## 2 Materials and Methods
### Final Irrigation Technique  
According to the final irrigation protocol, the teeth were assigned to 5 groups on a random basis, as follows: conventional syringe irrigation (CSI), EA, PUI, ErCr:YSGG laser, and SWEEPS.  
In the CSI group, an irrigation needle (30-gauge) (Ultradent, South Jordan, UT, USA) was used to administer 5 mL of 2.5% NaOCl and 5 mL of 17% ethylene diamine tetra acetic acid (EDTA) into the canal for 30 s each. The irrigation needle was positioned 1 mm above the established WL and moved in a 1-2 mm motion in an in-and-out direction. Distilled water was used between these irrigation solutions and as a final rinse.  
In the second experimental group, an EA device (Dentsply Tulsa Dental Specialities, Tulsa, OK, USA) was employed to activate the NaOCl and EDTA irrigation solutions. Each irrigation solution, totaling 5 mL, was introduced into the canal and then activated using the EA handpiece set at 10,000 cycles/min, utilizing a medium polymer tip (size 25, taper 0.04). The tip was positioned 2 mm away from the WL and moved vertically in a 2-3 mm range from the root apex towards the crown for a duration of 20 s. This entire procedure was repeated three times for each irrigant. Then, distilled water was used between both solutions and as a final rinse.  
In the PUI group, the same irrigation solutions and volume that were used in the previous group were activated by PUI (Endo Soft Instruments (ESI); EMS, Le Sentier, Switzerland). This involved the insertion of a stainless-steel tip no. 20, 0.02 taper (IrriSafe tip, Satelec Acteon Group, Merignac, France), into the canal by 2 mm such that it was shorter than WL, which was followed by the activation of the PUI device at a frequency of 40 kHz for 20 s without touching the canal walls. Gentle irrigation was continued throughout the activation procedure. The process was conducted thrice for each irrigant. Distilled water was used to wash the canal between and after the PUI activation.  
In samples taken from the fourth group, the application involved the use of an Er,Cr:YSGG laser (Waterlase MD; Biolase, Irvine, CA, USA) emitting light at a 2780 nm wavelength. To activate the previously mentioned irrigation solutions, an RFT2 fiber tip (diameter of 275 lm and length of 21 mm; Endolase; Biolase Technology, San Clemente, CA, USA) was utilized. The setting used for the laser device was the hard tissue mode, and the parameters utilized were as follows: pulse duration of 140 \(\upmu\)s duration, 1 W of output power, 10 Hz repetition rate of 20 Hz, and 100 mJ of energy per pulse. As the process of activation occurred, the gold handpiece's coaxial water spray (Biolase Technology, Irvine, CA, USA) was deactivated. The RFT2 tip was only inserted into the coronal reservoir, and activation of the Er,Cr:YSGG laser lasted for 30 s, with a subsequent period of 30 s of rest, followed by 30 s of activation to end the procedure. Gentle irrigation was continued throughout the activation procedure. However, washing of the canal between and after the laser was performed using distilled water.  
In the final group, both irrigation solutions underwent activation using an Er:YAG laser employing a flat-end fiber tip known as SWEEPS (SWEEPS 600; Fotona, Ljubljana, Slovenia). The tip was positioned in the coronal reservoir of the canals, and the Er:YAG laser settings were configured at 0.3 W, 20 mJ/pulse, 15 Hz, and 50 us pulse duration. The air-and-water system was deactivated during this process. The activation duration was 30 s for each solution, and gentle irrigation was continued throughout the activation procedure. Finally, distilled water was used between these irrigation solutions and as a final rinse.