## 3 Results
### Impact of a Mixture of Alkylphenol Compounds on CWs Substrate Microbial Community  
For the 12 collected samples, a total of 877,881 of V4-V5 16S rRNA gene sequences were generated by Illumina MiSeq which decreased to a total of 870,156 after the quality filtering performed by the sequencing company. A total of 767,470 quality checked merged reads (around 88% of the total dataset) were then submitted and processed by SILVAngs automatic pipeline that clustered into 172,430 OTUs and classified them using the SILVA reference database. The total number of sequences per sample ranged from 28,965 to 92,696 with an average of 63,956 \(\pm\) 17,540 reads per sample. The number of OTUs per sample ranged from 7400 to 21,254 with an average of 14,369 \(\pm\) 3380 sequence variants per sample (see details in Table S3). Bacteria domain represented around 96% of the total dataset, _Archaea_ domain represented around 1% while the remaining 3% was classified into \"No Relative\" group (without any close relatives). The alpha diversity varied between control and doped CWs systems (Figure 4), but no significant differences (Kruskal-Wallis, \(p>0.05\)) were observed in terms of richness  
Figure 3: Removal percentage (%, mean and standard deviation, \(n=3\)) of ammonia (NH4^+), nitrite (NO2^-) and nitrate (NO3^-) ions in CWs, irrigated for eight weeks with Ribeira de Joane water not doped (control) or doped with PCP. * no NO3^- removal in CWs. The analyses of the cluster dendrogram (Figure 5a) showed two groups according to similarity: the group of T0 (beginning of the experiment) and the group of T8 (end of the experiment) samples. The samples in T0 (samples from plant root bed before being subjected to doped stream water) were more similar to each other than to the T8 samples. The same is observed based on NMDS analysis (Figure 5b), two groups are formed in function of the experimental time (T0 and T8). The existence of similarity between initial samples was expected as plant roots bed origin was the same. Over the experimental time the experimental conditions influenced the microbial community structure, which showed a tendency towards greater specificity. Taxonomic profile of the bacteria communities was performed at phylum, class and genus levels. At the phylum level, the three most abundant taxa were _Proteobacteria_ (abundance up to 41%), _Bacteroidetes_ (abundance up to 21%) and _Planctomycetes_ (abundance up to 10%) (Figure 6a), being very similar among samples. Relatively to class level, four classes represented more than 50% of those with abundance > 1%. Three of those classes belong to the _Proteobacteria_ phylum (classes _Gammaproteobacteria_, _Alphaproteobacteria_ and _Deltaproteobacteria_) and the other class to the _Acteroidetes_ phylum (class _Bacteridia_) (Figure 6b). When the analysis was made at the genus level, the 15 genera abundance across the samples represented up to 23% of genera community (Figure 6c) (average total number of genera per sample 453 \(\pm\) 51, cut-off of 0.90%). Figure 4: 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 NP and OP. 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). Multimonimal Species Classification Method (CLAM) analysis was performed to identify the potential specialist taxa present in T8 doped (Figure 7b) when compared with T8 control (Figure 7a). CLAM uses a multinomial model based of relative abundance of estimated OTUs for two distinct conditions (\"generalist\" and \"specialist\").