## 4 Discussion
The authors used a flow-through lab system to cultivate estuarine macroalgae _Ulva rotundata_ (formerly, _Ulva pseudorotunda_) in fish effluent ([PO4-] within the range of the effluent used in this work) and inferred that effluent flow rate can have a major contribution to algal growth and nutrients removal [50]. [PERSON] et al. [49], on the other hand, reported low efficiencies rates for _Ulva clathrata_, cultivated in shrimp effluent, under continuous and static flow. The authors also observed a regeneration of phosphate in the medium, consenting with the conclusions of [PERSON] et al. that an improper algal growth [50] can lead to P organic decay. By contrast to nitrogen, the chemical phosphorous cycle in seawater remains complex and rather unknown and deserves further investigation. STable 15N and 13C isotopes measured in cultivated _Ulva_ spp. reflected the uptake of nutrients and their assimilation in the algal tissue. 815N and 813C are effective indicators of nitrogen and carbon pollution in aquatic ecosystems but can also be utilised as a measure of nutrient removal. Nutri and organic material from fish waste are generally enriched in 15N and depleted in 13C [63]. 813C acts as time-integrative measure of sewage exposure, reflecting the contributions of available nutrient sources over the period of tissue generation and turnover. 815N, on the other hand, has been used to distinguish the two main N pollution sources, the main urban N-deposition form (NOx) and the main rural and agricultural N-deposition form (NHx). For example, NO3- from human and animal waste is enriched in 815N, with isotopic compositions of 10% to 22%, whereas nitrate in synthetic fertiliser, generally has much lower 815N, with values ranging from 3% to 3% [64]. In one's study, the initial 815N values for _Ulva_ spp. collected from the _Mondego_ estuary (d0) show that high nitrogen loads are entering in the system. Throughout the cultivation experiments, the 815N values measured in the _Ulva_ spp. reflect uptake of inputs of nitrogen from fish effluent. _Ulva_ spp. is a fast-growing macroalgae that responds rapidly to changes in nutrient conditions, particularly to short-term N supply, as the rate of turnover of the internal N content of this macroalgae is quite short (<2 d) [65]. Therefore, it is not surprising that the changes in 815N for _Ulva_ spp. match with the changes in nitrogen concentrations in the pilot system, particularly for ammonium. Regarding carbon, _Ulva_ spp. 813C at the end of all the experiments (except for AQUA2) was depleted in 13C, and this observed pattern was probably due to the presence of effluent-derived carbon in the water. Despite the enrichment of nitrogen, the content of crude protein of the cultivated _Ulva_ spp. decreased over the experimental periods. Published data on the protein contents of _Ulva_ spp. are very wide-ranging. For instance, higher contents of crude protein have been reported in autumn/winter in comparison to spring/summer [36, 66]. [PERSON] et al. [37], on the other hand, registered no clear temporal pattern of crude protein content of wild and farmed _Ulva_ species. In terms of total protein (Bradford method), the authors reported higher levels in wild _Ulva_ uncialis (formerly, _Ulva capensis_) and _Ulva rigida_ in spring and constant levels throughout the remaining seasons [37]. Herein, a similar trend was observed. Considering the registered depletion of nutrients and enrichment of nitrogen in the cultivated _Ulva_ spp., increased protein contents were expected. In spring (AQUA1), both coastal and estuarine _Ulva_ spp. specimens showed a significant loss of total and crude protein. In autumn (AQUA2), total protein content augmented and remained constant until the end of the experiment, although crude protein tended to decrease. Exposure to light saturating conditions can reduce nitrogen uptake in macroalgae tissue and the lower exposure to sunlight and temperatures during autumn can promote nitrogen uptake [36] which can justify the different trends registered in this work.