The contribution of the WWTP effluent to the flow of the receiving stream was highly variable (from < 1 to 100%) depending on the period of the year. This pattern is typically observed in many other intermittent streams across arid and semiarid regions (Arnon et al. 2015; Martí et al. 2010; Bicknell et al. 2020), because their hydrologic regime is characterized by extreme events (i.e., floods and droughts). This finding explains the strong temporal shifts in the impact that WWTP effluent inputs have on the receiving streams of these regions as shown in our study. During high flow conditions in winter and spring, landscape features and upstream conditions have a major influence on stream physical and chemical characteristics, whereas these characteristics become increasingly subjected to WWTP effluent inputs as the dilution capacity of the receiving stream decreases during summer low flow (Keller et al. 2014). In the study stream, the impact of the WWTP effluent was dramatic for both physical and chemical variables, especially during summer and autumn when the dilution factor was well below the 40% threshold. In particular, the stream commonly dried out upstream of the WWTP input for some weeks in summer; and thus, the dilution capacity of the stream was nil. Under these conditions, water temperature and electrical conductivity were higher and oxygen concentration was relatively lower than on dates when the stream dilution factor was > 40%. Low dissolved oxygen concentration in the receiving stream could be explained by the increases in ecosystem respiration that are usually observed downstream of WWTP inputs (Gücker et al. 2006; Bernal et al. 2020). In addition, mean SRP concentration increased between 4- and 8-fold during summer, and there was a shift towards N limitation as indicated by the low DIN:SRP molar ratios. These physicochemical changes suggest that in-stream net nutrient uptake could also show marked seasonal patterns because temperature, oxygen availability, and nutrient availability can strongly influence the metabolic activity, the demand of nutrients and the preferential biogeochemical pathways of microbial in-stream communities (Butturini and Sabater 1998; Dodds et al. 2002; von Schiller et al. 2008; Ribot et al. 2017). Moreover, the intermittent hydrological regime implies that, during low flow conditions, in-stream biogeochemical processing in the receiving stream may become essential to regulate nutrient concentrations in the water column since dilution from either upstream or groundwater sources is almost negligible (Bernal et al. 2020).
The longitudinal profiles in nutrient concentrations allowed examining the temporal variability in the N and P uptake and transformation capacity of receiving stream while allowing the evaluation of the influence of the WWTP contribution to the temporal dynamics. Results indicate higher in-stream bioreactivity for DIN forms than for SRP because longitudinal profiles exhibited more significant trends (i.e., indicative of net uptake or release) for the former element than for the latter. This finding is relatively common among WWTP-receiving streams (Martí et al. 2010). Concentration of SRP did not show any significant trend along the stream on most of the sampling dates, which suggests that uptake and release processes are counterbalanced. Only in very few dates we found net uptake of SRP, although the values of Vf for SRP were in general lower than those for DIN. Low or nil net uptake of SRP could be explained by the excess of SRP from the WWTP input that could saturate the demand by microbial assemblages as well as the buffering capacity of streambed sediments (House and Denison 1998; Haggard et al. 2005).
In contrast, despite DIN concentration did not show any significant longitudinal trends on most sampling dates, each particular DIN form (NH4+ NO2− and NO3−) did. Only on very few days we found a significant net uptake of DIN, but these dates did not follow any seasonal pattern. This finding suggests that the receiving stream acts more as a transformer than as a sink of N. Our results showed significant longitudinal decreases in the concentration of NH4+ in the study reach, an indication that this nutrient was highly processed along the stream and that uptake processes prevailed over release processes. Regardless of the season, declines in NH4+ concentration were accompanied by increases in NO2− and NO3− concentrations; supporting the idea that nitrification is a prevailing process in the receiving stream over time (Merseburger et al. 2005; Bernal et al. 2020). This pattern is consistent with previous results showing that nitrification can represent up to 90% of the uptake of NH4+ in the study stream (Bernal et al., 2017). Similar patterns have also been described in urban streams worldwide (Cébron et al. 2003; Marti et al. 2004; Gammons et al. 2011), suggesting that high in-stream nitrification rates might be a common phenomenon downstream of WWTP effluent inputs in urban streams. These high nitrification rates can be explained by the high inputs of both NH4+ and nitrifying bacteria from active sludge discharged into the receiving streams from the effluents of the WWTPs (Merbt et al. 2015). We also found evidence for the occurrence of other biogeochemical processes associated with N cycling within the stream. The slope between Vf for NO3− and Vf for NH4+ was clearly below 1 (Fig. 3), indicating that the biogeochemical demand of NH4+ uptake was higher than expected solely from nitrification. This result suggests that assimilatory NH4+ uptake by photoautotrophs and heterotrophs additionally contribute to the observed declines in NH4+ concentration downstream of the WWTP input. In contrast, net uptake of NO3− occurred on few dates mostly during spring. This suggests, that except for these dates, denitrification and assimilation of NO3− were overwhelmed NH4+ nitrification. The fact that uptake efficiency for NO3− tends to be lower than that for NH4+ could explain this finding (Ribot et al. 2017). Moreover, some studies have indicated that denitrification can be limited by availability of dissolved organic matter in receiving streams (Ribot et al. 2019)
While our results strongly support the idea of the receiving stream as a nitrification hot spot, we found that the magnitude of Vf for NH4+ varied widely over time. Yet, there were no statistical differences in Vf for NH4+ among seasons, suggesting that those environmental factors clearly fluctuating with season such as temperature or light availability were likely not strongly determining the temporal variability of NH4+ demand by biota. Yet, we found a strong and negative relationship between Vf for NH4+ and the PC1 scores suggesting that the temporal variability of in-stream net uptake of NH4+ was closely related to changes in hydrological conditions which determine the relative influence of the WWTP effluent to stream discharge and water chemistry. Nevertheless, in contrast to the idea that stream nutrient uptake efficiency increases with decreasing stream discharge and increasing water residence time (e.g. Peterson et al. 2001; Drummond et al. 2016), we found that the magnitude of Vf for NH4+ was higher during relatively high flow conditions (Fig. 4). This result could be explained by the large impact of the WWTP effluent on stream physicochemistry during low flows, which lead to decreased dissolved oxygen concentrations that likely inhibited the activity of nitrifiers, despite of increases in stream water temperature. In this sense, previous studies have shown that bacterial assemblages and associated microbial activity can experience dramatic shifts when dilution factors in WWTP impacted streams are < 50% (Romero et al. 2019). Moreover, high nutrient concentrations likely had a saturation effect on in-stream NH4+ demand during these low flow periods when the receiving stream had a small dilution capacity. A similar saturation effect has been reported for NO3− uptake in urban arid land streams, despite these streams tends to be strongly N limited under pristine conditions (Grimm et al. 2005). In this study, results from the relationship between Vf for NH4+ and PC1 supports the saturation idea, because Vf tends to decrease in these sampling case when the dilution factor is low and the effect of WWTP input on increases in NH4+ concentration is high. The concomitant reduction in NO2− net releases (i.e.,less negative values of Vf for NO2−) under these conditions further suggests a clear impact on the in-stream nitrification capacity. The characterization of NH4+ uptake kinetics of epilithic and episammic biofilms from this receiving stream additionally suggests that stream ambient NH4+ concentration can be above limitation (i.e., > than half saturation values) for NH4+ uptake and nitrification rates by microbial assemblages (Bernal et al. 2018). We further examined how NH4+ uptake demand varies with NH4+ concentration considering a wider set of pristine and human impacted streams, using the data set generated by Marcé at al. (2018) to verify if the saturation explanation holds. We found that variability in Vf for NH4+ from the study stream was relatively constrained and values were in the lower range of all the data set (Fig. 5) This comparison needs to be done with caution because the data set includes both gross and net uptake rates and there was no significant regression between Vf for NH4+ and NH4+ concentration (Fig. 5). However, the funnel-type pattern observed suggests that with increasing ambient NH4+ concentration, the biogeochemical reactivity of stream ecosystems, at least for NH4+, is seriously threatened, even under hydrological conditions favoring the interaction between nutrients and biota.
The hydrologic regime and the biogeochemical reactivity are essential factors to understand the variability of nutrient uptake in streams and understand the role of these ecosystems in the regulation of nutrient cycling and exports along the river networks (Battin et al. 2008; Acuña et al. 2019). Our results are concordant with the idea that biogeochemical reactivity can be equal or even more important that hydrological opportunity to drive nutrient cycling in stream ecosystems (Marcé et al. 2018), especially in highly perturbed streams receiving chronic nutrient inputs. In particular, this study shows the relative importance of bioreactive controls (i.e., nutrient saturation) over hydrologic controls (i.e., high water residence time) on the temporal variation of nutrient uptake in WWTP-receiving streams. Moreover, the study suggests that this is influenced by the interplay between the hydrologic regime and the WWTP influence (i.e the dilution capacity), especially in Mediterranean regions. Overall, this study contributes to emphasize the distinct biogeochemical heartbeat of streams under human pressure (Grimm et al. 2005). Therefore, a better understanding of the temporal variability in nutrient uptake capacity of the receiving streams, and especially of the biogeochemical processes prevailing during low flow conditions, is important for improving the management of urban streams impacted by point-sources. In this context, our study conveys with the perspective of current studies suggesting management strategies for WWTP-receiving streams (i.e., Bicknell et al. 2020). In particular, our study suggests that it is critical to design WWTP operation procedures taking into account both the dilution and the bioreactive capacity of receiving streams for an integrated management of water resources and their quality in urban landscapes, especially under water scarcity conditions.