*2.1. Quality of HRW*

The quality of drinking water is an important information for health and safety. The quality parameters of HRW, including dissolved H2 concentration, oxidation-reduction potential (ORP) value, and physical and chemical properties of the water samples, are shown in Table 1. The fresh HRW contained 1550 ppb H2 and had a negative ORP value (control water: +293 mv; HRW: −453 mv). HRW also had a higher pH than the control water. Ca2<sup>+</sup> concentrations were lower and Mg2<sup>+</sup> concentrations were higher in the HRW. Other parameters of water quality, including TDS, salt, electrolytic conductivity (EC), dissolved oxygen (DO), Na+, K+, Cl−, and SO42<sup>−</sup> levels, showed little or no difference between the control water and HRW.

The stability of dissolved H2 in HRW is shown in Figure 1.AH2 concentration of around 1500 ppb was maintained in water for 1 h, and the concentration remained around 1300 ppb for 2 h. After that, the H2 concentration in water gradually decreased over time. The remaining H2 concentration in HRW was around 200 ppb after the sample was set at 4 ◦C or 25 ◦C for 24 h. The ORP value in HRW increased gradually over time and, after 24 h, was around −100 mv. No significant differences in dissolved H2 or ORP values were observed in the HRW samples either at 4 ◦C or at 25 ◦C within 24 h.

It was noteworthy that water cluster size, determined by 17O NMR line-width, was comparable between the control water (55.8 Hz) and the HRW (54.9 Hz) (Figure 2). In a previous study, the water cluster size of tap water was shown to be greater than 110 Hz [14]. These results suggest that, after tap water is filtered through the first two filters, the water cluster size can be lowered by shear force. The final magnetized rod device may have strengthened the hydrogen bond network of the water and, thus, stabilized the water cluster [15].


**Table 1.** Quality of control water and hydrogen-rich water (HRW) a.

<sup>a</sup> All data were measured at 25 ◦C. The quality of tap water was as follows: H2, 0 ppb; ORP: 203.7 mv; TDS: 204.7 g/L; salt: 155 kU.m; EC: 310.7 ds/m; DO: 8.0 mg/L; pH: 8.0; Na+: 4.8 ppm; K+: 1 ppm; Ca2+: 51 ppm; Mg2+: 10 ppm; Cl−: 3.2 ppm; and SO4 <sup>2</sup>−: 42.0 ppm. Except for water cluster, all of the above parameters were expressed as the mean value of three determinations. In the previous study, the water cluster of tap water determined by the 17O NMR line-width method was 113 Hz [14]. DO, dissolved oxygen; EC, electrolytic conductivity; ORP, oxidation-reduction potential; TDS, total dissolved solids.

**Figure 1.** (**A**) Stability of dissolved H2 and (**B**) the ORP of hydrogen-rich water at 4 ◦C and 25 ◦C. ORP, oxidation-reduction potential.

**Figure 2.** 17O Nuclear magnetic resonance line-width of water samples: (**A**) Control water and (**B**) hydrogen-rich water.

#### *2.2. Body Weight, Tissue Weight, Water Drinking Volume, and Plasma Biochemical Parameters*

In this study, no significant effects were found on body weight, liver weight, kidney weight, or food intake in rats that ingested the control water or HRW for four weeks. The volume of drinking water ingested was mildly higher (+10.7%, *p* < 0.05) in the HRW group (81.8 ± 5.1 mL/day) than in the control group (73.9 ± 5.0 mL/day). The plasma biochemical parameters are shown in Table 2. Plasma glucose was mildly lower (−7.7%) in the HRW group than in the control group (*p* < 0.05). There were no significant differences in other plasma parameters including ions between the control and HRW groups.


**Table 2.** Effects of intake of HRW on plasma biochemical parameters in rats a.

<sup>a</sup> Results are expressed as the mean ± S.D. of eight rats in each group. ALT, alanine aminotransferase; BUN, blood urea nitrogen; GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances. \* Significantly different from the control group, *p* < 0.05.
