Nitrogen and phosphorus flux in wastewater from three productive stages in a hyperintensive tilapia culture

In this research, effect of productive stages in nitrogen and phosphorus excretion in wastewater from hyperintensive tilapia (Oreochromis niloticus) culture was evaluated. Fish were cultivated considering three development stages (fingerling of 1.79 g, juvenile of 36.13 g, and adult of 72.96 g). Nitrite, nitrate, ammonium, and phosphorus concentration were determined in order to know the amount of nutrients excreted per productive stage of the fish at a high stocking density. Biometric data were recorded during the experiment with the purpose of determining the growth behavior of fish, as well as the measurement of the aerobic metabolism. Results showed that survival, growth, and health of fish are not affected by hyperdensity of culture; as well, combined catabolism of proteins and lipids was presented as substrates for energy with value for O:N ratio ranging between 20 and 60. In addition, higher concentration in excretion of nitrogen compounds and phosphorus per gram of fish was recorded in wastewater from a hyperintensive culture in fingerlings than in juveniles and adults. These results suggest the use of this wastewater in the early stages of fish growth, aiming to enhance sustainable systems with maximum use of the resources, such as aquaponics systems.


INTRODUCTION
Human society faces the immense challenge of increasing food production; for this reason, work has been done for the production of food that optimizes natural resources and provides essential elements for human nutrition, an example is the production of fish (Avnimelech & Kochba ). Among the most cultured fish is the Nile tilapia (Oreochromis niloticus), which is the second largest group of cultured fish in the world with almost 5.6 million metric tons produced in 2015 (Fitzsimmons ). In recent years, there has been a great demand for Nile tilapia due to its high quality of protein, mainly due to its protein and carbohydrate digestibility, because of the species own metabolisms (Barreto-Curiel et al. ). To meet this demand, tilapia has been cultured in aquaculture production systems that are among the fastest growing animal food-producing sectors, accounting for almost half of the total food fish supply (FAO ).
One of the main problems of aquaculture production systems is their wastewater; these chemical-laden waters that are discarded by aquaculture operations have become a problem throughout the world due to their potential for environmental contamination (Chowdhury et al. ).
Aquaculture effluents are rich in dissolved solids and suspension containing mainly nitrogen and phosphorus generated from the excretion of fish, feces, and uneaten food (Summerfelt & Clayton ). Works reported in the literature show that 36% of the fish feed is excreted as organic waste, about 75% of the nitrogen and phosphorus consumed is unused and remains as a residue in the water.
Depending on the species and the type of crop, more than 85% of the phosphorus, 80-88% of the carbon, and 52-95% of the nitrogen that enters a fish culture system are lost in the environment (Zou et al. ). According to studies carried out (Zhang ; Hu et al. ; Endut et al. ; Vilhelm et al. ), the amount of dissolved nutrients that are released in a system depends on species, and quality of the food and management in the culture system.
Moreover, most recently, it was detected that fish growth rate and increasing feed input with water exchange rates resulted in highly variable proportions of the macro-nutrients during the aquatic production cycle ( Jia et al. ).
Until now, few studies have been carried out where the balance of nutrients and energy is recorded, as well as the use of water in highly productive tilapia systems. Thus, the works reported in the literature have been focused on flow of nitrogen and phosphorus, mainly. Schneider et al.
() studied nutrient conversions in intensive integrated aquaculture systems; the evaluation was made based on the balance of nitrogen and phosphorus with tilapia from 54 to 128 g. They concluded that integrated systems have their specific limitations, which are related to uptake kinetics, nutrient preference, unwanted conversion processes, and abiotic factors. Wahyuningsih et al. () presented a study about an aquaculture nitrogen waste reduction in an aquaponic system and indicated that integration of tilapia fish farming with organisms of 20 g, romaine lettuce, and bacteria can reduce inorganic nitrogen with the best removal efficiency.
Later, Vilhelm et al. () analyzed the influence of ration size on nitrogen retention in tilapia with an average body mass of 77.9 ± 1.7 g. The lowest rates of nitrogen excretion were observed for fish receiving meal sizes corresponding to 3% of their body mass. In contrast, fish fed ration sizes of 1% displayed a reduction in apparent digestibility of protein, nitrogen-free extract and dry matter, in addition to excreting a disproportionate amount of ingested nitrogen as ammonia and urea. In the same year, Cerozi & Fitzsimmons () quantified a phosphorus flow, phosphorus mass balance and evaluated phosphorus removal efficiency by hydroponic lettuce integrated with tilapia aquaculture of 20 g of weight. These authors report a high excretion of phosphorus by fish, which can be used by plant species, demonstrating that aquaponics is an excellent tool for the reuse of this nutrient to obtain a high quality culture.
In Osti et al. (), utilized environmental indicators as a quantitative method to evaluate and discuss the nitrogen and phosphorus flux in a tilapia system with fish initial average weight of 191 g. They proposed management oriented to a better use of feed to prevent waste production, making adaptations in food management to avoid loss of nutrients.
As observed in previous reports, studies have focused on adult fish, so the amount of nutrients in the wastewater of aquaculture systems is unknown according to the stage of development of the organism. Thus, the improvement of aquaculture crop efficiency requires detailed knowledge of the nutrient cycle (Adhikari et al. ). For this, it is necessary to understand the flow of nutrients in the aquaculture system, from its inputs to the outputs, as well as throughout the production cycle (Cerozi & Fitzsimmons ), from the sowing of the fingerlings, to the harvesting of the adult fish.
This allows us to identify the destination of nutrients to achieve waste reduction and to promote the use of them.
Thus, the present study was carried out to quantify nitrogen and phosphorus proportion in a hyperintensive tilapia aquaculture system through its life cycle to be a decision support system for nitrogen and phosphorus management in sustainable systems.

System design
In order to provide uniform environmental conditions for the culture of tilapia, the system design was located inside a poly- To control the temperature an Electro EVO 230 V Electric Engineering system was employed for a water flow of 1,000 to 17,000 L/h. To oxygenate water in all the tanks, a Topaz Airsep oxygen generator was used; this generator can produce at 6 LPM, at 9 psig with a purity of 93% ( Figure 1).

Fish culture conditions
The culture of tilapia consisted of 240 fingerlings, 240 juveniles, and 240 adults, with an initial average body weight of 1.79 g (15 days), 36.13 g (60 days), and 72.96 g (90 days), respectively, distributed in nine ponds. Fish were fed three times a day with commercial balanced food according to their stage at approximately 10% (fingerlings), 5% (juveniles), and 3% (adults) of body weight per day.
A temperature of 28 C and an oxygenation range of 4-6 mg/L of O 2 were maintained. Additionally, handling of tanks involved the removal of feces, along with a weekly partial water exchange (30%).
In order to monitor physical water quality, dissolved oxygen, pH, and temperature were measured daily during the experiment; this measurement was realized with a multiparameter instrument (Hach series HQ40d).

Nutrients' content analysis
Chemical determinations were carried out in three phases to know the behavior of nutrients over 24 hours, 5 days, and 60 days; each of the phases of chemical determinations was achieved for the three stages of fish development. All samples were taken in triplicate for each treatment. In the 24-hour phase, samples were collected every 4 hours; for the 5-day phase, samples were taken once a day; after that, samples were taken weekly until the 60 days of experimentation were completed.
The criterion for the period of creation of the determinations was based on the need to know the behavior during a 24-hour cycle of the fish in each of the stages.
After that, it was necessary to know the moment in which the nutrients reached toxic levels for the fish and to establish the periods and percentages of water changes.
Finally, the duration of 60 days of the experiment was considered so that each of the development stages reached the stage of the next treatment, in order to cover most of the development of the fish. Determinations of different forms of nitrogen present in water: ammonia (NH 3 À N), nitrates (NO 3 À ), and nitrites (NO 2 À ), as well as phosphorus (PO 4 À ), were made.
To carry out chemical determinations in water, 250 mL of water sample was taken manually from each tank. Water was collected, in triplicate, directly from the tanks in plastic containers, trying to obtain the sample from the same place to minimize variations. Analyses were carried out immediately after collection of the sample. In addition to the concentration of each of the nitrogen compounds and orthophosphates, the flow of nutrients was calculated, in the following equation: where L ¼ load of N and P-PO 4 (g day À1 ), [ ] ¼ nutrient con-

Metabolism analysis
Three individuals from each treatment were randomly selected to measure aerobic metabolism (oxygen

Growth performance
The following parameters were used to evaluate tilapia growth performance according to Kumar & Garg ():

RESULTS AND DISCUSSION
Nutrients content analysis This is explained by the difference in stocking density that was used for both researches.
The maximum values of nitrogenous and phosphorus excretion presented by adults indicated the amount and frequency of water exchange in the system. In this case, ammonia presented very high levels that could affect the development and survival of organisms; therefore, partial water changes were scheduled to avoid damage to fish. In the case of adult tilapia, this study used a lower average initial weight (72.96 g) than that used by Gichana et al.  Average values for each treatment followed by a superscript letter indicates that there is a significant difference (P < 0.05).  density, as well as the size of organisms and the quantity and quality of feed provided, among other factors; thus, making a comparison with other studies was difficult due to the difference in the density of the culture.
Calculation of the nutrient flow (Table 3)  Additionally, in order to know the amount of nutrients excreted per gram of fish in each of its productive stages at high cultivation densities, data of the concentration per gram of fish are presented. Figure 2 shows the nutrient concentration per gram of fish for three productive stages of tilapia in 24 hours, observing the same trend in all nutrients analyzed. Fingerlings have a higher concentration in nitrogen and phosphorus excretion, considered per gram of fish, followed by juveniles, and the lowest concentration of nutrient excretion is by adults. This shows that metabolism of fish in its early stages is more accelerated; hence, the concentration of excreted nutrients was higher. That condition may be a factor for the use of these nutrients in sustainable production systems, such as  () study, indicating that there is no effect on aerobic metabolism with increasing density. The difference in stages did not have a significant effect on the type of metabolic substrate used as the main source of energy ( Figure 4).  That is why this relationship is shown in the three productive stages of tilapia analyzed in this study ( Figure 5).

Growth performance
In the length-weight relationship, the regression coefficient b provides information about the type of growth; if b ¼ 3 the growth is isometric and when b ≠ 3 the growth is allometric.
In fingerlings and juveniles of the present study, growth was isometric, indicating a good physiological state.
Researchers have carried out studies regarding the weightlength relationship of O. niloticus under different conditions, resulting in most isometric growth conditions. This confirms the fact that tilapia is an adaptable species to different conditions. One of the studies was carried out by   Average values for each treatment followed by a superscript letter indicates that there is a significant difference (P < 0.05). The difference in length-weight relationship obtained during the three development stages of fish indicates that fish do not normally maintain the same shape or body conformity at different productive stages. This variation can also be attributed to the variation in sample size, growth stage, and environmental factors. There was no negative effect on physiological health derived from the hyperintensive density of culture that was administered in this study, which is greater than reported in the aforementioned studies.

CONCLUSION
In this research, a comparison of nitrogenous excretion (nitrites, nitrates, and ammonium) and phosphorus in residual water of a hyperintensive tilapia culture is presented. Based on the results, it might be concluded that a higher concentration per gram of fish is generated in tilapia fingerlings than in juveniles and adults in a hyperintensive culture of 270 fish/m 3 .
According to the data presented, the density of the culture did not affect the survival of the organisms in any of the productive stages, as well as the growth or health of the fish; in addition, combined catabolism of proteins and lipids was presented as substrates for energy. This research might contribute to the improvement in the use of wastewater from aquaculture systems towards plant production, with greater use in the fingerling stage. As the nutritive solution of hydroponic vegetable crops is an important economic factor, a way is sought to avoid the supplementation of these fertilizers, which is why the study provides important information on the flow of nitrogen and phosphorus with a view to their use in the generation of plant products, avoiding the pollution generated by the discharge of aquaculture effluents. In addition, it shows the carrying capacity of a hyperintensive culture, improving fish farming, by obtaining higher production, estimated at 80 kg/m 3 , without compromising the growth and quality of the fish.