## 3 Results and Discussion
### Microbiological Data  
The ability of CW to remove pathogens from wastewater has been reported by several authors [24, 27, 33]. In the present study, the numbers of total coliforms and _E. coli_ in wastewater collected from the inlet and outlet zones of the CW during the cold and hot seasons of three consecutive years are shown in Table 2.  
The CFU per ml of total coliforms in wastewater collected from inlet and outlet ranged between mean values of log\({}_{10}\) 4.3 (C3) and log\({}_{10}\) 5.1 (C1 and H3) and between log\({}_{10}\) 2.7 (C3) and log\({}_{10}\) 3.5 (H2), respectively; while _E. coli_ counts varied between mean values of log\({}_{10}\) 2.7 (C1) and log\({}_{10}\) 3.5 (H2) and between no viable counts (C1, C2, C3) and log\({}_{10}\) 2.5, in inlet and outlet zones, respectively. Despite the variation in the loads at the inlet, possibly due to the number of overnight guests, in general, no significant (\(p\) > 0.05) differences were found for both indicator organisms over the different sampling campaigns. A similar trend was  
Figure 2: Removal efficiency of the CW wastewater parameters for each sampling trial, during cold (C) and hot (H) seasons across three consecutive years (1, 2, 3). observed for total coliforms since no significant differences (\(p>0.05\)) were seen along the seasons. The mean outflow counts of indicator organisms are within values found in other reports in the literature [34]. In relation to _E. coli_ counts, in all cold seasons no viable cells were detected at the outflow. This could be attributed to the much lower occupation of the house verified in cold seasons, but also to the effective depruation of the CW. Seasonal variations and the associated edaphic parameters (e.g., temperature) have been identified as important factors in the removal of pathogens in CW [33]. It has been conveyed that as the reduction of the number of bacteria of anthropogenic origin is a complex process, comprising physical, chemical and biological factors [35], that can be influenced to different extents by operational parameters such as hydraulic regime and retention time, vegetation, seasonal fluctuation, and water composition [27]. There are though reported inconclusive results regarding the effect of season on pathogens removal efficiency, since several studies showed greatest removal of pathogens in warmer seasons [36; 37] while in others season variations did not induce any effect [38], as occurred in the present study. Nevertheless, a reduction of up to 3 log units for total coliforms and up to 2 log units for _E. coli_ was observed in the wastewater from the inlet to the outlet. Similar results were obtained in previous studies in the same CW for Enterobacteriaceae, _E. coli_ and total coliforms [1; 25]. [PERSON] [34] referred in a review that removal of total and fecal coliforms in CW is usually very high, (95-99%), a trend that was also verified in this study. Bacterial pathogens present in wastewater may be trapped both on the surface of substrate and roots, but they can also be internalized in plant tissues [25]. Several removal mechanisms have been reported and may comprise natural die-off due to starvation or predation, sedimentation and filtration, and adsorption [27]. In the present study, the prevalence of the indicator organisms, as well as of heterotrophic culturable bacteria was evaluated both associated to the substrate's surface and associated to plant roots over time (Table 3).