3.1. Water Quality
At the beginning of the experiment (d1), the water quality was similar between the treatment groups, with high DO and pH-values, low conductivity, high redox potential and very low levels of NH4+-N, NO2−-N NO3−-N, and PO43−-P, originating from the properties of the aerated tap water. Although the pH dropped over the course of the experiment, this change took place during the first 20 days in a continuing manner, allowing the fish to acclimatize to these changing conditions.
Addition of P
2O
5 did not simply result in proportional increase of PO
43−-P concentrations inside the water. This disproportional response was probably a result of precipitation of PO
43−-P with Ca
2+ and Mg,
2+ a phenomenon which is well understood [
22,
23]. This is supported by the increasing turbidity of the process water, significantly decreasing levels of dissolved Ca
2+ (
p < 0.05), and by the trend of decreasing levels of dissolved Mg
2+ after addition of P
2O
5. Using a protocol based on P
2O
5 dose and analytical response, the target levels of P40 and P80 were achieved. By trend, the levels of PO
43−-P in the group P120 mostly remained above the other concentrations with a high standard deviation, while the P0 group in maximum reached 8.9 mg L
−1 caused by natural excretion of the fish.
Only minor differences in the water quality parameters were observed between the groups. K
+, NH
4+-N and to lesser extent NO
2−-N started to accumulate from day 30, in all systems, indicating an overload of the biofilters. As regular water exchange remained, the water quality parameters were well appropriate for
C. gariepinus [
24,
25,
26,
27]. Given that our statistical analysis revealed significant differences in DO, temperature, pH-value and redox-potential between the groups, we cannot rule out minor influence on our results. However, due to the small magnitudes of these values, and because of the inevitable precipitation of Ca/Mg-phosphates, we estimate the observed effects to be monofactorial, resulting from the major differences in PO
43−-P.
3.2. Mortality and Growth Performance
The mortality during the experiment was very low (2.6%). Considering that of five dead fish three died in P0 and two in P80, the mortality was likely not a result of the increased PO43−-P concentrations in the rearing water.
For juvenile African catfish, we presumed an exponential growth (body weight
d0 × exp
(k × t), with k = specific growth rate and t = days of the experiment). From our own previously recorded experimental data [
20], we presumed a FCR of 1.0 and a daily feed ratio of 2.2. The FCR turned out to be lower (0.71–0.73), which can be considered as normal for smaller/younger fish [
11,
28]. After the acclimatization period, the fish always ate their scheduled ratio, but appeared to be close to maximum feed intake, which is important in commercial systems to maximize growth. The daily feed ratio was therefore also comparable with that of commercial systems, so we estimated it to be appropriate.
The dietary composition of the commercial feed, especially the levels of P, was adequate according to the literature [
4]. Although feed intake appeared to increase with elevated levels of PO
43−-P, this was not a result of increased appetite but in correspondence with the feeding curve and based on the initial body weight. Using the initial life weight as co-variant, no significant differences in TFI were observed. Consequently, optimum FCR (0.71) and SGR (2.66% d
−1) were by trend best in P40 and P80. Similar positive effects in response to moderately increased concentrations of PO
43−-P were also observed with turbot (
Psetta maxima) [
19] and tilapia (
Oreochromis niloticus) [
29].
The analysis of the whole fish revealed that body composition was in line with literature data, with only the levels of P, Ca and protein being higher, and the largest difference in direct comparison in the group P120 [
30,
31,
32]. The analysis of the fillets revealed a higher content of ash and protein in our study, a lower fat content, and minerals in a similar range [
33,
34,
35]. In our study, the analyzed fillets revealed higher proportions of ash, partially higher or comparable protein levels and similar or lower fat contents [
31,
32,
34,
35]. The levels of mineral contents were in range with literature data or even higher [
31,
32,
33]. For commercially produced African catfish (
C. gariepinus) fillet from intensive production in Germany, Wasenitz et al. [
36] found lower levels for ash and P
2O
5 4.2 g kg
−1 wet weight (ww) (value converted into P was 1.84 g kg
−1, in ww) and higher values for dry matter and protein. Wasenitz et al. [
36] determined almost double values for fat when compared to our data.
An interesting observation is the difference in P and Ca content between the juvenile fish at the beginning of the experiment (baseline) and the final body composition. The juvenile fish were fed with starter feed, containing higher levels of P, protein and lower levels of fat. The young fish had higher levels of Ca and P when compared with the final body composition of the fish at the end of the experiment, which was reduced by about 18% (Ca) in P0. They had neither access to a carnivorous natural diet with higher P/Ca contents when compared with the commercial growth out feed, soluble P, nor to sediments with organic/inorganic P deposits like fish in natural habitats. With increasing PO43−-P concentrations inside the water, the Ca/P content in the body also increased. This indicates that the P-content in the water influences the mineralization status of the fish bones, where most Ca/P is deposited. We also observed that, if PO43−-P increases, the fat content increases. This suggests that the fat content in the fillet can be manipulated via the levels of PO43−-P in the water. Our data demonstrate that the fish is able to uptake PO43−-P directly from the water, and requires adequate P concentrations to build up fat. The fish held in the P0 group with PO43−-P < 2.6 mg L−1 most likely mobilized P from the bones and were therefore unable to keep up with the growth when compared with the fish in P40 and P80. As a consequence, generally elevated levels of PO43−-P between 40–80 mg L−1 inside the rearing water increase the growth potential of African catfish RAS, because of higher feed efficiency. The content of P inside the fillets was rather constant at about 1% on a dry matter (dm) basis.
When challenged with elevated levels of PO
43−-P, African catfish increased the ANNUs of Ca and P by trend up to 120 mg L
−1, indicating higher bone mineralization [
15]. Likewise, the ANNU of protein slightly increased by trend from group P40 to P120.
Table 5 shows that the ANNU-efficiencies of Ca and P increase under elevated levels of PO
43−-P. Because the feed was the same in all groups, the difference of P between P0 and the other groups is a result of utilization of PO
43−-P from the water, allowing better mineralization of the bones, which is also evident in the higher utilization efficiency of Ca under elevated PO
43−-P. This effect obviously occurs under the use of P limited diets.
Growth can be defined as the assimilation of energy in the form of fat and protein. To be available for growth, the gross fat and protein from feed (net energy) must be: (1) digestible; (2) metabolizable; and (3) not required for maintenance energy expenditure [
2,
37]. Maintenance energy expenditure is the sum of energy used for swimming activity and the maintenance metabolism, the vital life functions [
38]. The partition of energy allocated to maintenance is substantial, as it accounts for about 15–30% of the gross energy intake [
39]. If growth is increased under restrictive feeding (our impression was that fish were not fully satiated), the observed trend in the P120 must result from reduced maintenance energy expenditure, resulting from elevated levels of PO
43−-P. We observed that the catfish significantly reduced their activity under increased concentrations of PO
43−-P (further discussed in
Section 3.3), possibly allowing the fish to utilize feed derived energy and nutrients for growth.
In fish, the maintenance metabolism is mainly dominated by ionic and osmotic regulation [
40]. Also the maintenance of phosphorus requires energy. If dietary P is deficient, increased effort (energy) has to be invested to maintain P-homeostasis. Fish are able to uptake P from the water [
41]. The primary and subsequent uptake pathways for organic or inorganic P in African catfish were not assessed, however, in trout these were identified in the proximal and distal intestine, subordinate uptake is provided by the gills [
42,
43]. Reabsorption and excretion of P from the urine is provided through the kidneys [
44]. It can be speculated that additional supply of P to the water allowed the animal to reduce the energy expenditure for P uptake from feed and water via intestine and the gills, and possibly for renal resorption. This energy could be incorporated as fat.
Considering that an increase in PO
43−-P also slightly increased protein assimilation efficiency (from P40 to P120), the elevated concentrations of body fat content under high PO
43−-P are unlikely a result of increased lipogenesis of dietary protein, but rather a direct deposition of dietary fat. Consequently, either the concentrations or the digestibility of the dietary P was too low (the feed specifications indicate only plant and animal P sources), or that the uptake via the intestine was exhausted. In either case, PO
43−-P will be used to supplement the needs of the animal. It is conceivable that the variation of PO
43−-P in the water prompted intrinsic responses of major players of P homeostasis. Indeed, studies in fish and mammals showed that the dietary P supply impacts on various tissue sites and body compartments such as gut, kidney and bone [
45,
46,
47]. However, molecular specificities of associated regulators, transporters, and endocrine and paracrine signals are largely unknown and yet to be elucidated in African catfish. It should be noted that levels of NH
4+ and glucose remained unaffected by treatments which indicate regular nutrient utilization and energy homeostasis. Serum Ca
2+ and NH
4+ remained unaffected by the treatment, therefore the homeostasis/excretion via the kidney was likely not impaired [
25,
44,
48].
3.3. Welfare
The welfare of fish is usually studied via functional and behavioral approaches. Functional approaches are used to evaluate whether an animal can cope physiologically with its environmental or husbandry conditions. Behavioral approaches are used to evaluate the ability to perform natural behavioral patterns [
49]. Environmental influences, primarily changes in water quality, can adversely affect fish welfare [
50,
51,
52]. Thus, it is possible that the physiology, in particular stress response reactions, is influenced [
53,
54]. Accordingly, behavioral changes may also occur.
We observed that with increasing concentrations of PO
43−-P, African catfish shifted towards a lighter grey tone of the skin. Catfish are able to alter their skin coloration to lighter or darker. Melanosomes inside the skin are responsible for this action, as they may change in number and size [
55,
56]. Different mechanisms causing this change are described, such as adaptation to the background color [
57,
58] and light regime [
59] of their environment, dietary composition [
60], hormones [
55] and environmental stress [
61]. A direct effect of PO
43−-P in the water on the skin coloration of fish is not described in literature. However, the skin of the scaleless African catfish could also be affected in regard to mineralization [
15]. It is known that the mineralization of the skin can be affected by dietary P [
62]. In this case, the lighter skin coloration would be the result of calcium-phosphates that precipitated inside the skin [
17,
63], to be tested in subsequent studies.
Alterations of the cortisol level is often used as an indicator for stress in welfare investigations [
43], because it reflects acute and long-term (chronic) stress [
53,
64]. Cortisol production can be influenced by environmental impacts, such as pollutants in the water [
54]. To our knowledge, there is no study addressing cortisol changes resulting from increased PO
43−-P concentrations in the rearing water available. In our study, we did not find significant differences in the plasma cortisol response between the treatment groups. Slightly elevated plasma cortisol levels due to netting stress occurred over the course of the sampling procedure. Under this consideration, the plasma cortisol amplitude increased regularly with ongoing sampling in every group but was not affected by the PO
43−-P concentration in the rearing water. This also demonstrated that an alteration of the cortisol level due to PO
43−-P concentration did not occur. Alterations of the blood glucose concentrations were similar in all groups and in a typical range for
C. gariepinus [
65]. Further effects such as immuno- and growth-depression as a consequence of chronic hypercortisolism [
44] could not be observed in our study. Therefore, we suggest that the stress level following PO
43−-P exposure below 80 mg L
−1 can be considered as low.
In the P120 group, the fish altered their agonistic behavior, also seen in the highest number of biting wounds compared with P0 to P80. However, differences between P40, P80 and P120, or P0 and P80 were not significant. This trend correlated with significantly less group air breathing and swimming activity from P0 towards P120, leading to the assumption that the observed elevation in the number of biting wounds was due to behavioral changes. One explanation is an adaption to the water conditions, in particular regarding turbidity. African catfish have only poorly developed eyes [
66] and are nocturnal. It can be speculated, that inside the turbid water, the fish react differently on each other; therefore interaction (visual, tactile sense) only occurs when the animals are very close, triggering a more aggressive response, such as biting (discussed in Baßmann et al. [
65]). African catfish are normally territorial animals. Under high stocking density, this territorial behavior is suppressed, and the fish tend to school up and spend much time resting, provided that feed and DO are adequate. Under high concentrations of DO, the African catfish is described as a faculatative air breather [
67]. If however, the fish returns from air breathing at the surface, finding a resting place may disrupt the school and trigger an aggressive response in other fish. Because African catfish are more active at twilight and/or night, Britz and Pienaar [
68] demonstrated a link between light intensity, swimming activity, air breathing and agonistic behavior, resulting in injuries and cannibalism. When exposed to bright light, the animals in these studies were less active and reduced air breathing, but spend more time resting, were more territorial and had increased numbers of injuries. This is corresponding to our results. Possibly, the diffuse light conditions during the day in our experiments in combination with the increased water turbidity under high PO
43−-P is perceived brighter than without turbidity by the fish. To find out which exact mechanism (e.g., the visual or tactile sense) caused the increased number in biting wounds under high PO
43−-P concentrations, the experiment would have to be repeated under total darkness.