EnvironmentAsia The international journal published by the Thai Society of Higher Education Institutes on Environment Available online at www.tshe.org/EA Available online at www.tshe.org/EA EnvironmentAsia 27(1) (2014) 81-88 EnvironmentAsia (2009) 50-54 Genotoxicity Assessment of Mercuric Chloride in the Marine Fish Therapon jaruba Nitrogen and Phosphorus Removal in the Recirculating Aquaculture System Nagarajan Arumugam Kuppusamy Kumaraguru, Velmurugan JanakiCabbage Devi with Water Nagarani, Treatment Tank Containing Baked Clay Beads and Chinese and Chandrasekaran Archana Devi Aeknarin Thanakitpairin a, b, Wiboonluk Pungrasmi b and Sorawit Powtongsook c, d Center for Marine and Coastal Studies, School of Energy, Environment and Natural Resources, a Madurai Kamaraj University, Madurai-625021, India Department of Environmental Sciences, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chantaburi, Thailand b Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Thailand c Abstract National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand d Center of Excellence for Marine Biotechnology, Department of Marine Science, Faculty of Science, The aim of the present study was to Chulalongkorn standardize andUniversity, to assess Thailand the predictive value of the cytogenetic analysis by Micronucleus (MN) test in fish erythrocytes as a biomarker for marine environmental contamination Micronucleus frequency Abstract baseline in erythrocytes was evaluated in and genotoxic potential of a common chemical was determined in fish experimentally exposed in aquarium under controlled conditions Fish (Therapon jaruba) were exposed for 96 hrs a single heavy metal (mercuric Chromosomal determined Aquaculture as micronuclei frequency toThis research aims to describe thechloride) nitrogen and phosphorus damage removal was in Recirculating System (RAS)inby fish erythrocytes Significant increase in MN frequency was observed in erythrocytes of fish exposed to mercuric crop plants biomass production The experiment systems consisted of treatment (fish tank + baked clay beads + Chinese chloride of control-1 0.25 ppm (fish induced highest MN frequency (2.95 cells/1000 cells compared cabbage) Concentration and controls as tankthe only) and control-2 (fish tank + micronucleated baked clay beads), were performed With all to MNcell/1000 cells in control animals) The study revealed that micronucleus test, as an index of cumulative experimental RAS, Nile tilapia (Oreochromis niloticus) was cultured at kg/m density The baked clay beads (8-16 mm exposure, appears to beasa asensitive model to the evaluate genotoxictank compounds in fish under controlled conditions in diameter) were filled layer of 10 cm in water treatment of control-2 While in the treatment tank, Chinese cabbage (Brassica pekinensis) was planted at 334 plants/m in baked clay beads layer During 35 days of experiment, the Keywords: mercuric chloride; micronucleus average fishgenotoxicity; wet-weight in control-1, control-2 and treatment systems increased from 16.31±1.49, 15.18±1.28 and 11.31±1.49 g to 29.43±7.06, 28.65±3.12 and 27.20±6.56 g, respectively It was found that the growth rate of 0.45±0.15 g-wet weight/ day in a treatment tank was higher than in those controls, which were rather similar at 0.37±0.16 and 0.38±0.05 g-wet weight/day, respectively The fish survival rate of all experimental units was 100% The average Chinese cabbage wet-weight fieldallconditions 2006 Soumendra Introduction in treatment system increased from 0.15±0.02 g to 1.00±0.38 laboratory g For waterand quality, parameters In were within the acceptet al., made an attempt to detect genetic biomarkers able range for aquaculture The assimilation inorganic nitrogen in a treatment tank showed a slower rate and lower nitrite accumulation thosetons in control tanks The phosphorus balance Labeo analysisbata illustrated that most of the in two fish species, and Oreochromis In India,relative aboutto200 of mercury andnitrogen its and nitrogen and phosphorus input ininto all systems was from feed (82-87% and 21-87%) the final day of experiments, mossambica, by while MN atand binucleate (BN) compounds are introduced the environment nitrogen and in tilapia culture revealed at 15-19% and 4-13% The in accumulation of nitrogen phosphorus in erythrocytes the gill and kidneyand erythrocytes annually as phosphorus effluents from industries (Saffi, 1981) the water, up to 56% has and 70%, in control-1as while in the tank with bakedpower clay beads had discharge substantial lower exposed to thermal plant at Mercuric chloride been was usedfound in agriculture a water nitrogen and phosphorus concentration The most important part was unaccounted nitrogen and phosphorus as high as 60% Titagarh Thermal Power Plant, Kolkata, India fungicide, in medicine as a topical antiseptic and and 17% in treatment and 53% and 10% in control-2 systems Nitrogen and phosphorus incorporated in plant (treatment) The present study was conducted to determine disinfectant, and in chemistry as an intermediate in was only 1.31% and 0.11%, respectively It can be implied from the results that the assimilation in plant was a minor process the acute the heavy metal compound the production of in other mercury compounds for nutrient removal this RAS On the other hand, The the nitrification andgenotoxicity denitrificationofoccurred in the sediment layer of HgCl in static systems Mercuric chloride is toxic, contamination of aquatic ecosystems by heavy baked clay beads tank were the major treatment processes to maintain water quality in the recirculating system Without water it can theclay aquatic metals andbead, pesticides haswaste gained attention baked clay nitrogen wasincreasing accumulated as nitrate insolvable the waterinwhile in hence treatment tankpenetrate with backed beads, animals Mutagenic studies with native fish species in recent exposure to and nitrogen wasdecades significantlyChronic removed by denitrification process represent an important effort in determining the accumulation of these chemicals in aquatic biota Keywords: Aquaculture nitrogen potential removal; phosphorus nitrification; effects ofremoval; toxic agents Thisdenitrification; study was can result Recirculating in tissue burdens that System; produceRAS; adverse Chinese not cabbage carried out to evaluate the use of the micronucleus effects only in the directly exposed organisms, test (MN) for the estimation of aquatic pollution but also in human beings using marine edible fish under lab conditions Fish provides a suitable model for monitoring Introduction Common water treatment processes in the RAS, apart aquatic genotoxicity and wastewater quality from aeration, and are sediment Materials methodsremoval and nitrification because of its ability to metabolize xenobiotics and accumulated Recently,pollutants aquaculture industry is expanding processes In general, toxic nitrogen compounds such A micronucleus assay has rapidly duesuccessfully to an increase of the world food as ammonia nitrite derived from aquatic animal’s 2.1 Sample and Collection been used in several species (Dedemand Flora, Environmental friendly aquaculture system is therefore excretion, feed residues, and microbial degradation et al., 1993, Al-Sabti and Metcalfe, 1995) The essential for sustainable development The closed(Crab et al., 2007) must be regulated below 0.5 mg-N/L The fish species selected for the present study micronucleus (MN) test has been developed recirculating technology has been Highcollected ammoniafrom and Pudhumadam nitrite can cause adverse health was coast of Gulf of together withaquaculture DNA-unwinding assays as developing formethods decades, but under research effects in Southeast aquatic animals Mannar, Coastand ofcreate India.environmental Therapon perspective for mostly mass is monitoring of Recirculating Aquaculture System (RAS) water jarbua concernsbelongs if effluent notorder properly treated Apart to isthe Perciformes of from the clastogenicity and genotoxicity in fish anduses mussels treatment technologies to treat wastewater from nitrogen waste treatment, phosphorus accumulation family Theraponidae The fish species, Therapon (Dailianis et al., 2003) aquaculture and reuse the water for a long period in the RAS is also concerned but4-4.25 phosphorus removal jarbua (6-6.3 cm in length and g in weight) The MNtank tests have been successfully used as was selected for the detection of genotoxic effect a measure of genotoxic stress in fish, under both Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 requires sophisticated sequential anaerobic-aerobic 2.1 Recirculating aquaculture system process which is not yet commercial available (Burut-Archanai et al., 2013) In this research, we The experimental aquaculture system consisted studied the possibilities of combining wastewater of 38 x 58 x 31 cm3 fish tank (working volume 45 L) treatment with the production of crop plants biomass connected to the overlay water treatment tank The for phosphate removal which could not be treated by water treatment tank (plant tank) was 38 x 58 x 24 cm3 conventional treatment system The advantage of using plastic tank packed with 10 cm layer of spherical shape plant is that it is not only assimilating nitrogen waste baked clay beads (8-16 mm in diameter) and Chinese but it also remove phosphate from the water with the cabbage with approximately 4.83±0.35 cm height absorption of the root system (Beven, 2010) In an planted at 334 plants/m2 This bead packing performed aquaponic RAS with substrate support for planting, as suspended solids retainer and biological filtration the nitrogen assimilation process by plant and the media for inorganic nitrogen treatments (Fig 1) The nitrogen degradation process by nitrifying/denitrifying effluent from fish tank was pumped by submersible bacteria were combined for nitrogen removal from fish pump (Resun SP-6600) through PVC pipes lying over wastewater Unlike the aquaponic concept with floating the treatment tank Water was spray into treatment tank plants (Rakocy and Hargreaves, 1993; Timmons et al., at L/min for 10 minutes thereafter pump was pause 2002; Wilson, 2005), the proposed system applied for 60 minutes before the next pumping round Water baked clay beads layer in a tank, that was not only for from treatment tank was flow back to the fish tank by supporting plant root but also performed as the media gravity Continuous aeration in fish tank was provided for microbial nitrogen removal via nitrification and through diffusive stone aerators in order to maintain denitrification processes Moreover, the optimization proper environmental conditions for fish growth and of nitrogen and phosphorus removal processes was nitrifying process (i.e., well-mixed, DO > 4.0 mg O2/L, necessary for water quality control in fish tank and pH = 7-8 and alkalinity = 120-160 mg CaCO3/L by research, the possibilities of combining treatment with the production of yieldInofthis plant (Graberwe andstudied Junge, 2009) In our systems, addingwastewater sodium bicarbonate) plants biomasscabbage for phosphate removal which could nottilapia be treated conventional Nilecrop tilapia and Chinese were chosen for this Nile with by an average initialtreatment wet-weight system The advantage of using plant is that it is not only assimilating nitrogen waste but alsowas study as they are economical important species and fast of 14.27±1.42 g and length of 9.44±0.27it cm remove phosphate from the water with the absorption of the root system (Beven, 2010) In an of growth rates The experimental system was carried out in stocked in all fish tanks to obtain the initial density aquaponic RAS with substrate support for planting, the nitrogen assimilation process by plant and the partial controlled condition in which light, temperature, approximately kg/m Fish was fed twice daily at 8.00 by nitrifying/denitrifying combined for nitrogen removalfeed DO, nitrogen moisture,degradation nutrients andprocess pests were regulated to suit am bacteria and 3.00were pm with 25% protein commercial from fish wastewater Unlike the aquaponic concept with floating plants (Rakocy and Hargreaves, 1993;was for both fish and plant living pellets at 5% of total fish weight per day (feeding Timmons et al., 2002; Wilson, 2005), the proposed system applied baked clay beads layer in a tank, adjusted every week following fish biomass) Fish growth that was not only for supporting plant root but also performed as the media for microbial nitrogen Materials and Methods was monitored by length and weight measurement removal via nitrification and denitrification processes Moreover, the optimization of nitrogen and every week and the experimental period was 35 days phosphorus removal processes was necessary for water quality control in fish tank and yield of plant The experiment was conducted at the Center of Growth of Chinese cabbage was measured by weighing (Graber and Junge, 2009) In our systems, Nile tilapia and Chinese cabbage were chosen for this study Excellence Biotechnology, Department of growth at the rates initial The and experimental the end of 35system days experiment as they for are Marine economical important species and fast was carriedLeaf Marine Science, of condition Science, Chulalongkorn width, length canopynutrients size was every out in partial Faculty controlled in which light, temperature, DO,and moisture, andmeasured pests were University The treatment recirculating aquaculture week regulated to suit for both fish and plant living system consisted of fish tank growing Tilapia and plant2.tank packed and withMethods baked clay beads and Chinese 2.2 Water quality parameters and analytical methods Materials cabbage The fish tank without plant tank and fish tank + bakedThe clay experiment beads tank (no were assigned During the experiment, water samples were wasplant) conducted at the Center of Excellence for Marine Biotechnology, as control-1 and control-2, respectively (Table 1) All taken out daily for ammonia, nitrite, nitrate, alkalinity, Department of Marine Science, Faculty of Science, Chulalongkorn University The treatment experimental systems were performed replicates phosphate, total nitrogen totaltank phosphorus analysis recirculating aquaculture system with consisted of fish tank growing Tilapia andand plant packed with and placed in thebeads greenhouse following method for tank water+and wastewater baked clay and Chinese cabbage The fish tank withoutstandard plant tank and fish baked clay beads tank (no plant) were assigned as control-1 and control-2, respectively (Table 1) All experimental systems were performed with replicates and placed in the greenhouse Table Experimental systems performed in this study Experimental conditions Control-1 Control-2 Fish tank only Fish tank + Baked Clay Beads Treatment Fish tank + Baked Clay Beads + Chinese cabbage 2.1 Recirculating aquaculture system The experimental aquaculture system consisted of 38 x 58 x 31 cm3 fish tank (working volume 45 L) connected to the overlay water treatment tank The water treatment tank (plant tank) 82 was 38 x 58 x 24 cm3 plastic tank packed with 10 cm layer of spherical shape baked clay beads (8-16 mm in diameter) and Chinese cabbage with approximately 4.83±0.35 cm height planted at 334 Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 control and treatment units was 100% The average Chinese cabbage wet-weight in treatment system Figure Schematic diagram and photo of the treatment recirculating aquaculture system consisted of fish tank increased 0.15±0.02 g tobaked 1.00±0.38 g and increased and overlayfrom treatment tank with clay beads andlength Chinese cabbage.from 4.83±0.35 cm to 8.04±1.13 cm (0.11 cm/day) Leaf width increased from 0.77±0.10 cm to 3.26±0.54 cm (0.09 cm/day) and canopy size expanded fromwith 1.64±0.18 cm initial to 11.43±3.22 cm/day) The average growth rate of Nile tilapia an average wet-weightcmof (0.35 14.27±1.42 g and length of 9.44±0.27 cm was Chinese cabbage was equivalent to 0.02±0.01 g/day stocked in all fish tanks to obtain the initial density of approximately kg/m Fish was fed twice daily at 8.00 am and 3.00 pm with 25% protein commercial feed pellets at 5% of total fish weight per day Table Growth characteristics of Tilapia during the experiment (a, b shows was differed significantly) (feeding was adjusted every week following fish biomass) Fish growth was monitored by length and weight measurement every week and the experimental period was 35 days Growth of Chinese Parameter Control-1 Control-2 Treatment cabbage was measured by weighing at the initial and the end of 35 days experiment Leaf width, Initial tilapia wet-weight (g) 16.31±1.49 15.18±1.28 11.31±1.49 length and canopy size was measured every29.43±7.06 week Final tilapia wet-weight (g) 28.65±3.12 27.20±6.56 Initial tilapia length (cm) 9.96±0.23 9.46±0.27 8.89±0.32 11.92±0.62 11.37±1.02 Initial biomass density (kg/m3) 2.04 2.01 2.14 During the experiment, water samples Final biomass density (kg/m3) 3.68were taken out daily 3.81 for ammonia, nitrite, 5.11 nitrate, Total feed (g) 135.33 176.12 alkalinity, phosphate, total nitrogen and total phosphorus analysis following standard200.50 method for a a b Average growth (g/day) 0.37±0.16 0.38±0.05 0.45±0.15 water anddaily wastewater analysis (APHA, 2005) Suspended solids in the water was analyzed every three Feed conversion ratio; FCR including DO, pH, 2.06 1.96were measured using 1.69 portable days Physical parameters temperature and ORP Survival rate (%) 100 100 100in baked meters Nitrogen and phosphorus in feed, fish, suspended solids in fish tank, solid retained 2.2 parameters and analytical methods FinalWater tilapiaquality length (cm) 11.79±0.73 ORP (mV) clay beads tank, Chinese cabbage, and baked clay beads were determined at the initial and the end of It was found that growth of Chinese cabbage in this experiment was much samples slower were than the experiment for nitrogen and phosphorus budget analysis Nitrogen content in dried conventional vegetable planting in soil but comparable to aquaponic system by Graber and Junge analyzed by CHN analysis using dynamic flash combustion, CHNS-O analyzer Phosphorus was (2009) which the average 2.07plasma g of wet-weight and 18spectrometry, cm in lengthat after 55 days.Equipment This was analyzed usinghad inductively coupled optical emission the Scientific Results and Discussion analysis (APHA, 2005) Suspended solids in the and water probably due to the limitation of nutrients improper environmental condition in the experiment Center, Prince of Songkla University, Thailand Statistical analysis (ANOVA) between the controls wasLow analyzed every three days 980-25,410 Physical parameters light intensity Lux due to building and treatments was between calculated using Microsoft Excel 2007 shade on the experiment green house in 3.1 of fish and Chinese cabbagePotential including DO, pH, temperature and ORP were measured the afternoon was also another factor affecting growth Growth Decrease of Oxidation-Reduction using portable meters Nitrogen and phosphorus in (ORP) fromand +290 to +110 mV in baked clay bead layer indicated that accumulation of sediment in Results Discussion During 35bead dayslayer of experiment, feed,baked fish, suspended tank, solid retained clay beads solids causedinanfish increase of oxygen consumption in the (Fig 2) the average fish wet-weight in control-1, control-2 and treatment systems in baked clay beads tank, Chinese cabbage, and baked 3.1 Growth of fish and Chinese cabbage increased from 16.31, 15.18 and 11.31 g to 29.43, clay beads were determined at the initial and the end 350 28.65 and 27.20 respectively (Tableand 2).treatment It was found of the experiment for35nitrogen phosphorus During days ofand experiment, the budget average fish wet-weight in g,control-1, control-2 that treatment withg,baked clay beads and2) Chinese analysis Nitrogen in dried were 300 systems increasedcontent from 16.31, 15.18samples and 11.31 g to 29.43, 28.65 andtank 27.20 respectively (Table It cabbage had the highest growth of 0.45 g/day while analyzed by CHN analysis using dynamic flash was found that treatment tank with baked clay beads and Chinese cabbage had the highest growth of 250 analyzer fish growth in control-1 control-2 rather combustion, Phosphorus 0.45 g/dayCHNS-O while fish growth rate in control-1was and control-2 wererate rather similar atand 0.37 and 0.38were g/day, similar at 0.37 g/day, respectively These analyzed using inductively coupled plasma optical respectively These growth rates were within acceptable range due toand the0.38 proper fish density between 3 200at the fish 2-5 kg/m while growing at higher density e.g.growth 12 kg/m could daily growth rates werereduce within average acceptable range due to the emission spectrometry, Scientific Equipment 0.16±0.09 as reported by Azim and Little (2008) Statistical analysis indicated that3 while the fish in proper fish density between 2-5 kg/m growing Center, Princeg/day of Songkla University, Thailand 150 treatment tank had significantly higher growth rate than control-1 and control-2 Feed conversion ratio Statistical analysis (ANOVA) between the controls fish at higher density e.g 12 kg/m could reduce average (FCR) in treatment system wasMicrosoft 1.69 while FCR daily in control-1 control-2g/day wereas2.06 and 1.96, growth and to 0.16±0.09 reported by Azim and treatments was calculated using Excel 100 theLittle fish (2008) in treatment tanks Survival rate ofthat all the and Statistical analysis indicated 2007.respectively This indicated better feed utilization of 50 0 10 1583 20 Time (d) 25 30 35 Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 fish in treatment tank had significantly higher growth 3.2 Water quality rate than control-1 and control-2 Feed conversion ratio (FCR) in treatment system was 1.69 while FCR in According the Fig illustrates inorganic nitrogen control-1 and control-2 were 2.06 and 1.96, respectively and phosphate concentrations in control and treatment This indicated better feed utilization of the fish in systems High peak of total ammonia nitrogen (TAN) treatment tanks Survival rate of all control and treatment up to 1.2±0.3 mg-N/L was found in control-1 during the units was 100% The average Chinese cabbage wet- first days, while small TAN peaks at 0.31±0.1 mg-N/L control and treatment was 100% The average cabbage wet-weight treatment system weight in treatment systemunits increased from 0.15±0.02 g Chinese were found in control-2 and in treatment system which increased from 0.15±0.02 g to 1.00±0.38 g and length increased from 4.83±0.35 cm to 8.04±1.13 cm to 1.00±0.38 g and length increased from 4.83±0.35 cm was within the safety range [below 0.5 mg-N/L (Liao (0.11 cm/day) Leafcm/day) width increased from 0.77±0.10and cm Mayo, to 3.26±0.54 (0.09day cm/day) andaccumulation canopy to 8.04±1.13 cm (0.11 Leaf width increased 1972)].cm During 3-8, the size expanded from 1.64±0.18 cm to 11.43±3.22 cm (0.35 cm/day) The average growth rate ofpeaks from 0.77±0.10 cm to 3.26±0.54 cm (0.09 cm/day) and of nitrite was found in all tanks after TAN Chinese was equivalent to 0.02±0.01 g/day disappearance This indicated the occurrence of canopy size cabbage expanded from 1.64±0.18 cm to 11.43± 3.22 cm (0.35 cm/day) The average growth rate of nitrification process via ammonia oxidizing bacteria Table Growth characteristics of Tilapia during the experiment (a, b shows was differed significantly) Chinese cabbage was equivalent to 0.02±0.01 g/day The highest peak of nitrite, up to 3.84±0.8 mg-N/L, It was found that growth of Chinese cabbage in was also found in control-1, while smaller peaks were Parameter Control-1 Control-2 Treatment this experiment much (g) slower than conventional and treatment11.31±1.49 systems (0.86±0.1 Initial tilapia was wet-weight 16.31±1.49 found in control-2 15.18±1.28 vegetable planting in soil but comparable to aquaponic and 0.38±0.25 mg-N/L, respectively), Final tilapia wet-weight (g) 29.43±7.06 28.65±3.12 27.20±6.56and these system by Graber and Junge (2009) which had the concentrations were under the safety range [below Initial tilapia length (cm) 9.96±0.23 9.46±0.27 8.89±0.32 Final tilapia length (cm) 11.79±0.73 11.92±0.62 11.37±1.02 average 2.07 g of wet-weight and 18 cm in length mg-N/L (Hart and O,Sullivan, 1993)] The nitrification biomass (kg/m3) due to the limitation 2.04 was complete within 2.01 10 day when the 2.14accumulation afterInitial 55 days Thisdensity was probably Final biomass density (kg/m ) 3.68 3.81 5.11 of nutrients and improper environmental condition of nitrate, the end product of nitrification, occurred Total feed (g) 135.33 176.12 200.50 in the experiment Low light intensity between 980- a without nitrite accumulation At the end of the Average daily growth (g/day) 0.37±0.16 0.38±0.05a 0.45±0.15b 25,410 Lux due to building of nitrate1.69 was as high as Feed conversion ratio; FCRshade on the experiment 2.06 experiment, concentration 1.96 greenSurvival house rate in the afternoon was also another factor 100.37±5.6 mg-N/L in control-1 Accumulation of (%) 100 100 100 affecting growth Decrease of Oxidation-Reduction nitrate is generally found in closed aquaculture system in Potential (ORP) from +290that to +110 mVofin Chinese baked clay which water treatment process is nitrification It was found growth cabbage in the thismajor experiment was much slower than bead layer indicated that accumulation of sediment (Nootong et al., 2011; Nootong Powtongsook, 2012) conventional vegetable planting in soil but comparable to aquaponic system by and Graber and Junge in baked beads an increase was55higher (2009) clay which had caused the average 2.07 g of oxygen wet-weightThis andnitrate 18 cmconcentration in length after days than Thisthe wassafety consumption in the layer (Fig.of2).nutrients and improper concentration of 50condition mg-N/L insothe water exchange is probably due to bead the limitation environmental experiment Low light intensity between 980-25,410 Lux due to building shade on the experiment green house in the afternoon was also another factor affecting growth Decrease of Oxidation-Reduction Potential (ORP) from +290 to +110 mV in baked clay bead layer indicated that accumulation of sediment in baked clay beads caused an increase of oxygen consumption in the bead layer (Fig 2) 350 300 ORP (mV) 250 200 150 100 50 0 10 15 20 25 30 35 Time (d) Figure The variation of Oxidation-Reduction Potential in baked clay bead layer of control-2 ( ) and treatment () system 3.2 Water quality According the Fig illustrates inorganic84 nitrogen and phosphate concentrations in control and treatment systems High peak of total ammonia nitrogen (TAN) up to 1.2±0.3 mg-N/L was found in control-1 during the first days, while small TAN peaks at 0.31±0.1 mg-N/L were found in Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 therefore needed (Hart and O,Sullivan,1993).On generally known that plant can take up inorganic the other hand, nitrate in control-2 and treatment nitrogen and phosphorus compounds as nutrients for systems were 47.24±4.1 mg-N/L and 26.66±3.7 growth, however, phosphate concentration in treatment mg-N/L respectively The lower nitrate accumulation tank containing Chinese cabbage was slightly higher in control-2 and treatment systems indicated that than control-2 which had only baked clay beads Hence, baked clay beads played a significant role in nitrate the role of phosphorus uptake by plant in this experiment removal was still unclear and further detailed study is therefore It could be summarized that nitrification was the recommended major process for water quality control in fish tanks It was found that the baked clay beads tank was control-2 which was within not the only safetyremove range inorganic [below 0.5nitrogen mg-N/Lbut(Liao and without bakedand claytreatment beads andsystem plant This nitrification it also retain Mayo, 1972)] During day 3-8, the accumulation of nitrite was found in all tanks after TAN peaks activity occurred with the natural biofloc (suspended suspended solids (Fig 4) Water in the fish tank without disappearance This indicated theculture occurrence of nitrification process (control-1) via ammonia bacteria solids) that accumulated during fish (Nootong beads filtration hadoxidizing the suspended solids The highest peak of nitrite, up to 3.84±0.8 mg-N/L, was also found in control-1, while smaller peaks et al., 2011) In contrast, when baked clay beads tank concentration as high as 352.22±56.01 mg/L below were found in control-2 and treatment systems (0.86±0.1 and 0.38±0.25 mg-N/L, respectively), and was applied to the fish culture system, nitrate was the safety concentration of ,80 mg/L (Timmons et al., these concentrations were under the safety range [below mg-N/L (Hart and O Sullivan, 1993)] The significantly removed by denitrification process in the 2002) throughout the experimental period In general, nitrification was complete within 10 day when the accumulation of nitrate, the end product of anaerobic layer ofoccurred the bakedwithout clay bead tank.accumulation Moreover, suspended higher than 500 mg/L must be avoided nitrification, nitrite At the endsolids of the experiment, concentration of phosphate concentration was also low in control-2 due to it obstruct visibility while it was only 46.11±8.55 nitrate was as high as 100.37±5.6 mg-N/L in control-1 Accumulation of nitrate is generally found in and closed treatment system At the end of thethe experiment, mg/L and 55.00±22.55 mg/L in control-2 andettreatment aquaculture system in which major water treatment process is nitrification (Nootong al., accumulation of phosphate (8.84±0.4 mg-P/L) was nitrate system, respectively.was Hence, baked claythe beads tanks in 2011; Nootong and Powtongsook, 2012) This concentration higher than safety , found in control-1 Lower phosphate concentrations this experiment were successfully maintain suspended concentration of 50 mg-N/L so water exchange is therefore needed (Hart and O Sullivan, 1993) On werethefound control-2 and treatment solidswere in fish tank Other water quality parameters other in hand, nitrate in control-2 andsystems treatmentatsystems 47.24±4.1 mg-N/L and 26.66±3.7 mg-were 5.72±0.1 mg-P/L and 3.38±0.5 mg-P/L, respectively It is within the acceptable range for aquaculture (i.e., N/L respectively The lower nitrate accumulation in control-2 and treatment systems indicated thatpH = 8.23-8.55; alkalinity = 100-163.33 mg CaCO3/L; DO baked clay beads played a significant role in nitrate removal Figure The water quality analysis in fish tanks from control-1 (S), control-2 ( ) and treatment () systems (The horizontal dot lines indicate safety concentration for aquaculture) It could be summarized that nitrification was the major process for water quality control in fish tanks without baked clay beads and plant This nitrification activity occurred with the natural 85 fish culture (Nootong et al., 2011) In contrast, biofloc (suspended solids) that accumulated during when baked clay beads tank was applied to the fish culture system, nitrate was significantly removed It is generally known that plant can take up inorganic nitrogen and phosphorus compounds as nutrients for growth, however, phosphate concentration in treatment tank containing Chinese cabbage was slightly higher than control-2 which had only baked clay beads Hence, the role of phosphorus uptake by plant in this experiment was still unclear and further detailed study is therefore recommended Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 Total suspended solids (mg/L) 450 400 350 300 250 200 150 100 50 0 10 15 20 25 30 35 Time (d) Figure The suspended solids concentration in control-1 (S), control-2 ( ) and treatment () systems It was found that the baked clay beads tank was not only remove inorganic nitrogen but it also retain suspended solids (Fig 4) Water in the fish tank without beads filtration (control-1) had the suspended solids concentration Control-l as high as 352.22±56.01 mg/L below the safety concentration Control-2 Treatment of 80 mg/L (Timmons et al., 2002) throughout the experimental period In general, suspended higher Parameter Nitrogen per tank (g)* Nitrogen per tank (g)* Nitrogen solids per tank (g)* than 500 mg/L must be avoided due to it obstruct visibility while it was only 46.11±8.55 mg/L and In put at final day In put at final day In put at final day 55.00±22.55 mg/L in control-2 and treatment system, respectively Hence, baked clay beads tanks in Feed 7.15 (82.00%) 9.31solids (85.73%) 10.59 (86.45%) this experiment were successfully maintain -suspended in fish tank.- Other water quality parameters Fish 1.53 (17.54%) (17.89%) 1.51 1.59 (14.64%) 1.60 (13.06%) 2.28 (18.61%) were within the acceptable range for1.56 aquaculture (i.e.,(13.90%) pH = 8.23-8.55; alkalinity = 100-163.33 mg Ñ CaCO /L; DO = 7.07-9.07 mg/L; temperature = 26.50-30.07 C) TN in water 0.04 (0.46%) 4.83 (55.39%) 0.04 (0.37%) 2.18 (20.07%) 0.04 (0.33%) 1.21 (9.88%) Table The nitrogen balance in the recirculating aquaculture systems Suspended solid in fish tank - 0.56 (6.42%) - 0.07 (0.65%) - 0.08 (0.65%) Solid retained in baked clay beads tank - - - 1.28 (11.79%) - 1.19 (9.71%) 3.3 Nitrogen and phosphorus mass balance The nitrogen balance- analysis in Table shows -that nitrogen -input in 0.02 all systems Chinese cabbage (0.16%) was 0.16mostly (1.31%) from feed (82-87%) and fish (13-18%) while at the end of experiments; nitrogen in fish was between Baked clay beads 15-19% These results were comparable to the report of Avnimelech and Rityo (2003), which Unaccountedexplained that the input nitrogen (20.30%) was accumulate 5.74 7.33 (59.84%) and1.77 phosphorus in(52.85%) fish 22% and -16% respectively Moreover, in many research reports notified the proportion of ammonia nitrogen in RAS that 39.29% Total 8.72 (100%) 8.72 (100%) 10.86 (100%) 10.86 (100%) 12.25 (100%) 12.25 (100%) was from feed, 26-28% was from fish excretion while the final portion of 24% was accumulated in * CHNS-O Analyzer, CE Instruments Flash EA 1112 Series, Thermo Quest, Italy sludge suspended solids (Lin and Nash, 1996; Funge-Smith and Briggs, 1998).Accumulation of nitrogen in the water, up to 56%, was found in control-1 while water in the tank with baked clay beads had substantial lower nitrogen concentration The most important part was unaccounted nitrogen as = 7.07-9.07 temperature = 26.50-30.07ºC) was This accumulated in sludge highmg/L; as 53% in control-2 and 60% in treatment24% system was assumed as the suspended nitrogen gassolids loss 3.3 Nitrogen anddenitrification phosphorus mass balance (Lin2005; and Nash, 1996; Funge-Smith Briggs, 1998) through process (Rafiee and Saad, Funge-Smith and Briggs,and 1998) Nitrogen Accumulation nitrogen in theresults water,from up tothis 56%, was incorporated in Chinese cabbage (treatment system) was only of 1.31% Hence, study that nitrogen removal in 3our RAS was mainly by nitrification-denitrification processes Theillustrated nitrogen balance analysis in Table shows found in control-1 while water in the tank with baked while nitrogen by plant the minor role.substantial lower nitrogen concentration that nitrogen input in uptake all systems wasincorporated mostly fromwithclay beads had feed (82-87%) and fish (13-18%) while at the end of The most important part was unaccounted nitrogen as experiments; nitrogen in fish was between 15-19% high as 53% in control-2 and 60% in treatment system These results were comparable to the report of This was assumed as the nitrogen gas loss through Avnimelech and Rityo (2003), which explained that denitrification process (Rafiee and Saad, 2005; Fungethe input nitrogen and phosphorus was accumulate in Smith and Briggs, 1998) Nitrogen incorporated in fish 22% and 16% respectively Moreover, in many Chinese cabbage (treatment system) was only 1.31% research reports notified the proportion of ammonia Hence, results from this study illustrated that nitrogen nitrogen in RAS that 39.29% was from feed, 26-28% removal in our RAS was mainly by nitrificationwas from fish excretion while the final portion of denitrification processes while nitrogen uptake by plant 86 Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 Table The phosphorus balance in recirculating aquaculture systems Control-l Parameter Phosphorus per tank (g)* Nitrogen per tank (g)* Control-2 Phosphorus tank Nitrogen perper tank (g)*(g)* Treatment Phosphorus tank (g)* Nitrogen perper tank (g)* In put at final day In put at final day In put at final day Feed 0.55 (87.02%) - 0.50 (21.35%) - 0.56 (20.47%) - Fish 0.08 (12.66%) 0.08 (12.66%) 0.08 (3.42%) 0.10 (4.27%) 0.09 (3.29%) 0.10 (3.65%) TP in water TN 0.002 (0.32%) 0.439 (69.46%) 0.002 (0.08%) 0.298 (12.72%) 0.004 0.04 (0.15%) 0.186 (6.80%) Suspended solid in fish tank - 0.11 (17.41%) - 0.02 (0.85%) - 0.03 (1.10%) Solid retained in baked clay beads tank - - - 0.28 (11.96%) - 0.21 (7.68%) Chinese cabbage - - - - 0.001 (0.04%) 0.003 (0.11%) Baked clay beads - - 1.76 (75.15%) 1.41 (60.21%) 2.08 (76.05%) 1.76 (64.35%) Unaccounted - 0.003 (0.47%) - 0.234 (9.99%) - 0.446 (16.31%) 0.632 (100%) 0.632 (100%) 2.342 (100%) 2.342 (100%) 2.735 (100%) 2.735 (100%) Total * Optical Emission Spectrometer, Optima 4300 DV, Perkin Elmer Instruments, USA incorporated with the minor role The phosphorus balance in all systems is show in Table It was found that phosphorus input was mostly from feed (21-87%) and fish (3-13%) At the end of experiments, phosphorus in fish was between 4-13% which was slightly lower than previous report (15.98%) by Rafiee and Saad (2005), phosphorus accumulation in the water was up to 70% in control-1, while tanks with baked clay beads had substantial lower phosphorus concentration Unaccounted phosphorus was as high as 17% in treatment, but control-1 and control-2 system were lower at 1.0% and 10%, respectively Phosphorus in suspended solids ranged between 1-18% while phosphorus incorporated in plant (treatment) was only 0.11% Moreover, it was assumed that most of the nutrients were accumulated in suspended solids and solid deposited in baked clay beads tank Promotion and National Research University Project of Thailand, the Office of the Higher Education Commission (FW1017A) Additional support was also obtained from the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530068) Moreover, this research also received an extra funding from a master thesis grant of the National Research Council of Thailand for the year 2011 and received a partially financial support by the graduate thesis grant, Chulalongkorn University Equipments and facilities in this research were provided by the Center of Excellence for Marine Biotechnology, Department of Marine Science, Faculty of Science and Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn University, Thailand Some equipment (microplate spectrophotometer) is provided for by the Thai Government Stimulus Package2 (TKK2555) under the Project for Establishment of Comprehensive Center for Innovative Food, Health Products and Agriculture, Chulalongkorn University Conclusion References With the proposed RAS, toxic nitrogenous compounds such as ammonia and nitrite were maintained within the safety level for fish Significant amount of nitrogen compounds were removed mostly by degradation especially nitrification and denitrification processes while nutrients (nitrogen and phosphorus) assimilation in plant was the minor process This RAS concept has high potential for further development APHA Standard Methods for the examination of water and wastewater 21st ed APHA, AWWA, WPCF, Washington DC, USA 2005 Avnimelech Y, Rityo G Shrimp and fish pond soils processes and management Aquaculture 2003; 264: 140- 47 Azim ME, Little DC The biofloc technology (BFT) in indoor tanks: Water quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus) Aquaculture 2008; 283: 29-35 Beven S The Aquaponics Guidebook 3rd ed Aquaponic Europe, London, UK 2010 Burut-Archanai S, Eaton-Rye JJ, Incharoensakdi A, Powtongsook S Phosphorus removal in a closed recirculating aquaculture system using the cyanobacterium Synechocystis sp PCC 6803 strain lacking the SphU regulator of the Pho regulon Biochemical Engineering Journal 2013; 74: 69-75 Acknowledgements This research was financially supported by the Integrated Innovation Academic Center Chulalongkorn University Centenary Academic Development Project (CU56-FW14), with the partial supports from the Higher Education Research 87 Aeknarin Thanakitpairin et al / EnvironmentAsia 7(1) (2014) 81-88 Crab R, Avnimelech Y, Defoirdt T, Bossier P, Verstraete W Nitrogen removal techniques in aquaculture for a sustainable production Aquaculture 2007; 270: 1-14 Funge-Smith SJ, Briggs MRP Nutrient budgets in intensive shrimp ponds: implications for sustainability Aquaculture 1998; 164: 117-33 Graber A, Junge R Aquaponic systems: Nutrient recycling from fish wastewater by vegetable production Desalination 2009; 246: 147-56 Hart P, O’sullivan D Recirculating system: Design, construction and management Turtle Press Pty, Australia 1993 Liao PB, Mayo RD Salmonid hatchery water reuse system Aquaculture 1972; 1: 317-35 Lin CK, Nash GL Asian Shrimp News, Collected Columns Asian Shrimp Culture Council (1989-1995) 1996; 125 36 Nootong K, Pavasant P, Powtongsook S Effects of organic carbon addition in controlling inorganic nitrogen concentrations in a biofloc system Journal of the World Aquaculture Society 2011; 42(3): 339-46 Nootong K, Powtongsook S Performance evaluation of the compact aquaculture system integrating submerged fibrous nitrifying biofilters Songklanakarin Journal of Science & Technology 2012; 34(1): 53-59 Rakocy J, Hargreaves J Integration of vegetable Hydroponics with fish culture: A review In: Techniques for modern aquaculture, proceedings of aquaculture engineering conference, Washington on June 21-23, 1993 Rafiee GR, Saad CR Nutrient cycle and sludge production during different stages of red tilapia (Oreochromis sp.) growth in a recirculating aquaculture system Aquaculture 2005; 244: 109-18 Timmons MB, Ebeling JM, Wheaton FW, Summerfelt ST, Vinci BJ Recirculating aquaculture systems 2nd ed Cayuga Aqua Ventures, Ithaca, New York, USA 2002 Wilson G Australian barramundi farm goes aquaponic Aquaponics Journal 2005; 37: 12-16 Received 16 November 2013 Accepted December 2013 Correspondence to Mr Aeknarin Thanakitpairin Department of Environmental Sciences, Faculty of Science and Technology, Rambhai Barni Rajabhat University, Chantaburi, Thailand Tel: +668 4347 3739 E-mail: thanakitpairin_a@hotmail.com 88 ... Tilapia andand plant packed with and placed in thebeads greenhouse following method for tank water +and wastewater baked clay and Chinese cabbage The fish tank withoutstandard plant tank and fish baked. .. of the treatment recirculating aquaculture system consisted of fish tank increased 0.15±0.02 g tobaked 1.00±0.38 g and increased and overlayfrom treatment tank with clay beads andlength Chinese. .. wet-weight in control-1, control-2 and treatment systems in baked clay beads tank, Chinese cabbage, and baked 3.1 Growth of fish and Chinese cabbage increased from 16.31, 15.18 and 11.31 g to 29.43, clay