4.1. Abiotic Environment
In the present study an experimental brackish aquaponic system for Mediterranean fish (gilthead juvenile seabream) and halophytes (rock samphire) was investigated for a duration of 102 days, and to authors’ knowledge, this is the first such investigation. To date, only a few studies have been conducted on the use of brackish water in aquaponic systems [
5,
21,
24], whereas the use of freshwater in aquaponic systems has been widely studied [
5,
21,
24,
36]. A successful aquaponic system provides important benefits, such as water quality control, appropriate fish and plant growth performances, plant and fish disease management, eliminating environmental impacts and functioning as an RAS. In this way, such systems do not require large volumes of fresh water daily (less than 5% renewed water is needed due to evaporation or losses from daily functioning) [
37,
38].
Gilthead seabream is one of the most valuable commercial fish species of the Mediterranean, and it has shown very good adaptability to aquaculture [
39,
40]. The present study showed that the use of brackish water in aquaponics is efficient since the conditioning of the system is associated with the biological filter and achieved by minimizing ammonia and nitrite concentrations and increasing nitrate concentrations. The results of the study suggest that the nitrate concentration could maintain lower TAN concentrations during the experimental period and indicate that an equal amount of TAN oxidation to nitrate occurred for both treatments (8 ppt and 20 ppt). These results were suitable for rock samphire absorption and growth performance, which is consistent with previously reported results [
21,
24,
25], thus indicating that similar fluctuations in nitrate ions and pH were maintained within the recommended safety limits [
3]. The conditioning of the filter at the RAS occurred via the effectiveness of the filter bed, which reduced the ammonia and nitrite concentrations and produced higher levels of nitrates, which is similar to the values reported by Spotte [
6].
Previous studies [
30,
33] reported that the conditioning of the freshwater aquariums of two ornamental fish (cichlid zebra and angel fish) was achieved over the short term via biochemical nitrate production due to an increase in the abundance of nitrifying bacteria. The maintenance of water quality parameters is essential to ensuring the best aquaculture conditions for optimal fish performance and avoiding adverse conditions which could affect fish’s optimal growth, survival and health in an RAS system [
9,
10,
41,
42,
43].
The hydraulic retention time (HRT) of the present study for both salinities of the aquaponic system was 9.7 min/day, indicating that the biofilter performed efficiently (nitrification and denitrification rate) and removed ammonia. Timmons and Ebeling [
44] reported that in a saltwater RAS, an HRT of 2 h affected the denitrification process. In the same study, an increase of the HRT to 6 h decreased the denitrification rate. The HRT has an impact on the carrying capacity of the filter and especially affects the ammonia removal efficiency [
45,
46], alkalinity production [
47], sulphate production [
48] and C/N ratio in the denitrification process [
49]. Furthermore, the hydraulic loading rate (HLR) has an impact on fish and plant production and nutrient removal and is correlated with the daily food input in the system for the optimum plant ratio [
25,
50]. Chen et al. [
50] suggested that the best HLR for a freshwater aquaponic system is 1.28 m/day because it provides the best production performance for fish (SGR: 1.80%/day) and plant growth (1.75 cm/day). In the present study, the HLR (1.85 m/day) for both treatments was even higher, thus providing a higher growth performance for fish (SGR: 3.17%/day) and plants (PGR: 1.87 cm/day for 20 ppt and 0.99 cm/day for 8 ppt).
Concerning the C/N ratio, it has been reported to affect the oxidation capacity of the filter bed and depends on the daily feed supply and nutrient input to the system [
11]. Hence, a balance is required between bacterial abundance and fish and plant performance [
3]. Zhu and Chen [
51] reported that the load can affect the process of nitrification and the relationship with heterotrophic bacteria and autotrophic nitrifying bacteria in an aquaponic system. Additionally, according to Ohashi et al. [
52], the efficiency of the filter decreases when the C/N ratio increases, thus leading to a higher abundance of heterotrophic bacteria, and depends on the TAN concentration. High C/N ratios of heterotrophic bacteria reduce the diffusion of nitrogen and dissolved oxygen (DO) in autotrophic nitrifying bacteria, thereby adversely affecting the rate of nitrification [
53,
54]. Moreover, Zhu and Chen [
51] found that when the C/N ratio is less than 1, the biological process of nitrification is affected.
In this study, at the 20 ppt salinity, the C/N ratio was above 1, while at the 8 ppt salinity, the C/N ranged from 0.49 to 1 (
Figure 3b). Furthermore, at both salinities (20 and 8 ppt), an increase of the TN concentration was followed by a reduction in the C/N ratio. In the 20 ppt salinity treatment, the C/N ratio was 1.14 and the carrying capacity of the filter bed appeared to be affected. Additionally, the oxidation capacity of the filter was affected by the abundance of heterotrophic bacteria because of the increase in the total nitrogen concentration in the system, although the plant roots as well as the medium increased the surface for nitrification by ensuring the removal of TAN. Reports have shown that when the C/N varies from 1.5 to 2.5, TAN and nitrate removal is necessary for their absorption by the plants [
6]. Timmons and Ebeling [
40] also reported that the C/N ratio at 2.3 seems to ensure the optimal rate of denitrification by increasing the TN reduction rates, whereas in the present study, plants contributed to the reduction of nitrates via adsorption, which lead to a subsequent increase in plant height. Spotte [
6] has suggested that to initiate the process and set up a closed system, the C/N value should be close to 1. According to the results of the present study, the C/N ratio at 20 ppt (
Figure 3) increased from week one to week three and appeared to be inversely proportional to the reduction of TN. On the other hand, the C/N ratio at 8 ppt decreased from the first week to the end of the experiment (
Figure 3) and appeared to be inversely proportional to an increase in TN, which was probably because of the reduced nitrification rate associated with an increase in heterotrophic bacteria. Other studies by Gullian and Aramburu and Zhu and Chen [
41,
51] found that the increased C/N ratio was not inversely proportional to a reduction in the nitrification process due to the available DO in the RAS, although the C/N ratio was greater than 2.7 after the third week of experimentation.
In addition, in this study, the bacterial abundance was measured in two brackish aquaponic systems during the start-up period of system conditioning by generating constant conditions, such as daily feed input rates and daily ammonia production rates. Before the salinity was changed to 20 and 8 ppt, the abundance of bacteria did not show statistically significant differences (
p > 0.05). The results showed that the abundance of bacteria at the start of the experiment (when the salinity was set to the two requested salinities 20 ppt and 8 ppt) in all treatments was statistically higher (20.2 × 10
5 ± 0.92 cells/mL) at 20 ppt compared with 8ppt, where the abundance of cells was 1.08 × 10
5 ± 1.82 cells/mL. The average abundance of bacteria at the end of the experiment was higher (
p < 0.05) at 20 ppt salinity (18.6 ± 0.91 cells × 10
5/mL) than at 8 ppt salinity (6.8 ± 1.9 cells × 10
5/mL). The decreased bacterial abundance observed at 8 ppt in the present study may be related to salinity differences between both treatments and different C/N ratios and HLR, HRT and pH values [
3,
11,
55]. Previous studies [
30] reported that the bacterial abundance in two ornamental freshwater aquariums reached 29 × 10
5 cells/mL and 12 × 10
5 cells/mL for zebra cichlid fish and angelfish, respectively. In batch (closed) cultures, the increase in bacterial cell numbers is usually attributed to a few dominant species, although this was not the case in our study. The bacterial abundance continued to increase until the end of the experiment due to the lack of considerable grazing pressure [
56].
Previous studies conducted in RASs have shown similar increases in the bacterial abundance tendency relative to the results presented in this study and presented values below 0.005 × 10
5 cells/mL at RAS start up to 0.2 × 10
5 cells/mL at the end of the experiment [
11,
57,
58]. Somerville et al. [
3] suggested that in a freshwater aquaponic system, the system balance depends on the nitrate concentration, daily feed input, HLR, SSA of the filter, pH and bacterial abundance. Other studies [
59] suggested that the HRT in the RAS affects the preservation and maturation of bacteria in the filter. As the HRT of the water in the filter increases, bacterial maturation is more easily maintained, which leads to an increase in bacterial abundance. Alleman et al. [
60] reported that nitrifying bacteria exhibit increased resistance to salinity changes and short adaptation times. The bacteria shrink to salinities higher than 2 M [
61] due to electrostatic contraction of the cell and osmotic reactions.
4.2. Biological Elements
The results of the present study show that gilthead juvenile sea bream in a brackish aquaponic system presented a high growth performance (SGR = 3.17%/day), survival rate, and FCR. Khater et al. [
21] reported that tilapia cultured in a brackish aquaponic system showed a high survival rate and increased its weight by 0.78 g/fish/day for four months. Additionally, Pantanella and Colla [
24] and Waller et al. [
62] suggested that sea bass grew on average from 32 g to 54 g at an SGR of 1.5%/day and FCR of 0.93 in a 16 psu salinity aquaponic system over 35 days. Nozi et al. [
25] studied the quality of seabass (
Dicentrarchus labrax) in a brackish aquaponic system (continuous salinity changes from 35 ppt to 0 ppt) to determine the optimal plant growth performance via the addition of inorganic nutrients, such as iron, potassium and calcium and found that the fish were affected by the conditions created. The same study [
25] reported that the levels of saturated and polyunsaturated fatty acids were not similar at all salinity adjustments.
Rock samphire was chosen due to its high nutritional value, aromatic-pharmaceutical properties and natural tolerance to high salt concentrations [
27]. The results of the present study showed that rock samphire showed statistically significant better growth in terms of biomass, height and number of lateral branches at 8 ppt salinity than at 20 ppt salinity. Rock samphire is a facultative halophyte, meaning that it can grow in a wide range of salinity levels. While it thrives when the irrigation water contains up to 250 mM NaCl, it exhibits reduced growth rates when the salinity exceeds this limit [
28]. Nevertheless, rock samphire can survive in seawater [
63]. Previous studies indicated that the range of optimal performance in a hydroponics system occurs between 35 and 171 mM NaCl [
28], which corresponds to approximately 1.8–10 ppt. This finding is consistent with our results, where the optimal growth of rock samphire occurred at 8 ppt salinity. Nozi et al. [
25] cultivated
Beta vulgaris varcicla in a brackish water aquaponic system and found that it presented a statistically significant increase in the growth and length of roots. In addition, Kotzen et al. [
22] reported that eggplant, mint, celery and chili pepper were successfully grown in a brackish aquaponic system, whereas spinach, chives and tomato presented poor yields [
21,
22].
In addition, the two examined tissues of the gilthead seabream exposed to different salinities exhibit a differential HSP induction and MAPK activation with the esxeption of phosphor JNK in the intestine. The critical role and the significance of the HSPs expression and activation of MAPKs in relation to physiological functions when juvenile sea breams are exposed in different salinities remains to be investigated. In euryhaline fish, such as juvenile sea breams, the kidney plays an important role in osmoregulation. Sea bass, trout, herring, and juvenile sea bream can adapt to changes in salinity and are able to survive in both seawater and freshwater. According to Nebel et al. [
64], sea bass juveniles that were successfully adapted to freshwater showed smaller collecting ducts than those cultivated in seawater, which is consistent with our results. The kidney granulomas that were detected in both salinity groups are likely correlated with the long-term storage of formulated feeds or with ascorbic acid deficiency [
65,
66]. The absence or presence of mild histopathological alterations in the midgut, gills and liver of both groups (20 and 8 ppt) indicates that the fish were well adapted to freshwater. Similar results were reported by Giffard-Mena et al. [
67], Laiz-Carrión et al. [
68], and Masroor et al. [
69], thus indicating the high plasticity and gill remodelling of sea bass adapted from seawater to freshwater.