By Published On: June 3, 2024Categories: May - August 2023, Original Research

Menon Chandana, Mathew Josey, George Liza, Paul Sinju, Yacob Jeason Paul, Rajan Rishi

Department of Conservative Dentistry and Endodontics, Annoor Dental College and Hospital, Muvattupuzha

Running title – Antibacterial efficacy of Hydrogen water

Received: 09-05–2023

Revised: 15-05-2023

Accepted: 17-05-2023

Address for correspondence: Dr Chandana Menon, Parayil House, Sree Lakshmi

Elamakkara PO, Kochi-682026, Kerala

Email :


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How to cite this article: Menon C, Mathew J, George L, Paul S, Yacob J P, Rajan R. Comparative evaluation of Antibacterial efficacy of a novel root canal irrigant-Hydrogen water with conventional irrigants Sodium hypochlorite and Chlorhexidine gluconate : An In vitro Study. J Oral Biomed Sci 2023; 2(2):65-70


Background: Endodontic infections have complex microbial flora including E-Faecalis .Root canal treatment attempts to eliminate microbial flora from the root canals. Improved irrigants are needed for this. Hence, researchers are trying to develop newer irrigants with better properties. This study attempts to examine the antibacterial efficiency of a novel irrigant, hydrogen water (HW), toward E. faecalis while comparing it with the conventional irrigants, notably sodium hypochlorite (NaOCl) and chlorhexidine (CHX).

Materials and Methods: Top surface of Petri dishes containing Blood Agar is flooded with 100μl inoculum suspension of E. faecalis and desiccated for 15 minutes. Three equidistant wells punched, 50μl of irrigants were put into the wells followed by incubation at 37ºC for 14 days. Growth inhibition zones were measured at the end of day 2 and day 14. Paired t-test and One-way ANOVA was applied.

Results: The highest values of zones of inhibition were obtained for sodium hypochlorite followed by chlorhexidine gluconate and least by hydrogen water both on day 2 and day 14 with statistically significant difference. On comparison of zones of inhibition between between day 2 and day 14 of treatment within the group also showed a statistically significant difference.

Conclusion: Though hydrogen water has lesser antimicrobial potential in comparison with NaOCl and CHX, considering its advantages like biocompatibility. So HW may be considered as an alternative biocompatible irrigant in endodontics. Irrigant activation procedures may be employed to augment its antibacterial efficacy.

Keywords: Hydrogen water, E faecalis, Antibacterial effect


The purpose of endodontic treatment is to exterminate all the live and necrosed tissues, bacteria, and microbial metabolites from the system of root canals.1Irrigants used in combination with mechanical instrumentation helps in dissolving tissue, abolition of debris, and disinfectingng the root canal system. Sodium hypochlorite has organic tissue dissolving capacity and vast spectrum of antibacterial qualities and hence it is regarded as the gold standard irrigant. Often chelation solutions are indicated as supplemental irrigant for smear layer elimination or to limit its development on the surface of dentin.1

Endodontic infections are mainly caused by Enterococcus faecalis, one of the most resistant species which is an anaerobic gram-positive cocci that is typically a commensal in the oral cavity and has exhibited outstanding adaptation to such conditions with low nutrition along with low oxygen levels and complicated ecosystem. In situations of primary endodontic infection, E. faecalis is more probable to be related to cases that are asymptomatic rather than symptomatic ones.2

Potent antibacterial action with minimum friction during mechanical instrumentation along with accessibility and complete pulp dissolution are among the key attribute of a successful root canal irrigant. Sodium hypochlorite has largely been identified as an endodontic irrigant since its application was originally described in 1936 by Walker. It chiefly operates as an efficient antibacterial agent by excellent dissolution of living, necrotic, and fixed tissue. In spite of its appealing features, including ease of access and low cost, it has a few disadvantages, like a bitter taste, a tendency to bleach clothes and being possibly acidic. There is also a concern with regard to its destructive effects if extreme concentration of solutions are accidentally forced into the periapical tissues. Some arguments do exist pertaining to its antibacterial efficacy at lesser dosages, which are touted as an effort to decrease its detrimental impact.2

Chlorhexidine gluconate (CHX) has also been used for the disinfection of root canals. It has been proven that CHX exhibits powerful and substantial antibacterial action against the resistant Enterococcus faecalis. Although CHX is an admissible bacteriocidal agent, it is unable to dissolve the residual pulp tissues after the preparation of root canal. Since neither of the presently known irrigants fulfil all the parameters of an ideal irrigant, efforts are on to find an alternative biocompatible irrigant.3

Natural substances like hydrogen water have favorable antibacterial effects on oral microorganisms. It has exhibited antibacterial effectiveness against standard isolates of

  1. nucleatum, Actinobacillus actinomycetemcomitans,P.intermedia,P. gingivalis and the Treponema denticola species. Antibacterial efficiency of hydrogen water as root canal irrigant has never been examined in endodontics.4


Production of hydrogen water

Hydrogen water bottle was commercially procured (GOMAX-H7, Yongkang Industry, China).

It consisted of electrodes at the base that aided in the synthesis of hydrogen water, and distilled water was put into the hydrogen water bottle for it to undergo the process of electrolysis. Electrolysis started in 3 seconds after the electrical supply of the bottle was turned on. This decomposition reaction resulted in liberation of hydrogen (H2) at the cathode. Consequently after 5 min, hydrogen water was freshly generated. 4


In this investigation sodium hypochlorite, chlorhexidine and hydrogen water were taken for comparison of antibacterial effectiveness against E. faecalis. Strains were cultured from frozen stock cultures under aerobic conditions at 37°C for 24 hours of incubation in brain-heart infusion broth and assayed.Turbidity changes were noted which was indicative of microbial growth.Sterile saline was added to this for dilution so as to obtain a suspension of 0.5 turbidity on the McFarland scale.

The E. facecalis strain was treated to these irrigants for two lengths of time: 2 days and 2 weeks. The antibacterial effect of the irrigants was investigated by agar-well diffusion test.

Agar-well diffusion experiments were done on  Petri dishes of 90-mm-diameter with nutrient agar at 4mm depth. 100μl inoculum solution of the strain was poured onto the top surface of agar plate and then dried for 15 minutes at a temperature of 37°C . Three equidistant wells with a diameter of 5mm and depth of 4mm were drilled in the agar plate using a Pasteur pipette following which 50μl of each irrigant were inserted into the plate’s well. The agar media was cultured for 2 weeks aerobically at 37°C. The complete experiment was replicated to reach a sample size of 42.

The sizes of growth inhibition zones were evaluated on Day 2 and Day 14 for comparison. Growth inhibition zones surrounding each irrigant were evident by the absence of bacterial colonies seen as clearing of agar in proximity to each agar well, and they were measured using a millimeter ruler. All of the values exceeding 6 mm were assessed as demonstrating a notable reduction in the growth of bacteria. The inhibitory zones with higher width were indicative of increased antibacterial activity of the irrigants.

Statistical procedure

The acquired data was analyzed using statistical software, SPSS (version Comparisons between each group were done using one-way ANOVA and paired t-tests.


The zones of inhibition exhibited by each irrigants on the agar plate against E faecalis was recorded. On comparing the antibacterial efficacy of irrigants on Day 2 and Day 14, the mean values were higher on Day 14 than Day 2 for all the 3 irrigants (Table 1, Graph 1)

The mean values of the zones of inhibition were higher during 14 days for all the three root canal irrigants – Sodium Hypochlorite, Chlorhexidine and Hydrogen water when compared to day 2. This difference between day 14 and day 2 was statistically significant for all the irrigants. (p<0.001) (Table 2The highest mean value of the zone of inhibition was exhibited by sodium hypochlorite on both the days.

Comparison between the groups showed that there is significant difference in mean values of zones of inhibition with day 2 and day 14 among the groups. (p<0.001) (Table 3)

The study showed a higher mean values of zones of inhibition for sodium hypochlorite indicating the highest antibacterial efficacy of sodium hypochlorite on both Day 2 and Day 14 in comparison to other irrigants.

Chlorhexidine follows sodium hypochlorite in its antibacterial efficacy and hydrogen water exhibited the least antibacterial activity against E. faecalis.


Removal of damaged tissue, elimination of microorganism from the root canal system, and prevention of recontamination is considered as a crucial feature of endodontic therapy. It is necessary for the clinicians to be highly educated and have skill in this key component of endodontics. Chemomechanical preparation and profuse irrigation is vital to minimize the number of microbial flora inside the root canal to prevent periapical disease. It is also required for endodontic disinfection to be straightforward and cost-effective.

The result of this study shows the highest antimicrobial efficacy for sodium hypochlorite in comparison to other irrigants, This is in conformity with a research done by Arias et al., who demonstrated greatest antibacterial activity for 2.5% NaOCl against E. facecalis when used alone as well as in combination with HEBP.13 The antibacterial action is connected to better pulpal dissolution since sodium hypochlorite is highly basic in nature with pH of about 11 and it functions as a solvent for organic tissue, encouraging amino acid breakdown and hydrolysis via the formation of chloramine molecules.14 Though the antibacterial and tissue disintegration activity of hypochlorite grows with its concentration, there is a possibility of apical extrusion and toxicity.15

The antibacterial efficacy of sodium hypochlorite was followed by CHX in this research. 2% is recommended for mechanical irrigation of root canal. Previous studies reveal that it is strongly antibacterial, notably at pH 5.5–7.0. Chlorhexidine killed bacteria by a procedure in which the cell wall of bacteria was destroyed, resulting in a substantial drop in the colony viability of E. faecalis. Earlier research has proved that effective elimination of E. faecalis occurred from the outermost portions of the dentinal tubules when 2%CHX was used which is in accordance with our data. 8 Evans et al. established in their research that coupling of 1–2% CHX with calcium hydroxide resulted in effective eradication of E.faecalis. (R)

The key benefits of chlorhexidine are its decreased cytotoxicity and the absence of an unpleasant smell and unacceptable taste. Despite the features of chlorhexidine as an irrigating solution, it cannot be employed as a gold standard endodontic irrigant due to its failure to dissolve necrotic tissue. 15

It is expected that this research may uncover potential natural compounds with antibacterial effects in the field of endodontics. Since hydrogen water is one such natural molecule with antibacterial potential, it was chosen for this study. The findings of this investigation indicated lower antibacterial effectiveness for hydrogen water in contrast to sodium hypochlorite and chlorhexidine. This conclusion was contradictory to the study done by Aarati et al., who observed that hydrogen water demonstrated antibacterial action against clinical plaque specimens from individuals with chronic periodontitis. 16

The antibacterial capacity of the hydrogen water employed in this experiment is dependent on the products created during electrolysis. These products increased the bacterial cell membrane’s permeability thereby causing leakage of cellular components and annihilation of the bacteria. This is in keeping with work done by Zeng et al, who suggested the antibacterial mechanism of electrolyzed water processed against Staphylococcus aureus and Escherichia coli. (R)

The poor efficiency of hydrogen water against E. faecalis in our experiment can be attributed to its high pathogenicity and tough cell wall, and its capability to endure intra-canal treatments without the synergistic effects of other microorganisms. They have particular qualities that allow them to avoid instrumentation during endodontic treatment. These traits include the capacity to generate biofilms and emerge in complex locations distinct from the main canals, such as auxiliary canals, isthmuses and apical deltas. In addition, E. faecalis adopts several tactics for survival in severe settings. Those options include activating certain surviving genes, adopting different metabolic pathways, residing in a place with huge quantities of nutrients, and displaying synergism and bacterial aggregation skills.6


Endodontic lesions do not develop in the absence of bacteria. So it is imperative to debride the root canal system effectively to eliminate the microorganisms. In our study,though hydrogen water exhibited least antimicrobial potential in comparison to sodium hypochlorite and CHX, it should be considered for use due to its advantages like biocompatibility,and absence of bad taste and odour. Furthermore, it is considered to be safe if accidental apical extrusion occurs.  The antibacterial potency of hydrogen water can be augmented through various irrigant activation systems. Further investigations regarding this are required to obtain a conclusive evidence.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Source of support: Nil


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Hydrogen water

Fig 1 – Zone of inhibition of the irrigants on agar pate


Group Mean N Std. Deviation Std. Error Mean
Sodium Hypochlorite Day 2 4.962 42 0.091 0.014
Day 14 5.133 42 0.103 0.016
Chlorhexidine Day 2 3.640 42 0.077 0.012
Day 14 3.871 42 0.074 0.011
Hydrogen water Day 2 1.464 42 0.174 0.027
Day 14 3.740 42 0.066 0.010


Table 1: Mean values of irrigants on Day2 and Day 14

Group  Day Mean Std. Deviation Std. Error Mean t df Sig. (2-tailed)
Sodium Hypochlorite 2-14 -0.171 0.152 0.023 -7.315 41 0.000
Chlorhexidine 2-14 -0.231 0.109 0.017 -13.695 41 0.000
Hydrogen water 2-14 -2.276 0.186 0.029 -79.371 41 0.000


Table 2: Mean difference of irrigants on Day 2 and Day 14

Sum of Squares df Mean Square F Sig.
Day 2 Between Groups 262.014 2 131.007 8870.030 0.000
Within Groups 1.817 123 0.015
Total 263.831 125
Day 14 Between Groups 49.694 2 24.847 3637.312 0.000
Within Groups 0.840 123 0.007
Total 50.535 125


Table 3 : Intragroup mean values of irrigants


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