73±1.92a 26.83±2.94a 28.57±3.80a 25.00±2.43a

GPx 62.69±9.69a 63.07±6.34a 65.83±8.76a 64.15±8.13a

GSH 13.58±0.46a 12.60±1.22a 13.03±0.31a 13.25±0.83a

MDA 4.65±0.30a 4.88±0.28a 4.46±0.20a 4.72±0.39a

Values are mean ± SD, n = 4. Values that share the same superscript letter are not significantly different (P

< 0.05).

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Fig. 1 TEM images and XRD spectra of OH-MWCNTs nanoparticles before (A, C) and after (B, D)

ultrasonication.

Fig. 2 Thermogravimetric (TG) curves of pristine OH-MWCNTs.

Fig. 3 Adsorption isotherm for Cd ions at a concentration of 5.0 mg/l OH-MWCNTs. All the values are the

means of 2 replicates, and the standard deviations are sometimes smaller than the dot sizes.

Fig. 4 Cadmium concentration in different fish tissues (gill, liver and muscle) of Carassius auratus under

exposure to Cd, OH-MWCNTs and their mixture for 3 and 12 days. Values are the mean ± SD, n = 5.

Values that do not share the same superscript letter (a, b, c) are significantly different (P < 0.05).

Fig. 5 The GSH level (A) and MDA content (B) in liver of Carassius auratus after 3 and 12 d of exposure

to Cd, OH-MWCNTs and their mixture. Values are the mean ± SD, n = 5. Values that do not share the

same superscript letter (a, b, c) are significantly different (P < 0.05).

Fig. 6 Integrated biomarker response (IBR) of all parameters measured in liver tissue of Carassius auratus

for different exposure protocols.

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0 200 400 600 800 1000

Wei

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(%)

Temperature (oC)

Figure 2

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5

10

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qe (m

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Ce (mg/l)

Figure 3

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14 Control

Cd

OH-MWCNTs

Cd + OH-MWCNTs

3 days 12 days

Figure 6

metal accumulation and antioxidant defenses in the freshwater fish carassius auratus in response to...

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