Ozone Water Treatment

Introduction

Ozone (O₃) is a three-atom allotrope of oxygen held together by unstable bonds. A single oxygen atom breaks away easily and reacts with most of the organic and inorganic molecules that it contacts (Lawson 1995). This makes ozone a powerful oxidizing agent, reacting directly and indirectly with other compounds. Indirect reactions occur when ozone decomposes into radicals and these react with other compounds (Sugita et al. 1992; Lawson 1995). When free ozone and ozone decomposition products react with organic molecules, the reaction often takes place at bonds that are not easily oxidized by biological degradation. These compounds then degrade at a faster rate due to smaller molecular size and decreased numbers of higher-order covalent bonds (Summerfelt and Hochheimer 1997). These reactions make elimination of solids from the system easier.

Use of ozone in freshwater systems can be beneficial. Ozone is particularly well suited to aquaculture applications because it has a wide range of oxidizing uses, a rapid reaction rate, few harmful reaction by-products in freshwater, and oxygen production is an end product of reaction. Since many contaminants in aquaculture waters are oxidizable, ozone can be used in applications ranging from disinfection to general water quality control. Adding ozone to the recirculating system resulted in an overall improvement in water quality due to more complete oxidation of nitrite, color, organic material and suspended solids. Ozone reduced the concentration of suspended particulate and dissolved organics, water color, and nitrite in all trials when compared to the control (Summerfelt et al., 1997).

Ozone is a disinfection technique often used in aquaculture (Primavera et al., 1993, Dierberg and Kiattisimkul, 1996, Boyd, 1997, GESAMP, 1997, Boyd and Clay, 1998, Chang et al., 1998 and Funge-Smith and Briggs, 1998). Ozonation is sometimes used to disinfect hatchery water, but less frequently to disinfect water in grow-out ponds (GESAMP, 1997). The technique requires relatively large investments and is therefore more frequently used in the larger farms.

Ozone effectively destroys bacteria, viruses, fungi, algae and protozoa by disrupting cell membrane function, entering the cell and destroying the nuclear chemistry of the cell. The effectiveness of ozone as a disinfectant is a function of dosage and contact time (Lawson 1995). The target organism and water quality determine the required concentration of ozone and the necessary contact time (Lawson 1995; Summerfelt and Hochheimer 1997). Microbial reductions are limited by the ability to maintain a specific ozone concentration for the time needed (Summerfelt and Hochheimer 1997).

In fish crowding has been reported to be an aquaculture-related chronic stress factor which reduces growth and affects the inflammatory and immune responses (Montero et al., 1999). Stocking density is a major factor affecting fish growth under farming conditions (Bjornssono, 1994 and Irwin et al., 1999). Chang (1988) reported that, fish stocking density is an important factor use in aquaculture as it can affect natural food availability, the efficient of food resource and total fish yield in ponds. Also, Ridha (2006) reported that the lower density (125 fish/m³) had significantly (P<0.05) increased mean weight, daily growth rate and specific growth rate of Nile tilapia than the higher density (200 fish /m³).

The objective of the present study was to investigate the interactive effects of fish stocking density and ozone treatments on growth performance, feed efficiency, blood chemistry and water quality of Nile tilapia (Oreochromis niloticus).

Materials and methods

The present study was carried out at the wet Laboratory of the Animal Production Department, Faculty of Agriculture, Zagazig University, Egypt. The experimental period lasted 84 days from June to September, 2011. The experimental fish (average weight 5.0 g after two weeks acclimation under normal laboratory conditions) were randomly distributed into 12 glass aquaria (35 X 40 X 70 cm) in 4 treatments (3 replicates per treatment).

The experiment was based on a 2 x 2 factorial design with two stocking density; low level (10 fish/aquarium - 135 fish/m³) and high level (20 fish/aquarium - 270 fish/m³) and two levels of ozone treatment at the levels 0 or 0.5 mg/l, each replicated three time. Ozone was generated (Ozone Generator, Matra Co., Model RAIT, China) and injected throw the water. All fish groups were fed on basal pelleted diet consistent of fish meal 30.0%, soybean meal 20.0%, corn 20.0%, wheat bran 15.0%, alfalfa hay 10.0%, sunflower oil 2.5%, minerals mixture 0.5%, vitamin mixture 1.0% and carboxymethyl cellulose 1.0%. The chemical composition of the diet was crude protein 40.12%, ether extract 6.40%, crude fiber 5.32% and gross energy 4280.0 Kcal/Kg.

Fish were fed at the rate of 3% of body weight per day and it offered three feedings at 8.00, 12.00 and 17.00 hours. The fish in each aquarium were weighed every 2 weeks, and the feed weight was adjusted after each fish weighing. Fish wastes were siphoned out at the level 30% of the water in each aquarium was removed every two days and replaced with fresh new water treated with ozone (0.5 mg/l for two minutes).

All fish were individually weighed to the nearest 0.1 g at the beginning of the experiment and biweekly intervals throughout the experimental period. Food consumption was then calculated as g/fish/day by dividing the amount of food consumed each day by the number of fish in the aquarium. Fish faeces and residual were removed by siphoning by using plastic tube. Feed conversion ratio (FCR) was calculated according to Berger and Halver (1987) according to the following equation: FCR = Cumulative feed delivered to aquarium/Fish biomass gain. Gain percent was calculated according to the following equation: Gain% = ((Final weight - Initial weight) / Initial weight) X 100.

Blood samples were collected from the caudal vein of three randomly selected fish with a heparinized syringe and the plasma separated by centrifugation at 3000 rpm for 20 min and stored at -20°C until further biochemical analysis. Total protein, albumin (Sundeman, 1964), urea-N, creatinine (Henery, 1974) and plasma transaminase enzymes (AST; aspartate amino transferase and ALT; alanine amino transferase (Reitman and Fankel, 1957) were determined by using the commercial kits.

Water quality parameters were monitored at 4 and 8 weeks before replacing the water in the aquarium during the experimental period. Water temperature was 28 °C. Different methods of water chemical analysis were carried out according to APHA (1990).

Economic evaluation was calculated as: Margin = Income from body gain weight - Feed cost. Other overhead costs were assumed constant. Price of one kg of diet was 4.20 LE (Egyptian pound = 0.167 US$) and price of selling of one kg live body weight of fish was 10.0 LE.

The data were statistically analyzed with SAS (2002) according to the following model:

Y_ij = µ + D_i+ O_j+ DO_ij+ e_ij

Where, µ is the overall mean, D is the fixed effect of stocking density (I = 1 ...2), O is the fixed effect of ozone treatment (j = 1 ...2), DO_ij is the interaction effect of stocking density and ozone treatment, e_ijis random error. Differences between treatments were tested with Duncan´s multiple range test (Duncan, 1955).

Results

Growth performance:

The non significant differences between the experimental groups for initial live body weight indicated that the groups at the beginning of the experiment were homogenous (Table 1).

Stocking density significantly affected final body weight, daily gain (P<0.01), daily feed offered (P<0.001), while feed conversion ratio insignificantly affected (Table 1). Increasing fish density to 270 fish/m³ reduced final body weight, daily gain, daily feed offered and survival rate by 3.19, 3.88, 3.66 and 0.83%, respectively, while feed conversion impaired by 1.54% when compared with fish groups stocked at 135 fish/m³ (Figure 1). Fish group stocked at the lower density recorded higher gain% (348.45%) when compared with those stocked at the high level.

Treatment water with ozone significantly affect final body weight, daily gain and daily feed offered (P<0.001), while feed conversion insignificantly affected (Table 1). Fish groups reared in water treatment with ozone increased survival rate and gain percent. Treatment aquarium water with ozone increased final body weight, daily gain and daily feed offered by 10.83, 13.62 and 8.83%, respectively, when compared with those reared in untreated water with ozone (Figure 2). Fish group reared in aquarium treated with ozone recoded gain% 360.30%, while fish group in control group recoded 322.09%.

The interaction between fish stocking density and ozone treatment did not show any significantly effect on body weight, daily gain, daily feed offered and feed conversion (Table 1). Within each stocking density, ozone treatment group recorded higher final body weight and growth rate and the best feed conversion. Fish group reared at low density and water treated with ozone obtained the higher growth rate (Figure 3).

Physiological parameters:

Fish stocking density was significantly P<0.01 or 0.05) affected all blood components except ALT (alanine amino transferase) insignificantly affected (Table 2). Increasing fish density decreased the plasma total protein, albumin, globulin, AST and ALT with 8.52, 11.50, 3.60, 5.10 and 2.55%, respectively, while the concentrations of urea-N and creatinine increased with 7.33 and 8.46%, respectively, when compared with those reared at the low density level (Figure 4).

Ozone treatments was significantly (P<0.001, 0.01 or 0.05) affected all blood components except plasma globulin and ALT insignificantly affected (Table 2). Treatment aquarium water with ozone increased the plasma total protein, albumin, globulin and ALT with 18.15, 20.40, 14.73 and 3.09%, respectively, while the concentrations of urea-N, creatinine and AST decreased with 11.90, 11.92 and 12.59%, respectively, when compared with those reared at the water without ozone treatment (Figure 5).

The interaction between fish stocking density and ozone treatment did not show any significantly effect on all studied blood parameters (Table 2). Within each stocking density, ozone treatment group recorded higher values of plasma total protein and its fractions, while the concentrations of plasma urea-N, creatinine, AST and ALT recorded lower values (Figure 6).

Water quality:

Fish stocking density was significantly affected water ammonia, nitrite (P<0.001) and dissolved oxygen (P<0.05) at 4 week of the experimental period, while pH and nitrate insignificantly affected (Table 3). At 8 week of the experimental period fish stocking density was significantly affected water ammonia (P<0.05), nitrite and nitrate (P<0.01), while pH and dissolved oxygen insignificantly affected (Table 4). The concentrations of ammonia, nitrite and nitrate in aquarium water increased with increasing fish density in units, on the other hand the concentration of dissolved oxygen decreased.

Ozone treatment was significantly (P<0.001) affected water ammonia, nitrite, nitrate and dissolved oxygen at 4 and 8 weeks of the experimental period (Tables 3 and 4). The concentrations of ammonia, nitrite and nitrate decreased in water when treated with ozone, while dissolved oxygen increased.

The interaction between fish stocking density and ozone treatment did not show any significantly effect on all studied water parameter quality, except the nitrite at 4 and 8 weeks and ammonia at 4 week of the experimental period (Tables 3 and 4). Within each stocking density, ozone treatment group recorded higher water quality.

Profit analysis:

Treated water with ozone increased feed cost, return from body gain and final margin (Table 5). Fish group reared at the low density and treated with ozone recorded higher feed cost, return from body gain and final margin than fish group reared at the high density and treated with ozone, when compared with the other experimental groups (Figure 7).

Discussion

Aquaculture systems generally employ intensive culture methods involving dense populations of fish. These can produce large quantities of waste often released into the water. Final live body weight and daily body gain of Nile tilapia fish decreased with increasing stocking density. Also, gain % decreased with increasing stocking density. Decreasing fish density stocking improved the feed conversion. Blood total protein, albumin, globulin, AST and ALT decreased in fish groups reared at high stocking density, when compared with those reared at low stocking density, while the concentration of urea-N and creatinine increased. The concentrations of dissolved oxygen in water decreased with increasing fish density, while the concentrations of ammonia, nitrite and nitrate increased. The present results indicated that increasing stocking density decreased the individual growth rate of fish. The similar trend was obtained by Ayyat and Abbas (2003), who reported that daily weight gain decreased with 26.0% and feed conversion impaired by 20.82% in fish reared at 150 fish/m³, than those reared at 100 fish/m³. Serum total protein, albumin, globulin, creatinine and AST were significantly decreased with increasing stocking density, while ALT decreased. Also, Uddin et al. (2007) and Youssouf et al. (2007) found that the growth rate was found to be lower at the higher stocking densities. On the other hand, Rowland et al. (2006) found that the stocking density did not affect final weight, specific growth rate or absolute growth rate, but feed conversion ratios of fish stocked at 25 or 50 fish/m³ were significantly higher (P<0.01) than at other densities (100 or 200 fish/m³). Also, the results of Aksungur et al. (2007) revealed that stocking density had a significant effect on growth and survival rates of turbot. Fish held at the highest density exhibited lowest growth rate and survival rates.

The high stocking densities, associated fish stress and increased nutrient loads an ideal environment for fish pathogens. In response to an additional acute crowding stressor, the levels of cortisol and non-esterified fatty acids were significantly higher and of glucose significantly lower in sea bass reared at 45 kg/m3 compared to fish kept at 15 and 30 kg/m3. Results indicate that stocking density at 45 kg/m3 for 6 weeks did affect the energetic status of sea bass and their sensitivity to a subsequent crowding stressor (Marco et al., 2008)

Ozone is a powerful oxidizing agent and is widely used for disinfection and improvement of water quality in aquaculture system. Highly significant (P<0.001) differences in the final body weight and daily gain weight of the Nile tilapia were obtained by the use of ozone. Ozone supplementation significantly increased plasma total protein, albumin and decreased the urea-N and creatinine. Adding ozone to the water resulted in an overall improvement in water quality reducing ammonia, nitrite and nitrate levels. Adding ozone to the water of the aquarium resulted in an overall improvement the growth rate, feed conversion and water quality due to more complete oxidation of ammonia and nitrite. Ozone reduced the concentration of suspended particulate dissolved organics, and nitrite in all experimental period when compared to the control.

The addition of ozone in the culture system can thus be used on one hand to protect from toxicity / accumulation of nitrite. The reduction of nitrite in the culture supplemented with ozone can be explained by the oxidative properties of ozone that rapidly oxidizes ammonia to nitrite and oxidizes nitrite to nitrate (Rosenthal and Kruner, 1985 and Parka et al., 2011).

In the present study ozone supplementation in culture system insignificantly pH, while dissolved oxygen increased significantly. Ozone provides additional oxygen to the aquarium as well as killing bacteria and parasites by eating away at their cell membranes. Suantika et al. (2001) found that the supplementation of ozone did not affect pH and dissolved oxygen levels. Besides the positive effect of ozone to the nitrification process, a better removal of suspended solids was noticed as well. The use of ozone also reduced the number of bacteria in the culture water. In general terms, it can be stated that supplementation of ozone in a closed recirculation system for rotifers considerably improves water quality and controls bacterial proliferation.

Table 1. Growth performance, feed efficiency and survival rate of Nile tilapia fish as affected by fish density, water ozone treatment and their interaction.

Treatments	Initial weight (g)	Final weight (g)	Daily gain (g/day)	Gain %	Daily feed offered (g/day)	Feed conversion (g food/g gain)	Survival rate (%)
Effect of fish density
Low	5.067±0.042	22.733±0.542	0.210±0.006	348.45± 7.65	0.396±0.009	1.883±0.025	98.33
High	5.017±0.031	21.767±0.533	0.199±0.006	333.93±10.62	0.380±0.007	1.912±0.032	97.50
Effect of ozone treatments
Without ozone	5.000±0.036	21.100±0.259	0.192±0.003	322.09±5.58	0.370±0.003	1.934±0.027	97.50
With ozone	5.083±0.031	23.400±0.273	0.218±0.003	360.30±3.88	0.406±0.005	1.862±0.021	98.33
Interaction between fish density and ozone treatment
Fish density	Ozone
Low	Without	5.000±0.058	21.600±0.2526	0.198±0.002	332.01±2.04	0.376±0.004	1.902±0.039	96.67
With	5.133±0.033	23.867±0.348	0.223±0.004	364.89±4.22	0.416±0.002	1.865±0.034	100.00
High	Without	5.000±0.058	20.600±0.153	0.186±0.002	312.16±7.30	0.365±0.002	1.966±0.034	98.33
With	5.033±0.033	22.933±0.186	0.213±0.002	355.70±6.04	0.396±0.002	1.858±0.031	96.67
Statistical analysis (P value):
Fish density	0.3198	0.0044	0.0048	0.0252	0.0003	0.4293	-------
Ozone treatment	0.1151	0.0001	0.0001	0.0001	0.0001	0.0687	-------
Interaction	0.3198	0.8956	0.7411	0.3431	0.1080	0.3312	-------

Table 2. Blood components of Nile tilapia fish as affected by fish density, water ozone treatment and their interaction.

Treatments	Total Protein (g/100 ml)	Albumin (g/100 ml)	Globulin (g/100 ml)	Urea-N (mg/100 ml)	Creatinine (mg/100 ml)	AST (IU)	ALT (IU)
Effect of fish density
Low	5.750±0.191	3.582±0.125	2.168±0.112	8.735±0.225	1.135±0.027	24.503±0.913	14.533±0.415
High	5.260±0.269	3.170±0.200	2.090±0.094	9.375±0.259	1.231±0.047	23.255±0.644	14.163±0.428
Effect of ozone treatments
Without ozone	5.047±0.195	3.063±0.165	1.983±0.010	9.427±0.287	1.258±0.036	25.483±0.534	14.130±0.443
With ozone	5.963±0.103	3.688±0.079	2.275±0.058	8.683±0.145	1.108±0.020	22.275±0.300	14.567±0.392
Interaction between fish density and ozone treatment
Fish density	Ozone
Low	With	5.373±0.159	3.313±0.048	2.060±0.194	8.953±0.413	1.187±0.015	26.373±0.648	14.317±0.751
Ozone	6.127±0.125	3.850±0.058	2.277±0.113	8.517±0.186	1.083±0.029	22.633±0.502	14.750±0.500
High	Without	4.720±0.241	2.813±0.267	1.907±0.079	9.900±0.132	1.330±0.032	24.593±0.461	13.943±0.618
With	5.800±0.104	3.527±0.039	2.273±0.065	8.850±0.208	1.133±0.023	21.917±0.267	14.383±0.696
Statistical analysis (P value):
Fish density	0.0182	0.0188	0.5434	0.0380	0.0055	0.0339	0.5838
Ozone treatment	0.0006	0.0021	0.0457	0.0204	0.0004	0.0002	0.5195
Interaction	0.3530	0.5463	0.5603	0.2684	0.1064	0.3083	0.9960

Table 3. Water Quality of Nile tilapia fish as affected by fish density, water ozone treatment and their interaction at 4^th week of the experimental period.

Treatments	pH	Ammonia (mg/l)	Nitrite (mg/l)	Nitrate (mg/l)	Dissolved oxygen (mg/l)
Effect of fish density
Low	7.82±0.07	0.0039±0.0011	0.0305±0.0066	0.0295±0.0072	8.333±0.369
High	7.74±0.05	0.0046±0.0013	0.0358±0.0079	0.0327±0.0083	8.050±0.399
Effect of ozone treatments
Without ozone	7.76±0.06	0.0069±0.0003	0.0495±0.0017	0.0483±0.0017	7.350±0.111
With ozone	7.81±0.07	0.0015±0.0001	0.0168±0.0008	0.0138±0.0006	9.033±0.088
Interaction between fish density and ozone treatment
Fish density	Ozone
Low	Without	7.80±0.08	0.0063±0.0001	0.0453±0.0015	0.0457±0.0015	7.533±0.145
With	7.84±0.12	0.0014±0.0001	0.0157±0.0012	0.0133±0.0009	9.133±0.145
High	Without	7.71±0.09	0.0075±0.0002	0.0537±0.0023	0.0510±0.0023	7.167±0.088
With	7.77±0.08	0.0017±0.0001	0.0180±0.0006	0.0143±0.0009	8.933±0.088
Statistical analysis (P value):
Fish density	0.4374	0.0007	0.0004	0.0678	0.0461
Ozone treatment	0.6085	0.0001	0.0001	0.0001	0.0001
Interaction	0.9177	0.0070	0.0111	0.1866	0.5077

Table 4. Water Quality of Nile tilapia fish as affected by fish density, water ozone treatment and their interaction at 8^th week of the experimental period.

Treatments	pH	Ammonia (mg/l)	Nitrite (mg/l)	Nitrate (mg/l)	Dissolved oxygen (mg/l)
Effect of fish density
Low	7.78±0.08	0.0040±0.0011	0.0292±0.0064	0.0285±0.0074	8.283±0.288
High	7.79±0.08	0.0046±0.0013	0.0355±0.0082	0.0327±0.0082	8.083±0.334
Effect of ozone treatments
Without ozone	7.81±0.07	0.0070±0.0003	0.0485±0.0025	0.0480±0.0016	7.517±0.108
With ozone	7.76±0.09	0.0016±0.0001	0.0162±0.0009	0.0132±0.0007	8.850±0.096
Interaction between fish density and ozone treatment
Fish density	Ozone
Low	Without	7.76±0.11	0.0065±0.0001	0.0433±0.0015	0.0450±0.0015	7.667±0.145
With	7.80±0.13	0.0014±0.0001	0.0150±0.0011	0.0120±0.0006	8.900±0.115
High	Without	7.85±0.10	0.0075±0.0003	0.0537±0.0018	0.0510±0.0012	7.367±0.120
With	7.72±0.14	0.0017±0.0001	0.0173±0.0012	0.0143±0.0009	8.800±0.173
Statistical analysis (P value):
Fish density	0.9569	0.0139	0.0021	0.0051	0.1922
Ozone treatment	0.7064	0.0001	0.0001	0.0001	0.0001
Interaction	0.4891	0.1071	0.0222	0.1320	0.4967

Table 5. Profit analysis of Nile tilapia fish as affected by the interaction between fish density and water ozone treatment.

Treatments	Feed intake (g)	Feed cost (LE)	Total gain (g)	Return from gain (LE)	Final margin (LE)
Fish density	Ozone
Low	Without	31.584	0.133	16.632	0.166	0.033
With	34.944	0.147	18.732	0.187	0.040
High	Without	30.660	0.129	15.624	0.156	0.027
With	33.264	0.140	17.892	0.179	0.039

Effect of ozone and stocking fish density rates on growth performance, blood components and water quality of Nile tilapia (Oreochromis niloticus) - Image 1

Figure 1. Growth performance and feed efficiency index of Nile tilapia as affected by stocking density, when considering the low stocking density values as 100%.

Figure 2. Growth performance and feed efficiency index of Nile tilapia as affected by ozone treatment, when considering the values of control group (without ozone) as 100%.

Figure 3. Growth performance and feed efficiency index of Nile tilapia as affected by the interaction between stocking density and ozone treatment, when considering the values of group low stocking density and without ozone as 100%.

Where, LD = Low density, HD = High density, W = Without ozone treatment and O = Ozone treatment.

Figure 4. Blood components index of Nile tilapia as affected by stocking density, when considering the low stocking density values as 100%.

Figure 5. Blood components index of Nile tilapia as affected by ozone treatment, when considering the values of control group (without ozone) as 100%.

Figure 6. Blood components index of Nile tilapia as affected by the interaction between stocking density and ozone treatment, when considering the values of group low stocking density and without ozone as 100%.

Where, LD = Low density, HD = High density, W = Without ozone treatment and O = Ozone treatment.

Figure 7. Profit analysis index of Nile tilapia as affected by the interaction between stocking density and ozone treatment, when considering the values of group low stocking density and without ozone as 100%.

Where, LD = Low density, HD = High density, W = Without ozone treatment and O = Ozone treatment.

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Ozone Water Treatment

Effect of ozone and stocking fish density rates on growth performance, blood components and water quality of Nile tilapia (Oreochromis niloticus)

Yeast postbiotics strengthen preventive management of S. agalactiae and A. hydrophila outbreaks in tilapia farming

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