School of Chemical and Life Sciences, Nanyang Polytechnic, Singapore *Corresponding author

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1 0 Differential Effect of Solar Light in Increasing the Toxicity of Silver and Titanium Dioxide Nanoparticles to a Fish Cell Line and Zebrafish Embryos Saji George # *, Hannah Gardner, Seng Eng Khuan, Hengky Chang, Chunyan Wang, Crystal Hay Yu Fang, Mark Richards, Suresh Valiyaveettil, Chan Woon Khiong # Centre for Sustainable Nanotechnology, School of Chemical and Life Sciences, Nanyang Polytechnic, Singapore 0 School of Engineering, Nanyang Polytechnic, Singapore 0 Department of Chemistry, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore # Equal contribution *Corresponding author Summary Number of pages: Figures: Tables: 0 0 Simulated Solar Light (SSL)

2 0 We used a solar light simulator (IVT Solar Singapore Pte Ltd, model- VS00 XES) for exposing NPs and biological systems to SSL. This solar simulator uses a 00W xenon lamp with an irradiance uniformity meeting ASTM - Class A and IEC 0- Class A requirements. The terrestrial solar spectrum was simulated using optical filters to achieve Air Mass (AM). with a spectral match that is consistent with ASTM G-0 and IEC 00- :00. Air Mass is the optical path length taken by sunlight through atmosphere where AM. is the internationally accepted standard spectral condition to simulate solar light. Since, the total irradiance of the sunlight is defined as 000 Wm - for the standard Sun measurement, the light energy received on the surface of the platform (on which the test plates were kept) was ensured to be 000 Wm - by calibrating the lamp power using a monocrystalline silicon cell certified by Fraunhofer ISE. The wavelength spectra of SSL and its comparison with ASTMG- the standard wavelength spectrum of solar light is given in supplementary information, Figure S. 0 Figure S. Wavelength of light used in this study (SSL) in comparison with ASTMG- reference spectra for solar light (provided by instrument supplier). High Content

3 Screening: Image and Data Analysis The high content screening for multi-parametric analysis on cytotoxicity was conducted as detailed previously., Cellular fluorescence images were acquired using an automated 0 0 ImageXpress Micro XLS Widefield High Content Screening System (Molecular Devices, USA). Representative images of cells from different treatment groups stained with the three dye cocktails are shown in figure S. The images were analyzed by Meta-Xpress software (Molecular Devices, USA) to score the percent positive responses for each cytotoxicity parameter. Hoechst dye was used to count the total number of cells using the following blue channel settings: minimum width = µm (~ pixels), maximum width = 0 µm (~ pixels), threshold intensity above background = 0 gray levels. The area of the Hoechst stained region was measured in order to measure the nuclear area. The corresponding red channel settings for PI and MitoSox Red were: minimum width = µm (~ pixels), maximum width = 0 µm (~ pixels), threshold intensity above background = 0 gray levels and that for JC was minimum width = µm (~ pixels), maximum width = 0 µm (~ pixels), threshold intensity above background = 0 gray levels. Cells with fluorescence intensity above the threshold levels were scored positive for that cytotoxicity response and the percentage of cells positive for particular cytotoxicity response was generated by the software based on total number of cells. Each experiment was repeated two times with quadruplet replicates. The data on % cells affected for each cytotoxicity response were used for statistical analysis, using a strictly standard mean difference (SSMD) parameter to calculate the statistically significant difference between test and control samples according to the formula given below, β = µ σ sample sample µ + σ control control

4 where µ and σ denote the mean and standard deviation of the sample (quadruplicate response measurements for a NP concentration and/or SSL) or negative control population (cell population not exposed to NPs and/or SSL). The SSMD is a suitable statistical metric for HTS data analysis since it avoids the potential overestimation of the difference between sample and control responses. It is noted that, SSMD has a clear statistical meaning indicated by its positive correlation with the d + -probability (i.e., probability of the difference being greater than 0). For example, SSMD> indicates that the sample response is different from the response of negative control with probability of difference >.%. A heat map was constructed based on the SSMD values to summarize the multi-parametric data Hoechst+PI Hoechst+MitoSox Hoechst+JC Negative control Ag NPs TiO NPs Figure S. Representative images from high content screening of Ag and TiO NPs treated BF cells stained with different dye cocktails.

5 High Content Imaging of Zebrafish Embryos We used a total of Zebrafish embryos ( doses of NPs, embryos per tested dose, exposure conditions (dark and SSL), NPs and replicates) for this study. Healthy zebrafish embryos at hour post fertilization (hpf) were transferred to -well plate and were exposed to incremental concentrations of NPs at hpf. We used transparent U-bottom well plates as this type of wells positions embryo in the center. The images of embryos were acquired using ImageXpress Micro XLS Widefield High Content Screening System (Molecular Devices, USA). The plates containing zebrafish embryos treated with NPs with/without SSL exposure were loaded on the fully automated stage of the microscope and the bright-field phase 0 contrast images were taken using X objective lens. Since the embryo and the larvae could be at different focus planes, we used Z-stack function of ImageXpress to capture image at the best focused z-plane. Representative image of zebrafish embryos at different developmental stages treated with NPs with/without SSL exposure is shown in figure S. Figure S. Plate images of zebrafish embryos at two different stages of development obtained using high content screening

6 0 Figure S. Dissolved Fraction of Silver Enhanced the Cytotoxicity of Ag NPs We observed an increase in the cytotoxicity of Ag NPs when co-exposed with SSL (Figure A & C). We also noticed that SSL exposure of Ag NPs suspended in PBS could cause ~.% increase the dissolved fraction of silver (Figure A). Therefore, we tested if the dissolved fraction of Ag NPs could % cell viability AgNPs AgNPs+ AgNO * * * * explain the increase in toxicity when the Concentrations (µg/ml) 0 cells are co-exposed with Ag NPs and SSL. We subjected one set of BF cells to incremental concentrations of Ag NPs and another set to incremental concentration of Ag NPs wherein.% of the total concentration was constituted by AgNO (i.e. equivalent of µg/ml of Ag NPs will be 0. µg of Ag NPs+. µg of AgNO in ml). After h, the cell viability was assessed using resazurin assay as detailed for figure C. Our results showed that, presence of dissolved fraction could significantly enhance the cytotoxic potential of Ag NPs, a trend that was similar to our observation with Ag NPs under SSL exposure. Interestingly, however, the increase in cytotoxicity was not as high as that was observed when cells were co-exposed to Ag NPs and SSL (Figure C). This suggested that, in addition to the dissolution of Ag NPs, other factors such as reduced primary particle size, destabilization and increased agglomeration size of Ag NPs in exposure media may also contribute to its enhanced toxicity under SSL exposure. Destabilization and agglomeration, of Ag NPs is thought to perturb the effective concentration because of the sedimentation of Ag NPs onto the BF cells.

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