Toxicological effect of MPA–CdSe QDs exposure on zebrafish embryo and larvae

Chemosphere 89 (2012) 52–59

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Toxicological effect of MPA–CdSe QDs exposure on zebrafish embryo and larvae Wei Zhang a,b,c,⇑, Kuangfei Lin a,b,c,⇑, Xue Sun a,b,c, Qiaoxiang Dong d, Changjiang Huang d, Huili Wang d, Meijin Guo e, Xinhong Cui f 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 e State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China f Shanghai Institute of Landscape Gardening, Shanghai 200233, PR China

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Article history: Received 3 October 2011 Received in revised form 11 March 2012 Accepted 4 April 2012 Available online 15 May 2012 Keywords: MPA–CdSe QDs Zebrafish embryo Zebrafish larvae Developmental toxicity Behavioral toxicity

a b s t r a c t Cadmium selenium (CdSe) quantum dots (QDs) are semiconductor nanocrystals that hold wide range of applications and substantial production volumes. Due to unique composition and nanoscale properties, their potential toxicity to aquatic organisms has increasingly gained a great amount of interest. However, the impact of CdSe QDs exposure on zebrafish embryo and larvae remains almost unknown. Therefore, the lab study was performed to determine the developmental and behavioral toxicities to zebrafish under continuous exposure to low level CdSe QDs (0.05–31.25 mg L 1) coated with mercaptopropionic acid (MPA). The results showed MPA–CdSe exposure from embryo to larvae stage affected overall fitness. Our findings for the first time revealed that: (1) The 120 h LC50 of MPA–CdSe for zebrafish was 1.98 mg L 1; (2) embryos exposed to MPA–CdSe resulted in malformations incidence and lower hatch rate; (3) abnormal vascular of FLI-1 transgenic zebrafish larvae appeared after exposure to MPA–CdSe including vascular junction, bifurcation, crossing and particle appearance; (4) larvae behavior assessment showed during MPA–CdSe exposure a rapid transition from light-to-dark elicited a similar, brief burst and a higher basal swimming rate; (5) MPA–CdSe induced embryos cell apoptosis in the head and tail region. Results of the observations provide a basic understanding of MPA–CdSe toxicity to aquatic organisms and suggest the need for additional research to identify the toxicological mechanism. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Quantum dots (QDs) hold considerable promise during the last decades in solar cell, electronics, life sciences, biomedical imaging, diagnostics, and photovoltaics applications because of their physical size, bright fluorescence, narrow emission spectra, broad absorption coefficients spectra, and high photostability (Chan and Nie, 1998; Jr et al., 1998; Wang et al., 2004; Michalet et al., 2005; Bhatt and Tripathi, 2011). QDs cores consist of a variety of metal complexes, which may exert detrimental consequences not only by their nanoscale properties but also by chemical interactions with cells at relatively low concentrations. To render them biologically compatible/active, newly synthesized QDs are given secondary coatings, which improves water solubility, core durability, and suspension characteristics (Lovric et al., 2005; Medintz et al., 2005; Wang et al., 2008). However, QDs with various organic ⇑ Corresponding authors. Address: Box 563, 130 Meilong Road, Shanghai 200237, PR China. Tel.: +86 21 64253244; fax: +86 21 64253988. E-mail addresses: [email protected] (W. Zhang), kfl[email protected] (K. Lin). 0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2012.04.012

coatings would be subject to modification or degradation under environmental conditions, and hence would become toxic (Kim et al., 2010). Cadmium selenium (CdSe) QDs are semiconductor nanocrystals that hold wide range of applications and substantial production volumes. Initial reports suggested dihydrolipoic acid-capped CdSe QDs lacked toxicity toward mammalian (HeLa) and slime mold (AX2) cells (Jaiswal et al., 2003). However, subsequent studies on surface-coated CdSe QDs demonstrated some toxicity in various cell lines (Derfus et al., 2004; Kim et al., 2010). CdSe QDs are increasingly found in environmental water samples via waste streams from industries that synthesize or use CdSe and through clinical and research uses (Aitken et al., 2006; Hardman, 2006). However, the impact of CdSe QDs exposure on water environment remains largely unknown. Therefore, there is an urgent need to determine the CdSe QDs induced ecotoxicological effects to aquatic organisms and to identify convenient biomarkers for early signaling. The potential toxicity of engineered nanoparticles (NPs) to aquatic organisms is poorly understood (Buffet et al., 2011). In

W. Zhang et al. / Chemosphere 89 (2012) 52–59

literatures, animal studies were the most prevalent methods for NPs toxicity assay. The zebrafish as a valuable vertebrate model for investigating the developmental toxicity and neurotoxicity to our understanding of the potential ecotoxicological impacts of NPs releases to aquatic environments has received increased popularity in recent years, and results obtained with zebrafish have relevance to human health and feral fish populations (Spitsbergen and Kent, 2003; Hill et al., 2005; Griffitt et al., 2007; Henry et al., 2007; Lee et al., 2007; Zhu et al., 2007; Fent et al., 2010; Wang et al., 2011a,b). However, there has been no report of employing zebrafish model for CdSe QDs toxicity studies. In this study, our objective was to use the zebrafish to identify the developmental and behavioral toxicities associated with CdSe QDs exposure. A number of physiological parameters were assessed to define the toxic responses following QDs exposure. There were some impacts on overall fitness such as mortality, hatch rate, malformation, swimming behavior, vascular patterning and cell apoptosis.

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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 6.5–7.5), and instant ocean salt was added to the water to raise the conductivity to 450–1000 uS cm 1. The adult fish were fed twice daily with live Artemia (Jiahong Feed Co., Tianjin, China) and dry flake diet (Zeigler, Aquatic Habitats, Apopka Florida, USA). The development status of zebrafish embryos and larvae were observed with an Inverted Microscope (8–50, 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 (Westerfield, 1995). 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). 2.4. Exposure protocols

2. Materials and methods 2.1. Chemicals Water-soluble CdSe QDs and Cadmium chloride (CdC12
2.5H2O) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Merck (Darmstadt, Germany). All chemicals used in the present study were analytical grade. 2.2. Stock solutions of MPA–CdSe QDs CdSe QDs were synthesized as previously described and coated with mercaptopropionic acid (MPA) (Huang et al., 2004; Kim et al., 2010). These coatings were selected to examine the extent to which ligand chain length and terminal functional group affected uptake and toxicity. QDs solutions used in 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 MPA and placed in distilled water at pH 10 for 4 h at 20 °C to remove any traces of labile cadmium and selenium. MPA–CdSe QDs were observed to have an irregular shape with a typical size of approximately 3.5 nm by TEM (Fig. 1A). The concentration of MPA–CdSe QDs produced in the present study was approximately 1.24  10 5 mol L 1, and the stock solutions were prepared by dissolving it in 60 mg L 1 ocean salt. In preliminary experiments, we found that ocean salt did not result in toxic effect on zebrafish. MPA–CdSe were observed to have excellent stability about QDs properties (i.e., shape and size) after addition to the salt water by TEM (Fig. 1B) and spectrum characteristic with narrow and sharp peak by FS during the entire exposure period. 2.3. Fish husbandry and embryo collection Adult zebrafish of wild-type strain (AB) were raised and kept at standard laboratory conditions of 28 ± 0.5 °C with a 14:10 dark/

Fig. 1. (A) The TEM image of the original MPA–CdSe QDs; and (B) the TEM image of the MPA–CdSe QDs in the zebrafish media solution after 6 d exposure period.

To explore MPA–CdSe stability, QD test solutions (12.15 and 31.25 mg L 1) were exposed for 120 h and then passed through a filtration membrane (10 kDa cutoff, AmiconÒ Ultra-15 Centrifugal Filter Devices, Millipore Corporation, Billerica, MA, USA) to determine the amount of Cd released from intact QDs. Cd2+ was measured using graphite furnace AAS (novAA400, Analytik Jena AG, Germany) following Creed et al. (1994). In the present study, exposure concentrations and periods, and endpoints of zebrafish used for each experiment were listed in Table 1. To determine the LC50, normal embryos were exposed to 0, 0.05, 0.15, 0.45, 1.35, 4.05, 12.15, and 31.25 mg L 1 MPA–CdSe and CdC12
2.5H2O from 6 to 120 hpf respectively. Embryos were kept in sterile 96-well plates, with one embryo per well, containing 200 lL treatment or control solutions. Plates were covered with sealing films to prevent evaporation. For each exposure treatment, 30 embryos were performed in light-controlled incubator. The mortality of zebrafish at 120 hpf were observed, and then the LC50 were determined. Normal embryos at 6 hpf were exposed into seven treatment groups (60 mg L 1 ocean salt control, and 0.05, 0.15, 0.45, 1.35, 4.05, and 12.15 mg L 1 MPA–CdSe) from 6 to 72 hpf. For each batch of zebrafish embryo were used for assessing the hatch rate at 60 and 72 hpf. After normal embryos at 6 hpf exposure to ocean salt control (60 mg L 1) and MPA–CdSe (12.15 mg L 1) for 3 d (72 hpf), the malformation of zebrafish from 12 to 72 hpf were observed with an Inverted Microscope (Nikon, Japan). 2.5. Behavior assessment There were two trials in this experiment. Zebrafish embryos were exposed to 0, 0.15, 0.45, and 1.35 mg L 1 MPA–CdSe from 6 to 72 hpf and further subjected to behavior assessment at 120 hpf in our study. In the first trial, after a 10 min adaption, locomotion monitoring of morphologically normal larvae were assessed in visible light for 20 min; in the second trial, zebrafish larvae were allowed to adapt for 10 min and swimming speed were recorded when they responded to a 60-min (10 min for each period) light-to-dark transition stimulation (Burgess et al., 2010; Huang et al., 2010; Wang et al., 2011a,b). The test was monitored with the Zebralab Video-Track system (Videotrack, version 3.5) equipped with a Sony one-third inch Monochrome camera (Model DR2-HIBW-CSBOX, 30Fps) and an infrared filter. The entire record hardware is linked to the computer control program and kept insulated from environmental conditions in a sealed opaque plastic box (Zebrabox, ViewPoint Life Science, France). The data (movement

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W. Zhang et al. / Chemosphere 89 (2012) 52–59

Table 1 Experimental layout. Exposure concentration (mg L 0, 0, 0, 0, 0,

1

)

0.05, 0.15, 0.45, 1.35, 4.05, 12.15, 31.25 12.15 0.15, 0.45, 1.35 4.05 0.15, 0.45, 1.35

Exposure period (hpf)

Endpoints

Results

6–120 6–72 6–72 6–96 6–24

Mortality (LC50) (120 hpf) Malformation (12–72 hpf) Swimming speed (120 hpf) Vascular patterning (96 hpf) Cell apoptosis (24 hpf)

Fig. Fig. Fig. Fig. Fig.

frequency, travelled distance and total movement duration) were collected every 60 s and further analyzed using custom Open Office.Org 2.4 software. 2.6. Vascular patterning detection 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. FLI-1 larvae were exposed into two treatment groups (60 mg L 1 ocean salt control and 4.05 mg L 1 MPA–CdSe) from 6 to 96 hpf, and their vascular patterning at 96 hpf were further observed by Fluorescence Inverted Microscope (Nikon TE2000-U, Japan) (Huang et al., 2010; Wang et al., 2011b). 2.7. In vivo cellular apoptosis To visualize embryo cell apoptosis, the control (60 mg L 1 ocean salt) and MPA–CdSe (0.15, 0.45, and 1.35 mg L 1) exposed embryos were incubated with acridine orange (Usenko et al., 2007). Embryos exposed from 6 to 24 hpf were dechorionated via enzymatic digestion with 0.5 mg mL 1 protease E in 50 mm Petri dishes for 6 min at room temperature. Ten embryos from each treatment were rinsed twice with embryo medium (EM) and incubated with 5 lg mL 1 acridine orange dissolved in EM for 1 h at room temperature in the dark. The embryos were then washed with EM three times for 5 min each. Before examination, the live animals were anesthetized with 0.02% MS-222 for 5 min and mounted laterally on a microscope slide. All embryos at 24 hpf were observed with a Fluorescence Microscope (Nikon, Japan) and representative pictures were shown. 2.8. 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 concentration–response curves which were required to determine LC50 values was completed using origin 8.0 (OriginLab, Northampton, MA, USA). One-way ANOVA was applied to calculate statistical significance followed by 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) and P value of less than 0.05 was considered to be statistically significant. The data were shown as mean ± standard error (SE). 3. Results 3.1. Cd2+ and MPA–CdSe affected zebrafish mortality and hatch rate For MPA–CdSe QD test solution, Cd2+ concentrations of 0.43 and 1.08 mg L 1 were detected in the filtered solution after the incubation of MPA–CdSe of 12.15 and 31.25 mg L 1 for 120 h, which indicated that only about 3.5% of Cd appeared to be released from the

2 3 4 5 6

MPA–CdSe core. The LC50 of CdCl2
2.5H2O at 120 hpf was calculated to be 1.12 mg L 1 (Fig. 2A). In addition, in previous study, we found that MPA did not result in obvious toxic effect on zebrafish. The controls and 0.05, 0.15 mg L 1 MPA–CdSe exerted no detectable toxicity to zebrafish, and percent survival were more than 90% at 120 hpf whereas other dose of MPA–CdSe were obviously toxic (Fig. 2B). The concentration–response curve suggests zebrafish developmental toxicity got stronger with the increase of MPA–CdSe concentration. For example, percent mortality was above 91% for 12.15 mg L 1 MPA–CdSe treatment group, and especially all zebrafish even died for the treatment groups exposed to the dose of 31.25 mg L 1 MPA–CdSe. The LC50 of MPA–CdSe at 120 hpf was calculated to be 1.98 mg L 1. MPA–CdSe can affect the hatch rate of zebrafish embryos (data not shown). At 60 hpf there was a concentration-dependent decrease in hatch rate, and no difference was observed between the control and 0.05, 0.15 mg L 1 MPA–CdSe treatment groups, whereas 0.45, 1.35, 4.05, and 12.15 mg L 1 showed significant differences (P < 0.05). In contrast, for 72 hpf treatments, embryos in each group except 12.15 mg L 1 were nearly all hatched (above 90%) with no significant difference between treated groups and the control, which indicated that 60 hpf exposure affected embryos hatch more significant than 72 hpf. 3.2. MPA–CdSe induced zebrafish malformation Zebrafish were exposed to 12.15 mg L 1 MPA–CdSe from 6 to 72 hpf and were monitored daily for the malformation until 72 hpf (Fig. 3). The vehicle control (60 mg L 1 ocean salt) was not toxic to embryos (no observed malformation), while treatment groups exhibited significant increases in malformation when compared with controls. Additionally, there was a pattern of concentration dependence and increase in severity and type of malformation after exposure to 4.05 and 12.15 mg L 1 MPA–CdSe. Malformations such as oosperm coagulation, eyespots and melanin developmental inhibition, pericardial edema, bent spine, unhatched and tail without extension were observed to be dominant in all the treatment groups. Total observed cumulative malformations displayed that pericardial edema often occurred and several kinds of malformations types could appear together. 3.3. MPA–CdSe affected zebrafish swimming behavior As displayed in Fig. 4A, the average swimming speed of zebrafish larvae were detected at 120 hpf after exposure to MPA–CdSe at various concentrations from 6 to 72 hpf. Compared to the controls (2.35 mm s 1), 0.15 and 0.45 mg L 1 treatment groups exhibited no significant differences. In contrast, exposed to 1.35 mg L 1 MPA–CdSe (0.95 mm s 1) showed significant (P < 0.05), which was obviously slower than that in controls and even decreased 59.57%. The results indicated that high dose MPA–CdSe can elicit neurobehavior alternations in zebrafish larvae. In the light stimulation trial, zebrafish larvae at 120 hpf with normal morphology derived from the control or 0.15, 0.45, and 1.35 mg L 1 MPA–CdSe exposure groups were subjected to the light stimulation motor behavior test. As Fig. 4B shown, a 60-min

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A

120

100 80 60

y=-84.6739*exp(-x/1.41453)+88.37295

40

LC50 =1.12 mg L-1

R2=0.9736

20 0 0

5

10

15

20

25

30

Mortality at 120 hpf (%)

Mortality at 120 hpf (%)

120

B

100 80 60

y=-89.25041*exp(-x/2.6595)+92.3451

40

R2=0.9949

-1

LC50 =1.98 mg L

20 0

35

0

-1

5

10

15

20

25

30

35 -1

Concentraion of CdCl 2.2.5H 2O (mg L )

Concentraion of MPA-CdSe (mg L )

Fig. 2. Effect of exposure concentration on zebrafish mortality at 120 hpf (n = 30 embryos): (A) CdCl2
2.5H2O and (B) MPA–CdSe. Values represent the mean ± standard error of three replicates.

Fig. 3. The malformation types of zebrafish after exposure to 12.15 mg L 1 MPA–CdSe. (A) Oosperm coagulation at 12 hpf; (B) eyespots developmental inhibition at 24 hpf; (C) eyespots and melanin developmental inhibition at 36 hpf; (D) pericardial edema at 48 hpf; (E) unhatched embryo at 60 hpf; (F) pericardial edema at 60 hpf; (G) bent spine at 60 hpf; (H) tail without extension at 72 hpf; (I) pericardial edema at 72 hpf. Scale bar = 0.5 mm.

3.0

A

B

Control 0.15 mg L-1 0.45 mg L-1 1.35 mg L-1

2.5 500

2.0

*

1.5 1.0 0.5

Swimming distance (mm (5 mins)-1)

Swimming speed (mm s-1)

600

400 300 200 100

0.0 0

0.15

0.45

1.35

Concentration of MPA-CdSe (mg L-1)

0 16:00 16:05 16:10 16:15 16:20 16:25 16:30 16:35 16:40 16:45 16:50 16:55 17:00

Time

Fig. 4. The swimming speed of zebrafish larvae in persistent visible light (A) and when subject to a 60-min light-to-dark photoperiod stimulation (B) after exposure to different concentrations of MPA–CdSe at 120 hpf (n = 30 larvae). Asterisks indicate a statistically significant difference from control (⁄P < 0.05). Values represent the mean ± standard error of three replicates.

photoperiod stimulation test began in light (10 min), followed by two cycles of darkness (10 min) and light (10 min). A rapid transi-

tion from light-to-dark resulted in a similar, brief burst of larvae swimming in all groups (both exposed and control). Larvae

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duration of MPA–CdSe exposure elicited a higher basal swim rate than the controls, moreover, all the treated groups exhibited no significant differences compared to the controls. The results showed that zebrafish larvae were sensitive to sudden darkness and MPA–CdSe exposure stress. 3.4. MPA–CdSe affected the vascular patterning of FLI-1 transgenic zebrafish The vascular patterning of FLI-1: EGFP transgenic zebrafish larvae in the control and 4.05 mg L 1 MPA–CdSe exposure groups were detected by Fluorescence Inverted Microscope at 96 hpf (Fig. 5). Compared to the control (Fig. 5A), after exposure to MPA–CdSe from 6 to 96 hpf treatment groups also had almost integrated blood circulation system, while the vascular exhibited abnormal phenomenon. Furthermore, we found that higher dose of MPA–CdSe exposure were more toxic to blood circulation system (data not shown). Vascular abnormal patterning mainly included vascular junction (Fig. 5B), vascular bifurcation (Fig. 5C), vascular crossing (Fig. 5D), QDs particle and aggregation appearance (Fig. 5E and F). The TEM images confirmed that the QDs transported and accumulated in the zebrafish (Fig. 5G–I). 3.5. MPA–CdSe induced zebrafish cellular apoptosis Cellular apoptosis assays were performed to determine if MPA– CdSe exposure affected cell death in specific cells or tissues. Dechorionated embryos exposed MPA–CdSe from 6 to 24 hpf were exposed to acridine orange for 1 h. For the AO assay, more apoptotic cells in embryos would be observed to display more luminous green by Fluorescence Microscope. As observed (Fig. 6), there was no dramatic increase in the controls (A and E) or the 0.15 mg L 1 MPA–CdSe treatment groups (B and F), while strongest labeling for apoptotic cells was consistently found in the head (C

and D) and tail (G and H) region of embryos treated with 0.45 or 1.35 mg L 1 MPA–CdSe. As Fig. 6 shown, on the whole, there was a concentration-dependent increase in cellular apoptosis of zebrafish embryos (D > C > B > A; H > G > F > E). Compared to the controls, exposure groups showed more blurry and many green fluorescence particles appeared both in the head and tail region, which indicated that MPA–CdSe induced more cellular apoptosis at 24 hpf. We assume that cellular apoptosis can act as a more sensitive and early toxicological endpoint than other parameters of non-lethal effect. 4. Discussion QD core metals (e.g., Cd, lead, zinc, tellurium, and mercury), could be released by oxidative and photolytic conditions, and many core materials are known to be toxic to vertebrate systems at relatively low concentrations (Hardman, 2006). Therefore, Cd, the QD core material, seems to be an important factor in determining MPA–CdSe QDs toxicity. However, in this study, the level of Cd ions release could not support the hypothesis that Cd is the major or unique contributor to the observed zebrafish toxicity. As shown in Fig. 2, the released amount of free Cd2+ (0.43 or 1.08 mg L 1) in the MPA–CdSe treatment groups (12.15 or 31.25 mg L 1) for 120 h was not enough to explain approximately 91% (12.15 mg L 1) or 98% (31.25 mg L 1) zebrafish mortality, because of considering that 0.43 or 1.08 mg L 1 Cd2+ just resulted in zebrafish mortality less than 35% or 50% (the 120 h LC50 of CdCl2
2.5H2O 1.12 mg L 1). King-Heiden found that zebrafish larvae showed clear signs of Cd2+ toxicity, while the nanoscale properties were even more potent and produced end points of toxicity distinct from that of Cd2+ (King-Heiden et al., 2009). These observations strongly suggest that QDs scale effect might act as more important function for its toxic effect. By using MT gene induction as an indicator of Cd2+ release, future work should detect the breakdown of MPA–CdSe so as to provide more

Fig. 5. The vascular patterning of FLI-1 transgenic zebrafish larvae in the control and 4.05 mg L 1 MPA–CdSe exposed groups at 96 hpf by Fluorescence Inverted Microscope (200). (A) normal vascular (control); (B) vascular junction (4.05 mg L 1); (C) vascular bifurcation (4.05 mg L 1); (D) vascular crossing (4.05 mg L 1); (E) QDs particle appearance (4.05 mg L 1); (F) QDs aggregation appearance (4.05 mg L 1); (G) normal vascular without QDs (before exposure); (H) QDs transport in vascular (during exposure); (I) aggregated QDs at the same site (after exposure). The vascular defects are shown in the red box/circle.

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Fig. 6. Apoptotic cells in zebrafish embryos after exposure to different concentrations of MPA–CdSe at 24 hpf. (A and E) Control; (B and F) 0.15 mg L (D and H) 1.35 mg L 1. Scale bar = 200 lm.

evidence to determine which played a more important role in toxicological effect on zebrafish between MPA–CdSe and released Cd2+. Moreover, in further experiments we should study simultaneously the toxicity of Cd2+ and MPA–CdSe to the hatch rate, malformation, swimming, vascular patterning and cellular apoptosis of zebrafish in order to discuss the contribution of Cd. In the present study, MPA–CdSe induced various malformations including oosperm coagulation, eyespots and melanin developmental inhibition, bent spine, pericardial edema, unhatched and tail without extension. Likely no single mechanism can account for the malformations, but hypotheses have been proposed. Cheng et al. (2000) attributed the spinal deformities to a reduction in both myosin and myotome formation necessary for the healthy musculo-skeletal system development. We hypothesize that the bent spine may relate to the muscle or skeleton as we have observed broken, disorganized, and loosen array muscle fibers in the previous research, however, this needs to be confirmed by further investigation. Cell apoptosis of embryos at 24 hpf induced by MPA–CdSe was consistently found in the head and tail region, which may partially contribute to behavioral disruption as head and tail are all important regions for fish behavior response. For example, hindbrain cell apoptosis could reflect the ongoing morphogenesis of the cerebellum, which plays an important role in adjusting muscle tension and maintaining body balance on zebrafish behavior (Cole and Ross, 2001). Behavioral analysis often serves as a sensitive tool for detecting sublethal chemical effects (Kane et al., 2004). We found MPA–CdSe can also disturb the neurobehavior of zebrafish larvae. For example, swimming behavior was affected by MPA–CdSe exposure where animals exhibited decreased activity upon introduction to new surroundings. In this study, 120 hpf zebrafish larvae derived from 1.35 mg L 1 MPA–CdSe exposure swam at a much slower speed at an inactive stage (0.95 mm s 1) in a 20 min light period. A rapid transition from light-to-dark resulted in a similar, brief burst of swimming in all treatment groups and larvae duration of MPA– CdSe exposure elicited a higher basal swim rate than the controls. Zebrafish showed a biorhythm which illustrated that larvae became active after exposure to sudden darkness and contamination stress

1

; (C and G) 0.45 mg L

1

;

for several minutes and then slowed down as was shown in a previous study (Prober et al., 2006; MacPhail et al., 2009). The typical behavior of low activity in normal conditions and hyperactivity when stressed in these larvae suggests that MPA– CdSe exposure can alter neurobehavior of zebrafish larvae. How MPA–CdSe affected the neural circuits and mechanisms at the physiological or biochemical levels underlying locomotion behaviors in response to light-to-dark stimulation in this study remains still unclear. Recent published studies suggest a significant involvement of motor neurons and muscle fiber in the overall locomotor behavior (Drapeau et al., 1999; Lanagan-Steet et al., 2005; Levin et al., 2009). Zebrafish larvae possess two types of skeletal muscle fibers: slow (red) fibers and fast (white) fibers, which could contribute to their behavioral response (Greek-Walker and Pull, 1975; Buckingham and Ali, 2004). Functionally, the red fibers are derecruited during fast burst swimming, while the white fibers are inactive during slow swimming episodes (Buss and Drapeau, 2002; Sylvain et al., 2010). So we conjecture that the white fibers played an important role for burst swimming during photoperiod stimulation. However, this is still needs to be confirmed by further investigation. Future studies are necessary to reveal the underlying mechanism for MPA–CdSe induced physiological or neurochemical levels changes and explore further the stress-related behavioral responses on zebrafish larvae, in particular their relationship with whole-body cortisol level, because it is the main mediator of physiological response to stress in fish (Egan et al., 2009). After exposure to MPA–CdSe from 6 to 96 hpf the vascular patterning of FLI-1: EGFP transgenic zebrafish larvae exhibited abnormal phenomenon such as vascular junction, bifurcation, crossing and particle appearance. At the same time, pericardial edema malformation was also observed, so we presume there could have some relationship between the two results, which is needed in future studies. Our preliminary investigation of MPA–CdSe elimination suggested that the clearance was negligible (data not shown), but a more thorough analytical approach to MPA–CdSe pharmacokinetics such as the determination of body burden, metabolism and elimination kinetics with both higher and lower, environmentally relevant exposures is needed to explored in future.

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5. Conclusions This study demonstrates the utility of zebrafish as a model for assessing potential risks of MPA–CdSe QDs. We continuously exposed zebrafish to MPA–CdSe from embryo to larvae stage. Such an exposure scheme could more closely simulate the real environmental exposures of aquatic animals dwelling in a MPA–CdSe contaminated environment. Cd2+ release from MPA–CdSe core was measured, and the concentration in a filtered solution was too low to be fully responsible for the observed zebrafish toxicity; therefore, QDs scale effect also acts as important function. In addition, to the best of our knowledge, this study for the first time demonstrated that MPA–CdSe exposure (ranging from 0.05 to 31.25 mg L 1) can affect zebrafish development and behavior. The calculated LC50 of MPA–CdSe at 120 hpf was 1.98 mg L 1, and embryo mortality was reported a dose-dependent increase. The results showed that MPA–CdSe exposure decreased embryos hatch rate, and induced malformation and cellular apoptosis. We also found that MPA–CdSe exposure altered the swimming speed and vascular patterning of zebrafish larvae. Such studies will be particularly important and valuable in the assessment of MPA– CdSe ecological risks. In summary, the determination of developmental toxicity will help to establish water-quality standards to protect aquatic life. However, further studies are needed to investigate the mechanisms underlying the developmental and behavioral changes. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (40901148, 40871223), the Major State Basic Research Development Program of China (2011CB200904), the Scientific Project on Treatment and Control of Water Pollution (2012ZX07115), the National Forestry Public Welfare Science and Technology Research Program of China (201104088), the Fundamental Research Funds for the Central Universities (WB0911011), the Natural Science Foundation of Shanghai (11ZR1409400), and Zhejiang Provincial Science Foundation of China (Y5100333). We also would like to thank the anonymous referees for their helpful comments on this paper. References Aitken, R.J., Chaudhry, M.Q., Boxall, A.B., Hull, M., 2006. Manufacture and use of nanomaterials: current status in the UK and global trends. Occup. Med. 56, 300– 306. Bhatt, I., Tripathi, B.N., 2011. Interaction of engineered nanoparticles with various components of the environment and possible strategies for their risk assessment. Chemosphere 82, 308–317. Buckingham, S.D., Ali, D.W., 2004. Sodium and potassium currents of larval zebrafish muscle fibers. J. Exp. Biol. 207, 841–852. Buffet, P.E., Tankoua, O.F., Pan, J.F., Berhanu, D., Herrenknecht, C., Poirier, L., AmiardTriquet, C., Amiard, J.C., Bérard, J.B., Risso, C., Guibbolini, M., Roméo, M., Reip, Paul., Valsami-Jones, E., Mouneyrac, C., 2011. Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles. Chemosphere 84, 166–174. Burgess, H.A., Schoch, H., Granato, M., 2010. Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation. Curr. Biol. 20, 381–386. Buss, R.R., Drapeau, P., 2002. Activation of embryonic red and white muscle fibers during fictive swimming in the developing zebrafish. J. Neurophysiol. 87, 1244– 1251. Chan, W.C.W., Nie, S., 1998. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016–2018. Cheng, S.H., Wai, A.W.K., So, C.H., Wu, R.S.S., 2000. Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos. Environ. Toxicol. Chem. 19, 3024–3031. Cole, L.K., Ross, L.S., 2001. Apoptosis in the developing zebrafish embryo. Dev. Biol. 240, 123–142. Creed, J.T., Martin, T.D., O’Dell, J.W., 1994. Determination of Trace Elements by Stabilized Temperature Graphite Furnace Atomic Absorption. US Environmental Protection Agency. Derfus, A.M., Chan, W.C.W., Bhatia, S.N., 2004. Probing the cytotoxicity of semiconductor quantum dots. Nano. Lett. 4, 11–18.

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