## 3 Results
### Impact of PCP on CWs Substrate Microbial Community  
For the 12 collected samples, a total of 887,135 of V4-V5 16S rRNA gene sequences were generated by Illumina MiSeq which decreased to a total of 883,920 after the quality filtering performed by the sequencing company. A total of 789,722 quality checked merged reads (around 89% of the total dataset) were then submitted and processed by SILVAngs automatic pipeline that clustered into 185,748 OTUs and classified them using the SILVA reference database. The total number of sequences per sample ranged from 52,216 to 95,767 with an average of 65,810 \(\pm\) 13,681 reads per sample. The number of OTUs per sample range from 11,316 to 20,710 with an average of 15,479 \(\pm\) 2847 sequence variants per sample (see details in Table S4). Bacteria domain represented around 94% of the total dataset, _Archaea_ domain represented around 2% while the remaining 4% was classified into \"No Relative\" group (without any close relatives). The alpha diversity varied between systems (Figure 8), but no significant differences (Kruskal-Wallis, \(p\) > 0.05) were observed in terms of richness (number of observed OTUs) or dominance (Berger-Parker index). In terms of diversity (Shannon diversity index),  
Figure 7: Relative abundance of the specialist genera in plant root bed substrate samples: (**a**) in control CWs and (**b**) in doped CWs collected at the end of the experiment (after 8 weeks). Systems irrigated along eight weeks with Ribeira de Joane water not doped (control) or doped with NP and OP. Only genera with abundance > 1% are shown. significant differences were observed among samples from control and from doped CWs systems at the end of the experiment (Kruskal-Wallis; \(p\) < 0.05), showing that the presence of PCP leads to a decrease in the microbial diversity. The analyses of the cluster dendrogram (Figure 9a) showed two groups according with similarity, the group of T0 (beginning of the experiment) and the group of T8 (end of the experiment) samples. The samples in T8 were more similar to each other than to the T0 samples. T0 samples refer to plant root bed before being subjected to doped stream water. The same is observed based on the NMDS analysis (Figure 9b) and two groups are formed in the function of the experimental time (T0 and T8). The existence of similarity between initial samples was expected as plant root bed origin was the same. Over the experimental time, the experimental conditions influenced similarly the microbial community, which showed a tendency towards greater specificity. For the bacteria domain, the three most abundant phyla were the same (Figure 10a): _Proteobacteria_, _Bacteroidetes_ and _Plancomycetes_, being the percentage composition very similar among samples. Relatively to class level, four classes represented more than 50% of the group with abundance >1%. Three of those classes belong to _Proteobacteria_ phylum (classes _Gammaproteobacteria_, _Alphaproteobacteria_ and _Deltaroteobacteria_) and the other to _Acteroidetes_ phylum (class _Bacteridia_) (Figure 10b). When the analysis was made at genus level, the fifteen genera more abundance represented between 20 to 26% of the total number of genera (Figure 10c) (average total number of genera per sample 799 \(\pm\) 61, cut-off of 0.95%). Figure 8: Alfa diversity index of the microbial community in the different CWs systems, irrigated for eight weeks with Ribeira de Joane water not doped (control) or doped with PCP. Samples collected at the beginning (T0) and at the end of the experiment (T8) (\(n\) = 3). (**a**) Richness (number of observed OTUs); (**b**) dominance (Berger–Parker index); (**c**) diversity (Shannon diversity index). Looking more specifically at samples collected at the end of the experiment (T8), in samples from control CWs 25 different genera specialists were observed while in samples from doped CWs only 22 different genera specialists were observed, Figure 11a,b, respectively.