2.1. Physicochemical Analysis of Thermal Water
Ultra-pure water was produced on a Milli Q Gradient A10 system, from Millipore (Molsheim, France).
The conductivity was determined with a CyberScan CON 200, supplied by Eutech Instruments (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the calibration and control of the equipment was made with several conductivity standards (84, 147, 1413, and 12880 μS/cm, 25 °C) supplied by Reagecon (Shannon, Co. Clare, Ireland).
The pH was measured using a pH meter, Crison model micropH 2002 (Barcelona, Spain) with a glass electrode (Crison, Barcelona, Spain), calibrated using the pH standards (pH 4, pH 7, pH 6 and pH 9) supplied by the same company.
The turbidity was determined with a EUTECH TN-100/T-100, supplied by Eutech Instruments (Thermo Fisher Scientific Inc.), and the calibration and control of the equipment was made with several conductivity standards (0.02, 20, 100 and 800 UNT) supplied by Eutech Instruments (Thermo Fisher Scientific Inc, Waltham, MA, USA).
The dissolved oxygen was determined with an inoLab® Laboratory Oxygen Meter, Models Oxi 740, WTW (VWR, Portugal).
The total dissolved solids were determined by gravimetric method at 180 °C using a Memmert oven, model ULM-700 (Memmert, Germany).
Absorption spectrophotometry was used to determine the color and the concentrations of nitrate, nitrite, ammonia, sulphate, phosphate, sulfide, zinc, iron, and silicon, using a double beam UV/Visible spectrophotometer HITACHI U-2000 (Tokyo, Japan).
Fluoride was analyzed by a potentiometric method with a fluoride combination ion selective electrode from Thermo Scientific, model 9609BNWP (Thermo Fisher Scientific Inc, Waltham, MA, USA) and a Crison potentiometer, pH meter model GLP22 (Crison, Barcelona, Spain).
Trace elements (aluminum, antimony, arsenic, barium, beryllium, cadmium, lead, cobalt, copper, chromium, manganese, mercury, nickel, selenium, and vanadium) were determined by electrothermal atomic absorption spectrometry (EAAS) with a Perkin Elmer Zeeman Atomic Spectrometer 4110ZL and Autosampler AS-72 from Perkin Elmer (Norwalk, CT, USA).
Sodium and potassium were analyzed by flame photometry using a 400 Flame Photometer Corning 450 (Corning Limited, Essex, UK).
Hardness, alkalinity, dissolved carbon dioxide, permanganate index, and calcium were analyzed by titrimetric methods.
All analytical methods for water analysis were standard procedures described in the Standard Methods for Examination of Water and Wastewater Analysis (SMEWWA) and in official procedures [11
2.3. Skin Biometrics Evaluation
The moisture content in the stratum corneum is important for cosmetic applications. The skin hydration was evaluated by epidermal capacitance and transepidermal water loss (TEWL) using a Corneometer CM 825 (Courage + Khazaka Electronic GmbH) and a Tewameter TM 210 (Courage + Khazaka Electronic GmbH, Germany). These are indispensable parameters for the evaluation of the water barrier function of the skin and are basic measurements in all kinds of applications.
In this study, the three cream formulations (not diluted) were tested in forearms of healthy volunteers randomly selected in an experimental protocol. A control area in the arm where no cream was applied was chosen as the control. Before use, all formulations under study were transferred to opaque white tubes with a corresponding identification code. Thus, neither the volunteers nor the responsible investigator had any knowledge of which cream was being tested.
The distribution of the cream on the forearms was defined according to a simple procedure of alternating between the right and left arm. The creams were applied to the corresponding forearm twice a day for 28 consecutive days. In the days of skin biometrics evaluation, the cream was applied after the measurement. Volunteers evenly applied about 5 mg/cm2 of cream in an area of 10 × 5 cm, simulating a normal use of the product massaging in 3 circular movements. During the study, the volunteers also kept all their hygiene habits, avoiding any changes in the type of cleaning products used.
A minimized and double-blind study was performed. This protocol was approved by the local Ethical Committee and respected the Helsinki Declaration and Good Clinical Practice studies on cosmetic products. The study was conducted under the supervision of a dermatologist who participated in the evaluation of irritation/allergic reactions to the tested formulations. All participants gave their informed consent. A minimum of 12 volunteers were defined as acceptable for this study. The inclusion criteria in this study were: age range of 18–65 years, feminine gender, skin phototype between I–IV, no skin diseases in the arms and forearms, no marks (tattoos, scars) in the arms and forearms, and arms and forearms with little hair. This study also had exclusion criteria, such as volunteers with systemic disease, pregnancy, autoimmune pathology, serious pathology in the 6 months prior to the beginning of the study (including cancer, myocardial infarction, diabetes), use of any topical medication at the site of assessment, use of any anti-inflammatory systemic medication in the period up to 1 month prior to study initiation or 2 weeks in case of antibiotics, fever for more than 24 h on the 8 days before the beginning of the study, use of drugs or alcohol, beach sunbathing during the course of the study, and participation in other cosmetic or clinical studies in the previous two weeks. On the day of the skin biometrics evaluation, the volunteers had an acclimatization period of 20–30 min in the laboratory (21 °C, 50% relative humidity), so that the skin parameters were measured under the same conditions throughout the study.