Toxic effects of copper ion in zebrafish in the joint presence of CdTe QDs

Environmental Pollution 176 (2013) 158e164

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Toxic effects of copper ion in zebrafish in the joint presence of CdTe QDs Wei Zhang a, b, c, *, Youna Miao a, b, c, Kuangfei Lin a, b, c, *, Lin Chen a, b, c, Qiaoxiang Dong d, Changjiang Huang d a

State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China University of Science and Technology, Shanghai 200237, PR China b Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, PR China c School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, PR China d Zhejiang Provincial Key Lab for Technology and Application of Model Organisms, Wenzhou Medical College, Wenzhou 325035, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 September 2012 Received in revised form 24 January 2013 Accepted 25 January 2013

Quantum dots (QDs) have strong adsorption capacity; therefore, their potential toxicity of the facilitated transport of other trace toxic pollutants when they co-exist to aquatic organisms has become a hot research topic. The lab study was performed to determine the developmental toxicities to the zebrafish after exposed to the combined pollution of Cadmium-telluride (CdTe) QDs and copper ion (Cu2þ) compared to the single exposure. Our findings for the first time revealed that: 1) CdTe QDs facilitated the accumulation of Cu2þ in zebrafish, 2) the higher mortality, lower hatch rate, and more malformations can be clearly observed, 3) the diverse vascular hyperplasia, turbulence, and bifurcation of the exposed FLI-1 transgenic zebrafish larvae appeared together, 4) the synergistic effects played more important role during joint exposure. These observations provide a basic understanding of CdTe QDs and Cu2þ joint toxicity to aquatic organisms. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Quantum dots Heavy metal Zebrafish Facilitated transport Toxicological effect

1. Introduction Quantum dots (QDs) have been employed in many recent applications, such as solar cells, light-emitting devices, biological and medical imaging because of their small size, bright fluorescence, narrow emission spectra, broad absorption spectra, and high photostability (Nowack and Bucheli, 2007; Wang et al., 2010). The QDs’ potential biological toxicity has become a health concern due to their inherent chemical composition and nanoscale properties (Worms et al., 2012). Literature reports show that the expression patterns of miRNAs are widely affected after Cadmium-telluride (CdTe) or Cadmium-selenium (CdSe) QDs exposure, resulting in the apoptosis-like cell death (Derfus et al., 2004; Li et al., 2011). CdTe QDs hold the greatest commercial potential (Zhao et al., 2009). Although there is an increasing amount of research on the ecotoxicity of CdTe QDs, little information is currently known about their potential influence on the transfer and toxicity of other coexisting pollutants in the aquatic environment. Previous studies indicated that the large surface area, crystalline structure, and reactivity of some nanoparticles may facilitate the transport of toxic chemicals (Zhang and Masciangioli, 2003; Zhang et al., 2007).

* Corresponding authors. E-mail addresses: [email protected] (W. Zhang), kfl[email protected] (K. Lin). 0269-7491/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2013.01.039

Hence, how and to what extent the emerging QDs may influence the transfer and ecorisk of toxic pollutants should be addressed as an important research hotspot. Moreover, the aggregation characteristics in the organisms can further deepen the toxic effects (Rispoli et al., 2010; Clément et al., 2013). The contamination of heavy metal in natural environment has always been a great concern because they are toxic and nonbiodegradable. Copper (Cu) is an abundant trace element found in a variety of rocks and minerals and has received much attention due to its widespread use, persistent nature, tendency to accumulate and toxicity to aquatic organisms (Sloman and Wilson, 2006). There are many documented physiological effects of waterborne Cu2þ exposure in a variety of fish species. Johnson investigated the effects of Cu2þ on the morphological and functional development of zebrafish embryos (Johnson et al., 2007). In excess, Cu2þ will target the gills, gut and sensory systems causing higher mortality and more malformation (Grosell et al., 2003; Shaw and Handy, 2006). The colloid-facilitated transport of heavy metal pollutants has been implicated in a number of studies (Grolimund et al., 1996). Sun and Zhang found that when cadmium (Cd) or arsenite (As) co-existed with titanium dioxide (TiO2) nanoparticles, their bioaccumulation into carp increased significantly due to the carrying effect of TiO2 nanoparticles, and there was a positive correlation between Cd/As and TiO2 concentration (Sun et al., 2007; Zhang

W. Zhang et al. / Environmental Pollution 176 (2013) 158e164

et al., 2007). However, there is no data indicating if the emerging QDs may facilitate the accumulation and toxicity of heavy metal in the environment. CdTe QDs and Cu contents might increase in environmental water samples via waste streams from industries that synthesize or use and through research uses (Boeck et al., 2006; Gagné et al., 2008; Zhang et al., 2009; Bhuiyan et al., 2010; Wang et al., 2012). During long-term exposure period due to its larger surface area and stronger electrostatic attraction Cu2þ might be expected to associate with QDs and then jointly enter into aquatic organisms. Therefore, there is an urgent need to address several critical CdTe QDs and Cu2þ associated joint exposure toxicity to aquatic organisms, as well as identification of biomarkers for early signaling (Zhao et al., 2009; Yan et al., 2011; Li et al., 2012). Zebrafish has increasingly been employed as a model vertebrate for investigating the developmental toxicity of the potential ecotoxicological impacts of nanoparticles releases to aquatic environments (Usenko et al., 2007; Zhu et al., 2007). Embryo-larval (EL) toxicity tests are generally more sensitive than toxicity tests with juvenile and adult fish. For many pollutants, EL toxicity tests are as sensitive or almost as sensitive as chronic (life cycle) toxicity tests (McKim, 1977). However, there has been no report of employing zebrafish embryos and larvae for CdTe QDs and Cu2þ joint toxicity. In the present study, zebrafish was employed to examine the potential of CdTe QDs to facilitate the transport of Cu2þ, and then study the toxic responses associated with different doses of CdTe QDs and Cu2þ joint exposure such as mortality, hatch rate, malformation, and vascular patterns. 2. Material and methods 2.1. Chemicals Thioglycolic acid (TGA) is a classical short and straight-chain stabilizing agent and its small steric hindrance can make surface passivation of QDs more effective than the long and branched-chain one, and according to the research report TGAcoated CdTe QDs are highly stable (Tian et al., 2009; Jhonsi and Renganathan, 2010; Fan et al., 2011). Water-soluble TGA-CdTe QDs were purchased from Nanosquare Inc. (Tokyo, Japan). Copper chloride (CuC12) and other chemicals were purchased from Merck (Darmstadt, Germany). All chemicals used in this study were analytical grade. 2.2. Stock solutions of TGA-CdTe QDs TGA-CdTe QDs solutions used in the treatments were centrifuged at 2000 rpm for 5 min, dialyzed by 10 kDa membrane dialysis pores (Shanghai Green Bird Science & Technology Development Co., China) against TGA and placed in distilled water at pH 10 for 5 h at 25  C to remove any traces of cadmium and Telluride. After purification, TGA-CdTe QDs were observed to have an irregular shape with a typical size of approximately 3.5 nm by Transmission Electron Microscopy (TEM) (Fig. 1A). The stock solution (1150 mg/L) was prepared by dissolving TGA-CdTe QDs in 60 mg/L ocean salt which hardly resulted in toxic effect on zebrafish in previous study (Miao, 2010); TGA-CdTe solution were highly stable and hardly resulted in the aggregation in terms of QDs properties (i.e., shape and size) during the entire exposure period (Fig. 1B).

159

2.3. Fish husbandry and embryo collection Adult zebrafish of wild-type strain (AB) were raised and kept at 28  0.5  C with a 14:10 dark/light photoperiod (lights on at 8:00 a.m.) in a recirculation system according to standard zebrafish breeding protocols (Westerfield, 1995). Water supplied to the system was filtered by reverse osmosis (pH 6.5e7.5), and instant ocean salt was added to the water to raise the conductivity to 450e1000 uS/cm. Zebrafish were fed twice daily with 20 g of live Artemia (Jiahong Feed Co., Tianjin, China) and dry flake diet (Zeigler, Aquatic Habitats, Apopka Florida, USA). The developmental status of zebrafish embryos and larvae were observed with an Inverted Microscope (8e50, Olympus, Japan). Zebrafish embryos used for chemical exposure were obtained from spawning adults in tanks overnight with the sex ratio of 1:1. Embryos were collected within 1 h after the light was switched on and rinsed in embryo medium. The fertilized and normal embryos were inspected and staged for the following experiment under a stereomicroscope (Nikon, Japan) according to Kimmel’s descriptions (Kimmel et al., 1995). FLI-1: EGFP transgenic zebrafish larvae were purchased from Molecular Toxicology Research Center of Oregon State University (USA) and can exhibit green fluorescence under fluorescence spectroscopy (Huang et al., 2010; Wang et al., 2011). 2.4. Zebrafish toxicity test In order to determine the exposure concentrations, based on previous toxicity information about these compounds, we had carried out a great deal of preliminary experiments. To determine the LC50, normal embryos were exposed to the TGA-CdTe solutions (0.12, 3, 6, 12, 24, 36, 48 mg/L), and the 120 h LC50 of TGA-CdTe was calculated to be 22.31 mg/L, and after zebrafish exposed to 6 mg/L level the mortality was only 10.26% (Zhang et al., 2012). Moreover, 100 mg/L of Cu2þ single exposure just resulted in 10.67% zebrafish mortality. Primary tests results showed that 6 mg/L of TGA-CdTe and 100 mg/L of Cu2þ joint exposure can result in significant difference or obvious symptom compared to the controls for each toxicological endpoint. So we chose the 6 mg/L of TGA-CdTe QDs, 0, 0.1, 10, and 100 mg/L of Cu2þ as exposure concentration. Exposure period of each experiment were dependent on zebrafish developmental stage. Such experimental methods had literature support (Zhu et al., 2007; Huang et al., 2010; Wang et al., 2011). Normal embryos were kept in sterile 96-well plates, with one embryo per well containing 200 mL solutions. Plates were covered with sealing films to prevent evaporation. For each treatment, 30 embryos were performed in light-controlled incubator. After exposure the body burden, mortality, hatch, malformation, and vascular patterning of zebrafish were observed, respectively. The exposure concentrations and period, and toxicological endpoint of zebrafish used for each experiment were listed in Table 1. Cu2þ concentrations in the digested samples was measured using graphite-furnace atomic-absorption spectrophotometry (Hitachi Zeeman Z-8200) following Chen’s descriptions (Chen et al., 2011). Normal embryos were jointly exposed to TGA-CdTe QDs (6 mg/L) and CuCl2 (0, 0.1, 10, 100 mg/L) from 6 to 120 hpf (hours post fertilization), and then the body burden and mortality of zebrafish at 120 hpf, the hatch rates of embryos at 72 hpf, the malformation of zebrafish at 96 hpf, were observed and recorded. Moreover, FLI-1: EGFP transgenic zebrafish larvae were jointly exposed to TGA-CdTe QDs (6 mg/L) and CuCl2 (100 mg/L) from 6 to 96 hpf; and their vascular patterns at 96 hpf were further observed by Fluorescence Inverted Microscope (Nikon TE2000-U, Japan). 2.5. Data statistical analysis Each treatment was replicated three times; the results were reported as the average of three parallel determinations of the mixture of three replicated samples. The figures were completed using origin 8.0 (OriginLab, Northampton, MA, USA). One-way ANOVA was applied to calculate statistical significance followed by

Fig. 1. A) The TEM image of the original TGA-CdTe QDs; B) The TEM image of the TGA-CdTe QDs in the zebrafish media solution after 120 h exposure period.

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Table 1 Experimental program of the toxicity study. Exposure concentration 6 6 6 6 6

mg/L mg/L mg/L mg/L mg/L

TGA-CdTe; TGA-CdTe; TGA-CdTe; TGA-CdTe; TGA-CdTe;

0, 0.1, 10, 100 mg/L 0, 0.1, 10, 100 mg/L 0, 0.1, 10, 100 mg/L 0, 0.1, 10, 100 mg/L 100 mg/L CuCl2

CuCl2 CuCl2 CuCl2 CuCl2

Kind of fish

Exposure period

Toxicological endpoints

Results

Normal embryo (n ¼ 30) Normal embryo (n ¼ 30) Normal embryo (n ¼ 30) Normal embryo (n ¼ 30) FLI-1 transgenic larvae (n ¼ 30)

6e120 hpf 6e120 hpf 6e72 hpf 6e96 hpf 6e96 hpf

Body burden (120 hpf) Mortality (120 hpf) Hatch rate (72 hpf) Malformation (96 hpf) Vascular pattern (96 hpf)

Fig. Fig. Fig. Fig. Fig.

Dunnett’s test as a post hoc test to independently compare each exposure group to the control group. All statistical analyses were run separately by performed using SPSS 16.0 software (SPSS, Chicago, USA). The data were shown as mean  standard error.

3. Results 3.1. The body burden determination of Cu2þ in zebrafish larvae in the presence of TGA-CdTe QDs at 120 hpf As displayed in Fig. 2, during the entire exposure period, the body burden of Cu2þ in zebrafish larvae in the presence of TGACdTe QDs generally increased with increasing Cu2þ concentration following certain doseeresponse relationships. Compared with the single exposure of Cu2þ, the presence of TGA-CdTe QDs can result in higher concentration of Cu2þ in all the treated groups. And when Cu2þ concentration was 100 mg/L, the difference even showed significant level (P < 0.05) compared to Cu2þ single treatment. 3.2. The joint effects of Cu2þ and TGA-CdTe QDs on zebrafish mortality at 120 hpf

indicated between joint treatments and TGA-CdTe QDs single exposure groups. 3.3. The joint effects of Cu2þ and TGA-CdTe QDs on zebrafish hatch rate at 72 hpf As illustrated in Fig. 4, the hatch rate gradually declined with the increase of Cu2þ concentration and the doseeresponse relationships were obvious. Additionally, TGA-CdTe QDs can clearly aggravate the trend. Compared to Cu2þ single exposure, lower level of QDs þ Cu treatments did not result in significant difference, while obviously reduced the hatch rate. Moreover, in the 6 mg/L of TGA-CdTe QDs and 100 mg/L of Cu2þ joint exposure, the hatch rate exhibited highly significant toxicity from the TGA-CdTe QDs (6 mg/ L) or Cu2þ (100 mg/L) single treatment at P < 0.01 level, and the hatch rate was only 28.47%. 3.4. The joint effects of Cu2þ and TGA-CdTe QDs on zebrafish malformation at 96 hpf As shown in Fig. 5A, with the increase of Cu2þ concentration, the malformation rates of single and joint exposure indicated the growing trends. Additionally, at the same Cu2þ level groups, the abnormity of combined contamination was higher and it can be observed that the two chemicals are a synergy effect. Moreover, compared to the TGA-CdTe QDs or Cu2þ (same concentration) single exposure, the malformation rates of 6 mg/L of TGA-CdTe QDs and 0.1 or 10 mg/L of Cu2þ joint treatments exhibited no significant differences during 96 h exposure period. However, quite to the contrary, 100 mg/L of Cu2þ and QDs joint exposure group indicated

Cu

2+

Cu 2+ Cu +CdTe

80

50 40 30 20 10 0

**

90

60

Mortality at 120 hpf (%)

2+ Body burden (ng Cu / mg wet weight)

The Fig. 3 data indicated that the ascendant trend of zebrafish mortality became more notable with increased Cu2þ dose whether in single or joint treatments. The presence of TGA-CdTe QDs can lead to the sharp growth of the mortality. Particularly for 10 and 100 mg/L of Cu2þ treatments, the joint exposure displayed highly significant toxicity (P < 0.01) compared to the treated groups single exposed to Cu2þ (same concentration), and the mortality were 34.17% and 63.33%, respectively, whereas the controls (Cu2þ single exposure) were only 6.86% and 10.67%. The same trend was

2 3 4 5 6

2+

##

2+

C u +C dTe

70 60

** ##

50 40 30 20 10 0

0

0.1

10

100

2+ Cu concentration (µ Fig. 2. Body burden determination of Cu2þ in zebrafish larvae in the presence of TGA-CdTe QDs at 120 hpf (n ¼ 30). * Indicates a statistically significant difference from the Cu2þ single exposure (100 mg/L) at P < 0.05 level. Values represent the mean  standard error of three replicates.

0

0.1

10

100

2+ Cu concentration (µ Fig. 3. Effect of exposure concentration on zebrafish mortality at 120 hpf (n ¼ 30). Double ## indicate a statistically significant difference from the TGA-CdTe QDs single exposure (6 mg/L) at P < 0.01 level. Double ** indicate a statistically significant difference from the Cu2þ single exposure (10 and 100 mg/L) at P < 0.01 level. Values represent the mean  standard error of three replicates.

W. Zhang et al. / Environmental Pollution 176 (2013) 158e164

161

hyperplasia (Fig. 6A), vascular turbulence (Fig. 6B), vascular bifurcation (Fig. 6C) and QDs aggregation appearance (Fig. 6D). 3.6. The interactive effects of Cu2þ and TGA-CdTe QDs on zebrafish The interactive effects of Cu2þ and TGA-CdTe QDs are presented in Table 2. We can observe synergistic effects were dominant in almost all the treatments, indicating that TGA-CdTe QDs can adsorb other pollutants or induce the cell membrane wound, which might result in more Cu2þ into the zebrafish body and further elicit higher toxicity. 4. Discussion Due to small particle size, large specific surface area and the presence of high surface energy compared to normal materials, QDs have strong adsorption capacity for other trace pollutants. To simulate the actual exposure of aquatic animals dwelling in a CdTe QDs and Cu2þ combined contamination environment, the zebrafish was jointly exposed to different doses of QDs and Cu2þ from the embryonic stage. In this study the CdTe QDs were synthesized from aqueous solution and were stabilized by using water soluble TGA. In addition to serving as stabilizer, TGA serves to: 1) passivate the surface of QDs and thereby removing the surface traps which lower the photoluminescence efficiency of the quantum dots, 2) prevent the non-radiative recombination of electron and hole, 3) control growth kinetics thereby prevent aggregation via steric hindrance (Jhonsi and Renganathan, 2010). And then the bioaccumulation, mortality, hatch rate, malformation, and vascular pattern of zebrafish were observed. The results showed the presence of QDs can obviously affect the toxic effects of Cu2þ in zebrafish body during the exposure period. The FT-IR spectra of TGA-CdTe QDs indicated that the thiol moiety strongly interacted with the surface of CdTe QDs through its carboxylic ligands, the carboxylic ligands donating its electrons to the surface of QDs and made it negatively charged. Oxidation potential of CdTe QDs was observed at 0.6 V, and free energy change values were calculated to be 0.9 V, indicating the electron transfer process studied was thermodynamically favorable (Miao, 2010). In the present study, the body burden of Cu2þ in zebrafish was accumulated higher in the joint presence of TGA-CdTe QDs than that after single exposure to Cu-contaminated water, which indicated that more Cu2þ entered into the larvae body and the carrying

Fig. 4. Effect of exposure concentration on zebrafish hatch rate at 72 hpf (n ¼ 30). Double ## indicate a statistically significant difference from the TGA-CdTe QDs single exposure (6 mg/L) at P < 0.01 level. Double ** indicate a statistically significant difference from the Cu2þ single exposure (100 mg/L) at P < 0.01 level. Values represent the mean  standard error of three replicates.

247.3% or 140.2% malformation rate increase respectively, and displayed significantly developmental inhibition (P < 0.05) compared to the TGA-CdTe QDs (6 mg/L) or Cu2þ (100 mg/L) single exposure. Zebrafish were jointly exposed to TGA-CdTe QDs (6 mg/L) and Cu2þ (100 mg/L) from 6 to 96 hpf and the malformations were observed at 96 hpf (Fig. 5B). After exposure several malformation patterns (i.e., melanin decrease, pericardial edema, vitelline cyst, bent spine, bent tail, and somite decrease) were observed to be predominant in almost all the treated groups, and vitelline cyst and pericardial edema might be the most important malformation because of relatively higher probability of occurrence. 3.5. The joint effects of Cu2þ and TGA-CdTe QDs on the vascular pattern of FLI-1 transgenic zebrafish larvae at 96 hpf The vascular patterns of FLI-1: EGFP transgenic zebrafish larvae in the joint exposure groups (6 mg/L of TGA-CdTe QDs and 100 mg/L of Cu2þ) were observed and recorded by Fluorescence Inverted Microscope at 96 hpf. Compared to the QDs and Cu2þ single treatments (data not shown), joint exposure resulted in more obvious and diverse abnormal vascular patterns such as vascular

Malformation rate at 96 hpf (%)

A

B

*

25

#

Cu

MD

BS

C u +CdTe

20 15

VC

10 5

VC

PE

BT

0 0.1

0 Cu

2+

concentration (µ

10

100

SD

Fig. 5. A): Effect of exposure concentration on zebrafish malformation rate at 96 hpf (n ¼ 30). # Indicate a statistically significant difference from the TGA-CdTe QDs single exposure (6 mg/L) at P < 0.05 level. * Indicate a statistically significant difference from the Cu2þ single exposure (100 mg/L) at P < 0.05 level. Values represent the mean  standard error of three replicates. B): Malformation patterns of zebrafish after joint exposure to 6 mg/L of TGA-CdTe and 100 mg/L of CuCl2 at 96 hpf (n ¼ 30). i.e., Melanin decrease (MD), Pericardial edema (PE), Vitelline cyst (VC), Bent tail (BT), Bent spine (BS), Somite decrease (SD). Scale bar ¼ 0.5 mm.

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Fig. 6. The vascular pattern of FLI-1 transgenic zebrafish larvae after joint exposure to 6 mg/L of TGA-CdTe QDs and 100 mg/L of CuCl2 at 96 hpf (n ¼ 30) by Fluorescence Inverted Microscope (200). A): Vascular hyperplasia, B): Vascular turbulence, C): Vascular bifurcation, D): Aggregation appearance. The vascular defects are shown in the red circle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

effect of QDs was obvious. The Fig. 2 data showed that Cu2þ concentration increased significantly than that without QDs addition at 120 hpf and reached 52.25 ng/mg (100 mg/L of Cu2þ and QDs joint treatment), suggesting that the presence of TGA-CdTe QDs greatly enhanced the accumulation of Cu2þ in zebrafish. Based on several literature and our previous observation (Sun et al., 2007; Zhang et al., 2007; Miao, 2010; Thein et al., 2011), potential reasons and mechanism hypotheses are as follows: 1) Cu2þ might be adsorbed onto TGA-CdTe QDs due to large surface area and electrostatic attraction, and then entered into the zebrafish with the accumulation of QDs (TGA-CdTe QDs enhanced the body burden of Cu2þ 19.3% in 100 mg/L of Cu2þ joint exposure group), 2) the physical properties (i.e., small particle size and strong penetrability) of TGACdTe QDs can induce more cell membrane wound, which elicited the transmembrane transport of Cu2þ through the wound and enhanced the cellular uptake of Cu2þ. This research provides powerful evidence of facilitated bioaccumulation of the toxic pollutant by QDs in the aquatic organisms. Hence, further research should focus on the validation of transport mechanism. Mixing the QDs and metals, the electrostatic interaction can occur and then exhibit the changes in fluorescence spectra of QDs (Yang et al., 2008). An explanation on the electrostatic forces and the mechanism of two electrostatic assembles between QDs and Cu ions through attraction should be proposed. Although QDs and heavy metal (including Cu2þ and Cd) both contributed to the ecotoxicity (the mortality, hatch or malformation, etc), in previous study we observed that the influence of Cd, Te or TGA release from TGA-CdTe QDs on zebrafish can be neglected (Miao, 2010; Zhang et al., 2012). On the whole, in this research TGACdTe QDs (6 mg/L) or Cu2þ (0.1, 10, and 100 mg/L) single exposure also hardly resulted in distinct toxic effects on the mortality, hatch or malformation rate of zebrafish. From the results it is no doubt that joint presence of TGA-CdTe QDs and Cu2þ will bring higher toxicity. For example, the mortality in the TGA-CdTe QDs (6 mg/L) and Cu2þ (100 mg/L) joint exposure group were 7.6 or 5.9 times higher than that in single treatments respectively. The oxidative stress responses were often used as biomarkers (Almeida et al., 2002). Some scholars reported that CdSe QDs can cause a marine

microalga oxidative stress and ROS accumulation and further affect its growth and mortality (King-Heiden et al., 2009; Hu et al., 2011; Morelli et al., 2012). In order to explore the toxicity mechanism, the phenomenon of oxidative stress should be observed after QDs and Cu2þ joint exposure. The joint exposure of TGA-CdTe QDs and Cu2þ can induce various malformations due to one or more possible mechanism. Results of observations suggested that during the different phase of the fish development the vitelline cyst and pericardial edema would often occur and might be the most important malformation, indicating the similar trend as Usenko’s description (Usenko et al., 2008). Moreover, the toxic effect would result in several malformation patterns at the same time. We hypothesize that the bent spine was related to the muscle or skeleton based on the broken, disorganized, and loosen array muscle fibers observed in the previous research, and such presupposition is consistent with Huang’s observation (Huang et al., 2010). Moreover, to gain insight into potential mechanisms of these malformations, we observed the toxic effects following single exposed to CuCl2 (100 mg/L) or TGACdTe QDs (6 mg/L), and the results indicated that single exposure can result in pericardial edema, vitelline cyst, bent spine, while unrelated to malformed tail, so joint effects of the two chemicals might be the contributors to bent tail. Blechinger and King-Heiden also reported similar observation (Blechinger et al., 2002; KingHeiden et al., 2009). After jointly exposed to TGA-CdTe QDs and Cu2þ from 6 to 96 hpf, the vascular patterns of FLI-1: EGFP transgenic zebrafish larvae showed more abnormalities such as vascular hyperplasia, turbulence and bifurcation. In previous experiment TGA-CdTe QDs were found to aggregate in back and abdominal vascular of zebrafish resulting in vascular obstruction, moreover, the aggregation between nanoparticles and heavy metals because of reciprocal reaction were also found (Jiang et al., 2009; Hamaguchi et al., 2010; Miao, 2010; Sun, 2010; Zhao et al., 2010), and in this research the phenomenon was also visibly observed. This aggregation might have contributed to the observed abnormalities. On the whole, as for the interactive action of Cu2þ and TGA-CdTe QDs, the experimental results indicated that the synergistic effects

Table 2 The interactive effects of Cu2þ and TGA-CdTe QDs joint exposure. Exposure pattern

Toxicological endpoints Mortality rate (%)

Cu2þ (0.1, 10, 100 mg/L) single exposure TGA-CdTe QDs (6 mg/L) single exposure TGA-CdTe QDs (6 mg/L) and Cu2þ (0.1, 10, 100 mg/L) joint exposure Interactive effecta a

Un-hatch rate (%)

1.67 8.37 14.75

6.86

10.67

34.17

þ

þ

“þ” or “e” represents synergistic or antagonistic effect respectively.

Malformation rate (%)

30.39

33.50

63.33

27.53 25.62 36.84

6.27

6.42

71.53

2.14 4.44 7.23

42.36

12.27

15.42

þ

e

e

þ

þ

þ

þ

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played more important role. Future study should not only observe the fate and toxicity of QDs themselves, but also to the facilitated transport and joint toxicity of other pollutants when they co-exist in the same environment, which is favorable for overall understanding of the ecological risks that QDs might cause. Additionally, our preliminary observation of TGA-CdTe QDs or Cu2þ elimination suggested that the clearance was negligible during 120 hpf period (data not shown), but a more thorough analytical approach to TGACdTe QDs and Cu2þ joint exposure such as the metabolism and elimination kinetics is needed to explore in future in order to evaluate the toxic effects more comprehensively. 5. Conclusions During the entire exposure period, the joint effects of TGA-CdTe QDs and Cu2þ on the body burden of Cu2þ (120 hpf), mortality (120 hpf), hatch rate (72 hpf), and malformation rate (96 hpf) of the zebrafish were dose-dependent of greater toxicity at increasing dose. Additionally, the exposure can obviously alter the vascular patterns (96 hpf). As a whole, the joint toxicities of the two toxicants were synergistic. Further studies are needed to investigate the mechanisms underlying the developmental changes. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (40901148), the Science and Technology Committee Research Program of Shanghai (12DZ0502700), the Environmental Protection Science and Technology Research Program of Shanghai (2012-03), the Major State Basic Research Development Program of China (2011CB200904), and the National Forestry Public Welfare Science and Technology Research Program of China (201104088). References Almeida, J.A., Diniz, Y.S., Marques, S.F.G., 2002. The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environment International 27, 673e679. Blechinger, S.R., Warren Jr., J.T., Kuwada, J.Y., Krone, P.H., 2002. Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environmental Health Perspectives 110, 1041e1046. Boeck, D.G., Vander, V.K., Hattink, J., Blust, R., 2006. Swimming performance and energy metabolism of rainbow trout, common carp and gibel carp respond differently to sublethal copper exposure. Aquatic Toxicology 80, 92e100. Bhuiyan, M.A.H., Islam, M.A., Dampare, S.B., Parvez, L., Suzuki, S., 2010. Evaluation of hazardous metal pollution in irrigation and drinking water systems in the vicinity of a coal mine area of northwestern Bangladesh. Journal of Hazardous Materials 179, 1065e1077. Chen, D.S., Zhang, D.Q., Yu, J.C., Chan, K.M., 2011. Effects of Cu2O nanoparticle and CuCl2 on zebrafish larvae and a liver cell-line. Aquatic Toxicology 105, 344e354. Clément, L., Hurel, C., Marmier, N., 2013. Toxicity of TiO2 nanoparticles to cladocerans, algae, rotifers and plants-Effects of size and crystalline structure. Chemosphere 90, 1083e1090. Derfus, A.M., Chan, W.C.W., Bhatia, S.N., 2004. Probing the cytotoxicity of semiconductor quantum dots. Nano Letters 4, 11e18. Fan, X.Q., Peng, J.J., Yan, S.G., Wang, L., He, Y.Q., 2011. Study on the interaction of CdTe quantum dots with ferulic acid and protocatechuic aldehyde by optical spectroscopy. Journal of Luminescence 131, 2230e2236. Gagné, F., Auclair, J., Turcotte, P., Fournier, M., Gagnon, C., Sauvé, S., Blaise, C., 2008. Ecotoxicity of CdTe quantum dots to freshwater mussels, impacts on immune system, oxidative stress and genotoxicity. Aquatic Toxicology 86, 333e340. Grolimund, D., Borkovec, M., Barmettler, K., Sticher, H., 1996. Colloid-facilitated transport of strongly sorbing contaminants in natural porous media, A laboratory column study. Environmental Science and Technology 30, 3118e3123. Grosell, M., Wood, C.M., Walsh, P.J., 2003. Copper homeostasis and toxicity in the elasmobranch Raja erinacea and the teleost Myoxocephalus octodecemspinosus during exposure to elevated water-borne copper. Comparative Biochemistry and Physiology C 135, 179e190. Hamaguchi, K., Kawasaki, H., Arakawa, R., 2010. Photochemical synthesis of glycinestabilized gold nanoparticles and its heavy-metal-induced aggregation behavior. Colloids and Surfaces A: Physicochemical and Engineering Aspects 367, 167e173.

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