The application of phytoremediation Lemna perpusilla to increase the production performance of Nile tilapia Oreochromis niloticus in a recirculation system

This study aimed to evaluate the production performance and physiological of Nile tilapia using Lemna perpusilla as a phytoremediator in a recirculation system. A completely randomized design with two treatments and three replications was applied. The treatments were Nile tilapia cultured with L. perpusilla (TL) and without L. perpusilla (L). The experimental fish in this study was the juvenile of Nile tilapia with a body length of 9.98 ± 0.08 cm and an average weight of 36.27 ± 1.07 g. The stocking density was 46 fish/pond and the container size was 275×100×60 cm3 and was separated in two areas using a fiber separator screen of 55.9% area for fish culture and 44.1% for L. perpusilla. The Nile tilapias were reared for 60 days, fed with commercial diet and fresh L. Perpusilla with amount 2% and 1% of biomass, respectively. They were fed three times a day with fresh L. perpusilla at noon and commercial diet in the morning and afternoon. The results showed that the Nile tilapia reared with L. perpusilla phytoremediation had normal physiological condition and production performance. The predominances of this system were lower feed conversion ratio value, more optimal values of feeding efficiency, and higher coefficient of weight uniformity.


INTRODUCTION
The intensive tilapia Oreochromis niloticus rearing will also support by increased feeding.The common problem on Nile tilapia rearing is an excessive waste, especially nitrogen (N) and phosphorus (P), caused by uneaten feed and metabolism waste (Lazzari & Baldisserotto, 2008).The waste in rearing media will pollute water quality and growth performance so that it will lead to profit loss.One of the solution to overcome kind of situation is using recirculation system.The recirculation system potentially reduces water renewal, rearing waste, and overcome water and land availability limitation (Delong et al., 2009;Martins et al., 2010;Zhang et al., 2011;Van Rijn, 2012).The principal of recirculation system is reuse of the rearing media by recirculating the rearing media in the certain system.The effect of using recirculation system is waste accumulation along with fish growth will cause dissolved oxygen decreasing (Schreier et al., 2010).The solution to reducing the side effect of using recirculation system is combining recirculation system and phytoremediation.
Phytoremediation application using aquatic plant has already used in aquaculture (Velichkova & Sirakov, 2013).One of the aquatic plant used as phytoremediator is Lemna perpusilla (Ferdoushi, 2008;Amalia et al., 2014).In this study, L. perpusilla is applied as phytoremediator and additional feed for Nile tilapia.The study by Tavares et al. (2010) reported that utilization of L. valdiviana Phil. in red tilapia rearing as water quality treatment and additional feed.L. perpusilla biomass was applied as an alternative feed for Nile tilapia because it potentially substituted commercial feed as many of 25% with the protein content of 38.10% (Ilyas et al., 2014).This study aimed to evaluate production performance and physiology of Nile tilapia with phytoremediator L. perpusilla application in a recirculation system.The outcome of this study is expected to be applied in eco-friendly Nile tilapia rearing.

Experimental design
This study was conducted using randomized complete design with two treatments and three replication.The treatments were TL (without L. perpusilla) and L (phytoremediation using L. perpusilla) and the container scheme is showed in Figure 1.

Rearing container
The Nile tilapia rearing container was concrete pond covered by a 275×100×60 cm 3 tarp.The pond was cleaned, covered with a tarp and then filled with 0.9 m 3 of reservoir water each pond.Water renewal was not conducted, however, to maintain water height caused by evaporation, water addition was conducted.Each pond was equipped with aeration installation and internal recirculation.A plastic bulkhead separator was placed inside the pond at a distance about 44.1% of the total pond extent (Amalia et al., 2014).

The experimental fish and aquatic plant
The experimental fish was the Nile tilapia juvenile with average length 9.98 ± 0.08 cm and an average weight 36.27 ± 1.07 g.The stock density was 50 fishes/m 3 (Gibtan et al., 2008).The Nile tilapia juvenile was acquired from the experimental pond of Aquaculture Department, Faculty of Fisheries and Marine Science, Bogor Agricultural University.The experimental aquatic plant was L. Perpusilla with initial stock density based on the placement of the plastic bulkhead separator at 44.1% of total pond extent (Amalia et al., 2014).The aquatic plant L. perpusilla was also used as additional feed.According to proximate analysis, the protein content of commercial diet and L. perpusilla were 33.07%and 31.86%,respectively.

The rearing activity
The rearing activity of the Nile tilapia was conducted for 60 days.During the rearing activity, the Nile tilapia was fed using commercial feed (pellet) and fresh L. perpusilla with feeding rate 1% and 2% from the fish biomass, respectively (Gibtan et al., 2008).The feeding frequency was done three times a day, in the morning, in the afternoon, and in the evening.In the morning and evening, the fishes were fed using pellet, while in the afternoon using the fresh L. perpusilla.

The experimental parameter
The experimental parameter in this study consisted of water quality, physiological respond, and growth performance of the experimental fish.The water quality was measured in situ every day and the parameters were temperature, pH, and dissolved oxygen (Zhou et al., 2009).The weekly measured water quality consisted of the brightness, total organic matter (TOM), turbidity, hardness, and alkalinity (Azim & Little, 2008) (Table 1).
The sample collection for the growth and physiological respond (blood glucose and lysozyme activity) was conducted every 20 days (Braun et al., 2010).The blood glucose level analysis was done using CHOD-PAP (enzymatic colorimetric test for glucose method with deproteinization) with glucose liquicolor kit in brand Human.The equation to analize the blood glucose is showed below: The lysozyme activity analysis was started with liquid suspension addition of Micrococcus lysodeikticus (Sigma) bacteria as many of 100 μL (0.4 mg/mL) in 0.1 M of phosphate buffered saline pH 6.2 into blood plasma sample (100 μL) in 25 °C.The absorbant was read twice in 450 nm wavelength on the microplate reader for 30 seconds, 15 minutes, 30 minutes, 45 minutes, and 60 minute of the blending.The lysozyme activity unit would be limited in certain amount of enzyme which caused the absorbant decreasing 0.001/minute.The equation of lysozyme activity is showed below: The production performance parameter was measured through the fish growth (fish length and body weight) every 20 days (Braun et al., 2010).The production performance parameter consisted of the absolute length, feed convertion ratio, feeding efficiency, survival rate, and the coefficient of length and body weight variance.

Specific length growth rate
The measurement of specific growth rate was calculated using below equation (Effendie, 1997)

Feed conversion ratio (FCR)
FCR is calculated using the equation according to Goddard (1996): FCR=F/((Wt+Wd)-Wo) Note : FCR : feed convertion ratio Wt : final fish biomass (g) Wo : biomass of dead fish during the experiment (g) Wd : initial fish biomass (g) F : total mount of feed during the experi ment (g)

Survival rate (TKH)
The survival rate of experimental fish was calculated using the equation below (Goddard, 1996):

Data analysis
All the datas were analyzed using t-test with confidence interval 95% in Minitab 16.0.Descriptive analysis was used to analyzed the water quality parameter.

Growth peroformance
Data analysis using t-test showed that L. perpusilla application as phytoremediator on recirculation system had significant difference towards FCR, EPP, and CV of weight (P<0.05).
The other production performance parameters consisted of specific length growth, absolute length growth, absolute growth rate, and length coefficient of variance, showed no significant difference among treatments (P>0.05;Table 2).

Blood glucose
The result of t-test indicated that the addition of L. perpusilla did not affect blood glucose level of Nile tilapia significantly (Figure 2).The blood glucose level of Nile tilapia in TL and L treatments ranged from 81.09-110.87mg/dL and 69.81-99.01mg/dL respectively.

Lysozyme activity
The result of t-test showed that L. perpusilla addition did not affect lysozyme activity significantly.The increasing of lysozyme activity occurred at all treatments in the day-20, then the lysozyme activity at all treatments decreased until the end of the experiment (Figure 3)

Temperature and pH level oof rearing media
The L treatment had higher pH level and temperature compared to the TL treatment.The pH level range of TL and L treatments were 7.32-7.79and 7.51-7.79respectively.The temperature of TL treatment was ranged from 28.07-30.60°C, while the temperature of L treatment was ranged from 28.27-30.17°C.The decreasing of rearing media temperature has the similar tendency with the decreasing of pH level.The decreasing of temperature was occurred at day-14 until day-28, while at day-42 until the end of the experiment (Figure 4).

Water brightness and turbidity
The water brightness in the L treatment was higher than the TL treatment, while the turbidity in the L treatment was lower than the TL treatment.The water brightness and turbidity have an inversely proportional correlation with the water brightness parameter.The higher turbidity gets, the water brightness will decrease.In the L treatment, the turbidity fluctuation was more stable than the TL treatment (Figure 5)

Dissolved oxygen and total organic matter (TOM)
Overall, the dissolved oxygen in all treatments showed a decreasing trend during the rearing period.The TL treatment had the higher dissolved oxygen (4.74-5.87mg/L) compared to the L treatment (4.21-5.83mg/L).The TOM concentration in both treatments had inversely proportional correlation with the dissolved oxygen (Figure 6).

Hardness and alkalinity
The water hardness and alkalinity during the rearing period were increasing in both treatments.Overall, the TL treatment had a lower range in the alkalinity and hardness parameters compared with the L treatment (Figure 7).The hardness of the TL treatment ranged from 5.40-13.35mg/L, while the L treatment ranged from 5.40-16.95mg/L.The alkalinity of TL treatment ranged from 39.69-89.31mg/L CaCO3 which lower than the L treatment which ranged from 39.69-121.37 mg/L CaCO3.

Total ammonia nitrogen (TAN)
The total ammonia nitrogen (TAN) in the rearing media was decreased since day-14 until day-42 and tend to decrease until the end of the experiment.The range of TAN value in TL treatment (0.113-0.616 mg/L) was lower than the L treatment (0.081-0.646 mg/L) (Figure 8).

Discussion
The production performance using L. perpusilla as phytoremediator in the recirculation system did not affect the absolute and specific growth rate, length growth rate (specific and absolute), and length coefficient of variance were not also affected significantly (P>0.05).The predominance of recirculation system had lower FCR, feeding efficiency, and higher in the weight uniformity.It was complied with the experiment by Velichkova and Sirakov (2013) which stated that Lemna minor and Wolffa arrhiza application in a recirculation system resulted in better FCR and increased the growth performance of Nile tilapia.
The physiological respond of the fish by using L. perpusilla as phytoremediator in a recirculation system did not affect lysozyme activity and blood glucose level significantly.There were no disease-infected fishes during the rearing period.
It was an indicator that the lysozyme activity between treatments was not significantly different.Lysozyme held an important role in nonspecific immunity or innate immunity (Saurabh & Sahoo, 2008).The blood glucose level was also no significant difference between treatments.It was assumed that environmental factors, such as temperature and dissolved oxygen, were slightly on the similar range.The blood glucose level of Nile tilapia will increase significantly along with the water temperature increasing (Mirea et al., 2013).The increasing of blood glucose will occur when the fish is lack of oxygen (EL-Khaldi, 2010).
The blood glucose level of the experimental Nile tilapia in the TL and L treatments were still in normal condition.Roques et al. (2010) reported that the blood glucose of the experimental Nile tilapia in normal condition ranges from 32-137 mg/dL.The result of blood sugar level of the Nile tilapia described that all the experimental fishes were not suffered from stress condition.Sanches et al. (2015) also reported that the decreasing of blood glucose in Nile tilapia is secondary stress respond indicator.
The predominance of using L. perpusilla as phytoremediator in a recirculation system was supported by the other water quality parameter.The pH value and temperature in the TL and L treatment were within the tolerance range.The optimum pH value and temperature during a rearing period were 7-8 and 27-32 °C respectively (El-Sherif & El-Feky, 2009;Pandit & Nakamura, 2010).
The turbidity value describes the turbid level of an aquatic environment.The higher turbidity gets, the waste load of rearing activity is also get higher.The turbidity value in the TL treatment was higher than the L treatment.It is in accordance with the experiment by Vanitha et al. (2015) which reported that the application of macrophyte could decrease the water turbidity level.The brightness level has inversely correlation with the turbidity level.The brightness level in the L treatment was higher than the TL treatment.It was in accordance with the experiment by Estlander et al. (2009) which stated that the water brightness was influenced by the density of macrophyte, if the macrophyte density was low, the water brightness would also low.
The dissolved oxygen in the L treatment was lower than the TL treatment.It was caused by total organic matter (TOM) in the L treatment was higher than the TL treatment.It was caused by the decomposing process by L. perpusilla because it needs a high level of dissolved oxygen (Zhu et al., 2011).The organic matter in aquaculture system consisted of rotten algae, uneaten feed, and bacteria community (Boyd et al., 2016).Nevertheless, the dissolved oxygen condition in the L and TL treatment was in optimum condition.The optimum dissolved oxygen to support the survival of Nile tilapia was ranged from 3-5.6 mg/L (Colt et al., 2011).
The water hardness described the concentration of Ca 2+ and Mg 2+ cation, while the alkalinity described the concentration of ion HCO3 − , CO3 2− , and OH − (Boyd et al., 2016).The concentration of hardness and alkalinity in all treatments during the rearing period was in the optimum condition.According to De Holanda Cavalcante et al. (2009), the optimum water hardness and alkalinity to support the growth performance of Nile tilapia were >50 and >140 CaCO3 mg/L.The high level of alkalinity in the L treatment was caused various factors, such as high level pH and ammonium.The increasing of pH value will follow by ion CO3 2− and HCO3 − (Boyd et al., 2016).The range of total ammonia nitrogen in all treatments is 2.58-2.63mg/L, compared to the experiment by Nakphet et al. (2017).The total ammonia nitrogen in the L treatment was decreased at the end of the experiment.It was assumed that total ammonia nitrogen was utilized by L. perpusilla.Chaudhary and Sharma (2014) also Zhao et al. (2014) stated that aquatic plant L. minor was potentially reduced ammonium waste (NH4 + ) in water and utilized it to grow.

CONCLUSION
Application of L. perpusilla as phytoremediator in a recirculation system was able to generate a better production performance with a normal physiological condition with some of the predominances were a lower feed conversion ratio, better in feeding efficiency, and high level of uniformity in weight.

Figure 2 .
Figure 2. Blood glucose level of Nile tilapia on TL (without L. perpusilla) and L (with L. perpusilla) during the rearing period.The similar superscript indicates no significant difference through t-test with 5% of significance level.

Figure 3 .
Figure 3. Lysozyme activity on Nile tilapia with TL (without L. perpusilla) and L (phytoremediation using L. perpusilla) during the rearing period.The same superscript letter at the same observation time indicates non significant result through t-test with 5% of the significant level.

Figure 7 .
Figure 7. Water hardness (a); and alkalinity (b) in the TL (without L. perpusilla) and L treatment (phytoremediation with L. perpusilla) during the rearing period

Table 1 .
The water quality parameter : (Schulz et al., 2005) length growth (cm) Pt : average body length in certain time (cm) Po : initial average body length (cm) Specific growth rate (SGR) Specific growth rate is stated through the equation below(Schulz et al., 2005):

Table 2 .
The production performance of Nile tilapia in TL (without L. perpusilla) and L (with L. perpusilla) treatment.The same superscript indicates no significant difference between treatments (P>0.05)