Examination of molecular hydrogen (H${}_2$) treatment and electromagnetic field (EMF) radiation effects on the growth of animal (zebrafish embryos) and plant (Chlorella vulgaris)

In this study, the beneficial effects of molecular hydrogen (H${}_2$) and electromagnetic field (EMF) stimulation have been analysed and demonstrated by observing the influence on the regular tissue development of intact-fin and tissue regeneration of amputated-fin of zebrafish larvae using neutrophil expressed transgenic line Tg(lyzC:DsRed). Zebrafish larva is a perfect model to study tissue regeneration since the caudal-fin fold is easily accessible and its ability to rapidly regenerate the fin via wound healing responses without causing a detrimental effect on the organism. Furthermore, the tissue repair responses by neutrophils in zebrafish larva can be monitored via in vivo imaging. The beneficial effect of molecular H${}_2$ is also investigated on Chlorella vulgaris. C. vulgaris species is a fast-differentiating, photosynthetic, unicellular green microalga. The beneficial effect on C. vulgaris cell is determined based the cell density and bioactive compounds quantification. The pre-treatment of medium with pure H${}_2$ generated by PEM water electrolysis system saturates ~1.0 mM (1.6 - 2.0 mg/L) of H${}_2$ enriched medium. The H${}_2$ treated larvae have 29.0% and 43.0% of higher tissue growth of intact-fin meanwhile 21.0% and 38.0% of higher tissue regeneration of amputated-fin compared to air and N${}_2$ treated larvae, respectively, on average. The neutrophil count at the fin-amputated site of zebrafish larvae has shown that molecular H${}_2$ treated larvae have significantly lower neutrophil count at 3 and 48 hpa compared to the control (N${}_2$-treated). In this study, further confirmed that H${}_2$ generated together with O${}_2$ by PEM water electrolysis system saturates the medium by 0.4 mM (0.8 mg/L). The H${}_2$-O${}_2$ gas mixture treated larvae showed significantly higher intact-fin development and amputated-fin regeneration compared to the control (O${}_2$-levelled). Meanwhile, Brown's gas generated by an alkaline water electrolysis system with intercalated electrodes which is claimed to have "magical" components and "special" beneficial health effects by some researchers, has a similar H${}_2$ and O${}_2$ proportion as H${}_2$-O${}_2$ gas mixture. The Brown's gas treated larvae also showed significantly higher intact-fin development and amputated-fin regeneration compared to the control (O${}_2$-levelled). However, the beneficial health response stimulated by the Brown's gas is most probably due to the presence of 0.4 mM of dissolved H${}_2$ in the Brown's gas enriched medium. In other respect, the EMF radiation was expected to dissociate water into H${}_2$ and O${}_2$ but this is not able to be determined. Nevertheless, Tesla oscillator with 144 MHz EMF indeed regulated the growth response negatively, in zebrafish larvae. The Tesla oscillator causes tissue growth retardation in both intact-fin and amputated-fin of zebrafish larvae. The Tesla oscillator radiation exposed intact-fin and amputated-fin grow 33.2% and 12.7% slower on average compared to the untreated larvae, respectively. The Tesla oscillator treatment also induced a constantly higher number of neutrophils on 3, 24 and 48 hpa larvae compared to the controls on average. Meanwhile, WLAN radiation does not show any impact on larvae. On the other hand, the molecular H${}_2$ as well as H${}_2$-O${}_2$ gas mixture and Brown's gas, do not bring any benefits to the C. vulgaris cell growth density or to the quantification of cell resources such as chlorophyll, protein and starch. While the EMF treatment for 28 days on C. vulgaris cell culture showed an obvious negative result. Even though the cell density and chlorophyll content were not affected but the quantification of protein is reduced by 21.6% and 17.1% for Tesla oscillator and WLAN radiation exposed culture compared to the untreated C. vulgaris cell culture, respectively. Meanwhile, the quantification of starch of Tesla oscillator and WLAN radiation exposed culture were 38.0% and 33.37% lower than untreated C. vulgaris culture, respectively.