Evaluation of Time-Dependent Chemical Alterations in Sodium Hypochlorite Solution Kept on the Unit Tray During an Average Root Canal Treatment Period

Introduction

Given the irregular and complex structure of the root canal system, effective irrigation using a suitable solution is necessary to remove pulp, microorganisms, and dentin residues in conjunction with mechanical instrumentation. An ideal irrigation agent should dissolve vital and necrotic pulp tissue while neutralizing microorganisms and their byproducts [1,2]. Sodium hypochlorite (NaOCl), with its exceptional tissue-dissolving properties, antimicrobial efficacy, and ability to neutralize toxic by-products, stands as the cornerstone of irrigation solutions in endodontics [3]. The effectiveness of this solution is influenced by various factors, including concentration, active chloride content, pH, and surface tension [4–6].

NaOCl demonstrates its antibacterial and tissue-dissolving activity by engaging the active chlorine in its content with bacteria, biofilm, and the organic contents of pulp tissue [7]. It has been observed that the available chlorine level in the solution can decrease when exposed to air, light, certain temperatures, and organic and inorganic pollutants [8]. Similarly, a change in pH value can lead to significant changes in the chemical properties of the solution by altering the ratio of hypochlorite (OCl−) and hypochlorous acid (HOCl−) in the NaOCl solution [9]. A decrease in pH value enhances the antibacterial activity of the solution by increasing the ratio of HOCl−, a potent antiseptic. However, it can also lead to a significant decrease in the solution’s tissue-dissolving capacity, which can impact the thorough cleaning of the root canal system, thereby emphasizing the importance of maintaining a balanced pH value of the solution [10–12]. Conversely, an increase in the pH value can have the opposite effect [13]. Therefore, it is crucial to maintain the pH level of the solution at values that can provide this balance, to ensure both the antibacterial and tissue-dissolving effects.

Another factor that influences the effectiveness of irrigation solutions is the solution’s surface tension and contact angle properties, which determine its penetration into the dentin surface and tubules [14]. Research has revealed that reducing the surface tension of NaOCl can increase its penetration into irregular areas of the root canal system and tubule depths [15]. This significant finding emphasizes the need for further research on the environmental factors that may affect the surface tension value of NaOCl. While there are numerous studies in the literature on the chemical stability and shelf life of NaOCl solutions [16–21], to the best of our knowledge, there is no study that evaluates the changes that can occur depending on the conditions under and duration for which the solution remains on the unit table. Chairside time for a typical non-surgical root canal appointment commonly falls within 60 to 120 minutes, with clinical studies reporting approximately 60 to 75 minutes for single-sitting procedures and up to approximately 112.5 minutes total treatment time in randomized trials [22–24]. Therefore, in this study, we aimed to determine changes in pH, active chlorine amount, and surface tension of NaOCl solutions stored in transparent open-lid containers over 1- and 2-hour intervals.

Material and Methods

PREPARATION OF NAOCL SAMPLES:

A commercially available 5% NaOCl solution (Mikrovem AF, Istanbul, Türkiye) for dentistry was used for this study. To minimize batch-related variability, all samples were prepared from a single bottle (same batch/lot) opened at the beginning of the experiment. Three experimental groups were established: fresh NaOCl analyzed immediately (T₀), NaOCl exposed to ambient conditions for 1 hour (T₁), and NaOCl exposed to ambient conditions for 2 hours (T₂). Each group consisted of 10 independent samples (20 mL per sample) placed in standardized, unused transparent polyethylene terephthalate (PET) cups with open lids.

Environmental conditions during exposure were those of a typical endodontic operatory. Room temperature was maintained at approximately 22 °C (air-conditioned clinical room). Light intensity (lux), relative humidity, and airflow were not instrumentally recorded. No direct sunlight was allowed on the samples.

PH MEASUREMENTS:

The pH values of NaOCl samples were measured using a digital pH meter (HANNA Instruments, Romania) (Figure 1). To ensure homogeneity, each sample was placed on a magnetic stirrer in a beaker. The pH meter was calibrated for accuracy prior to measurement. While the stirrer was operating, the pH probe was immersed in the solution, and readings were recorded after 1 minute of stabilization. All measurements were performed by a single trained operator. Each sample was measured once. This process was systematically applied to all 3 sample groups: fresh NaOCl (n=10), NaOCl stored for 1 hour (n=10), and NaOCl stored for 2 hours (n=10).

SURFACE TENSION MEASUREMENTS:

The density of NaOCl solutions was determined using a precision 10-mL pycnometer (Çalışkan LG025.31.0010, Ankara, Türkiye). After thorough cleaning with ethyl alcohol, the pycnometer was dried and weighed with its stopper on a Denver Instrument SI 234 balance. Then, the tare weight was recorded. The pycnometer was filled with freshly prepared distilled water, ensuring no air gaps, and the stopper was secured. Thereafter, the exterior was dried, and the combined mass of the filled and empty pycnometer was recorded. The difference between these 2 weightings was the mass of distilled water. Subsequently, the volume of the pycnometer was computed by dividing the mass of the water by its density, taken as 0.997 g/cm3 at approximately 25°C [25]. After rinsing and drying the pycnometer, it was filled with the NaOCl solution, and the mass was rerecorded. Density was calculated as the difference in mass between the full and empty pycnometer divided by the pycnometer’s volume. This process was replicated for all NaOCl samples, including fresh samples (n=10) and those kept open for 1 hour (n=10) and 2 hours (n=10).

For measuring the surface tension of NaOCl samples, a 3.5-mL Traube stalagmometer (Çalışkan, LG028.20.00, Ankara, Türkiye) and the drop counting method were used. All stalagmometric measurements were performed by a single calibrated operator to minimize operator-dependent variability. Measurements were conducted at room temperature (≈22 °C). Each sample was measured once. The surface tension was calculated based on the number of drops falling from the stalagmometer, the density of the NaOCl samples measured with a pycnometer, and the surface tension of the reference liquid.

In addition, freshly prepared distilled water was used as the reference liquid for this study. Before beginning the experiment with the prepared NaOCl solutions, the number of drops formed by the distilled water with known surface tension in the determined volume V was counted and noted (Figure 2). The drop-counting process began when the liquid level reached point A. The last drop was taken at point B to determine the number of drops counted in the total volume, V. First, the number of drops of water was determined; thereafter, the number of drops of NaOCl solutions in fresh, 1-hour, and 2-hour samples were determined and noted, respectively.

The surface tension of water was taken as approximately σW: 72 dyn/cm at around 25°C [26]. Since the surface tension is directly proportional to the drop mass and inversely proportional to the number of drops, the surface tension of the NaOCl solution in the samples was calculated using the following formula:

where σ_L=surface tension of the liquid, σ_W=surface tension of water, ρ_L=density of the liquid, ρ_W=density of water, N_w=number of drops in water, and N_L=number of drops in liquid.

ACTIVE CHLORINE MEASUREMENTS:

Purified water (1 L) was boiled for 5 minutes and then allowed to cool. Thereafter, 12 g of sodium thiosulfate pentahydrate (Na₂S₂O₃·5H₂O; molar mass: 248 g/mol) was dissolved in the water, followed by the addition of 0.01 g of sodium carbonate (Na₂CO₃). The mixture was stirred until fully dissolved and then transferred to an opaque bottle for storage.

Thereafter, a 2% starch solution was prepared. Approximately 0.32 g of dried potassium iodate (KIO3) was dissolved in a 250 mL graduated flask. To 50 mL of this solution, 2 g of potassium iodide (KI) and 10 mL of hydrochloric acid (HCl) were added. This mixture was titrated with the standardized sodium thiosulfate (Na2S2O3) solution until a light-yellow endpoint was reached. Following this, 3 mL to 5 mL of starch solution was added, and titration was continued until the blue color disappeared (Figure 3). The concentration of the Na2S2O3 solution was calculated using the following relevant reactions and equations:

At the equivalence point, 6*moles of iodate=mole Na₂S₂O₃.

A graduated burette was filled with Na₂S₂O₃ solution, and the initial volume was recorded. Approximately 10 mL of NaOCl was measured into a tared 50 mL beaker, weighed to the nearest 0.1 mg, and then transferred to a 250-mL conical flask. After adding 5 mL of glacial acetic acid, approximately 2 g of KI was introduced and mixed. The solution was allowed to stand for approximately 5 minutes in the dark to ensure reaction completion. Once a brown color developed, titration with the Na₂S₂O₃ solution was conducted using a magnetic stirrer until the color transitioned to light yellow. Upon adding 3 mL of starch solution to this solution, a blue-colored complex was formed. This solution was then titrated with sodium thiosulfate (Na₂S₂O₃) until it became colorless, thus marking the endpoint. The final volume of thiosulfate utilized was recorded, and the active chlorine (AC) percentage was calculated using the specified formula [AC% = (V1 × M1 × 3.546*)/m, where V1: volume of standardized sodium thiosulphate solution; M1: molar concentration of standardized sodium thiosulphate solution; and m: weight of the sample analyzed (*1 mL of 0.1 mol/L sodium thiosulfate pentahydrate equals 3.546 mg active chlorine)]. The data obtained were documented for statistical analysis. All titrations were performed by a single trained operator. Evaporation during the 1- to 2-hour exposure period was not directly quantified.

STATISTICAL ANALYSIS:

Descriptive statistics for continuous variables, which are among the characteristics under consideration, were expressed as median, mean, standard deviation (SD), minimum (min), and maximum (max) values. For continuous variables, the Kolmogorov-Smirnov test was used to assess normality, and the Levene test was used to assess homogeneity of variances. Following these tests, a one-way analysis of variance (ANOVA) was performed to compare group means for normally distributed characteristics. Duncan’s multiple-comparison test was used to identify differences among groups after the ANOVA. The statistical significance level was set at 5%, and the SPSS (version 21) statistical package program was used for the calculations.

Results

The mean, SD, min, max, and P values of the pH levels of fresh, 1-hour, and 2-hour NaOCl samples are presented in Table 1. A statistically significant difference was determined between the pH values of the groups (P<0.05). The pH value of the T1 samples was significantly lower than that of the T0 samples (P<0.05), while the pH value of the T2 samples was significantly lower than that of the T0 and T1 samples (P<0.05).

Table 2 presents the mean, SD, min, max, and P values of the surface tension values determined by the Traube stalagmometer for the 3 NaOCl groups. No statistically significant difference was observed among the surface tensions of the T0, T1 and T2 solution groups (P>0.05).

The mean, SD, min, max, and P values of the active chlorine percentages of NaOCl samples determined by iodometric titration are presented in Table 3. The mean active chlorine percentages of the NaOCl solution groups revealed a statistically significant difference. According to the paired comparisons, no statistically significant difference was found between the active chlorine contents of the T0 and the T1 samples (P>0.05). In contrast, the active chlorine percentage in the T2 group was significantly higher than that in the T0 and T1 groups (P<0.05).

Discussion

LIMITATIONS:

This in vitro study evaluated physicochemical parameters (pH, active chlorine, and surface tension) and did not directly assess antimicrobial efficacy or pulp tissue dissolution under clinical conditions. Organic load (eg, dentin or pulp tissue), canal anatomy, irrigation dynamics, and clinically relevant interactions with other irrigants were not simulated. Environmental parameters that can affect evaporation and degradation (light intensity, relative humidity, and airflow) were not instrumentally quantified. In addition, evaporation (mass/volume loss) during the exposure of 1 and 2 hours was not measured, so the mechanism underlying the observed change in active chlorine percentage cannot be confirmed. Moreover, stalagmometry and iodometric titration are manual, operator-dependent methods; therefore, although measurements were performed by a single trained operator using a standardized protocol, some degree of operator-related variability cannot be excluded. Finally, measurements were performed without technical replication per sample and only 1 commercial NaOCl product was tested, which may limit generalizability. The relatively small sample size (n=10 per group) may have limited statistical power to detect small between-group differences.

Conclusions

Within the limitations of this in vitro study, keeping NaOCl in transparent open-lid containers for 1 to 2 hours was associated with a decrease in pH and a statistically significant increase in the measured active chlorine percentage at 2 hours (compared with fresh and 1-hour samples), while no statistically significant change was observed in surface tension. Because the present study did not compare container opacity or different dispensing bottles, conclusions should be limited to the tested condition (transparent open-lid cups). From a practical standpoint, clinicians may consider minimizing unnecessary open-tray exposure time and using dispensing practices that reduce air and light contact (eg, smaller aliquots and light-protective storage) to help limit time-dependent physicochemical changes. Future ex vivo and in vivo studies incorporating organic load and direct testing of antimicrobial and tissue-dissolving efficacy are needed to clarify the clinical relevance of these changes.