Toxicity of N-Nitrosodimethylamine and SiO2 nanoparticles to HaCaT and caco-2 cell lines found in waste water treatment

Nanoparticles (NPs) offer the possibility of safe removal of pollutants and microbes in water treatment and purification. Nowadays, NPs are used in the detection and purification of water from chemicals and biological substance such as metals (cadmium, copper, lead and zinc) nutrients (nitrate, nitrite ammonia and phosphate) bacteria, viruses, parasite and antibiotics. Metal containing NPs are among the four classes of particles used commonly in water treatment. Membrane technologies such as Ultrafiltration (UF), Nanofiltration (NF) and Reverse Osmosis (RO) have been widely used all over the world especially for water treatment and desalination. NPs after have been incorporated into membranes gained attention due to its ability to enhance membrane permeability, mechanical properties and selectivity in some cases. However, these membranes are suspected to fouling causing NPs and other contaminate to reach waterways. In this study, we tested the toxicity of SiO2 NPs synthesised by Stöber method and N-Nitrosodimethylamine (NDMA) as potent known carcinogens forms during chlorination on HaCaT and caco-2 cell lines for 4, 24 and 48h using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays also the morphology and size of SiO2 determined by Scanning electron microscopy (SEM). After exposure to SiO2 NPs (concentrations between 0.05-2 mg/mL), the concentration of 2 mg/mL inactivated LDH in both cell lines; however, it did not reduce the metabolic activity of both cell lines when MTT assay used. SEM revealed spherical and uniformed SiO2 particles with size 200 nm in diameter. NDMA (concentrations between 0.1-1000 μg/mL) inactivated LDH leakage in HaCaT and caco-2 also reduced the metabolic activity of HaCaT cell line at 48 hours exposure. The outcome of this study suggest that a concentration of SiO2<2 mg/mL used in water treatment can reduce the risk of nanmaterials toxicity to human and possibly the ecosystem. Our results urgefor more studies on the effect of nanomaterials to the aquatic environment and human exposure to NPs.


Background
The rapid development of nanoparticles (NPs) applicationshave resulted in enhancing the process of products manufacturing, developed products that reduce the concentrations of toxic compounds and assisted in the attainment of water quality standards and health advisories [1]. Therefore, it is necessary to investigate the possible risks related to these small particles to human in environmental exposure. Microfiltration (MF) and Ultrafiltration (UF) are one of the most effective methods for raw water treatment [2]. The majority of UF membranes are formed by hydrophobic polymers such as polysulfone (PS), polyethersulfone (PES), polypropylene (PP), polyethylene (PE) and polyvinylidenefluoride (PVDF) [2]. PES is the most used as UF membrane due to its excellent thermal, mechanical properties and chemical resistance. However, the PES is hydrophobic in nature and is suspected to fouling [3]. The main interest in the clearing efficiency of nanoparticles within sewage plant is to prevent nanomaterials from reaching the waterways [4]. Recently, possible toxic effect of carbon nanotubes to aquatic species such as larval Zebrafish and DaphinaMagna has been studied [5,6]. Another study investigated the physiological changes and toxicity to DaphinaMagna exposed to Titanium CrossMark ← Click for updates doi: 10.7243/2056-3779-4-4 dioxide (TiO 2 ) NPs which is one of the most important metal oxide nanoparticles [7]. These studies urge for clarifying the entrances of nanomaterials into aquatic environment. So far, little is known about the nanomaterials reaching the waterways [8]. Some studies criticised the standard wastewater treatment as it poorly suited to capture Nanomaterials [8]. Moreover, Wiesner et al. (2008) reported that waterways contaminated with nanomaterials water desalination plants may become problematic [9]. Due to the lack of experimental data, the method of nanomaterials disposal is not understood clearly [9,10]. Obviously, this will be a problematic because there is no sufficient data explaining the risk of inorganic nanoparticles dispersed in the environment or public sewage treatment plants. N-nitrosodimethylamine (NDMA) is well known for its carcinogenic properties [11,12]. For many years, NDMA was used in pesticide (nematicide), plasticizer for rubber, battery component, antioxidant, lubricant additive, and in polymers [13]. Recently, some studies revealed that water treatment polymers such as poly (epichlorohydrin dimethylamine) (polyamine) and poly (diallyldimethylammonium chloride) (polyDADMAC) may form NDMA if contacted with chloramine water [14]. In addition, NDMA is mainly used in research to induce cancer in mice [15]. Thus, the health concerns of this compound has been the focus for some industries after recognised as disinfection of drinking and wastewater containing chlorine and chloramines [16]. Recently, successful attempts have been made to add nanoparticles to polymers in membrane synthesis for removing water contaminants [17,18].
Ceramic membranes have been made with catalytic nanoparticles for synergistic effects on the membrane enhancement [17]. However, other studies suggest that silica polymerisation in the presence of polyvalent cations and anions in Reverse Osmosis (RO) systems are the cause of membranes fouling [19]. RO is by far the most effective method in removing dissolved silica from brackish water. The present study investigates the toxicity of NDMA as a known water contaminate and silica NPs as newly NPs used in membranes separationand found in high concentrations in brackish water [20]. NDMA and silica NPs exposed to human skin keratinocytes (HaCaT) and human colon carcinoma (caco-2), the toxicity determined using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Lactate dehydrogenase (LDH) assays also the particle morphology was identified by Scanning Electron Microscope (SEM).

Preparation of SiO 2 Nanoparticles
Silica NPs were synthesized by Stöber method [21]. A silica precursor of TEOS (1 mL) was added rapidly to a mixture of 95% ethyl alcohol (10 mL) and Milli-Q water (3 mL). The reaction was allowed to proceed for 30 min with mild stirring at 50°C to obtain a homogeneous mixture. Subsequently, ammonia solution (30%) (1 mL) was injected drop-wise over an 8 min period with stirring. The mixture gradually changed from transparent to milky as the nanoparticles formed. The mixture was then stirred for a further 2 h. The resultant precipitate was then centrifuged at 101890 G and washed with Milli-Q water four times. Finally, the silica particles were dried overnight at 75°C.

Nanoparticle characterization
Scanning electron microscopy (SEM) was performed Silica NPs. The samples were sputter coated with a 10 nm layer of platinum using a Quorumtech K757X Sputter coater. The images of the sample were collected using a CAMScan MX2500 SEM with a field emission source at an accelerating voltage of 10 kV.

Cell culture
HaCaT and caco-2 cell lines were obtained from American Type Culture Collection (ATCC). HaCaT cells were grown in RPMI and caco-2 cells in DMEM medium with 10% fetal bovine serum (FBS) and incubated at 37°C, 5% CO 2 in a humidified incubator. Cells growth started from 2x10 6 cells/ml and subcultured when confluence reached 60-70% every 2-3 days.

For toxicity assays
HaCaT and caco-2 cells were seeded at 10,000 cells/well in a 96-flat plate then incubated for 18h to allow adherence. NDMA and SiO 2 were diluted in fresh medium. After incubation, cells were treated with 6 concentrations of silica nanoparticles (0.05-2 mg/mL) and five concentration of NDMA (0.1-1000 µg/mL) for 4, 24 and 48h. The solution was removed and the cells were washed twice with phosphate buffered saline (PBS) twice to remove excess SiO 2 residue.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay Experimental Procedure
The toxicity of SiO 2 and NDMA was determined by MTT assay as described in literature [22,23]. 1x10 4 cells were seeded in volume of 100 µl into 96-well flat bottom plate. MTT was doi: 10.7243/2056-3779-4-4 added to each well at 0.5 mg/mL, and then plates were incubated at 37°C for 4h, then 80 µl of 20% SDS in 0.02 M HCl was added to each well. The plates were kept in the dark at room temperature for overnight.Absorbance was read at a primary wavelength of 570 nm and a reference wavelength of 630 nm by ELISA reader. In each experiment a standard curve was run to convert the OD values to cells/well.

Lactate Dehydrogenase leakage assay
The leakage of LDH in HaCaT and caco-2 cells was detected by LDH assay. Cells were plated into the 96-well plates and exposed to concentrations of SiO2 and NDMA as described previously. At the end of exposure time, 50 μl of aliquot cell medium was used for LDH activity analysis. The absorption was measured using ELISA reader at primary wavelength 490 nm and 630 nm as a reference wavelength. LDH assay experiments were programed in triplicate.

Statistical analysis
The data were expressed as mean +/-SD of at least three independent experiments using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons test using SPSS software to compare exposure groups. All comparisons were considered significant level p<0.05.

Result and discussion
Exposure of monodisperse spherical SiO 2 (size 200 nm in diameter) to HaCaT and caco-2 cell lines for 4, 24 and 48h did not interrupt the cells growth. LDH leakage was clearly low at concentrations <2mg/mL in both cell lines. Similarly, all concentrations did not reduce the cell growth using MTT assay in both cell lines compared to positive control Figure 1. Until now, researchers cannot decide with each other on dose and physiochemical properties of NPs that could cause biological respond [24]. Some related the toxicity of NPs to the particle total weight, others to the number of particle per volume [25]. Recently, some studies suggested that the logical way to define particles toxicity is to calculate the dose based on the NPs total surface area also other studies on SiO2 NP S found that the shape of particle is the main cause of particle pathogenesis of lung diseases [26][27][28][29]. The ecotoxicological impact of SiO 2 NPs on freshwater fish has not been completely understood [30]. Therefore, the haematological, ionoregulatory and enzymological toxicity effect of silica NPs concentrations 1, 5 and 25 mg L −1 were evaluated on freshwater teleost fish Labeorohita [30]. The exposure to Silica NPs concentrations has altered the values of haematological parameters such as haemoglobin (Hb) red blood cells, white blood cells, and plasma electrolytes. The plasma electrolytes such as potassium, sodium, chloride levels and Na + /K + ATPase activity were shifted by all Silica NPs concentrations through all study exposure times. Moreover, the effect of the Silica NPs was found to be time and concentration dependent [30]. In another study, twelve fish cell lines taken from six species which is rainbow trout, zebrafish, fathead minnow, haddock and American eel tested for metabolic activity using Alamar Blue assay [31]. The toxicity of SiO 2 NPs (16, 24, and 44 nm in diameter)was related to physiochemical properties such as the size and temperature also dose dependent and tissue specific. SiO 2 NPs (size 44 nmin diameter) concentrations greater than 100 µg/mL for 24h exposure reduced the fish cell lines viability by 50% [31]. Smaller NPs (size 16 nm) were found to induce more toxicity than larger NPs. In addition to size related toxicity, cells derived from external lining epithelial tissues (skin, gills) were more effected by SiO 2 NPs than cells taken from internal tissues (brain, liver, intestine, gonads) [31]. SEM image of the SiO 2 synthesised by Stöber method showeda monodisperse spherical silica particle with a size 200 nm in diameter On the other side, exposure of NDMA (concentrations between 0.1-1000 µg/mL) to HaCaT and caco-2 cell lines activated LDH leakage in all concentrations but mostly observed at 4h exposure. The LDH leakage by NDMA reduced after 24 and almost 5% at 48h. Comparably, NDMA interacted the metabolic activity of HaCaT cell line at concentration 1µg/mL and higher after 48h exposure, which show clearly the toxic impact. NDMA formation potential (FP) test is a fast and simple method to detect NDMA precursor concentrations in wastewater [32]. This method was applied on a tertiary wastewater treatment plants using an Ultrafiltration-Reverse Osmosis membranes. The results revealed that the NDMA FP from a variety of water samples were at the ranges between 350 to 1020±20 ng/L [32]. Furthermore, the fate of NDMA precursors was also investigated over two different points of advanced water treatment plants. The final results showed that more than 98.5±0.5% of NDMA precursors were removed by RO [32]. In California, one of the most used herbicides are phenylurea herbicide diuron has been continuously found in California's water sources, and ithad a structure that might be an NDMA precursor. NDMA precursors then consistently were found even in the absence of added ammonia which is a cause of nitro-nitrogen formation during chloramination. This is due to the both nitrogen atoms were donated by diuron during chlorination even where there is no ammonia exist.

Conclusion
In summary, SiO 2 NPs synthesised by Stöber method resulted in a monodisperse spherical particle with size 200 nm in diam-doi: 10.7243/2056-3779-4-4 eter analysed by SEM. Due to multiple factors the membrane can foul and nanoparticles can reach the waterways causing adverse effect on ecosystem. In this study, we tested the toxicity of SiO 2 NPs used in water filtration and NDMA as a member of a family of extremely potent carcinogens found in drinking water contaminant resulting from reactions occurring during chlorination. After 4, 24 and 48h exposure of SiO 2 (concentrations range between 0.05-2 mg/mL) to HaCaT and caco-2 cell lines using MTT and LDH assays only 2 mg/mL increased LDH leakage compared to positive control. NDMA activated LDH leakage in both cells and interrupted the metabolic activity of HaCaT cell line at most concentrations. Our finding suggests that SiO 2 imposed less environmental risk to humans and animals at the mentioned concentrations.