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Toxicity comparison of nanopolystyrene with three metal oxide nanoparticles in nematode Caenorhabditis elegans
Chemosphere 245 (2020) 125625
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Toxicity comparison of nanopolystyrene with three metal oxide nanoparticles in nematode Caenorhabditis elegans Dan Li, Jie Ji, Yujie Yuan, Dayong Wang* Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School, Southeast University, Nanjing, 210009, China
h i g h l i g h t s We compared toxicity of nanopolystyrene with three metal-NPs in nematodes. Nanopolystyrene could induce more severe toxicity than SiO2-NPs. Nanopolystyrene could not induce severe toxicity comparable to Al2O3-NPs or TiO2-NPs. Cellular basis of toxicity difference of nanopolystyrene with metal-NPs was examined. Our data will be helpful for understanding exposure risk of nanopolystyrene on organisms.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2019 Received in revised form 29 November 2019 Accepted 9 December 2019 Available online xxx
Using Caenorhabditis elegans as an animal model, we compared the toxicity between nanopolystyrene and three metal oxide nanoparticles (NPs) (Al2O3-NPs, TiO2-NPs, and SiO2-NPs). After exposure from L1larvae to adult day-1, nanopolystyrene (100 mg/L) reduced brood size and induced severe germline apoptosis, and nanopolystyrene (10e100 mg/L) decreased locomotion behavior, induced obvious reactive oxygen species (ROS) production, and activated noticeable mitochondrial unfolded protein response (mt UPR). Using several endpoints (lethality, development, reproduction, and/or locomotion behavior), we found that nanopolystyrene could induce more severe toxicity than SiO2-NPs, although nanopolystyrene did not cause the toxicity comparable to that in Al2O3-NPs or TiO2-NPs exposed nematodes. Our data will be useful for understanding the exposure risk of nanopolystyrene on environmental organisms. Moreover, the detected toxicity difference between nanopolystyrene and three metal oxide NPs were associated with the differences in both induction of oxidative stress and activation of mt UPR in exposed nematodes. © 2019 Elsevier Ltd. All rights reserved.
Handling editor: Tamara S. Galloway Keywords: Nanoparticles Toxicity comparison Nanopolystyrene Caenorhabditis elegans
1. Introduction Microplastics are defined as the polymers with the sizes less than 5 mm. Miroplastics are widespread and abundant in various environments, including both aquatic and soil environments (Burns and Boxall, 2018; Chae and An, 2018; Zhang et al., 2018). Recently, bioavailability and adverse effects of microplastics have been examined in different environmental organisms, such as zooplankton (Botterell et al., 2019; Li et al., 2019). Moreover, the microplastics could be further detected in drinking water and sources of drinking water (Koelmans et al., 2019). In the environment, microplastics can be potentially degraded
* Corresponding author. E-mail address: [email protected] (D. Wang). https://doi.org/10.1016/j.chemosphere.2019.125625 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
into nanoplastics (nano-sized plastics) (Mattsson et al., 2015). Besides the microplastic pollution, the existence, fate, and pollution of nanoplastic in different ecosystems have also received the attention (da Costa et al., 2016; Wan et al., 2018; Ng et al., 2018; Alimi et al., 2018; Enfrin et al., 2019). The release of nanoplastics in human food production chain and the environment has implied their potential implications for human health (Bouwmeester et al., 2015; Lehner et al., 2019). Nevertheless, the toxic degree of nanoplastics on organisms is still largely unclear. Nanopolystyrene is a frequently examined example to determine the toxic effects of nanoplastics on organisms (Balbi et al., 2017; Pitt et al., 2018; Lim et al., 2019). Reduction in lifespan, deficit in development, suppression in reproduction, decrease in behaviors, and oxidative stress could be detected in nanopolystyrene exposed organisms, such as zebrafish and Daphnia (Della Torre et al., 2014; Jeong C et al., 2016; Rist et al., 2017;
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Gambardella et al., 2017; Chen et al., 2017). Model animal of Caenorhabditis elegans is sensitive to environmental exposure, and has been proven to be useful for evaluating the toxicity of various nanomaterials, including nanoparticles (NPs) (Wang, 2018; Yang et al., 2018; Liu et al., 2019a, 2020; Zhao et al., 2019a, 2019b; Starnes et al., 2019; Moon et al., 2019; Eom and Choi, 2019). Recently, C. elegans has been further employed to assess the toxicity of nanopolystyrene on animals at various aspects, such as neurotoxicity, intestinal toxicity, and reproductive toxicity (Lei et al., 2018; Hanna et al., 2018; Qu et al., 2019a, 2019b; Kim et al., 2019; Qu and Wang, 2019; Zhao et al., 2019c). The toxicity of nanopolystyrene was charge-dependent and enhanced by surface amino modification in nematodes (Qu et al., 2019c). Some molecular signals, such as insulin, p38 MAPK, and Wnt signals, in response to nanopolystyrene have also been identified in nematodes (Shao et al., 2019a, 2019b; Shao and Wang, 2019; Qu et al., 2019d; Qu et al., 2019e; Yang et al., 2019). To further evaluate the toxic degree of nanopolystyrene, we employed the C. elegans as an animal model to compare the toxicity of nanopolystyrene with three metal oxide NPs (Al2O3-NPs, TiO2NPs, and SiO2-NPs). Among the examined three metal oxide NPs, exposure to Al2O3-NPs could cause the very severe neurotoxicity (Li et al., 2013), and exposure to TiO2-NPs caused the more severe toxicity than the exposure to SiO2-NPs in nematodes (Wu et al., 2013). These backgrounds imply the toxicity difference among Al2O3-NPs, TiO2-NPs, and SiO2-NPs in nematodes. Our results demonstrated the toxicity order among nanopolystyrene and the examined three metal oxide NPs. Our data will be helpful for our evaluating the possible risk of nanopolystyrene on environmental organisms. 2. Materials and methods 2.1. Characterizations of nanopolystyrene and three metal oxide NPs The sizes of nanopolystyrene and three metal oxide NPs were all 30 nm. Nanopolystyrene was from Janus New-Materials Co. (Nanjing, China), Al2O3-NP was from Shanghai Macklin Biochemical company (Shanghai, China), TiO2-NP was from Nano Applied Research Center of Nanjing University of Technology (Nanjing, China), and SiO2-NP was from Wan Jing New Material Co. Ltd. (Hangzhou, China). Purities of nanopolystyrene, Al2O3-NPs, TiO2NPs, and SiO2-NPs were >99.9%, >99.9%, >99%, and >99.7%. Images of transmission electron microscope (TEM) show the morphology and the aggregation state of nanopolystyrene and three metal oxide NPs in K-medium after sonication at 100 W and 40 kHz for 30 min (Fig. S1). The aggregation sizes of nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs were 98.7 ± 9.8, 516.3 ± 45.2, 398.5 ± 65.2, and 105.4 ± 8.9 nm (Fig. S1). Based on the dynamic light scattering (DLS) analysis, zeta-potentials of nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs were 20.5 ± 2.47, 38.12 ± 3.77, 13.1 ± 1.21, and 63.32 ± 3.11 mV. The Raman spectrum of nanopolystyrene was determined by Raman spectroscopy (Renishaw Invia Plus laser Raman spectrometer, Renishaw, UK). Fig. S2 shows the Raman spectrum of nanopolystyrene. 2.2. Animal maintenance and exposure Nematode strains (wild-type N2 and transgenic strains of CF1553/muIs84 [SOD-3:GFP] and SJ4100/zcIs13 [HSP-6:GFP]) were fed with Escherichia coli OP50 on nematode growth medium (NGM) plates (Brenner, 1974). Exposure to nanopolystyrene or metal oxide NPs was performed from L1-larvae to adult day-1. Exposure was
carried out in liquid solutions of nanopolystyrene or metal oxide NPs. The working solutions of nanopolystyrene or metal oxide NPs were added with OP50 (~4 106 colony-forming units (CFUs)). During the exposure, the nanoparticle solutions were refreshed daily. After the exposure, the nematodes were washed with M9 buffer for three times. After that, the nematodes were used for toxicity assessment using different endpoints. All the toxicity assessments for each of the examined endpoints were performed for three replicates. To prepare age-synchronous L1-larvae, the mature gravid nematodes were treated with bleaching mixture solution (0.45 M NaOH, 2% HOCl) to release the eggs and let them further hatch on NGM plates. 2.3. Lethality and growth Lethality was assessed by the endpoint of % survival of nematodes (Wu et al., 2013). After exposure, we scored the inactive animals, which were judged if they did not respond to stimulus using a small, metal wire. For each treatment, one hundred nematodes were examined. The growth of nematodes was assessed by the endpoint of body length (Wu et al., 2013). The body length was determined after the measurement of flat surface length using an Image-Pro® Express software. For each treatment, thirty nematodes were examined. 2.4. Reproduction Reproduction was evaluated by the endpoints of brood size and germline apoptosis (Qu et al., 2019c; Qu et al., 2019f). The brood size refers to the number of offspring at all stages beyond the egg. For each treatment, ten animals were examined. To analyze the germline apoptosis, the animals were stained with acridine orange (AO, 25 mg/mL) for 1-h in darkness. The germline apoptosis was examined under an epifluorescence microscopy (excited light, 395 nm; emitted light, 509 nm). For each treatment, forty animals were examined. 2.5. Locomotion behaviors After exposure, two endpoints (head thrash and body bend) were used to assess the possible neurotoxicity on locomotion behaviors in nematodes (Kong et al., 2019). A head thrash is considered as an alteration in bending direction at body mid-region, and a body bend is considered as an alteration in posterior bulb direction. For each treatment, thirty animals were examined. 2.6. Activation of oxidative stress To determine the activation of oxidative stress, the reactive oxygen species (ROS) production was determined (Liu et al., 2019b). After exposure, the nematodes were labeled with CM-H2DCFDA (1 mM) in darkness for 3 h. The nematodes were then examined for both excitation wavelength at 488 nm and emission filter at 510 nm under a laser scanning confocal microscope. In nematodes, the strongest ROS fluorescent signals can be detected in the intestine (Wang et al., 2007). ROS signals in the intestine were examined by relative fluorescence units and normalized to autofluorescence. For each treatment, forty animals were examined. Besides the ROS production, transgenic strain of muIs84 was further used to examine the alteration in SOD-3:GFP expression (Zhao et al., 2019d). SOD-3 is a mitochondrial superoxide dismutase, which provides an antioxidation defense system against the oxidative stress (Hunter et al., 1997). SOD-3 is constitutively expressed in pharyngeal and intestine (Henderson et al., 2006). Relative fluorescence of SOD-3:GFP signals in intestine was
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examined. For each treatment, forty animals were examined.
nematodes (Fig. 1B).
2.7. Mitochondrial unfolded protein response (mt UPR)
3.3. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing brood size and in inducing germline apoptosis
HSP-6 is a marker for mt UPR (Haynes et al., 2007). Transgenic strain of zcIs13 was used to assess the mt UPR in nematodes. HSP-6 is broadly expressed in the intestine (Sanders et al., 2017). Relative fluorescence of HSP-6:GFP signals in intestine was examined. For each treatment, forty animals were examined.
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Using lethality as an endpoint, all the examined 4 NPs at concentrations of 0.1 and 1 mg/L did not induce the lethality (Fig. 1A). Additionally, TiO2-NPs, SiO2-NPs, and nanopolystyrene at the concentration of 10 mg/L, as well as SiO2-NPs and nanopolystyrene at the concentration of 100 mg/L, also did not induce the lethality (Fig. 1A). In contrast, only exposure to Al2O3-NPs (10e100 mg/L) and TiO2-NPs (100 mg/L) could induce the obvious lethality in nematodes (Fig. 1A).
Using brood size to reflect the reproductive capacity, we found that all the examined 4 NPs at the concentration of 0.1 mg/L did not affect brood size (Fig. 2A). Al2O3-NPs and TiO2-NPs at concentrations of 1 and 10 mg/L could significantly reduce the brood size, whereas SiO2-NPs and nanopolystyrene at concentrations of 1 and 10 mg/L did not influence the brood size (Fig. 2A). All the examined 4 NPs at the concentration of 100 mg/L could significantly reduce the brood size (Fig. 2A). Moreover, Al2O3-NPs (100 mg/L) or TiO2-NPs (100 mg/L) caused the more severe toxicity in reducing brood size than nanopolystyrene (100 mg/L), and nanopolystyrene (100 mg/L) resulted in the more severe toxicity in reducing brood size than SiO2-NPs (100 mg/L) (Fig. 2A). In nematodes, germline apoptosis acts as an important cellular basis for the reduction in brood size (Qu et al., 2019c). Considering that only exposure to nanopolystyrene (100 mg/L) could reduce the brood size, we next compared the induction of germline apoptosis in nematodes exposed to the examined 4 NPs at concentrations of 10 and 100 mg/L. Al2O3-NPs (10 mg/L) or TiO2-NPs (10 mg/L) could induce the obvious increase in germline apoptosis; however, SiO2NPs (10 mg/L) or nanopolystyrene (10 mg/L) did not induce the increase in germline apoptosis (Fig. 2B). All the examined 4 NPs at the concentration of 100 mg/L caused the significant increase in germline apoptosis (Fig. 2B). Al2O3-NPs (100 mg/L) or TiO2-NPs (100 mg/L) caused the more severe toxicity in inducing germline apoptosis than nanopolystyrene (100 mg/L), and nanopolystyrene (100 mg/L) resulted in the more severe toxicity in inducing germline apoptosis than SiO2-NPs (100 mg/L) (Fig. 2B).
3.2. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing body length
3.4. Toxicity comparison of nanopolystyrene with three metal oxide NPs in decreasing locomotion behaviors
Using body length as an endpoint to reflect the development, we observed that all the examined 4 NPs at concentrations of 0.1 and 1 mg/L did not obviously affect the body length (Fig. 1B). TiO2-NPs, SiO2-NPs, and nanopolystyrene at the concentration of 10 mg/L, as well as SiO2-NPs and nanopolystyrene at the concentration of 100 mg/L, also did not obviously influence the body length (Fig. 1B). Different from these, exposure to Al2O3-NPs (10e100 mg/L) and TiO2-NPs (100 mg/L) could significantly reduce the body length in
Using head thrash and body bend as endpoints for locomotion behavior, we found that Al2O3-NPs and TiO2-NPs at concentrations of 0.1 and 1 mg/L significantly decreased both head thrash and body bend, whereas SiO2-NPs and nanopolystyrene at concentrations of 0.1 and 1 mg/L could not obviously affect both head thrash and body bend (Fig. 3). All the examined 4 NPs at concentrations of 10 and 100 mg/L significantly decreased both head thrash and body bend (Fig. 3). Moreover, we observed that exposure to Al2O3-NPs (10 mg/
2.8. Statistical analysis Statistical analysis was carried out using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were analyzed with one-way analysis of variance (ANOVA). Two-way ANOVA analysis was performed for multiple factor comparison. Probability level of 0.01 (**) was considered to be statistically significant. 3. Results 3.1. Toxicity comparison of nanopolystyrene with three metal oxide NPs in inducing lethality
Fig. 1. Toxicity comparison of nanopolystyrene with three metal oxide NPs in inducing lethality (A) and in reducing body length (B) in nematodes. Al-NPs, Al2O3-nanopaticles; TiNPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; and polystyrene-NPs, polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control.
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Fig. 2. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing brood size (A) and in inducing germline apoptosis (B) in nematodes. Al-NPs, Al2O3nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
Fig. 3. Toxicity comparison of nanopolystyrene with three metal oxide NPs in decreasing locomotion behaviors in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; and polystyrene-NPs, polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
L) or TiO2-NPs (10 mg/L) caused the more severe neurotoxicity in decreasing head thrash and body bend than nanopolystyrene (10 mg/L), and exposure to nanopolystyrene (10 mg/L) resulted in the more severe neurotoxicity in decreasing head thrash and body bend than SiO2-NPs (10 mg/L) (Fig. 3). Similarly, exposure to Al2O3NPs (100 mg/L) or TiO2-NPs (100 mg/L) induced the more severe neurotoxicity in decreasing head thrash and body bend than nanopolystyrene (100 mg/L), and exposure to nanopolystyrene (100 mg/L) also induced the more severe neurotoxicity in decreasing head thrash and body bend than SiO2-NPs (100 mg/L) (Fig. 3). 3.5. Toxicity comparison of nanopolystyrene with three metal oxide NPs in activating oxidative stress Activation of oxidative stress is an important cellular basis for toxicity induction of various nanomaterials in nematodes (Wang, 2018). Using induction of ROS production as an endpoint for assessing activation of oxidative stress, we compared the toxicity of
nanopolystyrene with three metal oxide NPs in activating oxidative stress. Exposure to Al2O3-NPs (0.1e1 mg/L) or TiO2-NPs (0.1e1 mg/L) induced the significant ROS production; however, exposure to SiO2NPs (0.1e1 mg/L) or nanopolystyrene (0.1e1 mg/L) did not cause the significant induction of ROS production (Fig. 4A). Exposure to all the examined 4 NPs at concentrations of 10e100 mg/L resulted in the significant induction of ROS production (Fig. 4A). Moreover, in the range of 10e100 mg/L, exposure to Al2O3-NPs or TiO2-NPs caused the more severe induction of ROS production than nanopolystyrene, and exposure to nanopolystyrene could result the more severe induction of ROS production than SiO2-NPs (Fig. 4A). We also examined the effect of nanopolystyrene and three metal oxide NPs in activating oxidative stress by investigating expression of SOD-3, a mitochondrial SOD. Considering that only exposure to nanopolystyrene (10e100 mg/L) induced the significant ROS production, we further compared the expression of SOD-3:GFP in nematodes exposed to the examined 4 NPs at concentrations of 10 and 100 mg/L. All the examined 4 NPs at the concentrations of
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Fig. 4. Effects of nanopolystyrene and three metal oxide NPs on induction of ROS production (A) and activation of SOD-3:GFP expression (B) in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
10e100 mg/L caused the significant increase in SOD-3:GFP expression (Fig. 4B). Moreover, in the range of 10e100 mg/L, Al2O3-NPs or TiO2-NPs caused the more pronounced increase in SOD-3:GFP expression than nanopolystyrene, and exposure to nanopolystyrene resulted in the more pronounced increase in SOD3:GFP expression than SiO2-NPs (Fig. 4B). 3.6. Comparison of nanopolystyrene with three metal oxide NPs in activating mt UPR
nematodes against the environmental stresses (Wang, 2019). Using HSP-6:GFP as a marker of mt UPR, we further compared the effect of nanopolystyrene and three metal oxide NPs in activating mt UPR. All the examined 4 NPs at the concentrations of 10e100 mg/L caused the significant increase in HSP-6:GFP expression (Fig. 5). More importantly, in the range of 10e100 mg/L, Al2O3-NPs or TiO2-NPs caused the more pronounced increase in HSP-6:GFP expression than nanopolystyrene, and exposure to nanopolystyrene resulted in the more pronounced increase in HSP-6:GFP expression than SiO2-NPs (Fig. 5).
In the mitochondrion, mt UPR is also an important response for
Fig. 5. Effects of nanopolystyrene and three metal oxide NPs on HSP-6:GFP expression in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
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4. Discussion To examine the toxicity order among nanopolystyrene and three metal oxide NPs (Al2O3-NPs, TiO2-NPs, and SiO2-NPs) in nematodes, we selected the nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs with the same size (30 nm) to perform this toxicity comparison. For the selected three metal oxide NPs, exposure to Al2O3-NPs (10 mg/L) could induce obvious lethality and reduced body length, exposure to TiO2-NPs (100 mg/L) caused obvious lethality and body length reduction, and exposure to SiO2-NPs at all the examined concentrations (0.1e100 mg/L) did not induce lethality or affect body length (Fig. 1). That is, the toxicity order of the selected three metal oxide NPs was Al2O3-NPs > TiO2NPs > SiO2-NPs in nematodes. Using lethality and body length as endpoints, the toxicity of nanopolystyrene was less than Al2O3-NPs or TiO2-NPs (Fig. 1). Nevertheless, we could not make a distinction between nanopolystyrene toxicity and SiO2-NPs toxicity using lethality and body length as endpoints, since both nanopolystyrene and SiO2-NPs at the examined concentrations did not induce lethality and affect body length (Fig. 1). Using reproduction (brood size and germline apoptosis) and locomotion behaviors (head thrash and body bend) as endpoints, we further found that exposure to nanopolystyrene could induce the more severe toxicity than exposure to SiO2-NPs (Figs. 2 and 3). These observations suggested that the endpoints reflecting reproduction and locomotion behavior were sensitive than lethality and endpoints reflecting development in nematodes. More importantly, our data suggested that the nanopolystyrene toxicity was between TiO2-NPs and SiO2-NPs. In nematodes, SiO2NPs (5 mg/L) could cause the moderate toxicity on locomotion behavior and induce the significant ROS production, and SiO2-NPs (50 mg/L) could moderately but significantly reduce the brood size (Wu et al., 2013). Therefore, the potential of nanopolystyrene (30 nm) on environmental organisms might be more than the toxicity of 30 nm SiO2-NPs (5 mg/L). In this study, we observed that 30 nm nanopolystyrene (100 mg/ L) could reduce the brood size and induce the obvious germline apoptosis (Fig. 2). Meanwhile, 30 nm nanopolystyrene (10 mg/L) could significantly decrease both head thrash and body bend (Fig. 3). The predicted environmental concentrations of nanopolystyrene (50 nm) are in the range 15 mg/L (Lenz et al., 2016; AlSid-Cheikh et al., 2018). Our data imply that long-term exposure to nanopolystyrene (30 nm) at predict environmental concentration may at least potentially induce the neurotoxicity on locomotion behaviors in nematodes. In this study, we observed that the nanopolystyrene was more toxic than SiO2-NPs, but less toxic than Al2O3-NPs and TiO2-NPs. For the observed difference of toxicity between nanopolystyrene and the examined three metal oxide-NPs, we here raised two underlying cellular mechanisms. Firstly, the observed toxicity difference might be associated with the differences in induction of oxidative stress between nanopolystyrene and metal oxide NPs exposed nematodes. In the range of 10e100 mg/L, the differences in induction of ROS production and SOD-3:GFP expression were consistent with the differences in reproductive toxicity and/or neurotoxicity on locomotion behaviors between nanopolystyrene and three metal oxide NPs (Figs. 2e4). Moreover, the detected toxicity difference might be also associated with the differences in activation of mt UPR between nanopolystyrene and metal oxide NPs exposed nematodes. In the range of 10e100 mg/L, the differences in HSP6:GFP expression were also consistent with the differences in reproductive toxicity and/or neurotoxicity on locomotion behaviors between nanopolystyrene and three metal oxide NPs (Figs. 2e3 and 5). Therefore, both oxidative stress response and mt UPR response may contribute to the formation of toxicity differences between
nanopolystyrene and the examined three metal oxide NPs in nematodes. 5. Conclusions In conclusion, using C. elegans as an animal model, we here compared the toxicity of nanopolystyrene (30 nm) with three metal oxide NPs (Al2O3-NPs, TiO2-NPs, and SiO2-NPs). With the aid of several endpoints (such as reproduction and locomotion behavior), our results suggested that long-term exposure to nanopolystyrene could induce more severe toxicity than SiO2-NPs. Nevertheless, the nanopolystyrene could not induce the severe toxicity comparable to that observed in Al2O3-NPs or TiO2-NPs exposed nematodes. The observed differences of toxicity between nanopolystyrene and the examined three metal oxide NPs were largely associated with the differences in induction of oxidative stress and activation of mt UPR in exposed nematodes. Our observations will be helpful for our understanding the degree of exposure risk for nanopolystyrene on organisms, such as nematodes. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125625. Author contributions Dan Li, Jie Ji, and Yujie Yuan: Performed the experiments.Dayong Wang: Designed the research and wrote the manuscript. References Alimi, O.S., Farner Budarz, J., Hernandez, L.M., Tufenkji, N., 2018. Microplastics and nanoplastics in aquatic environments: aggregation, deposition, and enhanced contaminant transport. Environ. Sci. Technol. 52, 1704e1724. Al-Sid-Cheikh, M., Rowland, S., Stevenson, K., Rouleau, C., Henry, T.B., Thompson, R.C., 2018. Uptake, whole-body distribution, and depuration of nanoplastics by the scallop pecten maximus at environmentally realistic concentrations. Environ. Sci. Technol. 52, 14480e14486. Balbi, T., Camisassi, G., Montagna, M., Fabbri, R., Franzellitti, S., Carbone, C., Dawson, K., Canesi, L., 2017. Impact of cationic polystyrene nanoparticles (PSNH2) on early embryo development of Mytilus galloprovincialis: effects on shell formation. Chemosphere 186, 1e9. Botterell, Z.L.R., Beaumont, N., Dorrington, T., Steinke, M., Thompson, R.C., Lindeque, P.K., 2019. Bioavailability and effects of microplastics on marine zooplankton: a review. Environ. Pollut. 245, 98e110. Bouwmeester, H., Hollman, P.C., Peters, R.J., 2015. Potential health impact of environmentally released micro- and nanoplastics in the human food production chain: experiences from nanotoxicology. Environ. Sci. Technol. 49, 8932e8947. Brenner, S., 1974. Genetics of Caenorhabditis elegans. Genetics 77, 71e94. Burns, E.E., Boxall, A.B.A., 2018. Microplastics in the aquatic environment: evidence for or against adverse impacts and major knowledge gaps. Environ. Toxicol. Chem. 37, 2776e2796. Chae, Y., An, Y.J., 2018. Current research trends on plastic pollution and ecological impacts on the soil ecosystem: a review. Environ. Pollut. 240, 387e395. Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., Hollert, H., 2017. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584e585, 1022e1031. da Costa, J.P., Santos, P.S.M., Duarte, A.C., Rocha-Santos, T., 2016. Nano)plastics in the environment - sources, fates and effects. Sci. Total Environ. 566e567, 15e26. Della Torre, C., Bergami, E., Salvati, A., Faleri, C., Cirino, P., Dawson, K.A., Corsi, I., 2014. Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus. Environ. Sci. Technol. 48, 12302e12311. Eom, H.J., Choi, J., 2019. Clathrin-mediated endocytosis is involved in uptake and toxicity of silica nanoparticles in Caenohabditis elegans. Chem. Biol. Interact. https://doi.org/10.1016/j.cbi.2019.108774. e, L.F., Lee, J., 2019. Nano/microplastics in water and wastewater Enfrin, M., Dume treatment processes - origin, impact and potential solutions. Water Res. 161, 621e638. Gambardella, C., Morgana, S., Ferrando, S., Bramini, M., Piazza, V., Costa, E., Garaventa, F., Faimali, M., 2017. Effects of polystyrene microbeads in marine planktonic crustaceans. Ecotoxicol. Environ. Saf. 145, 250e257.
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Toxicity comparison of nanopolystyrene with three metal oxide nanoparticles in nematode Caenorhabditis elegans Dan Li, Jie Ji, Yujie Yuan, Dayong Wang* Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School, Southeast University, Nanjing, 210009, China
h i g h l i g h t s We compared toxicity of nanopolystyrene with three metal-NPs in nematodes. Nanopolystyrene could induce more severe toxicity than SiO2-NPs. Nanopolystyrene could not induce severe toxicity comparable to Al2O3-NPs or TiO2-NPs. Cellular basis of toxicity difference of nanopolystyrene with metal-NPs was examined. Our data will be helpful for understanding exposure risk of nanopolystyrene on organisms.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2019 Received in revised form 29 November 2019 Accepted 9 December 2019 Available online xxx
Using Caenorhabditis elegans as an animal model, we compared the toxicity between nanopolystyrene and three metal oxide nanoparticles (NPs) (Al2O3-NPs, TiO2-NPs, and SiO2-NPs). After exposure from L1larvae to adult day-1, nanopolystyrene (100 mg/L) reduced brood size and induced severe germline apoptosis, and nanopolystyrene (10e100 mg/L) decreased locomotion behavior, induced obvious reactive oxygen species (ROS) production, and activated noticeable mitochondrial unfolded protein response (mt UPR). Using several endpoints (lethality, development, reproduction, and/or locomotion behavior), we found that nanopolystyrene could induce more severe toxicity than SiO2-NPs, although nanopolystyrene did not cause the toxicity comparable to that in Al2O3-NPs or TiO2-NPs exposed nematodes. Our data will be useful for understanding the exposure risk of nanopolystyrene on environmental organisms. Moreover, the detected toxicity difference between nanopolystyrene and three metal oxide NPs were associated with the differences in both induction of oxidative stress and activation of mt UPR in exposed nematodes. © 2019 Elsevier Ltd. All rights reserved.
Handling editor: Tamara S. Galloway Keywords: Nanoparticles Toxicity comparison Nanopolystyrene Caenorhabditis elegans
1. Introduction Microplastics are defined as the polymers with the sizes less than 5 mm. Miroplastics are widespread and abundant in various environments, including both aquatic and soil environments (Burns and Boxall, 2018; Chae and An, 2018; Zhang et al., 2018). Recently, bioavailability and adverse effects of microplastics have been examined in different environmental organisms, such as zooplankton (Botterell et al., 2019; Li et al., 2019). Moreover, the microplastics could be further detected in drinking water and sources of drinking water (Koelmans et al., 2019). In the environment, microplastics can be potentially degraded
* Corresponding author. E-mail address: [email protected] (D. Wang). https://doi.org/10.1016/j.chemosphere.2019.125625 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
into nanoplastics (nano-sized plastics) (Mattsson et al., 2015). Besides the microplastic pollution, the existence, fate, and pollution of nanoplastic in different ecosystems have also received the attention (da Costa et al., 2016; Wan et al., 2018; Ng et al., 2018; Alimi et al., 2018; Enfrin et al., 2019). The release of nanoplastics in human food production chain and the environment has implied their potential implications for human health (Bouwmeester et al., 2015; Lehner et al., 2019). Nevertheless, the toxic degree of nanoplastics on organisms is still largely unclear. Nanopolystyrene is a frequently examined example to determine the toxic effects of nanoplastics on organisms (Balbi et al., 2017; Pitt et al., 2018; Lim et al., 2019). Reduction in lifespan, deficit in development, suppression in reproduction, decrease in behaviors, and oxidative stress could be detected in nanopolystyrene exposed organisms, such as zebrafish and Daphnia (Della Torre et al., 2014; Jeong C et al., 2016; Rist et al., 2017;
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Gambardella et al., 2017; Chen et al., 2017). Model animal of Caenorhabditis elegans is sensitive to environmental exposure, and has been proven to be useful for evaluating the toxicity of various nanomaterials, including nanoparticles (NPs) (Wang, 2018; Yang et al., 2018; Liu et al., 2019a, 2020; Zhao et al., 2019a, 2019b; Starnes et al., 2019; Moon et al., 2019; Eom and Choi, 2019). Recently, C. elegans has been further employed to assess the toxicity of nanopolystyrene on animals at various aspects, such as neurotoxicity, intestinal toxicity, and reproductive toxicity (Lei et al., 2018; Hanna et al., 2018; Qu et al., 2019a, 2019b; Kim et al., 2019; Qu and Wang, 2019; Zhao et al., 2019c). The toxicity of nanopolystyrene was charge-dependent and enhanced by surface amino modification in nematodes (Qu et al., 2019c). Some molecular signals, such as insulin, p38 MAPK, and Wnt signals, in response to nanopolystyrene have also been identified in nematodes (Shao et al., 2019a, 2019b; Shao and Wang, 2019; Qu et al., 2019d; Qu et al., 2019e; Yang et al., 2019). To further evaluate the toxic degree of nanopolystyrene, we employed the C. elegans as an animal model to compare the toxicity of nanopolystyrene with three metal oxide NPs (Al2O3-NPs, TiO2NPs, and SiO2-NPs). Among the examined three metal oxide NPs, exposure to Al2O3-NPs could cause the very severe neurotoxicity (Li et al., 2013), and exposure to TiO2-NPs caused the more severe toxicity than the exposure to SiO2-NPs in nematodes (Wu et al., 2013). These backgrounds imply the toxicity difference among Al2O3-NPs, TiO2-NPs, and SiO2-NPs in nematodes. Our results demonstrated the toxicity order among nanopolystyrene and the examined three metal oxide NPs. Our data will be helpful for our evaluating the possible risk of nanopolystyrene on environmental organisms. 2. Materials and methods 2.1. Characterizations of nanopolystyrene and three metal oxide NPs The sizes of nanopolystyrene and three metal oxide NPs were all 30 nm. Nanopolystyrene was from Janus New-Materials Co. (Nanjing, China), Al2O3-NP was from Shanghai Macklin Biochemical company (Shanghai, China), TiO2-NP was from Nano Applied Research Center of Nanjing University of Technology (Nanjing, China), and SiO2-NP was from Wan Jing New Material Co. Ltd. (Hangzhou, China). Purities of nanopolystyrene, Al2O3-NPs, TiO2NPs, and SiO2-NPs were >99.9%, >99.9%, >99%, and >99.7%. Images of transmission electron microscope (TEM) show the morphology and the aggregation state of nanopolystyrene and three metal oxide NPs in K-medium after sonication at 100 W and 40 kHz for 30 min (Fig. S1). The aggregation sizes of nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs were 98.7 ± 9.8, 516.3 ± 45.2, 398.5 ± 65.2, and 105.4 ± 8.9 nm (Fig. S1). Based on the dynamic light scattering (DLS) analysis, zeta-potentials of nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs were 20.5 ± 2.47, 38.12 ± 3.77, 13.1 ± 1.21, and 63.32 ± 3.11 mV. The Raman spectrum of nanopolystyrene was determined by Raman spectroscopy (Renishaw Invia Plus laser Raman spectrometer, Renishaw, UK). Fig. S2 shows the Raman spectrum of nanopolystyrene. 2.2. Animal maintenance and exposure Nematode strains (wild-type N2 and transgenic strains of CF1553/muIs84 [SOD-3:GFP] and SJ4100/zcIs13 [HSP-6:GFP]) were fed with Escherichia coli OP50 on nematode growth medium (NGM) plates (Brenner, 1974). Exposure to nanopolystyrene or metal oxide NPs was performed from L1-larvae to adult day-1. Exposure was
carried out in liquid solutions of nanopolystyrene or metal oxide NPs. The working solutions of nanopolystyrene or metal oxide NPs were added with OP50 (~4 106 colony-forming units (CFUs)). During the exposure, the nanoparticle solutions were refreshed daily. After the exposure, the nematodes were washed with M9 buffer for three times. After that, the nematodes were used for toxicity assessment using different endpoints. All the toxicity assessments for each of the examined endpoints were performed for three replicates. To prepare age-synchronous L1-larvae, the mature gravid nematodes were treated with bleaching mixture solution (0.45 M NaOH, 2% HOCl) to release the eggs and let them further hatch on NGM plates. 2.3. Lethality and growth Lethality was assessed by the endpoint of % survival of nematodes (Wu et al., 2013). After exposure, we scored the inactive animals, which were judged if they did not respond to stimulus using a small, metal wire. For each treatment, one hundred nematodes were examined. The growth of nematodes was assessed by the endpoint of body length (Wu et al., 2013). The body length was determined after the measurement of flat surface length using an Image-Pro® Express software. For each treatment, thirty nematodes were examined. 2.4. Reproduction Reproduction was evaluated by the endpoints of brood size and germline apoptosis (Qu et al., 2019c; Qu et al., 2019f). The brood size refers to the number of offspring at all stages beyond the egg. For each treatment, ten animals were examined. To analyze the germline apoptosis, the animals were stained with acridine orange (AO, 25 mg/mL) for 1-h in darkness. The germline apoptosis was examined under an epifluorescence microscopy (excited light, 395 nm; emitted light, 509 nm). For each treatment, forty animals were examined. 2.5. Locomotion behaviors After exposure, two endpoints (head thrash and body bend) were used to assess the possible neurotoxicity on locomotion behaviors in nematodes (Kong et al., 2019). A head thrash is considered as an alteration in bending direction at body mid-region, and a body bend is considered as an alteration in posterior bulb direction. For each treatment, thirty animals were examined. 2.6. Activation of oxidative stress To determine the activation of oxidative stress, the reactive oxygen species (ROS) production was determined (Liu et al., 2019b). After exposure, the nematodes were labeled with CM-H2DCFDA (1 mM) in darkness for 3 h. The nematodes were then examined for both excitation wavelength at 488 nm and emission filter at 510 nm under a laser scanning confocal microscope. In nematodes, the strongest ROS fluorescent signals can be detected in the intestine (Wang et al., 2007). ROS signals in the intestine were examined by relative fluorescence units and normalized to autofluorescence. For each treatment, forty animals were examined. Besides the ROS production, transgenic strain of muIs84 was further used to examine the alteration in SOD-3:GFP expression (Zhao et al., 2019d). SOD-3 is a mitochondrial superoxide dismutase, which provides an antioxidation defense system against the oxidative stress (Hunter et al., 1997). SOD-3 is constitutively expressed in pharyngeal and intestine (Henderson et al., 2006). Relative fluorescence of SOD-3:GFP signals in intestine was
D. Li et al. / Chemosphere 245 (2020) 125625
examined. For each treatment, forty animals were examined.
nematodes (Fig. 1B).
2.7. Mitochondrial unfolded protein response (mt UPR)
3.3. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing brood size and in inducing germline apoptosis
HSP-6 is a marker for mt UPR (Haynes et al., 2007). Transgenic strain of zcIs13 was used to assess the mt UPR in nematodes. HSP-6 is broadly expressed in the intestine (Sanders et al., 2017). Relative fluorescence of HSP-6:GFP signals in intestine was examined. For each treatment, forty animals were examined.
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Using lethality as an endpoint, all the examined 4 NPs at concentrations of 0.1 and 1 mg/L did not induce the lethality (Fig. 1A). Additionally, TiO2-NPs, SiO2-NPs, and nanopolystyrene at the concentration of 10 mg/L, as well as SiO2-NPs and nanopolystyrene at the concentration of 100 mg/L, also did not induce the lethality (Fig. 1A). In contrast, only exposure to Al2O3-NPs (10e100 mg/L) and TiO2-NPs (100 mg/L) could induce the obvious lethality in nematodes (Fig. 1A).
Using brood size to reflect the reproductive capacity, we found that all the examined 4 NPs at the concentration of 0.1 mg/L did not affect brood size (Fig. 2A). Al2O3-NPs and TiO2-NPs at concentrations of 1 and 10 mg/L could significantly reduce the brood size, whereas SiO2-NPs and nanopolystyrene at concentrations of 1 and 10 mg/L did not influence the brood size (Fig. 2A). All the examined 4 NPs at the concentration of 100 mg/L could significantly reduce the brood size (Fig. 2A). Moreover, Al2O3-NPs (100 mg/L) or TiO2-NPs (100 mg/L) caused the more severe toxicity in reducing brood size than nanopolystyrene (100 mg/L), and nanopolystyrene (100 mg/L) resulted in the more severe toxicity in reducing brood size than SiO2-NPs (100 mg/L) (Fig. 2A). In nematodes, germline apoptosis acts as an important cellular basis for the reduction in brood size (Qu et al., 2019c). Considering that only exposure to nanopolystyrene (100 mg/L) could reduce the brood size, we next compared the induction of germline apoptosis in nematodes exposed to the examined 4 NPs at concentrations of 10 and 100 mg/L. Al2O3-NPs (10 mg/L) or TiO2-NPs (10 mg/L) could induce the obvious increase in germline apoptosis; however, SiO2NPs (10 mg/L) or nanopolystyrene (10 mg/L) did not induce the increase in germline apoptosis (Fig. 2B). All the examined 4 NPs at the concentration of 100 mg/L caused the significant increase in germline apoptosis (Fig. 2B). Al2O3-NPs (100 mg/L) or TiO2-NPs (100 mg/L) caused the more severe toxicity in inducing germline apoptosis than nanopolystyrene (100 mg/L), and nanopolystyrene (100 mg/L) resulted in the more severe toxicity in inducing germline apoptosis than SiO2-NPs (100 mg/L) (Fig. 2B).
3.2. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing body length
3.4. Toxicity comparison of nanopolystyrene with three metal oxide NPs in decreasing locomotion behaviors
Using body length as an endpoint to reflect the development, we observed that all the examined 4 NPs at concentrations of 0.1 and 1 mg/L did not obviously affect the body length (Fig. 1B). TiO2-NPs, SiO2-NPs, and nanopolystyrene at the concentration of 10 mg/L, as well as SiO2-NPs and nanopolystyrene at the concentration of 100 mg/L, also did not obviously influence the body length (Fig. 1B). Different from these, exposure to Al2O3-NPs (10e100 mg/L) and TiO2-NPs (100 mg/L) could significantly reduce the body length in
Using head thrash and body bend as endpoints for locomotion behavior, we found that Al2O3-NPs and TiO2-NPs at concentrations of 0.1 and 1 mg/L significantly decreased both head thrash and body bend, whereas SiO2-NPs and nanopolystyrene at concentrations of 0.1 and 1 mg/L could not obviously affect both head thrash and body bend (Fig. 3). All the examined 4 NPs at concentrations of 10 and 100 mg/L significantly decreased both head thrash and body bend (Fig. 3). Moreover, we observed that exposure to Al2O3-NPs (10 mg/
2.8. Statistical analysis Statistical analysis was carried out using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were analyzed with one-way analysis of variance (ANOVA). Two-way ANOVA analysis was performed for multiple factor comparison. Probability level of 0.01 (**) was considered to be statistically significant. 3. Results 3.1. Toxicity comparison of nanopolystyrene with three metal oxide NPs in inducing lethality
Fig. 1. Toxicity comparison of nanopolystyrene with three metal oxide NPs in inducing lethality (A) and in reducing body length (B) in nematodes. Al-NPs, Al2O3-nanopaticles; TiNPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; and polystyrene-NPs, polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control.
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Fig. 2. Toxicity comparison of nanopolystyrene with three metal oxide NPs in reducing brood size (A) and in inducing germline apoptosis (B) in nematodes. Al-NPs, Al2O3nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
Fig. 3. Toxicity comparison of nanopolystyrene with three metal oxide NPs in decreasing locomotion behaviors in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; and polystyrene-NPs, polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
L) or TiO2-NPs (10 mg/L) caused the more severe neurotoxicity in decreasing head thrash and body bend than nanopolystyrene (10 mg/L), and exposure to nanopolystyrene (10 mg/L) resulted in the more severe neurotoxicity in decreasing head thrash and body bend than SiO2-NPs (10 mg/L) (Fig. 3). Similarly, exposure to Al2O3NPs (100 mg/L) or TiO2-NPs (100 mg/L) induced the more severe neurotoxicity in decreasing head thrash and body bend than nanopolystyrene (100 mg/L), and exposure to nanopolystyrene (100 mg/L) also induced the more severe neurotoxicity in decreasing head thrash and body bend than SiO2-NPs (100 mg/L) (Fig. 3). 3.5. Toxicity comparison of nanopolystyrene with three metal oxide NPs in activating oxidative stress Activation of oxidative stress is an important cellular basis for toxicity induction of various nanomaterials in nematodes (Wang, 2018). Using induction of ROS production as an endpoint for assessing activation of oxidative stress, we compared the toxicity of
nanopolystyrene with three metal oxide NPs in activating oxidative stress. Exposure to Al2O3-NPs (0.1e1 mg/L) or TiO2-NPs (0.1e1 mg/L) induced the significant ROS production; however, exposure to SiO2NPs (0.1e1 mg/L) or nanopolystyrene (0.1e1 mg/L) did not cause the significant induction of ROS production (Fig. 4A). Exposure to all the examined 4 NPs at concentrations of 10e100 mg/L resulted in the significant induction of ROS production (Fig. 4A). Moreover, in the range of 10e100 mg/L, exposure to Al2O3-NPs or TiO2-NPs caused the more severe induction of ROS production than nanopolystyrene, and exposure to nanopolystyrene could result the more severe induction of ROS production than SiO2-NPs (Fig. 4A). We also examined the effect of nanopolystyrene and three metal oxide NPs in activating oxidative stress by investigating expression of SOD-3, a mitochondrial SOD. Considering that only exposure to nanopolystyrene (10e100 mg/L) induced the significant ROS production, we further compared the expression of SOD-3:GFP in nematodes exposed to the examined 4 NPs at concentrations of 10 and 100 mg/L. All the examined 4 NPs at the concentrations of
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Fig. 4. Effects of nanopolystyrene and three metal oxide NPs on induction of ROS production (A) and activation of SOD-3:GFP expression (B) in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2-nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
10e100 mg/L caused the significant increase in SOD-3:GFP expression (Fig. 4B). Moreover, in the range of 10e100 mg/L, Al2O3-NPs or TiO2-NPs caused the more pronounced increase in SOD-3:GFP expression than nanopolystyrene, and exposure to nanopolystyrene resulted in the more pronounced increase in SOD3:GFP expression than SiO2-NPs (Fig. 4B). 3.6. Comparison of nanopolystyrene with three metal oxide NPs in activating mt UPR
nematodes against the environmental stresses (Wang, 2019). Using HSP-6:GFP as a marker of mt UPR, we further compared the effect of nanopolystyrene and three metal oxide NPs in activating mt UPR. All the examined 4 NPs at the concentrations of 10e100 mg/L caused the significant increase in HSP-6:GFP expression (Fig. 5). More importantly, in the range of 10e100 mg/L, Al2O3-NPs or TiO2-NPs caused the more pronounced increase in HSP-6:GFP expression than nanopolystyrene, and exposure to nanopolystyrene resulted in the more pronounced increase in HSP-6:GFP expression than SiO2-NPs (Fig. 5).
In the mitochondrion, mt UPR is also an important response for
Fig. 5. Effects of nanopolystyrene and three metal oxide NPs on HSP-6:GFP expression in nematodes. Al-NPs, Al2O3-nanopaticles; Ti-NPs, TiO2-nanoparticles; Si-NPs, SiO2nanoparticles; polystyrene-NPs, and polystyrene nanoparticles. Exposure was performed from L1-larvae to adult day-1. Bars represent means ± SD. **P < 0.01 vs control (if not specially indicated).
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4. Discussion To examine the toxicity order among nanopolystyrene and three metal oxide NPs (Al2O3-NPs, TiO2-NPs, and SiO2-NPs) in nematodes, we selected the nanopolystyrene, Al2O3-NPs, TiO2-NPs, and SiO2-NPs with the same size (30 nm) to perform this toxicity comparison. For the selected three metal oxide NPs, exposure to Al2O3-NPs (10 mg/L) could induce obvious lethality and reduced body length, exposure to TiO2-NPs (100 mg/L) caused obvious lethality and body length reduction, and exposure to SiO2-NPs at all the examined concentrations (0.1e100 mg/L) did not induce lethality or affect body length (Fig. 1). That is, the toxicity order of the selected three metal oxide NPs was Al2O3-NPs > TiO2NPs > SiO2-NPs in nematodes. Using lethality and body length as endpoints, the toxicity of nanopolystyrene was less than Al2O3-NPs or TiO2-NPs (Fig. 1). Nevertheless, we could not make a distinction between nanopolystyrene toxicity and SiO2-NPs toxicity using lethality and body length as endpoints, since both nanopolystyrene and SiO2-NPs at the examined concentrations did not induce lethality and affect body length (Fig. 1). Using reproduction (brood size and germline apoptosis) and locomotion behaviors (head thrash and body bend) as endpoints, we further found that exposure to nanopolystyrene could induce the more severe toxicity than exposure to SiO2-NPs (Figs. 2 and 3). These observations suggested that the endpoints reflecting reproduction and locomotion behavior were sensitive than lethality and endpoints reflecting development in nematodes. More importantly, our data suggested that the nanopolystyrene toxicity was between TiO2-NPs and SiO2-NPs. In nematodes, SiO2NPs (5 mg/L) could cause the moderate toxicity on locomotion behavior and induce the significant ROS production, and SiO2-NPs (50 mg/L) could moderately but significantly reduce the brood size (Wu et al., 2013). Therefore, the potential of nanopolystyrene (30 nm) on environmental organisms might be more than the toxicity of 30 nm SiO2-NPs (5 mg/L). In this study, we observed that 30 nm nanopolystyrene (100 mg/ L) could reduce the brood size and induce the obvious germline apoptosis (Fig. 2). Meanwhile, 30 nm nanopolystyrene (10 mg/L) could significantly decrease both head thrash and body bend (Fig. 3). The predicted environmental concentrations of nanopolystyrene (50 nm) are in the range 15 mg/L (Lenz et al., 2016; AlSid-Cheikh et al., 2018). Our data imply that long-term exposure to nanopolystyrene (30 nm) at predict environmental concentration may at least potentially induce the neurotoxicity on locomotion behaviors in nematodes. In this study, we observed that the nanopolystyrene was more toxic than SiO2-NPs, but less toxic than Al2O3-NPs and TiO2-NPs. For the observed difference of toxicity between nanopolystyrene and the examined three metal oxide-NPs, we here raised two underlying cellular mechanisms. Firstly, the observed toxicity difference might be associated with the differences in induction of oxidative stress between nanopolystyrene and metal oxide NPs exposed nematodes. In the range of 10e100 mg/L, the differences in induction of ROS production and SOD-3:GFP expression were consistent with the differences in reproductive toxicity and/or neurotoxicity on locomotion behaviors between nanopolystyrene and three metal oxide NPs (Figs. 2e4). Moreover, the detected toxicity difference might be also associated with the differences in activation of mt UPR between nanopolystyrene and metal oxide NPs exposed nematodes. In the range of 10e100 mg/L, the differences in HSP6:GFP expression were also consistent with the differences in reproductive toxicity and/or neurotoxicity on locomotion behaviors between nanopolystyrene and three metal oxide NPs (Figs. 2e3 and 5). Therefore, both oxidative stress response and mt UPR response may contribute to the formation of toxicity differences between
nanopolystyrene and the examined three metal oxide NPs in nematodes. 5. Conclusions In conclusion, using C. elegans as an animal model, we here compared the toxicity of nanopolystyrene (30 nm) with three metal oxide NPs (Al2O3-NPs, TiO2-NPs, and SiO2-NPs). With the aid of several endpoints (such as reproduction and locomotion behavior), our results suggested that long-term exposure to nanopolystyrene could induce more severe toxicity than SiO2-NPs. Nevertheless, the nanopolystyrene could not induce the severe toxicity comparable to that observed in Al2O3-NPs or TiO2-NPs exposed nematodes. The observed differences of toxicity between nanopolystyrene and the examined three metal oxide NPs were largely associated with the differences in induction of oxidative stress and activation of mt UPR in exposed nematodes. Our observations will be helpful for our understanding the degree of exposure risk for nanopolystyrene on organisms, such as nematodes. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125625. Author contributions Dan Li, Jie Ji, and Yujie Yuan: Performed the experiments.Dayong Wang: Designed the research and wrote the manuscript. References Alimi, O.S., Farner Budarz, J., Hernandez, L.M., Tufenkji, N., 2018. Microplastics and nanoplastics in aquatic environments: aggregation, deposition, and enhanced contaminant transport. Environ. Sci. Technol. 52, 1704e1724. Al-Sid-Cheikh, M., Rowland, S., Stevenson, K., Rouleau, C., Henry, T.B., Thompson, R.C., 2018. Uptake, whole-body distribution, and depuration of nanoplastics by the scallop pecten maximus at environmentally realistic concentrations. Environ. Sci. Technol. 52, 14480e14486. Balbi, T., Camisassi, G., Montagna, M., Fabbri, R., Franzellitti, S., Carbone, C., Dawson, K., Canesi, L., 2017. Impact of cationic polystyrene nanoparticles (PSNH2) on early embryo development of Mytilus galloprovincialis: effects on shell formation. Chemosphere 186, 1e9. Botterell, Z.L.R., Beaumont, N., Dorrington, T., Steinke, M., Thompson, R.C., Lindeque, P.K., 2019. Bioavailability and effects of microplastics on marine zooplankton: a review. Environ. Pollut. 245, 98e110. Bouwmeester, H., Hollman, P.C., Peters, R.J., 2015. Potential health impact of environmentally released micro- and nanoplastics in the human food production chain: experiences from nanotoxicology. Environ. Sci. Technol. 49, 8932e8947. Brenner, S., 1974. Genetics of Caenorhabditis elegans. Genetics 77, 71e94. Burns, E.E., Boxall, A.B.A., 2018. Microplastics in the aquatic environment: evidence for or against adverse impacts and major knowledge gaps. Environ. Toxicol. Chem. 37, 2776e2796. Chae, Y., An, Y.J., 2018. Current research trends on plastic pollution and ecological impacts on the soil ecosystem: a review. Environ. Pollut. 240, 387e395. Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., Hollert, H., 2017. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584e585, 1022e1031. da Costa, J.P., Santos, P.S.M., Duarte, A.C., Rocha-Santos, T., 2016. Nano)plastics in the environment - sources, fates and effects. Sci. Total Environ. 566e567, 15e26. Della Torre, C., Bergami, E., Salvati, A., Faleri, C., Cirino, P., Dawson, K.A., Corsi, I., 2014. Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus. Environ. Sci. Technol. 48, 12302e12311. Eom, H.J., Choi, J., 2019. Clathrin-mediated endocytosis is involved in uptake and toxicity of silica nanoparticles in Caenohabditis elegans. Chem. Biol. Interact. https://doi.org/10.1016/j.cbi.2019.108774. e, L.F., Lee, J., 2019. Nano/microplastics in water and wastewater Enfrin, M., Dume treatment processes - origin, impact and potential solutions. Water Res. 161, 621e638. Gambardella, C., Morgana, S., Ferrando, S., Bramini, M., Piazza, V., Costa, E., Garaventa, F., Faimali, M., 2017. Effects of polystyrene microbeads in marine planktonic crustaceans. Ecotoxicol. Environ. Saf. 145, 250e257.
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