3.3.1. Plant Development
The agronomic results of plant sampling are shown in
Table 4. As already stated, plant species in the SFCW changed over the course of time due to the different conditions requested by some projects that the CER Land Reclamation Consortium participated in. The contained variation of plant biomass observed in the 2004–2006 period, ranging from 4.68 to 5.40 kg·dw·m
−2, has to be considered in the context of the equilibria among the different aquatic plant species inhabiting the system. In this period, TN and TP that entered the CW through agricultural drainage water enabled plant development. The observed above-ground biomass is in line with that reported by other studies, for example, [
22] recorded 0.37–1.76 kg·dw·m
−2 and [
23] 1.2–1.4 kg·dw·m
−2.
Over the years, there was a decrease in the ratio between above and below-ground biomass. While in 2004 it was 1.3, in 2009 it dropped to 0.2, meaning that below-ground biomass had 5 times greater weight than above-ground biomass. This observation is likely due to the fact that above-ground biomass was never harvested, so withered plants from the previous year blocked the sunlight for sprouting plants, took space and consequently limited development of new shoots [
23].
The visible drop of all biomass parameters, including biomass, average height and number of shoots, in 2007 (
Table 4) is mainly due to the removal of above-ground biomass from the first two meanders, as clearly indicated by the low value of the above-ground/below-ground biomass ratio, as well as the interruption of water inflow and nutrient input into the system to allow for the poplar and willow plantation/rooting. After 2007, the agronomic parameters indicated the establishment of new plant consortia.
3.3.2. Nutrients’ Content of Biomass and Soil
Owing to the removal of above-ground biomass of aquatic plants from the first two meanders in autumn 2006, no nutrient distribution can be estimated between soil and vegetation. Nevertheless, some considerations on their occurrence are still possible.
The plant biomass sampled was analysed for its TN and TP content, as shown in
Figure 5. In the 2004–2009 period, the content of both of these elements in the biomass show some fluctuations that could be related to the variation in inflow water (
Figure 3) and nutrient loads (
Table 2), as well as to the change in plant species that inhabited the SFCW. The plantation of willow and poplar trees in 2006 surely increased plant competition, thus modifying the equilibria among plant and microorganism species.
Only partial TP data were available for the same period. Nevertheless, the content of plant biomass peaked in 2005 at 1.5 g·kg
−1 (
Figure 5). Similarly, the biomass TN content peaked in the 2005–2006 period at about 11.5 g·kg
−1 (
Figure 5). This value is a bit lower than the TN reported by [
22] in a study where a SFCW received municipal wastewater that was more polluted than the agricultural drainage water used in this study.
TN and TP concentrations in the SFCW top soil are reported in
Figure 5. Although no values for nutrients or for OMC (organic matter content) in CW soil are available from before 2004 to be considered as a reference, it is possible to observe that TN concentration slowly increased over the years, reaching a value of 2.1 g·kg
−1 in 2017, almost twofold higher than that of 1.2 g·kg
−1 measured in 2004. TP, on the other hand, did not accumulate in the top soil, as is evident by its quite constant level over the 13-year-long observation period. This can be explained by the low nutrient loads of the influent (
Table 2) and the specific flush-out events already described in the
Section 3.2.1.
As far as the OMC of the SFCW soil is concerned, an increase in the first 15 cm was observed since the beginning of the monitoring. While the OMC in 2004 was 19.6 g·kg
−1, it increased more than 2.5 times in 2017, reaching a value of 49.8 g·kg
−1. This positive trend suggests constant organic matter production and its sedimentation into the CW during the 14 years of the system’s operation. The higher OMC increase in other SFCW soils reported in the literature is typical of applications of water containing higher amounts of organic matter and nutrients than those contained in our agricultural drainage water. For example, [
24] reported a tenfold increase of TOC over a period of 5 years, but in this case several applications of slurry were made to the SFCW.
3.3.3. Boron and Heavy Metals in Biomass and Soil
Additional information on the state of the CW can be obtained by considering the metal content of the biomass and soil, as these elements, apart from being naturally contained in the soil in background levels, can enter the SFCW by water inflow and can be subsequently uptaken by plants or accumulated in soil. Cu and Zn are present in several plant products, either as active ingredients themselves or as counterions of organic products [
25], so their inflow to the SFCW can be seasonal or constant, depending on their administration frequency. B is a plant micronutrient usually applied to crops as fertiliser [
26]. Cd, Ni and Pb are considered potentially toxic elements (PTE) and can occur in wastewater and be accumulated in soil through different anthropogenic activities [
27].
Figure 6 gives the content of these metals in the above- and below-ground biomass for the period 2004–2006, before the plantation of new trees in the SFCW. As a general trend, it is possible to observe that the metals were found to mostly have accumulated in the below-ground plant tissues, and only a small portion was transferred to the above-ground parts. These findings are in accordance with [
22], which also reported accumulation of heavy metals in below-ground biomass.
Furthermore, high variation was observed among the amounts of heavy metals retained by plant tissues. The metal accumulated the most by plants was Fe, with a maximum concentration of more than 4000 mg·kg
−1 in 2006. Cd was never higher than 0.8 mg·kg
−1, Cu ranged between 31 and 8 mg·kg
−1, while Zn was never less than 30 mg·kg
−1 (
Figure 6). Similar results were reported by [
28] for
Phragmites australis from different horizontal flow CWs. For example, slightly higher Zn levels and a few times higher Cu concentration in
Typha were reported after a 15-day-long experiment [
13]. As far as B is concerned, this nutrient was found to be well retained by plant root apparatus (27 mg·kg
−1 on average) in the 2004–2006 period.
Some considerations can be drawn by comparing the concentrations of heavy metals in the biomass with their levels in the top soil (
Table 5) for the period 2005–2006, when a complete set of data is available. In fact, even though the root system of aquatic plants can enter the soil deeper than 15 cm, the level of heavy metals in the top soil gives some indications as to their accumulation in the system and their potential bioavailability to plants.
In general, the concentrations of heavy metals in the first 15 cm of the CW soil were on average lower than, but still in the range of, other studies, such as, for example, that reported by [
29], who studied heavy metal removal using a horizontal flow CW from road runoff in Ireland.
In detail, in the 2005–2006 observation period, Cd was present at very low concentrations in both soil and biomass, whereas the average Pb level in the top soil of 25.3 mg·kg
−1 and its relatively low concentration in the biomass (1.7 mg·kg
−1,
Table 5) are in accordance with the fact that it is a fairly immobile element [
27], as well as its known low bioavailability to plants [
30]. The other heavy metals, including Ni, Cu and Zn, are considered of medium bioavailability to plants in aerated soil [
30]. In the system reported in this study, the average biomass concentrations of Ni, Cu, and Zn (6.2, 9.4 and 28.7 mg·kg
−1, respectively) were lower than, but still in the range of, their levels in the top soil (54.3, 33.8 and 97.3 mg·kg
−1, respectively), thus indicating moderate availability to plants.
The Fe level in the biomass (2550.5 mg·kg
−1) was very high, and the top soil concentration of 35.2 mg·kg
−1 cannot justify such a high accumulation at the roots. Specific root uptake mechanisms have to be considered for this metal, as already defined by [
31], who have reported that aquatic plants may modify the rhizosphere by facilitating the formation of iron oxide plaques that immobilise and concentrate heavy metals.
Under conventional operational conditions, the aquatic plants of the system studied act as Fe accumulators and Cu, Pb, Ni and Zn bio-indicators. These findings could be of interest for further study on metal bioavailability for aquatic plants and phytoremediation mechanisms against PTE.
Finally, the actual SFCW state was monitored as well, in order to evaluate how the 17 years of operation had affected the distribution of nutrients and heavy metals along the soil profile (
Table 6). As expected, the level of total organic carbon, TN, TP and B decreased from the top soil to the deeper layers, even though significant differences (
p < 0.05) were shown only for the first two parameters (
Table 6). This highlights the surface accumulation of nutrients and organic material produced by aquatic plants. The average pH value of the soil (measured in water) was found to be 8.51 ± 0.04 (20).
No visible accumulation of heavy metals can be seen along the vertical soil profile, and there were no significant differences among them (
Table 6). Most probably, this is due to the fact that the root systems of aquatic plants can accumulate large amounts of heavy metals [
32] and occupy large soil volumes. Moreover, when compared to their legal limits, as introduced by the Italian law [
33], all of the heavy metals resulted at concentrations below the lowest admitted threshold (limit A: soil suitable for private and public green areas). In addition, the data for Cr, Ni, Zn, Cu, Pb, and Sn of CW soil still safely fall within the range of the local anthropic-natural available background data (≤75, ≤120, ≤75; ≤60, and ≤50 mg·kg
−1, respectively [
34]), thus indicating that no important changes have affected the soil heavy metal content. Therefore, even after 17 years of functioning, the SFCW can still be considered to be a bio-filter with no visible accumulation of PTE in either the top or the deep soil layers (up to 60 cm depth), and with levels below the current legal limits.