3.2.1. Effect of the Water Level Height
The conventional depth of HF CWs is 50–60 cm. However, different authors have observed that shallow HFs (<27 cm deep) achieved better removals of COD, BOD
5, NH
4+-N and DRP. The better performance of shallower HF CWs could be explained by considering that: (i) water can be forced to go through the rooting zone of the plants, and (ii) shallower substrates displayed higher redox potential and slightly higher dissolved oxygen concentrations [
15,
27]. Thus, the effect of water height was considered a key parameter in this study. According to the results for the entire experimental period (
Table 3), HF10 was, in general, more efficient than HF40, and was significantly better for turbidity.
As can be observed, both treatment lines were very efficient at the removal of organic matter (BOD
5 and COD), turbidity and TSS. The average removals of (HF40: 63%, HF10: 53%) and NH
4+-N (HF40: 57%, HF10: 61%) for all the experimental period fall in the upper limit of the range provided for CWs (40–60%) by Vymazal [
28]. Those of TP (20–37%) and DRP (18–14%) were clearly lower. However, the removals of NH
4+-N and DRP were strongly affected by HLR, as will be discussed in the following section.
The effect of a design parameter on the performance of CWs is usually measured by comparing the obtained average removals [
29], but the high dispersion of the data can obscure the results and lead to ambiguous conclusions [
27]. Another way to compare the efficiencies of HF40 and HF10 is to determine their capability to meet the legislation by measuring the percentage of samples with concentrations below the legal limits.
Table 4 shows the degree of compliance with the Spanish National Regulation as the percentage of samples with values below those established by the Royal Decrees that regulate the limits for: (i) COD regarding the discharge of treated wastewater into the environment [
30] and (ii) TSS, turbidity and
E. coli, considering the possible reuse (urban, agricultural, industrial, recreational and environmental) of treated wastewater [
31].
As can be seen, the degree of compliance of HF10 was higher than that of HF40 for COD (90% vs. 73%), TSS (54% vs. 100%), turbidity (2 NTU: 61% vs. 25%, 10 NTU: 100% vs. 83%).
3.2.2. Effect of HLR
To determine the effect of SLR on the efficiency of the HF CWs, the influent pump working time was doubled on 30 March 2016. Thus, two different periods can be regarded.
Table 3 summarizes the SLRs of HF10 and HF40 for the periods of high and low SLRs.
As can be observed in
Table 5, the SLRs of both treatment lines were quite similar (
p > 0.05) for each period and the removals can be compared.
Figure 2 illustrates the removals of COD, NH
4+-N, DRP, turbidity,
E. coli and total coliforms in HF40 and HF10 and
Table 6 summarizes the average SLRs and the removals achieved.
The results from
Figure 2 and
Table 6 indicate that augmenting the SLR reduced the removals of NH
4+-N, DRP and fecal indicators but those of COD and turbidity remained high.
Figure 3 shows the mass removal rates (g/m
2d) of COD, NH
4+-N and DRP and
E. coli in the first stages (HF40-and HF10-1) and second stages of the HF CWs (HF40-2 and HF10-2) during the periods of high and low HLR. In the case of COD, the first stages achieved the highest COD mass removal rates in both periods.
In the period of low HLR, the average removals of NH
4+-N were high for HF CWs (HF40: 67%, HF10: 75%,
Table 6), suggesting the availability of enough dissolved oxygen to support nitrification. This could be partially due to the fact that the first unit of the CWs was placed above the other ones and the influent was intermittently dosed. Thus, a fill and drain effect was obtained that would favor the aeration of the substrate. Unfortunately, the role of the plants in terms of N removal was not considered in this study. When the HLR was increased, the removal of NH
4+-N decreased (HF40: 48%, HF10: 49%) and became more unstable, as shown by the higher standard deviations observed (
Table 6 and
Figure 3). Unlike the results of this study, Nivala et al. [
27] found negligible NH
4+-N removal in shallow HF CWs, though the shallowest systems achieved the lowest mean effluent NH
4+-N concentrations. In this study, in the low HLR period, similar surface removal rates were observed in both parts of the HFs. However, when the HLR increased, the surface removal rates also increased in both stages, being slightly better in the first stage, as observed for COD (
Figure 2).
The average DRP removals in the period of low HLR were statistically similar (HF40: 60%, HF10: 53%,
p > 0.05) and were high for HF CWs. Nonetheless, the high HLR had a notoriously negative effect, since DRP removals became negative (HF40: −16%, HF10: −20%). Dierberg et al. [
32] found that TP retention efficiency decreased at high HLR due to lower HRT, as it favored preferential flow and affected the P diffusion and sorption processes. It is widely accepted that the main DRP removal mechanism in CWs is the adsorption/precipitation on the substrate, as plant uptake and microbial activity are less important [
33]. It was expected that the sand used in the final unit of HF10 and HF40, with a high carbonate content, and thus in Ca
2+ and Mg
2+ ions, could help in the removal of DRP by precipitation [
34]. However, the porous media used in these experiments (palm mulch, gravel and sand) did not have any removal phosphorus removal capacity, as observed in lab experiments. This can explain why there was no difference in the DRP surface removal rate removal in both stages of HF10 and HF40 (
Figure 3). Thus, the results of this study indicate that plant uptake and microbial activity had a more relevant role than expected. In fact, plant uptake is considered to be the second most important mechanism of P removal [
35]. Additionally, microbial P removal by means of polyphosphate-accumulating organisms can be an important mechanism [
36]. These authors found that an intermediate HRT provided the best removal of TP in VFs, as it provided a balance between P adsorption on the substrate and release under anaerobic conditions. This effect can explain the negative DRP removals observed in the high HLR period of the present study.
The main mechanisms of pathogen removal in CWs are to kill them off by starvation or predation, sedimentation, filtration and adsorption [
37]. Many different variables play a role in the removal of pathogens in CWs: temperature, influent composition, HRT and HLR, water flow type (surface, sub-surface, vertical or horizontal), the presence and type of macrophytes or the substrate [
38]. Additionally, Morató et al. [
23] observed that water depth had also an important effect on disinfection. The results from the present study do not reveal a significant role of water depth regarding
E. coli and total coliform elimination (
Table 6). During the low SLR period,
E. coli and total coliform removals of almost five orders of magnitude were registered for both treatment lines (
Table 6). Other authors have found similar
E. coli and pathogen removals in CWs using fine soil [
39] and sand [
40] as substrates, but such high removals are not usually found in CWs [
38].
In the case of
E. coli and total coliforms, a clear removal reduction was observed after increasing the HLR for HF40 with respect to HF10 (
Figure 2). This fact suggests the higher robustness of the latter. The analysis of surface removal rates for the first and second stages of HF40 and HF 10 (
Figure 3) reveals a similar behavior of that of COD: a higher average SLR removal of both parameters at higher SLR and a stronger retention in the first part of the system (H40-1 and HF10-1). The results of this study suggest that the presence of the sand in the last treatment units of HF10 and HF40 did not play a key role in the removal of pathogens, since SLR removals were not better in the second stages of the treatment lines. This fact suggests that the presence of the plants and the good aeration of the substrate (as shown by the good NH
4+-N removals observed) are the main pathogen removal mechanisms. Macrophytes can improve pathogen removal by increasing HRT with their root system [
41] and providing a larger surface area for microbe attachment, favoring the formation of biofilms, oxygen seepage through roots, the secretion of plant exudates, etc. [
38].
3.2.3. Hybrid CWs: Sand vs. Mulch Substrates for the VF Stage
The hybrid CWs achieved similar average removals without significant differences, as can be seen in
Table 7. However, the preliminary results of the first 3 months of operation of Hybrid CW 1 with sand, were especially good and better than those of Hybrid CW 2, with removals of NH
4+-N and
E. coli greater than 90% and 4–5 log units, respectively. The
E. coli concentrations in the effluent were 40–540 CFU/100 mL, although HLRs as high as 426 L/m2d were used in the VF stage (
Table 2). However, after about 3 months of operation, the first symptoms of clogging began to be observed, i.e., ponding on the surface of the sand-based VF. In consequence, performance was dramatically reduced. Initially, maintenance strategies such as resting periods of several days and scraping the surface were enough to unclog the filter, but after about 9 months of operation it became irreversibly clogged. It was necessary to remove a 0.10 m layer on its surface to recover an acceptable hydraulic conductivity, but the initial high performances were not observed again. Clogging is the main cause of malfunction in vertical sand filters and is the cause of the oxygen transfer reduction in vertical flow filters [
42]. This can explain the obtained results, since nitrification is very sensitive to oxygenation and
E. coli removal is faster in aerobic conditions. The results of the present study partially agree with those of de Oliveira Cruz [
43] who found clogging problems after only 4 months of operation in a sand filter. However, the full restoration of its function was recovered by scraping off a shallow layer on its surface.
Nevertheless, Hybrid CW 2 (only mulch) remained unclogged throughout the whole experimental period. The only maintenance required was the addition of mulch because of the reduction in the height of the substrate, which occurred more rapidly at the beginning of the experimental period, falling by 20% in the first months. The substrate was added, and the subsequent reductions were minimal. Regarding clogging, woodchips were also preferred to sand as substrates for filters treating soiled dairy water [
44].