Methods

Experimental Methods

Grouping and Treatment of Lifespan Observation: Daphnia pulexs born at the same time were randomly divided into pure water group, mineral water group, tap water group, hydrogen-rich water group and filtered water group. Forty daphnia pulex were set up in each group. The culture temperature of each group was controlled to be the same, between 16 ℃ and 25 ℃. 3ml yeast liquor (30ml/L) was fed every 24 hours and count the number of survival daphnia pulex, the death and newly bred fleas were sucked out with disposable straw.

Determination of Water Quality Parameters of the Tested Water: Each drinking water collecting water samples from 10 pieces, each piece of measuring cylinder accurately measuring 100mL, with a multifunctional water quality analyzer determination, detection of indicators include: pH, total dissolved solids(TDS), oxidation - reduction potential(ORP). The determination of H₂SiO₃ was carried out by silicon-molybdenum yellow spectrophotometry. The main metal elements were determined in 10 samples of each drinking water by ICP-MS.

Statistical Analysis Method: The experimental data were expressed as x±s, and SPSS 20.0 statistical software was used for data processing and analysis. Kaplan-meier method was used to describe the distribution of survival time. If there was no crossover of survival curve, Log rank test was directly used for comparison between groups. If the survival curves were intersected, survival at each time point was compared using the test or Fisher’s exact probability method. P<0.05 was considered statistically significant.

Results

The Parameter of Five Kind of Dranking Water:As shown in Table 1, hydrogen-rich water, filtered water and mineral water are alkaline water, pure water is neutral water, tap water is weak acid; The content of H₂SiO₃ is filtered water > mineral water > tap water, and there is little H₂SiO₃ in pure water and hydrogen-rich water; The ORP of hydrogen-rich water and filtered water was negative, indicating that the tow kinds of water are reductive, and the five kinds of water showed hydrogen-rich water > filtered water > mineral water > pure water > tap water. Pure water and hydrogen-rich water contain only a very small amount of TDS. The content of TDS in tap water is close to which in filtered water ,more than 80mg/L but not more than 100mg/L. The content of TDS in mineral water is the highest, reaching more than 500mg /L.Table 2 shows that there are little contents of Se, Be, Co, Tl and Cr in five kinds of water, which can’t be detected by ICP-MS test. The distribution of other elements in five kinds of water is as follows:Ca: pure water ≈ hydrogen-rich water < detection limit < tap water ≈ mineral water < filtered water; Zn: pure water ≈ hydrogen-rich water < detection limit < mineral water < filtered water < tap water; Sr and Li are: pure water ≈ hydrogen-rich water < detection limit < tap water ≈ filtered water < mineral water; Fe: pure water ≈ hydrogen-rich water < tap water ≈ filtered water < mineral water; Mg: pure water ≈ rich hydrogen water < detection limit < mineralwater < tap water < filtered water; Cu and Mn are: mineral water ≈ pure water ≈ rich hydrogen water < detection limit < tap water < filtered water.

Table 1: Comparison of general indicators of drinking water quality (n=10, x±s).

Note: In table 1, pure water(PW), tap water(TW), mineral water(MW), hydrogen-rich water(HW) and filtered water(FW).

Table 2: Comparison of main metal elements in five kinds of drinking water (n=10, μg/L, x±s).

Note: In table 2, pure water(PW), tap water(TW), mineral water(MW), hydrogen-rich water(HW) and filtered water(FW). Undetected results are recorded as less than or equal to the detection limit.

Differences in Lifespan of Daphnia Pulex: Synthesize Figure 1, which is corresponding to daphnia pulex’s survival curve, Table 3, timetable and Table 4, Statistical results of pairwise comparison of survival between groups, we can find that the lifetimes of daphnia pulex in different kind ofwater aren’t exactly the same. It showed : pure water group ≈ tap water group < hydrogen-rich water group≈filtered water group < mineral water group (analysis has no statistically significant differences between groups expressed in “≈”). The average survival time and median survival time of the mineral water group were the longest, up to more than 35 days.

Table 3: Survival schedules of daphnia magna.

Table 4: Statistical results of pairwise comparison of survival between groups.

Note: In table 4, “0, 1, 2, 3 and 4” respectively means “pure water group, tap water group, hydrogen-rich water group, mineral water group and filtered water group”.“N” means “NO”,“Y” means “NO”.

Figure 1: Survival curve of daphnia pulex.

Discussion

Today,there are various types of water in our daily life. As the development of the study on dranking water in longevity area,we found that there are various factors that decide water quality,such as pH, ORP,TDS, content of H₂SiO₃ and content of mineral elementand so on,had become important factors affecting the quality of drinking water. Immortality has always been the goal of mankind. In ancient times, The emperor tried different pills aimed to be immortal.Today,people are still searching for the secret of longevity in order to prolong human life. In structure, caenorhabditis elegans is Simpler than daphnia pulex, but at present,it has been widely studied as a biological model in life span research, and the research results have been applied to mammals, which has great significance inextending human life span and improving human life quality[16-20]. Compared withcaenorhabditis elegans, daphnia pulex has a more complete morphological structure and lives in water. Therefore, It’ s reasonable in this study that used daphnia fleas as a model to investigate the effects of five different types of drinking water on the lifespan of daphnia pulex.The parameter analysis results of five different types of water, including: pure water, tap water, filtered water, hydrogen-rich water and mineral water, showed that mineral water, filtered water and hydrogen-rich water are weakly alkaline and have low ORP, among which, the ORP of filtered water and hydrogen-rich water are negative mean they are with reducibility. The results of fivegroupsof daphnia pulex showed that the effects of pure water and tap water on the lifetime of daphnia daphnia were similar.Compared with the pure water group and the tap water group, mineral water, filtered water and hydrogen-rich water extended the lifespan of daphnia daphnia in different degrees. It is preliminarily believed that alkaline water and low ORP water may play a important role in prolongation of life due to its antioxidant activity.Researchers from the world health organization (WHO) and other agencies have discovered that the secret to a long and healthy life lies in drinking raw water rich in mineral nutrients.

In this study, it was found that high iron and high strontium mineral water significantly extended the survival period of daphnia pulex, which reached the longest in the five experimental groups, and the difference was statistically significant. However,there had no advantage in pH,H₂SiO₃ content, ORP for mineral water, it can be seen that strontium and iron seem really play great role in the extended life. From thereportabout investigation of longevity area we can usually read the discovery of strontium in common water. But whether iron in water plays a role in extending life is unknown,which need further research. The effects of tap water and pure water on the survival of daphnia magna were similar and the differencewasnot statistically significant. However, the results of metal element analysis showedthat the zinc in tap water was significantly higher than that in the pure water group,possibly because the main nutritional role of zinc was to promote growth and development andenhanceimmunity[21].

It has little effect on life extension or the dose of 98.43μg/L is too small. It may also due to the high ORP of tap water, which has strong oxidation, can accelerate the aging, which is offset by the effect of zinc. Similarly, there was no statistically significant difference in the lifespan of daphnia pulex between the filtered water group and the hydrogen-rich water group. Both the hydrogen-rich water and the filtered water showed weak alkalinity and strong reduction. The difference was that compared with hydrogen-rich water, the filtered water obviously contained calcium, zinc, strontium and iron. According to the analysis of mineral water group, we found strontium and iron in water are likely to play a great role in the life extension. However, the lifespan of daphnia pulex in filtered water did not longer than which in hydrogen-rich water obviously. It may due to the low dose ,so,we speculate that the role of strontium and iron in prolonging life is dose dependent,which need a further study. As for calcium, it may do little to prolong life. In summary, the order of the effects of five drinking water types on the lifespan of D.pulex was as follow, pure water group ≈ tap water group < hydrogen rich water group ≈ filtered water group < mineral water group (“ ≈ “means the difference in lifespan has no statistical significance). Strontium and iron in water may play an important role in prolonging the lifespan, which is affected by dose. Whether zinc prolongs the life of daphnia pulex is unclear. Calcium may do little to prolong life.

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