# Chaves Thermal Spring Water Impact on Skin Health: Potential Cosmetic Application
## 3 Results
### Evaluation of Skin Parameters and Skin Microbiota  
The skin biometric parameters, including the hydration levels, transepidermal water loss (TEWL), and pH values, were evaluated before (day 0) and after (days 8 and 16) the application of Chaves thermal spring water (Figure 4). The hydration levels of both inner forearms of 23 volunteers were similar when comparing day 0 with day 8 and day 16 (Figure 4a). To evaluate whether the hydration levels were influenced by gender, age, or the presence/absence of skin diseases, the results were analyzed based on these criteria, which demonstrated that the hydration levels were maintained during the time. Interestingly, when the volunteers were grouped based on their hydration levels at day 0 (before the application of Chaves thermal spring water), the group of volunteers with very dry skin (hydration levels \(<30\)) revealed a statistically significant increase in their hydration levels at day 8, maintaining this until day 16.  
On day 16, our results demonstrated that the application of Chaves thermal spring water significantly decreased the transepidermal water loss to values under 10 g/m\({}^{2}\)/h in comparison to day 0, meaning that this thermal water might help to restore the integrity of the skin barrier (Figure 4b). Additionally, the skin pH values of the volunteers increased significantly on days 8 and 16 in comparison to day 0. However, the volunteers presented similar skin pH values on days 8 and 16, demonstrating that Chaves thermal spring water allowed them to maintain a healthy skin pH (Figure 4c).  
Figure 3: Evaluation of anti-inflammatory potential of Chaves thermal water. The IL-6 levels were quantified in supernatants of HaCaT cells (**a**) without urban air pollution particles and (**b**) upon contact with urban air pollution particles. HaCaT cells were incubated with DMEM powder high-glucose medium plus Chaves thermal spring water (TW) and with DMEM powder high-glucose plus Mili-Q-type water (control). The pH values of both media were adjusted to 7.4, and the media were supplemented with FBS plus penicillin–streptomycin. Betamethasone (Beta) was used as a positive control for the anti-inflammatory effect. (**\(p<0.001\), ***\(p<0.0001\)).  
To further evaluate the impact of Chaves thermal spring water on skin health, we also characterized the skin microbiota in a subset of 13 volunteers at days 0 and 16. _Staphylococcus_, _Propionibacterium_, and _Corynebacterium_ are the main bacterial genera that compose the human skin microbiota [36]. Thus, their relative abundances were determined on the samples collected from the skin of the inner forearms of volunteers on day 0 (before the application) and day 16 (after the application), which presented similar amounts of these genera (Figure 5a-c). We also determined the relative abundances of _S. epidermidis_ and _P. acnes_ (skin health sentinels), demonstrating no statistically significant differences between the skin microbiota samples collected before and after the application of Chaves thermal spring water (Figure 5e,f). A similar result was obtained for the _Staphylococcus_ sp./_Propioniibacterium_ sp. and _S. epidermidis_/_P. acnes_ ratios when comparing both time points of collection. Moreover, the comparison of samples collected on day 0 and those collected on day 16 showed that the relative abundance of _Malassezia_ genus was similarly detected at both time points (Figure 5d).