## 4 Discussion
Theoretically, a higher frequency results in an increased flow velocity, which allows the irrigant to reach otherwise inaccessible regions inside the complex root canal system and increases the shear stress that disrupts debris [33; 34]. However, it is worth noting that potent ultrasonic irrigation may also entail some limitations. It has been shown that even in noncomplicated root canal geometries, ultrasonic instruments, in the mean, come in contact with the wall during 20% of the activation time [35]. File-to-wall contact dampens the energy and constrains the file movement, which may lead to the accidental removal of small amounts of dentin, changing root canal morphology, even with a noncutting design [36]. Instead, sonic activation with soft polymer tips minimizes the risk of unintentional dentin removal and root canal alterations [37]. The resulting surfacealterations may also hamper the proper adhesion of filling materials and offer favorable niches for bacterial adhesion and proliferation. Microbial biofilms play a central role in the development of pulpal and periapical diseases and, accordingly, the reduction of the microbial load in the root canal is a major clinical aim and a relevant parameter to be evaluated in experimental studies of endodontic irrigation [38; 39]. In this context, _E. faecalis_ is the primary pathogen isolated, in part due to its ability to bind to dentin and invade dentinal tubes, where it can survive for long periods [19]. Perseverance of microorganisms within the root canal system is the major cause of post-treatment failure [40]. In the present investigation, both sonic and ultrasonic activation of the irrigant presented a significantly higher capability in the elimination of a mature biofilm of _E. faecalis_ than manual irrigation, regardless of the apical size, which is in accordance with published data [10]. These differences may be explained by the fact that conventional irrigation provides far lower fluid dynamics compared to the investigated activation techniques [41]. For an apical size of \(35/.06\), antibacterial efficacy was similar for both sonic and ultrasonic activation procedures, which is in accordance with other studies [14]. However, for an apical size of \(50/.06\), ultrasonic activation exhibited significantly improved results, compared to sonic activation. The enhanced effectiveness of ultrasonic activation has been associated with its acoustic streaming and cavitation effects that increase shear stress and, consequently, enhances the disruption of the biofilm [14]. Overall, an improved performance in eliminating a 21-day biofilm was attained for the larger apical size for all the assayed procedures, in accordance with other studies that advocate the enlargement of the apical sizes for a more significant reduction in the root canal biomass and increased effectiveness of the irrigation procedure [12; 42]. The experimental models used in this study have some limitations. The use of straight, single-rooted teeth precludes possible variations found in clinical scenarios, such as WL loss or nonstandard preparation and irrigation in curved root canals. However, this experimental system provided a standardized and reproducible setup to determine the potential relationship between root canal preparation size and irrigating procedure, through an integrative analysis of clinically relevant parameters. The next step will involve investigating the replicability of the results of the present study in multi-rooted teeth, with curved roots and/or more complex root canal anatomies.