## 4 Discussion
### Quantification of the Generated (Reactive) CAP Species
#### 4.1.2 Generation of ROS/RNS  
As mentioned before (Section 3.1.2, Figure 3), H\({}_{2}\)O\({}_{2}\) was only detected in the CAP treated water for some of the examined combinations of plasma characteristics, i.e., for DBD-0.0% (_v/v_)-21.88 V and DBD-1.0% (_v/v_)-21.88 V. It was therefore concluded that the influence of the plasma characteristics on the formation of H\({}_{2}\)O\({}_{2}\) was rather limited. Previous studies (e.g., [21; 36]) indicated that the generation of ROS such as H\({}_{2}\)O\({}_{2}\) was especially influenced by the gas composition, with the highest concentration measured when the feed gas contained oxygen. Within the presented study, however, the opposite trend was observed while applying the DBD electrode at the highest input voltage. Few possible explanations can be given for this observed phenomenon. First of all, it should be mentioned that the input voltage of 21.88 V resulted in the highest dissipated plasma power when no oxygen was added to the gas flow (Table 1). Consequently, this stronger plasma can result in a more efficient generation of CAP species which serve as a precursor for the generation of H\({}_{2}\)O\({}_{2}\). One of these precursors is the hydroxyl radical, which can be generated in the presence of water vapor originating from the treated water (or from the biofilm samples) [37]. Secondly, when no oxygen is added to the gas flow, the quenching effect of the gas is comparatively low. Hence, the (limited) amount of generated ROS originating from impurities from the environment and the presence of water vapor can still reach the sample at sufficiently high concentrations. The high amounts of generated ROS in the presence of oxygen are, on the other hand, more vulnerable to quenching. As a result, their higher generation rate is (partially) nullified [38]. Thirdly, the absence of H\({}_{2}\)O\({}_{2}\) molecules following 10 min of CAP treatment does not necessarily mean that this species was not formed over the course of the treatment since H\({}_{2}\)O\({}_{2}\) can further react with other molecules to form other reactive CAP species. As an example, H\({}_{2}\)O\({}_{2}\) can react with nitrite to form nitrate and peroxynitrite at acidic conditions [39; 40]. The presence of nitrate for some treatment conditions (Figure 3) can (partially) confirm this hypothesis. In future studies, it could be of interest to examine the formation of H\({}_{2}\)O\({}_{2}\) as function of the CAP treatment time. Finally, the small water volume served as a model for the biofilm, which in reality contains a high variety of components such as proteins, polysaccharides, extracellular DNA, water, and nutrients. The complexity of the biofilms will therefore also have an effect on the generation of certain ROS by, for example, serving as an oxygen scavenger [41]. In future research, the complexity of the water model could be increased by adding some proteins, polysaccharides, and eDNA, provided that the composition of the biofilm matrix is fully known. Based on Figure 5, it was concluded that the intracellular ROS concentration (significantly) increased when oxygen was added to the gas flow. This means that the oxygen level of the gas flow has an influence on the concentration and/or the type of generated (intra- and extracellular) ROS species. The latter is of high importance as this can have an influence on their ability to penetrate into the biofilm matrix and to interact with the biofilm-associated cells and the EPS matrix. The research of [PERSON] et al. [42], for example, reported that only 5% of the generated ROS species was able to penetrate a tissue with a thickness of 500 uM. Within the presented study, the obtained CLSM images (Figure 9) indicated that all examined treatment conditions resulted in cell death in the deeper biofilm layers. However, this cell damage can also be the result of other reactive species (e.g., RNS).