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50. Lin, C. K., P. Ruamthaveesub, and P. Wanuchsoontorn. 1993. Integrated culture of green mussel (Perna viridis) in wastewater from an intensive shrimp pond: concept and practice. World Aquaculture 44:187-200. 51. M.S.M. Jetten, M. Wagner, J. Fuerst, M.C.M. Van Loosdrecht, G. Kuenen, M. Strous, Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process, Curr. Opin. Microbiol. 12 (2001) 283–288. 52. Marti´nez-Cordova, L. R., M. A. Porchas-Cornejo, H. Villareal-Colmenares, J. A. Caldero´n-Perez, and J. E. Naranjo-Paramo. 1998. Evaluation of three feeding strategies on the culture of white shrimp Penaeus vannamei Boone 1931 in low water exchange pond. 53. Mazik, P.M., Hinman, M.L., Winkelmann, D.A., Klaine, S.J., Simco, B.A., Parker, N.C., 1991. Influence of nitrite and chloride concentrations on survival and hematological profiles of striped bass. Trans. Am. Fish.Soc. 120, 247- 254. 54. Meincke, M., Krieg, E., Bock, E., 1989. Nitrosovibrio sp., the dominant ammonia-oxidizing bacteria in building sandstone. Appl. Env. Microbiol. 55 (8), 2108-2110. 55. Ministry Marine Affairs and Fisheries-Directorate General of Aquaculture in USDA foreign Agricultural service, Global Agriculture Information Network (GAIN) Report number: ID7024, Indonesia Fishery Products Shrimp Report 2007, 2007. 56. Pa´ez-Osuna, F., M. Hendrickx-Reners, and R. Cortes-Altamirano. 1994. Efecto de la calidad del agua y composicion biologica sobre la produccion en granjas camaronicolas. Informe final CONACYT proyecto 0625-N9110. 445 pp. (in Spanish). 57. Pa´ez-Osuna, F., S. R. Guerrero-Galva´n, A. C. Ruiz-Ferna´ndez, and R. Espinoza-Angulo. 1997. Fluxes and mass balances of nutrients in a semiintensive shrimp farm in north-western Mexico. Marine Pollution Bulletin 34:290 -297. 58. Pa´ez-Osuna, F., S. R. Guerrero-Galva´n, and A. C. Ruiz-Ferna´ndez. 1998. The environmental impact of shrimp aquaculture and the coastal pollution in Mexico. Marine Pollution Bulletin 36:65-75. 59. Pa´ez-Osuna, F., S. R. Guerrero-Galva´n, and A. C. Ruiz-Ferna´ndez. 1999. Discharge of nutrients from shrimp farming to coastal waters of the Gulf of California. Marine Pollution Bulletin 38:585-592. 60. Pambrun, V., Paul, E., Sperandio, M. Control and modeling of partial nitrification of effluents with high ammonia concentrations in sequencing bath reactor, Chem. Eng. Process. (2007) 65

61. Panswad T. and Anan C., (1999b). Specific oxygen, ammonia, and nitrate uptake rates of a biological nutrient removal process treating elevated salinity wastewater. Bioresour. Technol. 70 237-243. 62. Payne, W.J., 1973. Reduction of nitrogenous compounds by microorganisms. Bacteriol. Rev. 37 (4), 409-452. 63. Phillips, M. J. 1994. Aquaculture and the environment-striking a balance. Pages 29-31 in Proceedings of Infofish Aquatech 94, 29-31 August 1994, Colombo, Sri Lanka. 64. Pierce et al., 1993 R.H. Pierce, J.M. Weeks and J.M. Prappas, Nitrate toxicity to five species of marine fish, J. World Aquacult. Soc. 24 (1993), pp. 105-107 65. Rivera-Monroy, V. H., L. A. Torres, N. Bahamon, F. Newmark, and R. R. Twilley. 1999. The potential use of mangrove forests as nitrogen sinks of shrimp aquaculture pond effluents: The role of denitrification. Journal of the World Aquaculture Society 30:1 66. Robertson, A. I., and M. J. Phillips. 1995. Mangroves as filters of shrimp pond effluent: Predictions and biochemical research needs. Hydrobiologia 295:649-666. 67. Rodriguez-Valera F., Ruiz-Berraquero F. and Ramos-Cormenzana A. (1981) Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Micro. Ecol. 7, 235-243. 68. Rosa, M. F., Albuquerque, R. T., Ferna´ndez, J. M., Leite, S. G., and Medronho, R. A. (1997). ‘‘Nitrification of saline effluents.’’ Braz. J. Chem. Eng., 14(2), 151–158. 69. Rosenberry, B. About Shrimp Farming, ShrimpNews, August 2004 70. Rosenberry, B. 1998. World shrimp farming. Shrimp News International, San Diego, California, 328 pp. 71. Rosenthal and Otte, 1979 H. Rosenthal and G. Otte, Adaptation of a fish recirculating fish culture system to salinity change, Wissenschaftlichen Konnission fuer Meeresforschung 27 (1979), pp. 203–206. 72. Ruffier, P.J., Boyle, W.C., Wleinschmidt, J., 1981. Short-term acute bioassays to evaluate ammonia toxicity and effluent standards. J. Water Pollut. Control Fed. 53, 367- 377 73. Sandifer, P. A. and J. S. Hopkins. 1996. Conceptual design of a sustainable pond-based shrimp culture system. Aquacultural Engineering 15:41-52. 66

74. Schwedler and Tucker, 1983 T.E. Schwedler and C.S. Tucker, Empirical relationship between percent methemoglobin in channel catfish and dissolved nitrite and chloride in ponds, Trans. Am. Fish. Soc. 112 (1983), pp. 117-119. 75. Shishehchian, F., F. M. Yusoff, H, Omar, and M. S. Kamarudin 1999. Nitrogenous excretion of Penaeus monodon postlarvae fed with different diets. Marine Pollution Bulletin 39:224-227. 76. Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with the variables of substrate concentration and population. Appl. Environ. Microbiol. 47, 1299–1306. 77. Supangat, A., 2000, Sistem Biofilter Untuk Meningkatkan kualitas Air Tambak Udang, JTM, vol VII, no. 3/2000. 78. Suzuki, L., Dular, U., Kwok, S.C., 1974. Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts. J. Bacteriol. 120, 556-558. 79. S.Y. Wang, D.W. Gao, Y.Z. Peng, P. Wang, Q. Yang, Nitrification– denitrification via nitrite for nitrogen removal from high nitrogen soybean wastewater with on-line fuzzy control,Water Sci. Technol. 49 (2004) 121– 127. 80. Tacon, A. G. J., Thematic Review of Feeds and Feed Management Practices in Shrimp Aquaculture, World Bank/NACA/WWF/FAO Consortium Program on Shrimp Farming and the Environment, 2002. 81. Talaro, K.P. Foundations in Microbiology sixth edition, Mc Graw Hill, Boston, 801 pp, 2006. 82. Timmons, M.B., Ebeling, J.M., Wheaton, F.W., Summerfelt, S.T., Vinci, B.J., 2002. Recirculating Aquaculture Systems, 2nd Edition. Cayuga Aqua Ventures, New York. 769 pgs. 83. Tomasso, 1994 J.R. Tomasso, Toxicity of nitrogenous wastes to aquaculture animals, Rev. Fish. Sci. 2 (1994), pp. 291-314. 84. Tunvilai, D., P. Songsanginda, and K. Chaiyakaj. 1993. Pollution loading of effluent from intensive tiger shrimp culture ponds. Technical paper 4/1993. National Institute of Coastal Aquaculture. Department of Fisheries. Kao Saen, Muang District, Songkhl 85. USDA foreign Agricultural service, Global Agriculture Information Network (GAIN) Report number: ID7024, Indonesia Fishery Products Shrimp Report 2007, 2007.

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APPENDIX A ANALYTICAL METHODS

A.1. COD (Chemical Oxygen Demand) Titrimetric Method Most types of organic matter are oxidized by a boiling mixture of chromic and sulfuric acids. A sample is refluxed in strongly acidic solution with a known excess of potassium dichromate (K2Cr2O7). After digestion, the remaining unreduced K2Cr2O7 is titrated with ferrous ammonium sulfate to determine the amount of consumed and the oxidizable organic matter is calculated in terms of oxygen equivalent. The ratios of reagent weights, volume, and strengths are kept constant when sample volume was other from 50 ml. The standard 2-h reflux time may be reduced if it has been shown that a shorter period yields the same results. The following apparatus were used for COD analysis: borosilicate digestion tubes, heating block, oven, ampule sealer, and buret. The reagents used for COD analysis include: Potassium dichromate 0.05 N, FAS standard 0.05 N, digestion solution, and sulfuric acid reagent. The samples were digested in a heating block at 150oC for 2 hours. The samples were cooled and titrate with FAS until the color change from green into red. The COD was calculated using the following formula:

COD as mg O2 /L =

(A-B)* M * 8000 mL sample

where : A = ml FAS used for blank, B = ml FAS used for sample, and M = molarity of FAS. A.2. NO2- Colorimetric Method Nitrite (NO2-) was analyzed by colorimetric method (ASTM, 1989). Nitrite in the sample forms a reddish purple azo dye produced at pH 2.0 to 2.5 by coupling diazotized sulfanilamide with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride). The range of the method for spectrophotometric measurements was 10 to 1000 µg NO2-N/L. The color developed was read in spectrophotometer at 543 nm wavelength. The sample containing high nitrite concentration was measured by diluting sample with deionized water.

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The reagents used for the analysis include nitrite free water, color reagent, sodium oxalate 0.025M, FAS 0.05 N, and stock nitrite solution. The color was developed by adding diazotized sulfanilamide with N-(1-naphthyl)ethylenediamine dihydrochloride (NED dihydrochloride) to 50 ml of sample. The color was read using specthrophotometer set at wavelength 543 nm. Standard curve was prepared using known NO2--N concentration solutions. A.3. NO3- Ultraviolet specthrophotometric Screening Method Nitrate (NO3-) was analyzed by colorimetric method (ASTM, 1989). The NO3calibration curve follows Beer's law up to 11 mg N/L. Measurement of UV absorption at 220 nm enables rapid determination of NO3-. Because dissolved organic matter also may absorb at 220 nm and NO3- does not absorb at 275 nm, a second measurement made at 275 nm may be used to correct the NO3- value. The sample containing high nitrite concentration was measured by diluting sample with deionized water. The reagents used for the analysis include nitrate free water, HCl 1N, and stock nitrite solution. Standard curve was prepared using known NO2--N concentration solutions. For samples and standards, subtract two times the absorbance reading at 275 nm from the reading at 220 nm to obtain absorbance due to NO3. A.4. NH3 Phenate Method An intensely blue compound, indophenol, is formed by the reaction of ammonia, hypochlorite, and phenol catalyzed by a manganese salt. The reagents used for the analysis include, ammonia free water, Hypochlorous acid reagent, manganese sulfate solution, 0.003M, phenate reagent, and stock ammonium solution. The color was developed by adding phenate reagent and hypochlorous acid to 50 ml of sample. The color was read using spectrophotometer set at wavelength 630 nm. Standard curve was prepared using known NH3-N concentration solutions. A.5. Total Solids Analysis A well-mixed sample is evaporated in a weighed dish and dried to constant weight in an oven at 103 to 105°C. The increase in weight over that of the empty dish represents the total solids. The following apparatus were used for used for total solids analysis, evaporating dishes, desiccators, oven, and analytical balance. Evaporating dish was ignited at 103 to 105°C for 1 h, cooled in dessicator, and then weighed immediately. 2,5 ml of sample was transferred to preweighed

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dish and evaporated to dryness on a drying oven at least 1 h in an oven at 103 to 105°C, cooled in dessicator, then weighed. The total solid was calculated using the following formula:

mg total solids/L =

(A-B)X 1000 sample volume, mL

where: A = weight of dried residue + dish, mg, and B = weight of dish, mg A.6. Total Suspended Solids Analysis A well mixed sample is filtered through a weighed standard glass-fiber filter and the residue retained on the filter is dried to a constant weight at 103 to 105°C. The increase in weight of the filter represents the total suspended solids. If the suspended material clogs the filter and prolongs filtration, the difference between the total solids and the total dissolved solids may provide an estimate of the total suspended solids. The following apparatus were used for total solids analysis, evaporating dishes, desiccators, oven, glass-fiber filter disks, filter paper whatman 40, and analytical balance. Evaporating dish and filter paper were ignited at 103 to 105°C for 1 h, cooled in dessicator, then weighed immediately. 2.5 ml of sample was filtered, then the filter paper was transferred to preweighed dish and evaporated to dryness on a drying oven at least 1 h in an oven at 103 to 105°C, cooled in dessicator, then weighed. The total suspended solid was calculated using the following formula:

mg total suspended solids/L =

(A-B)X 1000 sample volume, mL

where: A = weight of filter + dried residue, mg, and B = weight of filter, mg

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APPENDIX B ANALYSIS FLOW CHART

B.1 Chemical Oxygen Demand

Preparation of Digestion Solution

Preparation of Sulfuric Acid Reagent

5.108 g K2Cr2O7

2.75 g Ag2SO4

Heat to 103oC For 2 h

Add 0.5 kg (283 mL) cone H2SO4

Add 83.5 mL cone H2SO4 Let 1 to 2 days Add 16.65 g HgSO4

Sulfuric acid Reagent

Dissolve, cool to room temperature, dilute to 500 mL

Digestion solution

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Preparation of Primary standard K2Cr2O7 0.05 N

Preparation of FAS Solution 0.05 N

9.8035 g FAS

0.6129 g K2Cr2O7

Add 10 ml H2SO4

Dilute to 250 mL

Dilute to 500 mL

K2Cr2O7 Standard

FAS standard 0.05 N Preparation of Ferroin Indicator 0.7425 g 1,1-0fenantrolin monohydrate

Add 0.3475 g FeSO4.7H2O

Dilute to 50 mL

Ferroin indicator

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Sample Analysis

Sample, blank @ 2.5 mL Put each into the tube which was washed with 20 % H2 SO4

Add 1.5 mL digestion solution

Add 3.5 mL Sulfuric Acid Solution

Close culture tube and shake well Place in oven preheated to 1500C and reflux for 2h Cool to room temperature Add 2 drops of ferroin indicator

Titrate with FAS

COD concentration

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B.2 NO2- Colorimetric Method Preparation of FAS Solution 0.05 N

Preparation of Color Reagent

25 mL 85 % phosphoric acid

9.8035 g FAS

Add 2.5 g sulfanilamide

Add 10 ml H2SO4

Add N-(1-naphtyl)ethlenediamine dihydrochloride

Dilute to 500 mL

Dilute to 250 mL FAS standard 0.05 N

Color reagent

Preparation of Stock Nitrite Preparation of Potassium Permanganate Solution

Solution

0.616 g NaNO2

4 g KMnO4

Dilute to 500 mL Dilute to 500 mL Add 1 mL CHCl3 KMnO4 standard 0.05 N

Stock Nitrite 1 mL=250 µg N

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Preparation of Intermediate Nitrite Solution

Standardization of Stock Nitrite Solution

NO2stock

50 mL KMnO4 standard 0.05 N

Calculate 12.5/ NO2- stock concentration

Add 5 mL cone H2SO4

Add 50 mL NO2Stock solution

Dilute to 250 mL

Shake gently

NO2Intermediate solution 1 mL= 50 µg

Add 10 mL FAS 0.05 N

Preparation of Standard Nitrite Solution

Titrate with KMnO4

Make a blank

10 mL NO2intermesiate solution

NO2- stock Concentration

Dilute to 1000 mL

NO2- standard solution 1 mL= 0.5µg

76

Preparation of Standard Curve

Sample Analysis

NO2- standard solution With volume variation

Adjust pH 5-9

Dilute to 50 mL

Add 2 mL color reagent

Read Absorbance at 543 nm Plot A versus NO2concetration

Standard curve

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B.3 NO3_ Analysis Preparation of Stock Nitrate Solution

Sample Analysis

2 mL Wastewater

KNO3

Remove suspended solid

Dry at 105 0C 24 h

Dilute to 50 mL, add 1 mL HCl 1 N, mix

Dissolve 0.18045 g dilute to 250 mL

Read A at 220 and 275 nm Dilute to 250 mL Add 2 mL CHCl3

Calculate NO3concentration =2 A275-A220

Nitrate stock solution

Plot A versus NO2concetration

Preparation of Standard Curve

NO3- concetration

Several volume of stock nitrate

Dilute to 50 mL, add 1 mL HCl 1 N, mix Read A at 220 and 275 nm Calculate NO3concentration =2 A275-A220 Plot A versus NO2concetration Standard curve

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B.4 NH3 Phenate Method Preparation of Phenate Reagent

Preparation of Hypochlorous Acid Reagent

2.5 g NaOH

10 mL 5 % NaOCl

Add 40 ml water

Add 10 g Phenol

Dissolve in 100 mL water

Adjust pH to 6.5 to 7 with HCl

Phenate reagent

Hypochlorous acid reagent

Preparation of Stock Ammonium solution

Preparation of Manganous Sulfate Solution

381.9 mg anhydrous NH4CL

50 mg MnSO4.H2O

Dried at 1000C

Dissolve in 100 mL water

Dissolve in 1000 mL

MnSO4 0.003 M

Stock Ammonium Solution 1 ml = 122 µg

79

Preparation of Standard curve

Sample Analysis

Sample

Stock Ammonium Solution

Add 1 drop (0.05 mL) MnSO4 solution

Prepare 5 different concentrations 0.1 to 5µg

Add 0.5 mL hypochlorous acid reagent

Read the Absorbance at 630 nm

Immediately add 0.6 mL phenate reagent

Calibration curve

Read absorbance at 630 nm

Ammonia concentration

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B.5 Total Solid Analysis Sample Analysis

Prepararation of Evaporating Dish

Sample

Cleaned dish

Transfer to preweighed dish

Heat to 103105oC For 1 h

Evaporate

Cool in desiccator

Heat to 103105oC For 1 h

Weigh

Cool in desiccator

Weight of dish

Weigh

Repeat the cycle of drying, cooling, desiccating, and weighing until i h i Weight of (dish + residu) - Weight of dish

Weight of total solids

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B.6 Suspended Solids Analysis

Filtering apparatus with suction

Filter the sample

Wash 3 times with 10 ml distilled water

Transfer filter to dish

Dry at 103-105oC For 1h

Cool in the desiccator

Weigh

Repeat the cycle of drying, cooling, desiccating, and weighing until weight is constant Suspended solids concentration

82