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Figure 4.1 show the temperature and DO concentration in EM treated water sample and control water sample. The reading has been taken once in 2 days by using YSI 52 CE DO meter (S.N 06L1446) for 26 days in a row in unit mg/L (ppm). The optimum range of DO concentrations for tilapia culture is between 5.00 ppm - 8.30 ppm. Lower than 5.00 ppm, the fish will distress and if the concentration of DO less than 2.00 ppm the potency of fish dies is high.
Figure 4.1: Concentration of DO in EM Tank and Control Tank
Base on figure 4.1, the concentration of DO starting from day 0 till day 26th not shown an obvious changed in both water samples. At day 0, the DO concentration for EM treated water sample is 5.06 ppm and at the end of the study which is at day 26th, the concentration of DO in EM treated water sample is 5.15 ppm. The highest concentration of DO in was appeared at day 24th which is 5.92 ppm. In contrast, the lowest concentration of DO in EM treated water sample is 4.79 ppm which at day 10th. For EM treated water, the DO concentration at day 8th (4.86 ppm) and at day 10th (4.79 ppm) were recorded below the optimum concentration.
Conversely, the highest concentration of DO in Control water sample is 5.83 ppm which at day 8th. On the contrary, the lowest concentration was recorded at day 20th which is 4.88 ppm. Furthermore, the DO concentration at day 4th (4.97 ppm) and day 20th (4.88 ppm) appeared at low then optimum concentration. Even though the concentrations of DO at both days are slightly low then optimum concentration, this was not affected the Tilapia culture.
Theoretically, the temperature of water sample will affect the concentration of DO. Oxygen becomes less soluble in water as the temperature increases (Geer and Kamila, 2005). Meaning that, warm water is less capable in dissolving the gases like oxygen while cold water has greater ability in holding oxygen. How ever, those days which the DO concentration were shown not in the optimum range, the temperature was lower (24°C) compared to others (Figure 4.1). However, this fact was assumed to show negative result due to the deepness of the probe when DO concentration reading. More deep of water from the surface, the temperature and DO are lower compare to the temperature and DO at the surface. Therefore, the more deep the probe of DO meter goes from the surface during reading, the lower the temperature and the DO concentration.
In conclusion, the concentrations of DO for both tanks are almost the same. No significantly changed in DO concentrations due to the aeration system that has been used to supply the oxygen. Therefore, the hypothesis can be made as the used of EM are not affecting the levels of DO in Tilapia culture. The level of DO is mainly control by aeration system. Even the surrounding temperature can affect the DO concentration, but it is not indicate an obvious change to the DO concentration.
Temperature also acts as an important parameter which needs to be checked in fish culture. The temperature of EM treated water samples were in range of 24-27°C and in control water samples were in the range of 24-26 °C. From figure 4.2, the data had shown there was no significant change through out the whole study. More over, the temperature of the water for both tanks was not away from the optimum range which is between 20 and 35 °C.
Figure 4.2: Temperature in EM Tank and Control Tank
For EM treated water sample, the lowest temperature recorded was 24°C, while the highest temperature was 27°C. In contrast, the lowest temperature for control water sample was same with EM treated water sample which is 24 °C but the highest temperature recorded in control water sample is 26 °C.
In general, the temperature for both tanks was control by the environment. Due to the location of the culture tanks which was under the roof, the heat from the sun are not directly affect the temperature. Moreover, in rainy day the temperature reading is not dropped significantly because the rain water does not drop into the culture tank and the volume of water in culture tanks remain the same.
Total Ammonia Nitrogen NH3-N (TAN)
Figure 4.3 show the concentration of Total Ammonia Nitrogen (TAN) in EM treated water sample and control water sample. Starting from day 0 to day 10th, the TAN concentration in both water samples increased quite in the same way. At day 0, the TAN concentration in EM treated water sample is 1.64 ppm and rose to 4.85 ppm at day 10th. On the other hand, the TAN concentration in control water sample rose from 1.65 ppm at day 0 to 4.90 ppm at day 10th. However, the concentrations of TAN in both water samples show major differences starting from day 12th to day 26th. The TAN concentration in control water sample started to acumulate at higher rate compared to EM treated water sample from 4.92 ppm at day 12th to 5.25 ppm at day 26th. On the contrary, TAN concentration in EM treated water sample show in decreased (33.70%) from 4.57 ppm at day 12th to 3.03 ppm in day 26th.
Figure 4.3: Concentrations of Total Ammonia NH3-N
The waste from fish pallet was proven to be the main contributor to the TAN concentration in water. For that reason, the concentration of TAN in control water sample was significantly increased by time. The ammonia cycle in control tank was not effective enough to reduce the concentration of TAN. This fact is mainly due to the lack of bacteria that utilized ammonia. In contrast, the decreased concentration of TAN starting from day 12th to day 26th in EM treated water sample was mainly due to the used of EM treatment. The microorganisms in EM were proven to be effective in reducing the TAN concentration. Moreover, the microorganisms such as Nitrosomonas and Nitrobacter that can make use of ammonia as energy source may be include in the consortium of microorganisms in EM.
Besides, the concentration of TAN will affect the pH of the water (refer chapter 4.1.4) and the concentration of Un-ionized ammonia (refer chapter 4.1.5). Since ammonia is alkaline, it will indicate higher pH value when its concentration increases. Moreover, the greater the concentration of TAN, the higher concentration of Un-ionized ammonia will produce in the water system (Ruth and Craig, 2005)
The value of the pH has an effect toward the toxicity of ammonia and increasing in pH will increase the toxicity of ammonia. The recommended value for Tilapia culturing is between 6 - 8 ppm (Tilapia culturing technique, Lembaga Kemajuan Pertanian MADA). If pH readings are beyond this range, fish growth is reduced and at values below 4 or above 10, mortalities will occur.
Figure 4.4: The pH Values in EM Tank and Control Tank
Figure 4.4 shows the pH value for EM treated water sample and control water sample. The pH values for both tanks were increased for the first 10 days. At day 10th where the EM solution was introduced to the EM tank, the pH values for EM treated water samples were started to decrease. On the contrary, the pH values for control water sample still show an increase.
For the first 10 days, the pH value for EM treated water sample increase from 7.13 ppm to 7.46 ppm. While, pH value in control water sample had been increase from 6.98 ppm to 7.81 ppm. After 2.5 L cultured of EM was added to the EM tank at day 10th, the pH value in EM treated water sample has slightly decreased from 7.46 ppm to 7.27 ppm. While, the pH values in control water sample still increased until 8.31 ppm toward the end of the study (day 26th). For EM treated water sample, the value of pH remain in the recommended pH value and still suitable for the culture of Tilapia. Whereas, the pH values in control tank at day 14th, 16th, 18th, 22nd, 24th and 26th (figure 4.4) has go beyond the recommended range of pH value for Tilapia culture (6-8 ppm).
These results proved that the use of EM in aquaculture will reduce the pH level to suitable pH range for the Tilapia culture. Since the pH value is related with concentration of total ammonia in water, the used of EM will solve both problems. Hence the production cost will decrease and the productivity of Tilapia will increase by using EM.
Un-ionized Ammonia NH3-N
Figure 4.5 shows the concentration of un-ionized ammonia (UIA) in both EM treated water sample and control water sample. UIA is a toxic form and the toxicity begins as low as 0.05 ppm. If the UIA is higher than 0.05 ppm, the fish gill is being damaged. As the concentration rises above 0.05 ppm it causes more and more damage and at 2.0 ppm fish will die. The UIA can be calculated from the concentration of TAN multiply by the Fraction Factor (Appendix 7). Prior to the calculation of the UIA, the pH and temperature of the water sample need to be determined.
Figure 4.5: Concentrations of Un-ionized Ammonia (UIA)
It was shown that the concentration of UIA in EM treated water sample shown an increase from day 0, 0.0098 ppm to 0.0686 ppm at day 12th. The concentration of UIA is related to the concentration of TAN. Since there was an increasing in TAN concentration in EM treated water sample for the first 10 days (Figure 4.3), therefore, the UIA in EM treated water sample was increased from day 0 to day 12th. However, after EM has been introduced, the concentration of UIA was slightly decreased (57.58%) from 0.0686 ppm at day 12th to 0.0291 ppm at day 26th. This happened because the decreasing of TAN (33.70%) in EM treated water sample from day 12th to day 26th (Figure 4.3).
On the other hand, the concentration of UIA in control water sample was increased with time. The concentration of UIA at day 0 is 0.0099 ppm and rose significantly to 0.4620 ppm at day 26th. This observable fact was due to the increasing of TAN in control water sample. In general, the higher the concentration of TAN, the grater the concentration of UIA in water sample.
Figure 4.6 show the concentration of nitrite in EM treated water sample and control water sample. Started from day 0 to day 10th, the nitrite concentration in both water samples increased in the similar manner. At day 0, the nitrite concentration in EM treated water sample is 0.036 ppm and rose to 0.427 ppm at day 10th. On the other hand, the nitrite concentration in control water sample rose from 0.042 ppm at day 0 to 0.453 ppm at day 10th. But after addition of EM into EM treated tank, the concentration of nitrite in both water sample show noticeably differences starting from day 12th to day 26th. The nitrite concentration in control water sample started to increase at higher rate compared to EM treated water sample from 4.92 ppm at day 12th to 5.25 ppm at day 26th. In contrast, nitrite concentration in EM treated water sample show in decreased from 0.299 ppm at day 12th to 0.193 ppm in day 26th (35.45%).
Figure 4.6: Concentration of Nitrite NO2-N in EM Treated Water Sample and Control Water Sample
Since nitrite is the product of the ammonia metabolisms in nitrogen cycle, the concentration of TAN will affect the concentration of nitrite. In conclusion, EM was proven in reducing the concentration of nitrite due to the reducing of TAN concentration.
Microwave Distillation Solid Phase Micro Extraction (MD-SPME)
Gas Chromatography Mass Selective (GC-MS) detector should be used in this analysis. Due to some technical problem that cannot be accounted, alternatively, Gas Chromatography with Flame Ionized Detector (GC-FID) has been used. However, the chromatograph did not show the peak of interest but show a lot of unknown peaks.
This problem was believed due to the GC type that has been used. The GC-MS is more sensitive in detection because it detection is based on the mass of the compounds. In contrast, GC-FID detection is based on ionization. More over, GC-FID that being used is equipped with ultra-5 column which is semi-polar instead of ultra-1 column which is non-polar that needs to be used. Since GEO and MIB is semi volatile polar compound, the used of ultra-5 as a column is not good for separation of these compounds. Further more, the method used is well-matched with GC-MS but the same method has been used when running GC FID.
Conversely, sample preparation also one of the factor that contributed to this problem. Methanol has been used as a solvent in dilution of the standard samples which are GEO and MIB. Even though the solvent has been filtered with 0.45 µm and 0.2 µm filter membrane, but there were still impurities or contaminant occurs in the standard samples. This impurities and contaminant has shown in the chromatograph as unknown peak (Appendix 14-16). How ever, after methanol (solvent) or blank has been injected in GC, all the unknown peaks have been identified from the solvent itself (Appendix 16).
Therefore, as an alternative, the sensory evaluation method has been used to investigate the effect of EM in elimination of off-flavor in Red Tilapia.
Figure 4.7 and figure 4.8 show the graph of Average Score of Sensory Evaluation for EM treated fish sample and control fish sample. The four attributes that has been judged for 5 evaluation session were texture of the fish fillet, earthy taste of the fish, moisture and acceptability. The major attribute focused in this study was earthy taste in fish.
The evaluation session was carried out once in 3 days. It was started with first session that was held on day 13th followed by second session on day 17th, third session on day 21st, fourth session on day 25th, and last session on day 29th. At each session, six fish samples have been introduced to the panelist consisted of EM treated fish samples and control fish samples.
Figure 4.7: Average Score of Sensory Evaluation in EM Treated Fish Sample
Figure 4.8: Average Score of Sensory Evaluation in Control Fish Sample
The first attribute that has been study is texture of fish sample. The score vary from 1 for hard to 5, soft. Based on figure 4.9, the average score for EM treated fish sample and control fish sample not show significant differences. For the 1st session till the 3rd session, the average score given by the panelist for EM treated fish sample and control fish sample are almost the same. It was found that the 4th session of evaluation, the average score for EM treated fish sample is 4.2 while for Control fish sample, the average score is 3.5. On the other hand, at 5th session, the average score for EM treated fish sample is 4.1 compared to control sample which is 3.6.
Figure 4.9: Average Score in texture for EM treated fish sample and control fish sample According to Evaluation Session
In general, the fish samples that had treated with EM have higher quality of texture compare to control sample (Zulkafli A. R. Pemahaman asas-asas mutuair: panduan mudah untuk penternak. Unpublished note, Pusat Penyelidikan Perikanan Airtawar). Further study need to carry out to confirm this fact because the effect of EM on fish texture is time consuming process and need longer study period to see the result. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 16).
Figure 4.10 show the distribution of earthy taste in EM treated fish sample and Control fish samples. The score vary from 1 (very dislike), 2 (dislike), 3 (neither dislike nor like), 4 (like) and 5 (very like). From the figure 4.12, the earthy tastes in EM treated fish sample show drastically increase in quality. In 1st session, the average score is 2.7 which mean the panelist not sure whether they like or dislike. But at the end of the study, at 5th session almost the entire panelist agreed that the earthy taste in EM treated fish sample has decreased and give an average score 4.5 (almost very like).
Figure 4.10: Average Score in Earthy Taste for EM treated Fish Sample and Control fish Sample According to Evaluation Session
On the other hand, the control fish samples did not show in drastic improvement on earthy taste. The average score are varying from 2.1 (dislike) for 1st session to 2.8 (neither dislike nor like) at the last session. The panelist agreed that the earthy taste is still in the fish sample after 5 session of evaluation.
As a hypothesis, the used of EM will reduce the earthy taste in the fish tissues. The earthy taste had change from dislike to almost like very much after treatment of EM to the fish sample. Based on this study, 16 to 20 days after treatment with EM was enough to reduce the earthy taste in fish sample. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 17).
Figure 4.11 show the average score for moisture in EM treated fish samples and control fish samples. The scores vary from 1 (dry) to 5 (wet). For the 1st session, the average score for EM treated fish sample is 3.6 while for control fish sample, the average score is 3.3. Moreover, the average score for control fish samples at 2nd and 3rd session are similar which is 3.2. Towards the end of the evaluation session, the average score for EM treated fish sample is 4.1 compared to control fish sample 3.9.
Figure 4.11: Average Score in Moisture for EM treated Fish Sample and Control fish Sample According to Evaluation Session
In conclusion, the effects of using EM toward the moisture content in fish sample are not clearly defined in this study. Since there is no significant different in moisture content between EM treated fish sample and control fish sample, the hypothesis can be made as EM was not affect the moisture contain in fish tissue. May be other factors such as the genetic of the fish, aging or the way of sample preparation will affect the moisture contain in fish. Therefore, further study must be carry on by using different methods to determine the moisture content in fish. From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 18).
Figure 4.12 show the average score for acceptability in EM treated fish sample and control fish sample. For this attribute, the score vary from 1 (worse), 2 (bad), 3 (fair), 4 (good) and 5 (best). These attributes were judged to know the level of satisfactoriness toward the fish samples. In other word, this attribute was indicated that either the fish sample is satisfied to eat or not.
Figure 4.12: Average Score in Acceptability and Earthy Taste for EM treated Fish Sample and Control fish Sample According to Evaluation Session
Figure 4.12, shown that the average score for EM treated fish sample always higher compared to control fish sample. In 1st session, average score for EM treated fish sample was 3.2 whereas control fish sample was 2.3. Towards the last session of the evaluation session, the average score for EM treated fish sample also higher than control fish sample. Overall, the average score for EM treated fish sample has change from Fair (3.2) to Good (4.3) toward the end. On the other hand, for control fish sample not show drastically change. For the 1st session the average score was bad (2.3) and at the lass session of evaluation the score is still at the same score (2.7) but a bit higher toward Fair score.
Moreover, figure 4.12 shows the relationship involving acceptability and earthy taste. The change in average score for acceptability for both sample were affected by the earthy taste in the fish sample. The less of earthy taste in the both fish samples or in other word the higher the average score in earthy taste indicate higher average score in acceptability.
Therefore, the earthy taste was the main problem contributed to the rejection of Tilapia in the local and global market. Even though there are other attribute that has been judged in this study, but the main attribute contributed to the negative response of tilapia was earthy taste. The used of EM has changed the panelist tolerability toward fish sample from fair to good (figure 4.12). From the Analysis of Variances (ANOVA), the data was significantly differ with p < 0.005 (Appendix 19).
Table 4.1 shows the comparison of the average CFU between the EM treated water sample and the control water sample. From the spread plate results, control soil sample recorded an average 6.0 x 107 CFU/mL at day 14th, 1.6 x 108 CFU/mL at day 19th, and 1.2 x 108 CFU/mL at day 24th. In general, the average microorganism colonies for EM treated water sample are 2.7 x 108 CFU/mL at day 14th, 5.7 x 108 CFU/mL at day 19th, and 6.5 x 108 CFU/mL at day 24th. Furthermore, by combining all these results, the average microorganism colonies for control water sample is 1.13 x 108 CFU/mL while the average microorganism colonies for EM treated water sample is 4.97 x 108 CFU/mL which is 4.398 times (339.82%) higher. In simple word, the application of EM has vitally increased the number of beneficial microorganisms in the water.
Table 4.1: Comparison of CFU
Average Colonies (CFU/mL)
2.7 x 108
6.0 x 107
5.7 x 108
1.6 x 108
6.5 x 108
1.2 x 108
4.97 x 108
1.13 x 108