Lethal and sub-lethal effects of chlorination on green mussel perna viridis in the context of biofouling control in power plant cooling water system. Int J Marine Environmental Research, 53(1), p65-76, (2002).

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Lethal and sub-lethal effects of chlorination on green mussel perna viridis in the context of biofouling control in power plant cooling water system. Int J Marine Environmental Research, 53(1), p65-76, (2002).
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  Lethal and sub-lethal effects of chlorination ongreen mussel  Perna viridis  in the context of biofouling control in a power plant coolingwater system Gunasingh Masilamoni a, *, K.S. Jesudoss a , K. Nandakumar b ,K.K. Satapathy b , J. Azariah a , K.V.K. Nair b a Department of Zoology, Madras University, Chennai 600 025, Tamil Nadu, India b Water and Steam Chemistry Laboratory, BARCF Indira Gandhi Center for Atomic Research campus,Kalpakkam, Tamil Nadu 603 102, India Received 8 October 2000; received in revised form 18 May 2001; accepted 11 June 2001 Abstract Continuous chlorination is a widely followed cooling water treatment practice used in thepower industry to combat biofouling. The green mussel  Perna viridis  is one of the dominantfouling organisms (>70%) in the Madras Atomic Power Station. Mortality pattern as well asphysiological responses such as oxygen consumption, filtration rate, byssus thread productionand faecal matter production of three different size groups of this mussel were studied at dif-ferent chlorination concentrations. At 0.7 mg l  1 residual chlorine, 3–4 cm size musselsshowed 100% mortality in 553.3 h while 8–9 cm size group mussels died within 588 h. At arelatively high level of residual chlorine (9.1 mg l  1 ), 100% mortality in 3–4 cm and 8–9 cmsize groups took 94 and 114 h, respectively. All physiological activities studied showed aprogressive reduction as chlorine residuals were increased from 0 to 0.55 mg l  1 . The dataindicated that the green mussel can sense a residual chlorine level as low as  <  0.15 mg l  1 andcomplete valve closure occurs only at 0.55 mg l  1 . The paper also shows that the sub-lethalphysiological responses are better indices than lethal responses in planning chlorinationstrategies. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Perna viridis ; Biofouling; Chlorination; Mortality; Oxygen consumption; Filtration rate;Byssus thread production; Faecal matter productionMarine Environmental Research 53 (2002) 65–76www.elsevier.com/locate/marenvrev0141-1136/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.PII: S0141-1136(01)00110-6* Corresponding author. Present address. National Institute of Ocean Technology, (Department of Ocean Development Government of India, Velachery-Tambaram Main Road, Narayanapuram Village,Pallikaranai (Post) Chennai—601 302, Tamil Nadu India. E-mail address:  gunasingh@hotmail.com (G. Masilamoni).  1. Introduction Screening of the cooling water flow and cleaning the plant are effective foulingcontrol strategies as is thermal recirculation. Chemical dosing at sub-lethal levels is,at least in the temperate zone, regarded only as a means of deferring a physicalclean-up. Continuous low level chlorination is widely considered to be the mosteffective dosing strategy for controlling fouling in power plant cooling water con-duits. Chlorine was first used for fouling control in 1924 at the Northwest powerplant in Chicago, United States of America (Holmes, 1970). In recent years, the useof chlorine for fouling control in power plant cooling water systems has becomesubject to increasing regulatory pressures due to possible ecological impacts of therecirculating water (Jenner, Taylor, van Donk, & Khalanski, 1997). In India,Madras Atomic Power Station (MAPS) practises continuous low dose chlorinationto control fouling growth. Chlorine is used as a biocide in the cooling systems forthe following reasons: (1) direct toxic effect on adult organisms (2) inhibition of thesettlement and growth of larvae (3) weakening of the mechanisms by which organ-isms remain attached to the substratum (4) availability at a reasonably low cost (5)simple feed system, and control at low capital cost, and (6) minimal feed systemmaintenance (Claudi & Mackie, 1994). The study reported here was based upon anexperimental laboratory system having a once-through seawater flow in whichchlorine concentrations can be controlled. The objective of this study was to under-stand the lethal and sub-lethal responses of green mussels subjected to varyingchlorine concentrations. 2. Materials and methods Green mussels ( Perna viridis ) were collected from the concrete pillar near theintake area of MAPS and immediately transported to the laboratory. Mussels wereplaced in a 100-l aquarium tank containing a well aerated continuous flow of sea-water. Mussels were segregated into three groups based on their tissue dry weightand shell size such: group I 0.43  0.11 g (3–4 cm), Group II 0.75  0.09 g (5–6 cm),Group III 1.25  0.21 g (8–9 cm). Mussels were acclimated in seawater (34.3  0.1 % salinity, 8.2  0.1 pH and 31.3  0.3   C) for 48 h under laboratory conditions. Foreach experiment animals were taken from this acclimated stock. Epizoic organismsand byssus threads were removed before experimentation. 2.1. Experiment 1. Time to 100% mortality The time taken for 100% mortality in the three size groups of mussels was testedin five different residual chlorine concentrations (0.72  0.21, 1.36  0.28, 3.17  0.26,5.22  0.34 and 9.7  0.31 mg l  1 ). The experiments were conducted in the con-tinuous once-through flow system with a seawater (34.3  0.1 ppt salinity, 8.4  0.1pH and 30.7  0.3   C ) flow rate of 120 ml min  1 . Each experimental tank contained25 l of seawater with 25 animals belonging to one of the size groups. The seawater 66  G. Masilamoni et al./Marine Environmental Research 53 (2002) 65–76  and chlorine flow to the individual tanks was supplied through a multichannelperistaltic pump. Chlorine stock was prepared by mixing sodium hypochlorite indistilled water. Residual chlorine concentrations in the tanks were measured hourlyby iodometric titration (White, 1972) by collecting water from the outlet. The sen-sitivity of the method is 0.05 mg/l. Animals were considered dead if they failed torespond to external stimuli. The number of animals that died during each experi-ment was recorded. The experiment was repeated four times for each size group andat each residual chlorine concentration. 2.2. Experiments 2–5: sub-lethal physiological responses2.2.1. Oxygen consumption Oxygen consumption by the organisms was determined by the method outlined bySasikumar, Nair, and Azariah (1992), Masilamoni, Jesudoss, Nandakumar, Sat-pathy, Azariah, and Nair (1997). In each experimental set the oxygen concentration,both initial and after 1 h, and the residual chlorine levels were monitored. The oxy-gen content was determined using the Winkler method (Strickland & Parsons, 1972).Four sub-lethal residual chlorine concentrations such as 0.12  0.04, 0.29  0.07,0.39  0.09, 0.53  0.08 mg l  1 were tested ( n =5 for each treatment). Controls (nochlorine) were run in parallel with the experimental set with mussels of the same sizegroup. The effect of chlorination on the rate of oxygen consumption by the animalsin the experimental set was thus determined. 2.2.2. Filtration rate Filtration rate was estimated following the method of Coughlan (1969). Thismethod is based on the rate of absorption of Neutral Red by the mussels. Con-centration of neutral red (1 mg l  1 ) was prepared in Millipore (0.45  m m) filteredseawater. The test organisms were introduced into a 2-l beaker containing the dyealone (control) and four other sets ( n =5 for each treatment) with different sub-lethalconcentrations of chlorine and dye (0.15  0.04, 0.28  0.1, 0.41  0.09, 0.55  0.07mg l  1 ). Ten-millilitre aliquots of the seawater were drawn at 30-min intervals for 1h and the concentration of the dye in each aliquot was determined by measuring theoptical density at 530 nm in a Shimadzu Graphicord UV 240 Spectrophotometer.The chlorine concentration was determined by iodometric titration. The rate of fil-tration was calculated using the equation of Coughlan (1969): m  ¼  M = nt ð Þ  Log Co = C 1 ð Þ where,  M  =volume of the test solution;  n =number of mussels used;  t =time (h);Co= initial dye concentration; C1=concentration of dye after time  t ;  m =filtrationrate (litre mussel  1 h  1 ). 2.2.3. Byssus thread production The number of byssus threads produced by the animals was determined followingthe method outlined by Winkle (1970), Rajagopal, Venugopalan, Nair, and Azariah(1995), Masilamoni et al. (1997). Five mussels were kept in 2-l glass beakers with G. Masilamoni et al./Marine Environmental Research 53 (2002) 65–76  67  continuously flowing seawater (control) and at four different chlorine concentrations(residual chlorine concentration: 0.15  0.06, 0.31  0.08, 0.38  0.09, 0.45  0.1 mgl  1 ). The residual chlorine content was measured hourly and the number of byssusthreads produced was counted after 24 h using a hand lens and expressed as numberof threads mussel  1 h  1 . 2.2.4. Faecal matter production The faecal matter production was estimated following the method of Clark (1990),Masilamoni et al. (1997). Five mussels were introduced into a 2-l beaker with acontinuous sea water flow alone (control) and with chlorine (residual chlorine levels:0.14  0.03, 0.29  0.06, 0.41  0.1 and 0.47  0.09 mg l  1 ). The outlet from eachbeaker was covered with a 40- m  nylon mesh to prevent the escape of faecal matter.After 24 h, the faecal pellets were allowed to settle and were removed using a pasteurpipette. The material was then transferred on to a clean aluminum foil platform. Thefaecal pellets were rinsed several times with distilled water to remove the salt con-tent. This was then dried at 60   C for 24 h in an oven and weighed and expressed asmg dry weight mussel  1 day  1 . 2.3. Results2.3.1. Time to 100% mortality The time taken for 100% mortality in three different size groups of   P. viridis  inrelation to the different residual chlorine levels is shown in Fig. 1. Results showedthat the mortality rate depended on the size of the mussels. That is smaller animals(group I) died faster than that of the larger animals (group III). Similarly, as theresidual chlorine concentration increased from 0.72  0.21 to 9.7  0.31 mg l  1 the time to 100% mortality declined from 588 h to 95.5 h (Fig. 1). Thus, the timetaken for 100% mortality significantly depended on the size of the mussels as well asthe residual chlorine concentration (two-way ANOVA, Table 1). No mortality wasfound in the control. 2.3.2. Oxygen consumption The oxygen uptake by mussels declined with increasing chlorine concentrationand body size (Fig. 2). For example, group I mussels consumed 0.82  0.12 ml h  1 inthe control but only 0.39  0.05, 0.15  0.1 and 0.046  0.036 ml h  1 in 0.12  0.04,0.29  0.07 and 0.39  0.09 mg l  1 residual chlorine levels, respectively. Group I andII mussels ceased respiration at 0.53  0.08 mg l  1 residual chlorine, whilst only avery low rate of respiration (0.088  0.006 ml h  1 ) was observed in group III musselssubjected to the same regime. The results of a two-way ANOVA showed that sizeand chlorine concentration both significantly affected the oxygen consumption inmussels (Table 1). 2.3.3. Filtration rate The filtration rate of different size groups of   P. viridis  at various residualchlorine levels is shown in Fig. 3. Filtration rate varied according to size groups and 68  G. Masilamoni et al./Marine Environmental Research 53 (2002) 65–76  chlorine level. In the control group I animals filtered 0.71  0.09 l h  1 while groupIII mussels filtered 1.31  0.12 l h  1 . The filtration rate reduced markedly as resid-ual chlorine level increased. Group I mussels filtered 0.317  0.051 l h  1 and0.038  0.029 l h  1 when the chlorine concentrations were 0.15  0.04 and 0.41  0.09mg l  1 , respectively. The results of a two-way ANOVA showed that the filtrationrates were significantly different ( P < 0.0001) with respect to the size of the musselsand chlorine concentration (Table 1). 2.3.4. Byssus thread production The data on byssus thread production of mussels belonging to different sizegroups at different residual chlorine levels are presented in Fig. 4. Data showedthat the byssus thread production was inversely related to the size of the mussels.In the controls smaller sized animals (group I) produced more byssus (1.58  0.026h  1 ) than larger sized animals (group III; 1.23  0.067 h  1 ). This trend was entirelyreversed in the presence of chlorine. For instance, at 0.15  0.06 and 0.38  0.09mg l  1 residual chlorine levels the byssus thread production in group I was0.50  0.094 and 0.058  0.012 and in group III it was 0.81  0.046 and 0.27  0.039,respectively. Fig. 1. Time taken for 100% mortality in three different size groups of   Perna viridis  at different levels of chlorination. Vertical bars indicate standard deviation ( n =25). G. Masilamoni et al./Marine Environmental Research 53 (2002) 65–76  69
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