Dietary added bamboo charcoal can evoke Pangasianodon growth and can reduce ammonia from culture medium

Ninety-days feeding trial was conducted to determine the growth performances and reduction of ammonia by adding of dietary bamboo charcoal (BC) of pangasiid catfish, Pangasianodon hypophthalmus. Four levels of BC (0, 0.5, 1, and 2% treated as T0, T1, T2 and T3, respectively) were supplemented to the test diet composition. The growth performance and ammonia elimination were influenced by feed type. The mean value of ammonia were 1.5±0.08 mg/L, 0.67±0.16 mg/L, 0.25±0.06 mg/L and 0.42±0.08 mg/L; mean weight gain (g) of the P. hypophthalmus were 51.13±0.87, 68.27±0.88, 77.93±0.88, 68.60±0.58; average daily weight gain (g) were 0.57±0.01, 0.76±0.01, 0.86±0.01, 0.76±0.01; specific growth rate (SGR) (% day) were 1.05±0.003, 1.26±0.01, 1.32±0.01, 1.26±0.01; feed conversion ratio (FCR) were 2.59±0.02, 1.87±0.02, 1.83±0.02, 1.88±0.01; survival percentages were 87±0.58, 91±0.58, 94±1.15, and 92±1.15 in treatment T0, T1, T2 and T3, respectively. Water quality parameters especially ammonia elimination, weight gain, specific growth rate and survival rate of fish fed 1% BC diet (T2) were significantly (P< 0.05) higher than other compositions. Ammonia nitrogen excretion over a subsequent 12 h period decreased with increasing dietary BC. In conclusion, the diet supplemented with 1% BC was found to have a suitable level to fulfill the better growth performance and to decrease the ammonia nitrogen of P. hypophthalmus, under the conditions applied in this study.


INTRODUCTION
Aquaculture of the pangasiid catfish, Pangasianodon hypophthalmus, is one of the rapidly increasing industries because of its high market demand, and culture using a high stocking density is now common in Bangladesh (Begum et al., 2012).High density and semi-intensive culture of P. hypophthalmus in ponds can produce at a rate of as high as 25 to 30 tons/ha/year (BFRI and BARC, 2001).However, serious health problems are affecting the intensive culture of pangus, especially in a pond of high stocking density where high accumulation of *Corresponding author.E-mail: sadiqul1973@yahoo.comAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License nitrogenous waste products occurs.Due to high accumulation of nitrogenous waste products that is toxic to fish considered as a limiting factor for growth and survival of fish are affecting the culture of this species (Person-Le et al., 1995).The main source of ammonia in fish ponds is fish excretion.Other main sources of this ammonia are from aquaculture waste feed fed to the fish and diffusion from the sediment.An effective way to reduce the waste load is to modify aqua feeds with the aim of reducing excretion of nitrogen relative to fish growth (Cowey and Cho, 1991;Talbot and Hole, 1994).From the last few years, charcoal has been used in animal feed formulation as an additive because it absorbs ammonia and nitrogen and activates the intestinal function by eliminating the poisons and impurities from the gastrointestinal tract of land animals (Van et al., 2006;Mekbungwan et al., 2004).The term "charcoal" generally refers to the carbonaceous residue of wood, bamboo, cellulose, coconut shells or various industrial wastes left after heating the organic matter.This very fine odorless, tasteless black powder works as an adsorbent for many toxins, gases, and drugs without any specific action.The surface area of charcoal gives it countless bonding sites and its degree of adsorption depends on the dosages of charcoal and the amount of toxins present in the digestive tract (Banner et al., 2000;Bisson et al., 2001).
Several studies have reported about the effect of dietary charcoal supplementation on growth, feed efficiency ratio, specific growth rate, feed intake, nitrogen excretion and digestive functions of terrestrial animals (Banner et al., 2000;Kutlu et al., 2001;Samanya and Yamauchi, 2001;Mekbungwan et al., 2004;Van et al., 2006).Moreover, activated forms of charcoal have been widely used as an adsorbent or detoxicant in modern veterinary and medical science (Hoshi et al., 1991;Jindal and Mahipal, 1999).
Utilization of charcoal from wood or bamboo may provide an economical way to eliminate noxious substances because of their cheaper cost (Prasad et al., 2000).Moreover, bamboo charcoal (BC) is considered to have a higher adsorption capacity than wood charcoal because of the special structure of the micro pores of bamboo stem (Chung et al., 2004).Reports have clarified the ammonia adsorption effect of BC in aqueous solution (Asada et al., 2006), and dietary addition of BC effects on digestion, nitrogen retention, and excretion of growing goats (Van et al., 2006).However, very limited studies about BC in aquatic animal nutrition as a feed ingredient have been conducted.Preliminary study showed the efficacy of dietary BC supplementation on growth performances and nutrient utilization of stomach less tiger puffer, Takifugu rubripes (Moe et al., 2009).Accordingly, this study aimed to clarify the effects of BC in other species, such as pangasiid catfish, because the digestive and absorption mechanisms would be different between fish with and without a stomach.This study aimed to clarify the effects of dietary BC supplementation on growth performance, survival of P. hypophthalmus and water quality parameters specially ammonia excretion from the medium.

Study area
The experiment was conducted in twelve experimental ponds located in the fisheries field complex, Bangladesh Agricultural University, Mymensingh.Each experimental pond size was 30 m 2 and the water depth was maintained between of 1.0 and 1.3 m.The ponds were equal in size and similar in shape, depth, basin configuration and pattern type including water supply facilities.Three ponds were used for each treatment.

Pond preparation
For the preparation of the pond, water was drained out and pond bottom was dried in the sun.Aquatic weeds, undesirable fishes, insects and other aquatic organisms were removed manually and the grasses on the pond dykes were also pruned manually into very small size.Lime was applied at a rate of 0.5 kg/40 m 2 .

Collection and stocking of fry
The experimental P. hypophthalmus fry belonging to the same age group having average length and weight of 5±0.68 cm and 5.4±0.56 g, respectively were collected from the local hatchery (Digarikanda Fish Farm, Mymensingh, Bangladesh) and 100 fish/40 m 2 stocking density were used for each treatment.

Preparation of bamboo charcoal (BC)
Bamboo charcoal is made up of pieces of bamboo, which are taken from plants five years or older.Bamboo was then cut into small pieces and put into a tightly sealed container made of iron and then burned inside an oven at temperatures over 120°C.Once the fire was out, the container left to cool down completely before it opened.The BC then pounded into a fine powder and kept at cool and dry place.

Preparation of diet
Commercial fish feed obtained from commercial feed company (Mega Feed Co. Ltd., Bangladesh) was used as a basal feed supplemented with BC powder at 0, 0.5, 1.0 and 2% in T 0 , T 1 , T 2, and T 3 , respectively.Diets were prepared by mixing the dry ingredients and water (35% of the dry weight of ingredients) and then pellet-type diets were produced through a meat grinder with a diameter disc (size, 1.9 to 2.2 mm).The diets were later oven dried (40°C for 6 h) to approximately 11% moisture.After preparation, the diets were stored at refrigerator until used.

Feeding strategy
Feeding frequency in all treatments were two times a day at a rate of 5% of their body weight till the termination of the experiment.The feed was supplied by spreading method manually and half of the feed was supplied at 9:30 AM and rest of the feed was supplied at about 5:30 PM.

Sampling of fish
The experiment was conducted for three months.Fish sampling was done at fifteen days interval in the morning at around 8:30 AM to 9:30 AM.During each sampling, 10 fish from each pond was caught by cast net.The total length was measured by using ordinary scale graduated to tenth of centimeter and weight was taken by precision weighing balance Digital Scale KD-160 (Tanita Corporation, China) (accuracy up to 1 g).

Water quality parameters
The water quality parameters such as temperature, dissolved oxygen, pH and ammonia were recorded throughout the experimental period.Water samples were collected between 8:30 AM to 9:30 AM at fortnightly interval.The physico-chemical parameters like temperature (°C) was determined by a thermometer, dissolved oxygen (mg/L) was determined by DO meter (YSI Model-58, USA), pH was recorded by a pH meter (Corning pH meter, Model-445, UK) and ammonia (mg/L) was determined by HANNA ammonia test kit at fortnightly interval.

Growth parameters
The following analytical parameters were used to evaluate the growth of fish:

Data analysis
The final data were expressed as mean values ± standard error (SE) and analyzed by one-way analysis of variance (ANOVA).Percentage data were arcsine transformed before analysis of variance.Duncan's multiple range tests were analyzed among different group means.The significant level was set as P<0.05.All statistical analyses were performed using the SPSS11.0.

Physico-chemical parameters of the pond water
As shown in Table 1 during the experiment ammonia (mg/L) concentration was varied from 0.25±0.06 to 1.5±0.08mg/L.Maximum ammonia content was found in control (T 0 ) while minimum ammonia content was found in T 2 .Ammonia concentrations of different treatments were significantly different from each other.The dissolved oxygen content of the water varied from 7.12±0.01 to 7.96±0.01mg/L in the experimental ponds.The maximum dissolved oxygen was found in T 2 and minimum dissolved oxygen was found in T 0 .Again the range of pH values varied from 7.43±0.04 to 8.23 ±0.14 in the experimental ponds.However, there was no significant variation of temperature values under different treatments.

Weight gain
The mean initial weights of P. hypophthalmus in the treatments were 5.40±0.05g and the final average weight was 74.17±5.58g.The mean final weights of fish at the end of the experiments were 56.53±0.44 g, 73.67±0.88g, 83.33±0.88g and 74.00±0.57g in T 0 , T 1 , T 2 , and T 3, respectively.Weight gains of P. hypophthalmus in different treatments were significantly different to each other (Figure 1).

Average daily weight gain
Average daily weight gains of P. hypophthalmus at the end of the experiments were 0.57±0.01g, 0.76±0.01g,  0.86±0.01g and 0.76±0.01g in T 0 , T 1, T 2, and T 3, respectively.Average daily weight gains of different treatment were also significantly different (Figure 2).

Survival
The survival rates of P. hypophthalmus at the end of the experiment were 87±0.58,91±0.58,94±1.15 and 92±1.15 in T 0 , T 1 , T 2 , and T 3, respectively.Highest survival was obtained in T 2 (94%) and lowest was recorded in T 0 (87%) where no BC was added (Figure 6).

DISCUSSION
The present study demonstrated that BC can influence on Pangasianodon growth and it can reduce the ammonia from the aquatic environment.Based on the water quality parameters specially reduction of ammonia and growth, the optimum dietary BC supplementation level for the P. hypophthalmus was found 1% of the diet, which was far less than that found in the previous study, where the highest weight gain was obtained at 4% BC supplementation in Tiger puffer fish (Moe et al., 2009).In another study, the dietary BC supplementation level for the juvenile Japanese flounder Paralichthys olivaceus was 0.5% (Moe et al., 2010).These results indicated the species-related effect of dietary BC on growth, survival and water quality parameters, and it might be because of the differences in digestion and feeding behaviors of the stomach and stomach less characteristics of these three species.
The maximum growth enhancement was noticed at 1% BC supplementation level and it declined again in supplementation levels above 1%.The minimum ammonia concentration was noticed at 1% BC supplementation level and it increased again in supplementation levels above 1%.This result showed the dose-related effect of dietary BC on ammonia elimination and fish growth.However, fish groups that received dietary BC from 0.5 to 2% level showed higher values of weight gain, SGR, survival, DO and lower FCR, ammonia concentration in this study.These results indicated that the dietary BC supplementation could be a potential feed additive to enhance the ammonia elimination and growth of the P. hypophthalmus, and supports research in tiger puffer fish (Moe et al., 2009) and other studies that reported growth in goats (Van et al., 2006) and in broiler chicks (Kutlu et al., 2001).Moreover, similar results were reported by Yoo et al. (2005), who found that the suitable level of CV82 (80% charcoal and 20% vinegar) for optimum growth of juvenile Japanese flounder was within 0.5 to 1% of diet.
In the present experiment, the highest feed conversion ratio was recorded in the control (T 0 ) group (2.59) while the lowest was obtained in T 2 (1.83).Bamboo charcoal added feed (T 1, T 2 , and T 3 ) showed significantly lower FCR than the control feed (T 0 ), but no significant differences in FCR were observed among dietary BC treatments.However, the values obtained from fish fed diets containing 0.5 to 2% BC were lower in FCR than those previously reported for pangus (Sayeed et al., 2008).Survival rate of fish fed 1% BC diet were significantly (P<0.05)higher than those of fish fed the control diet (without BC).From the result of the experiment, it can be mentioned that BC added feed is more suitable compared to commercial feed.
Higher SGR and lower ammonia concentration in fish fed 1% BC diets may be because of the adsorbent effect of BC, which could be expected to have the potential to condition the intestinal cell membranes, reduce surface tension by eliminating gases and toxins or noxious substances along the intestine, and consequently can improve the utilization and absorption of nutrients across the cell membranes.Mekbungwan et al. (2004) reported that the wood charcoal and vinegar compounds (WCVC) could activate the intestinal function both at villus and cellular level, and it also increase the feed efficiency of piglets.Moreover, improved feed conversion ratio and activated morphological changes of intestinal villi were observed in chickens fed WCVC supplement diets (Samanya and Yamauchi, 2001).In this study SGR and survival were significantly increased in fish fed all levels of BC diets, but significant enhancements of dry matter digestibility were found at only 4% BC supplementation in puffer fish (Moe et al., 2009).These two results indicated that the effective levels of dietary BC on digestibility vary from species to species.
Another important objective of this study was to determine whether the total ammonia excretion could be reduced by dietary BC.The values obtained from fish fed diets containing 0.5 to 2% BC were lower in ammonia than those previously reported for pangasiid catfish (Ahmed et al., 1996).Similar effects of dietary BC on ammonia nitrogen excretion were found in the study with puffer fish (Moe et al., 2009).Moreover, Van et al. (2006) reported that adding BC at a level of 1 g/kg body weight induced significantly lower urine nitrogen content as compared with the control group in growing goats.
Overall, it is likely that the BC could be used for decreasing ammonia nitrogen by nitrogen retention in the fish body.However, this is the first report for the dietary BC effect on P. hypophthalmus and further investigation will be required to clarify the mechanism of dietary BC on nitrogen metabolism.

Figure 1 .
Figure 1.Final weight gains of P. hypophthalmus in different treatments.a, b, c means with different superscripts are significantly different from each other (P<0.05).

Figure 2 .
Figure 2. Average daily weight gains of P. hypophthalmus in different treatments.Different superscript alphabets in each treatment group are significantly different at P<0.05.

Figure 3 .
Figure 3. Percent weight gain of P. hypophthalmus in different treatments.Bars with different letters are significantly different (P<0.05).

Figure 4 .
Figure 4. SGR (%/day) of P. hypophthalmus in different treatments.a, b means with different superscripts are significantly different from each other (P<0.05).

Figure 5 .
Figure 5. FCR of P. hypophthalmus in different treatments.Bars with different letters are significantly different (P<0.05).

Figure 6 .
Figure 6.Survival of P. hypophthalmus in different treatments.Different superscript alphabets in each treatment group are significantly different at P < 0.05.

Table 1 .
Water quality parameters in four different treatments during the study period.
a Values are presented as mean ± SE.Values in the same row having different superscript letters are significantly different (P<0.05).