In vitro inactivation of penaeid shrimp larval pathogens, Vibrio iiarveyi and V

splendidus biovar 1, by free chlorine and the influence of organic matter on the

bactericidal activity of chlorine were assessed. More than 5 log unit (>99.99%) reduction

in luminous bacteria from >= log 6.00/ml within the first 60 sec of exposure to free

chlorine at 1 ppm level was observed. Chlorine was ineffective at <50 ppm levels to

inhibit luminous Vibrio spp in the presence of 0.1% peptone as interfering organic agent.

These results revealed that luminous bacteria are highly susceptible to chlorine but the

bactericidal activity of chlorine is affected by organic substance

Abraham, T. Jawahar

Dhevendaran, K.

Palamiappan, R.

Aquatic Commons

Bangladesh] Hsh. Res." 6(1), 2002: 53-58 
luminous · spp. by free chlorine 
T. J awahar Abraham*, R. Palamiappan1 and K. Dhevendaran2 
Fisheries College & Research Institute, Tamil Nadu Veterinary & Animal Sciences University 
Tuticorin 628 008, Tamil Nadu, India 
1SPK College, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India 
2Department of Aquatic Biology and Fisheries, University ofKerala, Trivandrum 695 007, India 
*Present address and correspondence: Department of Fishery Pathology & Microbiology 
Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences 
P.O. Krishi Viswa Vidyala, Mohanpur 741 252, West Bengal, India 
Abstract 
In vhro inactivation of penaeid shrimp larval pathogens, Vibrio iiarveyi and V 
splendidus biovar 1, by free chlorine and the influence of organic matter on the 
bactericidal activity of chlorine were assessed. More than 5 log unit (>99.99%) reduction 
in luminous bacteria from ~ log 6.00/ml within the first 60 sec of exposure to free 
chlorine at 1 ppm level was observed. Chlorine was ineffective at <50 ppm levels to 
inhibit luminous Vibrio spp in the presence of 0.1% peptone as interfering organic agent. 
These results revealed that luminous bacteria are highly susceptible to chlorine but the 
bactericidal activity of chlorine is affected by organic substance. 
Key words: Vibrio spp. , Chlorine 
Introduction 
Hatchery production and rearing of penaeid shrimp larvae require good quality 
water (Lavilla-Pitogo et a.J. 1990). Water treatment in shrimp hatcheries usually consists 
of sedimentation and filtration. However, in cases where microbial contamination is a 
problem, such physical treatment is inadequate and some form of disinfectant is needed. 
Chlorine is a powerful disinfectant that has long been used to control microorganisms in 
water. Laboratory and field studies demonstrated the biocidal effect of chlorine on viral 
(LeBlanc and Overstreet 1991) and bacterial pathogens (Sako et a.l. 1988, Pascho et al. 
1995) of aquaculture importance. Vibriosis caused by luminous Vibrio spp., particularly 
Vibrio harveyi, is a problem to shrimp hatchery operations globally (Lavilla-Pitogo et al 
1990, Karunasagar et ai 1994, lvlohney et a.l. 1994, Abraham et al. 1997a). The purpose of 
this work was to examine the jn vitro inactivation of penaeid shrimp larval pathogens 
such as V ~fJarveyi and spiendidus biovar 1 by chlorine and the influence of organic 
matter on the bactericidal activity of chlorine under laboratory condition. 
T.J. Abraham et al. 
Materials and methods 
Test organisms 
Two species of luminous bacteria viz., Vibrio harveyi SW57 and V. splendidus biovar 
1 SWD isolated from penaeid shrimp hatchery source water (Abraham et al. 1997b) were 
used. 
Chlorine solution 
A commercial preparation of sodium hypochlorite contammg available chlorine 
concentration of 20 mg/ml, as determined by the standard iodometric titration method 
(APHA/AWWA/WEF 1995), was used. This solution was diluted in seawater (salinity 35 
ppt), aged for more than 3 months and filtered, to provide 1-100 ppm free chlorine 
(target) concentrations when mixed with the inoculum. 
Interfering substance 
This was used to test the influence of organic substance on the efficacy of target 
chlorine levels. Peptone at 0.1% (w/v), dissolved in aged seawater, was used as interfering 
substance. The medium was adjusted to pH 7.8 and sterilized at 121 °C for 15 min. Half 
strength seawater made from 35 ppt aged seawater was used as diluent. 
Neutralizing solution 
A 0.01 mol/l sodium thiosulphate solution prepared in half strength-aged seawater 
was the neutralizing solution. 
Culture medium and preparation of cell suspensions 
Complex seawater (CSW) medium, with or without 1.5% agar, containing 75% aged 
seawater, 25% distilled water, 0.5% peptone, 0.3% yeast extract and 0.3% glycerol was 
used for the growth and maintenance of luminous Vibrio spp. The pH of the medium 
was 7.80. Cell suspensions of V. harveyi and V. splendidus biovar 1 was prepared 
separately as described in Abraham et al. (1997a). 
Susceptibility of luminous bacteria to free chlorine 
These experiments were designed to reproduce luminous bacterial inactivation, but 
not intended to simulate natural environments. Luminous bacterial inactivation was 
done in Erlenmeyer flasks containing 100 ml of sterile aged seawater (pH 8.0). Sodium 
hypochlorite solution was added to these flasks to get a concentration of 1, 5 and 10 ppm 
of free chlorine separately. The flasks were then inoculated with V. harveyi SW57 and/or 
V. splendidus biovar 1 SW1 at 106 - 107 cells/ml levels and incubated at 30±1 oc. The 
numbers of bacteria in each flask were determined after 1 min, 30 min and 24 h of 
exposure to chlorine and 24 h after neutralization. The effect of neutralizing agent, 0.01 
54 
Inactivation of luminous Vibrio spp. by free chlorine 
mol/1 sodium thiosulphate in half strength seawater, on luminous bacteria was evaluated 
by incubating the cells in neutralizing solution for 30 min. The numbers of bacteria 
before and after exposure to neutralizing agent were determined. 
Enumeration of luminous bacterial counts (LBC) was done by spread plating on 
CSW agar and/or by 5 tube most probable number (MPN) technique using CSW b:roth. 
Before enumeration, the residual chlorine present in one ml each of the samples drawn 
from chlorine treated flasks were neutralized by vigorous agitation in sterile 9 ml of 0.01 
mol/1 sodium thiosulphate solution (10-1 dilution). Subsequent ten fold serial dilutions 
were made in sterile half strength seawater. All the plates and tubes, after inoculation, 
were incubated at 30±1 °C for 24-72 h. Aliquots from CSW broth were streaked on to 
CSW agar and incubated for 48 h at 30±1 °C to record MPN value. Bacterial numbers 
were recorded as counts/mi. All experiments were repeated at least 3 times and the 
percentage survival calculated. 
Results and discussion 
Aquaculture is a large consumer of chlorine products. The use of chlorine has been 
recommended to eliminate shrimp pathogens in hatcheries (Baticodos and Pitogo 1990, 
LeBlanc and Overstreet 1991, Lewis et a11992) and as a disinfectant and sanitary agent 
for fish tanks, raceways, utensils, contaminated equipments and effluents at 200 mg/1 
level (LeBlanc and Overstreet 1990, Pascho et a11995). The results presented in Table 1 
showed that both V. harveyi and V. splendidus biovar 1 reacted in a more or less 
identical way to the bactericidal effect of chlorine. A reduction of more than 5 log unit 
(>99.99%) from >log 6.00/ml was achieved within the first 60 sec of exposure to free 
chlorine at 1 ppm level in the absence of any interfering agent, which in~icated that 
luminous bacteria are highly susceptible to free chlorine. At 1 and 5 ppm levels, 
luminous bacterial populations were completely eliminated and no recovery was possible 
even after neutralization with 0.01 mol/1 sodium thiosulphate and enrichment in CSW 
broth. Similar chlorine effect was reported for luminous bacteria (Baticados and Pitogo 
1990), V. anguillarum and V. ordalii (Sako et al. 1988), Renibacterium salmoninarum 
· (Pascho et al 1995) .. The mechanisms, of chlorine inactivation vary among 
microorganisms and are a result of general oxidation of reduced chemical species. The 
populations of V. harveyi (log 6.778/ml) and V. splendidus biovar 1 (log 6.873/ml) 
exposed to thiosulphate neutralizer for up to 30 min failed to show any difference from 
the respective counts of log 6. 781/ml and log 6.872/ml determined before exposure. The 
neutralizer was, therefore, assumed to exert no effect on results. 
55 
T.}. Abraham era!. 
Table l. Effect of sodium hypochlorite on the growth (counts/ml) of Vibrio harve_vi and V 
splendidus biovar l in sterile seawater 
Treatment/ Vibrio harveyi SW57 v splendidus biovar 1 swl 
Exposure time 
I 1 ppm Sppm 1 ppm 5 ppm 
Before chlorination 2.50xl06 4.45xl06 2.20xl06 2.80xl06 
After chlorination 
l min 1.00xl01 <l.00xl01 1.00xl01 <1.00 
30 min <1.00 <1.00 <l.OO <1.00 
24h <1.00 <1.00 <1.00 <1.00 
After neutralization 
24 h <1.00 <1.00 <1.00 <1.00 
Results on the interference of organic substance on the bactericidal activity of free 
chlorine, as shown in Table 2, revealed that chlorine is ineffective at <50 level to 
inhibit luminous bacteria ( V. harveyi and V. splendidus biovar 1) in the presence of 0.1% 
peptone. No bact~ricidal effect was seen at 1 ppm level. .At 5-20 ppm levels, the LBC 
reduced slightly immediately after chlorination and then increased to ;:::: log 8.00 ceHs/mi 
in 24 h. Chlorine effect was apparent at 50 ppm level. Chlorination and oxidation 
reaction between amino group and chlorine are most likely responsible for this effect. 
These results corroborate with the findings of earlier studies (Sae-Oui et al 1987, 
Chanratchakool 1995) . .According to Chanratchakool (1995), the minimum effective 
concentration of hypochlorite to inhibit V. harveyi and other Vibrio spp was 2-8 ppm 
and 2-16 ppm of active chlorine, respectively. About 16 ppm of.active chlorine was 
required for disinfection of water from the shrimp farm cont~i.ning .. > log 4.00 
organisms/ml. Sae-Oui et al. (1987), however, reported that f h_ajvejd could .. _be 
completely killed by treating with calcium hypochlorite at 20-30 ppm. Nevertheless, 
Karunasagar et aL (1995) using microcosm experiments demonstrated that chlorination 
would not kill V. harveyipresent in sediments and, the~;~fore, repopulation of the system 
occurs immediately after dechlorination: The result;S,, of the present in vitro study also 
confirmed that chlorine treatment not'ehminate shrimp larval pathogens such as 
V. harveyi and V. splendidus biovar 1 in a system where particulate and suspended 
organics are present. 
56 
Inactivation of luminous Vibrio spp. by free chlorine 
Table 2. Interference of 0.1% peptone on bactericidal effect of sodium hypochlorite: effect on. 
luminous bacteria 
Treatment/ Counts I ml 
Exposure time lppm 5ppm lOppm 20ppm 50ppm lOOppm 
Vibrio harveyi SW57 
Before chlorination 5.60xl06 7.80xl06 9.00xl06 6.00xl06 4.75xl06 5.20xl06 
After chlorination 
lmin 5.60xl06 6.90xl06 8.00xl06 2.80xl06 1.50xl01 <1.00 
30 min 5.65xl06 7.30xl06 8.30xl06 1.30xl04 <1.00 <LOO 
Vibrio splendidus 
biovar 1 swl 
Before chlorination 4.30xl06 4.70xl06 7.50xl06 6.10xl06 8.00xl06 5.00xl06 
After chlorination 
lmin 4.25xl06 3.45xl06 4.50xl06 l.50xl06 l.OOxl0 1 <1.00 
30 min 4.30xl06 3.55xl06 4.65xl06 6.10xl03 <1.00 <LOO 
The counts of Vibrio harveyi SW57 and V. splendidus biovar 1 SW1 were increased after 30 min in 1,5,10 and 
20 ppm levels. No growth of luminous bacteria was seen in 50 and 100 ppm levels even after 24 h. 
It is worth mentioning here that sodium hypochlorite was demonstrated to be 
effective at relatively high concentrations, 2:50 ppm, and for longer period of time for 
the control ofbaculovirus in shrimp mariculture facilities (Lewis et al 1992). In shrimp 
grow-out systems, chlorine disinfection is being followed in the reservoirs, although 
with variable success, to inactivate the causative agent of white spot viral disease and 
other bacterial pathogens. As majority of the components of source water of the shrimp 
aquaculture systems are particulate and suspended organics, these components must, 
therefore, be thoroughly removed by mechanical filtration or by sedimentation for 
effective hatchery disinfection protocol. 
References 
Abraham, T.J., R. Manley, R. Palaniappan and K. Dhevendaran, 1997a. Pathogenicity and 
antibiotic sensitivity of luminous Vibrio harveyi isolated from diseased penaeid shrimps. J 
Aquacult. Tropics~ 12: 1-8. 
Abraham, T.J., R. Palaniappan and K. Dhevendaran, 1997b. Epibiotic infestation of luminous 
bacteria in penaeid shrimp Penaeus indicus(H Milne Edwards) larvae. Indian] Mar. Sci, 26: 
209-212. 
APHAIAWWA/WEF, 1995. Standard methods for the examination of water and wastewater. 19'h 
edition (eds. A.D. Eaton, L.S. Clesceri and A.E. Greenberg). American Public Health 
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T.J. Abraham er al. 
Karunasagar, I., R. Pai, G.A. Malathi and Indrani Karunasagar, 1994. Mass mortality of Penaeus 
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Karunasagar, I., S.K. Otta and Indrani Karunasagar, 1995. Effect of chlorination on shrimp 
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Lavilla Pitogo, C.R., M.C.L Baticados, E.R. Cruz-Lacierda and LD. DelaPena, 1990. Occurrence 
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Mohney, LL, D.V. Lightner and T.A. Bell, 1994. An epizootic of vibrios1~ in Ecuadorian pond 
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(Manuscript received 10 September 2001) 
58 


Inactivation of luminous Vibrio spp. by free chlorine

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Inactivation of luminous Vibrio spp. by free chlorine

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