## 2 Materials and Methods
### In Vivo Toxicity  
A solution of GNPs in autoclaved potassium (K) medium composed of 2.36 g KCl and 3 g NaCl in 1 L of distilled water was prepared in a concentration of 1000 \(\upmu\)g/mL. The number of GNPs was first sterilized under UV light radiation for 1 h. After mixing with K medium, the solution was sonicated for 2 h at maximum frequency. After sonication, serial dilutions were carried out in order to prepare concentrations of 800, 500, 400, 300, 200, 100, 50, and 25 \(\upmu\)g/mL, considering that the final concentration would be half of the prepared one after mixing these nanomaterial dispersions with the growth medium. The maintenance and propagation of the N2 worms, supplied by the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA), were performed on OP50 _Escherichia coli_ seeded nematode growth medium (NGM) at 25 \({}^{\circ}\)C [20]. The nematodes and their eggs were washed off NGM plates with 5 mL of distilled water and collected in falcon tubes of 15 mL to produce an L1 stage-synchronized _C. elegans_ population. The centrifugation of the falcon tubes was performed at 1300 rp.m. (2209\(\times\)\(g\)) for 3 min and the supernatant was removed. The pellets of _C. elegans_' were resuspended in 100 \(\upmu\)L of deuterated water (dH\({}_{2}\)O) and placed in Eppendorf tubes with 700 \(\upmu\)L of a bleaching solution at 5%. Subsequently, incubation for 15 min while vortexing every 2 min was carried out. After that, centrifugation of the Eppendorf tubes was performed at 700\(\times\)\(g\) for 3 min. The supernatant was removed and washing of the pellets was carried out three times in 800 \(\upmu\)L of dH\({}_{2}\)O. Subsequently, the pellets were resuspended in 100 \(\upmu\)L of dH\({}_{2}\)O and introduced in NGM plates seeded with 100 \(\upmu\)L of an OP50 _E. coli_ culture. Egg incubation was carried out for 72 h at 25 \({}^{\circ}\)C. To pellet the L1 staged populations, centrifugation at 1300 rp.m. (2209\(\times\)\(g\)) for 3 min was performed, and, after that, it was resuspended in 3 mL of K medium. Plates with 48 wells were utilized to have wells with 62.5 \(\upmu\)L of a 1:250 suspension of cholesterol (5 mg/mL in ethyl alcohol) in sterile K medium, 62.5 \(\upmu\)L of a 50\(\times\) concentrated OP50 _E. coli_ culture with an optical density of 0.9, pelleted by centrifugation at 4000 r.p.m. (6797\(\times\)\(g\)) for 10 min and resuspended in K medium, 115 \(\upmu\)L of K medium and 250 \(\upmu\)L of the pertinent GNPs solution. A volume of K medium with 50-100 nematodes was then added. Parafilm was used to seal 48-well plates that were placed in an orbital shaker at 25 \({}^{\circ}\)C and 120 r.p.m. for 24 h, to study acute toxicity, or 72 h, to study chronic toxicity. After, mixing the GNPs dispersions with the growth medium, the final concentrations of the nanomaterials studied in this experiment were 500, 400, 250, 200, 150, 100, 50, 25, and 12.5 \(\upmu\)g/mL. In the plates with 48 wells, the volume of each well was divided into 10 drops of 50 \(\upmu\)L to be evaluated under a light microscope (Motic BA410E including Moticam 580 5.0 MP) and thus count the number of living and dead _C. elegans_ worms. The survival rate of the nematodes was also measured in the medium and in a toxic zinc chloride (1000 \(\upmu\)M) solution as a positive and negative control, respectively. These assays were performed in quintuplicate to ensure reproducible results.