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Chlorine demand and bacteria of low salinity shrimp pond sediment treated with different chlorine doses
Aquacultural Engineering 25 (2001) 165– 174 www.elsevier.nl/locate/aqua-online
Chlorine demand and bacteria of low salinity shrimp pond sediment treated with different chlorine doses Husnah, Chang-Kwei Lin * Agriculture and Aquatic Systems and Engineering Program, School of En6ironment, Resources De6elopment, Asian Institute of Technology, PO Box 4, Klong Luang, Pathumthani 12120, Thailand Received 16 March 2001; accepted 13 June 2001
Abstract An experiment was conducted to determine the chlorine demand and the efficacy of three chlorine doses to disinfect indigenous bacteria in the sediment of a shrimp (Penaeus monodon Fabricius) pond. Sediment cores were collected by inserting 48 PVC tubes 20 cm into the pond and these tubes were planted vertically into four circular concrete tanks containing clay soil. Sediment in those tubes were treated with active chlorine in overlying water at 300, 1200 and 2400 mg l − 1 as treatments. The residual chlorine concentration in each tube was determined at 0, 6, 24, 48, and 144 h and the chlorine demand of the sediment was calculated by the difference between initial dose and total residual chlorine. Chlorine efficacy to bacteria resided in the sediment was determined at a depth of 0 – 0.3, 0.9– 1.2 and 1.9– 2.1 cm in each core. Organic carbon, pH, and total Kjedhal nitrogen (TKN) in sediment were analyzed at the initial and end of experiment. Chlorine demand of sediment was 0.48 kg m − 2. Both organic carbon and TKN contributed to the loss of chlorine in the sediment. Chlorine at a dose of 300 mg l − 1 did not completely inactivate bacteria despite its free and residual chlorine in overlying water at the end of experiment still remained at 20 mg l − 1. Chlorine at a dose of 1200 and 2400 mg l − 1 inactivated 100% bacteria within 2 days of contact time. With high chlorine dose, chlorination was effective to inactivate bacteria only to a depth of 2.1 cm. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chlorine demand; Indigenous bacteria; Organic carbon; Nitrogenous compound; Shrimp pond
* Corresponding author. Tel.: + 66-2-5245458; fax: +66-2-5246200. E-mail address: [email protected] (H.C.-K. Lin). 0144-8609/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 4 - 8 6 0 9 ( 0 1 ) 0 0 0 8 0 - 2
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1. Introduction Chlorine is one of chemicals widely used to inactivate or to terminate pathogenic organisms and virus vectors causing white-spot and yellow-head diseases commonly occurred in cultured shrimp (Boyd, 1996; Cai, 1994; Hedge et al., 1996; Kongkeo, 1995). When chlorine dissolves in pond water, it forms free residual chlorine. Part of this free chlorine which reacts with organic and oxidizable substances is referred as chlorine demand, and the residual parts oxidize and damage nucleic acid and/or protein of microorganisms and cause lethal damage (Acher et al., 1997; Chanratchakool, 1995). High concentration of organic matter and suspended solids often occurred in water and sediment of shrimp pond (Dierberg and Kiattisimkul, 1996; Hopkin et al., 1993), causing high chlorine demand and reducing efficacy of chlorine. As a result, more chlorine is required to disinfect target organisms. It has been shown that the chlorine dose required to inactivate bacteria in water increased 100 times in the presence of 50 mg l − 1 organic matter and neutral pH condition (Harakeh, 1986). The study on the effect of chlorine on bacterial abundance in the sediment of catfish ponds in Alabama revealed that the significant chlorine concentration to partially inactivate bacteria was 600 and 1200 mg l − 1 (Potts, 1998). For shrimp culture in Thailand, farmers apply chlorine at a dose of 300 kg ha − 1 or 30 mg l − 1 of active chlorine during pond preparation (Lin and Nash, 1996; Kongkeo, 1995) and 0.1 mg l − 1 per week to control plankton and other organisms during the culture period (Boyd and Massaut, 1999). The present study aims to determine chlorine demand of pond sediment, and effective chlorine dose required to disinfect those organisms, particularly pathogenic microorganisms and their carriers.
2. Materials and methods
2.1. Sediment collection Sediment used for this study was collected from a 200-m2 pond with low salinity (3– 5 ppt) for rearing black tiger shrimp (Penaeus monodon Fabricius) at the Asian Institute of Technology in Thailand. Forty eight, PVC tubes of 4.4-cm diameter and 30-cm long were inserted 20 cm into the pond sediment. These soil filled tubes were transferred and inserted vertically into clayey soil in four circular concrete tanks with 12 tubes in each tank. The upper 10 cm of each tube connected with 1.5-l plastic jar was filled with distilled water. All tubes were aerated to maintain the dissolved oxygen above 5 mg l − 1. The tubes containing pond sediments in each concrete tank were divided into four sets with one set as a control and each of other sets treated with one chlorine concentration at 300, 1200 and 2400 mg l − 1, respectively. The chlorine solution of different concentrations was prepared by diluting 100 g l − 1 chlorine stock solution. Stock solution was made by dissolving calcium hypochlorite or High Test
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Hypochlorite (HTH) in distilled water and standardized with sodium thiosulphate (ASTM, 1992). The experiment was carried out indoors with air temperature at 27–28 °C under dark condition for 6 days (144 h). In addition to the 48 tubes mentioned earlier, other four PVC tubes with the size of 10-cm diameter and 40-cm long each were used to take soil samples near the sampling points of those 48 PVC tubes. Soil in each of these four tubes was separated into three layers at 0– 2.5, 2.5– 5.0 and 5.0–7.5 cm and dried in an oven at 60 °C for 1 day. Those soil samples were analyzed for soil texture, pH, and organic carbon (OC) using hydrometer, electroprobe, Walkley– Black methods, respectively (Boyd, 1995). Total Kjedhal nitrogen (TKN) was analyzed with semimicro Kjedahl method (Dewis and Freitas, 1970). At the end of 6-day chlorine treatment, triplicate soil samples were taken from three layers at 0– 0.3, 0.9– 1.2 and 1.8– 2.1 cm for analyses of above soil parameters.
2.2. Chlorine demand Concentrations of free and total residual chlorine (FRC and TRC) in overlying water in each sediment tube were analyzed at 0, 6, 24, 48 and 144 h after chlorine application. At each time interval, triplicate water samples were taken from four sets of tubes in one concrete tank by siphoning 100-ml water into 120-ml plastic bottles. Free and total residual chlorine were analyzed by DPD titration method (APHA, 1998). Chlorine demand of the soil was calculated as follows: Chlorine demand (kg Cl2 m − 2) =[{(Cl2 dose − TRC) ×10 − 6 kg}/{104/Tubes area in m2}]
(1)
2.3. Total bacteria count Total bacteria count in each soil sample was made in duplicates taken from tubes of replication 1 and 2 from tanks 1, 2, 3 and 4 at 0, 6, 24 and 48 h intervals, respectively. At day 6 (144 h), soil samples in the remaining replicate of each treatment in tank 1 and 2 were taken from three layers by inserting sterilized 1.0-ml micro syringes vertically into the soil column at depth of 0–0.3, 0.9–1.2 and 1.8–2.1 cm, respectively. Approximate 0.5-g soil sample was taken from each syringe and kept in sterilized buffer solution containing phosphate and magnesium chloride (MgCl2). The solution was shaken continuously on a mechanical shaker for 20 min prior to inoculation on a petri dish containing Bacto plate count agar media. The inoculated bacteria were incubated at temperature 28–30 °C and total bacteria count was made at 15 and 24-h intervals following Plate Count method with droplet modification (APHA, 1998). Results were expressed as colony forming unit per gram of soil (CFU per g soil). Results on the chlorine demand were analyzed by one-way analysis of variance (ANOVA) while organic carbon, pH, TKN and total bacteria count data obtained at different time intervals were analyzed by two-way ANOVA. These statistical analyses were run using a statistical software (Statistica version 5.0).
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3. Results
3.1. Chlorine demand of pond sediments Soil sediment used in this experiment was categorized as clayey soil (Boyd, 1995). The pH, organic carbon, and TKN concentration among three soil layers before chlorine treatment (Table 1) was not significantly different (P\ 0.05). Free residual chlorine (except before 24 h exposure) and total residual chlorine concentrations of the overlying water decreased over time in all chlorine treatments, while the soil chlorine demands increased (Fig. 1). Concentration of free residual chlorine at the end of experiment was 21, 242 and 367 mg Cl2 l − 1 and total residual chlorine was 58, 466 and 850 mg Cl2 l − 1 for initial chlorine dose of 300, 1200 and 2400 mg Cl2 l − 1, respectively. A whitish color was found covering the surface layer of soil sediment treated with high chlorine dose at 1200 and 2400 mg Cl2 l − 1. Fig. 1 shows that 50% of chlorine applied was consumed in the first 2 days in all chlorine doses, and at the end of the experiment the chlorine consumed was 80, 61 and 65% of the initial concentration of 300, 1200 and 2400 mg Cl2 l − 1 or 0.48, 1.45 and 3.06 kg of active chlorine m − 2 sediment, respectively. Throughout the experimental periods, chlorine demand of the sediment was significantly higher (PB 0.01) at chlorine doses of 1200 and 2400 mg Cl2 l − 1 than that in control and low chlorine dose (300 mg Cl2 l − 1). A clear pattern of chlorine demand in sediment was found on some chemical parameters of soil at layer 0– 0.3 cm at the end of experiment (Fig. 2). Increasing in chlorine dose resulted in increasing soil pH and decreasing concentration of organic carbon and TKN. Soil pH in high chlorine dose (2400 mg Cl2 l − 1) was 0.3 U higher than control while its organic carbon and TKN were 0.6 and 0.13% less than the control. Two-way ANOVA for these parameters among different chlorine doses and sediment layers show that chlorine concentration at 2400 mg Cl2 l − 1 significantly reduced content of organic carbon (PB 0.01) and TKN (PB 0.05) than those in the control. Table 1 Soil texture, organic carbon, pH and total Kjedhal nitrogen (TKN) of shrimp pond sediments before treated with different chlorine doses Soil parameters
Soil texture Clay (%) Silt (%) Sand (%) Organic carbon (%)a Soil pH (CaCl2 5:1)a TKN (%)a a
Mean and S.E.
Soil layers (cm) 0–2.5
2.5–5.0
5.0–7.5
75.83 14.28 9.88 4.059 0.04 7.069 0.06 0.1590.01
86.22 8.18 5.59 4.07 90.10 6.93 90.14 0.14 9 0.03
86.11 10.21 3.68 4.08 90.16 6.86 9 0.22 0.15 90.04
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Fig. 1. Concentrations of free and total residual chlorine (FRC and TRC) in water and chlorine consumption in sediment treated with different chlorine doses measured at 0 (black column), 6 (grey column), 24 (blank column), 48 (dotted column) and 144 h (striped column). Bars indicated standard error (S.E.).
Correlation analysis on chlorine demand with these three soil parameters at 0–0.3 cm layer at the end of experiment revealed that positive correlation with soil pH (r = 71), and negative correlation with organic carbon (r= 0.83) and TKN (r=0.83). For other soil layers, a weak correlation was found between chlorine demand and these three soil parameters.
3.2. Total bacteria count Inactivation of bacteria in pond sediment was influenced by the chlorine doses and exposure time (Fig. 3). Low chlorine dose (300 mg Cl2 l − 1) inactivated only
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part of bacteria population at the first hour of exposure, thereafter bacteria started to grow again. At the end of experiment, the bacterial count ranging from 105 to 3 × 105 CFU g − 1 wet soil was not significantly (P\ 0.05) different between control and treatment. However, in a treatment with chlorine dose at 1200 and 2400 mg Cl2 l − 1 the bacteria were inactivated immediately after exposure until the end of experiment. The chlorine dose at 1200 mg Cl2 l − 1 completely inactivated bacteria in 2 days for soil layer at 0– 0.3 cm and 6 days for soil layer at 0.9–1.2 cm. With chlorine dose at 2400 mg Cl2 l − 1, complete inactivation occurred in 6 h for soil layer 0–0.3 cm and 2 days for soil layer 0.9–1.2 cm. Bacterial numbers in these high chlorine doses were significantly smaller (PB0.01) than in control and in treatment of 300 mg Cl2 l − 1. At the end of experiment, no bacteria were found in
Fig. 2. The pH, organic carbon and total Kjedhal nitrogen (TKN) concentration in soil at layer of 0 – 0.3 cm (black column), 0.9 –1.2 cm (grey column) and 1.8 – 2.1 cm (blank column) of shrimp pond treated with different chlorine doses. Bars indicated S.E.
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Fig. 3. Total bacteria count in soil layer of 0 –0.3 cm (black column), 0.9 – 1.2 cm (grey column) and 1.8 – 2.1 cm (blank column) treated with different chlorine doses during the experiment period. Bars indicated S.E.
soil layer 0– 0.3 and 0.9– 1.2 cm of high chlorine doses, but in layer 1.8–2.1 cm bacteria count was 2.8× 104 CFU g − 1 wet soil and 1.4×104 CFU g − 1 wet soil for chlorine dose at 1200 and 2400 mg Cl2 l − 1, respectively. The results clearly show that the total bacteria count increased in deeper soil layer even at high chlorine doses at 1200 and 2400 mg Cl2 l − 1 from 24 to 144 h after chlorine exposure. Two-way ANOVA detected a significantly lesser number of bacteria in soil layer 0– 0.3 cm than that in soil layer 0.9–1.2 and 1.8–2.1 cm (PB 0.01). Total bacteria in three soil layers correlated negatively with their chlorine demand, with correlation coefficient of 0.77, 0.62 and 0.52 for soil layer 0–0.3, 0.9 –1.2 and 1.8– 2.1 cm, respectively.
4. Discussion The high percentage of chlorine consumption in pond water occurred during the first 2 days of chlorine exposure in both treatments of low and high chlorine doses. This could be largely related to the process of chlorine degradation, which rate depends on the type of compounds present in the pond water and sediment. As
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chlorine react first with reactive organic compounds (more oxidizable matters) and then with less reactive organic compounds or inorganic decomposition of chlorine (Wong and Davidson, 1977). The presence of greater concentration of organic and nitrogenous compounds in the top soil layer of 0– 0.3 cm consumed more chlorine than in layers of 0.9– 1.2 and 1.8–2.1 cm. Negative correlation (r= 0.83) was found between the chlorine demand with concentration of organic carbon and TKN in soil layers 0– 0.3 cm in all chlorine treatments at the end of the experiment support this explanation. Second, it could be related to the initial concentration of chlorine applied. In treatment with higher chlorine concentration applied, the more chlorine demand was resulted which was partly because of the loss of the undissolved chlorine through precipitating and adsorbing to the surface of sediment. Acher et al. (1997) stated that the efficiency of chlorine application was governed by the chlorine dose and the contact time. These explanations are also the reason for the presence of free and total residual chlorine in low chlorine dose at the end of the experiment. There should be no free and total residual chlorine in chlorine dose at 300 mg Cl2 l − 1 since soil sediment could consumed more than 1500 mg Cl2 l − 1. Results show that lower chlorine dose did not completely inactivate bacteria in the sediment even the free and total residual chlorine were in the range that could kill other aquatic organisms (Boyd, 1996). The presence of high proportion of deposited particles commonly found in bottom of shrimp ponds may harbor bacteria from chlorine effect. A shorter time needed to inactivate bacteria in layer 0–0.3 cm than in deeper layers of high chlorine doses is related to their initial free and total residual chlorine concentrations. Concentration of free and total residual chlorine in surface layer was higher than in deeper layers since chlorine diffusion to deeper layers takes longer time. This also explains that a shorter time was needed for bacteria inactivation in higher chlorine dose of 2400 Cl2 l − 1 than in 1200 mg Cl2 l − 1. Total bacteria number in treatments with chlorine dose of 300 and 1200 mg Cl2 l − 1 ranged from 0 to 3×105 CFU g − 1 of wet soil. The number was smaller than total bacteria number in catfish pond soil tested (106 CFU g − 1) with same chlorine dose (Potts, 1998). In Potts experiment, chlorine dose of 1200 mg Cl2 l − 1 did not completely inactivate bacteria. The different results were probably due to difference in sediment depth and chlorine demand. In Potts experiment, bacteria sample was taken from 5-cm deep sediment while in this experiment the sediment depth used was 2.5-cm where bacteria were completely inactivated at high chlorine dose. In practice, shrimp farmers in Thailand commonly apply a chlorine dose of 30 mg Cl2 l − 1 which may achieve the purpose of disinfecting organisms in pond water, but its certainly inadequate to terminate micro-organisms in the pond bottom. Although the free and total residual chlorine remaining in the water was still sufficiently high to kill aquatic organisms, it was ineffective to treat bottom dwellers. At chlorine dose of 2400 mg Cl2 l − 1, 100% bacteria within 2.1-cm sediment depth was inactivated in 2-day exposure. With this dose the amount of chlorine needed to treat the sediment was 2.11 kg Cl2 m − 2 with free and total residual chlorine in the overlying water at 850 and 1300 mg Cl2 l − 1, respectively. If this dose was applied to the shrimp pond with the assumption of little chlorine loss
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from exposure to sun light, it would require an estimated amount of 2 ton ha − 1 of active chlorine or 3 ton ha − 1 of HTH to treat shrimp ponds effectively. This level of chlorine treatment is neither economically nor environmentally practical. An alternative method should be investigated and found for effective disinfection of shrimp pond from pathogenic microorganisms harbored in pond bottom. We recommend a practical procedure for chlorine application in shrimp ponds. The first step is to apply high chlorine dose to pond sediment with a shallow overlying water of 2– 5-cm depth for 2 days to inactivate the bacteria in the sediment; the second step is to raise the water to a depth for shrimp culture (usually 1.0–1.5 m depth) to dilute the remaining residual chlorine to a level that can inactivate bacteria in the overlying water column. With this procedure, the estimated amount of chlorine needed to disinfected sediment and water for 1 ha of shrimp pond with 2–cm overlying water is 480 kg ha − 1 active chlorine or 740 kg ha − 1 HTH. Acknowledgements The study was supported by a doctoral scholarship (IBRD Loan No. 3886-IND) from Agricultural Research and Management Project Phase II, Agricultural ministry of Indonesia. Authors would like to thank Weena Koeypudsa and Paria Rakhmati, for assistance with this study. References Acher, A., Fischer, E., Turnheim, R., Manor, Y., 1997. Ecologically friendly wastewater disinfection techniques. Water Res. 31 (6), 1398 –1404. American Public Health Association, (Alpha), 1998. Standard methods for the examination of water and waste water, (20th edition) Eaton, A.D., Clesceri, L.S., Greenberg, A.E., Washington, DC. American Society for Testing and Materials (ASTM), 1992. Standard Practice for estimation of chlorine requirement or demand of water, or both. In: Fazio, P.C., Fisher, D., Gutman, E.L., Hsia, C.T., Kaufman, S.L., Kramer, J., Lane, M. (Eds.), Annual Books of ASTM Standards: Water and Environmental Technology, Vol 11.01, American Society for Testing and Materials, Philadelphia. pp. D1291 –89. Boyd, C.E., 1995. Bottom Soils, Sediment, and Pond Aquaculture. Chapman and Hall, New York. Boyd, C.E., 1996. Chlorination and water quality in aquaculture ponds. World Aquacult. 27, 41 – 45. Boyd, C.E., Massaut, L., 1999. Risks associated with the use of chemicals in pond aquaculture. Aquacult. Eng. 20, 113 –132. Cai, W., 1994. Fish disease. In: Li, S., Mathias, J. (Eds.), Freshwater Fish Culture in China: Principles and Practice. Elsevier, Netherland, pp. 387 – 424. Chanratchakool, P., 1995. White patch disease of black tiger shrimp (Penaeus monodon). AAHRI Newsletter 4 (1), 3. Dewis, J., Freitas, F., 1970. Physical and chemical methods of soil and water analysis. Soil Bulletin no. 10. FAO. Rome. 275 pp. Dierberg, F.E., Kiattisimkul, W., 1996. Issue, impacts, and implications of shrimp aquaculture in Thailand. Environ. Man. 20 (5), 649 –666. Harakeh, M.S., 1986. Factors influencing chlorine disinfection of wastewater effluent contaminated by rotaviruses, enteroviruses and bacteriophages. In: Jolley, R.L., Bull, R.J., Davis, W.P., Katz, S., Robert, M.H. Jr, Jacobs, V.A. (Eds.), Water Chlorination: Chemistry, Environmental Impact and Health Effects, vol. 4. Lewis Publisher Inc, USA, pp. 681 – 690.
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Hedge, A., Anthony, J., Rao, S., 1996. Inactivation of viruses in aquaculture systems. Infofish Int. 6, 40 – 43. Hopkin, J.S., Hamilton, R.D. II, Sandifer, P.A., Browdy, C.L., Stokes, A.D., 1993. Effect of water exchange rate on production, water quality, effluent characteristics and nitrogen budgets of intensive shrimp ponds. J. World Aquacult. Soc. 24 (3), 304 – 320. Kongkeo, H., 1995. How Thailand made it to the top. Infofish Int. 1, 25 – 31. Lin, C.K., Nash, G., 1996. Asian Shrimp News collected volume, 1989 – 1995. Asian Shrimp Culture Council, Bangkok. Potts, A.C., 1998. Effects of chlorination on water quality, pond bottom soils and fish production in aquaculture ponds. Fisheries and Allied Aquacultures, Auburn University, Alabama. 93 pp (Ph.D. Dissertation). Wong, G.T.F., Davidson, J.A., 1977. The fate of chlorine in sea-water. Water Res. 11, 971 – 978.
Chlorine demand and bacteria of low salinity shrimp pond sediment treated with different chlorine doses Husnah, Chang-Kwei Lin * Agriculture and Aquatic Systems and Engineering Program, School of En6ironment, Resources De6elopment, Asian Institute of Technology, PO Box 4, Klong Luang, Pathumthani 12120, Thailand Received 16 March 2001; accepted 13 June 2001
Abstract An experiment was conducted to determine the chlorine demand and the efficacy of three chlorine doses to disinfect indigenous bacteria in the sediment of a shrimp (Penaeus monodon Fabricius) pond. Sediment cores were collected by inserting 48 PVC tubes 20 cm into the pond and these tubes were planted vertically into four circular concrete tanks containing clay soil. Sediment in those tubes were treated with active chlorine in overlying water at 300, 1200 and 2400 mg l − 1 as treatments. The residual chlorine concentration in each tube was determined at 0, 6, 24, 48, and 144 h and the chlorine demand of the sediment was calculated by the difference between initial dose and total residual chlorine. Chlorine efficacy to bacteria resided in the sediment was determined at a depth of 0 – 0.3, 0.9– 1.2 and 1.9– 2.1 cm in each core. Organic carbon, pH, and total Kjedhal nitrogen (TKN) in sediment were analyzed at the initial and end of experiment. Chlorine demand of sediment was 0.48 kg m − 2. Both organic carbon and TKN contributed to the loss of chlorine in the sediment. Chlorine at a dose of 300 mg l − 1 did not completely inactivate bacteria despite its free and residual chlorine in overlying water at the end of experiment still remained at 20 mg l − 1. Chlorine at a dose of 1200 and 2400 mg l − 1 inactivated 100% bacteria within 2 days of contact time. With high chlorine dose, chlorination was effective to inactivate bacteria only to a depth of 2.1 cm. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Chlorine demand; Indigenous bacteria; Organic carbon; Nitrogenous compound; Shrimp pond
* Corresponding author. Tel.: + 66-2-5245458; fax: +66-2-5246200. E-mail address: [email protected] (H.C.-K. Lin). 0144-8609/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 4 - 8 6 0 9 ( 0 1 ) 0 0 0 8 0 - 2
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1. Introduction Chlorine is one of chemicals widely used to inactivate or to terminate pathogenic organisms and virus vectors causing white-spot and yellow-head diseases commonly occurred in cultured shrimp (Boyd, 1996; Cai, 1994; Hedge et al., 1996; Kongkeo, 1995). When chlorine dissolves in pond water, it forms free residual chlorine. Part of this free chlorine which reacts with organic and oxidizable substances is referred as chlorine demand, and the residual parts oxidize and damage nucleic acid and/or protein of microorganisms and cause lethal damage (Acher et al., 1997; Chanratchakool, 1995). High concentration of organic matter and suspended solids often occurred in water and sediment of shrimp pond (Dierberg and Kiattisimkul, 1996; Hopkin et al., 1993), causing high chlorine demand and reducing efficacy of chlorine. As a result, more chlorine is required to disinfect target organisms. It has been shown that the chlorine dose required to inactivate bacteria in water increased 100 times in the presence of 50 mg l − 1 organic matter and neutral pH condition (Harakeh, 1986). The study on the effect of chlorine on bacterial abundance in the sediment of catfish ponds in Alabama revealed that the significant chlorine concentration to partially inactivate bacteria was 600 and 1200 mg l − 1 (Potts, 1998). For shrimp culture in Thailand, farmers apply chlorine at a dose of 300 kg ha − 1 or 30 mg l − 1 of active chlorine during pond preparation (Lin and Nash, 1996; Kongkeo, 1995) and 0.1 mg l − 1 per week to control plankton and other organisms during the culture period (Boyd and Massaut, 1999). The present study aims to determine chlorine demand of pond sediment, and effective chlorine dose required to disinfect those organisms, particularly pathogenic microorganisms and their carriers.
2. Materials and methods
2.1. Sediment collection Sediment used for this study was collected from a 200-m2 pond with low salinity (3– 5 ppt) for rearing black tiger shrimp (Penaeus monodon Fabricius) at the Asian Institute of Technology in Thailand. Forty eight, PVC tubes of 4.4-cm diameter and 30-cm long were inserted 20 cm into the pond sediment. These soil filled tubes were transferred and inserted vertically into clayey soil in four circular concrete tanks with 12 tubes in each tank. The upper 10 cm of each tube connected with 1.5-l plastic jar was filled with distilled water. All tubes were aerated to maintain the dissolved oxygen above 5 mg l − 1. The tubes containing pond sediments in each concrete tank were divided into four sets with one set as a control and each of other sets treated with one chlorine concentration at 300, 1200 and 2400 mg l − 1, respectively. The chlorine solution of different concentrations was prepared by diluting 100 g l − 1 chlorine stock solution. Stock solution was made by dissolving calcium hypochlorite or High Test
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Hypochlorite (HTH) in distilled water and standardized with sodium thiosulphate (ASTM, 1992). The experiment was carried out indoors with air temperature at 27–28 °C under dark condition for 6 days (144 h). In addition to the 48 tubes mentioned earlier, other four PVC tubes with the size of 10-cm diameter and 40-cm long each were used to take soil samples near the sampling points of those 48 PVC tubes. Soil in each of these four tubes was separated into three layers at 0– 2.5, 2.5– 5.0 and 5.0–7.5 cm and dried in an oven at 60 °C for 1 day. Those soil samples were analyzed for soil texture, pH, and organic carbon (OC) using hydrometer, electroprobe, Walkley– Black methods, respectively (Boyd, 1995). Total Kjedhal nitrogen (TKN) was analyzed with semimicro Kjedahl method (Dewis and Freitas, 1970). At the end of 6-day chlorine treatment, triplicate soil samples were taken from three layers at 0– 0.3, 0.9– 1.2 and 1.8– 2.1 cm for analyses of above soil parameters.
2.2. Chlorine demand Concentrations of free and total residual chlorine (FRC and TRC) in overlying water in each sediment tube were analyzed at 0, 6, 24, 48 and 144 h after chlorine application. At each time interval, triplicate water samples were taken from four sets of tubes in one concrete tank by siphoning 100-ml water into 120-ml plastic bottles. Free and total residual chlorine were analyzed by DPD titration method (APHA, 1998). Chlorine demand of the soil was calculated as follows: Chlorine demand (kg Cl2 m − 2) =[{(Cl2 dose − TRC) ×10 − 6 kg}/{104/Tubes area in m2}]
(1)
2.3. Total bacteria count Total bacteria count in each soil sample was made in duplicates taken from tubes of replication 1 and 2 from tanks 1, 2, 3 and 4 at 0, 6, 24 and 48 h intervals, respectively. At day 6 (144 h), soil samples in the remaining replicate of each treatment in tank 1 and 2 were taken from three layers by inserting sterilized 1.0-ml micro syringes vertically into the soil column at depth of 0–0.3, 0.9–1.2 and 1.8–2.1 cm, respectively. Approximate 0.5-g soil sample was taken from each syringe and kept in sterilized buffer solution containing phosphate and magnesium chloride (MgCl2). The solution was shaken continuously on a mechanical shaker for 20 min prior to inoculation on a petri dish containing Bacto plate count agar media. The inoculated bacteria were incubated at temperature 28–30 °C and total bacteria count was made at 15 and 24-h intervals following Plate Count method with droplet modification (APHA, 1998). Results were expressed as colony forming unit per gram of soil (CFU per g soil). Results on the chlorine demand were analyzed by one-way analysis of variance (ANOVA) while organic carbon, pH, TKN and total bacteria count data obtained at different time intervals were analyzed by two-way ANOVA. These statistical analyses were run using a statistical software (Statistica version 5.0).
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3. Results
3.1. Chlorine demand of pond sediments Soil sediment used in this experiment was categorized as clayey soil (Boyd, 1995). The pH, organic carbon, and TKN concentration among three soil layers before chlorine treatment (Table 1) was not significantly different (P\ 0.05). Free residual chlorine (except before 24 h exposure) and total residual chlorine concentrations of the overlying water decreased over time in all chlorine treatments, while the soil chlorine demands increased (Fig. 1). Concentration of free residual chlorine at the end of experiment was 21, 242 and 367 mg Cl2 l − 1 and total residual chlorine was 58, 466 and 850 mg Cl2 l − 1 for initial chlorine dose of 300, 1200 and 2400 mg Cl2 l − 1, respectively. A whitish color was found covering the surface layer of soil sediment treated with high chlorine dose at 1200 and 2400 mg Cl2 l − 1. Fig. 1 shows that 50% of chlorine applied was consumed in the first 2 days in all chlorine doses, and at the end of the experiment the chlorine consumed was 80, 61 and 65% of the initial concentration of 300, 1200 and 2400 mg Cl2 l − 1 or 0.48, 1.45 and 3.06 kg of active chlorine m − 2 sediment, respectively. Throughout the experimental periods, chlorine demand of the sediment was significantly higher (PB 0.01) at chlorine doses of 1200 and 2400 mg Cl2 l − 1 than that in control and low chlorine dose (300 mg Cl2 l − 1). A clear pattern of chlorine demand in sediment was found on some chemical parameters of soil at layer 0– 0.3 cm at the end of experiment (Fig. 2). Increasing in chlorine dose resulted in increasing soil pH and decreasing concentration of organic carbon and TKN. Soil pH in high chlorine dose (2400 mg Cl2 l − 1) was 0.3 U higher than control while its organic carbon and TKN were 0.6 and 0.13% less than the control. Two-way ANOVA for these parameters among different chlorine doses and sediment layers show that chlorine concentration at 2400 mg Cl2 l − 1 significantly reduced content of organic carbon (PB 0.01) and TKN (PB 0.05) than those in the control. Table 1 Soil texture, organic carbon, pH and total Kjedhal nitrogen (TKN) of shrimp pond sediments before treated with different chlorine doses Soil parameters
Soil texture Clay (%) Silt (%) Sand (%) Organic carbon (%)a Soil pH (CaCl2 5:1)a TKN (%)a a
Mean and S.E.
Soil layers (cm) 0–2.5
2.5–5.0
5.0–7.5
75.83 14.28 9.88 4.059 0.04 7.069 0.06 0.1590.01
86.22 8.18 5.59 4.07 90.10 6.93 90.14 0.14 9 0.03
86.11 10.21 3.68 4.08 90.16 6.86 9 0.22 0.15 90.04
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Fig. 1. Concentrations of free and total residual chlorine (FRC and TRC) in water and chlorine consumption in sediment treated with different chlorine doses measured at 0 (black column), 6 (grey column), 24 (blank column), 48 (dotted column) and 144 h (striped column). Bars indicated standard error (S.E.).
Correlation analysis on chlorine demand with these three soil parameters at 0–0.3 cm layer at the end of experiment revealed that positive correlation with soil pH (r = 71), and negative correlation with organic carbon (r= 0.83) and TKN (r=0.83). For other soil layers, a weak correlation was found between chlorine demand and these three soil parameters.
3.2. Total bacteria count Inactivation of bacteria in pond sediment was influenced by the chlorine doses and exposure time (Fig. 3). Low chlorine dose (300 mg Cl2 l − 1) inactivated only
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part of bacteria population at the first hour of exposure, thereafter bacteria started to grow again. At the end of experiment, the bacterial count ranging from 105 to 3 × 105 CFU g − 1 wet soil was not significantly (P\ 0.05) different between control and treatment. However, in a treatment with chlorine dose at 1200 and 2400 mg Cl2 l − 1 the bacteria were inactivated immediately after exposure until the end of experiment. The chlorine dose at 1200 mg Cl2 l − 1 completely inactivated bacteria in 2 days for soil layer at 0– 0.3 cm and 6 days for soil layer at 0.9–1.2 cm. With chlorine dose at 2400 mg Cl2 l − 1, complete inactivation occurred in 6 h for soil layer 0–0.3 cm and 2 days for soil layer 0.9–1.2 cm. Bacterial numbers in these high chlorine doses were significantly smaller (PB0.01) than in control and in treatment of 300 mg Cl2 l − 1. At the end of experiment, no bacteria were found in
Fig. 2. The pH, organic carbon and total Kjedhal nitrogen (TKN) concentration in soil at layer of 0 – 0.3 cm (black column), 0.9 –1.2 cm (grey column) and 1.8 – 2.1 cm (blank column) of shrimp pond treated with different chlorine doses. Bars indicated S.E.
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Fig. 3. Total bacteria count in soil layer of 0 –0.3 cm (black column), 0.9 – 1.2 cm (grey column) and 1.8 – 2.1 cm (blank column) treated with different chlorine doses during the experiment period. Bars indicated S.E.
soil layer 0– 0.3 and 0.9– 1.2 cm of high chlorine doses, but in layer 1.8–2.1 cm bacteria count was 2.8× 104 CFU g − 1 wet soil and 1.4×104 CFU g − 1 wet soil for chlorine dose at 1200 and 2400 mg Cl2 l − 1, respectively. The results clearly show that the total bacteria count increased in deeper soil layer even at high chlorine doses at 1200 and 2400 mg Cl2 l − 1 from 24 to 144 h after chlorine exposure. Two-way ANOVA detected a significantly lesser number of bacteria in soil layer 0– 0.3 cm than that in soil layer 0.9–1.2 and 1.8–2.1 cm (PB 0.01). Total bacteria in three soil layers correlated negatively with their chlorine demand, with correlation coefficient of 0.77, 0.62 and 0.52 for soil layer 0–0.3, 0.9 –1.2 and 1.8– 2.1 cm, respectively.
4. Discussion The high percentage of chlorine consumption in pond water occurred during the first 2 days of chlorine exposure in both treatments of low and high chlorine doses. This could be largely related to the process of chlorine degradation, which rate depends on the type of compounds present in the pond water and sediment. As
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chlorine react first with reactive organic compounds (more oxidizable matters) and then with less reactive organic compounds or inorganic decomposition of chlorine (Wong and Davidson, 1977). The presence of greater concentration of organic and nitrogenous compounds in the top soil layer of 0– 0.3 cm consumed more chlorine than in layers of 0.9– 1.2 and 1.8–2.1 cm. Negative correlation (r= 0.83) was found between the chlorine demand with concentration of organic carbon and TKN in soil layers 0– 0.3 cm in all chlorine treatments at the end of the experiment support this explanation. Second, it could be related to the initial concentration of chlorine applied. In treatment with higher chlorine concentration applied, the more chlorine demand was resulted which was partly because of the loss of the undissolved chlorine through precipitating and adsorbing to the surface of sediment. Acher et al. (1997) stated that the efficiency of chlorine application was governed by the chlorine dose and the contact time. These explanations are also the reason for the presence of free and total residual chlorine in low chlorine dose at the end of the experiment. There should be no free and total residual chlorine in chlorine dose at 300 mg Cl2 l − 1 since soil sediment could consumed more than 1500 mg Cl2 l − 1. Results show that lower chlorine dose did not completely inactivate bacteria in the sediment even the free and total residual chlorine were in the range that could kill other aquatic organisms (Boyd, 1996). The presence of high proportion of deposited particles commonly found in bottom of shrimp ponds may harbor bacteria from chlorine effect. A shorter time needed to inactivate bacteria in layer 0–0.3 cm than in deeper layers of high chlorine doses is related to their initial free and total residual chlorine concentrations. Concentration of free and total residual chlorine in surface layer was higher than in deeper layers since chlorine diffusion to deeper layers takes longer time. This also explains that a shorter time was needed for bacteria inactivation in higher chlorine dose of 2400 Cl2 l − 1 than in 1200 mg Cl2 l − 1. Total bacteria number in treatments with chlorine dose of 300 and 1200 mg Cl2 l − 1 ranged from 0 to 3×105 CFU g − 1 of wet soil. The number was smaller than total bacteria number in catfish pond soil tested (106 CFU g − 1) with same chlorine dose (Potts, 1998). In Potts experiment, chlorine dose of 1200 mg Cl2 l − 1 did not completely inactivate bacteria. The different results were probably due to difference in sediment depth and chlorine demand. In Potts experiment, bacteria sample was taken from 5-cm deep sediment while in this experiment the sediment depth used was 2.5-cm where bacteria were completely inactivated at high chlorine dose. In practice, shrimp farmers in Thailand commonly apply a chlorine dose of 30 mg Cl2 l − 1 which may achieve the purpose of disinfecting organisms in pond water, but its certainly inadequate to terminate micro-organisms in the pond bottom. Although the free and total residual chlorine remaining in the water was still sufficiently high to kill aquatic organisms, it was ineffective to treat bottom dwellers. At chlorine dose of 2400 mg Cl2 l − 1, 100% bacteria within 2.1-cm sediment depth was inactivated in 2-day exposure. With this dose the amount of chlorine needed to treat the sediment was 2.11 kg Cl2 m − 2 with free and total residual chlorine in the overlying water at 850 and 1300 mg Cl2 l − 1, respectively. If this dose was applied to the shrimp pond with the assumption of little chlorine loss
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from exposure to sun light, it would require an estimated amount of 2 ton ha − 1 of active chlorine or 3 ton ha − 1 of HTH to treat shrimp ponds effectively. This level of chlorine treatment is neither economically nor environmentally practical. An alternative method should be investigated and found for effective disinfection of shrimp pond from pathogenic microorganisms harbored in pond bottom. We recommend a practical procedure for chlorine application in shrimp ponds. The first step is to apply high chlorine dose to pond sediment with a shallow overlying water of 2– 5-cm depth for 2 days to inactivate the bacteria in the sediment; the second step is to raise the water to a depth for shrimp culture (usually 1.0–1.5 m depth) to dilute the remaining residual chlorine to a level that can inactivate bacteria in the overlying water column. With this procedure, the estimated amount of chlorine needed to disinfected sediment and water for 1 ha of shrimp pond with 2–cm overlying water is 480 kg ha − 1 active chlorine or 740 kg ha − 1 HTH. Acknowledgements The study was supported by a doctoral scholarship (IBRD Loan No. 3886-IND) from Agricultural Research and Management Project Phase II, Agricultural ministry of Indonesia. Authors would like to thank Weena Koeypudsa and Paria Rakhmati, for assistance with this study. References Acher, A., Fischer, E., Turnheim, R., Manor, Y., 1997. Ecologically friendly wastewater disinfection techniques. Water Res. 31 (6), 1398 –1404. American Public Health Association, (Alpha), 1998. Standard methods for the examination of water and waste water, (20th edition) Eaton, A.D., Clesceri, L.S., Greenberg, A.E., Washington, DC. American Society for Testing and Materials (ASTM), 1992. Standard Practice for estimation of chlorine requirement or demand of water, or both. In: Fazio, P.C., Fisher, D., Gutman, E.L., Hsia, C.T., Kaufman, S.L., Kramer, J., Lane, M. (Eds.), Annual Books of ASTM Standards: Water and Environmental Technology, Vol 11.01, American Society for Testing and Materials, Philadelphia. pp. D1291 –89. Boyd, C.E., 1995. Bottom Soils, Sediment, and Pond Aquaculture. Chapman and Hall, New York. Boyd, C.E., 1996. Chlorination and water quality in aquaculture ponds. World Aquacult. 27, 41 – 45. Boyd, C.E., Massaut, L., 1999. Risks associated with the use of chemicals in pond aquaculture. Aquacult. Eng. 20, 113 –132. Cai, W., 1994. Fish disease. In: Li, S., Mathias, J. (Eds.), Freshwater Fish Culture in China: Principles and Practice. Elsevier, Netherland, pp. 387 – 424. Chanratchakool, P., 1995. White patch disease of black tiger shrimp (Penaeus monodon). AAHRI Newsletter 4 (1), 3. Dewis, J., Freitas, F., 1970. Physical and chemical methods of soil and water analysis. Soil Bulletin no. 10. FAO. Rome. 275 pp. Dierberg, F.E., Kiattisimkul, W., 1996. Issue, impacts, and implications of shrimp aquaculture in Thailand. Environ. Man. 20 (5), 649 –666. Harakeh, M.S., 1986. Factors influencing chlorine disinfection of wastewater effluent contaminated by rotaviruses, enteroviruses and bacteriophages. In: Jolley, R.L., Bull, R.J., Davis, W.P., Katz, S., Robert, M.H. Jr, Jacobs, V.A. (Eds.), Water Chlorination: Chemistry, Environmental Impact and Health Effects, vol. 4. Lewis Publisher Inc, USA, pp. 681 – 690.
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