This study was approved by Ethics Committee of Ankara University Faculty of Dentistry, Turkey, in accordance with the Declaration of Helsinki (Reference number:36290600/121).
For this study, forty-eight (N = 48) freshly extracted single-rooted human mandibular premolar teeth with single canals were collected from the Clinic of the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Gazi University of Ankara, Turkey. The teeth with straight root canals of similar size were included to reduce the effects of canal size and curvature on the extrusion of the irrigant. Radiographic images from the buccal and proximal aspects for each sample were exposed. The teeth with an open apex in the radiographic images were excluded from the study.
Following extraction, the teeth were stored for two days in 3 % NaOCl at room temperature to remove organic debris. They were scaled with ultrasonics, washed with distilled water to remove any calculus or soft tissue debris, and then immersed in 10 % formalin solution until use.
The teeth were decoronated to obtain root segments of 14-mm in length. A 10-K file (Antaeos; Vereinigte Dentalwerke GmbH & Co, Munich, Germany) was placed into the canal until it was visible at the apical foramen. The working length was established as 0.5 mm short of this length.
The root canal preparation was performed using rotary instruments (ProTaper, Dentsply, Maillefer, Ballaigues, Switzerland) with a crown down technique. Apical instrumentation was completed with a F3 file (ISO size 30, taper 0.09-0.05). Between the instruments, each canal was irrigated with 2 mL of 2 % NaOCl solution using a syringe and 27-gauge needle. Apical patency was checked with a size 10-K file between each instrument. The teeth were secured with self-curing acrylic resin through the lid of an Eppendorf tube filled with 1.0 mL distilled water to collect the apically extruded irrigating solution. 27-gauge needle was inserted into the Eppendorf tube to equalize the pressure inside and outside the tube (Fig. 1)
The volume of the irrigating solution was kept constant as 4 mL for all control and experimental groups.
Non-activated syringe irrigation (Control group): 4 mL of 2 % NaOCl was injected into the root canals in 60 s with a 27-gauge open-end tip needle, which was placed 2-mm short of the working length without binding (with a constant fluid flow rate of 0.7 mL/sec).
LAI using diode laser (Pocket Diode Laser, Orotig, Verona, Italy): the diode laser (915 nm, 1.2 W, 200 μm fiber optic tip, continuous wave) was activated for a total of 21 s (3 X 7 sec).
LAI using Nd:YAG laser (Pulse Master 600 IQ, Texas, USA): Nd:YAG laser (120 mJ, 20 Hz, 2.4 W, 320 μm fiber optic tip, pulsed mode) was activated for 20 s.
PUI (Satelec, Acteon Group, Merignac, France): PUI was performed with a piezoelectrical ultrasonic unit with power setting 5. A stainless steel size 15-K file (Satalec) was inserted into the root canal and the irrigating solution was ultrasonically activated for 60 s [23].
After LAI using diode laser, LAI using Nd:YAG laser and PUI, the root canals were irrigated with 2 mL of 2 % NaOCl for 30 s with a 27-gauge needle, which was placed 2 mm short of the working length (with a constant fluid flow rate of 0.7 mL/sec).
The quantitiy of hydrochloride ion in an aqueous sample can be determined by finding out how much iodine it can produce by oxidizing an iodide ion [24]. The amount of the extruded NaOCl in the Eppendorf tubes was determined by this chemical reaction of OCl− with I− (iodide) in acidic solution.
$$ \begin{array}{l}\\ {}\begin{array}{ll}\mathbf{2}{\mathbf{e}}^{-} + \mathbf{2}{\mathbf{H}}^{+} + \mathbf{O}\mathbf{C}{\mathbf{l}}^{-}\hfill & \kern0.36em \mathbf{C}{\mathbf{l}}^{-} + {\mathbf{H}}_{\mathbf{2}}\mathbf{O}\hfill \\ {}\mathbf{2}{\mathbf{I}}^{-}\hfill & \kern0.36em {\mathbf{I}}_{\mathbf{2}} + \mathbf{2}{\mathbf{e}}^{-}\hfill \\ {}\mathbf{2}{\mathbf{H}}^{+} + \mathbf{O}\mathbf{C}{\mathbf{l}}^{-} + \mathbf{2}{\mathbf{I}}^{-}\hfill & \kern0.36em {\mathbf{I}}_{\mathbf{2}} + \mathbf{C}{\mathbf{l}}^{-} + {\mathbf{H}}_{\mathbf{2}}\mathbf{O}\hfill \end{array}\end{array} $$
Following the chemical reaction, the amount of NaOCl was determined by spectrophotometric assessment of the change in color due to the formation of iodine (I2) in an aqueous solution with a spectrophotometer (Unicam UV2-100 UV/Visible Spectrometer, Aberdeen, UK). The statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc tests (α = 0.05).