Danielle E. Que1, Wen-Che Hou This email address is being protected from spambots. You need JavaScript enabled to view it.1, Micah Belle Marie Yap Ang2, Chih-Chung Lin3

1 Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan
2 R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Taoyuan 32023, Taiwan
3 Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan


 

Received: April 19, 2020
Revised: June 28, 2020
Accepted: July 19, 2020

 Copyright The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.


Download Citation: ||https://doi.org/10.4209/aaqr.2020.04.0157  

  • Download: PDF


Cite this article:

Que, D.E., Hou, W.C., Ang, M.B.M.Y. and Lin, C.C. (2020). Toxic Effects of Hydroxyl- and Amine-functionalized Silica Nanoparticles (SiO2 and NH2-SiO2 NPs) on Caenorhabditis elegans. Aerosol Air Qual. Res. 20: 1987–2002. https://doi.org/10.4209/aaqr.2020.04.0157


HIGHLIGHTS

  • Toxic effects of SiO2 and NH2-SiO2 NPs were evaluated using C. elegans.
  • SiO2 NPs showed higher chronic toxicity as compared to NH2-SiO2 NPs.
  • SiO2 NPs showed higher toxicity in different C. elegans endpoints.
  • Functionality is an important indicator of toxicity in nanosafety evaluations.
 

ABSTRACT


Silica nanoparticles (SiO2 NPs) are engineered nanomaterials (ENMs) that have a wide range of application. Increased use in manufacturing has led to concerns about their environmental impact and possible adverse health effects. We conducted a comparative toxicity assessment of bare SiO2-NPs and amine-functionalized SiO2 NPs (NH2-SiO2 NPs) utilizing the Caenorhabditis elegans (C. elegans) in vivo model. L1 nematodes were exposed to exposure concentrations of 0.25, 0.5, 2.5, and 5 mg mL–1 until the worms reached the L4 stage. The chronic lethality and lifespan assays revealed a significant decrease in survival rate and lifespan at 2.5 and 5 mg mL–1 for nematodes exposed to bare SiO2 NPs (89% and 88%; 22 days, p < 0.05 and 14 days, p < 0.05) and at 5 mg mL–1 for the NH2-SiO2 NPs-exposed group (86%; 20 days, p < 0.001). Exposure to all SiO2 NP concentrations reduced progeny production to 79–60% while exposure to 2.5 and 5 mg mL–1 of NH2-SiO2 NPs significantly reduced the brood size to 64–63%. Neurobehavioral toxicity was also observed in the SiO2 NP-exposed worms, which displayed significantly decreased head thrashing for up to 92–71% and in the NH2-SiO2 NPs-exposed worms which showed significantly reduced head thrashing movement for up to 91–85% at concentrations of 0.5-5 mg mL–1. Body bending movements were also significantly reduced at 0.5–5 mg mL–1 SiO2 NPs (71–34%) and 2.5–5 mg mL–1 NH2-SiO2 NPs (94–66%). Significant shortening of body size was also observed in nematodes exposed to 0.5–5 mg mL–1 for both SiO2 NPs (93–81%) and NH2-SiO2 NPs (94–88%). Overall, bare SiO2 NPs were observed to be more toxic due to the negatively charged surface OH groups, which may have disrupted protein homeostasis, resulting in the observed toxicities. We suggest that functionality is an important indicator in nanosafety evaluations.


Keywords: Caenorhabditis elegans; Silica nanoparticles; Nanotoxicity; Amine-functionalized silica nanoparticles; Hydroxyl-functionalized silica nanoparticles.


INTRODUCTION


Silicon dioxide nanoparticles (SiO2 NPs) are engineered nanomaterials that can be categorized as solid, porous, or mesoporous (Esim et al., 2019). The ease of modification of the surface chemistry of SiO2 NPs has been widely exploited to control their interactions with biological systems used for drug delivery and to serve their purpose related to the remediation of environmental contaminants (Farrukh et al., 2014; Gomes et al., 2016; Mao et al., 2020). It has been reported that surface modification of SiO2 NPs enhances colloidal stability, biocompatibility, and target specificity as well as improving cellular uptake, all of which are necessary components of drug delivery and bioimaging applications (Besic Gyenge et al., 2011; Kralj et al., 2012; Liberman et al., 2014). The alteration of the surface chemistry of SiO2 NPs has also been found to be beneficial in water treatment applications such as in nanofiltration processes (Ang et al., 2019) as well as other types of environmental contaminant remediation methods (Huang et al., 2003; Kim et al., 2005; Walcarius and Delacôte, 2005; Chen et al., 2019; Lin et al., 2019). SiO2 NPs are also utilized as food additives, to improve building and construction materials, in microelectronics manufacturing, and as an ingredient in agricultural mixtures (Kang et al., 2011; Go et al., 2017; Palla et al., 2017; Shang et al., 2019; Park et al., 2019).

Synthesized SiO2 NPs commonly have hydroxyl groups (OH) on their surfaces, which essentially only require basic silane chemistry for further required functionalization (Acharya et al., 2017). The OH groups provide colloidal stability due to the negatively charged surface in the physiological environment as well as enhance linking interactions between the SiO2 NP surfaces and target biomolecules (Osseo-Asare and Arriagada, 1999; Haensch et al., 2010). In addition, the contaminant adsorption of SiO2 NPs can be improved through the control of their surface chemistry (Li et al., 2019). Aside from the OH group-containing SiO2 NPs, amine-functionalized SiO2 NPs (NH2-SiO2 NPs) are also popular for surface modification purposes, such as in processes that involve the attachment of a variety of organic groups (Ghosh et al., 2013). Crosslinkers are easily coupled with the amino group and the positively charged surface of the particles enables gene therapy applications due to strong binding with DNA (Hsiao et al., 2019). Aside from the surface chemistry, the end use of SiO2 NPs is also dependent on the synthesis parameters that control their other features, including structure, stability, particle size, and porosity (Wilczewska et al., 2012).

In general, the wide-range of applications of SiO2 NPs has increased their rate of manufacture, where in 2014, the global production volume was reported to be 185–1400 kilotons (Keller and Lazareva, 2013; Pulit-Prociak and Banach, 2016). The dispersion of SiO2 NPs into the environment can be attributed to their end use (Piechulek et al., 2019), and their surface functionalization plays an important role in their environmental fate and transport (Jarvie et al., 2009). Most engineered oxide nanoparticles such as SiO2 NPs have been observed to be introduced into the environment through industrial wastewater and sewage discharges (Boxall et al., 2007). Surface-functionalized SiO2 NPs as compared to uncoated SiO2 NPs have been shown to be removed more easily during primary wastewater treatment through the sedimentation process (Jarvie et al., 2009). Other probable routes for SiO2 NPs released into the air include direct introduction of the particles or through vehicle exhaust, while aerial deposition, leakage, and spills release SiO2 NPs into surface waters (Bahadar Zeb et al., 2018; Piechulek et al., 2019). Overall, soils and sediments have been observed to be the major environmental sinks of these releases (Piechulek et al., 2019). The median SiO2 NPs levels predicted in central European surface waters had values of 3.5 µg L–1 while natural and urban soils, sewage sludge-treated soil, and landfill waste had values of 160, 390, and 490 µg kg–1, respectively (Wang et al., 2016; Wang and Nowack, 2018).

Agricultural formulations containing SiO2 NPs that are extensively used, are considered as major routes to which they are introduced into the food chain (Iavicoli et al., 2017; Shang et al., 2019). SiO2 NPs can also be introduced into the terrestrial and aquatic food chains through the uptake process of plants and crops as well as through via trophic transfer (Unrine et al., 2012; Skjolding et al., 2014; Gupta et al., 2016). Furthermore, the usage of SiO2 NPs as food additives is also a straightforward route for exposure. The United States Food and Drug Administration (FDA) and the European Unions have imposed standards on the allowable concentrations of silica additives in food (2% and 1% per weight), respectively (EU, 2011; FDA, 2015). Around 80% of silica food additives were found to be in the nano-size range during the digestion process in the intestines where the gut epithelium is most likely to be affected (Peters et al., 2012). The workplace has been reported to provide a major risk of exposure in humans (Kim et al., 2014; Oh et al., 2014).

Although SiO2 NPs are nontoxic, previous studies have reported adverse effects in in vitro and in vivo models, including inflammatory effects, cytotoxicity, and hemolysis (Park and Park, 2009; Slowing et al., 2009; Al-Rawi et al., 2011; Panas et al., 2013; Panas et al., 2014). The toxicity of unmodified SiO2 NPs stems from the available OH groups on their surfaces and their strong interactions with the components of biological membranes such as lipids and proteins (Slowing et al., 2009). Other findings have suggested that SiO2 NP exposure is associated with respiratory and cardiovascular illnesses, multiple organ damage, oxidative stress, and cellular- and molecular-level damage (Chen et al., 2004; Chen and von Mikecz, 2005; Lin et al., 2006; Chen et al., 2008; Shang et al., 2009; Choi et al., 2010; Lu et al., 2010; Yang et al., 2010; van der Zande et al., 2014; Niu et al., 2016). Amine functionalized amorphous SiO2 NPs have demonstrated less toxicity as compared to bare amorphous SiO2 NPs in both previous in vitro and in vivo studies (Marzaioli et al., 2014; Nagano et al., 2017), although other studies have reported otherwise (Yu et al., 2011; Kurtz-Chalot et al., 2014). The varying toxicities of NH2-SiO2 NPs may be attributed to the exposure conditions (Boussif et al., 1995; Xia et al., 2008; Hsiao et al., 2019) as well as the extent of amino group surface coverage (Yu et al., 2011; Morris et al., 2016) and steric hindrance brought about by the linker structure which binds the amino groups to the SiO2 NP surface (Hsiao et al., 2019). Amino groups can be protonated at low pH conditions such as in the endolysosomal compartment of cells which triggers the “proton sponge effect” (rupture of lysosomes due to water influx) as demonstrated in amine-modified polystyrene (PS-NH2)-exposed human and murine macrophages (Boussif et al., 1995; Xia et al., 2008). The interaction between the reactive OH groups and the cell membrane may be reduced depending on the degree of amino group surface substitution (Yu et al., 2011; Morris et al., 2016) and linker-related steric hindrance (Hsiao et al., 2019). Hsiao et al. (2019) demonstrated that exposure conditions reduced the toxicity and improved the biocompatibility of amine functionalized amorphous SiO2 NPs rather than the degree of amino group surface substitution.

Caenorhabditis elegans (C. elegans), a nematode living in soil, has been established as an in vivo model for various applications such as in drug, nanomaterial, and chemical assessments in the biomedical field as well as for studying the toxicity of environmental contaminants such as metals, chemicals, fine particulate matter, and nanomaterials (Chung et al., 2019; Queiros et al., 2019). Its advantages over other in vivo models include a short life cycle and lifespan, easy maintenance, completely sequenced genome, large brood size, and a small, transparent body (Brenner, 1974; Shen et al., 2018). In addition, the C. elegans features can act as an intermediate between in vitro and mammalian testing (Hunt, 2017). C. elegans are potential targets for NP deposition since their habitat primarily overlaps with the known major sinks of NPs in the environment such as sludge treated soils and sediments found along riverbanks (Mikecz, 2018; Sinis et al., 2019). Only a few studies have investigated the toxicity of SiO2 NPs utilizing C. elegans in vivo model (Pluskota et al., 2009; Scharf et al., 2013; Jung et al., 2015; Acosta et al., 2018; Eom and Choi, 2019; Piechulek et al., 2019; Li et al., 2020). Our study is aimed towards comparing the toxicity of dense SiO2 NPs and NH2-SiO2 NPs through an evaluation of various C. elegans toxicological endpoints such as chronic lethal toxicity, lifespan, brood size, body length, and locomotion, as well as to contribute to the existing global data on possible indicators for nanosafety. To the best of our knowledge, this is the first comparative study on the toxicity of OH- and NH2-functionalized silica-NPs using the in vivo model, C. elegans.

 
METHODS



Sample Preparation and Exposure Concentrations

Dense SiO2 and NH2-SiO2 NPs were purchased from Merck KGaA, Darmstadt, Germany. These particles were dispersed in ddH2O (pH = 7) to prepare stock solutions with a concentration of 25 mg mL–1. Each NP type was diluted using ddH2O to exposure concentrations of 0.25, 0.5, 2.5, and 5 mg mL–1 (pH = 7). The exposure method was carried out by adding 200 µL of each of the exposure concentrations on bacterial lawns with a mean area of 5.05 cm2 resulting in particle per loading area concentrations of 9.9 (0.25 mg mL–1), 19.8 (0.5 mg mL–1), 99 (2.5 mg mL–1), and 198 (5 mg mL–1) µg cm–2, respectively (Pluskota et al., 2009), while the control group fed on a bacterial lawn that was solely supplemented with water. To achieve a homogeneous dispersion, the samples were sonicated for 1 hr before conducting the exposure experiments.

 
Characterization

The chemical structure of the NP samples was verified using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (Perkin Elmer Spectrum 100 FTIR Spectrometer, Waltham, MA, USA). The NPs were then pelletized at a fixed weight of 100 mg. The samples were then placed on an ATR crystal stage with the samples being scanned 16 times at a resolution of 4 cm–1. The morphology and size distribution of the NPs were verified using field emission scanning electron microscopy (FESEM, S-4800 Hitachi Ltd. Tokyo, Japan). The samples were then transferred onto the sample holder using double-sided carbon tape and then coated with platinum (Pt) under vacuum conditions of 15 mA for 150 seconds. The FESEM acceleration voltage was maintained at 3 kV. The charges of the NP samples were verified using the dynamic light scattering (DLS) instrument (Zeta Nano ZS, Malvern, UK). The dispersion of the particles was performed using ultrasonication in deionized water with a stock solution concentration of 20 mg mL–1. The samples were then placed in the sample cells to determine the charge. Using a thermal gravimetric analysis (TGA, Q500, TA Instrument, USA), the decomposition temperature of the samples was determined under a nitrogen atmosphere. Samples of 2-5 mg each were transferred onto a Pt pan and then placed on the TGA furnace.

 
C. elegans Cultivation and Exposure Conditions

The nematodes were maintained in nematode growth medium (NGM) plates seeded with OP50 E. coli. At a temperature of 20℃. Age synchronization was achieved using the alkaline bleaching method (Porta-de-la-Riva et al., 2012). Eggs collected using the age synchronization method were maintained in M9 medium and incubated until they hatched into L1 worms for experimentation. The NGM plates contained bacteriological agar and bactopeptone, which were obtained from Laboratories Conda (S.A., Spain) and NaCl obtained from Honeywell Fluka™ (New Jersey, USA). Additional ingredients including CaCl2, K2HPO4, and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, USA) while MgSO4 was acquired from Avantor Performance Materials, Ltd. (Gyeonggi-do, South Korea). OP50 E. coli cultures were acquired from the Bioresources Collection and Research Center (Hsinchu, Taiwan) and the Luria-Bertani broth was obtained from Sigma-Aldrich (St. Louis, MO, USA). For the bleaching solution, NaOCl was obtained from J.T. Baker (Central Valley, PA), and KOH was obtained from Duksan Pure Chemicals (Gyeonggi-do, South Korea). The KH2PO4 used for the phosphate buffer was acquired from Avantor Performance Materials, LLC (Radnor, PA, USA), and the Na2HPO4 used for the M9 buffer was obtained from Honeywell Fluka™ (New Jersey, USA). All physiological observations were done under a dissecting microscope (Olympus, SZX10, Waltham MA, USA).

 
Chronic Lethality Assay

The age-synchronized nematodes were exposed to the different concentrations of SiO2 NPs and NH2-SiO2 NPs at the L1 stage at a constant temperature of 20℃ until they reached their gravid adult stage. The lethal chronic toxicity of the samples was assessed immediately after the exposed worms reached the L4 stage. The nematodes were scored with regard to whether they were dead or alive by gently prodding them using a worm picker. Any worms that responded after prodding were scored as alive while worms that did not respond to touch were scored dead. Three biological replicates were performed, and a total of 150 worms were assayed.

 
Lifespan Assay

Using the different exposure concentrations of the SiO2 NPs and NH2-SiO2 NPs, age-synchronized worms were exposed at the L1 stage until they reached their gravid adult stage. After exposure, the nematodes were transferred to fresh plates for the first 4–5 days of egg-laying and were transferred every other day to fresh plates after the egg-laying period. The exposed worms were assessed for their lifespan and were scored as either dead or alive every day. Three biological replicates and a total of 150 worms were evaluated. The temperature was maintained at 20℃

 
Reproductive Assay

After chronic exposure, the age-synchronized worms were assessed for 4–5 days of egg-laying at 20℃. The nematodes were exposed to the various exposure concentrations of SiO2 NPs and NH2-SiO2 NPs at the L1 stage, and the L4 worms were later assayed for their progeny production. One worm was transferred to each plate, and each worm was again transferred to fresh plates during the egg-laying period. Each of the old plates containing the laid eggs were incubated, and the progeny worms were counted. Three biological replicates and a total of 60 worms were examined in this assay.

 
Locomotion Assay

The neurobehavior of the nematodes was assessed using two toxicological endpoint assays: head thrashing and body bending. The nematodes were exposed using the same exposure concentrations of SiO2 NPs and NH2-SiO2 NPs starting at the L1 stage at 20℃ until the worms reached the L4 stage. For the head thrashing assessment, the worms were individually transferred on a droplet of M9 placed on a glass slide, and the movements were recorded using the dissecting microscope camera and then measured for a duration of 1 minute. Three biological replicates were performed, and a total of 30 worms were evaluated. For the body bending assay, individual worms were placed on a fresh NGM plate without any bacterial lawn and then left to adjust in the incubator at the same exposure temperature. The worms were then carefully assessed under a dissecting microscope with a mounted camera recording the movement for 20 seconds. The number of body bends was then determined for all of the non-exposed and exposed groups. Three biological replicates were performed, and a total of 60 worms were evaluated.

 
Body Size Measurement

For the body size measurement, the L1 worms were exposed to the different exposure concentrations of the target samples and then allowed to reach adulthood. L4 worm images were captured using a dissecting microscope (Olympus, SZX10, Waltham MA, USA) at a 1.5X magnification, and the sizes were measured using the Java-based open source imaging software, ImageJ (http://imagej.nih.gov/ij/) (Wisconsin, USA). Three biological replicates were performed, and a total of 150 worms for each experimental group were measured.

 
Statistical Analysis

The test for normality was performed on all the data. Normal and non-normal distribution were determined using the Shapiro-Wilk test. Normally distributed data were examined for significance using a one-way ANOVA, and non-normal distributed data were examined using the non-parametric test, the Kruskal-Wallis H test. The Mann Whitney U test was used to determine the significant differences between two independent samples. A survival plot or the Kaplan-Meier plot was constructed to evaluate the effects of the different concentrations of SiO2 NPs and NH2-SiO2 NPs on the lifespan or ageing of the nematodes. All figures for the toxicological endpoint data were made using GraphPad Prism 6 (San Diego, California, USA). All statistical analyses were carried out using SPSS version 23 (International Business Machines Corp., New York, USA).

 
RESULTS AND DISCUSSION


 
Characterization of SiO2 NPs and NH4-SiO2 NPs

Figs. 1(A) to 1(D) show the morphology and size distribution of two commercial NPs, SiO2 and NH2-SiO2. The SiO2 NPs were determined to have an average size distribution of 31 ± 4 nm, and the NH-SiO2 NPs had an average size distribution of 37 ± 15 nm. As compared to the NH2-SiO2 NPs, a narrower size distribution was observed in the SiO2 NPs. Although a frequency of 35 nm particles was observed in NH2-SiO2 NPs, both types showed a similar average size, which is important when comparing the toxicity of both NPs in terms of their functionalization.

Fig. 1. Morphology and size distribution of (A and B) SiO2 and (C and D) NH2-SiO2 NPs
 Fig. 1. Morphology and size distribution of (A and B) SiO2 and (C and D) NH2-SiO2 NPs.

Fig. 2(A) shows the ATR-FTIR spectra of the NPs. The peak at 805 cm–1 in the case of both NPs corresponds to the Si-O-Si stretching while peaks at 1030 cm–1 for the SiO2 NPs and at 1080 cm–1 for the NH2-SiO2 NPs represent the siloxane vibrations of the (SiO)n groups. The O-H stretching band for the SiO2 NPs was observed at 3360 cm–1, and the peak at 1640 cm–1 can be attributed to the adsorbed water molecules. The peak at 1622 cm–1 for the NH2-SiO2 NPs represents the N-H bending of the amine groups. The CH band of the propyl group of NH2-SiO2 NPs can be located at 2919 cm–1 (Bois et al., 2003; Azarshin et al., 2017). Thus, this confirms that the two NP samples have different functionalization. Due to their differences in functionalization, their particle charge was also different (Fig. 2(B)). At pH = 7, the charges of the two NPs are as follows: SiO2 = –31.57 ± 1.06 mV, and NH2-SiO2 = 16.73 ± 0.83. The OH group of the SiO2 NPs could deprotonate, which resulted in a negative total net charge, whereas the amine groups on the NH2-SiO2 NPs could protonate, which provided a positive charge. The degradation behavior of the nanoparticles was determined in a temperature range of 50℃–850℃ (Fig. 2(C)). The NH2-SiO2 and SiO2 NPs showed a total loss of 2.05 and 2.32 wt.%, respectively. The SiO2 NPs had a higher total loss value due to the higher content of organic compounds such as OH groups and adsorbed water, which relatively decomposed within the temperature range. The water content in both types of NPs was removed starting at 50℃–120℃, wherein the SiO2 and NH2-SiO2 NPs lost about 0.35 and 0.06 wt.%, respectively. The OH groups on the SiO2 NP surfaces and the NH2 groups on the NH2-SiO2 NP surfaces degraded at temperatures starting at 120℃–550℃.

Fig. 2. (A) ATR-FTIR spectra, (B) particle charge, and (C) TGA of SiO2 and NH2-SiO2 NPs.Fig. 2. (A) ATR-FTIR spectra, (B) particle charge, and (C) TGA of SiO2 and NH2-SiO2 NPs.

 
Effects of Exposure Conditions and Chronic Lethality of SiO2 NPs and NH4-SiO2 NPs

In our study, the nematodes’ exposure at the L1 stage until adulthood to the different concentrations of the target samples is considered to be chronic exposure due to the relatively short life cycle of the nematodes (Comber et al., 2008). Although environmental dispersion concentrations of SiO2 NPs do not fall within the indicated exposure concentrations in this study, other scenarios that present clusters of local particle accumulation, such as surface coating local leaching, leakage and accidental spills, as well as occupational environments, permit the possibility of such concentration levels being present specifically (Scharf et al., 2013). Additionally, C. elegans thrive in the soil, which is a major sink for released nanomaterials in the environment (Sinis et al., 2019); therefore, supplementation of the target NPs on the bacterial lawn shows relevance to actual conditions where they are exposed to NPs. Before interpreting any results, Sinis et al. (2019) emphasized the importance of taking into account the exposure conditions used. Factors such as homogeneity, monodispersity, and colloidal stability can be altered when using conventional liquid media (e.g., M9, K-medium) due to the high ionic strength coupled with high chloride content, which may promote aggregation and precipitation of NPs and thereby compromise the size integrity of the NPs. Salt exposure may also cause some toxicity in nematodes (Sinis et al., 2019). Chronic exposure with starvation of the nematodes is not suggested due to outcomes being affected by the additional biological surface and unprecedented NP interactions with the supplied bacterial food in liquid medium. In addition, NPs mixed in solid NGM may present a homogeneous distribution of NPs (Kim et al., 2012); however, nematodes are only known to be in close contact with the NGM surface. We chose to allocate our exposure concentrations on a bacterial lawn to improve the distribution of NPs as well as to reduce stress and provide adequate food for the nematodes (Pluskota et al., 2009; Contreras et al., 2013; Contreras et al., 2014).

As shown in Fig. 3, nematodes exposed to the 2.5 and 5 mg mL–1 of SiO2 NPs showed a significzant decrease in survival rate (89% and 88%) immediately after exposure (p < 0.05) while only the highest concentration showed significant lethality in the nematodes exposed to NH2-SiO2 NPs (86%, p < 0.01).

Fig. 3. Chronic lethality effects of exposure to bare SiO2 NPs and NH4-SiO2 NPs. The bars in the figures represent means ± SD. *p < 0.05, **p < 0.01 with respect to the control group unless otherwise specified.Fig. 3. Chronic lethality effects of exposure to bare SiO2 NPs and NH4-SiONPs. The bars in the figures represent means ± SD. *< 0.05, **< 0.01 with respect to the control group unless otherwise specified.

In a study conducted by Eom and Choi (2019), L4 nematodes exposed to an SiO2 NP (60–100 nm size distribution) concentration of 500 mg L–1 in K-medium for 24 hours did not show any significant mortality while exposure to a lower concentration of 50 mg L–1 in the same medium for 48 hours induced significant mortality with a calculated LC50 of 123 mg L–1. Li et al. (2020) investigated the lethal effects of SiO2 NPs at 30 nm in worms exposed from the L1 stage to adulthood in K-medium and observed no lethality at any of the exposure concentrations, which ranged from 0.1–100 µg L–1. The supplementation of SiO2 NPs on a bacterial lawn may affect the lethality in C. elegans in comparison to liquid exposure methods; thus our study cannot be directly compared to the studies mentioned above. Overall, our results exhibited slightly higher lethality in worms exposed to SiO2 NPs as compared to NH2-SiO2 NPs.


Lifespan Effects

The toxic effects of exposure to the SiO2 NPs and NH2-SiO2 NPs on the aging of C. elegans were also evaluated. Day 1 of the life span was designated as the day after the exposure was conducted and the worms already reached adulthood. The exposed worms were then transferred to fresh NGM plates within the egg-laying period and were scored every day. In the case of bare SiO2 NPs, a significant decreasing trend in the lifespan and the average lifespan of the nematodes were observed at exposure concentrations of 2.5 (p < 0.05) and 5 (p < 0.05) mg mL–1 (Fig. 4(A)), with the average lifespans significantly shortening to 22 and 14 days (Fig. 4(C) and Table 1), respectively. In addition, the total population of the C. elegans was observed to decrease below 50% at days 12 and 6. Nematodes exposed to NH2-SiO2 NP concentrations of 5 mg mL–1 exhibited a significantly reduced lifespan (Fig. 4(B)), with an average lifespan of 20 days (Fig. 4(D) and Table 1) and a below 50% decrease in the total population at day 6.

Fig. 4. Lifespan assay on C. elegans exposed to SiO2 NPs and NH4-SiO2 NPs shows the (A and B) survival rate of the exposed worms for the duration of their lifespan and their calculated (C and D) average lifespan.Fig. 4. Lifespan assay on C. elegans exposed to SiO2 NPs and NH4-SiONPs shows the (A and B) survival rate of the exposed worms for the duration of their lifespan and their calculated (C and D) average lifespan.

 
Table 1. Summary of the average lifespans of nematodes exposed to SiO2 and NH2-SiO2 NPs.

Although in the present study, the same exposure technique and similar exposure concentrations were used as in Pluskota et al. (2009), whose results showed that SiO2 NPs did not significantly reduce the worm lifespan. The dissimilar outcome may be attributed to their exposure being performed on L4 worms, which excluded any developmental-related function degeneration. However, in this study, L1 worms were exposed until adulthood, thus suggesting the possibility of overlapping effects of developmental- and age-related organ degeneration on ageing in C. elegans. The younger stage of the nematodes in their study led to greater sensitivity and resulted in more obvious outcomes during chronic exposure (Tyne et al., 2013; Luo et al., 2017). Additionally, in comparison with our study, the size distribution of the SiO2 NPs was relatively smaller (30 nm), which may have contributed to the higher toxicity in the lifespan of the worms. Acosta et al. (2018) demonstrated that the nano-sized SiONPs (338 nm) significantly decreased the lifespan of the worms at concentrations of 0.5, 5, and 50 µg L–1 as compared to their harmless micro-sized counterparts (1930 µm and 1114 µm). Although a study by Acosta et al. (2018) utilized a larger NP size distribution and sublethal or environmentally-relevant exposure concentrations as compared to those used in the present study, which thus showed a more pronounced toxicity toward the longevity of the nematodes, the NP exposure done in liquid media may have contributed to higher toxicity as compared to when the target NPs were supplemented on a bacterial lawn in the present study. Age-related degeneration of nematode functions such as neurobehavior and egg-laying processes can be associated with long term exposure to NPs (Mikecz, 2018; Piechulek et al., 2019), particularly in worms exposed during adulthood (Pluskota et al., 2009). Sublethal concentrations of SiO2 NPs have been reported to have no significant effects on the nematode’s longevity via a high-throughput analysis but rather were found to accelerate age-related degeneration of reproductive and behavioral fitness (Jung et al., 2015; Mikecz, 2018). Our study presented the higher toxicity of bare SiO2 NPs as compared to NH2-SiO2 NPs at high concentrations.


Reproductive Toxicity

Oral uptake of nanomaterials may lead to their distribution to secondary organs such as the reproductive tract due to their ability to penetrate and translocate from primary organs such as the intestinal epithelial cells, which results in defects in the reproduction of C. elegans (Pluskota et al., 2009; Acosta et al., 2018; Sinis et al., 2019). Furthermore, nanomaterials may find entrance through the vulvar slit and consequently interact with reproductive components such as the plasma membrane of the vulvar cells and spermathecae (Scharf et al., 2013). Therefore, evaluation of reproductive-related endpoints such as the brood size is necessary to discern a possible translocation process of the nanomaterials in the nematode. After exposing the worms from the L1 stage into the L4 stage, the exposed adult worms were individually transferred to an NGM plate, and each worm, allowed to lay eggs for a duration of 4–5 days. Each worm was transferred to a new NGM plate every day, keeping the old plates in the incubator for the eggs to hatch and then counted to determine the total brood size (Fig. 5(A)). The nematodes exposed to all the bare SiO2 NP exposure concentrations experienced significantly reduced progeny production in a range of 79–60% as compared to the unexposed worms (p < 0.001). A significant decrease in the progeny number was only observed in the NH2-SiO2 NPs-exposed nematodes at concentrations of 2.5 and 5 mg mL–1 (p < 0.001) at 64–63%. As compared to NH2-SiO2 NPs, SiO2 NPs showed higher reproductive toxicity even at lower concentrations (Fig. 5(B)). Eventually, both types exhibited the same toxic intensity at higher exposure concentrations of 2.5 and 5 mg mL–1.

Fig. 5. Measurement of the (A) brood size of NH2-SiO2 and SiO2 NPs-exposed C. elegans (B) comparison between the two samples at different concentrations using a statistical analysis to determine any significant differences. The bars in the figures represent means ± SD. ***p < 0.001 with respect to the control group unless otherwise specified.Fig. 5. Measurement of the (A) brood size of NH2-SiO2 and SiO2 NPs-exposed C. elegans (B) comparison between the two samples at different concentrations using a statistical analysis to determine any significant differences. The bars in the figures represent means ± SD. ***< 0.001 with respect to the control group unless otherwise specified.

Pluskota et al. (2009) observed that L4 nematodes exposed to all SiO2 NP concentrations with a size distribution of 50 nm showed a significant decrease in progeny production. In addition, an increase in the bag-of-worms (BOW) phenotype, an egg-laying deficiency characterized by eggs hatching inside the body, was observed in 39-55% of worms exposed to 2.5 mg mL–1 of the SiO2 NPs. This was attributed to movement deficiencies in the vulvar muscles rather than vulvar developmental abnormalities since L4 worms already exhibited developed vulvas. Therefore, the results reported by Pluskota et al. (2009) represented an age-related degeneration of the reproductive capability of the nematodes exposed to the bare SiO2 NPs rather than a developmental defect. In a study conducted by Scharf et al. (2013), L1 worms exposed to 2.5 mg mL–1 of SiO2 NPs supplemented on a bacterial lawn until adulthood were shown to have developmentally defective egg-laying, which was determined to be the result of SiO2 NP-induced protein aggregation, causing serotonin presynaptic accumulation, which also leads to reduced neurotransmission capacity. To that end, such conclusions might explain the similar observations that were found in our study with regards to the observed developmentally related defects in brood production. As compared to the bare SiO2 NPs, the NH4-SiO2 NPs showed less toxicity toward the egg-laying capacity of C. elegans. The disturbance in protein homeostasis described by Scharf et al. (2013) can be explained by the fact that the presence of the negatively charged OH groups on the surface can lead to strong interactions with cellular membrane components such as positively charged lipids and proteins (Slowing et al., 2009) while the presence of positively charged amino groups are more likely to interact with negatively charged genetic materials such as the DNA (Hsiao et al., 2019). Although the degree of amino group surface substitution and steric hindrance due to the presence of linker have been associated with the reduction of toxicity and improvement of biocompatibility of amine-functionalized silica-NPs in previous studies (Yu et al., 2011; Morris et al., 2016; Hsiao et al., 2019), the exposure condition is more likely the probable cause as demonstrated by Hsiao et al. (2019). In their study, they observed a significant reduction in toxicity and increased biocompatibility of amine functionalized amorphous SiO2 NPs in the presence of serum proteins as compared to when the serum proteins were absent, regardless of the degree of amino group surface substitution.

At a molecular level, genes such as the msp (Major Sperm Protein), which is related to reproduction in C. elegans, has been reported to be downregulated after exposure to SiO2 NPs, an outcome that was attributed to the clathrin-mediated endocytosis pathway uptake mechanism (Eom and Choi, 2019). Although a detailed study of the mechanism of NH2-SiO2 NPs-induced reproductive toxicity in C. elegans is yet to be conducted, the possibility of toxic effects at a molecular level, particularly on the reproductive genes, is possible, but is likely only at higher concentrations as compared to bare SiO2 NPs. Acosta et al. (2018) reported lower toxicity of mesoporous silica (MSP) at concentrations of 0.5, 5.0, and 50 µg mL–1 in an M9 buffer with size distributions of > 50 nm and in MSPs functionalized with a starch derivative, emphasizing the effects of NP properties such as the size and functionality. Li et al. (2020) observed that SiO2 NPs with size distributions of 30 nm significantly reduced brood size and induced germline apoptosis in nematodes exposed to SiO2 NP concentrations of 100 µg L–1. Overall, the functionality, size, and dosage of silica-NPs are found to be important indicators of the extent of their toxicity on nematodes, particularly in terms of their reproductive ability.

 
Neurobehavioral Toxicity

Two of the commonly used locomotion tests for the neurobehavior of C. elegans were performed in this study, the head thrashing and body bending assays. Head thrashing movements are defined as changes in the bending of the mid-body while body bends are defined as a directional change in the part of the worm corresponding to the posterior bulb of the pharynx with respect to the y axis while the nematode is traveling along the x axis (Nouara et al., 2013). Head thrashes were counted within the duration of 1 minute, and body bends were counted within intervals of 20 seconds. Nematodes exposed to the bare SiO2 NPs concentrations of 0.25 (p < 0.01), 0.5 (p < 0.001), 2.5 (p < 0.001), and 5 (p < 0.001) mg mL–1 were found to exhibit significantly decreased head thrashing movements of between 92 and 71% (Fig. 6(A)). The NH2-SiO2 NP-exposed nematodes were found to have significantly reduced head thrashing movements at exposure concentrations of 0.5 (p < 0.05), 2.5 (p < 0.001), and 5 mg mL–1 (p < 0.001), with reduction percentages of 91%, 88%, and 85%, respectively. As compared to the NH2-SiO2 NPs, bare SiO2 NPs appeared to affect the head thrashing movement more, with an observed significant difference at the highest concentration (p < 0.01) (Fig. 6(B)). The body bending of the exposed nematodes was assessed by letting them crawl on an NGM plate with a freshly spread thin OP50 lawn and counting the number of times the nematode part corresponding to the posterior bulb of the pharynx changed in direction with respect to the y axis while travelling along the x axis in an interval of 20 seconds (Nouara et al., 2013). A significant reduction in the body bending movement was observed at SiO2 NP concentrations of 0.5–5 mg mL–1, with the body bending number reduced to a range of 71-34%. NH2-SiO2 NPs concentrations of 2.5 and 5 mg mL–1 significantly decreased the nematode body bending movement by 94–66% (Fig. 6(C)). Significant differences in both sample types were observed at concentrations of 0.5, 2.5, and 5 mg mL–1 (all with p values < 0.001), with SiO2 NPs showing higher toxicity (Fig. 6(D)).

Fig. 6. Neurobehavioral testing comprising the (A) measurement of head thrashes and (B) body bends were performed to evaluate neurotoxicity. In addition, a comparison between the two silica-NPs was also performed by determining their significant differences at different concentrations for the (C) head thrashing and (D) body bending tests. The bars in the figures represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 with respect to the control group unless otherwise specified.Fig. 6. Neurobehavioral testing comprising the (A) measurement of head thrashes and (B) body bends were performed to evaluate neurotoxicity. In addition, a comparison between the two silica-NPs was also performed by determining their significant differences at different concentrations for the (C) head thrashing and (D) body bending tests. The bars in the figures represent means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 with respect to the control group unless otherwise specified.

As compared to the lifespan assay, the reproductive and the locomotion tests appeared to be more sensitive towards SiO2 NP exposure, which indicates evident neurotoxic properties. Scharf et al. (2013) attributed such neurodegenerative effects to accumulated ubiquitinated insoluble proteins and nuclear amyloid, which damage the neural circuits responsible for the neurobehavior of worms. This was supported by their investigation on the proteomic characteristics of exposed nematodes, which showed that the aggregation of SiO2 NPs mainly affects the protein homeostasis pathway-related proteins. In addition, both the negative effects on locomotion and reproductive behavior were also evident in behavioral phenotypes treated with anti-amyloid compounds. Aside from presynaptic serotonin accumulation and egg-laying defects, the sensitivity of serotonergic HSN motor neurons towards SiO2 NPs can also result in neurotransmission problems. Acosta et al. (2018) demonstrated the toxicity of nano-sized mesoporous silica-based particles (338 nm) at concentrations of 5 and 50 µg mL–1 on 2-day and 9-day adult nematodes and reported a significant dose-dependent reduction in their body bending rates (p values of 0.08 and 0.01). In contrast, starch functionalization enhanced biocompatibility and reduced the SiO2 NPs toxicity in the worms. At a concentration of 10–100 µg L–1, L1 to adult day-1 nematodes exposed to SiO2 NPs (30 nm) showed significant slowing in head thrashing and body bending movements (Li et al., 2020). Overall, previous studies have demonstrated SiO2 toxicity based on factors such as functionality, size, and dosage. Although this is the first comparative study to compare the toxicity of NH2-SiO2 NPs with SiO2 NPs and to study the toxicity mechanism, the former had less toxic effects as compared to the latter, which was attributed to the negatively charged nature of the OH groups, which are more likely to interact with proteins (Hsiao et al., 2019). However, increasing the dosage of NH2-SiO2 NPs has been found to lead to significant toxic effects comparable to its counterpart. 


Growth Effects

Developmental toxicity in C. elegans can be represented by its capacity to grow (Li et al., 2020). After exposure, the exposed nematodes were measured for body size to evaluate the effects of the target samples on their growth. C. elegans exposed to bare SiO2 NP concentrations of 0.5, 2.5, and 5  mg mL–1 were found to have body sizes significantly reduced to 93% (p < 0.001), 86% (p < 0.001), and 81% (p < 0.001), respectively, while NH2-SiO2 NPs induced significant decreases in growth, which was evident in the reduced body lengths of 94% (p < 0.001), 89% (p < 0.001), and 88% (p < 0.001) at similar exposure concentrations (Fig. 7(A)). Although both target samples showed reduced growth at similar concentrations, bare SiO2 NPs showed higher toxicity at concentrations of 2.5 (p < 0.01) and 5 mg mL–1 (p < 0.001) (Fig. 7(B)). The mean body size measurements of the worms exposed to 0.25, 0.5, 2.5, and 5 mg mL–1 of SiO2 NPs were 1020, 969, 896, and 842 µm while the NH4-SiO2 NP-exposed nematodes showed mean body sizes of 1027, 976, 927, and 909 µm, respectively.


Fig. 7. Body size measurement of the unexposed and exposed nematodes. Significant differences in body size measurements were determined between the (A) control group and the exposed group and between (B) the two different samples at different concentrations. The bars in the figures represent means ± SD. ***p < 0.001 with respect to the control group unless otherwise specified.Fig. 7. 
Body size measurement of the unexposed and exposed nematodes. Significant differences in body size measurements were determined between the (A) control group and the exposed group and between (B) the two different samples at different concentrations. The bars in the figures represent means ± SD. ***< 0.001 with respect to the control group unless otherwise specified.

Piechulek et al. (2019) observed a reduction in the body length of L4 nematodes exposed to 200 µg mL–1 of silica-NPs synthesized using different methods (12nm HTFH, 25 nm Hartlen and 50nm Stoeber silica-NPs) and BULK silica (500 nm) for 72 h. Nematodes exposed to BULK silica had a normal body size (1233 µm) in comparison to the untreated group (1194 µm). In contrast, regardless of the synthesis method, nematodes exposed to the different silica-NPs showed a mean body length of 736 µm, which is relatively similar to that of the pep-2 deletion mutants, which lack the OPT-2/PEP-2 peptide transporter (838 µm). This result indicated that although the OPT-2/PEP-2 peptide transporter is active in nematodes exposed to silica-NPs, nutrient peptides reportedly trapped in vesicles formed when intestinal cells absorbed the reporter β-Ala-Lys-AMCA. This prevented essential proteins such as di- and tri-peptides from undergoing further hydrolysis and amino acid metabolism (Smith et al., 2013). Our study demonstrated that nematodes exposed to SiO2 NPs exhibited the same reduced body size phenotypes resulting from a disturbance in protein homeostasis. We concluded that the OH groups on the bare SiO2 NP surfaces contributed more to the disruption of protein homeostasis effects as compared to the amine groups on the surface of the NH2-SiO2 NPs, which resulted in the observed toxicity. The interaction of silica-NPs with biomolecules can be credited to their differences in terms of charge, with the negatively charged OH groups having higher affinity to lipids and proteins and positively charged amine groups having higher affinity to genetic materials.

 
CONCLUSION


The present study is the first comparative analysis of the toxic effects of two commonly utilized silica-NPs (SiO2 and NH2-SiO2 NPs) in terms of functionality and dosage in the in vivo model, C. elegans. Our target concentrations were relatively higher as compared to the reported predicted concentrations of silica-NPs in the environment from previous studies. However, we cannot ignore the possibility that local particle accumulation resulting from untoward accidents such as local leaching of surface coating, occupational environments, and leakage and accidental spills may occur. Although significant chronic toxicity was only observed at higher exposure concentrations of both silica-NP types, developmental toxicities were observed. Nematodes exposed to both silica NP types were shown to have significantly reduced average lifespans, brood size, locomotion, and body size. However, the functionality assessment differentiated their toxic intensities, with SiO2 NPs exhibiting negatively charged OH groups on the surface being more toxic due to their higher interaction with biomolecules, particularly proteins resulting in a disturbance in protein homeostasis as compared to the positively charged amino groups on the NH2-SiO2 NP surface. Protein homeostasis disruption has been associated with egg-laying and neurotransmission defects as well as to reductions in body size, all of which may contribute to a reduced lifespan in nematodes. To that end, our study suggests functionality as an important indicator for assessing the toxicity of nanoparticles.

 
ACKNOWLEDGEMENT


This study was supported by a grant from the Ministry of Science and Technology (MOST 106-2221-E-020-001-MY3). We gratefully want to give thanks to Professor How-Ran Chao and Assistant Professor Ming-Hsien Tsai from National Pingtung University of Science and Technology (NPUST) for study design and providing instruments and the laboratory. We would like to express our deepest gratitude to Professor Kuair-Rarn Lee of Chung-Yuan Christian University (CYCU) for supporting the experiment and for providing the materials for the study. We acknowledge Professor Lemmuel L. Tayo and Ms. Mariene-Syne Edisa P. Cortez from Mapua University and Ms. Lala Mariam Dabo, Ms. Nosizwe Haru Kunene, and Mr. Yo-Hsien Su from NPUST for assisting us with maintaining and culturing of the C. elegans. We would also like to thank Dr. Chang-Shi Chen from NCKU and Dr. Wen-Li Hsu from Kaohsiung Medical University for their advice and help in attaining the C. elegans culture.

 
CONFLIC OF INTEREST


The authors declare no conflicts of interest.


REFERENCES


  1. Acharya, G., Mitra, A.K. and Cholkar, K. (2017). Chapter 10 - Nanosystems for diagnostic imaging, biodetectors, and biosensors. In Emerging nanotechnologies for diagnostics, drug delivery and medical devices, Mitra, A.K., Cholkar, K. and Mandal, A. (Eds.), Elsevier, Boston, pp. 217–248.

  2. Acosta, C., Barat, J.M., Martínez-Máñez, R., Sancenón, F., Llopis, S., González, N., Genovés, S., Ramón, D. and Martorell, P. (2018). Toxicological assessment of mesoporous silica particles in the nematode Caenorhabditis elegans. Environ. Res. 166: 61–70. https://doi.org/10.1016/j.envres.2018.05.018

  3. Al-Rawi, M., Diabate, S. and Weiss, C. (2011). Uptake and intracellular localization of submicron and nano-sized SiO₂ particles in HeLa cells. Arch. Toxicol. 85: 813–826. https://doi.org/10.1007/s00204-010-0642-5

  4. Ang, M.B.M.Y., Pereira, J.M., Trilles, C.A., Aquino, R.R., Huang, S.H., Lee, K.R. and Lai, J.Y. (2019). Performance and antifouling behavior of thin-film nanocomposite nanofiltration membranes with embedded silica spheres. Sep. Purif. Technol. 210: 521–529. https://doi.org/10.1016/j.seppur.2018.08.037

  5. Azarshin, S., Moghadasi, J. and Aboosadi, Z.A. (2017). Surface functionalization of silica nanoparticles to improve the performance of water flooding in oil wet reservoirs. Energy Explor. Exploit. 35: 685–697. https://doi.org/10.1177%2F0144598717716281

  6. Bahadar Zeb, B., Khan Alam, K., Armin Sorooshian, A., Blaschke, T., Ahmad, I. and Shahid, I. (2018). On the morphology and composition of particulate matter in an urban environment. Aerosol Air Qual. Res. 18: 1431–1447. https://doi.org/10.4209/aaqr.2017.09.0340

  7. Besic Gyenge, E., Darphin, X., Wirth, A., Pieles, U., Walt, H., Bredell, M. and Maake, C. (2011). Uptake and fate of surface modified silica nanoparticles in head and neck squamous cell carcinoma. J. Nanobiotechnol. 9: 32. https://doi.org/10.1186/1477-3155-9-32

  8. Bois, L., Bonhommé, A., Ribes, A., Pais, B., Raffin, G. and Tessier, F. (2003). Functionalized silica for heavy metal ions adsorption. Colloids Surf., A 221: 221–230. https://doi.org/10.1016/S0927-7757(03)00138-9

  9. Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B. and Behr, J.P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. PNAS 92: 7297–7301. https://doi.org/10.1073/pnas.92.16.7297

  10. Boxall, A.B.A., Tiede, K. and Chaudhry, Q. (2007). Engineered nanomaterials in soils and water: how do they behave and could they pose a risk to human health? Nanomedicine 2: 919–927. https://doi.org/10.2217/17435889.2.6.919

  11. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77: 71–94.

  12. Chen, M. and von Mikecz, A. (2005). Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles. Exp. Cell. Res. 305: 51–62. https://doi.org/10.1016/j.yexcr.2004.12.021

  13. Chen, M.Y., Tsai, Y.C., Tseng, C.F., Lin, H.P. and Hsi, H.C. (2019). Using rice-husk-derived porous silica modified with recycled cu from industrial wastewater and ce to remove Hg0 and NO from simulated flue gases. Aerosol Air Qual. Res. 19: 2557–2567. https://doi.org/10.4209/aaqr.2019.09.0468

  14. Chen, Y., Chen, J., Dong, J. and Jin, Y. (2004). Comparing study of the effect of nanosized silicon dioxide and microsized silicon dioxide on fibrogenesis in rat. Toxicol. Ind. Health 20: 21–27. https://doi.org/10.1191/0748233704th190oa

  15. Chen, Z., Meng, H., Xing, G., Yuan, H., Zhao, F., Liu, R., Chang, X., Gao, X., Wang, T. and Jia, G. (2008). Age-related differences in pulmonary and cardiovascular responses to SiO2 nanoparticle inhalation: Nanotoxicity has susceptible population. Environ. Sci. Technol. 42: 8985–8992. https://doi.org/10.1021/es800975u

  16. Choi, J., Zheng, Q., Katz, H.E. and Guilarte, T.R. (2010). Silica-based nanoparticle uptake and cellular response by primary microglia. Environ. Health Perspect. 118: 589–595. https://doi.org/10.1289/ehp.0901534

  17. Chung, M.C., Tsai, M.H., Que, D.E., Bongo, S.J., Hsu, W.L., Tayo, L.L., Lin, Y.H., Lin, S.L., Gou, Y.Y., Hsu, Y.C., Hou, W.C., Huang, K.L. and Chao, H.R. (2019). Fine particulate matter-induced toxic effects in an animal model of Caenorhabditis elegans. Aerosol Air Qual. Res. 19: 1068–1078. https://doi.org/10.4209/aaqr.2019.03.0127

  18. Comber, S.D.W., Rule, K.L., Conrad, A.U., Höss, S., Webb, S.F. and Marshall, S. (2008). Bioaccumulation and toxicity of a cationic surfactant (DODMAC) in sediment dwelling freshwater invertebrates. Environ. Pollut. 153: 184–191. https://doi.org/10.1016/j.envpol.2007.07.032

  19. Contreras, E.Q., Cho, M., Zhu, H., Puppala, H.L., Escalera, G., Zhong, W. and Colvin, V.L. (2013). Toxicity of quantum dots and cadmium salt to Caenorhabditis elegans after multigenerational exposure. Environ. Sci. Technol. 47: 1148–1154. https://doi.org/10.1021/es3036785

  20. Contreras, E.Q., Puppala, H.L., Escalera, G., Zhong, W. and Colvin, V.L. (2014). Size-dependent impacts of silver nanoparticles on the lifespan, fertility, growth, and locomotion of Caenorhabditis elegans. Environ. Toxicol. Chem. 33: 2716–2723. https://doi.org/10.1002/etc.2705

  21. Eom, H.J. and Choi, J. (2019). Clathrin-mediated endocytosis is involved in uptake and toxicity of silica nanoparticles in Caenohabditis elegans. Chem. Biol. Interact. 311: 108774. https://doi.org/10.1016/j.cbi.2019.108774

  22. Esim, O., Kurbanoglu, S., Savaser, A., Ozkan, S.A. and Ozkan, Y. (2019). Chapter 9 - Nanomaterials for drug delivery systems. In New developments in nanosensors for pharmaceutical analysis, Ozkan, S.A. and Shah, A. (Eds.), Academic Press, pp. 273–301.

  23. European Union (EU) (2011). Commission Regulation (EU) No 1129/2011 of 11 November 2011 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council by establishing a Union list of food additives (Text with EEA relevance). Official Journal of the European Union, OJ L 295, 12.11.2011.

  24. Farrukh, A., Akram, A., Ghaffar, A., Tuncel, E., Oluz, Z., Duran, H., Rehman, D.H. and Yameen, B. (2014). Surface-functionalized silica gel adsorbents for efficient remediation of cationic dyes. Pure Appl. Chem. 86: 1177–1188. https://doi.org/10.1515/pac-2014-0105

  25. Ghosh, S., Goswami, S.K. and Mathias, L.J. (2013). Surface modification of nano-silica with amides and imides for use in polyester nanocomposites. J. Mater. Chem. A 1: 6073–6080. https://doi.org/10.1039/C3TA10381A

  26. Go, M.R., Bae, S.H., Kim, H.J., Yu, J. and Choi, S.J. (2017). Interactions between food additive silica nanoparticles and food matrices. Front. Microbiol. 8: 1013–1013. https://doi.org/10.3389/fmicb.2017.01013

  27. Gomes, M., Cunha, A., Trindade, T. and Tomé, J. (2016). The role of surface functionalization of silica nanoparticles for bioimaging. J. Innovative Opt. Health Sci. 9: 1630005. https://doi.org/10.1142/S1793545816300056

  28. Gupta, G.S., Kumar, A., Shanker, R. and Dhawan, A. (2016). Assessment of agglomeration, co-sedimentation and trophic transfer of titanium dioxide nanoparticles in a laboratory-scale predator-prey model system. Sci. Rep. 6: 31422. https://doi.org/10.1038/srep31422

  29. Haensch, C., Hoeppener, S. and Schubert, U.S. (2010). Chemical modification of self-assembled silane based monolayers by surface reactions. Chem. Soc. Rev. 39: 2323–2334. https://doi.org/10.1039/B920491A

  30. Hsiao, I.L., Fritsch-Decker, S., Leidner, A., Al-Rawi, M., Hug, V., Diabaté, S., Grage, S.L., Meffert, M., Stoeger, T., Gerthsen, D., Ulrich, A.S., Niemeyer, C.M. and Weiss, C. (2019). Biocompatibility of amine-functionalized silica nanoparticles: The role of surface coverage. Small 15: 1805400. https://doi.org/10.1002/smll.201805400

  31. Huang, H.Y., Yang, R.T., Chinn, D. and Munson, C.L. (2003). Amine-grafted MCM-48 and silica xerogel as superior sorbents for acidic gas removal from natural gas. Ind. Eng. Chem. Res 42: 2427–2433. https://doi.org/10.1021/ie020440u

  32. Hunt, P.R. (2017). The C. elegans model in toxicity testing. J. Appl. Toxicol. 37: 50–59. https://doi.org/10.1002/jat.3357

  33. Iavicoli, I., Leso, V., Beezhold, D.H. and Shvedova, A.A. (2017). Nanotechnology in agriculture: Opportunities, toxicological implications, and occupational risks. Toxicol. Appl. Pharmacol. 329: 96–111. https://doi.org/10.1016/j.taap.2017.05.025

  34. Jarvie, H.P., Al-Obaidi, H., King, S.M., Bowes, M.J., Lawrence, M.J., Drake, A.F., Green, M.A. and Dobson, P.J. (2009). Fate of silica nanoparticles in simulated primary wastewater treatment. Environ. Sci. Technol. 43: 8622–8628. https://doi.org/10.1021/es901399q

  35. Jung, S.K., Qu, X., Aleman-Meza, B., Wang, T., Riepe, C., Liu, Z., Li, Q. and Zhong, W. (2015). A multi-endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis elegans. Environ. Sci. Technol. 49: 2477–2485. https://doi.org/10.1021/es5056462

  36. Kang, Z., Liu, Y. and Lee, S.T. (2011). Small-sized silicon nanoparticles: New nanolights and nanocatalysts. Nanoscale 3: 777–791. https://doi.org/10.1039/C0NR00559B

  37. Keller, A. and Lazareva, A. (2013). Predicted releases of engineered nanomaterials: From global to regional to local. Environ. Sci. Technol. Lett. 1: 65–70. https://doi.org/10.1021/ez400106t

  38. Kim, B., Kim, H. and Yu, I.J. (2014). Assessment of nanoparticle exposure in nanosilica handling process: including characteristics of nanoparticles leaking from a vacuum cleaner. Ind. Health 52: 152–162. https://doi.org/10.2486/indhealth.2013-0087

  39. Kim, S., Ida, J., Guliants, V.V. and Lin, Y.S. (2005). Tailoring pore properties of MCM-48 silica for selective adsorption of CO2. J. Phys. Chem. B 109: 6287–6293. https://doi.org/10.1021/jp045634x

  40. Kim, S.W., Nam, S.H. and An, Y.J. (2012). Interaction of silver nanoparticles with biological surfaces of Caenorhabditis elegans. Ecotoxicol. Environ. Saf. 77: 64–70. https://doi.org/10.1016/j.ecoenv.2011.10.023

  41. Kralj, S., Rojnik, M., Romih, R., Jagodič, M., Kos, J. and Makovec, D. (2012). Effect of surface charge on the cellular uptake of fluorescent magnetic nanoparticles. J. Nanopart. Res. 14: 1151. https://doi.org/10.1007/s11051-012-1151-7

  42. Kurtz-Chalot, A., Klein, J.P., Pourchez, J., Boudard, D., Bin, V., Alcantara, G., Martini, M., Cottier, M. and Forest, V. (2014). Adsorption at cell surface and cellular uptake of silica nanoparticles with different surface chemical functionalizations: Impact on cytotoxicity. J. Nanopart. Res. 16: 2738. https://doi.org/10.1007/s11051-014-2738-y

  43. Li, C.M., Wang, X.P., Jiao, Z.H., Zhang, Y.S., Yin, X.B., Cui, X.M. and Wei, Y.Z. (2019). Functionalized porous silica-based nano/micro particles for environmental remediation of hazard ions. Nanomaterials 9: 247. https://doi.org/10.3390/nano9020247

  44. Li, D., Ji, J., Yuan, Y. and Wang, D. (2020). Toxicity comparison of nanopolystyrene with three metal oxide nanoparticles in nematode Caenorhabditis elegans. Chemosphere 245: 125625. https://doi.org/10.1016/j.chemosphere.2019.125625

  45. Liberman, A., Mendez, N., Trogler, W.C. and Kummel, A.C. (2014). Synthesis and surface functionalization of silica nanoparticles for nanomedicine. Surf. Sci. Rep. 69: 132–158. https://doi.org/10.1016/j.surfrep.2014.07.001

  46. Lin, C.J., Chang, C.L., Tseng, C.F., Lin, H.P. and Hsi, H.C. (2019). Preparation of Cu-Mn and Cu-Mn-Ce oxide/mesoporous silica via silicate exfoliation for removal of NO and Hg0. Aerosol Air Qual. Res. 19: 1421–1438. https://doi.org/10.4209/aaqr.2018.10.0389

  47. Lin, W., Huang, Y.W., Zhou, X.D. and Ma, Y. (2006). In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicol. Appl. Pharmacol. 217: 252–259. https://doi.org/10.1016/j.taap.2006.10.004

  48. Lu, X., Tian, Y., Zhao, Q., Jin, T., Xiao, S. and Fan, X. (2010). ntegrated metabonomics analysis of the size-response relationship of silica nanoparticles-induced toxicity in mice. Nanotechnology 22: 055101. https://doi.org/10.1088/0957-4484/22/5/055101

  49. Luo, X., Xu, S., Yang, Y., Zhang, Y., Wang, S., Chen, S., Xu, A. and Wu, L. (2017). A novel method for assessing the toxicity of silver nanoparticles in Caenorhabditis elegans. Chemosphere 168: 648–657. https://doi.org/10.1016/j.chemosphere.2016.11.011

  50. Mao, J., Ma, Y., Zang, L., Xue, R., Xiao, C. and Ji, D. (2020). Efficient adsorption of hydrogen sulfide at room temperature using fumed silica-supported deep eutectic solvents. Aerosol Air Qual. Res. 20: 203–2015. https://doi.org/10.4209/aaqr.2019.10.0520
  51. Marzaioli, V., Aguilar-Pimentel, J.A., Weichenmeier, I., Luxenhofer, G., Wiemann, M., Landsiedel, R., Wohlleben, W., Eiden, S., Mempel, M., Behrendt, H., Schmidt-Weber, C., Gutermuth, J. and Alessandrini, F. (2014). Surface modifications of silica nanoparticles are crucial for their inert versus proinflammatory and immunomodulatory properties. Int. J. Nanomed. 9: 2815–2832. https://doi.org/10.2147/IJN.S57396

  52. Mikecz, A. (2018). Lifetime eco-nanotoxicology in an adult organism: Where and when is the invertebrate C. elegans vulnerable? Environ. Sci.: Nano 5: 616–622. https://doi.org/10.1039/C7EN01061C

  53. Morris, A.S., Adamcakova-Dodd, A., Lehman, S.E., Wongrakpanich, A., Thorne, P.S., Larsen, S.C. and Salem, A.K. (2016). Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs. Toxicol. Lett. 241: 207–215. https://doi.org/10.1016/j.toxlet.2015.11.006

  54. Nagano, T., Nagano, K., Nabeshi, H., Yoshida, T., Kamada, H., Tsunoda, S.I., Gao, J.Q., Higashisaka, K., Yoshioka, Y. and Tsutsumi, Y. (2017). Modifying the surface of silica nanoparticles with amino or carboxyl groups decreases their cytotoxicity to parenchymal hepatocytes. Biol. Pharm. Bull. 40: 726–728. https://doi.org/10.1248/bpb.b16-00917

  55. Niu, Y.M., Zhu, X.L., Chang, B., Tong, Z.H., Cao, W., Qiao, P.H., Zhang, L.Y., Zhao, J. and Song, Y.G. (2016). Nanosilica and polyacrylate/nanosilica: A comparative study of acute toxicity. Biomed. Res. Int. 2016: 353275. https://doi.org/10.1155/2016/9353275

  56. Nouara, A., Wu, Q., Li, Y., Tang, M., Wang, H., Zhao, Y. and Wang, D. (2013). Carboxylic acid functionalization prevents the translocation of multi-walled carbon nanotubes at predicted environmentally relevant concentrations into targeted organs of nematode Caenorhabditis elegans. Nanoscale 5: 6088–6096. https://doi.org/10.1039/C3NR00847A

  57. Oh, S., Kim, B. and Kim, H. (2014). Comparison of nanoparticle exposures between fumed and sol-gel nano-silica manufacturing facilities. Ind. Health 52: 190–198. https://doi.org/10.2486/indhealth.2013-0117

  58. Osseo-Asare, K. and Arriagada, F.J. (1999). Growth kinetics of nanosize silica in a nonionic water-in-oil microemulsion: A reverse micellar pseudophase reaction model. J. Colloid Interface Sci.       218: 68–76. https://doi.org/10.1006/jcis.1999.6232

  59. Palla, R., Karade, S., Mishra, G., Sharma, U. and Singh, L.P. (2017). High strength sustainable concrete using silica nanoparticles. Constr. Build. Mater. 138: 285–295. https://doi.org/10.1016/j.conbuildmat.2017.01.129

  60. Panas, A., Comouth, A., Saathoff, H., Leisner, T., Al-Rawi, M., Simon, M., Seemann, G., Dossel, O., Mulhopt, S., Paur, H.R., Fritsch-Decker, S., Weiss, C. and Diabate, S. (2014). Silica nanoparticles are less toxic to human lung cells when deposited at the air–liquid interface compared to conventional submerged exposure. Beilstein J. Nanotechnol. 5: 1590–1602. https://doi.org/10.3762/bjnano.5.171

  61. Panas, A., Marquardt, C., Nalcaci, O., Bockhorn, H., Baumann, W., Paur, H.R., Mulhopt, S., Diabate, S. and Weiss, C. (2013). Screening of different metal oxide nanoparticles reveals selective toxicity and inflammatory potential of silica nanoparticles in lung epithelial cells and macrophages. Nanotoxicology 7: 259–273. https://doi.org/10.3109/17435390.2011.652206

  62. Park, E.J. and Park, K. (2009). Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro. Toxicol. Lett. 184: 18–25. https://doi.org/10.1016/j.toxlet.2008.10.012

  63. Park, H., Hwang, E. and Yoon, C. (2019). Respirable crystalline silica exposure among concrete finishing workers at apartment complex construction sites. Aerosol Air Qual. Res. 19: 2804–2814. https://doi.org/10.4209/aaqr.2019.05.0251

  64. Peters, R., Kramer, E., Oomen, A.G., Rivera, Z.E., Oegema, G., Tromp, P.C., Fokkink, R., Rietveld, A., Marvin, H.J., Weigel, S., Peijnenburg, A.A. and Bouwmeester, H. (2012). Presence of nano-sized silica during in vitro digestion of foods containing silica as a food additive. ACS Nano 6: 2441–2451. https://doi.org/10.1021/nn204728k

  65. Piechulek, A., Berwanger, L.C. and von Mikecz, A. (2019). Silica nanoparticles disrupt OPT-2/PEP-2-dependent trafficking of nutrient peptides in the intestinal epithelium. Nanotoxicology 13: 1133–1148. https://doi.org/10.1080/17435390.2019.1643048

  66. Pluskota, A., Horzowski, E., Bossinger, O. and von Mikecz, A. (2009). In Caenorhabditis elegans nanoparticle-bio-interactions become transparent: Silica-nanoparticles induce reproductive senescence. PLoS One 4: e6622. https://doi.org/10.1371/journal.pone.0006622

  67. Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A. and Cerón, J. (2012). Basic Caenorhabditis elegans methods: Synchronization and observation. J. Vis. Exp. 10: e4019. https://doi.org/10.3791/4019

  68. Pulit-Prociak, J. and Banach, M. (2016). Silver nanoparticles – A material of the future…? Open Chem. 14: 76–91. https://doi.org/10.1515/chem-2016-0005

  69. Queiros, L., Pereira, J.L., Goncalves, F.J.M., Pacheco, M., Aschner, M. and Pereira, P. (2019). Caenorhabditis elegans as a tool for environmental risk assessment: emerging and promising applications for a "nobelized worm". Crit. Rev. Toxicol. 49: 411–429. https://doi.org/10.1080/10408444.2019.1626801

  70. Scharf, A., Piechulek, A. and von Mikecz, A. (2013). Effect of nanoparticles on the biochemical and behavioral aging phenotype of the nematode Caenorhabditis elegans. ACS Nano 7: 10695–10703. https://doi.org/10.1021/nn403443r

  71. Shang, Y., Zhu, T., Li, Y. and Zhao, J. (2009). Size-dependent hydroxyl radicals generation induced by SiO2 ultra-fine particles: The role of surface iron. Sci. China, Ser. B Chem. 52: 1033–1041. https://doi.org/10.1007/s11426-009-0141-9

  72. Shang, Y., Hasan, M.K., Ahammed, G.J., Li, M., Yin, H. and Zhou, J. (2019). Applications of nanotechnology in plant growth and crop protection: A review. Molecules 24: 2558. https://doi.org/10.3390/molecules24142558

  73. Shen, P., Yue, Y., Zheng, J. and Park, Y. (2018). Caenorhabditis elegans: A convenient in vivo model for assessing the impact of food bioactive compounds on obesity, aging, and Alzheimer's disease. Annu. Rev. Food Sci. Technol. 9: 1–22. https://doi.org/10.1146/annurev-food-030117-012709

  74. Sinis, S.I., Gourgoulianis, K.I., Hatzoglou, C. and Zarogiannis, S.G. (2019). Mechanisms of engineered nanoparticle induced neurotoxicity in Caenorhabditis elegans. Environ. Toxicol. Pharmacol. 67: 29–34. https://doi.org/10.1016/j.etap.2019.01.010

  75. Skjolding, L.M., Winther-Nielsen, M. and Baun, A. (2014). Trophic transfer of differently functionalized zinc oxide nanoparticles from crustaceans (Daphnia magna) to zebrafish (Danio rerio). Aquat. Toxicol. 157: 101–108. https://doi.org/10.1016/j.aquatox.2014.10.005

  76. Slowing, II, Wu, C.W., Vivero-Escoto, J.L. and Lin, V.S. (2009). Mesoporous silica nanoparticles for reducing hemolytic activity towards mammalian red blood cells. Small 5: 57–62. https://doi.org/10.1002/smll.200800926

  77. Smith, D.E., Clemencon, B. and Hediger, M.A. (2013). Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications. Mol. Aspects Med. 34: 323–336. https://doi.org/10.1016/j.mam.2012.11.003

  78. Tyne, W., Lofts, S., Spurgeon, D.J., Jurkschat, K. and Svendsen, C. (2013). A new medium for Caenorhabditis elegans toxicology and nanotoxicology studies designed to better reflect natural soil solution conditions. Environ. Toxicol. Chem. 32: 1711–1717. https://doi.org/10.1002/etc.2247

  79. United States Food and Drug Administration (FDA) (2015). Code of Federal Regulations Title 21, 21cfr172. 480. 2015.

  80. Unrine, J.M., Shoults-Wilson, W.A., Zhurbich, O., Bertsch, P.M. and Tsyusko, O.V. (2012). Trophic transfer of Au nanoparticles from soil along a simulated terrestrial food chain. Environ. Sci. Technol. 46: 9753–9760. https://doi.org/10.1021/es3025325

  81. van der Zande, M., Vandebriel, R.J., Groot, M.J., Kramer, E., Rivera, Z.E.H., Rasmussen, K., Ossenkoppele, J.S., Tromp, P., Gremmer, E.R. and Peters, R.J. (2014). Sub-chronic toxicity study in rats orally exposed to nanostructured silica. Part. Fibre Toxicol. 11: 8. https://doi.org/10.1186/1743-8977-11-8

  82. Walcarius, A. and Delacôte, C. (2005). Mercury(II) binding to thiol-functionalized mesoporous silicas: critical effect of pH and sorbent properties on capacity and selectivity. Anal. Chim. Acta 547: 3–13. https://doi.org/10.1016/j.aca.2004.11.047

  83. Wang, Y., Kalinina, A., Sun, T. and Nowack, B. (2016). Probabilistic modeling of the flows and environmental risks of nano-silica. Sci. Total Environ. 545–546: 67–76. https://doi.org/10.1016/j.scitotenv.2015.12.100

  84. Wang, Y. and Nowack, B. (2018). Dynamic probabilistic material flow analysis of nano-SiO2, nano iron oxides, nano-CeO2, nano-Al2O3, and quantum dots in seven European regions. Environ. Pollut. 235: 589–601. https://doi.org/10.1016/j.envpol.2018.01.004

  85. Wilczewska, A.Z., Niemirowicz, K., Markiewicz, K.H. and Car, H. (2012). Nanoparticles as drug delivery systems. Pharmacol. Rep. 64: 1020–1037. https://doi.org/10.1016/S1734-1140(12)70901-5

  86. Xia, T., Kovochich, M., Liong, M., Zink, J.I. and Nel, A.E. (2008). Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS Nano 2: 85–96. https://doi.org/10.1021/nn700256c

  87. Yang, X., Liu, J., He, H., Zhou, L., Gong, C., Wang, X., Yang, L., Yuan, J., Huang, H. and He, L. (2010). SiO2 nanoparticles induce cytotoxicity and protein expression alteration in HaCaT cells. Part. Fibre Toxicol. 7: 1. https://doi.org/10.1186/1743-8977-7-1

  88. Yu, T., Malugin, A. and Ghandehari, H. (2011). Impact of silica nanoparticle design on cellular toxicity and hemolytic activity. ACS Nano 5: 5717–5728. https://doi.org/10.1021/nn2013904 

Aerosol Air Qual. Res. 20 :1987 -2020 . https://doi.org/10.4209/aaqr.2020.04.0157  


Share this article with your colleagues 

 

Subscribe to our Newsletter 

Aerosol and Air Quality Research has published over 2,000 peer-reviewed articles. Enter your email address to receive latest updates and research articles to your inbox every second week.

5.9
2020CiteScore
 
 
81st percentile
Powered by
Scopus

2020 Impact Factor: 3.063
5-Year Impact Factor: 2.857

Aerosol and Air Quality Research (AAQR) is an independently-run non-profit journal that promotes submissions of high-quality research and strives to be one of the leading aerosol and air quality open-access journals in the world. We use cookies on this website to personalize content to improve your user experience and analyze our traffic. By using this site you agree to its use of cookies.