The biofloc was made in a fermenter fiberglass tank (300 L) at the Inland Water Branch of the National Institute of Oceanography and Fisheries (NIOF), using filtered water from River Nile, the Delta Barrage located in the Kalubiya Governorate, Egypt, following the protocol presented by Helal et al. (2024). Briefly, in a beaker, sugarcane molasses was diluted with water before being added to the fermentor. The tank bottom was continuously cleaned, and the evaporated water was replaced. To prevent the biofloc from settling, continuous aeration was provided. Sugarcane molasses was used as a carbon source over 30 days, and daily adjustments were made to keep the C/N ratio at 1:10, as previously mentioned by Avnimelech (1999). Each day, the potential volume of biofloc was added to each group after correctly incorporating biofloc, ensuring that the Nile tilapia juveniles received a natural diet. Following the methods previously outlined by Avnimelech (1999), the required volumes of biofloc were examined weekly using the Imhoff cone. Once a week, biofloc volumes were recorded in culture water using an Imhoff cone, which involved dumping 1 L of water from each tank for 15 to 20 minutes. The proximate compositions of biofloc were performed following the recommended protocol by AOAC (2003) based on the dry matter content (%), total protein (44.27%), ether extract (5.29%), ash (4.77%), crude fiber (4.69%), and nitrogen-free extract (40.58%), while gross energy (482,225 kcal kg⁻¹) was calculated.

Using the standard procedures of American Public Health Association (APHA), as reported by Beutler et al. (2014), a variety of water quality variables were measured during the experimental period. Un-ionized ammonia (NH₃), nitrite (NO₂), nitrate (NO₃), total alkalinity (T-Al), and total dissolved solids (TDS) were assessed weekly, whereas dissolved oxygen (DO), pH, and temperature were measured daily (at midday).

Nile tilapia (O. niloticus) juveniles, with an average initial weight of 13.3 g and length of 9.5 cm, were obtained from a private Nile tilapia hatchery and transported to the Inland Water Branch of the National Institute of Oceanography and Fisheries (NIOF) for acclimation. The fish were acclimatized for 15 days, during which time they were manually fed twice a day with a commercial diet from ALER Aqua Egypt Company (28% protein, 6% ether extract, 7% ash, 4% crude fiber, and 55% nitrogen-free extract, with a gross energy of 452,600 kcal kg⁻¹). After 15 days of acclimatization, the fish were randomly chosen and restocked in 18 fiberglass tanks of 500 L, with a rate of 40 fish/tank (three replicates/tank), filled with filtered agricultural water.

In this study, six biofloc groups (with three replicates per group) were tested and supplemented to the water culture for a 60-day rearing period. These selected groups are based on our previous work (Helal et al., 2024). The first group (control) did not have any biofloc addition per volume in a water culture (T₀ = 0 g L⁻¹), while in groups 2–5, biofloc was supplemented, as follows: T₁ = 14.2 g L⁻¹, T₂ = 28.4 g L⁻¹, T₃ = 42.6 g L⁻¹, T₄ = 56.8 g L⁻¹, and T₅ = 71 g L⁻¹. There was no daily water exchange in the fish tanks, except for cleaning the tank’s bottom of remaining food and fish feces. Every day, biofloc volumes for all groups were established by applying the Avnimelech (1999) guidelines. The volumes were then supplemented with new biofloc volumes from the fermentation tank to keep each group’s biofloc levels at the suggested levels. The fish dietary rates were adopted at 3% of the total biomass and changed once a week, following the weekly weighing of randomly selected specimens of fish (Azim and Little, 2008).

The initial and final length (IL and FL, respectively) and weight (IW and FW, respectively) of fish were determined to calculate daily weight gain (DWG), weight gain rate (WGR), and length gain rate (LGR). Moreover, specific growth rate (SGR), survival rate (SR), and feed conversion ratio (FCR) were calculated based on the recommended equations as previously reported.

Following the guidelines of AOAC (2003), the proximate compositions of fish were determined. At the termination of the experiment, random fish samples (n = 5) were chosen from each tank. After fish euthanization, samples were homogenized using a blender, oven-dried, ground, and stocked at −20°C until the analysis. Dry matter, total protein, ether extract, and ash (based on dry matter content %) were determined.

To determine serum biochemical composition, blood samples from 15 fish/treatment (5 fish/replicate) were collected at the termination of the experiment. Using a syringe containing 15 units mL⁻¹ of heparin, blood samples were taken from the caudal artery. Serum was obtained by centrifuging the remaining blood samples for 20 minutes at 585 × g. Following the guidelines recorded by Wootton et al. (1982) and Lowry (1951), the total albumin (ALB) and total serum protein (TP) were determined, respectively. The total globulin (GLB) was calculated by subtracting the albumin value from the total serum protein value. The serum creatinine and urea were determined following the protocols by Larsen (1972) and Henry et al. (1974), respectively. The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined following the procedures by Reitman and Frankel (1957).

In this study, the zooplankton were identified and counted at three time points: at the start of the experiment (D-0), the middle of the experiment (D-30), and the end of the experiment (D-60). This was to investigate the impact of biofloc water supplementation on the zooplankton community, abundance, and structure, as well as the culture of these small invertebrates as potential prey for cultured Nile tilapia during the experiment period.

To achieve these objectives, a zooplankton net (55-µm mesh size) was used to filter 5 L of water from each subsurface layer of the tank. The samples were then immediately transferred to plastic jars containing a 5% formalin solution for preservation. A 1-mL sub-sample was brought into a Sedgwick Rafter Cell for counting purposes in the laboratory and investigated using a binocular microscope. Using the identification guides by Koste (1978); Einsle (1996), and Smironov (1996), zooplankton organisms were identified at the species levels. Each replication (n = 12) had its standing crop counted every 4 weeks on D-0, D-30, and D-60. The standing crop was computed using the equation given by Santhanam and Srinivasan (1994).

The consistency (Fasano and Franceschini, 1987), homogeneity assumptions (Levene, 1960), and normality (Mudholkar et al., 1995) were estimated before the statistical analysis was carried out. All data percentages were arc-sin transformed (Zar, 1984). The one-way analysis of variance (ANOVA) followed by Tukey’s test was performed, using the SPSS Statistics software, to compare the mean values (means ± standard deviation) at a significance level of 0.05. Moreover, using the Paleontological Statistics software (PAST4.17), the principal component analysis (PCA) and Pearson’s correlation coefficient confidence (PCCC) were performed to analyze the given data. However, the polynomial regression was conducted, using Excel Software, to assess the individual effects of biofloc supplementation levels and zooplankton, WG, SGR, and FCR.

Table 1 shows water quality parameters during the experimental period. The recorded values of water quality for all groups were within the recommended values for fish (El-Sayed, 2006). In all groups, no significant differences (p < 0.05) were observed for T-Al, temperature, and DO. However, the pH, EC, NO₂, NO₃, NH₃, and TDS values showed significant differences (p < 0.05) among the control and all biofloc groups. In the current study, the recorded pH and EC values in T₀ (the control group) were lower than those in all biofloc groups (T₁–T₅), meaning that the biofloc application tended to increase the alkaline and EC. These findings were similar to the findings previously reported by Ekasari et al. (2014) and Mohammady et al. (2023). In Nile tilapia culture, the concentrations of TDS are generally advised to be between 2,000 and 5,000 mg L⁻¹. Nile tilapia exposed to TDS levels of more than 5,000 mg L⁻¹ had slower development rates, higher stress levels, and a lower adaptive response (Nhan et al., 2006). The current study showed that the increase in biofloc volume tended to gradually increase the TDS values between groups, ensuring that all biofloc groups (T₅ > T₄ > T₃ > T₂ > T₁) were significantly (p < 0.05) higher than the control group (T₀), as well as ensuring that all studied biofloc group and the control group were in the recommended values for Nile tilapia. Our findings were confirmed by Mohammady et al. (2023), who concluded that, compared to the control diet, the enrichment of Nile tilapia by bioflocs gradually increased the TDS concentration in the water culture.

NO₂ and NO₃ are produced as a consequence of nitrification. According to Luo et al. (2014), under controlled laboratory conditions, the NO₃ generated in the biofloc condition system is capable of partial denitrification to produce NO₂ and dissimilatory reduction of NO₃ to un-ionized ammonia (NH₃). Nitrite is a transitional phase of oxidation that lies between ammonia (low oxidation states) and nitrate (higher oxidation states). The metabolic oxygenation of ammonia (nitrification) and reduction of nitrate (denitrification) within the water body are the primary processes that produce nitrite (Magouz et al., 2021a). In freshwater and marine aquaculture, the recommended range of NO₂ should not increase more than 0.2 and 0.125 mg L⁻¹, respectively. Nitrate is a non-toxic form of N for fish in freshwater or marine aquaculture. It is a non-toxic form of N for fish if less than 90 mg L⁻¹ (Luo et al., 2014).

In this study, Table 1 show that the biofloc-containing groups (T₁–T₅) significantly reduced NO₂, NO₃, and NH₃ levels compared to the control group (T₀) (p < 0.05). Regardless of significance, group T₃ achieved the lowest values of NO₂, NO₃, and NH₃ compared to the control group (T₀) or the other biofloc groups (T₁, T₂, T₄, or T₅). Our results are not in agreement with Mohammedy et al. (2021), who concluded that biofloc supplementation significantly (p < 0.05) increased NO₂, NO₃, and NH₃. This differentiation may be attributed to different scenarios such as i) the differences in biofloc volumes, ii) experimental conditions, iii) fish age, and iv) stock density.

Table 2 shows the growth performances and the efficiency of nutrient utilization of Nile tilapia fed different biofloc concentrations during a 60-day experiment. In all biofloc groups (T₁–T₅), the FW, FL, DWG, WGR, and SGR values were significantly (p < 0.05) higher than those in the control group (T₀). Interestingly, the FW, FL, DWG, WGR, LGR, and SGR values of the fish in group T₃ were significantly higher (p < 0.05) compared to those in all other groups, while the FCR was significantly lower (p < 0.05) in group T₃, compared to the other groups. Our results are confirmed by the conclusions of several reports that the addition of biofloc significantly enhances the growth performances and overall nutrient utilization efficiency of several species such as Nile tilapia, O. niloticus (Kishawy et al., 2020; El-Hawarry et al., 2021; Souza et al., 2019), African catfish, Clarias gariepinus (Chen et al., 2020; Fauji et al., 2018), Indian shrimp, Penaeus indicus (Panigrahi et al., 2020; Das et al., 2022), and whiteleg shrimp, Litopenaeus vannamei (Mansour et al., 2022a, 2022b).

Figures 1A–C show the polynomial regression of FW, SGR, and FCR, reporting that with the increase of biofloc supplementation levels of the experimented groups (T₀ “control”, T₁, T₂, T₃, T₄, and T₅: 0, 14.2, 28.4, 42.6, 56.8, and 71 g L⁻¹, respectively), the WG and SGR polynomial regression ( Figures 1A, B , respectively) were increased (r² = 0.7963 and 0.8218, respectively), while FCR polynomial regression ( Figure 1C ) was decreased (r² = 0.8735). These findings indicated that the biofloc supplementation levels improve the final weight, specific growth rate, and feed conversion ratio of Nile tilapia O. niloticus (Helal et al., 2024).

Table 3 shows the whole-body biochemical composition of Nile tilapia fed different biofloc concentrations after 60 days. The dry matter percentage was significantly higher in fish reared in T₃, followed by T₂, T₁, T₅, and T₄, while the lowest was reported by fish reared in the control group (T₀). The ether extract percentage was significantly higher in fish reared in T₃, followed by T₄, T₅, T₂, T₀, and T₁ ( Table 3 ). The percentage of ash was significantly higher in fish reared in T₅, followed by T₄, T₀, T₂, T₁, and T₃. Table 3 shows that increasing biofloc concentration inclusions significantly increased protein percentage, while the lowest protein percentage was reported in T₀. The current findings showed that the biofloc groups significantly improved dry matter, total protein, and ether extract, compared to the control group. These findings may be attributed to biofloc supplementation, which has a high total protein and lipid content (44.27% and 5.29%). Our findings were previously confirmed by Long et al. (2015), who concluded that the application of biofloc technology (BFT) in Nile tilapia culture showed increasing trends in protein, lipid, and ash content (%).

However, Aliabad et al. (2022) investigated the impact of feeding limitation and stocking density on the body composition of Nile tilapia fry. It was observed that in response to a reduction in the feeding rate, lipid content (%) decreased significantly, while protein (%), ash (%), and moisture (%) increased. The difference between our findings and the findings reported by Aliabad et al. (2022) may be attributed to different scenarios such as experimental design, fish age, feeding rate, stocking density, and water quality parameters.

Biochemical characteristics are frequently employed to assess the fish health, nutritional status, and capacity for environmental adaptation (Mansour et al., 2022a). Table 4 shows the biochemical profile of Nile tilapia fed biofloc for 60 days. It found that the highest significant TP was observed in fish reared in groups T₀ (control group), T₁, T₃, and T₅, followed by T₂, while the lowest was observed in T₄. The highest significant GLB was observed in fish reared in groups T₀ (control group), T₂, and T₅, followed by T₁ and T₃, while the lowest was observed in T₄. The highest significant ALB was observed in fish reared in group T₀ (control group), followed by T₁, T₃, T₂, and T₅, while the lowest was observed in T₄ ( Table 4 ). The fish reared in group T₃ showed the highest significance (p < 0.05) of creatinine, while the fish reared in groups T₀ (control group) and T₁ showed the highest significance (p < 0.05) of urea content. The highest significant (p < 0.05) values of AST and ALT were reported in groups T₁ and T₀ (control group).

Fish with higher levels of serum protein, globulin, and albumin are thought to have a stronger innate immune response, as these proteins are critical for the immunological response (Mohammady et al., 2023). The present results showed significant differences in serum protein, globulin, and albumin between the control group and biofloc groups. These findings revealed that the serum total protein, globulin, and albumin values reported in fish reared in T₃ were the most similar to those in the control group (T₀), while other biofloc groups (T₁, T₂, T₄, and T₅) had equal to or lower than those in the control group. Our findings were in agreement with those in Haghparast et al. (2020) study, which found that the addition of biofloc did not impact the biochemical parameters of common carp juveniles, contrary to the findings of Mohammady et al. (2023).

ALT and AST activities are helpful markers of liver integrity and function. When there are notable increases of these enzymes in the blood, it suggests that there is liver injury or tissue necrosis (Huang et al., 2015). In the current study, fish reared in biofloc groups had ALT values significantly lower than those of the control group. In addition, fish reared in biofloc groups had AST values significantly lower than or the same as those in the control group. Based on this finding, the current results concluded that the biofloc supplementation did not have harmful effects on liver tissue. Our results were also previously confirmed by several works (Hersi et al., 2023; Haraz et al., 2023, Ahmed et al., 2019; Aliabad et al., 2022). Examining biochemical serum markers including urea and creatinine is an approach for monitoring the prospective harmful adverse effects and the toxicity of the kidney (Abdel-Khalek et al., 2020). In our study, regarding creatinine and urea concentrations, fish rearing in biofloc groups had significant values lower than or the same as the control group. The biofloc supplementation levels also had no harmful effects on the kidneys of Nile tilapia (Haghparast et al., 2020).

The present examination of zooplankton was identified and counted on D-0, D-30, and D-60 to investigate the effects of adding biofloc water supplementation on zooplankton (community, quantity, abundance, and structure) and to explore the aquaculture potential of these small invertebrates, in water culture, which will be grown to serve as prey for Nile tilapia during the experimental period under biofloc strategy. Figure 2 shows the standing crop of zooplankton (structure and community) on day 0 (D-0, the initial of the experiment), which is mainly Rotifera.

On day 0, in all groups, all identified species belonged to the Rotifera family only, with an average total number of 24,000 Ind. L⁻¹. A total of six species comprised 100% of the total community: Lecane lunaris (11,000 Ind. L⁻¹, 45.83%), Monostyla sp. (11,250 Ind. L⁻¹, 46.88%), Annuropsis fissa (1,100 Ind. L⁻¹, 4.58%), Asplanchna sp. (450 Ind. L⁻¹, 1.88%), Synchaeta sp. (50 Ind. L⁻¹, 0.21%), and Trichocerca sp. (150 Ind. L⁻¹, 0.63%).

Figures 3A, B show the culture of small prey invertebrates after 30 days (D-30) and 60 days (D-60) of the experiment. For D-30 and D-60, the total number of zooplankton were 46,501 and 24,537 Ind. L⁻¹, respectively, comprises 100% of the total community, and belongs to four families: Rotifera, Copepoda, Cladocera, and free-living nematodes. This reduced number may be attributed to the high consumption of these invertebrates as live feed by Nile tilapia during the experiment.

All investigated small invertebrate groups were positively utilized as prey by Nile tilapia. Our findings are consistent with those of Tesfahun and Temesgen (2018), who observed that all the zooplankton species identified in the present study are common natural prey consumed by Nile tilapia in African lakes.

For D-30, the total Rotifera, Copepoda, Cladocera, and free-living nematodes were 81.66% (37,973 Ind. L⁻¹), 8.46% (3,932 Ind. L⁻¹), 8.79% (4,087 Ind. L⁻¹), and 1.09% (509 Ind. L⁻¹), respectively ( Figure 3A ). For D-60, the total of Rotifera, Copepoda, Cladocera, and free-living nematodes were 93.89% (23,038 Ind. L⁻¹), 5.65% (1,386 Ind. L⁻¹), 0.16% (39 Ind. L⁻¹), and 0.30% (74 Ind. L⁻¹), respectively ( Figure 3B ).

In both D-30 and D-60, the first dominant family was Rotifera (81.65% and 93.89%, respectively). In all our experimental groups, the results recorded that Rotifera was the most dominant prey. This result was confirmed by previous studies conducted by Rao et al. (2015) and Maciel De Lima et al. (2022). For Cladocera, it was the second dominant family on D-30 while the third dominant family on D-60 (8.79% and 0.16%, respectively). However, Copepoda was the third dominant family on D-30 and the second dominant family on D-60 (8.46% and 5.65%, respectively). However, these small invertebrates have been reported previously as good small invertebrates that are excellent feed and prey for aquatic animals (Abo-Taleb et al., 2021b; Helal et al., 2024).

For the free-living nematodes, it was represented in small amounts on both D-30 and D-60 (1.09% and 0.30%, respectively). It was concluded that the biofloc reduced the amount of free-living nematodes after 60 days of culture. The present result is consistent with those of several earlier studies that found that adding biological compounds to the amount of aquaculture water greatly reduced the number of Nematoda (Gallardo-Collí et al., 2019; Aboseif et al., 2022).

Table 5 shows the influence of biofloc supplementation levels on the small prey invertebrates’ culture, community, structure, and abundance in the water culture of Nile tilapia after day 30 and day 60.

As presented in Table 5 , during this feeding trial period, 13 different small invertebrate forms were identified in different representations between all the groups (T₀–T₅). Rotifera was associated with eight species: L. lunaris, Monostyla sp., A. fissa, Asplanchna sp., Synchaeta sp., Trichocerca sp., Brachionus sp., and Polyarthra sp. Cladocera was reported with two species: Alona sp. and Ceriodaphnia sp. Copepoda was represented by two forms: copepodite stage and nauplius larvae. Finally, the Nematoda was represented by their free-living form.

In the case of D-30, the highest total number of individuals was reported in group T₂, followed by T₁, T₅, T₃, T₀, and T₄. The highest percentage of Rotifera was recorded in group T₂, followed by T₄, T₃, T₅, and T₀, and finally T₁. The highest Copepoda percentage was recorded in group T₁, followed by T₅, T₃, T₀, and T₄, and finally T₂. Cladocera was found only in groups T₁, T₃, T₄, and T₅. Finally, free-living nematodes were found only in groups T₀, T₅, T₂, and T₃ ( Table 5 ).

In the case of D-60, this stage was the linked stage with data from Nile tilapia on growth performance, nutrient utilization efficiency, whole-body composition, and biochemical indicators. However, the highest total number of individuals was reported in group T₄, followed by T₅, T₁, and T₂. The lowest total number of individuals was reported in groups T₀ and T₃. For the other groups, the highest percentage of Rotifera was recorded in groups T₅, T₄, T₂, T₁, and finally T₀. The highest percentage of Copepoda was recorded in groups T₀, T₄, T₅, T₂, and T₁. No Copepoda was counted in T₀. Cladocera was found only in groups T₃ and T₂. However, free-living nematode individuals were found only in groups T₁, T₄, and T₅ ( Table 5 ).

The findings of the present study demonstrated that increasing the inclusion levels of biofloc greatly enhanced the structure, diversity, culture, and abundance of small prey invertebrates. This result was in line with several previous studies on shrimp (Da Silva et al., 2022) and Nile tilapia (Helal et al., 2024). According to Nguyen et al. (2021), the biofloc supplementation levels greatly enhanced the plankton diversity and community structure and Nile tilapia performance across growth and feed consumption compared to a clear-water technology. Our results are consistent with those of Said and Taha (2022), who revealed that, in comparison to the clear system, all biofloc administrations had greater zooplankton counts. However, our results were inconsistent with those of Aboseif et al. (2022), who indicated that fish cultured on a control diet showed the highest species diversity and zooplankton abundance when compared to the biofloc groups. The variations in the water quality, fish stock density, and experimental settings could be the explanation for such variance. Stated differently, regarding D-60, the greater biological diversity of small invertebrates in the T₁, T₂, T₄, and T₅ groups may suggest that the fish in these tanks are dependent on an artificial feed diet while it is readily available, meet their daily needs, and save the effort of catching swimming prey items. However, the reduced diversity of small invertebrates in group T₃ may be an indication that the fish are indeed reliant on prey for food. This decrease in prey, particularly in this biofloc group (T₃), may be explained by the FCR value of this group (1.68), which is the lowest one in all experimented groups. This will be clarified, nevertheless, by the statistical analysis of PCA, and PCCC was carried out.