Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans

Environmental Pollution xxx (2016) 1e9

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Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans* Chi-Wei Huang 1, Shang-Wei Li 1, Vivian Hsiu-Chuan Liao* Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2016 Received in revised form 20 October 2016 Accepted 28 October 2016 Available online xxx

ZnO nanoparticles (ZnO-NPs) are emerging contaminants that raise the concerns of potential risk in the aquatic environment. It has been estimated that the environmental ZnO-NPs concentration is 76 mg/l in the aquatic environment. Our aim was to determine the aquatic toxicity of ZnO-NPs with chronic exposure at environmentally relevant concentrations using the nematode Caenorhabditis elegans. Two simulated environmentally relevant mediumsdmoderately hard reconstituted water (EPA water) and simulated soil pore water (SSPW)dwere used to represent surface water and pore water in sediment, respectively. The results showed that the ZnO-NPs in EPA water has a much smaller hydrodynamic diameter than that in SSPW. Although the ionic release of Zn ions increased time-dependently in both mediums, the Zn ions concentrations in EPA water increased two-fold more than that in SSPW at 48 h and 72 h. The ZnO-NPs did not induce growth defects or decrease head thrashes in C. elegans in either media. However, chronic exposure to ZnO-NPs caused a significant reduction in C. elegans body bends in EPA water even with a relatively low concentration (0.05 mg/l); similar results were not observed in SSPW. Moreover, at the same concentrations (50 and 500 mg/l), body bends in C. elegans were reduced more severely in ZnO-NPs than in ZnCl2 in EPA water. The ATP levels were consistently and significantly decreased, and ROS was induced after ZnO-NPs exposure (50 and 500 mg/l) in EPA water. Our results provide evidences that chronic exposure to ZnO-NPs under environmentally relevant concentrations causes metabolic and locomotive toxicities implicating the potential ecotoxicity of ZnO-NPs at low concentrations in aquatic environments. © 2016 Elsevier Ltd. All rights reserved.

Keywords: ZnO-NPs Emerging contaminants Caenorhabditis elegan Chronic exposure Environmentally relevant concentrations Metabolic toxicity Locomotive toxicity

1. Introduction Engineered nanoparticles (NPs) are nanoscale particles that are produced by human beings. Due to their unique physicochemical properties, engineered NPs are useful for advanced industrial materials and processes as well as consumer applications. However, the rapid development of nanotechnology and increased use of NPs has raised concerns about environmental risk when NPs are disposed and released into the environment (Sajid et al., 2015). The ZnO nanoparticles (ZnO-NPs) are emerging contaminants that are widely used. ZnO-NPs are often used in toothpaste, beauty products, sunscreens, and textiles (Aitken et al., 2006; Serpone

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This paper has been recommended for acceptance by B. Nowack. * Corresponding author. E-mail address: [email protected] (V. Hsiu-Chuan Liao). 1 Both authors contributed equally to this work.

et al., 2007). The widespread application has increased the potential of ZnO-NPs to be released into the environment. The estimated environmental ZnO-NPs concentration is 76 mg/l in the aquatic environment (Boxall et al., 2007), and it is expected to increase 0.49e3.33 mg/kg per year in sediment (Gottschalk et al., 2009). A recent study found that Zn-containing NPs are present in drinking water at ~105 particles/ml (Donovan et al., 2016). The environmental levels of ZnO-NPs are expected to increase with increasing use of NPs. This might pose a threat to the ecosystems (Batley et al., 2013). Due to the significant dissolution effect of ZnO-NPs, some studies have suggested that ZnO-NP toxicity is mainly from dissolved Zn ions (Aruoja et al., 2009; Kasemets et al., 2009; Wong et al., 2010). However, several studies suggest that particleinduced toxic effects represent another important mechanism of toxicity for ZnO-NPs (Ma et al., 2013; Poynton et al., 2011). A typical aquatic environment contains both ZnO particles and dissolved Zn

http://dx.doi.org/10.1016/j.envpol.2016.10.086 0269-7491/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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ions, which might contribute to ZnO-NPs toxicity. Therefore, a detailed characterization of NPs in exposure media such as ionic release and aggregation size are needed for proper interpretation of toxicity data (Ma et al., 2013). Furthermore, due to dynamic behavior of NPs in aquatic environment, toxicity from long-term exposure at low concentrations might be more relevant for real environmental exposure and for further ecotoxicity studies of NPs. The nematode Caenorhabditis elegans is a vital animal model for toxicity assays due to its short lifecycle, compact genome, ease of maintenance, and available transgenic and mutant strains. C. elegans reproduces every 3e4 days under optimal conditions (http://www.wormbook.org/). Because of the short lifecycle of C. elegans, an examination from the first larval stage (L1) through adulthood can be used to study chronic exposure (Comber et al., 2008). Moreover, nematodes are ecologically significant because of their abundance in soils and sediments rich in rotting plant materials. Therefore, C. elegans is widely used as a model for ecotoxicological assays (Ma et al., 2013). Several sub-lethal endpoints are well established in C. elegans including growth, locomotive behaviors, and intracellular reactive oxygen species (ROS) levels (Bischof et al., 2006; Leung et al., 2008; Tejeda-Benitez and OliveroVerbel, 2016). Furthermore, a bioluminescence-based ATP assay for intracellular ATP measurements has been recently developed in C. elegans to assess several toxins including heavy metals and urban sludge (Lagido et al., 2008; McLaggan et al., 2012). ATP levels can reflect metabolic toxicity. Several studies have shown that silica, silver, and polymer nanoparticles decrease the microbial and cellular ATP levels indicating that NPs might cause metabolic toxicity (AshaRani et al., 2009; Bhattacharjee et al., 2012; Lok et al., 2007; Sibag et al., 2015). While the toxic effects of NPs in vitro and in vivo have been investigated, the consequences of chronic exposure of NPs at low concentrations needs to be further elucidated. Chronic effects of ZnO-NPs on the lethality, reproduction, and bioaccumulation have been suggested in Daphnia magna and Folsomia candida (Collembola) (Adam et al., 2014; Kool et al., 2011). However, the chronic ecotoxicity effects of ZnO-NPs remain unclear. In C. elegans, the toxicities of ZnO-NPs on growth and locomotive behaviors have been evaluated at high concentrations of ZnO-NPs or deionized water or K-medium, which are far from ecological conditions (Khare et al., 2015; Wang et al., 2009; Wu et al., 2013). Here, we studied the ecotoxicity of ZnO-NPs under chronic exposure at environmentally relevant media: moderately hard reconstituted water (EPA water) (USEPA, 2002) and simulated soil pore water (SSPW) (Tyne et al., 2013). In addition, sublethal endpoints including growth, locomotive behaviors, metabolic ATP level, and ROS levels were examined to investigate the chronic toxicity of ZnO-NPs under environmentally relevant concentrations in the aquatic environment. 2. Materials and methods 2.1. Chemicals and preparation of ZnO-NPs suspension All chemicals were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA) unless otherwise described. The particle size of ZnO-NPs (assay grade, >97% purity) in present study is less than 50 nm as confirmed by transmission electron microscopy (TEM). The ZnO-NPs suspension was freshly prepared each time prior to use via sonication (40 kHz, 150 W) to suspend ZnO-NPs powder in deionized water for 30 min followed by a series of dilutions to homogeneously suspend the ZnO-NPs. The ZnCl2 used in this study was assay grade 98.6% purity. The concentrations of ZnCl2 (mg/l) were prepared based on weight of Zn metal (mg Zn/l), which is equivalent to ZnO-NPs.

2.2. Characterization of ZnO-NPs The morphological feature of ZnO-NPs suspension was analyzed by TEM (JEM1200EXII; Jeol Ltd., Tokyo, Japan). A few drops of prepared ZnO-NPs suspension (5 mg/l) were dried on copper grid before analysis. The hydrodynamic diameter of the ZnO-NPs (5 mg/ l) suspension was determined by dynamic light scattering (DLS) (Delsa Nano C; Beckman Coulter, CA, USA). The release of Zn ions from ZnO-NPs was analyzed in EPA water and SSPW (STable 1.) at different time intervals. The concentration of analyzed ZnO-NPs suspension was 5 mg/l and was immediately subjected to analysis. The samples were filtered through Amicon ultra 15 ml centrifugal filter unit (3 kDa cutoff z 0.9 nm, Millipore, Billerica, MA, USA) to remove un-dissolved ZnO-NPs, and the Zn ions in aqueous phase were measured using a Zn colorimetric assay kit (MAK032, Sigma-Aldrich) with spectrophotometer at different time points including < 0.5, 4, 7, 24, 48, and 72 h. 2.3. C. elegans strains maintenance and growth condition The C. elegans strains used here were wild-type N2 and PE254 (feIs4). The PE254 (feIs4) strain labels sur-5 with GFP and luciferase (Lagido et al., 2008; McLaggan et al., 2012) was a kind gift from Dr. Lagido, The University of Aberdeen, UK. The wild-type N2 and Escherichia coli OP50 strain were originally obtained from Caenorhabditis Genetics Center (CGC) (University of Minnesota, MN, USA). All strains were maintained on nematode growth medium (NGM) agar plates freshly seeded with a lawn of E. coli OP50 at 20  C unless otherwise stated. 2.4. C. elegans growth assays The growth of C. elegans was measured across the body length of C. elegans. Wild-type N2 synchronized L1 nematodes were exposed to various concentrations of ZnO-NPs (0, 0.05, 0.5, 5, 10, 50, 100, and 500 mg/l) in EPA water or SSPW in the presence of E. coli OP50 at 109 cells/ml as a food source at 20  C. Deionized water was used as the solvent. After 72 h, the worms' body length was measured. Worms were first washed with EPA water or SSPW three times and dropped onto NGM plates. After the plates were dried, the plates were placed under the microscope with a 10 objective lens. The images were collected with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA) and analyzed by Fiji (Schindelin et al., 2012) with a worm sizer plugin (Moore et al., 2013). The growth assay was performed at least for three independent experiments and at least fifty worms were measured per treatment in each replicate. 2.5. C. elegans locomotive behaviors assays Wild-type N2 nematodes were cultured as above described. In short, synchronized L1 nematodes were exposed to various concentrations of ZnO-NPs or ZnCl2 in EPA water or SSPW with E. coli OP50 at 109 cells/ml as food source at 20  C. Deionized water was used as the solvent control. After 72 h exposure to ZnO-NPs or ZnCl2, the worms were washed three times with the corresponding incubation medium before being subjected to locomotive behavioral assays. The behavior analysis used the body bend and head thrash frequency. All assays were repeated in at least three independent experiments and at least twenty nematodes were scored per treatment in each replicate. The body bends frequency assay was adapted from previous studies (Li et al., 2013; Tsalik and Hobert, 2003; Tseng et al., 2013). The worms were randomly selected and placed in unseeded NGM plates. Assuming the worm was crawling along the X-axis, a body bend was determined as a change in direction of the part of the

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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worm corresponding to the posterior bulb of the pharynx along the Y-axis. The number of body bends was counted per 20 s. The head thrash frequency assay was also adapted from previous studies (Li et al., 2013; Tsalik and Hobert, 2003; Tseng et al., 2013). Each nematode was placed into a 60 ml K-medium (32 mM KCl, 53 mM NaCl) (Williams and Dusenbery, 1990) on top of the agar prior to performing the assay. After a recovery period of 1 min, the head thrashing movements were recorded for 60 s to count the changes in the direction of bending at the mid body. A head thrash was defined as a change in the direction of bending at the mid body. 2.6. Analysis of metabolic ATP level with a transgenic strain PE254 Metabolic toxicity was determined by changes in bioluminescence reflecting the metabolic ATP level in the transgenic strain PE254 (Lagido et al., 2008; McLaggan et al., 2012). Exposure conditions were basically the same as described above with slight modifications on exposure time. The conditions were chosen based on the above results. Synchronized L1 PE254 transgenic worms were exposed to 50 and 500 mg/l of ZnO-NPs or ZnCl2 in EPA water with E. coli OP50 as the food source at 109 cells/ml at 20  C. Deionized water was the solvent control. After 66e67 h exposure to ZnO-NPs or ZnCl2, changes in bioluminescence were analyzed on young adult nematodes just before egg formation (Lagido et al., 2008). Worms were washed with EPA water three times before the assay. Nematodes were transferred into a 96-well plate, and twenty worms were placed into each well containing citratephosphate buffer 100 ml with D-luciferin (0.1 mM) (Lagido et al., 2008). The bioluminescence was quantified with a FLx800 Microplate Fluorescent Reader (Bio-Tek Instruments, Winookski, VT, USA), and data were normalized with luciferase GFP signal at 485 nm excitation and 520 nm emission. The ATP assay was performed in at least three independent experiments; there were six technical replicates per treatment in each replicate.

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error of the mean (SEM) (unless otherwise stated). The p values were calculated by one-way ANOVA followed by LSD post hoc test, and p < 0.05 was considered to be significantly different among populations. 3. Results and discussion 3.1. Characterization of ZnO-NPs in EPA water and SSPW Several studies have reported that the exposure media might have a significant influence on the toxicity of the NPs (Tyne et al., 2013; Yang et al., 2012). To better reflect natural fresh water and soil solution conditions, simulated environmental water such as EPA water and SSPW have been developed (Tyne et al., 2013; USEPA, 2002). Thus far, the medium effect is not clear on ZnONPs in simulated environmental water samples. Thus, before investigating the toxicity of ZnO in different mediums, we extensively characterized the ZnO-NPs used here. TEM images of ZnO-NPs showed that the aggregated particle size in EPA water was much smaller than that in SSPW (Fig. 1). Similar results for particle size were also shown in Table 1. The hydrodynamic diameter in EPA water increased time-dependently and reached 1000 nm at 24 h (Table 1). However, the hydrodynamic diameter in SSPW quickly increased to 1062.2 nm and remained about 1000 nm within 7 h (Table 1). Clearly, the hydrodynamic diameter in SSPW was larger than that in EPA water at each time point. Ionic release from ZnO-NPs can also affect toxicity. Several studies have shown that Zn ions are rapidly released from ZnO-NPs

2.7. C. elegans intracellular reactive oxygen species measurement Wild-type N2 nematodes were cultured as above described. Synchronized L1 nematodes were exposed to 50 and 500 mg/l of ZnO-NPs or ZnCl2 in EPA water with E. coli OP50 as a food source at 109 cells/ml at 20  C. Deionized water was used as the solvent control. After 72 h ZnO-NPs exposure, adult worms were washed with EPA water three times, and the intracellular ROS were stained with 50 mM 2’,7’-dichlorodihydrofluoroscein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR, USA) for 2.5 h at 20  C in a dark incubator (Morgan et al., 2010). Before microscopic examination, the worms were washed three times with phosphate buffered saline (PBS). At least thirty worms from each treatment group were mounted onto microscope slides with 3% agarose gel on top. After anaesthetizing the animals with 100 mM sodium azide, the slides were covered with coverslips. Fluorescence images of ROS stained by H2DCFDA were acquired with an epifluorescence microscope (Leica, Wetzlar, Germany) with a suitable filter set (excitation, 485 ± 20 nm; emission, 530 ± 20 nm) and a cooled charge coupled device (CCD) camera. The images were captured with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA). Quantified fluorescence signal represented the amount of ROS in nematodes. Intracellular ROS examination was performed at least for three independent experiments and at least thirty worms were analyzed per treatment in each replicate. 2.8. Statistical analysis Statistical analysis used SPSS Statistics 22.0 Software (IBM, Inc., New York, USA, 2013). The data are presented as mean ± standard

Fig. 1. TEM images of ZnO-NPs in EPA water and SSPW. (A) 5 mg/l ZnO-NPs in EPA water. (B) 5 mg/l ZnO-NPs in SSPW.

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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Table 1 Characterization of ZnO-NPs used in this study. Duration (h)

<0.5 4 7 24 48 72 a b

Hydrodynamic diameter (nm)a

Released Zn ions (mg/l)b

EPA water

SSPW

EPA water

SSPW

295.2 ± 119.4 327.9 ± 90.7 617.2 ± 163.6 1332.8 ± 309.6 e e

1062.2 1663.4 1048.1 2201.8 e e

273 ± 24 736 ± 26 936 ± 21 1230 ± 53 1865 ± 27 3511 ± 61

397 ± 34 788 ± 5 887 ± 44 993 ± 196 952 ± 54 1624 ± 20

± ± ± ±

150.5 292.5 132.7 217.5

Determined by dynamic light scattering (DLS). Initial concentration of ZnO-NPs: 5 mg/l.

and play an important role in toxicity (Ma et al., 2009; Miller et al., 2010; Wong et al., 2010). Therefore, the concentrations of free Zn ions in EPA water and SSPW were measured over 72 h (Table 1). The released Zn ions in the EPA water and SSPW both increased timedependently but with different concentrations. Within the first 24 h, the difference between EPA water and SSPW was not significant. However, the Zn ionic concentration in EPA water increased two-fold more than in SSPW at 48 h and 72 h (Table 1). Ionic strength is a major factor that influences the size of nanoparticles (Bizmark and Ioannidis, 2015; Domingos et al., 2013; El Badawy et al., 2010). High ionic strength can result in rapid aggregation (Basnet et al., 2013). The ionic strength of EPA water (4.58 mM) was much lower than that of SSPW (10.3 mM) (Majedi et al., 2014; Tyne et al., 2013; USEPA, 2002). This causes smaller aggregates of ZnO-NPs in the EPA water (Table 1). The dissolved Zn ions in the ZnO-NPs in EPA water were much higher than that in SSPW (Table 1). This might be due to the smaller size of the aggregates in the EPA water (David et al., 2012). Thus, ZnO-NPs are quite different in EPA water and SSPW. The smaller size of the aggregates and higher amount of dissolved Zn might increase the toxicity of the ZnO-NPs. 3.2. Effects of ZnO-NPs on C. elegans growth in EPA water and SSPW Growth is a general endpoint for toxicity (Bischof et al., 2006; Tejeda-Benitez and Olivero-Verbel, 2016). In C. elegans, growth

can be determined by measuring the body length (Tuck, 2014). In addition, due to the short life cycle of C. elegans, an examination from larval L1 through adult (~72 h at 20  C) can be regarded as chronic exposure (Comber et al., 2008). The estimated environmental ZnO-NPs concentration is 76 mg/l in aquatic environments (Boxall et al., 2007). Herein, synchronized wild-type N2 L1 larvae were exposed to a range of environmentally relevant concentrations of ZnO-NPs (0, 0.05, 0.5, 5, 10, 50, 100, 500 mg/l) for 72 h at 20  C in EPA water and SSPW, respectively. The body length of each nematode was measured. The results showed that prolonged exposure to ZnO-NPs did not decrease the body length of worms in either EPA water or SSPW at the examined concentrations (Fig. 2). Nematodes successfully grew to mature adults with a normal body length of about 1.2 mm in all groups (Fig. 2). Therefore, the results indicated that ZnO-NPs did not exert growth defects under the examined ZnO-NPs concentrations. A prolonged exposure to 50 mg/l ZnO-NPs has been shown to decrease the body length of worms (Wu et al., 2013). In contrast, our data indicated that even 500 mg/l ZnO-NPs did not cause growth defects in C. elegans. This discrepancy can be due to different ZnONPs particle sizes in the exposure medium as well as the exposure conditions. In our study, we used EPA water and SSPW as a simulated environmental medium throughout the toxicity assay whereas the K-medium was used in their study (Wu et al., 2013). In addition, Ma et al. (2009) compared buffered and unbuffered Kmedium and found that unbuffered K-medium led to precipitation of Zn and increased the toxicity of ZnO-NP as the concentration increased (Ma et al., 2009). In sum, the differences in exposure media might affect the properties and stability of ZnO-NPs and thus probably underlies the different toxicity outcomes. 3.3. Effects of ZnO-NPs on C. elegans locomotive behaviors in EPA water and SSPW C. elegans locomotive behaviors including body bends and head thrashes have been used to evaluate the sub-lethal neurotoxicity of toxins (Tejeda-Benitez and Olivero-Verbel, 2016). Here, we studied whether prolonged exposure to ZnO-NPs at environmentally relevant concentrations would induce locomotive toxicity.

Fig. 2. Effects of ZnO-NPs on C. elegans growth in EPA water and SSPW. L1-larvae and adults were exposed to EPA water and SSPW. The concentrations of ZnO-NPs (mg/l) were prepared based on the weight of the Zn metal (mg Zn/l). The results are represented as the mean ± standard error of the mean (SEM). The statistical significance of the differences was determined using a one-way ANOVA and LSD post hoc test. Different lower letters (in EPA water) or upper letters (in SSPW) represented statistically significant differences between groups (p < 0.05).

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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Synchronized wild-type N2 L1 larvae were exposed to a range of ZnO-NPs (0, 0.05, 0.5, 5, 10, 50, 100, and 500 mg/l) for 72 h at 20  C in EPA water and SSPW, respectively, and the body bends and head thrashes of each nematode were measured. The results showed that ZnO-NPs in EPA water reduced the body bends of C. elegans in a dose-dependent manner (Fig. 3A). The estimated aquatic environmental ZnO-NPs concentration was 76 mg/l (Boxall et al., 2007). We found that the body bends of C. elegans were significantly decreased even when exposed to 0.05 mg/l ZnO-NPs in EPA water versus a non-exposed control (Fig. 3A). Conversely, the same concentrations of ZnO-NPs in SSPW did not result in any significant reduction in C. elegans body bends (Fig. 3A). This discrepancy in toxicity agreed with the characterization of ZnO-NPs as shown in Fig. 1 and Table 1. In addition to the body bends, head thrashes of

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C. elegans were also evaluated. Fig. 3B showed that the head thrashes of worms at examined ZnO-NPs concentrations were comparable to that of non-exposed control in both EPA water and SSPW suggesting that head thrashes of C. elegans are a less sensitive endpoint for ZnO-NPs locomotive toxicity assays. Intriguingly, the same concentrations of ZnO-NPs exposure gave rise to different outcomes between the body bends and head thrashes in C. elegans (Fig. 3A and B). This might suggest different modes of action in neurotoxicity induced by ZnO-NPs. It has been reported that there are molecular differences between crawling (body bends) and swimming (head thrashes) (Pierce-Shimomura et al., 2008). Physical characterization revealed unique kinematics between these two gaits (Pierce-Shimomura et al., 2008). Crawling is mainly modulated by T-type calcium channel while swimming

Fig. 3. Effects of ZnO-NPs on C. elegans locomotive behaviors in EPA water and SSPW. (A) Effects of ZnO-NPs on head thrash in EPA water and SSPW; (B) Effects of ZnO-NPs on body bends in EPA water and SSPW. The test used L1-larvae to mature adults in EPA water and SSPW (72 h). The concentrations of ZnO-NPs (mg/l) were prepared based on weight of Zn metal (mg Zn/l). The results are represented as mean ± standard error of the mean (SEM). The statistical significance of differences was determined using one-way ANOVA and LSD post hoc test. Different lower letters (in EPA water) or upper letters (in SSPW) represented statistically significant differences between groups (p < 0.05).

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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Fig. 4. Comparison of effects of ZnCl2 and ZnO-NPs on C. elegans body bends. Exposures were performed from L1-larvae to mature adults in EPA water (72 h). The concentrations of ZnCl2 (mg/l) were prepared based on the weight of Zn metal (mg Zn/l), which is equivalent to ZnO-NPs. The results are represented as the mean ± standard error of the mean (SEM). The statistical significance of the differences was determined using one-way ANOVA and LSD post hoc test. Different lower letters (in EPA water) represented statistically significant differences between groups (p < 0.05).

depends on NCA-type ion channel, which is a sodium “leak” channel (Pierce-Shimomura et al., 2008). Thus, the body bend defect observed in Fig. 3A is probably due to affected T-type calcium channel in cholinergic motor neurons. Moreover, the transition between two gaits and the maintenance in specific gait is reportedly related to two universal neurotransmitters: dopamine and serotonin (Vidal-Gadea et al., 2011). The impairment in crawling also indicates possible interactions between ZnO-NPs and neurotransmitters. However, a detailed mechanism remains unknown, and there might be interactions between ZnO NP and reception of neurotransmitters, but this requires further study. 3.4. Comparison of effects of ZnO-NPs and ZnCl2 on C. elegans body bends Previous studies suggested that the observed toxicity induced by ZnO-NPs is mainly due to Zn ions released from the nanoparticle (Franklin et al., 2007; Johnson et al., 2015). The results in Fig. 3A characterize the ZnO-NPs in EPA water (Fig. 1 and Table 1). We further investigated whether the C. elegans body bends observed in Fig. 3A are elicited by Zn ions. Therefore, we compared C. elegans body bends by ZnCl2 and ZnO-NPs in EPA water. We chose 50 mg/l and 500 mg/l ZnO-NPs or ZnCl2 for the following experiments because the estimated aquatic environmental ZnO-NPs concentration was 76 mg/l (Boxall et al., 2007). Synchronized wild-type N2 L1 larvae were exposed to 50 mg/l and 500 mg/l ZnO-NPs or ZnCl2 for 72 h at 20  C in EPA water. The body bends of each nematode were measured. The results showed that nematodes exposed to 50 mg/l and 500 mg/l of ZnCl2 exerted less body bends of C. elegans than that of ZnO-NPs (Fig. 4) suggesting that different modes of toxic action might be involved between ZnO-NPs and ZnCl2 during prolonged exposure. One study reported that Zn ions rather than ZnO-NPs induced cell death because the toxic effect was ameliorated after Zn ions were scavenged by EDTA (Johnson et al., 2015). However, a recent study suggested that the toxicity of ZnO-NPs were size-dependent, and the toxicity of ZnO-NPs was much worse than ZnO ions (Khare et al., 2015). It has been reported that Zn ions play an important role

in ZnO-NPs-induced toxicity, but particulate effects cannot be excluded either (Liu et al., 2016). Ma et al. (2009) found no significant difference in locomotion in C. elegans between ZnO-NPs and ZnCl2 for a 4-hr acute exposure in adults. In contrast, chronic exposure to C. elegans shows a significant difference in body bends between ZnO-NPs and ZnCl2 (Fig. 4) providing evidence for the ZnO-NPs toxic particulate effects. Although the contribution of ZnO-NPs to toxic effects is still debatable, our results suggest that ZnO-NP themselves play an important role in C. elegans body bend toxicity aside from Zn ions. Taken together, this study indicated that Zn ions only partially contribute to the ZnO-NP induced C. elegans body bend toxicity. This is probably via a distinct pathway. 3.5. Effects of ZnO-NPs and ZnCl2 on metabolic ATP level in C. elegans It has been suggested that the change in intracellular ATP levels can be a useful chronic endpoint for ecotoxicity assessment because they reflect the metabolic status of organisms (Lagido et al., 2008, 2009; McLaggan et al., 2012). To test whether a prolonged exposure to ZnO-NPs at environmentally relevant concentrations caused metabolic toxicity, we investigated the relative ATP levels in response to ZnO-NPs using the transgenic strain PE254 as a bioindicator (Lagido et al., 2008; McLaggan et al., 2012). The bioluminescence of firefly luciferase in strain PE254 indicates relative ATP levels and overall metabolic health, and thus this signal could be used to monitor toxins (Lagido et al., 2008; McLaggan et al., 2012). In addition, ZnCl2 at the same concentrations were also used to compare the toxic effects of released Zn ions and nanoparticles. The ZnO-NP concentrations were chosen based on the results of Fig. 4. Synchronized L1 PE254 transgenic worms were exposed to 50 and 500 mg/l of ZnO-NPs or ZnCl2 in EPA water for 66e67 h at 20  C, and changes in bioluminescence were measured. The results showed that ATP levels was decreased 10e15% in the presence of ZnO-NPs or ZnCl2 (50, 500 mg/l) versus non-exposed controls (Fig. 5). In contrast to the body bend results (Fig. 4), there was no statistical difference between the treatments of ZnONPs and ZnCl2 (Fig. 5).

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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Fig. 5. Effects of ZnO-NPs and ZnCl2 on metabolic ATP level in C. elegans. Exposures were performed from L1-larvae to young adults in EPA water (66e67 h). The concentrations of ZnCl2 (mg/l) were prepared based on weight of Zn metal (mg Zn/l) that is equivalent to ZnO-NPs. Transgenic strain PE254 was used as a luminescent intracellular ATP sensor to show the metabolic toxicity of ZnO-NPs and ZnCl2. The data are the mean ± standard error of the mean (SEM). The statistical significance of the differences was determined using one-way ANOVA and LSD post hoc test. Different lower letters (in EPA water) show statistically significant difference between groups (p < 0.05).

ATP is synthesized by ATPase in cells, and metabolic ATP levels can be determined by ATPase activity. However, this method does not reflect real-time ATP levels in vivo. Stress-induced ATP changes are often rapid, and ATPase activity assays are not suitable in stress response experiments (Corton et al., 1994; Lagido et al., 2008). Most studies investigated NP-induced ATP changes in bacteria and cell cultures (AshaRani et al., 2009; Bhattacharjee et al., 2012; Lok et al., 2007; Sibag et al., 2015). Our study further investigated the metabolic toxicity of ZnO-NPs in the multicellular animal model C. elegans. Previous studies showed that luciferase can report ATP levels both in vitro and in vivo (de Wet et al., 1987; Lagido et al., 2008).

Metabolic ATP level could be affected by multiple factors including ROS, mitochondria dysfunction, and high energetic cost of stress-responsive mechanisms (Lagido et al., 2009). Intracellular Zn ions might trigger heavy metal detoxification mechanisms causing extra energetic cost and affecting mitochondria function. Our results showed that ZnO-NPs or ZnCl2 caused similar ATP reductions suggesting that the Zn ions play an important role in the metabolic toxicity of C. elegans. Some in vitro studies reported that Zn ions influence mitochondrial function and inhibit ATP production (Dineley et al., 2003; Lemire et al., 2008), which agrees with our results (Fig. 5).

Fig. 6. Effects of ZnO-NPs and ZnCl2 on intracellular ROS level in C. elegans. Exposures were performed from L1 to mature adults in EPA water (72 h). The concentrations of ZnCl2 (mg/l) were prepared based on weight of Zn metal (mg Zn/l) which is equivalent to ZnO-NPs. The results are represented as mean ± standard error of the mean (SEM). The statistical significance of the differences was determined using one-way ANOVA and LSD post hoc test. Different lower letters (in EPA water) show statistically significant difference between groups (p < 0.05).

Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086

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3.6. Effects of ZnO-NPs and ZnCl2 on intracellular ROS level in C. elegans

Conflict of interest The authors declare that no competing interests exist.

Several studies have reported that intracellular ROS is a valuable indicator of biological oxidative stress, and that ROS might affect metabolic ATP levels (AshaRani et al., 2009; Gutterman, 2005). The ZnO-NPs produced a large amount of ROS by photo-reactions and increased intracellular ROS levels were found in cell lines and C. elegans (Li et al., 2012; Ma et al., 2011; Vandebriel and De Jong, 2012; Wu et al., 2013). Herein, we investigated intracellular ROS level in C. elegans under environmentally relevant medium (EPA water) and concentrations in the presence of ZnO-NPs and ZnCl2. The EPA water and ZnO-NPs concentrations were chosen based on the results of Figs. 4 and 5. Synchronized wild-type N2 L1 larvae were exposed to 50 mg/l and 500 mg/l ZnO-NPs or ZnCl2 for 72 h at 20  C in EPA water, and the intracellular ROS level of each nematode was measured. As shown in Fig. 6, intracellular ROS levels were significantly increased when worms were exposed to 50 mg/l and 500 mg/l ZnCl2 and ZnO-NPs. The ROS production did not show significant differences between ZnCl2 and ZnO-NPs (Fig. 6). The photocatalytic ROS generation by ZnO-NPs has been reported to be low under ambient artificial laboratory light versus natural sunlight (Ma et al., 2011). In this study, C. elegans was exposed to ZnO-NPs at relatively low concentrations (50 mg/l and 500 mg/l) and under laboratory conditions. Thus, the increased intracellular ROS might not be due to photo reactions. Another ROS generation mechanism of ZnO-NPs is probably induced by Zn ions (Ma et al., 2013). Studies have suggested that intracellular Zn ions from dissolved ZnO-NPs could damage mitochondria and lead to ROS generation (Song et al., 2010; Xia et al., 2008). The ROS induced by ZnO-NPs can be scavenged by adding antioxidants such as ascorbic acid into the incubation medium. In vitro data showed that L-ascorbic acid can significantly reduce the intracellular ROS level with no cytotoxicity in human immune cells (Shen et al., 2013). This suggests that ROS only played a partial role in ZnO-NPs-induced toxicity. Based on the body bend data (Fig. 4), there was a significant difference between ZnO-NPs and ZnCl2exposed worms, but this was comparable between ZnO-NPs and ZnCl2 exposures in ROS level. This also suggests that ROS only partially contribute to the toxic effects and implies that different mechanisms are involved in the ZnO-NP toxicity.

4. Conclusions In summary, our study shows that the toxicity of ZnO-NPs is largely different between two environmentally relevant media: synthetic surface water (EPA water) and soil pore water (SSPW). The locomotive toxicity of ZnO-NPs on C. elegans body bends is more significant in EPA water than SSPW. The higher toxicity caused by ZnO-NPs might be due to the smaller aggregate size of ZnO-NPs in this medium as well as the higher concentrations of dissolved Zn ions. Our study indicated that the ZnO-NPs and ZnCl2 in EPA water decrease ATP levels and similarly increase the intracellular ROS indicating that the metabolic toxicity is mainly due to dissolved Zn ions. Our results suggest that chronic exposure to ZnONPs under environmentally relevant concentrations caused metabolic and locomotive toxicities; this highlights the potential ecotoxicity of ZnO-NPs at low concentrations in aquatic environments. Finally, our study shows that sub-lethal metabolic and locomotive toxicity endpoints of C. elegans model including body bends, ATP levels, and ROS levels in EPA water can assess the potential ecotoxicity of nanoparticles in aquatic environments.

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Please cite this article in press as: Huang, C.-W., et al., Chronic ZnO-NPs exposure at environmentally relevant concentrations results in metabolic and locomotive toxicities in Caenorhabditis elegans, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.10.086