INTRODUCTION

The increasing global population and the limiting global capture fisheries undeniably increase the demand of aquaculture. On the other hand, those will also bring about limitation to aquaculture expansion in particular of land and water utilization. Therefore, productivity enhancement in term of total production per input used becomes one of the major priority in the development of aquaculture (Avnimelech et al., 2008).

Aquaculture is the culture of aquatic organisms under controlled conditions (FAO, 2016). Among these organisms, tilapia (Oreochromis niloticus L.) is the second main species cultured in the world, and the common carp Cyprinus carpio (Linnaeus, 1758) is the first one (FAO, 2018a). However, the development of aquaculture has faced challenges because of lack of land and water source, feed costs and degradation of environment (Rahman, 2012).

The common approaches to maintain water from deterioration and avoid nitrogen increases are through water exchange, using nitrifying biofilters and the microorganisms that grow while using a carbon source (Avnimelech and Kochba, 2009).For decades, re-circulating system (RAS) has been considered as the main application for intensive rearing of several species. However, operating and implementation costs of all structure considered high. For tilapia culture, BFT was more effective in terms of cost–benefit than RAS (Luo et al., 2014).

The biofloc technology system (BFT) provides the intensive aquaculture with no or minimum water renewal reducing its environmental impact (Poli et al., 2019). In this system, the management of the microbial community is determinant to keep the water quality, especially the development of heterotrophic bacteria, through the complementary carbon source, which stimulates its growth and improves the process of removing inorganic nitrogen from water, besides allowing its transformation into bacterial biomass (Robinson et al., 2019).

The stocking density in a BFT depends on the manipulation of the physicochemical variables that determine the quality of the water. The amount of dissolved oxygen (DO) is the first limiting factor to increase the density of the culture and the second one is the ammonia nitrogen that comes from the metabolism of food in fish (Timmons et al., 2002). In this context, the objective of this experiment was to study the effect of BFT application on water quality, growth performance and feed utilization of Nile tilapia fingerlings (Oreochromis niloticus) cultured at different densities comparing with a control group.

MATERIALS AND METHODS

Study site

The experiment was conducted at the experimental fish Lab, Department of Animal and Fish Resources, Faculty of Agriculture, Suez Canal University, Egypt (30° 37’ 08” N 32° 16’ 19” E) in 7/2019 To 10/2019.

ExperimentalFish

Monosexnile tilapia, O. niloticus fingerlings obtained from private fish Hatchery (Khalil Saad Khalil Hatchery) Tell El Kebir Center, Ismailia Governorate (30° 18’ 45” N 31° 53’ 02” E). Fish was transported in containers and used of pure bottled oxygen during fish transport,Fish R. Prior to the start of the experiment, fish was acclimatized to laboratory conditions for two weeks and fed twice daily with commercial diet (38%CP).

Experimental design

After the acclimatization period, all tanks were stocked with Nile tilapia fingerlings with an initial body weight of 20.35±0.35 grams andbody length 10±0.25cm at three different stocking densities. Completely randomized designed experiment with three different stocking densities (200, 300 and 400 Fish/m³) under Biofloc system and Clear system (control) representing six experimental treatments in triplicate. Experiment was carried out in 18 cylindrical fiberglass tanks with water volume 50 litre, in the experimental fish Lab.

Experimental conditions

The tanks were supplied with well water source. Aeration was continuously provided using an air blower (220 Watt). Fish were held under natural light (12:12 h, light: dark schedule). In the tanks representing the control treatments (clear system); water was exchanged daily, while for experimental BFT tanks, no water exchange was done (zero water exchange) except the evaporation compensate.

Experimental Diets

The experiment established for 75 days, for six days week^-1 fish was fed different Commercial diet (30%) ofSkrettingCompanyunder clear and /or biofloc systems. The daily ration was 3% of the total stocked biomass divided into two equal amounts and offered two times a day (9.00 and 14.00). Fish in each replicate aquaria was weighed every 15 days and the amount of the daily allowance feed was accordingly adjusted.

Starch was used in biofloc treatments as an external carbon source, it was added at the same amount of feeding ration to maintain the optimal C: N ratio for activate heterotrophic bacteria growth (>N10:1) (Avnimelech, 1999). Starch was completely mixed in a glass beaker with tank water sample and spread to the tank surfaces at the afternoon time(Azim and Little, 2008).

Water quality assessment

Water temperature and dissolved oxygen using a portable oxygen meter (OxyGuard meter) were recorded weekly. pH values were recorded twice a week using a portable pH meter (OxyGuard meter). Ammonia nitrogen (TAN), nitrite and nitrate were detected biweekly, Total suspended solid (TSS) values were measured twice during the experimental period using Spectrophotometer model (LKB Bichrom UV visible spectrophotometer) according to (APHA, 1992; Mullin and Riley, 1955).

Water sample (50ml) were collected from each tank and filtered by filter papers for analysed Total ammonium nitrogen (TAN), Total suspended solid (TSS), Nitrite-N (NO₂-N) and Nitrate-N (NO₃-N) using spectrophotometer model. Total bacterial count (TBC) was determined according to (AOAC, 1995; APHA, 1992) then reported as CFU mL^-1. Biofloc volume (water settleable solids) was determined weekly using Imhoff cone to monitor the developing of biofloc(Avnimelech and Kochba, 2009).

Growth parameters

Fish Weight gain (WG), Weight gain % (WG%), Specific growth rate (SGR) and Survival rate% (SR) were calculated according to (Recker, 1975; Castell and Tiewes, 1980) using the given formula below:

- Weight gain (WG) = Final body weight (g) - Initial body weight (g)

- Weight gain % (WG%) = (Weight gain (g) / Initial body weight(g)) ×100.

- Specific growth rate % (SGR) = [(Ln FBW - Ln IBW) /day of experiment] ×100. (where: FBW is final body weight (g); IBW is initial body weight (g); ln= natural logarithmic).

- Survival rate % (SR) = (Final number of fish / Initial number of fish) ×100.

Feed utilization parameters

Feed intake (g/fish) is the amount of feed given or supplied during the experimental period for each fish per gram, and used for calculating the following equation:

- Feed conversion ratio (FCR) = Feed intake (g)/Weight gain (g)

- Feed conversion efficiency (FCE) = Weight gain (g)/ Feed intake (g)

- Protein efficiency ratio (PER) = weight gain (g)/protein intake (g)

Statistical analysis

The data were analyzed by Two-way ANOVA using Statistical Analysis System (SAS) version 9.0.0 (2004) program at P<0.05 level to test the effects of stocking densities (different stocking densities 200, 300 and 400 Fish/m³) and system Condition (under clear and biofloc system) supplementation, as well as their interactions The ANOVA was followed by Duncan test (1955) at P<0.05 level of significant.

RESULTS AND DISCUSSION

Water quality

Table (1) present water quality criteria of monosex as affected by stocking density which reared in tanks under clear and biofloc system. High stocking densities combined with highly nitrogenous diets in intensive fish culture negatively affect the water quality. especially the accumulation of inorganic nitrogen forms (NH₃ and NO₂) (Hargreaves and Tucker, 2004). As Means of the water quality parameters recorded during the trial were as followings; temperature (28.3 ± 1.10), pH (7.3 ± 0.03), DO (5.4 ± 0.1), TAN (0.34 ± 0.02), NO₂ (0.21 ± 0.02), NO₃ (5.04 ± 0.02) and TSS (286± 67.5)Table (1). Only stocking density had significant effect on water characteristics including pH,DO, TAN and NO₂. TAN and NO₂ values increased significantly (P < 0.05) as the density increased and recorded the highest values in group (400 fish/m³) over the low density (200 fish/m³) while DO decreased following the same trend for both clear and biofloc system in agreement with Gibtan et al. (2008).

In contrast, some studies suggested that stocking density had no effect on water quality parameters as reported by Li et al. (2012).Higher stocking densities normally resulted in poor water quality which is the main stress factor in the aquaculture ponds (De Oliveira et al., 2012). Oxygen required for fish is generally affected by nitrite and ammonia levels in the culture system (Remen et al., 2008). Exchanging the water in the culture system is essential to maintain the properties of water quality from deterioration. Frequently, using techniques with minimum or zero-water exchange increases nitrogen levels in water (Randall and Tsui, 2002). In the current study, the level of inorganic nitrogen concentrations was increased in BF tanks with increasing the stocking density (Labib, 2016). These findings agree with some studies demonstrating that the in situ biofloc formation accelerates the nitrification process in the water of tanks (Labib, 2016). The bacteria formed in BF system is helping to keep the nitrogen at safe levels for fish rearing by utilizing nitrogen in situ microbial protein and increasing the nitrification process to maintain the ammonia and nitrite at safe levels for Nile tilapia (Mansour and Esteban, 2017a, b).

Table (1). Effect of stocking density on water quality parameters (Mean ± SE) of Nile tilapia (O. niloticus) fingerlings throughout under clear and biofloc system throughout the experimental period (75 days).

In contradiction with our results, Abdel-Tawwab (2012) reported that unionized ammonia was significantly affected by rearing density which in agreement with Ayyat et al. (2011) who reported that the fish stocking density significantly affected the concentrations of dissolved oxygen and pH. As they decreased with increasing fish density, while the concentrations of ammonia, nitrite, and nitrate increased for both clear and biofloc system. Total ammonia nitrogen (TAN) values were in the softy range (0.35±0.10 mg/L). Also, Nitrite (NO₂) during the experimental period showed values around the normal rang (0.5 mg/L). Nitrate (NO₃) was recorded for higher values than normal range (5 mg/L) for all experimental treatments. The present results of TAN and NO2 concentrations at biofloc treatments were lower compared to the control (P<0.05), which was also agreed with other researchers such as (Gaona et al., 2011; Kuhn et al., 2009; Wasielesky et al., 2013).

The concentration of solids tends to increase with the increase in the stocking density due to the increase in the increment of food, feces and carbon source (De Silva et al., 2015). The present results were similar to that in Rajkumar et al. (2016) study, who found that TSS was within the recommended level of <500 mg/L (Samocha et al., 2007).

Biofloc volume and bacterial counts

BF volume and bacterial counts were significantly (P<0.05) lower (23 mg L⁻¹ and 3.7× 10⁶ CFU L⁻¹, respectively) at 200fish/m³and increase with increasing stocking level where the highest values were 34 mg L⁻¹ and 5.8×10⁶ CFU L⁻¹, respectively at 400fish/m³(Table 2).In the present study, higher values of BF volume and bacterial counts were recorded in fish reared at 300 and 400fish/m³as compared to the low density (200fish/m³). The surface of BF and its particle size can increase the surface area required for bacterial growth to increase the produced BF (Mansour and Esteban, 2017a, b). Increasing the substrate surface area in BF tanks resulted in increased bacterial growth and improvement of water quality as well as food availability (Ferreira et al., 2016; Nunes Caldini et al., 2015). The increased amounts of BF can be also attributed to the increased levels of nitrogen emissions as a result of increasing the stocking density. The differences may be due to the differences in fish size, duration and experimental conditions.

Table (2). Effect of stocking density on Biofloc volume and total bacterial counts under biofloc system throughout the experimental period (75 days).

Tilapia growth performance

Growth performance parameters of Nile tilapia in different stocking density of 200, 300 and 400 fish/m³ and management conditions (CS, BS) (p<0.05) are presented in Table (3). The highest final body weight (FBW), weight gain (WG), weight gain percent and specific growth rate (SGR) values recorded for fish at density 200 fish/m³under biofloc system noticed for the highest FBW, WG and SGR, while the lowest results noticed for 400 fish/m³under clear system (CS).

Generally, growth parameters improved under biofloc system. It could assume that starch addition to biofloc tanks activate growth of bacterial floc and algae which in turn act as secondary protein source for fish under those treatments. These result are in agreement with the finding of (Burford et al. 2003) who suggested that adding starch helps to develop and control of dense heterotrophic microbial bioflocs in the water column. Carbohydrate addition leads to elevate the C/N ratio which helps to convert inorganic nitrogen into organic nitrogen as dense floc. In agreement with (Avnimelech et al., 2008). Azim et al., (2007) concluded that growth performance for Nile tilapia in biofloc system improved and it contributed 43% of growth compared with system without BFT.

Similar results were suggested by Zhao et al., (2012) who reported that the bioflocs technology significantly increased the individual shrimp weight at harvest. Contrastively, some other studies suggest that biofloc led to the level of production well be low commercially viable levels(Little et al., 2008). However, there might be several reasons attributed to the poor fish growth and production. Increased turbidity due to biofloc reduces the visibility and hence artificial feed intake. Even when floc separator was used, it was not easy to maintain 500 mg L⁻¹ TSS level, and very often the level reached to 1000 mg L⁻¹ TSS. Maintaining optimum floc levels was also identified as a critical issue in managing BFT systems (Little et al., 2008). Another reason for the poor fish growth and production, water quality parameters were not stable, high fluctuation of pH and alkalinity, high concentrations of inorganic nitrogen species might have chronic effects on fish health (Azim and Little, 2008).

Corroborating to this work, other studies show that regardless of the growth stage, the increase in stocking density tends to reduce the growth of fish in recirculating water systems and ponds ( Ferdous et al., 2014), cages (Moniruzzaman et al., 2015) and BFT ( Zaki et al., 2020).

The growth of tilapia (O. niloticus ) depends on the stocking density, food quality, energy content of the diet, its physiological status, reproductive state and environmental factors such as temperature, pH, etc. (Lovell, 1989). In this study, there was a significant difference in growth (P <0.05) with increasing stocking density in all feed treatments. This result is in agreement with the findings of Tadesse (2007), who studied the effects of stocking density (50, 100, 150 and 200 fish m⁻³ cages) for the same species, and found that the fish size and production were significantly affected by the stocking density. Ouattara et al. (2003) also studied the effects of stocking density (20, 50, 100 and 150 fish m⁻³ cages) for tilapia, and found that fish size and production were significantly affected by stocking density. Canario et al. (1998) also studied the effect of stocking density (0.35, 1.3 and 3.2 kg m⁻³) on the growth of gilthead sea-bream, S. aurata , and found that fish in the highest density group grew 25% slower than fish in the lowest density group.

Table (3). Growth performance of tilapia fed different dietary protein levels under clear and biofloc system.

Some studies have demonstrated that the stocking density can affect fish growth performance even though water quality in aquaculture systems was kept in good conditions (Tolussi et al., 2010). High stocking density of Amur sturgeon (Acipenser schrenckii) has negative effects on growth where, a significant decrease in specific growth rate was observed as stocking density was increased; high stocking density might inhibit fish growth through decreasing food conversion efficiency (Li et al.,2012). A stocking density of 200 fish/m³ produced the best growth performance for tilapia fish. In the same context, increase of stocking density inversely affected the growth of Nile tilapia compared with high stocking density where, final body weight and weight gain percent were significantly (P < 0.05) higher at lower stocking density. Abdel-Tawwab (2012) reported that fish growth was inversely affected by rearing density, thus density of 200 fish/m³ produced the best growth performance. Same results for Nile tilapia were showed by (El-Sayed, 2002; Ayyat et al., 2011).

Normally, stress due to agglomeration, competition for food, and social interactions can reallocate metabolic energy intended for growth to restore homeostasis ( Martínez-Porchas et al., 2009). In this context, the increase in the fish stocking density tends to increase the time needed for tilapia to reach harvest weight (Garcia et al., 2013), as was observed in the present study. Studies of stocking density of tilapia in BFT are usually done for a short period of growth using fish at larvae or juvenile stages (Zaki et al., 2020).

Moreover, other fish species Chinook salmon Oncorhynchu stshawytscha (Walbaum) (Martin and Wertheimer, 1989), African catfish (Haylor, 1991) and Arctic chirr, S. alpinus (Jørgensen et al., 1993) also showed an inverse relationship between stocking density and growth parameters difference of the effect of stocking density can be obtained from the results of specific growth rates Table (3). Silva et al., (2000) also studied the effect of stocking density (2, 3 and 4kg m⁻³) on the growth of tetra hybrid red tilapia, and found that the final body weight gain was significantly higher at densities of 2 and 3kg/m³, while the largest biomass and feed consumption were observed at a density of 4kg/m³.

In this regard, Studies of stocking density of tilapia in BFT are usually done for a short period of growth using fish at larvae or juvenile stages ( Liu et al., 2018; Zaki et al., 2020). The growth parameters and feed utilization were better in BF units than the control groups. These results are in consistence with those obtained with Nile tilapia reared in BF system (Mansour and Esteban,2017a, b). The increased growth performance in fish reared in BF with stocking density (200 fish per m³) resulted from the optimum water quality values and availability of BF for the fish (Qasem, 2016; Yoo and Lee, 2016). The groups reared at high stocking level (400 fish / m³) performed less growth which is in consistent with the results obtained by (Sorphea et al., 2010).

Feed Utilization

Feed utilization of different treatment is presented in Table (4). Regards to stocking density, tilapia stocked at 200 fish/ m³under biofloc showed the highest values for Feed intake (FI), and the best feed conversation ratio (FCR). While the highest protein efficiency ratio (PER) noticed for tilapia stocked at 200 fish/m³. Tilapia fed under biofloc condition noticed for the Best feed utilization parameters. With increasing stocking density, depression in feed utilization parameters were recorded. These results in agreements with the finding of (Li et al.,2012) who suggested that with increasing stocking density depression in feed utilization parameters was noticed. Same was recorded by (Abdel- Tawwab, 2012), It was assumed that chronic stress, due to the high density, increases the fish's overall energy demand, which is then unavailable for growth (WendelaarBonga, 1997). Moreover, fish density can affect the efficiency of feed utilization; as the number of fish stocked in a pond increases, the amount of feed available to each fish decreases (Chang, 1988). Moreover, the food conversion ratio trend that was seen in this experiment is in agreement with that obtained by As compared with the work of Ouattara et al. (2003), This enhancement in the food conversion ratio suggests efficient food utilization through the extraction of more nutrients from the food and converting it into flesh (Bhijkajee and "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x", "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x" Gobin, 1997). The food conversion ratio value obtained in the 50F treatment was better than the best food conversion ratio (12.64) reported by Ouattara et al. (2003) for tilapia at the same stocking density (50 fish m⁻³). Similarly, in the other treatments (100 and 150F), the food conversion ratios of 5.64, and 6.24 obtained, respectively, were better than the food conversion ratios of 12.49 and 13.44 reported by Ouattara et al. (2003) for tilapia at a similar stocking density. In this study, the food conversion ratio increased significantly (P <0.05) under low stocking density than high stocking density and the best food conversion ratio for O. niloticus was obtained for 50F (under low density). This result is in agreement with those obtained by Ouattara et al. (2003) and Bolivar et al. (2006),who reported that the food conversion ratio decreased with increasing stocking density.

Moreover, the food conversion ratio trend that was seen in this experiment is in agreement with that obtained by Khattab et al.(2004) . As compared with the work of Ouattara et al. (2003), 12.49, 13.44 and 12.65 for 50, 100 and 150 fish m⁻³ cage, the findings of food conversion ratio in this experiment (2.48, 5.64 and 6.24 for 50, 100 and 150F respectively) were high.

On the contrary, Siddiqui et al., (1989) reported no difference in the growth or the food conversion ratio of O.niloticus (40.3g average weight) reared in a brackish water (3.5–3.9ppt) tank for 164 days fed on supplementary feeding at densities of 16, 32 and 42.6fish/m³. Watanabe et al., (1990) also reported that feed conversion of Florida red tilapia fed supplementary feed did not differ at densities ranging from 100 to 300 m⁻³. The considerable variations in the results of the present study with those recorded earlier by Siddiqui et al., (1989) and Watanabe et al., (1990) for food conversion ratio might be due to the variations in fish size and age, stocking density, food quality, hygiene and environmental conditions or other unknown factors, whose synergy of effects has not been adequately studied (Diana et al., 1996). There was a strong trend for total production increment with increasing stocking density. These findings are in agreement with those reported by Cruz and Ridha (1991), Alemu (2003) and Watanabe et al., (1990) for tilapia (O. niloticus ). Similar production scenarios were also obtained with many other species such as catfish (Engle and "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x" and "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x" Valderrama, 2001). The positive relationship between stocking density and yield has been noted in culture-based fisheries in reservoirs (Phan and "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x", "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x" de Siliva, 2000; Sugunan and "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x", "https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2109.2008.02021.x" Katiha, 2004).

Table (4). Effect of stocking density on Feed utilization of Nile tilapia under clear and biofloc system.

With increasing stocking density under biofloc system a decrease in PER values were noticed. Avnimelech et al. (1994) estimated that feed utilization is higher under biofloc system. For Tilapia better values for feed conversion ratio, protein efficiency ratio and protein productive value were observed in all the bioflocs treatments compared to those treatments without biofloc (Avnimelech, 2007). Same trend was noticed for tilapia feed utilization under biofloc system by Azim and Little (2008). These observations suggested also for shrimps by Zhao et al. (2012) who reported that the biofloc treatment for shrimp resulted in 12.0% higher protein efficiency ratio, and 7.22% lower feed conversion rate comparing with treatments without biofloc. reduction in inorganic nitrogen (Wahab et al., 2003).Low toxic inorganic nitrogen levels and utilization of microbial cells are demonstrated to be an effective potential food source for tilapia and shrimp (Avnimelech, 2007). Furthermore, lower ammonia nitrogen in the sediment positively influenced the food intake and health of the shrimps (Avnimelech and Ritvo, 2003). Another reason for the improvement of feed utilization under biofloc system is that the increased activities of digestive proteinases indicated enhanced digestive capabilities of the feed (Xu et al., 2012). As a massive number of live microorganisms existed in the bioflocs, they could transit through the stomach into the intestine and interfere with resident intestinal micro flora balance which plays an important role in the production or secretion of digestive enzymes (Xu et al., 2012).

We obtained a significant (P<0.05) differences in feed conversion ratio between treatments. However, feed conversion ratio was 1.62, 1,76 and 1.96 for stocking densities (200, 300 and 400 Fish/m³) respectively. The better food efficiency ratio of tilapia was found at lower stocking densities ( Moniruzzaman et al., 2015). According to Ellis et al. (2002), the increased energy demand associated with stress has a negative effect on feed conversion ratio, a fact not observed in this study. However, it is possible that if the fish from treatments of higher stocking densities were allowed more time to reach the same final body weights as from the treatments of lower stocking densities, the feed conversion ratio would be worse.

CONCLUSION

It could be concluded that that stocking density (200 fish/m³) was the best under biofloc system in terms of growth performance, feed utilization, water quality and the total count of zooplankton under these experimental conditions. This system can play a key role in developing a sustainable aquaculture via better water quality maintenance decrease in feed requirements, reduced use fishmeal or other protein sources costly in feeding and higher production to achieve more profit in fish farming in Egypt.