Group I: the root canal was irrigated with 1 ml of 17% EDTA (Microvem, Istanbul, Turkey), and the solution was kept in the canal for 1 minute. The canal was subsequently irrigated with 2 ml of distilled water and 3 ml of 2.5% NaOCl for 1 minute.

Group II: HEDP (DualRinse HEDP, Medcem, Vienna, Austria), containing 0.9 mg of etidronic acid per capsule, was added to 10 ml of 2.5% NaOCl in a sterile container according to the manufacturer's instructions. The solution, which was prepared just before use in the canal, was mixed for 1 min to ensure that the powder was fully dissolved. The canal was then irrigated with 4 ml of the 2.5% NaOCl - 9% HEDP combined solution for 2 minutes.

Group III: A similar irrigation protocol to that of Group I was followed, with an additional 1-minute sonic activation using the EA device on 3 ml of 2.5% NaOCl. The tip of the EA was placed 2 mm away from the WL and the irrigant was activated with 10,000 cycles per minute with a tip size 0.04/35.

Group IV: Similar to Group II, the canal was filled with 4 ml of the 2.5% NaOCl - 9% HEDP combined solution for 1 minute. The tip of the EA was placed 2 mm away from the WL, followed by 1 minute of sonic activation with the EA device (10,000 cycles per minute with a tip size 0.04/35).

After cavity preparation with sterile burs, rubber dam components were applied to the tooth. The root canal filling material was removed via retreatment files. After the working length was determined, in all the groups, the canals were filled with distilled water. S1were taken from the canal after retreatment. Irrigation protocols were applied for 4 groups. S2 were taken from the canal. Intracanal medicament was placed in the root canal, the cavity was securely sealed. After 14 days,the medicament was removed with 10 mL of 17% EDTA and subsequently irrigated with 5 ml of distilled water, S3 was taken by sterile paper points. The final irrigation was performed according to the group to which the tooth belonged, and S4 was collected as before. the root canals were dried with sterile paper points and filled gutta-percha master cones and sealer.

In all the groups, A sterile paper point of size R50 was placed at the working length (WL) and left in the canal for 1 minute. Three paper points were then placed into an Eppendorf tube containing phosphate-buffered saline (PBS). The samples taken from the canal after retreatment were recorded as the S1. After root canal preparation and irrigation protocols, S2 sample was taken. After dried protocol, medicament was placed in the root canal.After 14 days,the intracanal medicament was removed with 17% EDTA. The root canal was irrigated with 5 ml of distilled water.S3 was taken by sterile R50 paper points. The final irrigation was performed according to the group to which the tooth belonged,the S4 was collected as before.Each sample obtained from the patient was placed into culture medium.The inoculated plates were incubated at 37°C for 48 hours. After incubation, the total bacterial counts were determined by calculating the number of colony-forming units (CFU/ml) on the plates.