Assessment of Daphnia, Moina and Cylops in Freshwater Ecosystems and the Evaluation of Mixed Culture in Laboratory Md

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American International Journal of Available online at http://www.iasir.net Research in Formal, Applied & Natural Sciences ISSN (Print): 2328-3777, ISSN (Online): 2328-3785, ISSN (CD-ROM): 2328-3793

AIJRFANS is a refereed, indexed, peer-reviewed, multidisciplinary and open access journal published by International Association of Scientific Innovation and Research (IASIR), USA (An Association Unifying the Sciences, Engineering, and Applied Research)

Assessment of Daphnia, Moina and Cylops in Freshwater Ecosystems and the Evaluation of Mixed Culture in Laboratory Md. Faruque Miah, Somit Roy, Ekhtiar Jinnat and Zobada Kanak Khan Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh.

Abstract: This study was carried out to observe abundance and monthly variation of three zooplanktons in four different aquatic ecosystems of Sylhet during the period of March to June 2013. Cyclops, Daphnia and Moina was considered for this study and the abundance of these zooplanktons was recorded highest in April and lowest in March in all the experimental ecosystems. Among four water samples, planktonic abundance were recorded maximum in pond 1 (Daphnia 47.1%, Moina 26.1% and Cyclops 11.9%), followed by pond 2 (Daphnia 30.9%, Moina 20.1% and Cyclops 10.4%) and pond 3 (Daphnia26%, Moina 17.8% and Cyclops 8.5%) whereas minimum zooplankton was recorded in river water (Daphia 20.1%, Moina 14.9% and Cyclops 2.8%) respectively. The relationship of three zooplanktons with physico-chemical parameters (pH, temperature, CO2, COD, BOD, DO, Alkalinity, NO2-N, NO3-N) were also measured considering their monthly variation.

During this study, mixed culture of these three zooplanktons (Daphnia, Moina and Cyclops) was practiced in the laboratory conditions. Culture was done at two aquariums using yeast extract for 21 days of experiment. Final abundance was recorded 1360±20 zooplankton/liter in aquarium1 and 1015±20 zooplankton/liter in aquarium 2, while initial recruitment of zooplanktons was 55/litre in both aquariums. This result suggests that zooplanktons culture in laboratory could be most potential for aquaculture.

Key words: Zooplankton, pond, river, physico-chemical factors, culture

I. Introduction Zooplanktons are the main sources of natural food for fish and shellfish which is directly related to the survival and growth and these are the base of food chains and food webs in all aquatic ecosystems. They also play a major role in recycling nutrients as well as cycling energy within their respective environments. Zooplanktons are an essential food item of omnivorous and planktivorous fishes [1] and the most essential for larvae culture [2]. The abundance of zooplankton in a water body is regarded as an indicator of productivity. Both the qualitative and quantitative abundance of zooplankton in a fish pond are of great importance in managing the successful aquaculture operations, as they vary from location to location and pond to pond within the same location even within similar ecological conditions [3]. In order to fisheries development and to increase the present production level, proper and scientific management is essential in which the knowledge of water quality and natural productivity plays an important role.

Though huge number of zooplankton is available in freshwater and all of them are highly important in aquaculture, however, Moina, Cyclops and Daphnia are found in diverse natural environments especially in freshwater [4]. These three zooplanktons with higher protein contents are essential for fish larval growth as well as contain a broad spectrum of digestive enzymes such as proteinases, peptidases, amylases, lipases and even cellulase that can serve as exo-enzymes in the gut of the fish larvae. They are the finest and most universally accepted live foods for most fish. Different zooplanktons are being culturing both in laboratory as well as small environments for using in fish and shrimp larvae culture in several countries but no available practice of zooplankton culture in Bangladesh. Normally Artemia are being used in shrimp industries import from Europe and USA as dry cysts. Zooplankton culturing certainly has the potential to reduce the need of purchasing frozen or live fish feeds like brine shrimp, bloodworms, glassworms, Tubifex, or California blackworms as well as cost effective and time consuming for mass production.

Vast number of zooplanktons is available in freshwater which are easy to culture and use in aquaculture industry. Most of the researches were done on these three experimental genera of zooplankton in the world in different field of researches but in Bangladesh available research was not found particularly in Sylhet region. Few researches have been done in Bangladesh about different zooplanktons [5], [6], [7], [8]. Due to their necessary applications in aquaculture both in nature and farms especially in fish and shrimp larvae culture, therefore, this research was carried out to observe the monthly abundance associated to the physico-chemical parameters of Daphnia, Moina and Cyclops of ponds and river ecosystems in Sylhet, Bangladesh. Further goal of this study was to establish the mixed culture of these zooplanktons in laboratory environment.

II. Materials and Methods Collection and Identification The water samples were collected from upper surface of three ponds of the campus of Shahjalal University of Science and Technology and Surma River of Sylhet for identification and observation of the abundance of three experimental zooplanktons (Daphnia, Moina and Cyclops). The experiments were conducted in the period of March to June, 2013 and the samples were collected by using plankton net, kept them in plastic container and brought to the fisheries biotechnology laboratory of the Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology (SUST), Sylhet for further analyses. The water quality parameters were studied in the Water Supply Management Lab in the Department of Civil and Environmental Engineering, Shahjalal University of Science and Technology (SUST), Sylhet, Bangladesh. The water samples were measured and poured into 250ml beaker. The water samples were observed drop by drop on the slides and the slides was set up under microscope and check with 4x, 10x and 40x. Cyclops was identified by Todd [9] while Daphnia and Moina were identified according to Charles [10].

Abundance Study Different methods has been established by different authors, however, one simple method was used to calculate these three zooplanktons (Table 1). This technique was followed 50 times for each water sample to calculate the abundance of zooplankton per litre. Table1: Observation of abundance of three zooplanktons Drop of water Zooplankton Number Drop of water Zooplankton Number 1st drop Daphnia 3 11th drop Moina 4 2nd drop Daphnia,Moina 3,2 12th drop Daphnia 6 3rd drop Daphnia 2 13th drop Daphnia 3 4th drop Daphnia,Moina 4,2 14th drop Moina 3 5th drop Daphnia 1 15th drop Nil 0 6th drop Moina 3 16th drop Daphnia,Moina 4,4 7th drop Daphnia,Moina 4,3 17th drop Daphnia 5 8th drop Daphnia 3 18th drop Moina,Cyclops 3,1 9th drop Daphnia 2 19th drop Cyclops 1 10th drop Daphnia,Cyclops 4,2 20th drop Daphnia 2 Measurements of physico-chemical parameter of the water Different sophisticated simple equipments and essential chemicals were used for the measurements of different water parameters. Various types of physico-chemical parameters such as temperature, pH, DO, BOD, COD, Alkalinity, CO2, NO2-N, NO3-N were measured. Surface water was taken in a plastic container and recorded its temperature immediately by dipping the thermometer for about one minute and water pH was measured by a bench top electrometric pH meter. The water samples were fixed up by using alkaline iodide and manganous sulfate and after few times, concentrated H2SO4 was added into the BOD bottle for measurements of Dissolved Oxygen (DO). Two drops methyl orange indicator was added into 10 ml of water sample and H2SO4 solution was taken in the burette. In the titration of alkalinity, light pink color was observed at the end point. 0.1N NaOH was taken in the burette and phenolphthalein indicator (5 drops) was added to the solution. Permanent pink color was observed at the end point. Two drops starch solution was added to the fixed up sample (20 ml) and Na2SO3 was taken in burette. The titration was continued to first disappearance of blue color. The following formula was used to calculate alkalinity of the waters: Alkalinity=T×10 ppm (Here T=Total Sulfuric acid used). CO2 of the water was measured with titration method where taken 100 ml of water sample into a beaker and same quantity of distilled water into another beaker. 10 drops of phenolphthalein indicator into each sample was added and if no pink colour is seen, added N/44 solution hydroxide from a burette to the sample and stir gently until a slight permanent pink colour appears as compared with the distilled water. The following formula was used to calculate CO2 of the waters, CO2mg/l = ML of N/44 ×10 and the data was recorded the ml of sodium hydroxide used. For determination of Biological Oxygen Demand (BOD), water sample was taken in an 8–oz glass coppered bottle carefully to avoid

AIJRFANS 13-308; © 2013, AIJRFANS All Rights Reserved Page 2 F. Miah et al., American International Journal of Research in Formal, Applied & Natural Sciences, 4(1), September-November, 2013, pp. 01-07 contact of the sample with air. Manganous and alkaline potassium iodide solution was added immediately with 1 ml of each. Insert the stopper and mixed by inverting the bottle several times. Allow the precipitated to the settle halfway and mixed again and 1 ml of concentrated sulfuric acid was added and remained with 5 minutes. 100 ml of that solution was transferred into a conical flask and 0.025N sodium thiosulfate was added immediately drop by drop from a burette until the yellow colour almost disappears. Further, about 1 ml of starch solution and sodium thiosulfate was also added continuously until the disappeared of blue colour. Sodium thiosulfate used was recorded by ml and finally dissolve oxygen present in sample was calculated in mg/l=ML of 0.025N sodium thiosulfate used × 2 BOD in mg/l=(Di-df)/P, where P=decimal volumetric fraction of sample used. Chemical Oxygen Demand (COD) was determined with 100 ml of the water into a 250 ml conical flask. Diluted Sulfuric acid and standard potassium permanganate were added with 10 ml of each, and kept it in the boiling water above the level of the solution in the flask for exactly 30 minutes. When solution became faintly colour, repeated the above using a smaller sample diluted to 100 ml with distilled water. After 30 min, in the water bath, 10 ml of standard ammonium oxalate was added. Titrate while hot with standard potassium permanganate to the first pink coloration and the ml of potassium permanganate used was recorded. The formula of the calculation of COD is, Oxygen consumed (COD) in mg/l=ML of KMnO4 used in step no. 6 × 100/ML of sample used. Nitrate nitrogen was determined with filtering 30 to 35 ml of the water sample through a filter paper. Evaporate 25 ml of the filtrate to dryness on a water bath. Moisten the residue with 1ml of phenol-di-sulfonic acid. Dilute to about 20 ml with distilled water where 50% solution of sodium hydroxide was added until the maximum yellow color was developed. Filter into a 100ml Nessler tube, raised the dish and paper with distilled water. Added the filtered rinsing to the filtrate and make up to the mark with distilled water. When the permanent standards were not available, made up temporary standards by placing 0.2, 0.4, 0.6, 0.8, 1,1.5, 2, 3, 4 and 5 ml of standard sodium nitrate solution in 100 ml, Nessler tubes and adding 2ml of 50% sodium hydroxide. Dilute to the mark with distilled water and the color was compared and recorded the standard having color nearest to that of the sample. Nitrate nitrogen was calculation by (NO3-N)=ML of standard NaNO3/ML of sample and nitrite nitrogen was determined with measurement of NO2-N is similar as measurements of NO3-N. In this case only NaNO2 was used instead of NaNO3. Statistical analysis Statistical analysis was done to find the correlation between total zooplanktons with physico-chemical parameters.

Correlation (r) was observed by following formula: Correlation Coefficient, r = , here, x=X , x=total zooplankton, y= , y=Physico-chemical parameters

Laboratory Culture Technique Two aquariums with 4 litres of distilled water in each were used for culturing zooplankton in the laboratory condition. Culture duration was 21 days and as initial steps 220 zooplanktons were added in each aquarium including 110 Daphnia, 70 Moina and 40 Cyclops. Culture was done at room temperature and pH values lied between 7.3-7.6. Low aeration was supply into the aquarium by aerator. In aquarium1, baker’s yeast was supplied 0.8 ml/day from 1-7 days, 0.9 ml/day from 8-14 days and 1 ml from 15-21 days. On the other hand, baker’s yeast was used as 0 .4 ml/day from 1-7 days, 0.5ml/l from 8-14 days and 0.6 ml/l for 15-21 days in aquarium 2. III. Result Abundance of zooplankton This study showed the monthly variation of three zooplanktons both qualitatively and quantitatively. During the study period, three genera such as Moina, Cyclops and Daphnia were observed and Daphnia was dominant during the experimental period (March to June, 2013) followed by Moina and Cyclops respectively (Table 2). The maximum abundance of these zooplanktons was found in the month of April and minimum in the month of March. Daphnia abundance was highest in all the aquatic ecosystems in experimental periods whereas, abundance of Daphnia, Moina and Cyclops was found maximum in the month of April respectively. But minimum number of these zooplanktons was found in the month of March. Table 2: Monthly density of three zooplanktons from March-June Zooplankton March April May June P 1 P 2 P 3 R P 1 P 2 P 3 R P 1 P 2 P 3 R P 1 P 2 P 3 R Daphnia 31 202 295 169 597 400 290 241 563 345 272 189 412 289 182 204 2 Moina 21 166 180 127 380 255 172 136 253 212 206 176 200 171 155 157 0 Cyclops 10 123 115 18 177 129 108 42 143 101 64 23 51 61 54 28 6 P1=Pond Water 1, P2= Pond Water 2, P3= Pond Water 3, R= River Water

The average number of these three zooplanktons was estimated (Table 3) where the abundance of Daphnia was 47.1% followed by Moina 26.1% and Cyclops 11.9% in pond water 1. In pond water 2, the abundance of Daphnia was found 30.9%, Moina 20.1% and Cyclops 10.4% respectively whereas the large quantity of Daphnia was observed 26%, Moina 17.8 % and Cyclops 8.5% in pond 3. However, lower quantity of zooplanktons such as 20.1% Daphnia, 14.9% Moina and 2.8% Cyclops were recorded in River water.

Table 3: Average number of three Zooplanktons (org/l) in four ecosystems by 4 months

Zooplankton P1 P 2 P 3 R Max Min Avg ±10 Max Min Avg ±10 Max Min Avg. ±10 Max Min Avg ±10 Daphnia 597 312 471 400 202 309 295 182 260 241 169 201 Moina 380 200 261 255 166 201 206 155 178 176 127 149 Cyclops 177 51 119 129 61 104 115 54 85 42 18 28

From the above statement, Daphnia abundance was found highest in all aquatic ecosystems in the experimental period which is followed by Moina and Cyclops (Table 3). The distribution of these three zooplankton communities were found maximum in pond water 1 (Daphnia 47.1%, Moina 26.1% and Cyclops 11.9%) and minimum in river water (Daphnia 20.1%, Moina 14.9% and Cyclops 2.8%) (Figure1). The avarage composition of these three experimental planktons were recorded and Daphnia is mostly abundant than Moina and Cyclops respectively (Figure 2).

45 40 35 30 25 Daphnia Daphnia 20 Moina Moina 15 Cyclops Cyclops 10

Average DistributionAverage % in 5 0 P1 P2 P3 R Four ecosystems

Figure 1: Distribution of three zooplanktons in four ecosystems. Figure 2: Average composition of three zooplanktons in 4 ecosystems.

Abundance related to Physico-chemical parameters of experimental ecosystems Some selected physico-chemical parameters were studied and average value of these parameters were recorded (Table 4). pH was observed throughout the study period which was fluctuated from 9.9 to 6.54 while highest (9.9) and lowest pH (6.54) was observed at river water in March and June respectively. In this study temperature was varied from 25.C to 31.2ºC and minimum (25ºC) was observed at pond water 1 in March and maximum (31.2ºC) at river water in June. Free Carbon dioxide was recorded from 2.2mg/l to 5 mg/l whereas minimum (2.2) was observed at pond water 1 in April and maximum (5) was observed at river water in March. The value of COD varied from 2 to 4.5, however, minimum was (2) observed at pond water 3 in April and maximum was (4.5) at river water in April. The Dissolved Oxygen (DO) content of the water varied from 2 mg/l to 4.4 mg/l and minimum DO concentration (2 mg/l) was observed at pond water in April and maximum (4.77mg/l) at river water in March. The highest value of BOD was recorded maximum (4.2) at river water in March and minimum (2.4) was observed at pond water 1 in April. The minimum and maximum alkalinity was observed 50 and 196 at river water in March and June respectively. NO2-N Value fluctuated from 0.0069 at river water in March to 0.022 at pond water 1 in April. Finally NO3-N Value was recorded minimum 0.053 at river water in March and maximum 0.16 at pond water 1 in April.

Table 4: Physico-chemical parameters (mg/l) of four aquatic ecosystems from March-June, 2013 P March April May June P1 P2 P3 R P1 P2 P3 R P1 P2 P3 R P1 P2 P3 R pH 9 9.6 9.7 9.9 7.2 7.25 7.3 8 6.7 6.6 6.55 6.48 6.65 6.54 6.5 6.4 T 25 25.2 25.4 25.8 26.7 26.7 26.4 26.2 29.2 29.4 29.5 30 29.5 29.7 30 31.2 CO2 4.3 4.6 4.8 5 2.2 2.6 2.8 2.3 2.8 2.9 3.2 2.6 3.2 3.4 3.8 4 COD 2.3 4.2 2.6 4.5 2.1 2.8 2 4 2.2 3.8 2.4 4.3 2.4 4.2 2.5 4.4 DO 3.6 3.8 4.2 4.4 2 2.2 2.2 2.4 2.8 3.6 2.8 2.2 3 3.8 3.2 2.5 BOD 3.5 3.8 3.6 4.2 2.4 2.6 2.6 2.7 2.5 3.4 2.7 2.8 2.7 3.5 2.8 3.1 A 148 166 168 196 86 92 94 106 76 72 70 66 62 59 58 50 NO2-N .0094 .0085 .0082 .0069 .022 .021 .021 .018 .015 .019 .013 .010 .014 .013 .013 .012 NO3-N .064 .0613 .061 .053 .16 .15 .15 .14 .12 .11 .10 .098 .088 .086 .085 .077 P= Parameter, T= Temperature, A= Alkalinity

Correlation of coefficient Coefficient correlation of total zooplanktons of three species with different physico-chemical parameters were measured (Table 5) and abundance were negatively correlated with pH, temperature, DO, BOD, COD and alkalinity, but positively correlated with NO2-N and NO3-N. So, it means that NO2-N and NO3-N were positively significant for zooplankton abundance during this study.

Table 5: Value of Co-efficient of correlation of total zooplanktons with physico-chemical parameters Particulars Co-efficient of correlation Remarks Zooplankton Vs pH -0.99 Inversely related Zooplankton Vs Water Temperature -0.94 Inversely related Zooplankton Vs free CO2 -0.76 Inversely related Zooplankton Vs COD -0.68 Inversely related Zooplankton Vs DO -0.49 Inversely related Zooplankton Vs BOD -0.66 Inversely related Zooplankton Vs Alkalinity -0.95 Inversely related Zooplankton Vs NO2-N 0.16 Highly Significant Zooplankton Vs NO3-N 0.27 Highly Significant

Findings from laboratory culture The culture of these three zooplanktons was done at two aquariums with mixed population where after 21 days, 1360±20 zooplanktons/L was observed in aquarium 1 and 1015±20 zooplanktons/L was observed in aquarium 2. When it was started, 220 zooplanktons were released into each aquarium (Table 6) while high and low amount of baker’s yeast was used as food respectively in aquarium 1 and aquarium 2. During these experiments the water color was seen slightly green after 7 days and deep green after 14 and 21 days in aquarium 1 while water color was seen slightly green after 7, 14 and 21 days in aquarium 2. The abundance was found after 7 days with Daphnia 120/L, Moina 75/L and Cyclops 50/L and after 14 days including Daphnia 200/L, Moina 158/l, Cyclops 94/L while in final harvest at 21 days of experiments, Daphnia, Moina and Cyclops were found 290/L, 240/L and 138/L respectively in aquarium 1 (Table 6). During this culture, total zooplanktons after 21 days were 1360±20/L, among them, Daphnia was 610±15/L, Moina was 473±15/L and Cyclops was 282±10/L. However, in aquarium 2, the abundance was found after 7 days with 90/L Daphnia, 58/L Moina and 30/L Cyclops, where after 14 days Daphnia 155/L, Moina 118/L and Cyclops 50/L were observed. Finally after 21 days, Daphnia 220/L, Moina 190/L and Cyclops 104/L were harvested and total zooplanktons were recorded as Daphnia 465±15/L, Moina 366±15/L and Cyclops 184 ±10/L (Table 6).

Table 6: Observation of three zooplanktons in 21 days culture in aquarium 1and 2 (per L) Day Aquarium 1 Aquarium 2 Initiation Findings pH T Initiation Findings pH T D M C D M C D M C D M C 1-2 110 70 40 7.3 24 110 70 40 7.4 24 7 120 75 50 7.3 25 90 58 30 7.44 25 14 200 158 94 7.38 25 155 118 50 7.5 25 21 290 240 138 7.4 27 220 190 104 7.6 27 610±15 473±15 282±10 465±15 366±15 184±10 1360±20/L 1015±20/L D=Daphnia, M=Moina, C=Cyclops, T=Temperature

IV. Discussion In this experiment, the abundance of three important zooplanktons (Daphnia, Moina and Cyclops) with their monthly fluctuations related to physico-chemical parameters as well as their correlation with zooplanktons was recorded. Furthermore, mixed culture of these three zooplanktons was also observed at laboratory condition. This study showed that Daphnia abundance was dominated in all months in four aquatic ecosystems followed by Moina and Cyclops respectively. Total zooplankton abundance was found at maximum level in the month of April and minimum in the month of March. Pond water 1 contained more zooplankton communities than other three ecosystems. The fluctuation of these three zooplanktons was observed similar in different works by different resarchers [1], [11], [12], [13], [14], [15], [16]. The study of Halda River in Bangladesh showed similar plankton compositions where 11 genera of different groups of zooplankton were identified from the study area [17]. From the above findings the percentages of copepod, rotifera, cladocera, ostracoda and crustacean larvae brysozoa in brood fish culture system were 55%, 8%, 25%, 9%, 2% and 1% respectively. Among the various groups of zooplankton, the percentages of copepod, rotifer, cladocera, ostracoda and crustacean larvae in the culture system were 52%, 39%, 7%, 1% and 1% respectively. The above results agree with the present study whereas average zooplankton community were found maximum in pond water 1 (Daphnia 47.1%, Moina 26.1% & Cyclops 11.9%) and minimum in river water (Daphnia 20.1%, Moina 14.9% & Cyclops 2.8%). In pond water 2, the abundance of Daphnia was found 30.9%, Moina 20.1% and Cyclops 10.4% respectively whereas the quantity of Daphnia was observed 26%, Moina 17.8 % and Cyclops 8.5% in pond 3 ( Table 3).

Zooplankton communities in fish ponds are subjected to wide variations in environmental conditions in addition to the fish predation. Physico-chemical parameters of the water impact the fluctuation of zooplankton populations. The average values of water pH, temperature, DO, CO2, COD, BOD and alkalinity were negatively correlated with total zooplanktons (Table 5) and the present results agree with few previous findings [1], [14], [16], [17], [18], however, the water pH, DO and BOD disagree with a previous result [8] and the result of the CO2 was not resembled with another research [19]. An inversely proportional highly significant relation was recorded between zooplankton communities in water temperature [20] which is also the same as present findings. The average alkalinity was negatively correlated with total zooplankton which supports the earlier works [8], [19]. The average NO2-N was positively correlated (0.16) with total zooplankton which supports a previous finding [21] and that is directly significant for the richness of zooplankton. The average NO3-N was positively correlated (0.27) with total zooplankton which same the other prior works [8], [19], but doesn’t supports with other finding [21].

During this study, mixed culture of these three zooplanktons was simply studied in the laboratory environment by using baker’s yeast as food and Daphnia was found higher in number than Moina and Cyclops respectively. Total abundance of zooplanktons was found maximum in aquarium 1 than the aquarium 2. The variation of the findings occurred due to the amount of baker’s yeast added to each aquarium where more food was supplied in aquarium 1 than aquarium 2. The study observed that mixtures of both spirulina and chlorella algae added to either baker’s active dried yeast or ground dried split peas and paprika both performed very well in zooplankton [22]. Any time a dried algae product is used it adds considerable cost to zooplankton production. However, using a mixture of high cost algae combined with very low cost yeast or split peas gives rapid yields, with high harvests, at a medium cost to the hobbyist [22] whereas baker’s yeast was used only in the present study and almost same production of zooplanktons was found. This abundance depends not only the food supply, may be another factor affects the reproduction of zooplankton [23]. V. Conclusion From the above observation it is found that the zooplankton abundance has been varied from place to place and it showed direct or indirect relationships with the physico-chemical parameters. For successful mixed culture of zooplankton at laboratory it was required a good practice with special consideration should be taken for recycling system, good aeration, sufficient food supply, regular observation of culture, etc.

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