**3.2 Feeding effect**

The effect of feeding is almost always significant (**Figure 3**) on the toxicity of mixtures of contaminants on *D. magna*. Animals actively ingest nanoparticles during both fast and feeding conditions, but feeding condition reduces the effects of mixtures on the exposed animals. In young organisms, the only case in which the effect of food was not significant was due to the exposure to PE in the presence of n-TiO2.

In the case of adult individuals (**Figure 2**), the effect of feeding was significant in 5 out of 8 cases: in each experiment for PE+n-TiO2, partially following to exposure to Zn (with PE and PVC always at 96 hours) and with PVC+n-TiO2 at 24 hours.

It can be hypothesized that with the intake of food (phytoplankton) there is an increase in the metabolic rate, resulting in a better chance of detoxifying by *D. magna*, making the effects of toxic mixtures less evident. Concerning n-ZnO, another reason may be that, as the toxicity of metal-oxide nanoparticles reasonably due to the release of metal ions in water [33] some water chemical parameters, such as increasing pH and DOM (occurring during feeding with phytoplankton) could have reduced the concentration of free Zn2+ released from n-ZnO. Feeding could have affected these key parameters (DOM and pH in particular) following the addition of an external organic source (DOM) and due to changes induced by photosynthesis performed by algal activity on water chemical features as, also, previously hypothesized for single components of complex mixtures tested in this study [28].

#### **3.3 Aging effect**

The results achieved provides evidence that the level of toxicity of NPs+MPs mixtures depends on the age of the animals, confirming what yet seen following tests with several kinds of MPs [29]. The effect of aging (comparison between experiments carried out on young people and adults, **Figure 2**) was significant (affecting the possibility of remaining mobile or alive) in half of the experiments carried out. While results at 24, 48 and 72 hours appear controversial, the significance of a toxic effect clearly emerges at 96 hours, both for PE+n-ZnO and for PE+n-TiO2. Significant aging effect also for PE+n-TiO2 at 96 hours and PVC+n-TiO2 at 96 hours have been registered.
