*2.3. Dietary Transfer Assay*

The dietary transfer assay was adapted from a previous study with minor modifications [20]. *E. coli* OP50 treated with ZnO-NPs or ZnCl2 for 8 h were washed and re-suspended in deionized water. Subsequently, they were spread on the NGM plates onto which the *C. elegans* L1-larvae were placed. After 72 h of exposure, the worms were washed using M9 buffer for future assays.

To characterize the distribution and localization of ZnO-NPs in *C. elegans*, rhodamine B (RhoB) was used to label the ZnO-NPs, and unbound RhoB was removed by performing dialysis for 24 h in deionized water (regenerated cellulose dialysis tubing, MWCO: 6000–8000; Orange Scientific, Braine-l'Alleud, Belgium) [34]. The freshly prepared RhoB-labeled ZnO-NPs (RhoB/ZnO-NPs) were administered to the *E. coli* OP50 for 8 h. The RhoB and deionized water without ZnO-NPs (RhoB/deionized water) were treated using dialysis for 24 h and used as the control. Wild-type N2 *C. elegans* L1-larvae were fed with RhoB/ZnO-NPs or RhoB pre-treated *E. coli* OP50 for 96 h. After following the exposure and washing steps, the worms were anesthetized in 50 mM sodium azide on 2% agarose gel mounted on a glass slide. Fluorescent images of the worms were acquired using an epifluorescence microscope (Leica, Wetzlar, Germany) with a suitable filter set (excitation, 550 ± 30 nm; emission, 615 ± 45 nm) and a digital camera. Fluorescence intensities were analyzed and quantified using ImageJ [35]. The tests were conducted at least three times independently, and 25 worms were scored per treatment in each replicate.
